SGDEPARTMENT OF HEALTH AND HUMAN SERVICES

> FOOD AND DRUG ADMINISTRATION
>
> CENTER FOR FOOD SAFETY AND APPLIED NUTRITION
>
> **CONTAMINANTS AND NATURAL TOXICANTS SUBCOMMITTEE**
>
> **OF THE FOOD ADVISORY COMMITTEE**
>
> **VOLUME I**
>
> Tuesday, March 18, 2003
>
> 8:00 a.m.
>
> 4700 River Road
>
> Riverdale, Maryland
>
> PARTICIPANTS
>
> Francis Fredrick Busta, Ph.D. Co-Chair
>
> James E. Heubi. M.D. Co-Chair
>
> MEMBERS
>
> Alex D.W. Acholonu, Ph.D.
>
> Lawrence J. Fischer, Ph.D.
>
> Marion H. Fuller, D.V.M.
>
> Lawrence N. Kuzminski, Ph.D.
>
> Ken Lee, Ph.D.
>
> TEMPORARY VOTING MEMBERS
>
> James Anderson, Ph.D.
>
> Robert D. Baker, M.D., Ph.D.
>
> Larry R. Beuchat, Ph.D.
>
> Henry M. Blumberg, M.D., Ph.D.
>
> Margaret E. Briley, Ph.D., R.D.,L.D..
>
> Laurie J. Moyer-Mileur, Ph.D., R.D., C.D.
>
> Marguerite A. Neill, M.D.
>
> Virginia A. Stallings, M.D.
>
> Phillip Tarr, M.D.
>
> Patti Thureen, M.D.
>
> NON-VOTING INDUSTRY REPRESENTATIVE
>
> Toby L. Simon, M.D.
>
> TEMPORARY VOTING MEMBERS
>
> R. Bruce Tompkin, Ph.D.
>
> [C O N T E N T S]{.underline}
>
> [PAGE]{.underline}
>
> Welcome, Introduction and Charges 5
>
> Christine J. Taylor, FDA
>
> Administrative Issues 16
>
> Jeanne Latham, FDA
>
> Remarks by Chairpersons 21
>
> **Presentations by Guest Speakers**
>
> Current Marketing and Use of Powdered
>
> Infant Formula 27
>
> Sue Ann Anderson, FDA
>
> Tennessee Investigation 48
>
> Matthew Kuehnert, CDC \[via speakerphone\]
>
> Other Relevant Investigations 88
>
> Karl Klontz, FDA
>
> Clinical Consequences of E. Sakazakii Infections 121
>
> John Alexander, FDA
>
> General Microbiology - Ecology, Pathogenicity,
>
> Subtyping, Etc. 171
>
> Maria Nazarowec-White, Canada
>
> Microbial Detection\--Clinical and Food 197
>
> Don Burr, FDA
>
> Resistance\--Thermal and Other 221
>
> Robert Buchanan, FDA
>
> FDA Field Survey of Powdered Formula
>
> Manufacturing 246
>
> Don Zink, FDA
>
> Public Comments/Industry Speakers
>
> Jon A. Vanderhoof 320
>
> Les Smoot 329
>
> Russell Merritt 347
>
> Jatinder Bhatia, M.D. 351
>
> Preliminary Subcommittee Discussion on Clinical Presentations 388
>
> [P]{.underline} [R]{.underline} [O]{.underline} [C]{.underline}
> [E]{.underline} [E]{.underline} [D]{.underline} [I]{.underline}
> [N]{.underline} [G]{.underline} [S]{.underline}
>
> **Welcome, Introduction and Charges**
>
> DR. BUSTA: Good morning. We\'re here to convene the Contaminants and
> Natural Toxicants Subcommittee Meeting on Enterobacter sakazakii
> Contamination in Powdered Infant Formula.
>
> The first presentation this morning will be by Christine Taylor, who
> is the director of this group. And without going any farther, I will
> just turn this over directly to Christine from the start.
>
> DR. TAYLOR: Welcome and good morning. I am Christine Taylor, Director
> of the Office of Nutritional Products, Labeling and Dietary
> Supplements. Thank you for all for agreeing to participate in the
> advisory subcommittee meeting. You are an illustrious group of experts
> and we are looking forward to your discussions today and tomorrow.
>
> My job here today is to set the stage for the more in-depth
> presentations and white papers you will soon hear and to set the stage
> for the regulatory context that leads us to you ask you questions
> about Enterobacter sakazakii and powdered infant formula. As I am sure
> most of you know, FDA\'s mission is ensure a safe and adequate food
> supply. And recent findings have led us to ask questions about E. sak
> and powdered infant formula.
>
> I think it\'s important to recognize that within the Center we view
> not only the regulatory issue of powdered infant formula, but also it
> raises some interesting microbial questions. So, in pulling together
> this effort, many factors and groups within the Center were involved.
>
> I think the first point I want to make is a kind of tried and true
> one, which is that obviously one of the reasons we\'re here is that
> FDA has the mission of ensuring a safe and adequate food supply, and
> certainly infant formula falls into that. And the short summary\--the
> one sentence reason for being here\--is that recent findings have
> raised questions about Enterobacter sakazakii encountered in infant
> formula.
>
> \[Slide.\]
>
> Clearly, in addressing this issue, FDA needed extensive scientific
> input. It needed to be of the microbiological kind, the physiological
> and clinical kinds, particularly in the world of infants, and the
> manufacturing and processing of powdered infant formula. This required
> a diverse set of expertise, and the subcommittee that you see sitting
> amongst yourselves, before yourselves and in front of yourselves
> really include quite a wide range of expertise, including temporary
> voting members to round out expertise. And Ms. Latham later on will
> clarify various members\' roles.
>
> Could I have the next slide?
>
> \[Slide.\]
>
> As I\'ve mentioned before, our focus on infant formula. Infant formula
> is a product regulated by the Food and Drug Administration. And
> currently, in-place GMPs do not specify microbiological testing for E.
> sak. Infant formula, as probably many if not all of you know, comes in
> two forms: powdered and liquid, and the powdered, which is the subject
> of this advisory committee meeting, in terms of dollar sales, is about
> half of the infant formula sold.
>
> Just a little bit of regulatory context: there is an Infant Formula
> Act. It\'s part of the Food, Drug and Cosmetic Act. And this
> particular set of regulatory specifications does indicate that
> manufacturers must retain records pertaining to the microbiological
> quality and purity of their product. I do want to point out in the
> next slide, however, that Infant formula good manufacturing practices,
> while proposed, have not yet been finalized. And, more specifically,
> there are no current GMP regulations for powdered products. Many of
> you may know that liquid formula comes under the Novak and Tennessee
> provisions, but powdered formula have no such provisions.
>
> FDA has proposed GMPs to include practices to minimize opportunity for
> microbial contamination.
>
> Next slide.
>
> \[Slide.\]
>
> You\'ll note that despite listing a number of microbial issues, the
> agency did not specify, nor has it specified, provisions for
> Enterobacter sakazakii
>
> In short, what I\'ll try to do in the next few moments is tell the
> story of Enterobacter sakazakii in powdered infant formula, and then
> briefly go over the charges to the committee; the questions that FDA
> is asking.
>
> At that point, I will turn the meeting over to your two co-chairs. The
> Committee is chaired by two very prestigious individuals, and they
> will do a presentation of background information\--a series of white
> papers\--and then proceed to an open comment period, followed by a
> discussion set of recommendations.
>
> The staring point for the Enterobacter sakazakii was in April of 2001,
> an infant in Tennessee died of meningitis with E. sak. It was a
> premature infant in a neonatal intensive care unit or NICU. The
> Centers for Disease Control and Prevention investigated the situation
> and determined a link between the infant\'s death and E. sak in the
> powdered formula fed to the infant in the NICU. Because infant formula
> manufacturing is under the purview of FDA, the CDC notified FDA. In
> response to the CDC input, FDA undertook several activities. The
> Infant formula\--what was left of it on the market\--was recalled.
> And, in addition, FDA conducted outreach to health care professionals.
> The agency began to examine products and ingredients used to make this
> formula, and manufacturing, for the presence of E. sak, and we .
> conducted some related research.
>
> In the area of outreach, we obviously focused on health care
> professionals in hospitalized settings, because the incident at hand
> did occur in the NICU. We drafted and disseminated very widely a
> letter to health professionals. We created and posted an FDA \"talk
> paper\" on our website. And then in addition, CDC itself published its
> findings in reported the Enterobacter sakazakii in the MMWR.
>
> In our letter to health professionals we emphasized certain things.
> Powdered infant formula was a potential source of E. sak infections in
> infants, and we pointed out that the risk increased with prematurity
> and any underlying medical conditions. We indicated that low levels of
> E. sak can lead to infection and we also highlighted the CDC findings
> in Tennessee regarding the fatal meningitis case. In addition, we
> recommended that powdered infant formulas not be used in the NICU,
> unless there was no alternative available. Oftentimes powdered
> formulas are specialized formulas, and some physicians have indicated
> alternatives wouldn\'t be available And we also provided suggestions
> for reducing infection risks. These included the use of boiling water
> and certain feeding and related practices in hospitals.
>
> We did, later on, revise this letter to health professionals, and we
> focused largely on the question of boiling water preparation because
> possible problems associated with the loss of heat-sensitive
> nutrients, the physical changes to the formula, and perhaps even
> inadequate E. sak eradication, as well as injury to persons preparing
> the formula. And this was done in October of this year.
>
> A second part, in addition in addition to our letter and outreach to
> help health care professionals, involved FDA\'s actually going into
> plants and testing infant formula, as well as infant formula
> ingredients.. Our purpose then was to look for E. sak in powdered
> infant formula products and the raw ingredients. We sampled all
> domestic powdered infant formula facilities as well as all contract
> and manufacturing facilities. And, in addition, for transparency and
> clarity, we also posted the analytical method we were using on our
> website.
>
> FDA, as a result of this sampling, did find some positive low levels
> in both finished product and in the raw ingredients. It\'s been in
> positive finished products that we have identified that have already
> been marketed, and there were two such products that were recalled
> voluntarily by the manufacturers, and no known cases of E. sak
> infection resulted from the consumption of these products is known.
>
> In addition, we did conduct some related research. As I\'ve mentioned,
> we worked on the method for protecting from Enterobacter sakazakii in
> powdered formula. We also did various studies on heat \[inaudible due
> to technical difficulty\]. And also we conducted some studies to show
> effects of water temperature of nutrient levels in infant formula,
> which appear
>
> at this point to not have been finalized.
>
> In addition, there were related activities in response to the FDA
> outreach. Pediatric hospitals and laboratories also \[inaudible due to
> technical difficulty\].
>
> The industry itself conducted analyses and investigations, and as
> probably many of you know they worked hard not to distribute products
> that were found possibly to contain this particular strain.
>
> I\'m here largely to give you the story of why we\'re here, and what
> you\'ll find out after the opening of this meeting is the specifics of
> much of what I\'ve discussed, ranging from the specifics on the
> Tennessee case, to the FDA efforts in product sampling. And so, with
> your indulgence, I\'ll simply give an overview for now to you that
> later on this morning there will be a variety of white-papers
> presented, designed to give you the specifics on available information
> once we get the final documents.
>
> We\'ll begin with the current marketing and release of powdered infant
> formula, fun through the overview of the Tennessee investigation and
> other relevant investigations, in Enterobacter sakazakii. We\'ll
> provide for you information on the clinical consequences of the E. sak
> investigation, as well as some of the general microbiology and
> detection and \[inaudible due to technical difficulty\], and then end
> with the findings from our previous survey in the infant formula
> industry.
>
> Given all of that, there are two basic charges to this particular
> subcommittee. The first charge is a request to characterize the
> infants at risk, relative to Enterobacter sakazakii in powdered infant
> formula; if there is a risk, to identify the populations of infants at
> risk, with due consideration to whatever factors are relevant to
> morbidity, other related areas, such as age, on immune status.
>
> The second charge to the subcommittee begins: \"If there is a
> meaningful risk, how can this risk be addressed? What intervention
> strategy can be used in infant formula processing plans? Are there
> other intervention strategies? We\'ve taken a look at the possibility
> of labeling and directions for use. Is it possible, based on available
> information, to specify allowable lower levels of microbial detection
> of E. sak in infant formula, and do allowable levels vary by risk
> characteristics of the infant. And then, finally, what are the
> critical knowledge gaps and research priorities.
>
> Those are basically the two charges to the subcommittee.
>
> There is a final point relative to our efforts to finalize the infant
> formula final rule on GMPs. The agency will be reopening the comment
> period for this final rule. Any issues that may come from the
> discussions today and tomorrow that would be relevant to the final
> rule can be commented upon by interested parties at the time of that
> comment period.
>
> Again, I want to mention that I\'m so pleased and very proud of the
> fact that the agency was able to convince the important people here at
> the table to come and discuss the details of this very interesting
> issue, and also emphasize that I am telling an initial story, and many
> of you might have questions about, in terms of either the outbreak in
> Tennessee or the FDA\'s activities, will be both addressed and
> answered by others following me.
>
> And my role\--I\'m setting the regulatory context about FDA\'s
> responsibility in the area of Infant formula, and will end right
> there.
>
> Thank you all again. I\'m happy to take questions.
>
> DR. BUSTA: Any questions or clarification from the committee?
>
> \[No response.\]
>
> DR. TAYLOR: I also, sitting at the table here, will answer questions
> that may come up. Thank you.
>
> DR. BUSTA: The next item on the agenda is the administrative issues,
> and Jeanne Latham will cover those.
>
> **Administrative Issues**
>
> MS. LATHAM: Good morning. Is this on? I\'m Jeanne Latham. I will be
> replacing Dr. Henry Kim as the Executive Secretary of the FDA
> Contaminants and Natural Toxicants Subcommittee of the Food Advisory
> Committee for the purposes of this meeting.
>
> I want to welcome everybody, and I\'d like to read the conflict of
> interest statement for the record.
>
> By the authority granted under the Food Advisory Committee Charter of
> July 202, the following individuals have been appointed as temporary
> voting members by Joseph A. Leavitt, Director, Center for Food Safety
> and Applied Nutrition: James Anderson, Robert Baker, Larry Beuchat,
> Henry Blumberg, Margaret Briley, James Heubi, Laurie Moyer-Mileur,
> Marguerite Neill, Virginia Stallings, and Patti Thureen. Dr. Phillip
> Tarr is a special government employee to FDA Center for Drug
> Evaluation and Research, and is serving as a temporary voting member
> under the authority of Lender Ari Skledeny, Associate Commissioner for
> External Relations.
>
> The issues to be discussed at this meeting are issues of broad
> applicability. Unlike issues in which a particular sponsor\'s product
> is discussed, the matters at issue do not have a unique impact on any
> particular product or manufacturer, but may have widespread
> implications with respect to all infant formulas and their
> manufacturers. To determine if any conflicts of interest exist, the
> committee participants have been screened for interest in companies
> that make infant formula. As a result of this review, in accordance
> with Title 19 of the U.S. Code, Section 208(b)(3), Dr. Virginia
> Stallings has been granted a particular matter of general
> applicability waiver that permits her to participate fully in the
> matters at issue. A copy of the waiver statement may be obtained by
> submitting a written request to the agency\'s Freedom of Information
> Office, Room 12A30 of the Parklawn Building.
>
> We would also like to note for the record that Dr. R. Bruce Tompkin is
> participating at this meeting as the acting Industry Representative
> and a non-voting member of the committee. In the event that the
> discussions involve any other issues not already on the agenda for
> which FDA participants have a financial interest, the participant\'s
> involvement and their exclusion will be noted for the record.
>
> With respect to all other participants, we ask in the interest of
> fairness that they address any current or previous financial
> involvement with any firm which makes infant formula.
>
> In addition, I\'d like to take a few moments to very briefly refresh
> everyone\'s memory about Advisory Committee operations, particularly
> the roles and responsibilities of the Food Advisory Committee members
> and FDA staff. The committee members have been provided with a copy of
> a Committee Member Guide to FDA Advisory Committees, and there are
> copies of the member guide available at the registration desk for
> anyone who may be interested. The Member Guide is in need of updating,
> but by and large it provides a good operational overview.
>
> As a scientific regulatory agency, FDA needs access to highly
> qualified expert external advisors who can provide scientific and
> technical advise. Thus, FDA uses advisory committees to supplement the
> agency\'s internal expertise, and help the agency staff stay current
> with state-of-the-art technology. Committee members participate in an
> advisory capacity, and final decisions are ultimately made by agency
> officials.
>
> With respect to the committee members, the chair of the
> committee\--or, for purposes of this committee, the
> co-chairs\--preside at and conduct the meeting, and they may ask the
> FDA staff for clarification at any time during the meeting. Each
> committee member contributes their unique scientific and technical
> background, education and experience to the committee process. The
> standing committee members\--and, in this case, that is the
> Contaminants and Natural Toxicants Subcommittee\--generally are voting
> members, including a consumer representative who is voting, and an
> industry representative who is non-voting, as specified in the
> charter.
>
> All voting members of advisory committees are appointed by the
> Commissioner, and must be cleared as special government employees
> under FDA\'s conflict of interest regulations. Other advisory
> committee participants may include consultants, experts and guest
> speakers. At this meeting, the consultants are temporary voting
> members with expertise infant formula and pediatrics, who will
> participate in the discussions of the committee. The guest speakers
> will present pertinent information to the committee, and will not
> participate in committee discussions or vote.
>
> Additionally, the advisory committee process encourages public
> interaction with the agency in arriving at decisions. Not only is
> there a consumer representative\--in this case, a voting
> member\--appointed to serve on the committee, but the public is
> invited to appear before the committee during the open public comment
> period.
>
> FDA participates in a listening mode, and responds to questions or a
> need for clarification by the committee as needed through the chairs.
>
> Thank you very much.
>
> **Remarks by Chairpersons**
>
> DR. HEUBI: Good morning.
>
> We would like to have all of the committee members identify themselves
> and what their skill set is that they bring to the table that helps
> them be uniquely qualified to come today.
>
> I\'m Jim Heubi. I\'m a pediatric gastrologist. I run a clinical
> research center at the Children\'s Hospital, and I participate and
> actively pursue nutrition-related research.
>
> I think if we\'ll just go around the table, from Dr. Tompkin.
>
> DR. TOMPKIN: I\'m Bruce Tompkin, and I\'m retired now from ConAgra
> Foods, where I was Vice President of Product Safety. I am a
> food-market biologist, and basically that\'s all I\'ve done throughout
> the 39 years\' experience I\'ve had in the food industry.
>
> DR. BEUCHAT: I\'m Larry Beuchat. I\'m from the Center for Food Safety,
> University of Georgia. My area of research is in food-borne
> pathogens-mostly. I do also work with yeasts and molds, spoilage
> problems. I\'ve worked with a number of pathogens. I\'ve worked, on
> occasion, with infant foods, but not with E. sakazakii\--rather
> bacillus serius and, and never hemorrhagic before.
>
> DR. TARR: I\'m Phil Tarr. I\'m from Washington University in St.
> Louis. I\'m a pediatric gastroenterologist, and also have appointment
> in microbiology. My interest is in food-borne infections, particularly
> bacterial infections.
>
> DR. ACHOLONU: My name is Alex Acholonu. I\'m from Alcorn State
> University in Mississippi. I\'m a professor of biology. I\'m an
> epidemiologist in the area of microbiology and parasitology.
>
> DR. THUREEN: My name is Patti Thureen, and I\'m a neonatologist at the
> University of Colorado, and my particular research interests are in
> fetal and neonatal nutrition and metabolism.
>
> DR. FULLER: I\'m Marion Fuller. I\'m with the Florida Department of
> Agriculture and Consumer Services. I run the Division of Food Safety.
> I\'m a veterinarian by training, with certification in general
> toxicology.
>
> DR. LEE: I am Ken Lee. I chair the Food Science Department at Ohio
> State University. My work area is in the process-induced changes in
> nutrients.
>
> DR. MOYER-MILEUR: I\'m Laurie Moyer-Mileur. I\'m from the University
> of Utah. I\'m a research associate professor in pediatrics and a
> registered dietician with over 20 years of clinical experience in the
> NICU, monitoring babies with lots of feeding intolerance.
>
> DR. NEILL: Good morning. I\'m Dr. Peggy Neill. I\'m an adult
> infectious disease specialist, currently at the Brown University
> Medical School in Providence. I have background in public health
> epidemiology and food safety, with a particular interest in E. coli
> 0157 infections.
>
> DR. BAKER: I\'m Robert Baker. I\'m professor of pediatrics at
> University of Buffalo. I\'m a pediatric gastroenterologist at
> Children\'s Hospital of Buffalo. My particular research interest is in
> gut barrier function.
>
> DR. BLUMBERG: Good morning. My name is Henry Blumberg. I\'m in the
> Division of Infections Diseases at Emory University in Atlanta. I\'m
> also the hospital epidemiologist at Grady Memorial Hospital, and my
> interests include hospital and molecular epidemiology and clinical
> research training.
>
> DR. BRILEY: I\'m Margaret Briley, profession of nutritional sciences
> at the University of Texas at Austin, and my research is with children
> and pre-school and nutrition issues.
>
> DR. FISCHER: I\'m Larry Fischer, from Michigan State University. I
> direct the Institute for Environmental Toxicology there. I\'m a
> pharmacologist and a toxicologist. My research often deals with
> age-related changes in susceptibility to chemical toxins.
>
> DR. STALLINGS: I\'m Virginia Stallings. I\'m from Children\'s Hospital
> in Philadelphia, and professor of pediatrics, and at times have run a
> nutrition support service\--a large part of that service is in
> neonatal intensive care units.
>
> Most of my research is on children with chronic disease and in
> nutrition.
>
> DR. KUZMINSKI: My name is Larry Kuzminski. I\'m retired from the food
> processing industry. I was with Ocean Spray Cranberries, where I was a
> vice president of research and development and operations. And prior
> to that I was with the Kellogg Company, where I was vice president of
> science and quality in Canada, and director of food and research in
> the United States.
>
> My interests are in food safety, and in the application of HACCP
> principles in manufacturing processes.
>
> DR. BUSTA: And I\'m Frank Busta, and I\'m a professor emeritus a the
> University of Minnesota, food microbiologist and have worked my entire
> life, mostly, with the bad guys. And I\'m pleased to welcome all of
> you. It looks like a tremendous committee, and the entire group is
> covered.
>
> DR. HEUBI: Jeanne tells me that I should announce that Dr. Anderson is
> unable to attend because of family-related problem today. And I do
> want to remind you to use the microphones whenever you speak because,
> obviously, the audience can\'t hear unless we do, plus we can\'t hear
> ourselves.
>
> And I want to refer to your package that includes materials in this
> blue folder\--that includes the handout slides from the presenters.
>
> And we\'re going to make every effort to stay on time today, so that
> we can give full time and deliberations that they\'re asking us to
> make today.
>
> Yes?
>
> DR. STALLINGS: Who is our consumer representative?
>
> MS. LATHAM: Dr. Margaret Briley.
>
> DR. STALLINGS: Okay. Good. I just wanted to know for sure. Thank you.
>
> **PRESENTATIONS BY GUEST SPEAKERS**
>
> DR. BUSTA: So, our first presentation today is going to be on current
> marketing and use of powdered infant formulas, by Sue Ann Anderson.
>
> The guidelines state that we\'re to have 20-minute presentations, with
> 10 minutes for questions from the committee.
>
> **Current Marketing and Use of Powdered**
>
> **Infant Formula**
>
> DR. ANDERSON: Good morning. This morning I\'m going to talk about how
> powdered infant formula is currently marketed and used in the United
> States.
>
> If I could have the first slide, please? Next slide? There should be
> one before that. Well, in any case then, my talk will focus on three
> topics: the types and uses of formulas currently marketed in the
> United States; preparation of powdered formula for feeding; and
> feedings to infants in health-care settings.
>
> My remarks this morning will be brief and very general in nature.
> Greater detail can be found in the White Paper that is part of the
> briefing materials for the Committee for this meeting.
>
> In order to identify what the products are and who the consumers are,
> I should begin by defining \"infant formula\" and \"infants.\" Infant
> formulas are products intended for use solely as a food for infants.
> They are simulate human milk and are suitable as a complete or partial
> substitute for human milk. Next slide, please.
>
> \[Slide\]
>
> Infants are defined as persons not more than 12 months of age. This
> slide set is not my slide set. We\'ll go ahead and work with it,
> though.
>
> With those definitions in mind, I would like to provide a little
> information on the introduction of formula for infant feeding.
> Powdered infant formulas were introduced well over a hundred years
> ago, in the late 1800s. Liquid forms were not introduced until the mid
> 20^th^ century, with liquid concentrates coming onto the market in the
> early 1950s, and ready-to-feed products coming onto the market about
> 1960.
>
> It should be noted that powdered infant formula are not sterile
> products, however the liquid products are. After the liquid forms were
> introduced, the proportion of infant formula sold in powdered form
> decreased. The lowest proportion of sales of powdered infant formula
> occurred from about 1970 to 1980. Around 1980, technical advances in
> the processing of powdered infant formula resulted in products that
> were more readily dispersed in
>
> water, making it easier to reconstitute the powders for feeding. Since
> 1980, the proportion of powdered infant formula sales has risen and in
> 1999, sales of powdered infant formula accounted for about half of the
> dollar sales of infant formula products in the United States. This
> estimate was based on data collected from supermarket, drug store, and
> mass merchandiser scanners by
>
> Information Resources, Inc. I should also note that the proportion of
> infant formulas sold in powdered form in the United States differs
> from that of other countries. In most other countries, almost all
> infant formula is sold in the powdered form.
>
> I should also note at this point that the proportion of infant formula
> sold in powdered form in the United States is quite different from
> that in other countries. In most other countries, almost all infant
> formula is sold in powdered form.
>
> We\'ll see what the next slide is.
>
> \[Slide\]
>
> There is also a difference in the cost of feeding of the infant
> formulas, with the powdered infant formula being the least expensive,
> and concentrate intermediate, and ready-to-feed the most expensive
> product.
>
> Most infant formulas are formulated to meet the nutritional needs of
> infants with generally good health status.
>
> And, if you will bear with us for just a couple of minutes, we\'re
> going to see if we can find the right slides here.
>
> \[Pause.\]
>
> MS. LATHAM: This is Jeanne Latham. I just want to apologize for our
> technical difficulties. This is what happens when you try to perfect a
> presentation and you provide many, many different versions of it. So,
> hopefully, we\'re going to find the correct one.
>
> \[Pause.\]
>
> DR. ANDERSON: Okay,.why don\'t you advance and I\'ll tell you where we
> are. One back.
>
> Most infant formulas are formulated to meet the nutritional needs of
> infants with generally good health status. Included in this group are
> term and preterm infants, with term infants being those born at or
> later than 37 weeks gestational age, and preterms born before 37 weeks
> gestational age. These products are available in both powder and
> liquid form.
>
> Could I have the next slide, please?
>
> \[Slide\]
>
> Other infant formulas are designed to meet the needs of infants with
> special medical conditions such as genetic disorders of metabolism
> (for example, PKU) or other system disorders, for example
> gastrointestinal diseases. With the exception of protein hydrolysate
> formulas, this category is available only in powdered form.
>
> Could I have the next slide, please?
>
> \[Slide\]
>
> Okay. Because the infant population is at risk for nutrition insult
> and infection, proper preparation, storage, delivery, and disposal of
> their feedings are critical for prevention of food-borne infections.
> In order to provide consumers with accurate information for
> preparation and use of infant formulas, there are specific Federal
> regulations for infant formula labels. Preparation and use
> instructions required on infant formula labels include 1) product
> storage, 2) sterilization of water, bottle, and nipples, when
> necessary, and 3) dilution for powder and liquid concentrates showing
> major steps for preparation, including a required pictogram for
> illustration.
>
> Could I have the next slide?
>
> \[Slide\]
>
> This next slide shows the pictogram from the Code of Federal
> Regulations, depicting instructions for formula dilution. The wording
> on the slide is difficult to read, but it does say \"Sterilization is
> recommended. Your physician will decide if it is not required.\"
>
> Next slide.
>
> \[Slide\]
>
> Other information is also required on infant formula labels, and it\'s
> required to caution against improper preparation or use. Cautionary
> statements such as the following are required:\"The health of your
> infant depends on carefully following the directions for preparation
> and use;\" \"Use as directed by a physician.\" And, also, a \"Use by\"
> date required on the infant formula. The \"use by\" date is a date
> selected by the manufacturer on the basis of tests or other
> information showing that the formula, until that date, under
> conditions of handling, storage, preparation, and use prescribed by
> label directions, will contain not less than the quantity of each
> nutrient as specified on the label and will otherwise be of acceptable
> quality.
>
> Next slide.
>
> \[Slide\]
>
> Each manufacturer designs their product labels to conform to these
> regulations and the wording and pictograms on labels will vary for
> each manufacturer. For example, directors for preparation of water for
> reconstituting infant formulas generally fall into two categories. On
> this slide, you see the first category, which includes some direction
> to boil the water
>
> and to cool it before adding to the infant formula.
>
> Next slide.
>
> \[Slide\]
>
> This may be hard to see, too, but this is a sample from an infant
> formula label where it does say to boil the water. It says to boil the
> water for a minute, then cool; pour the cooled water into a bottle;
> add the powder and cap bottle, shake well and feed immediately.
>
> The second category \-\--next slide.
>
> \[Slide\]
>
> The second category of instructions for water preparation includes an
> instruction to consult with the doctor about the need to boil water.
>
> Next slide.
>
> \[Slide\]
>
> And a sample pictogram from a label with this sort of information
> says, \"Your baby\'s health depends on carefully following these easy
> directions. Ask your baby\'s doctor if you need to boil sterilized
> water for formula and bottle preparations, pour the desired amount of
> warm water into a bottle, add the powder, cap, shake very well and it
> does include a statement \"After feeding, throw away any formula
> remaining in the bottle.\"
>
> Next slide.
>
> \[Slide\]
>
> Instructions for feeding and storage direct the consumer to feed
> immediately or cover, refrigerate, and use, either within 48
> hours\--next slide\--
>
> \[Slide\]
>
> or, on some formulas, in 24 hours.
>
> Some labels\--next slide\--
>
> \[Slide\]
>
> \--also include instructions for disposing of the unused formula, such
> as \"Discard unused formula after feeding,\" or \"Throw away prepared
> formula left in the feeding bottle, or cap within one hour after
> feeding begins.\". Examples of such statements are shown on the next
> slide.
>
> \[Slide\]
>
> Use of commercially sterile infant formula products is not always
> feasible in health care settings and, in some situations, formulas
> must be prepared from nonsterile, powdered products for hospitalized
> infants. The American Dietetics Association (ADA) has developed
> guidelines for preparation of infant formulas in health care
> facilities. These guidelines include detailed recommendations intended
> to decrease the risk for microbial contamination and growth during
> infant formula preparation, storage, and use in health care
> facilities. The guidelines have been updated recently, and are
> available on line at the URL shown on the screen.
>
> I will not go into detail about the recommendations for preparation of
> formulas, but I should say that the ADA guidelines for delivery and
> disposal of infant formulas for nipple-fed and tube-fed infants
> \-\--there are guidelines for these groups in health care settings.
>
> Next slide.
>
> \[Slide\]
>
> For nipple-fed infants, the instructions are given to feed within 4
> hours of preparation, cover and refrigerated for up to 24 hours. If
> the formula is warmed, it should be warmed quickly, in less than 15
> minutes; and to discard any product remaining in the bottle one hour
> after feeding begins.
>
> Next slide.
>
> \[Slide\]
>
> For tube-fed infants given intermittent feedings, the formula should
> be, again, fed within four hours of preparation, covered and
> refrigerated for up to 24 hours if not used immediately. For
> intermittent feeding, it should be packaged in amounts for one feeding
> or for a 4-hour period and for continuous feedings, it should be given
> in a way so that the hang time would not exceed four hours.
>
> This has been a brief and very general overview of the current
> marketing and use of infant formulas. What I would like for you to
> remember\--if I can have the last slide\--
>
> \[Slide\]
>
> \--what I would like for you to remember as we proceed through
> today\'s agenda are the following points: About half of the market
> share\--the dollar sales in the United States, is for powdered
> products. This is based on 1999 dollar sales. Powders are not sterile
> products. There is a small percentage of infant formulas that are not
> available in commercially sterile, liquid forms, and methods given for
> reconstitution and delivery are variable.
>
> Thank you.
>
> DR. BUSTA: Thank you.
>
> Are there any questions?
>
> Robert.
>
> DR. BAKER: I\'m Baker. I have a clarification point.
>
> The labeling is required, is that right? But the exact wording, and
> sometimes even the content of the label is decided by the
> manufacturers.
>
> DR. ANDERSON: There are certain requirements\--there are certain
> elements that are required on the label, and there are certain
> statements that are required.
>
> Linda, if you would go back to\--about six or seven slides? Keep
> going.
>
> Okay. This one.
>
> \[Slide\]
>
> The statements that are in quotation marks\--the first two
> statements\--those statements are required.
>
> If you go back one more slide. One more. One more.
>
> \[Slide\]
>
> Information related to these topics is required, but there is no
> wording that is specified or directed by FDA. The manufacturers is
> given latitude in that regard.
>
> DR. HEUBI: I have a question\--Jim Heubi.
>
> Do we know, as an example\--first\--I have a two-part question. One,
> do the powdered formula labels include a statement that they\'re not
> sterile? And then, secondly, do we have any knowledge about how
> informed neonatologists are, or providers for preterm infants are in
> terms of understanding the non-sterile nature of powdered formulas
> that might be used in that venue?
>
> DR. ANDERSON: To answer your first question, it\'s not required that
> there be a statement on the label that says that the powders are not
> sterile products\--commercially sterile products. And I\'m trying to
> think quickly\--and I don\'t remember seeing a statement on a powdered
> product to that effect.
>
> With regard to knowledge of neonatologists about the nature of these
> products, I\'m not aware of any surveys that have been done to collect
> that information.
>
> DR. HEUBI: Is it the general sense that they understand this, or not.
> I guess that\'s a question that I have in the back of my mind right
> now.
>
> DR. ANDERSON: My inclination would be to say they have a better
> understanding of it now than they did before the E. sakazakii
> incidents occurred, but I don\'t really have a basis for saying how
> much they do or do not know.
>
> DR. HEUBI: Okay.
>
> DR. THUREEN: I polled our neonatologists at several hospitals in town,
> and one of about 25 had any clue how to prepare the infant formulas.
> It\'s usually done by a milk lab, and they really\--you know, it was
> totally out of their realm of knowledge. It just wasn\'t anything they
> thought about.
>
> DR. BEUCHAT: A question concerning the tube feeding\--
>
> DR. HEUBI: Please identify yourself.
>
> DR. BEUCHAT: Larry Beuchat.
>
> A question concerning tube feeding and perhaps also relates to some of
> the other recommendations: are there recommendations with regard to
> the tubes themselves, or any other vessels or containers into which
> the actual formula comes in contact before the baby consumes it? The
> re-use, the hygiene and so on?
>
> DR. ANDERSON: Those would be products that would be packaged for
> delivery of the formula, and those would have to meet guidelines from
> one of our sister centers. I\'m trying to figure whether it would be
> drugs or devices\--probably devices\--that they would have
> specifications for delivery systems\--for those types of products.
>
> DR. TARR: Is it possible for us to get a list of\--
>
> DR. HEUBI: Please identify yourself.
>
> DR. TARR: Phil Tarr, from St. Louis.
>
> Is it possible for us to get a list of the formulas that are not
> available as liquids?
>
> DR. ANDERSON: As specific products, or as types?
>
> DR. TARR: Probably both.
>
> DR. ANDERSON: We could certainly do it as types, and I could see if we
> could get the specifics for you.
>
> DR. MOYER-MILEUR: Moyer-Mileur.
>
> In regard to delivery systems, as far as tubing for continuous drip
> feedings, while the products themselves may be sterile when they\'re
> initially used, if they are reused then they\'re no longer that. And
> that would all be dependent on each individual NICU and their
> policies. So it would be really hard to\--for us to answer that or
> even to control that, I would think.
>
> Hopefully at a level-three\--in a level-three nursery the delivery
> systems would not be re-used, and that would not be their standard of
> practice. The worry would be if you had a level-two, or say even a
> level-one nursery where they\'re keeping babies who are 32, 33 weeks
> and feeding, and maybe not quite as knowledgeable about these
> products.
>
> DR. HEUBI: Heubi.
>
> Just as a point of clarification, do we have a general idea about how
> long NICUs utilize this equipment that is used for feeding? Is it 24
> hours, 48 hours, 72 hours? Is there any sense about this?
>
> DR. ANDERSON: I\'m not able to comment on that. I think\--I can say it
> varies among institutions, but further than that, I couldn\'t comment.
>
> DR. MOYER-MILEUR: You know, I can\'t either, because I think if
> you\'re dropping in a continuous drip-feeding tube, you leave it as
> long as possible.
>
> DR. HEUBI: I guess I was more thinking about the bag and the tubing
> that leads to the tube\--
>
> DR. MOYER-MILEUR: They should be changed every four hours.
>
> DR. HEUBI: The tubing should be as well?
>
> DR. MOYER-MILEUR: The connecting tube, not the actual\--
>
> DR. STALLINGS: Stallings.
>
> I would echo what Laurie was saying. I think in most level-three
> nurseries, the feeding system would be treated more like an IV feeding
> system, but with the\--you know, when you change the set you change
> the whole set.
>
> For a nasogastric feeding, of course the tube in the infant would not
> be changed four times a day and, in general, it would be left in I
> think it\'s about three days before it would be changed electively.
>
> So, you really do, in the NICU, when you\'re talking about these
> products, you\'re generally going to be thinking about feeding tubs.
> And then as the infants age and approach term, they\'ll go to
> nipple-feeding and more to the devices that we think of as usual
> infant feeding.
>
> But, you\'re right, we have to think about the plastic bag\--the
> formula being mixed, storage and all of that, but then the plastic
> back, the feeding tube, the connection to the feeding tube that\'s in
> the infant. So there are a number of different components.
>
> DR. HEUBI: Are there any additional questions? Thank you.
>
> Oh, I\'m sorry\--Dr. Beuchat.
>
> DR. BEUCHAT: Beuchat.
>
> Just one question\--for Virginia. What is, then\--what could be the
> length of time that the tube would be in the infant without removing
> it, and still feeding it?
>
> DR. STALLINGS: The nasogastric? It probably varies per center, but in
> the stressed neonate, I think it\'s about three days that because of
> the stress of moving a tube in and out of the baby, that you wouldn\'t
> be changing it every 24-hours unless it occluded, or there was some
> other reason.
>
> I really can\'t\--somehow three days sounds about right, but I can\'t
> remember exactly. But every nursery should have a protocol about
> electively changing that. But, again, as Laurie was saying, I think
> some of our concerns\--there\'s so much movement now to keeping more
> stable babies in second\--the different level nurseries that there\'s
> probably more nasogastric feeding that\'s being done in those settings
> than used to be done. So there is a site issue and an education issue
> here.
>
> DR. HEUBI: Heubi.
>
> And you have to realize that some of these babies have naso-duodenal
> tubes placed and that\'s the context. There\'s not a big move to
> replace them periodically. So they might be in for longer periods of
> time.
>
> Any additional discussion or questions?
>
> \[No response.\]
>
> All right. I think we have to move on to our multi-media presentation.
> Dr. Kuehnert is not available to present personally, but he\'s going
> to be available by telephone. He\'s involved with the investigation
> for the current respiratory outbreak in Asia, and we\'re pleased to
> have him available to present today for us. And as soon as we make our
> connection, we\'ll be ready to go.
>
> **Tennessee Investigation**
>
> DR. KUEHNERT: \[Via speakerphone\] Can you hear me okay?
>
> Thanks for inviting me to speak. I\'m sorry that I couldn\'t be there
> in person, but an emergent investigation precluded me coming. But I
> appreciate the opportunity to present by phone.
>
> What I wanted to do today is discuss primarily the case in Tennessee
> in 2001 that resulted in Enterobacter sakazakii meningitis and death
> in an infant who was hospitalized.
>
> MS. LATHAM: Dr. Kuehnert?
>
> DR. KUEHNERT: Yes.
>
> MS. LATHAM: This is Jeanne Latham. You are obviously not able to hear
> the mikes at the table very well, and I just wanted to remind you that
> we will be recording the presentation, and we will have to try to
> figure out how we\'re going to ask you questions, because Dr. Heubi
> was trying to get your attention, and we weren\'t able to, but we will
> work that out.
>
> So, go ahead and do your presentation. Thank you very much.
>
> DR. KUEHNERT: Okay. Okay.
>
> And, to discuss the case, discuss the resulting MMWR and interim
> recommendations, and also offer some discussion concerning the
> implications and possible activities going forward.
>
> Could I have the next slide, please.
>
> \[Slide\]
>
> So this should say \"Background.\" Enterobacter sakazakii is a gram
> negative rod that was classified as a yellow-pigmented variant of E.
> cloacae, until it was designated as separate species in 1980. And the
> reservoir is unknown, as far as the original source of the organism.
>
> Next slide, please.
>
> \[Slide\]
>
> Clinical characteristics. This is a pathogenic organism with a
> particular affinity for the nervous system. Complications are commonly
> quite serious, including necrotizing enterocolitis, sepsis,
> meningitis. And in addition to meningitis, cerebral abscesses, cysts,
> infarctions, fairly extensive cerebral damage. And outcome is poor. In
> most of these infections, there is an impaired neurological outcome.
> In fact, that\'s expected in the vast majority of cases that occur,
> and fatality rates has ranged, in the literature, between 40 and 80
> percent.
>
> Next slide, please.
>
> \[Slide.\]
>
> Potential sources of the organism\--as I said, the reservoir is
> unknown, but powdered infant formula has been associated in the past,
> before the case in 2001, with outbreaks of E. sakazakii infections in
> neonates. The organism has bene traced to a number of steps in the
> powdered formula preparation process. It\'s been found in freshly
> prepared or refrigerated powdered formula, in utensils and equipment
> that\'s used in formula preparation, such as blenders and other tools
> used to make formula, in the unreconstituted product, and also in
> unopened product.
>
> Powdered formula products, as I said, have been associated with these
> outbreaks, including meningitis, sepsis and necrotizing enterocolitis.
> Other studies have looked at powdered formulas for contamination by
> bacteria and there was one particular study that looked at
> Enterobacteriaceae in general, and found contamination at low levels,
> including over half of products from a number of different countries,
> including the United States, and specifically 14 percent of powdered
> formula samples were contaminated with E. sakazakii, and most of you
> are probably familiar with this literature. However, the
> concentrations of the organisms were quite low.
>
> Next slide, please.
>
> \[Slide.\]
>
> This is outline of presentation. Let\'s skip over this.
>
> Moving on to case description\--the next slide.
>
> So, on to the 2001 case. A male patient was admitted to the neonatal
> intensive care unit in April 2001. Gestational age was 33-1/2 weeks,
> delivered by C-section. APGAR scores were 4 and 7 at one and five
> minutes, and the birth weight was 1,270 grams.
>
> Patient was started on enteric feeding with powdered formula and
> breast milk, and then on day 11 of life, developed sepsis and
> neurologic symptoms.
>
> Lumbar puncture was performed which was consistent with meningitis.
> White cells and red cells were present, although at actually low
> numbers, but the protein was high, and the glucose was remarkably low.
>
> Cerebral spinal fluid culture grew Enterobacter sakazakii. The patient
> was treated with ampicillin and Cefoxitime, but did not do well
> despite full support, and became pulseless and was resuscitated with
> pressors.
>
> As the clinical course progressed, it was clear that the neurologic
> was severe, and that there was no significant brain activity. And so
> support was withdrawn, and the patient expired at day 20.
>
> Next slide.
>
> \[Slide\]
>
> The facility in which this occurred with the University of Tennessee
> at Knoxville. It\'s a regional referral and tertiary care center;
> fairly large facility\--360 beds. It has a level-three NICU with 55
> beds that\'s composed of two parts: and intensive care nursery with 27
> beds, and an intermediate care portion with 28 beds.
>
> They looked back\--because this was an unusual organism, the hospital
> epidemiologist looked back to see whether they had seen E. sakazakii
> in specimens in the recent past. In fact, they looked in the last
> three years and didn\'t find anything from 1998 to 2000. However, they
> did find two isolates detected in March 2001, in addition to this
> case. So this prompted a further investigation.
>
> Next slide.
>
> \[Slide\]
>
> So the study objectives were to ascertain whether there were
> additional cases of E. sakazakii infection or colonization that they
> had missed clinically to determine the source of the organism and to
> develop measures to prevent further infection.
>
> Next slide, please, that says \"Case Finding.\"
>
> \[Slide.\]
>
> A cross-sectional prevalence survey was performed, and what this was
> was basically looking at all patients in the NICU during the case
> patient with meningitis was ill, and that was defined as April 10^th^
> to 20^th^ 2001. I\'ll refer to that as the study period.
>
> They assessed all these patients for stool colonization. In addition,
> they looked for clinical reports from the microbiology laboratory for
> E. sakazakii and did a review to see what symptoms, if any, patients
> had. And a \"case patient\" was defined as any NICU patient with the
> E. sakazakii-positive culture during the study period, either from the
> stool or other sources.
>
> Next slide.
>
> \[Slide\]
>
> Case finding: 49 patients were hospitalized during the study period.
> There were nine case patients, and of these nine\--this slide shows
> the site of infection or colonization\--six E. sakazakii from the
> stool, two from tracheal aspirates, one from the urine, and one from
> cerebrospinal fluid, which was the index case.
>
> I would want to add here the footnote that you have 10 sites here, and
> that\'s because one patient had E. sakazakii in the stool and the
> urine.
>
> Next slide, please.
>
> \[Slide.\]
>
> Next, a cohort study was performed. These are the variables that we
> looked at, combining the literature concerning infant formula and also
> just nosocomial infection variables that are commonly looked at.
>
> Just to high the highlights, we looked at, obviously, modes of
> nutrition: split formula into powdered versus liquid versus
> ready-to-feed; also looked at whether the formula was given by
> continuous feed or by bolus. And that\'s pretty much what I wanted to
> highlight there.
>
> Next slide, please.
>
> \[Slide.\]
>
> These were the results of the cohort study. The only variable found to
> be significant was powdered formula use. So, just to walk you through
> this table, of the 30 patients that were exposed during the study
> period, nine were ill and were case patients. I\'m sorry\--of the 49
> in the cohort, 30 were exposed to powdered infant formula and, of
> those, nine had E. sakazakii and were cases. Of the remaining in the
> cohort, 19 did not have powdered formula, and none of those patients
> had been given powdered formula.
>
> There were some other things that I can\'t really say approached the
> statistical significance but were, I think, of some note, is that the
> continuous had a P-value of .16, and the absence of breast milk use
> was also\--had a P-value of .16.
>
> Next slide, please.
>
> \[Slide.\]
>
> Next, observational laboratory studies were performed in the facility;
> looked at formula preparation, storage and administration. I\'ll just
> briefly gloss over these; cultured the environment and materials for
> formula preparation in patient care, including the preparation area in
> the NICU, and cultured lots that were in use during the study period,
> including powdered formula from open containers.
>
> Next slide, please.
>
> \[Slide.\]
>
> So this should say \"Laboratory Studies.\" Laboratory studies were
> performed by CDC and included confirmation of isolates from the cohort
> study, cultures of open cans of formula. They were also able to obtain
> unopened cans of formula of an identical lot number that was supplied
> by the manufacturer, and we cultured that as well. Culture method was
> done according to a modified protocol of inisol, and all study
> isolates and selected historical isolates were compared by pulse-field
> gel electrophoresis.
>
> Next slide, please.
>
> \[Slide.\]
>
> Environmental and formula cultures\--these are the results\--showed
> that none of the on-site cultures that were performed, either in a
> preparation area or in the NICU, grew E. sakazakii. CDC cultures grew
> E. sakazakii from a single lot of powdered formula, and this was from
> both an unopened can and an open can. The PFGE patterns were
> indistinguishable between isolates of either the cerebrospinal fluid
> of the case patient that died from meningitis, and also the opened and
> unopened containers of powdered formula.
>
> And the other interesting sort of aspect of this is the PFGE results
> also suggested pattern diversity among other isolates from the cohort
> study compared with previously collected strains, and this is shown
> graphically in the next slide, which says \"PFGE Results.
>
> \[Slide\]
>
> And what you see here is a gel, and the PFGE results, and you see
> lanes two through six showing a CSF isolate, a respiratory isolate,
> stool isolate, urine isolate and the two formula isolates being
> identical. However, there were other patterns that are different, and
> these include some stool isolates from the cohort study, as well as
> historical isolates from isolates that were pulled from the freezer,
> from other previous investigations.
>
> The implications of this, I guess we can get into in the discussion.
>
> Next slide.
>
> \[Slide\]
>
> Observational studies. The hospital detected no breaches in infection
> control. The formula was prepared according to manufacturer\'s
> instructions on the label. It was mixed with sterile water,
> refrigerated for less than 24 hours. And their guidelines at the time
> were to use the mixed product within eight hours, and the hang time,
> although I\'m not sure of the exact time, they said it was around six
> hours.
>
> Next slide.
>
> \[Slide\]
>
> Intervention. The facility, at the time, prescribed a fair amount of
> powdered formula in the facility, to about 50 percent neonates in the
> NICU. As a result of this event, they changed the formula preparation
> site from the NICU to the pharmacy. The principal formula used was
> switched to liquid ready-to-feed, although they still had to use some
> powdered formula selectively for special formulations. They changed
> their allowable hang time for mixed feeds from eight to four hours.
> And, to date, they haven\'t seen any further E. sakazakii or clinical
> isolates.
>
> So, in conclusion, concerning this case the source of the case of
> Enterobacter sakazakii infection was traced to receipt of powdered
> infant formula. The only significant\--it was the only significant
> risk factor in epidemiologic study, and there were matching isolate
> patterns on PFGE concerning\--from the powdered formula and from the
> patient.
>
> So we concluded that powdered formula, which is a non-sterile product
> can be contaminated with E. sak and can cause fatal meningitis, and
> that use of powdered formula should be carefully considered in the
> neonatal health care setting.
>
> Next slide.
>
> \[Slide\]
>
> After this, Mead Johnson voluntarily recalled Portogen powder.
>
> Next slide.
>
> \[Slide\]
>
> And an MMWR was published which included summary interim
> recommendations for the NICU. This included to select formula products
> based on nutritional needs; that trained personnel should prepare
> products using aseptic techniques; to follow manufacturers
> recommendations for preparation of formula; that the administration of
> hang time should be less than four hours; and that there should be
> written hospital guidelines, including notification, reporting and
> follow-up available in the event of a product recall. And there are
> some aspects of that last bullet that I think are difficult for
> hospitals to follow, and I\'ll get into that a little bit in the
> discussion.
>
> Next slide.
>
> \[Slide\]
>
> We also asked that cases be reported, particularly concerning invasive
> infections in infants younger than 12 months to the state health
> departments, CDC and, of course, FDA Medwatch.
>
> Next slide.
>
> \[Slide\]
>
> So, just to conclude here, the questions I think that sort of come up
> with this case is when this case first emerged, well, the question was
> is whether this is an emerging pathogen. And in trying to answer that,
> there are a subset of questions that come up. Again, what is the
> reservoir of the organism? Is this\--has it changed? Is there
> something new as far as the reservoir? Has this been around all along?
> And what is the endemic rate of the E. sakazakii colonization or
> infection due to powdered infant formula? Since we don\'t have
> surveillance for it, we don\'t know, you know, how many cases there
> have been.
>
> Just as an aside, we have a nosocomial infection surveillance system,
> and we looked back at isolates\--at infections that have been reported
> due to E. sakazakii, and we found one where there was an isolate from
> the CSF in 10 years of surveillance. And this is approximately 300
> hospitals. So this is a very unusual organism for a hospital to find,
> and I don\'t think that this Tennessee facility\'s experience in not
> seeing an isolate for three years from NICU patients is unusual.
>
> And, next, what\'s the role of specific methods for preparation and
> use to promote growth and reach the threshold in clinical
> significance. So if it exists in formula, what are the factors that
> make it matter in causing clinical disease. And, as an adjunct to
> that, what are the predisposing risk factors for infection?
>
> Next slide.
>
> \[Slide\]
>
> And so I just sort of split sort of the separate issues, which I think
> encompass the spectrum of issues that cross agencies and different
> both clinical, laboratory and manufacturing arenas concerning the
> manufacture of the formula: screening during manufacture, issues about
> preparation and storage and use; treatment of infection if there\--you
> know, there\'s very little in the literature about treating E.
> sakazakii in general; and, finally, issues about case reporting and
> surveillance of infection, which I\'ve already touched on.
>
> Next slide.
>
> \[Slide\]
>
> So, concerning manufacturers specifically, I\'m sure what\'s going to
> be discussed today is going to be concerning processing and
> implementation of screening, which I understand has already begun to
> some extent. Concerning preparation, storage and use, development of
> guidelines or recommendations. The American Dietetic Association had
> had some recommendations previously that had been disseminated but was
> out of print at the time of this event, and I understand that they\'re
> developing revised guidelines.
>
> And, finally, case reporting and surveillance\--this was the issue I
> touched on before, which was about hospital record-keeping. They
> usually don\'t record whether\--they record, of course, in the orders
> whether formula has been given, but not necessarily whether it\'s
> liquid or powdered, and certainly not what lot number has been given.
> So, in trying to track\--do a trace-back investigation of the case,
> it\'s very, very difficult, because oftentimes the hospital doesn\'t
> now whether the patient even got powdered formula, or whether liquid
> ready-to-feed. And so this provides a particular challenge in the way
> formula documentation is currently set up.
>
> Next slide.
>
> \[Slide\]
>
> So, as far as future plans, what we\'ve currently been involved with
> FDA has been sporadic case investigations, and sort of case finding. I
> think a case series description would be very useful, and there are a
> couple people working on that. As far as policy on formula
> preparation, I mentioned the American Dietetics Association. They also
> have done surveys of preparation and use. I think that\'s important.
> And also, obviously, revising the guidelines.
>
> Concerning laboratory research, we\'ve been involved with some, and I
> know FDA has been as well, concerning the growth characteristics of E.
> sakazakii, and also, specifically, the effect of two things: one,
> competitive microbial flora. I mean, we\'ve done studies looking at E.
> sakazakii growth, but one of the questions is how does it grow
> differently when it\'s in sort of the milieu with different organisms,
> as it is with infant formula, and also about the effect of heat
> inactivation on growth.
>
> Next slide.
>
> \[Slide\]
>
> I\'d like to acknowledge many people who have given input over the
> last, actually, couple of years and, particularly, the hospital that
> originally detected the case in Tennessee.
>
> Next slide.
>
> \[Slide\]
>
> That\'s the end of my presentation. I\'d be happy to answer questions.
>
> DR. HEUBI: In order to answer questions, either we\'ll have to\--
>
> DR. KUEHNERT: Oh, yes, I\'m having a lot of trouble hearing you.
>
> DR. HEUBI: I think what we ought to do is each of us, maybe, should go
> and pose the question with the microphone to him, and then he can
> respond. That way we can all hear what he says. Do you want to do
> that? There\'s a microphone over there.
>
> So, if you\'ll queue up, that would be great.
>
> Now, please identify yourself.
>
> DR. STALLINGS: Hi. It\'s Virginia Stallings, from Philadelphia. Thank
> you for being with us.
>
> My question is: how hard is this bug to grow and identify in a busy
> microbiology clinical lab? Because that will give us some framework to
> consider the data we have at hand.
>
> DR. KUEHNERT: Yes. Yes. I heard the question.
>
> There are two aspects to that. One is whether E. sakazakii can be
> easily grown in a clinical specimen. I think that most laboratories
> are pretty well suited to that. We have had situations where they
> weren\'t sure whether it was run of the mill E. cloacae or E.
> sakazakii, because sometimes the yellow pigment isn\'t that strong.
> But it appears that from what I understand\--I\'m not a
> microbiologist\--but from my understanding, is that the methods used
> in most clinical microbiology laboratory should pick up most of them.
>
> Now, the issue concerning finding it in infant formula is different.
> You know, some hospitals that have suspected that they might have E.
> sakazakii in their formula have tried, you know, a number of different
> methods which really don\'t work, like putting the powdered formula
> just directly on media, or even trying to do\--trying to culture it,
> just using standard methods. And I\'m sure FDA will speak to this, but
> there are\--I mentioned a modified protocol, and it is a fairly
> intensive, somewhat complicated protocol that takes a number of days
> to grow it out of formula. And the biggest problem\--I don\'t want to
> belabor this too much, because I\'m sure it\'s going to be discussed
> later\--I mean, the big problem is that you\'ve got a lot of microbial
> flora in the formula that competes against the E. sakazakii, and so
> you have to pick\--it\'s sort of a needle in a haystack to pick it
> out. And that\'s some of the difficulty in trying to isolate it from
> formula.
>
> So, the short answer to the question is: yes, I think microbiology
> laboratories should pick it up in clinical specimen, but no, I think
> they\'re going to have difficulty from infant formula.
>
> DR. NEILL: Peggy Neill, from Brown University Medical School. I have
> two questions, and they\'re unrelated.
>
> The first one is that in your slides, it would probably be
> approximately \"9,\" for \"facility characteristics,\" at the bottom
> of the slide it indicates that there are two isolates of E. sakazakii
> detected in March 2001, but I\'m trying to discern from the text here
> that they appear not to have been either a clinical isolate at all? Or
> they were certainly not a clinical isolate from the NICU.
>
> DR. KUEHNERT: Right. Yes. Let me address that and try to remember what
> the significance of those isolates.
>
> If I remember right, one of those patients was included in the cohort
> study\--well, let me back up. The two isolates, I believe, were
> respiratory isolates. They were not thought to be causative for
> infection. One of the patients was included in the cohort study and, I
> believe, had a positive stool but did not have a clinical isolate
> during the study period.
>
> The other patient, I think, had already been discharged, and their
> illness was due to another issue, and they did not have the isolate
> for comparison\--they did not have either respiratory isolate for
> comparison at the time of the study. They had been discarded at the
> time that they noted the problem.
>
> DR. NEILL: Okay. Thanks.
>
> The second question pertains to your PFGE profiling?
>
> DR. KUEHNERT: Yes.
>
> DR. NEILL: And I suspect you know where I\'m going with this. You
> alluded to the fact that lanes two through six are clinical isolates
> that are identical, and they are from this particular outbreak
> investigation. But if I understood you correctly, you have other
> isolates farther to the right in that gel, but I don\'t know which
> lane, and that those have different pulsotypes?
>
> DR. KUEHNERT: Right. Yes. Let me try\--I was going to discuss this,
> and in the interest of time I did not. But I\'m glad you brought it
> up.
>
> Okay. There are two groups of isolates here. Unfortunately, I don\'t
> have them labeled, so I can\'t tell you which is which, but there are
> some\--there are stool isolates there from the cohort study that had
> different PFGE patterns from the index case and the isolate from
> formula. And then two or three of the other ones are from historical
> isolates from the CDC freezer.
>
> Now, concerning the stool isolates, I mean there are a couple of
> possibilities for explaining this. One is that the patients just
> happened to be colonized with E. sakazakii from the stool, which is
> possible\--although we don\'t usually look for it in clinical
> microbiology labs. So it might be there, but I think that\'s probably
> unlikely, because people do do screening for gram negatives in stool,
> and have not frequently found this.
>
> The other explanation\--which I think is more likely\--is that there
> were multiple\--there may have been multiple\--this is speculation on
> my part\--but there may be multiple isolates of E. sakazakii from
> that\--either that implicated lot, or other lots is possible, and that
> this represents isolates that were below our level of detection for
> the lot that we sampled.
>
> But, I mean, there\'s obviously got to be some explanation as to where
> these came from, and we don\'t have an answer for it.
>
> DR. HEUBI: Hi. Jim Heubi.
>
> After this initial case series was identified in the hospital, was
> there an effort made then to check any of the formulas given to these
> infants, later during the follow-up or new additional cases to know
> whether actually those formulas \[off mike\].
>
> DR. KUEHNERT: Could you repeat that? I\'m not sure I fully understood
> that?
>
> DR. HEUBI: Well, what you reported was that there were no additional
> cases after this series, including the nine that were colonized with
> one case. And then you made the comment about the fact that there\'s
> been no additional cases since that time.
>
> DR. KUEHNERT: Mm-hmm.
>
> DR. HEUBI: The question I had was: was there any effort made to try to
> make sure that the formula was or was not contaminated in any of those
> additional infants might have been exposed to after the initial
> reports?
>
> DR. KUEHNERT: Well, my understanding at the hospital is I don\'t think
> they\'ve done any further stool colonization studies, nor have they
> done any cultures of the formula. I think that they\'ve done\--I mean,
> I don\'t know\--a somewhat active surveillance, certainly in looking
> for E. sakazakii on clinical specimens. But as far as stool
> colonization, or directly culturing formula, I don\'t think that
> they\'ve done that.
>
> Again, I don\'t think they had the resources to do, you know, the
> modified protocol in the first place to culture formula. And so I
> don\'t think they\'ve been doing that on an ongoing basis, either.
>
> Does that answer your question?
>
> DR. HEUBI: Yes. I guess as the devil\'s advocate what I\'m saying is
> that the changes they implemented may or may not have had any impact
> on whether they have any future cases.
>
> DR. KUEHNERT: Yes. I think that\'s a valid statement.
>
> DR. HEUBI: Thank you.
>
> DR. BEUCHAT: Larry Beuchat, University of Georgia.
>
> I have two questions. One is a follow-up to Peggy\'s. Were the two
> March 2001 isolates, you know the PFGE patterns determined in this
> film\--were they similar or not from isolates that are shown on the
> gel?
>
> DR. KUEHNERT: Sorry, this is the March 2001 isolates?
>
> DR. BEUCHAT: Yes.
>
> DR. KUEHNERT: Yes, those were discarded before they knew there was a
> problem.
>
> DR. BEUCHAT: My second question is dealing with the four-hour hang
> time. What criteria\--what science-based information was used to
> determine and recommend that a four-hour hang time should be used?
>
> DR. KUEHNERT: That\'s a very good question. At the time that we made
> these recommendations, I think it was an educated guess. We weren\'t
> sure even that the hang time had a role in this. I think the previous
> question, you know, spoke to that, that they made these changes and
> didn\'t see any more isolates, and that doesn\'t mean that the changes
> they made resulted in fixing the problem. But also the flip side of
> that is we don\'t know whether the hang-time played role in it being a
> problem. But we knew that it made sense, you know, concerning
> microbial growth to shorten the time to as short as feasible. And I
> think four hours got to the limit of what we thought was feasible in a
> NICU.
>
> Since that time\--I\'m sorry I\'m not there, because I would have
> brought some extra slides, which I\'m looking at here\--our lab has
> done some studies on E. sakazakii growth in the implicated formula,
> Portogen with Iron. And what you see is a curve that shows very little
> growth until you get to about\--beyond six to eight hours, in which
> case, between eight hours and 24 hours you see a jump in growth from a
> log of 4 to about a log of 9. And also we\'ve done studies looking at
> not only that formula, but also other manufacturers formulas that have
> shown almost\--well, actually, identical, statistically significant
> differences in that growth.
>
> DR. BEUCHAT: On that growth curve that you were just describing, what
> was the initial number of E. sakazakii in the formula?
>
> DR. KUEHNERT: At t-0 hours, 3 logs\--so, 1,000 organisms were
> inoculated. And then you see about a log of 3 at t-2, t-4\--it goes to
> about a log of 4 at t-6 and t-8, and then it goes up to\--actually,
> it\'s log 8 at about 24 hours.
>
> I want to explain how this done. It was done under clinical
> conditions, so the formula was mixed with sterile water, and E.
> sakazakii was put in at that time. Then it was refrigerated for 24
> hours, and then hung. And that\'s t-0 was when it was hung. And so
> what I just described was under those conditions.
>
> DR. BEUCHAT: So the temperature would have been, perhaps, 70
> Fahrenheit? The room temperature?
>
> DR. KUEHNERT: Yes\--I don\'t have the breakdown to the exact
> temperature, but it was ambient temperature, which I\'d have to look
> at what actually she did. I think it was a little warmer than that, I
> think.
>
> DR. ACHOLONU: Doctor, my name is Alex Acholonu. I\'m from Alcorn State
> University in Mississippi.
>
> During your delivery, talking about the case findings, you talked
> about examining stool samples, tracheal aspirates, urine and CSF. May
> I know why blood is not checked as one of the fluids in the body?
>
> Number two question: we have been told that this disease is most
> prevalent in neonates, which mean children less than one month old.
> Did you, in order to rule out cases of prenatal infection, check the
> mothers of the babies?
>
> DR. KUEHNERT: That\'s a good question.
>
> We did not do cultures of the mothers or of siblings, or household
> contact at that time. Subsequent to this, there was another
> investigation in another hospital in Tennessee, where we did do those
> cultures\--vaginal cultures and other\--rectal cultures\--of the
> mother, and did not find it. But that\'s a very good point, and that
> was not done in this investigation\--to my knowledge.
>
> The first question, I didn\'t quite get. It was something about
> checking some site? What site was it?
>
> DR. ACHOLONU: Yes\--checking the blood. We are told that one of the
> effects of the disease is sepsis.
>
> DR. KUEHNERT: Right.
>
> DR. ACHOLONU: And I take it to be septicemia. May I know why blood
> samples were not checked.
>
> DR. KUEHNERT: Yes. Okay.
>
> That\'s sort of a complicated issue. I guess\--I mean, at the time it
> was sort of dealing with, certainly, an infection-control issue and an
> investigation. I\'m not sure\--I wasn\'t the hospital epidemiologist
> who had to make this decision. Inga Himmelreit was. But I mean, if I
> were her, I might be uncomfortable doing blood cultures where it\'s
> not clinically indicated. So I think that, you know, all these
> patients were clinically stable, and so I think that probably was the
> decision why they didn\'t do blood cultures. Although blood culture is
> obviously a low-risk procedure, it\'s not a no-risk procedure. And so
> I think that, you know, that may have been why it wasn\'t done. Also,
> the yield probably would have been pretty low.
>
> DR. ACHOLONU: My last question: since the infection is more in
> neonates in the intensive care unit, is it possible that some of the
> infections are nosocomial?
>
> DR. KUEHNERT: Do you mean that some of this\--
>
> DR. ACHOLONU: Picked up from the hospital, rather than coming from the
> baby formula?
>
> DR. KUEHNERT: Right. Right.
>
> Yes, I think that\'s a good point. I mean, as far as the stool\--I
> mean, we don\'t know\--okay, just looking at the cohort study, we
> can\'t tell whether these stool cultures represent transmission
> between the patients or that health care workers were a vector between
> patients. But the epidemiologic study, I think, clinches it, because
> though, again, looking back at that, those that were exposed to
> powdered formula were the only ones that had the stool cultures or
> cultures at other sites. Of all the patients who did not get powdered
> formula, none of those had E. sakazakii. And I think that sort
> of\--although there might have been something confounded exactly with
> powdered formula, I think that really leans against nosocomial
> infection. So that\'s what this investigation\--overall, I would say
> that it isn\'t a common nosocomial pathogen. I mean, I mentioned that
> NIS surveillance, and we just don\'t see it. And we\'ve also done some
> case finding, which I didn\'t have time to present, both clinical
> microbiology laboratories, ClinMicroNet, and also infectious disease
> consultants, and over a three-year period, there were very, very few
> isolates from NICUs. So, overall, it\'s probably pretty unusual, and I
> think the epi really points to the powdered formula rather than
> nosocomial transmission.
>
> DR. ACHOLONU: Thank you.
>
> DR. LEE: Hi. This is Ken Lee, from Ohio State.
>
> There was almost an offhanded reference to competing flora, and I\'m
> just wondering, is the competing flora a significant factor in E. sak
> infection, or is it even present? What\'s your best guess about other
> microorganisms that might be present?
>
> DR. KUEHNERT: Right. Well, \"guess\" is a good word as far as what
> I\'m going to say on that, as far as what the significance of it is. I
> mean, like the lab studies that I mentioned, I mean that was
> inoculating E. sakazakii into sterilized formula. We have not done
> studies yet, but I think it\'s important that we do, looking at sort
> of the in vivo environment of powdered formula, and how E. sakazakii
> behaves in that environment. We don\'t know if it\'s going to make it
> grow more slowly, grow faster. We don\'t know if other organisms are
> deleterious or actually whether it augments growth.
>
> It obviously is a complicated situation when you have, you know,
> multiple bacteria in the formula. And this also gets back to the
> difficulty in growing E. sakazakii out. I mean, again to just
> emphasize the obvious here, I mean formula is not sterile. When you
> try to grow\--when you try to isolate E. sakazakii you get all kinds
> of organisms growing from it\--you know, from bacillus, to other
> enterobacters, to other gram-negatives. And so the difficulty,
> actually, is in picking the E. sakazakii out. And what we don\'t know
> is whether the bacterial flora\--what role the other bacterial flora
> play in the growth of E. sakazakii.
>
> DR. TARR: Phil Tarr, Washington University.
>
> Is there any chance that these culture-positive cases are the tip of
> the iceberg? Was there any evidence of clustering of sterile CFS with
> the presence of cliacytosis that might be represented in partially
> treated cases of E. sakazakii?
>
> DR. KUEHNERT: Yes, that\'s a good question. We actually went back in
> the hospital and looked over a multi-month period at nosocomial
> infections of any kind; those due to enterobacter cloacae, because we
> thought, well, the point you bring up or touch on is that if formula
> has multiple organisms, could there be other organisms causing
> infection that maybe we don\'t notice because they\'re not as striking
> as a sentinel organism as E. sakazakii.
>
> We didn\'t see any association of nosocomial infections in general, or
> nosocomial meningitis associated with E. cloacae or with\--sorry, let
> me back up. We didn\'t see any association of powdered formula with
> either nosocomial infections or specifically nosocomial meningitis
> over the time period that we looked at at this hospital.
>
> I think it would be an interesting idea to look at that in a
> multi-center fashion. Again, it gets back to the difficulty and
> challenge of how hospitals record powdered formula. But if there were
> a way to get over that hurdle, I think this would be a very good idea
> to look at. Because we just don\'t know.
>
> DR. TARR: What particularly\--I\'m thinking of missed E. sakazakii, if
> it was partially treated prior to obtaining the CSF. Have sera been
> obtained from the survivors?
>
> DR. KUEHNERT: Ahh\--well, we have\--when we confirm organisms, we get
> the CSF and I\'m not sure if we have sera. We have the CSF stored, but
> I\'m not sure if we have sera.
>
> DR. TARR: Thank you.
>
> DR. BLUMBERG: This is Hank Blumberg from Emory University.
>
> There have been previous reports of E. sakazakii causing necrotizing
> enterocolitis, and I was wondering, in the outbreak that you
> investigated in Tennessee, were there any cases of necrotizing
> enterocolitis in the patients\--in the neonates\--who had positive
> stool cultures?
>
> DR. KUEHNERT: Yes. Thanks for that question, because it reminded me
> that\--no, the answer is no, we didn\'t see any NEC. And also, when we
> looked at that association between nosocomial infection and powdered
> formula, we also looked at NEC over the previous few years, and did
> not find an association.
>
> DR. BLUMBERG: Thank you.
>
> DR. TOMPKIN: Good morning. This is Bruce Tompkin, and I\'m the
> industry representative t this meeting.
>
> You mentioned a statistic that I hadn\'t heard of, where you\'d gone
> back to 300 hospitals over the past 10 years. Would you please repeat
> that, because I\'m not sure I heard it correctly.
>
> DR. KUEHNERT: Oh, yes. Sure.
>
> Umm\--this was not published anywhere. We did this case finding after
> this case, so this may be the first time you\'ve heard it. Let me just
> go through it again.
>
> We have a national nosocomial infection surveillance system, and this
> is a voluntary participation of hospitals in the United States. We
> looked back at\--and this is only nosocomial infections, you know,
> acquired in the health care setting\--and we looked back for E.
> sakazakii infection. This was during a 12-year period of surveillance.
> And we found one case of E. sakazakii meningitis in that time period.
>
> DR. TOMPKIN: And did you say it was 300 hospitals\--approximately?
>
> DR. KUEHNERT: Yes, it\'s about 300 hospitals.
>
> DR. TOMPKIN: Thanks.
>
> DR. KUEHNERT: Are there any other questions?
>
> DR. HEUBI: One comment. Jim Heubi.
>
> We would actually\--Dr. Busta and I were discussing the fac that we
> would actually appreciate it if you could provide us with the slide of
> the log growth for us to look at, if you have that.
>
> DR. KUEHNERT: Oh, sure.
>
> DR. HEUBI: I think that would be very valuable for us. And just to
> clarify\--this was E. sakazakii that was placed in sterile formula,
> and then hung and then cultured repetitively thereafter\--is that
> correct?
>
> DR. KUEHNERT: That\'s right. I\'m sorry\--so you were asking for me to
> e-mail it now, or\--
>
> DR. HEUBI: Right\--we\'ll actually\--someone from the FDA will contact
> you and we\'ll make arrangements to get this up here so we can have it
> for our books.
>
> DR. KUEHNERT: Okay.
>
> DR. HEUBI: Thank you for your presentation.
>
> \[Recess.\]
>
> DR. HEUBI: I think we\'ll reconvene.
>
> Dr. Karl Klontz is going to present other relevant investigations.
> Where\'s Dr. Klontz.
>
> **Other Relevant Investigations**
>
> DR. KLONTZ: Good morning. Karl Klontz, here.
>
> I want to spend the next 20 minutes or so\--oh, by the way, I\'m with
> the Epidemiology Team at the Center for Food Safety and Applied
> Nutrition.
>
> The title of my talk today is \"Enterobacter Sakazakii Case Reports
> and Outbreaks Involving Infants, As Reported in the Peer Reviewed
> Medical Literature.\"
>
> Next slide, please.
>
> \[Slide\]
>
> Now, before I go any further, you have probably\--you on the panel
> have a more extensive slide set than the one I\'m covering here. But I
> will, in the interests of time, just cover the most salient ones. And
> if there are questions on the others you have, I\'ll be happy to
> entertain those.
>
> Okay. For the purposes of summarizing the literature, we defined a
> case of Enterobacter sakazakii infection as an infant meeting at least
> one of the following criteria. First, E. sakazakii was recovered from
> one or more of the following normally sterile sites: blood,
> cerebrospinal fluid, brain tissue or urine.
>
> Second, an infant could have been classified or defined as a case of
> E. sakazakii if that infant was involved in an outbreak of necrotizing
> enterocolitis, and E. sakazakii was recovered from blood, stool or
> stomach aspirate of more than one infant in that outbreak.
>
> A third possible criterion was that the infant had bloody diarrhea and
> E. sakazakii was recovered from a stool specimen in pure culture; that
> is to say, no other pathogen was in the stool that could have
> accounted for the bloody diarrhea.
>
> So any infant that met one or more of these definitions, we called a
> \"case\" of Enterobacter sakazakii.
>
> Next slide, please.
>
> \[Slide.\]
>
> This slide summarizes the initial reports of Enterobacter sakazakii in
> the literature in chronological fashion. The first thing I\'d like to
> point out is that the first published report occurred in 1961, by
> Ermenye, et al. This was a report that described two fatal cases of
> neonatal meningitis. And, by the way, in some of these early reports,
> the name Enterobacter sakazakii, of course, wasn\'t used. That term
> was introduced\--proposed\--in 1980 by Dr. Farmer, et al. Some of the
> terms used earlier in these reports were \"yellow-pigmented
> Enterobacter cloacae\" and some other terms. But, nevertheless, for
> our purposes today, some of these following publications that came
> after the Ermenye, et al., study, described also infections in
> neonates or very young infants, and it really wasn\'t until 1982 that
> Jimenez, et al., reported the first case of Enterobacter sakazakii
> infection in an adult\--a 76 year old man with a history of rectal
> adenocarcinoma who had urosepsis and recovered after antibiotic
> therapy.
>
> Now the first case series was by Muytjens\--and, by the way, I\'m
> pronouncing this \"Moyt-yens,\" with a little bit of counseling from
> Dr. Muytjens himself. We were fortunate enough to reach him by e-mail
> in the last couple of weeks, and one of our questions, among other
> technical issues\--
>
> \[Laughter.\]
>
> \--was how to pronounce his name. And he was very kind to tell us
> \"Moyt-yens,\" although he did say, curiously, that his colleagues in
> the Netherlands, even in his area, have trouble pronouncing this name.
> So I\'m sure I\'m not doing a very good job here.
>
> But nevertheless, he published the first case series, and this was the
> first study to propose a possible link to powdered formula. And we\'ll
> go over this study in more detail, but\--let\'s go to the next slide.
>
> \[Slide\]
>
> I would like to stand back a bit now, and look at the literature as a
> whole, and point out that there were 27 references in the literature
> that described Enterobacter sakazakii infections, encompassing a total
> of 58 cases. As you can see, the majority of the cases were in
> infants; 17 percent in children over the age of one, or in adults. And
> in the bottom half of the slide, we\'ve broken down these cases by age
> group; the number of cases, and then the percent. And, as you can see,
> the majority, again, were in the first year of life\--83 percent. But
> even in that group, the majority were in the first month of life,
> underscoring\--at least from the literature standpoint\--that this
> appears to be largely a neonatal infection. That\'s not to say there
> aren\'t older cases. For example, there were six cases in individuals
> over the age of four. And if you look at their median age, they were
> actually at the other end of the spectrum\--median age 74 years of
> age.
>
> Okay. Let\'s go to the next slide, please.
>
> \[Slide\]
>
> Now this slide summarizes the 48 cases of Enterobacter sakazakii
> infection in infants, and selected clinical features of these cases.
>
> About half the cases were in males; half in females. Meningitis,
> however, was the predominant syndrome, accounting for 58 percent of
> the cases. Sepsis and bacteremia, 17 percent; and necrotizing
> enterocolitis, 29 percent.
>
> Overall, the case fatality rate was 33 percent. And, again, in the
> bottom half of this slide we\'ve broken down for you the birth weight,
> where reported, for these infants, and the number that were in each
> category, and then the case fatality weight. And, as you can see,
> there was really no increasing or decreasing trend of case fatality by
> birth weight. It\'s notable here that one of the stratum had a higher
> case fatality rate, but I think that\'s largely because a number of
> the cases in this group came from one paper\--and that\'s the Muytjens
> article that we\'ll discuss in just a minute\--which was a
> retrospective survey in the Netherlands, and probably more likely to
> have identified the most severe cases.
>
> Let\'s go to the next slide, please.
>
> \[Slide\]
>
> Now, this is the Muytjens article of 1983, the case series I
> mentioned. And it was an analysis of eight cases of neonatal
> meningitis and sepsis due to Enterobacter sakazakii. What they did
> here is they re-analyzed 20 Enterobacter strains that had been
> isolated from cerebrospinal fluid, going back six years; and 25
> strains from blood, going back two years\--all in the Netherlands.
>
> Now, their hypothesis was that Enterobacter sakazakii may not have
> been originally in this group of organisms because this organism
> produces its characteristic yellow pigment at temperatures generally
> lower than 36 degrees centigrade, which was the temperature that these
> strains were identified. So what they did is they used various
> biochemical and growth parameter systems, including a lower
> temperature, and they were able to identify eight of these organisms
> as actually being Enterobacter sakazakii. These were confirmed as
> being E. sakazakii by the Centers for Disease Control subsequently.
>
> And so the investigators then went back to medical records to obtain
> clinical data, retrospectively.
>
> Next slide, please.
>
> \[Slide.\]
>
> This slide now summarizes the clinical features of these eight cases
> of neonatal meningitis and sepsis. The first thing to point out is
> that three of the eight cases were delivered by Cesarean section, and
> this is notable, because in the literature, until this point, there
> had been some discussion about the possibility that Enterobacter
> sakazakii was acquired from the vaginal canal during the normal
> birthing process. But here we have three of the eight cases that had
> been delivered by Cesarean section, and one of which was delivered to
> a woman who gave birth less than 45 minutes after the membranes were
> ruptured.
>
> Now, as a rule, these infants progressed well in the first few days of
> life. The first signs of illness occurred between days four and eight.
> Two of the cases actually had necrotizing enterocolitis along with
> meningitis. The case fatality rate was 75 percent, and even the two
> survivors had significant neurologic sequelae.
>
> When death occurred, it did so within hours to several days after
> onset of illness. And the investigators actually used the term
> \"hemorrhagic encephalitis\" to describe the illnesses, because on
> autopsy, a number of these infants had large, swollen brains that were
> soft and showed signs of intra cerebral hemorrhage.
>
> Next slide, please.
>
> \[Slide.\]
>
> Same study\--now let\'s look at some of the epidemiologic features of
> these eight cases. Six out of eight had birth weights of less than
> 2,500 grams, with the lowest being 850 grams. Five of the eight cases
> were premature; that is, their gestations were less than or equal to
> 36 weeks.
>
> There was geographic clustering in this case series, in that five of
> eight cases occurred at the same general hospital in the Netherlands,
> whereas the other three cases were born at three other facilities. But
> there was also temporal clustering in this series, in that three of
> the five cases at the general hospital became ill within three months
> of each other, and even at the other hospitals, two of the three cases
> became ill within two months of each other.
>
> Next slide, please.
>
> \[Slide.\]
>
> Because of this geographic clustering, Muytjens and his colleagues did
> environmental sampling at the pediatric ward of the general hospital
> where five of the eight cases occurred, and they were able to recover
> Enterobacter sakazakii from prepared formula, from a dish brush, and
> from a stirring spoon\--but not from powdered formula itself, nor the
> water that was used to prepare that formula.
>
> Now, let me just say a word about the methods they used to test the
> powdered formula, because one of the questions we asked Dr. Muytjens
> in the last couple weeks\--because it\'s not stated in the
> article\--how much powdered formula did you look at? And he responded
> by saying it was somewhere between four and 10 grams of powdered
> formula\--which, in terms of today\'s methodologies is a relatively
> small amount.
>
> They did do plasmid profile analysis, and they concluded that \"three
> or four of the five isolates from the patients at the general hospital
> were probably the same strain.\"
>
> Now, it\'s important to note, though, that these plasmid: profiles
> differed between the isolates from the cases, versus the isolates from
> the prepared formula, but that may not be surprising, because keep in
> mind that this was a retrospective study. At the time of testing the
> prepared formula, some of the cases had become ill months, if not
> years, earlier
>
> Now, evidence that the formula, however, may have served as a vehicle
> for transmitting the infection was found in a 1990 letter to the
> editor by Muytjens, et al., to an infectious disease journal, in which
> he stated: \"No cases of Enterobacter sakazakii were observed at the
> general hospital since the powdered formula was replaced by liquid
> formula eight years ago.\" Again, liquid, of course, being a sterile
> product.
>
> Next slide, please.
>
> \[Slide.\]
>
> All right. Before I turn to three outbreak investigations, I would
> like to summarize, in this slide, some of the key developments that
> took place regarding Enterobacter sakazakii in powdered infant formula
> for the period 1961\--that is, the date of the first
> publication\--through the \'80s.
>
> The first thing to note is in 1980 Farmer, et al., in studying 57
> isolates of Enterobacter sakazakii, note in one of their tables that
> one of these isolates came from a \"pan of dried milk.\" This is a
> sample that came from a central public health laboratory in London,
> but there\'s no other details about it. We\'ve seen from the 1983
> Muytjens article that Enterobacter sakazakii was recovered from
> prepared formula, and that prompted the investigators to recommend, in
> fact, that sampling of \"milk\" take place as part of future
> investigations of Enterobacter sakazakii infections.
>
> And then in 1988 Muytjens and colleagues obtained 141 powdered infant
> formula products from 35 countries, tested them for the presence of
> Enterobacteriaceae and found 52 percent of the samples were positive;
> 14 percent of 20 powders were also positive for Enterobacter
> sakazakii.
>
> Important, thought, is to underscore that the concentration of
> Enterobacteriaceae in these powdered formulas was uniformly less than
> or equal to one colony-forming unit per gram.
>
> Next slide, please.
>
> \[Slide.\]
>
> Okay. What I\'d like to do now is use the second half of this talk to
> go over three particular outbreak articles that I think are salient to
> today\'s discussion. The first one is by Biering, et al., 1989. It was
> entitled, \"Three cases of Neonatal Meningitis Cause by E. Sakazakii
> in Powdered Formula.\"
>
> These illnesses occurred in 1986 and 1987 in Iceland\--two within a
> month of each other. Two of the infants were normal at birth. Their
> gestations are listed there. One baby was a Downs syndrome baby. All
> three did well until day five, when they became ill. The Downs baby
> died. The two other infants recovered, but with several neurologic
> sequelae. The investigators found that all three had been fed powdered
> infant formula, and this was the first study to show, at least in an
> illness setting, that Enterobacter sakazakii could be
> recovered\--albeit in low numbers\--from freshly prepared formula from
> a previously unopened can.
>
> Now, the investigators did not recover, or were not able to recover,
> the organism from the environment; specifically not from the milk
> kitchen, nor the utensils, nor the ward.
>
> Next slide, please.
>
> \[Slide.\]
>
> Staying with this same article by Biering, et al., they note that in
> addition to three ill cases, there was an infant with colonization,
> and that these four Enterobacter sakazakii strains that were isolated
> from the neonates had the same plasmid profile as 22 strains recovered
> from the formula. They also point out, however, that there was
> anecdotal evidence that the formula bottles had occasionally been kept
> at fairly warm temperatures\--35 to 37 degrees centigrade\--for
> extended periods in bottle heaters. However, they also note that in
> one instance they were able to recover the organism via direct plating
> from a bottle that had been refrigerated for an unknown amount of
> time.
>
> From this outbreak investigation, the investigators concluded: \"Milk
> powder can be the mode of transmission for Enterobacter sakazakii
> meningitis or sepsis in neonates.\"
>
> Next slide, please.
>
> \[Slide.\]
>
> The final two studies I\'d like to discuss with you I bring up because
> I think, importantly, can be discussed in the context of what we call
> \"historical cohort studies\" in epidemiology, which are very useful
> because they allow one to calculate rates of infection by exposure
> status\--whether infants ate the formula or did not eat the formula.
>
> And the two in particular I want to discuss with you are the Simmons,
> et al., article in \'89, and the Van Acker in 2001. But keep in
> mind\--we heard this morning from Dr. Kuehnert, the Tennessee outbreak
> of 2001 published in the MMWR, that too was handled as a historical
> cohort study.
>
> Okay, Next slide, please.
>
> \[Slide.\]
>
> The first study is the one by Simmons, et al., 1989, entitled,
> \"Enterobacter Sakazakii Infections in Neonates Associated with
> Intrinsic Contamination of Powdered Infant Formula.\" These illnesses
> occurred in February and March of 1988. There were two cases of
> bacteremia, one urinary tract infection, and one case of blood
> diarrhea. Enterobacter sakazakii was recovered from the stool of all
> four infants, which prompted the investigators to go back and look at
> the feeding practices in that neonatal intensive care unit. And they
> found that all four had been fed the same powdered formula.
>
> This formula had been prepared in a blender which was then rinsed with
> tap water between uses. On culturing, the blender yielded a heavy
> growth of Enterobacter sakazakii, but once a policy of sterilizing the
> blender was instituted, no further clinical isolates were obtained.
>
> They then conducted a historical cohort study in the neonatal
> intensive care unit to assess risk factors for infection and
> colonization. And, as you can see, this historical cohort study took
> place\--or covered a period of about five weeks in February and March
> of \'88.
>
> Next slide, please.
>
> \[Slide.\]
>
> All right. Here, then, are the results of the Simmons, et al.,
> historical cohort study. And it\'s just the standard setup for a
> two-by-two table, where we\'ve got \"infection and colonization\" up
> on the top\--\"yes/no;\" whether the infants ate the implicated
> formula, \"yes/no\" on the left.
>
> And, as we can see from the slide, in this outbreak setting, four of
> the five infants who consumed the implicated product developed
> infection or colonization, as opposed to zero of 40 infants who did
> not eat the implicated product. That yielded a highly statistically
> significant association between the implicated formula and
> infection/colonization.
>
> Next slide, please.
>
> \[Slide.\]
>
> Staying with the same outbreaks, they note that the implicated
> elemental formula had been given primarily to the most premature
> infants, largely because it required relatively little digestion. They
> obtained samples from an opened yet un-mixed can of powdered formula,
> and they cultured this powdered formula using the 1988 methods of
> Muytjens, et al., which called for at least 100 grams of powder to be
> sampled in each sampling frame, and then repeated three times for that
> matter.
>
> They were able to recover Enterobacter sakazakii from the powdered
> formula at a concentration of approximately eight colony-forming units
> per 100 grams. In addition, they cultured\--they recovered
> Enterobacter cloacae at a concentration of 48 colony-forming units per
> 100 grams. And, with respect to the Enterobacter sakazakii, they had
> the same plasmid and multi-locus enzyme profile\--both the isolates
> from the formula as well as the isolates from the patients.
>
> Next slide, please.
>
> \[Slide.\]
>
> All right, the final outbreak I would like to discuss with you is the
> 2001 Van Acker paper, entitled \"Necrotizing Enterocolitis Associated
> with E. Sakazakii in Powdered Formula.\" In this study there were 12
> cases of necrotizing enterocolitis that had been diagnosed in June and
> July of 1998 in Belgian neonatal intensive care unit. In this
> period\--June and July\--a total of 50 neonates were admitted to that
> neonatal intensive care unit, but with respect to the 12 cases of
> necrotizing enterocolitis, all 12 had birth weights of less than 2,000
> grams, and had been fed powdered infant formula. Six of the 12
> neonates with necrotizing enterocolitis had positive cultures for
> Enterobacter sakazakii\--and the specimens are listed up here\--versus
> zero of the 38 without necrotizing enterocolitis, and that was a
> statistically significant difference.
>
> In addition, 10 of the 12 neonates with necrotizing enterocolitis had
> been fed the same powdered formula, and the article states that this
> was a product called Alfare, a semi-elemental formula with a low
> osmolarity.
>
> Next slide, please.
>
> \[Slide.\]
>
> Here, using the same setup that we used before, are the results of the
> historical cohort study in this outbreak. And, as we can see, six of
> 14 infants who were fed the implicated formula developed Enterobacter
> sakazakii-positive necrotizing enterocolitis, versus zero of 36
> infants who did not\--were not fed that implicated formula. Again, a
> statistically significant association between the implicated formula
> and the development of necrotizing enterocolitis.
>
> Next slide, please.
>
> \[Slide.\]
>
> Van Acker and his colleagues noted that Enterobacter sakazakii was
> recovered from the prepared formula, as well as from unopened cans.
> They did molecular typing, using arbitrarily primed polymerase chain
> reaction, and they confirmed a partial strain similarity between the
> powdered formula and the patient isolates.
>
> There were no further cases of necrotizing enterocolitis observed
> after the implicated powdered formula was stopped in that neonatal
> intensive care unit. But it\'s interesting to note that during the
> outbreak period there was an inadvertent challenge test that took
> place as follows.
>
> The implicated formula was stopped on July 10^th^ of 1998, as soon as
> the investigators had a suspicion that there was an association
> between illness and that formula. But then the formula was released
> again 10 days later, when all cultures to that date had been negative.
> However, three days after releasing that formula, there was a new case
> of necrotizing enterocolitis linked to the same formula, and it was on
> that same day that the cultures came back showing intrinsic formula
> contamination.
>
> Next slide, please.
>
> \[Slide.\]
>
> In the same article, they also provide information regarding the
> manufacturers quality control data for the implicated formula, and
> that is as follows. Of five samples analyzed, one yielded 20 coliforms
> per gram; four yielded less than one coliform per gram. And these
> results fulfilled the requirements of Codex Alimentarius, which call
> for a minimum of four of five control samples to have less than three
> coliforms per gram, and a maximum of one in five samples that may have
> more than three, but less than or equal to 20 coliforms per gram.
>
> However, the implicated formula did not meet Belgian law, which called
> for less than one coliform per gram in all samples, and therefore the
> product was recalled.
>
> Next slide, please.
>
> \[Slide.\]
>
> I think this is my final slide, and I want to end it, again, on the
> Van Acker publication, because they note in the article that the
> facility that produced the implicated formula in the Netherlands was
> upgraded. More stringent standards were adopted for products;
> specifically, calling for less than .3 coliforms per gram, and zero
> Enterobacter sakazakii isolates per 10 grams.
>
> After these changes were instituted at the production facility, the
> implicated powdered formula was reintroduced to the very neonatal
> intensive care unit where the outbreak had taken place. Reintroduction
> took place in 1999, and Van Acker, et al., note that as of the
> publication date\--2001\--no further cases of necrotizing
> enterocolitis associated with E. sakazakii had taken place\--or had
> been diagnosed\--in that neonatal intensive care unit where the
> outbreak occurred.
>
> All right. That\'s my last slide, and I\'ll stop at this point.
>
> Do we want to take questions now? I saw by the schedule Dr. Alexander
> is coming up, but I defer to you as to what you\'d like to do.
>
> DR. HEUBI: I think we\'re going to take some questions now.
>
> DR. KUZMINSKI: Dr. Klontz\--Kuzminski, here\--just a question on your
> very last slide, please, if you could replace that on the screen.
> Thank you.
>
> Do you have information on the nature of the upgrades at the
> production facility?
>
> DR. KLONTZ: No. The article doesn\'t get into the specific upgrades
> that were made. The reference to this is in the discussion section,
> and it\'s fairly limited. No, I don\'t know exactly what took place
> there.
>
> DR. BEUCHAT: Larry Beuchat.
>
> Out of these cases that you\'ve summarized here, or other information
> that you might have, what is the minimum infectious dose of E.
> sakazakii, in terms of eliciting illness?
>
> DR. KLONTZ: The literature\--I don\'t think the literature answers
> that question. Certainly, not in the articles I read could one
> ascertain a definitive infectious dose. There are a lot of
> complicating factors here, as I\'m sure you\'re aware\--how much is in
> the powdered formula that leaves the plant, versus what sort of
> treatment that product received in the hospital setting, in terms of
> hang time and so on. So those are a lot of complicating factors. I
> don\'t think the literature tells us what the infectious dose is here.
>
> DR. ACHOLONU: You said that\--Alex Acholonu, from Alcorn State
> University in Mississippi.
>
> You said that you recovered the bacterial organism in unopened cans.
> My question is: what is the longevity of the bacterial organism; how
> long was it in the can when it was checked? And when we consider the
> fact that you\'re dealing with a powder in an unfavorable environment
> for the bacteria, is growth sustained while the organism is in the
> can?
>
> DR. KLONTZ: Again, the articles don\'t go into those details. That
> information is just not there. I saw no discussion whatsoever that
> would answer any of those questions\--how long the organization was in
> the can, or whether it reproduced in the can, how the environment
> could have affected its numbers and viability\--none of that
> discussion is in these articles. So I just can\'t answer that
> question.
>
> DR. ACHOLONU: But you are aware of the fact that they don\'t grow very
> well in dry conditions.
>
> DR. KLONTZ: That\'s what I hear. Right. That\'s what I hear.
>
> DR. ACHOLONU: So it is necessary to find out what is keeping them
> alive. They are not spore-forming bacterial organisms, as far as I
> know. So how do they stay in the can\--indefinitely\--until it is
> opened? Maybe this is food for thought; something we may have to think
> about.
>
> DR. KLONTZ: Yes. And I think as the day goes on we\'re going to have
> some more microbiologically-oriented discussions, and this would be a
> very relevant question to arise then.
>
> DR. NEILL: Peggy Neill.
>
> I was just trying to quickly go back through this, but it\'s probably
> faster just to ask you. In your literature review, then, are all the
> cases\--are any of the cases reported associated with non-powdered
> formula preparations?
>
> DR. KLONTZ: Let me start to answer that by saying\--no, I didn\'t see
> any cases that were linked to non-powdered formula preparations.
> However, a number of these articles don\'t even comment on source at
> all. There\'s just a question mark, when one finishes reading the
> article, as to the source.
>
> The ones I\'ve presented, I picked out largely because they focused,
> they commented on, or at least took effort to check the source. And,
> of those, they were all powdered produces that were implicated.
>
> DR. FISCHER: \[off mike\]
>
> DR. HEUBI: Please identify yourself.
>
> DR. FISCHER: Larry Fischer.
>
> You mentioned that several of the publications indicated that they
> fixed the problem by either using a sterile liquid, or they upgraded
> production facility. But did any of them say that they altered the
> procedures used to prepare the formula in the hospital and fix the
> problem?
>
> DR. HEUBI: No. From my reading of the literature, I didn\'t see any
> comment about that specific aspect of changing the way that they
> prepared it. There was actually relatively little discussion in these
> articles regarding\--after the outbreak, so to speak. And this is one
> area that I didn\'t see any comment, really, in terms of what they did
> in-house, in terms of their mechanism of dealing with powdered
> formula.
>
> DR. TARR: Tarr\--Seattle\--ah, St. Louis.
>
> \[Laughter.\]
>
> DR. TARR: Did the incidence of NEC total go down after these
> outbreaks, or was there inadequate data in the paper? And, also, you
> talked about powdered formula versus no powdered formula. Among the
> \"no powdered formula,\" was that heterogeneous or homogeneous, with
> respect to breast milk, or not eating anything at all?
>
> DR. KLONTZ: Regarding the first question, did necrotizing
> enterocolitis decrease? Do you mean in this particular outbreak
> setting, or just as a literature\--sort of an aggregate view?
>
> DR. TARR: Total cases of NEC in these facilities.
>
> DR. KLONTZ: Yes. The only article that commented, to my knowledge, on
> that was Van Acker, who said following the changes made at the firm,
> product reintroduced in 1999, and then until 2001, no further cases of
> necrotizing enterocolitis diagnosed in the facility.
>
> There\'s relatively\--there\'s very little sort of post-outbreak
> surveillance that\'s discussed in these articles.
>
> DR. TARR: \[off mike\] I\'m wondering about NEC not associated with\--
>
> DR. KLONTZ: They do not discuss that. I can\'t answer that.
>
> DR. TARR: And the second question is: the no powdered milk group, that
> was supposedly protected from the disorder, do we know\--is that
> NPO\--children no eating anything? Versus breast milk?
>
> DR. KLONTZ: They don\'t\--again, unfortunately, there\'s a lack of
> details on sort of other feeding practice attributes of the unexposed
> group. So I can\'t be too specific about that. Nevertheless, they
> shared the same environment. They were in the same place, over the
> same period of time that the exposed infants became infected. That\'s
> about as far as one can say.
>
> DR. KUZMINSKI: Thank you. Larry Kuzminski.
>
> Dr. Klontz, in one of your earlier slides of the Muytjens paper from
> 1983, there was a bullet that says, \"E. Sakazakii recovered from
> prepared formula but not from powdered formula and water.\" And you
> commented on that four to 10 grams of the powdered formula\--which you
> indicated was a small amount\--relatively small amount\--was used for
> the sampling here.
>
> And I relate that observation to what you have in the last slide here,
> on the second bullet: \"More stringent standards adopted for products.
> Zero E. sakazakii per 10 grams of product.\"\--a relatively small
> amount when you relate it back to your observation on the Muytjens
> sampling\--environmental sampling paper published in \'83.
>
> So I\'m just wondering: is, indeed, 10 grams, the more stringent
> sample\--quantity for a more stringent standard, when historically the
> literature would indicate it wasn\'t found with that amount sampled in
> the past, and perhaps not being found now? Just your comment.
>
> DR. KLONTZ: Yes, that\'s an interesting point. That\'s an interesting
> point. And, in fact, today, as some of the talks that we get into
> shortly on the microbiological aspect will underscore is that
> generally 100 grams of powder are taken, or 111 in three different
> sub-samples, and repeated three times. So much larger amounts than 10
> grams are used today , you know, in the process of looking for this
> organism.
>
> So one could argue that 10 grams is\--you\'re right\--a relatively
> small amount. And certainly today all I can say is larger amounts are
> used, in part because this organism may be uniformly distributed in
> dried powder. And if one is going to detect it, if it\'s indeed
> present there, one needs to look at larger amounts. It\'s a good
> point.
>
> DR. TOMPKIN: This is Bruce Tompkin.
>
> During the public open comments, there will be some information
> relative to the specific plant that was involved in the outbreak as
> Muytjens has reported. Okay. And could you clarify again, then,
> you\'re essentially summarizing all the data of cases reported
> internally, from across the world\--you mentioned 48\--but within the
> United States, there\'s a little confusion as to the number of cases,
> or clusters, that have occurred in the U.S. Could you\--
>
> DR. KLONTZ: In the\--I don\'t have the actual count. I believe you
> have a line list in your\--or, if you don\'t\--in your packet, that
> actually goes through each of the outbreaks and each of the single
> cases, as well, as to what country they were from and so on. So, if
> that\'s not in there we can get it to you.
>
> A quick count? I know that in terms of outbreaks, there was the
> Tennessee 2001 Dr. Kuehnert described. Simmons outbreak was four
> neonates. That was also the United States outbreak there. And then
> there have been some sporadic individual cases reported in the United
> States.
>
> But I think the bottom line is that this is truly an international
> phenomenon. And the Muytjens culture study of 1988 showed that, you
> know, formula from 35 different countries he tested and found a 14
> percent positivity for E. sakazakii. So I think international is
> really sort of the bottom line here.
>
> DR. HEUBI: Any additional questions?
>
> Thank you.
>
> Now, Dr. John Alexander is going to discuss clinical consequences of
> E. sakazakii infections.
>
> **Clinical Consequences of E. Sakazakii Infections.**
>
> DR. ALEXANDER: : Good morning. My name is John Alexander. I\'m an ID
> trained pediatrician who works in the Division of Anti-Infective Drug
> Products, in the Center for Drugs at FDA. And I was asked to give a
> presentation on what the clinical consequence of Enterobacter
> sakazakii infection in infants is.
>
> Next slide.
>
> \[Slide.\]
>
> So what I\'m going to do briefly is give a presentation talking about
> the three major manifestations of infection that have been identified
> in infants: neonatal meningitis, necrotizing enterocolitis and
> bacteremia and sepsis. And I\'m going to spend most of the time
> talking about neonatal meningitis, because that\'s\--we have the
> clearest information about it, in terms of the consequences of the
> disease and the infection.
>
> Next.
>
> \[Slide\]
>
> Now, overall, neonatal meningitis is a disease that occurs with an
> incidence of about .25 to 1 per thousand live births. But most of this
> is from the usual pathogens: Group B streptococci and Escherichia
> coli, with a small proportion cause by listeria monocytogenes, as the
> three major pathogens.
>
> There are, of course, a lot of other bacteria, other gram negative
> organisms, other types of bacteria that can cause neonatal meningitis.
> In one survey that was done in Dallas, Enterobacter species\--and I
> use \"species\" because of the fact that this was done before
> sakazakii was even separated from other Enterobacters\--accounted for
> less than 4 percent of the organisms. So this is talking about a rare
> organism in what is a rare disease.
>
> Next slide.
>
> \[Slide\]
>
> Now, the clinical manifestations of neonatal meningitis are often
> difficult, just as it is often difficult in neonates, to separate any
> type of infectious disease one from the other. There are a lot of
> findings that are non-specific and may represent multiple different
> infections, including fever or temperature instability, lethargy or
> just poor feeding, and some infants, as they become more ill, develop
> respiratory distress.
>
> The more specific findings of neonatal meningitis are infrequent,
> including a bulging fontanelle\--so the soft spot on the top of the
> head starts to raise up. Stiff neck, or epistotonus\--some type of
> posturing, or convulsions that develop from the infection.
>
> Next slide.
>
> \[Slide\]
>
> Now, the information that we have on E. sakazakii itself causing
> neonatal meningitis is based on the collective literature. And there
> is a predisposition for this occurring in neonates that are less than
> 2,500 grams, as a symbol of prematurity. So that it occurs more often
> in premature infants. And about half of the pediatric cases that have
> been reported, that were summarized by Lai, et al., in 2001, occurred
> in children that were less than one week of age, and almost 75 percent
> of all cases occurred in less than one month of age.
>
> But I would point out that this just represents what is collected in
> the literature as case series, and this predisposition may be just the
> fact that we are identifying through epidemiologic outbreaks in NICUs
> that might sort of push you towards thinking that this is much more of
> a disease in neonates and isolated to the neonatal intensive care
> unit. But there were a couple of cases that were reported in otherwise
> health term newborns, including a case that was reported of a
> five-week old who was healthy, left the hospital on time, who at five
> weeks of age developed neonatal meningitis. So that is important to
> keep in mind, that we aren\'t just talking about a NICU disease.
>
> Next slide.
>
> \[Slide\]
>
> One of the important manifestations that needs to be kept in mind with
> Enterobacter sakazakii infection is a predisposition towards
> developing these large cystic lesions that are believed to be brain
> abscesses in the brain. And this is important because in\--this is
> much more likely to represent severe disease, as opposed to other
> causes of meningitis. Other causes of neonatal meningitis that were
> listed\--the Group B, strep, E. coli and such\--actually cause brain
> abscess in a minority of cases, but from the cases that we have
> identified so far in the literature, approximately 75 percent or so
> are cases that involve brain abscess. And this is something that\'s
> recognized. There are other bacteria, including one called Citrobacter
> diversus, that\'s recognized to cause\--to have a predisposition to
> causing brain abscess, so that when it\'s there in the brain you have
> to look at CT scans to see whether these brain abscesses are present.
>
> Next slide.
>
> \[Slide\]
>
> Now, for neonatal meningitis, the outcome that we\'re concerned most
> about is certainly fatality. And for gram negative meningitis\--so,
> gram negative organisms of any variety that cause meningitis there\'s
> approximately a 17 percent case fatality rate. For E. sakazakii
> meningitis, from the reports, Lai, et al., had given about 45 percent,
> and the different reports that looked at the collective literature
> ranged from 40 to 80 percent.
>
> Now, it\'s a little difficult to take this as an indicator of what the
> overall case fatality rate is, because what you\'re doing is
> collecting the literature reports. But I do believe that we are
> talking about something that\'s at least 17 percent, and probably
> greater.
>
> Therapy for infants who have neonatal meningitis is usually at least
> three weeks of IV antibiotics, and would be longer for persistent
> positive cultures, which would be expected in those cases where you
> have brain abscesses. So you are talking about prolonged IV antibiotic
> treatment.
>
> Next slide.
>
> \[Slide\]
>
> Now, this study by Onhanand, et al.\--and I didn\'t check beforehand
> about how to pronounce that name\--
>
> \[Laughter.\]
>
> \--gives the sequelae for gram-negative meningitis overall in a his
> institution. And they had sequelae in approximately 58 percent of
> those children who survived. You have children with developmental
> delays, seizure disorders, cerebral palsy, hydrocephalus, and hearing
> loss as the main sequelae that are recognized from gram-negative
> meningitis. And, again, these are from a variety of other organisms,
> and I think it is appropriate to show these numbers because, if
> anything, they would probably be an underestimate of what Enterobacter
> sakazakii that causes these brain abscesses could do.
>
> Next slide.
>
> \[Slide\]
>
> So now we move on to necrotizing enterocolitis. And as an ID-trained
> pediatrician, I never thought I\'d be speaking to a bunch of
> gastroenterologists and nutritionists about this disease, but here
> goes.
>
> It\'s a disease of the GI tract that is seen mostly in premature
> infants. Up to 10 percent of NICU admissions, and approximately 10
> percent of affected infants, though, are term infants. So this is,
> again, mostly a disease that is seen and recognized in premature
> infants. It does, though, occur in some term infants, as well.
>
> And this is a multifactorial disease, so that there are other factors
> besides just the presence of organisms that are believed to play a
> role in this disease. But the reason that infection in some cases is
> considered important is because of the fact that there are the
> outbreaks that occur in association with infections.
>
> Next slide.
>
> \[Slide\]
>
> Now, it\'s important to remember that Enterobacter sakazakii is only
> one of a number of organisms that have been associated with
> necrotizing enterocolitis. These are lists from a pediatric infectious
> diseases textbook of the different bacteria and viruses that have been
> associated with outbreaks of necrotizing enterocolitis. So, in
> response to some of the questions that had been raised earlier about
> the effect on the incidence of NEC, I don\'t think it\'s that
> surprising that there may not be a change in the incidence of NEC
> overall from changing whether the exposure to E. sakazakii occurs.
>
> Next slide.
>
> \[Slide\]
>
> Now, the clinical manifestations of this disease\--again, there\'s a
> wide spectrum of disease, ranging from a sudden or to an insidious
> onset. Again, there are nonspecific findings, as you expect with
> neonates, of a bunch of different symptoms of infection or disease:
> feeding intolerance, temperature instability, lethargy. Some develop
> apnea and respiratory distress; evidence of metabolic acidosis, unable
> to maintain their blood glucose, and then a bunch of more specific GI
> findings, where you have infants who develop abdominal distension,
> tenderness and erythema, bilious emesis, blood in stools that\'s
> either grossly visible or microscopically tested for; and then a bunch
> of radiologic findings\--pneumatosis intestinalis being sort of a
> specific finding of air within the wall of the intestine that
> indicates that necrotizing enterocolitis is occurring. And then when
> you get to portal venal gas or pneumo turg, in the end you\'re talking
> about a\--usually a rupture of the intestinal wall and leakage of the
> gas into the systemic circulation or into the peritoneum.
>
> Next slide.
>
> \[Slide\]
>
> Briefly, therapy includes discontinuing feedings and nasogastric
> decompressions following serial radiographic examinations; blood
> cultures and antibiotics are usually given. IV fluid and supportive
> care when there are systemic manifestations of disease, so when the
> infant starts to appear shocky; and then for advanced disease,
> surgical intervention to remove part of the bowel that\'s infected.
>
> Next slide.
>
> \[Slide\]
>
> Now, the outcomes from both textbooks of neonatal medicine and a
> recent surgical article give overall mortality rates to NEC of around
> 9 to 28 percent. The survival is around 98 percent for medical
> management\--and medical management, again, is the less severe cases.
> It\'s the more severe cases that usually end up going to surgery.
>
> The surgical mortality rate was approximately 45 percent in the
> article by Grosfeld, et al., and ranged up to 60 percent from various
> sources. And, again, surgical mortality is inversely related to
> gestational age and size, so it\'s the younger and smaller infants
> that are going to have more problems overall and a higher risk of
> mortality.
>
> These are other complications of necrotizing enterocolitis: GI
> strictures occurring in 25 to 35 percent, whether after medical or
> surgical therapy; other gastrointestinal dysfunction;
> neuro-developmental sequelae\-\--whether that\'s directly related to
> the NEC or related to prematurity and NEC is difficult to tease out;
> and then short-gut syndrome, which is a syndrome where children have
> problems and require other forms of alimentation because of the fact
> that they don\'t absorb quite enough food from the feedings that they
> get.
>
> Next slide.
>
> \[Slide\]
>
> Now, for bacteremia and sepsis, just to give you a separation here,
> bacteremia is talking about bacteria in the blood, and it can occur
> with many different infections. A lot of the bacteremia that is
> reported with Enterobacter sakazakii is actually in association with
> the meningitis, which isn\'t surprising because you have to get the
> organism into the blood to get it to the brain; but also in cases of
> NEC.
>
> Sepsis is actually a clinical syndrome where you\'re talking about
> fever, systemic illness progressing to shock, and it is associated
> with morbidity and mortality. And from bacteremia to sepsis, there\'s
> actually a spectrum of disease, and different organisms can cause
> different things. Some organisms\--for instance pseudomonas\--are more
> likely to present with sepsis, even in normal hosts. Other organisms,
> like Enterobacter sakazakii, are more likely to be organisms that
> would be identified as bacteremia and in populations that are
> populations that are predisposed to developing the infection.
>
> Next slide.
>
> \[Slide\]
>
> So, what we\'re talking about here is basically an opportunistic
> pathogen; an organism that isn\'t usually something that\'s identified
> in children who are otherwise normal and healthy. The predisposing
> factors that have been recognized include the neonate and premature
> infants, where meningitis and NEC cases have been seen. And one of
> the\--what I\'m going through here is basically the reports that we
> have from the literature of infants who were septic, so there were the
> many reported cases with meningitis. There was also a separate report
> of a seven-day-old infant who developed fever and sepsis, but not
> meningitis, that was hospitalized and, I believe, recovered with
> antibiotic treatment.
>
> There are also other\--two other reports of children with bacteremia.
> One was a three-year-old with a rhabdomyosarcoma who recovered with
> antibiotics and removal of the lung from which the organism was
> identified, and what his exposure is is unknown.
>
> There\'s also the issues of altered host defense. The other case
> report of bacteremia is in a six-month-old who had had an intestinal
> resection. So that\'s probably a potential site of entry for the
> organism from the intestine. And this patient also recovered with
> antibiotics.
>
> So it\'s overall, in terms of talking about bacteremia, we have to
> recognize that it\'s difficult to just take the incidence of sepsis
> from other organisms and apply it to this disease. We are talking
> about, here, bacteremia with an opportunistic infection, so that
> you\'re talking about infants that are sickened in some way, have
> altered host defenses, or altered immunity that sort of predisposes
> them to developing infection with this organism.
>
> In conclusion, Enterobacter sakazakii is a cause of meningitis, NEC
> and bacteremia\--all diseases that are associated with serious
> morbidity and mortality, and it\'s found especially in neonatal
> disease but not exclusive to that population.
>
> DR. HEUBI: Questions?
>
> Heubi. My first questions would be: the last two\--the last slide you
> showed, the patient with rhabdomyosarcoma, and one with short-gut,
> were either of those patients on powdered formula during the course of
> their therapy; specifically, the short-gut patient?
>
> DR. ALEXANDER: I don\'t know.
>
> Actually, for the\--you\'re talking about the six-month-old who had
> the surgery?
>
> DR. HEUBI: Right. Right.
>
> DR. ALEXANDER: She had the surgery, for a jejunal atresia, actually.
> And I believe that she was receiving powdered formula. I\'d have to go
> back and check that.
>
> DR. HEUBI: Thank you.
>
> Virginia?
>
> DR. STALLINGS: Stallings.
>
> We haven\'t discussed antibiotic coverage. Is this a particularly
> difficult infection to treat once you\'ve identified it? Because the
> mortality rate seems to be high, in addition to the brain injury. What
> is it usually sensitive to, and why are the kids dying so fast?
>
> DR. ALEXANDER: Well, part of it is that meningitis, and especially
> meningitis that causes brain abscesses in premature neonates is more
> likely to be something that\'s going to be associated with mortality.
>
> Part of it, again, is that collecting this information from the case
> reports, we\'re more likely to be getting a snapshot of those patients
> with severe disease. We don\'t necessarily have a case series for the
> experience at one or a bunch of hospitals with this disease where we
> could feel more comfortable that we are sort of collecting all of the
> cases. So, there may be some of the\--there may be some bias towards
> seeing reports of higher mortality, and that\'s why we\'re dealing
> with that.
>
> In terms of the susceptibility of the organisms, it is susceptible to
> most of the immunoglycosides, which are typical treatment for
> neonates. So, I think that I saw one report for Enterobacter where
> gentamicin susceptibility for the genus as a whole is approximately 94
> to 95 percent. And from the case reports, I didn\'t see anything that
> really spoke to anybody having a problem with treatment, other than a
> very recent report, about a month or two ago, of an adult who had an
> Enterobacter sakazakii infection that was resistant to
> immunoglycoside.
>
> DR. HEUBI: Margaret? Oh. I thought you were going to ask a question.
>
> DR. BAKER: Baker.
>
> I wanted to just think a little bit about the susceptible populations.
> The original\--the FDA \"Dear Doctor\" letter was specifically for
> NICUs, and we\'ve seen that several of these outbreaks have been in
> NICUs, so our sort of\--the feeling was that this is a neonatal and
> maybe specifically for premature kids. On the other hand, we
> had\--there\'s been two things that have sort of contradicted that,
> and that is\--there was a slide earlier this morning which didn\'t
> show any greater susceptibility with decreasing birth weight. And then
> you talked about a five-day-old who was supposedly full term and was
> infected with E. sakazakii.
>
> DR. ALEXANDER: It was actually a five-week-old.
>
> DR. BAKER: Five-week-old\--so\--and then there have been some of these
> sporadic cases of older children and adults with E. sakazakii. So can
> we identify a susceptible population, and are there other susceptible
> populations outside the NICU? That\'s my question.
>
> DR. ALEXANDER: Well, I guess that for neonatal meningitis, and for the
> meningitis manifestations of Enterobacter sakazakii it really does
> seem to be something that is isolated to those infants that are within
> the first months or two of age, because for the reports that we\'ve
> received\--for the reports that I\'ve seen, at least, of Enterobacter
> sakazakii in anybody that\'s older than a month or two of age\--which
> is around the cutoff where you start transitioning from what would be
> considered the normal neonatal meningitis pathogens to
> community-acquired meningitis pathogens.
>
> After that transition period, you don\'t really see any reports of
> people who have meningitis, other than one report where I think there
> was an infection of the central nervous system in somebody who had
> some type of congenital malformation of the central nervous system.
> So, neonatal meningitis itself is something that is more likely to be
> seen early on, and more likely to be seen in more premature infants,
> and that may be why what we\'re seeing is mostly reports that are
> associated with the NICU.
>
> For the bacteremia, I think that we are talking about a population
> that has some altered host defense, so that you\'re not really talking
> about children who are otherwise normal and healthy, but it can be as
> simple as children who have had previous surgery, as well as children
> who have immunosuppression, or some other altered host defense system.
>
> DR. HEUBI: Phil?
>
> DR. TARR: Tarr.
>
> I\'d like to probe again about the issue of potentially missed cases.
> Are there data in NICUs, on a population basis, of culture-negative
> meningitis that could conceivably have been caused by E. sakazakii, or
> another organization? And are there cases of brain abscess caused by
> this organism in which the CSF was sterile?
>
> DR. ALEXANDER: I think, actually\--I wouldn\'t necessarily know from
> the literature whether there are going to be cases of culture-negative
> meningitis, or culture-negative brain abscess that could be related,
> because you wouldn\'t figure that out unless you got a culture that
> identified this organism. I do think that in most cases where somebody
> has a culture-negative infection, whether the spinal tap is negative
> or whether the source of an abscess doesn\'t grow our any bacteria,
> you do start looking for other potential causes, including
> tuberculosis, parasitic infections, different fungal infections that
> can manifest in that manner. So it\'s difficult to say how much of
> these other culture-negative things would be related to that.
>
> The other difficulty that I have is, again, we\'re dealing with the
> case reports. And we don\'t necessarily know whether that represents
> the tip of a large iceberg, or whether what we\'re seeing as a report
> is a lot, or a majority, of the cases that have been identified. I
> would think that it\'s more likely to be the tip of the iceberg,
> because I don\'t think that the literature reports represent all of
> the sporadic cases, and we certainly don\'t get investigations of
> individual sporadic cases to start looking at things like powdered
> formula, and what the source of infection would be.
>
> DR. STALLINGS: Stallings.
>
> Would you concur with the previous speaker that if you have properly
> obtained biological specimens\--the blood, the CSF, pathological
> tissue\--that we should be able to grow this bug and identify it, you
> know, in a U.S./Canadian setting?
>
> DR. ALEXANDER: I mean, I think that the cases where you\'re talking
> about isolation of the organism from CSF, or isolation of the organism
> from blood should be possible. I don\'t think that this Enterobacter
> sakazakii as a subspecies is going to be any more fastidious than
> normal Enterobacter, and our hospitals have plenty of experience with
> isolating those.
>
> The only caveat may be whether the testing is specific enough to
> separate Enterobacter sakazakii from other Enterobacter cloacae. So
> whether you\'re getting some cases that are reported out as
> Enterobacter cloacae that are actually this organism.
>
> DR. STALLINGS: But the issue of how useful\--if we wanted to go back
> and look at cultured-negative CSF, I mean in a neonatal setting, if
> you\'re working up a baby who\'s become ill, you\'re going to get, in
> the U.S. anyway, you\'re going to get blood and urine and CSF, and
> those will be managed in a way we ought to be able to identify that it
> was bacterial, at least, and at least to the species.
>
> DR. ALEXANDER: And certainly the other things that you look at in the
> CSF\--the white count, and glucose and things like that\--should be
> pointing you in the direction as to whether this is a likely bacterial
> infection or not.
>
> DR. BEUCHAT: Beuchat.
>
> The information that you and other speakers have given this morning on
> surveys of the dried powder for the presence of E. sakazakii indicate,
> at least in one instance, up to 52 percent of the samples were
> positive.
>
> Given that number of servings per annum is probably a billion or more,
> the question is why aren\'t we seeing more cases? What do we know
> about the level of virulence of these strains? Are some avirulent
> under any conditions, while others are highly virulent under all
> conditions? What do we know about the virulence and pathogenicity of
> E. sakazakii?
>
> DR. ALEXANDER: Well, I\'d say that overall what we can tell is that
> the virulence of the organism is likely low, until you get to a
> particular patient that may be predisposed. And I\'m not sure that
> there\'s any way, really, to identify all of those patients ahead of
> time. And then when you have an isolate that in neonates and young
> infants, specifically\--so children that we\'re probably talking about
> less than four to six weeks of age\--that they may be predisposed to
> developing meningitis. And I think that has something to do with the
> mechanism of neonatal meningitis overall, which we still have a lot
> of\--we still really aren\'t very clear about.
>
> DR. BEUCHAT: Are then all the strains equally virulent? Do we know
> that?
>
> DR. ALEXANDER: I don\'t know. I mean, so far all we\'ve done is sort
> of identify this organism as a particular species. If you\'re
> comparing the species of E. sakazakii to other species of Enterobacter
> cloacae, there are certainly indications that Enterobacter cloacae and
> other Enterobacter species may be more likely to cause bacteremia and
> sepsis in an older age population because of the fact that it\'s just
> isolated so infrequently. But, again, it\'s a question\--is it that
> it\'s isolated infrequently because of the fact that you need a
> certain exposure and you\'re only getting it from powdered formula? Or
> is it because of the fact that the organism is less pathogenic? Can\'t
> tell.
>
> DR. HEUBI: Larry?
>
> DR. KUZMINSKI: Larry Kuzminski.
>
> I think you\'ve answered partially, in your answer to the previous
> question, but I look back to part of our charge here\--based upon your
> survey\--and I apologize if I\'m asking a question that\'s been at
> least partially ask before\--but, in your opinion, what do you think
> are the populations of infants at risk to this?
>
> DR. ALEXANDER: Well, again, I certainly think that the infants who are
> in neonatal intensive care units, who are premature, are probably a
> population that\'s at the greatest risk. But I do think that there\'s
> also a somewhat lesser risk, but a risk of a fairly serious fatal
> disease\--neonatal meningitis\--that goes to term infants and probably
> out to a month to six weeks of age, which is the period that we see
> neonatal meningitis in. And then, again, for the older children and te
> bacteremia, it\'s certain children with altered host defense,
> immunosuppression, that are likely to be the ones who are at risk of
> developing bacteremia from the organism.
>
> So you have a population of infants that are susceptible because of
> cancer, because they have central lines for some other reason, that
> are fed powdered formula and then could develop a bacteremia due to
> the organism.
>
> DR. HEUBI: I think we\'re going to bring the rest of the presenters
> up, and we could actually pose these kind of questions to them
> collectively\--although if you have a question\--so if the remainder
> of the presenters from the morning, except for Dr. Kuehnert, could
> please come forward, we\'ll organize this, and have a little
> question-and-answer period.
>
> DR. BLUMBERG: Henry Blumberg.
>
> One question I had for you: do you know if FDA or CDC, or other groups
> are trying to do any surveillance to look at rates of necrotizing
> enterocolitis, specifically, you know, since the CDC report came out
> in the MMWR, at least at my institution, and I think probably others,
> there\'s been a shift away from using\--in the neonatal intensive care
> unit-from used powdered milk, and I think there\'s a lot more use of
> the liquid, sterilized product.
>
> And I was just curious if anybody has any data, is that impacting
> rates of NEC?
>
> DR. ALEXANDER: I\'m not aware of any.
>
> DR. HEUBI: So I think at this point\--thank you. I think at this point
> the committee members can pose questions to any of the presenters from
> this morning. And I think Larry\'s question is pretty relevant,
> because I think we\'re trying to define what populations may be at
> risk, because ultimately we\'re going to be asked to provide some
> counsel to FDA about which populations should be protected.
>
> DR. STALLINGS: Stallings. So, actually, thinking about that, and
> thinking about the clinical practice setting, I was sitting here
> going\--we\'ve talked about the pre-term infant in the NICU. And just
> to be sure that all of the people at the table may not be aware of how
> many term infants are NICUs, because of the different kinds of
> presentation. So not everybody there is a premature infant with all of
> the things that go with that, including less well-developed immune
> systems.
>
> So then I was thinking, well, the other couple of groups\--and I would
> just like for your response\--there\'s a whole subset of infants and
> lung children with poor gut function. And some of these are the ones
> that have got motility problems we don\'t understand. Some of them are
> post surgical. Some of them may have been the babies who had NEC and
> now have short-bowel syndrome\--and, again, a group that we\'ve often
> used special formulas in.
>
> And then the third group are children who we would recognize as
> immunosuppressed. So, discounting the premies, children who might have
> HIV, or cancer, or even of the new biological agents that are coming
> out to treat all sorts of diseases we\'re not sure. So there\'s a
> whole spectrum of children who might be at risk, as we open the
> umbrella from the case presentation, which was\--it was sort of a
> classic premie presentation.
>
> So, for the committee, just as we start to think about that, what do
> you think about those different scenarios, or other clinical ones you
> might think of from your experience, as well?
>
> DR. ALEXANDER: Well, again, I think it\'s difficult to try and define
> well a population that might be at risk. Now, it\'s easier to define
> the populations that are at risk for neonatal meningitis or for NEC,
> because what you are usually talking about are more often premature
> infants, but can occur in term infants. And then there\'s a defined
> window of time that we\'re usually looking at, something along the
> line of four to six weeks of age, which is when the neonatal
> meningitis would usually manifest\--whether early-onset, which is
> defined as just less than seven days, versus later-onset.
>
> And so for those two diseases\--and certainly neonatal meningitis
> being the most serious manifestation that we\'ve seen of Enterobacter
> sakazakii\--you have what would be an easily defined group that\'s
> based on what their chronological age is: about six weeks of age, or
> exposure within NICUs and prematurity. So it\'s potentially the case
> that you\'re talking about an infant who is very premature, who might
> go beyond that four to six week age period and then develop NEC at
> some point later on because of exposure.
>
> For bacteremia, when you get beyond that four to six week age
> population, you are really talking about populations that may have
> some form of immunosuppression, whether it\'s due to cancer treatment,
> whether it\'s due to an unrecognized immunologic deficiency\--and
> there are several different forms that it could take. And then altered
> host defenses. So if what\'s happening is that this organism is
> entering through the intestine, then potentially anybody who\'d had
> prior GI surgery or resection of either congenital anomalies or for
> treatment of NEC could be a population that\'s potentially exposed.
> And then also other populations that, for whatever reason, require a
> central line for treatment.
>
> DR. KLONTZ: Karl Klontz here. I\'d just like to comment briefly on
> that issue.
>
> In looking at the literature again, one of the things I did was to go
> through the line list of all the cases that were reported\--in
> infants\--n=48 cases.\--just to look at gestations. And of those 48,
> there were five reports that don\'t comment on how old the baby was,
> in terms of gestation. Then I think there were 13 cases that were
> described as either\--quote-unquote\--\"term,\" or gestations greater
> than 36 weeks, or\--some of the older reports\--\"normal baby\" at
> birth. So there were 13 of those, leaving 30 who fell into the
> category of prematurity.
>
> But beyond that, the articles really don\'t get into the sort of the
> molecular biology, so to speak, of immune systems. It\'s just
> insufficiently detailed to\--in my opinion\--to decipher clearly, from
> an immune status, who exactly is at risk. I don\'t think the
> literature does that for us.
>
> DR. ALEXANDER: Right. I think my comments, in terms of the bacteremia
> and sepsis are meant to be just an extrapolation of what you would
> think of as the populations at risk for an opportunistic pathogen to
> cause infection.
>
> DR. BEUCHAT: Beuchat.
>
> I tried to find it in the handouts, but can\'t. I\'ll ask\--I think it
> was Dr. Klontz\--you had indicated that four cases, median age 74
> years\--what was the
>
> DR. KLONTZ: Six cases, I believe it was.
>
> DR. BEUCHAT: What was the vehicle\--do we know what the food of the
> vehicle was, that may have been associated with those cases?
>
> DR. KLONTZ: No, I don\'t think any of the articles described what the
> vehicle was. So, no, I don\'t know the answer to that.
>
> DR. BEUCHAT: Presumably it wasn\'t infant food, but\--we don\'t know
> that.
>
> DR. KLONTZ: We don\'t know that. I mean, the gentleman, 76-year-old,
> with a history of rectal adenocarcinoma, presumably he was doing
> reasonably well, but then suddenly came down with, you know,
> urosepsis. No comment on dietary factors in that article nor, to my
> knowledge, the other five. I don\'t recall. I\'ll check during the
> break, but I don\'t recall any distinct dietary information given in
> those cases.
>
> DR. BEUCHAT: I guess what I\'m really asking: are there other foods
> that also may be vehicles of E. sakazakii, other than the powdered
> infant formula.
>
> DR. KLONTZ: I don\'t have enough information to answer that question.
> I don\'t know. I don\'t know the answer.
>
> DR. HEUBI: Dr. Fischer?
>
> DR. FISCHER: Fischer.
>
> I\'m not a physician, so I can ask this question. Why would you fed
> the powdered formula in the hospital instead of using the liquid
> formula? Under what circumstances would you want to take the risk of
> feeding the
>
> powdered formula?
>
> DR. ANDERSON: There are certain formulas that are only available in
> powdered form. In particular, the formulas for infants with metabolic
> disorders, the amino acid-based formulas, and some formulas for
> infants with special medical conditions are only available in powdered
> form.
>
> In addition, human milk fortifiers, which are added to human milk for
> premature infants are available in powder form. There is also one
> product on the market that\'s available in fluid form. The advantage
> to added the powder in this situation is that there is a minimal
> increment in volume that the infant must consume, and that\'s an
> important consideration for pre-term infants.
>
> DR. FISCHER: Well, let me follow up by asking what is the percentage
> of times that you need to use these special formulas, as opposed to
> the non-special powdered formula? You\'re saying the reason you\'re
> doing it is because it\'s a special case and special diet. What\'s the
> percentage of time that has to be used?
>
> DR. ANDERSON: I don\'t have specific information. It would be small,
> but I can\'t say further than that.
>
> DR. THUREEN: I can tell you that having talked to many of my
> colleagues across the country, that the use of powdered formulas has
> evolved over the last five to 10 years. Many infants can take the
> regular prepared formulas for preterm infants, but there\'s a
> perceived notion that better nutrition can be supplied if you
> supplement the existing liquid formulas with powdered formulas. Many
> times it\'s given to increase calories or proteins,
> calcium\--whatever\--but many units, individual, without any
> protocols, concoct a variety of different formulations using powdered
> formulas added to liquid formulas. And they don\'t just use more
> liquid formula, because many of these infants have fluid volume
> restrictions. So it\'s a widespread practice. And I contacted a number
> of units around the country when this first occurred, and about half
> of them stopped using powdered formulas, or had never really used them
> much and always used ready-to-feed formulas.
>
> But many other units have, and are continuing, to make their own
> concoctions, and it\'s highly variable as to what exactly they\'re
> making up, but they individualize it to different infants. So it\'s a
> very common practice.
>
> And why would they do it? Well, it\'s just the belief that if you
> strategically want a certain type of preparation for infant, you can
> make it using what\'s available on the market, and just mixing up what
> you think makes sense. It\'s a widespread practice.
>
> DR. HEUBI: Margaret?
>
> DR. BRILEY: \[Off mike.\]
>
> DR. HEUBI: Please identify yourself.
>
> DR. BRILEY: \[Off mike.\] Margaret Briley.
>
> DR. HEUBI: Speak up, please.
>
> DR. BRILEY: Margaret Briley.
>
> In regard the population that might be involved with this organism,
> there\'s been some part of our American adult group, as well as
> children, that are still trying to find raw milk products to consume,
> even though we do not advocate that, and we have rules against that.
> But there are rules in Texas where people can produce raw goat milk,
> for example, and sell it on their place. And we\'ve had some real
> serious situations about that.
>
> Does anybody here know if this organism is present in the raw state?
> Is that not where it\'s, maybe possibly, coming from?
>
> DR. ALEXANDER: I\'m not sure that we have clear idea about the source.
> I know that one of the reports\--I think it was Muytjens, et al., that
> tried to look at a bunch of environmental sources; so, looking at
> soil, looking at a bunch of sources, didn\'t isolate it from anywhere.
>
> I think there was something else that I had also seen about rice
> paddies as an environmental source of where this organism is able to
> grow. But I\'m not sure how that would be involved, and whether that
> would be the source at all, of the infant formula exposures that
> we\'re talking about.
>
> I don\'t know\--I can\'t speak to whether it\'s something that\'s
> identified from cow milk or not. But I don\'t think that that was
> considered to be the source, ultimately, of the organism.
>
> There was another interesting report, I think, for one of the adults,
> where it was actually found within the hospital environment, but it
> wasn\'t clear as to exactly where. So that there\'s the possibility
> that with the older adults, you may be talking about some sort of
> exposure through environmental\--through an organism that was present
> in the environment, but it\'s not clear how that happened, and there
> are certainly other reports with the different NICU outbreaks that
> didn\'t find the organism in the environments.
>
> DR. HEUBI: Dr. Stallings?
>
> DR. STALLINGS: \[Off mike.\] Stallings.
>
> I was just going to add \[inaudible\] so that just so\--there are some
> children with some diagnoses that would have to be on these things
> almost for their whole life. If you looked at something like PKU, or
> MSUD. But there are many children with GI diseases outside of infancy
> where we use all of these products, as you were saying, in whatever
> creative way that the group decides. So I think there is a lot of use.
> There\'s a lot of use beyond sort of the original intent. And, you
> know, again, the case, if I recall correctly, was a product that
> wasn\'t available in a liquid or ready-to-feed product.
>
> The other thing\--it has been my impression, although I don\'t know
> the data and maybe the manufacturing people will help us with
> this\--is there was an element of the dried products are cheaper and
> easier to store. It just takes up shelf space. So if you have a big
> setting that you\'re willing to have a formula room and that sort of
> thing to mix some of these things up.
>
> So there are a lot of different issues going on about who\'s exposed.
> But even if we trained all the neonatologists to do this perfectly,
> there are a lot of other people in pediatric practice who are using
> all these formulas to put together whatever they think they need for
> the patient.
>
> DR. ACHOLONU: Alex Acholonu.
>
> Has any survey been done on neonates outside the hospital environment,
> who are breast fed? And the reason why I ask this question is that
> they may be getting colostrum from their mother, and that is believe
> to have agglutinates that may be able to control the disease.
>
> DR. ALEXANDER: Again, I think that what we\'re dealing with in terms
> of what we can report about Enterobacter sakazakii is only the
> information that we have on these sporadic cases from the literature.
> I think it would be very difficult to try and identify and tease out
> specific factors as to whether children who are breast fed are less
> likely to have this organism, or more likely to have this
> organism\--and how do you necessarily separate that out from the fact
> that those children who are breast fed are less likely to be receiving
> powder or any type of formula at all.
>
> So, I don\'t think that there\'s anything out there that\'s going to
> be able to help use in terms of a survey, because I think we are still
> talking about a fairly rare organism.
>
> DR. BUSTA: Frank Busta.
>
> When I look at the Tennessee cohort study, which was given to us
> indirectly, seven of the nine were on continuous feeding, which I
> assume is tube feeding. Is there ever any discontinuous tube feeding?
>
> VOICE: Uh-huh.
>
> DR. BUSTA: So that the other two could have been a discontinuous tube
> feeding. Also, that same\--seven of the nine never received any breast
> milk.
>
> Is there a possibility that the infants that are infected are not
> exposed to the normal inoculum flora, like the bifidabacter, or
> lactobacilli, that normally-fed infants are exposed to, and colonize
> their digestive tract? Or is there any similar type of data in the
> other studies that you did internationally that would show that it\'s
> tube feeding, and maybe a lack of good competitive flora?
>
> DR. KLONTZ: No. Karl Klontz here.
>
> From the published literature there is really not that depth of
> discussion\--even in terms of how the formula was fed. I\'m thinking
> back to the Van Acker study, some of the major\--large outbreak
> publications, and I don\'t recall details on mechanism, or mode of
> delivering the prepared formula, nor were was there\--certainly, there
> were no features on sort of the microbiology of the intestines, to my
> knowledge, in those outbreaks.
>
> DR. ALEXANDER: I think part of the problem that you\'re going to get
> in trying to look at the idea of whether it might be associated with
> something like the tube feedings, or the plastic tubing, or things
> like that is when it comes to the disease of NEC, the infants that
> you\'re talking about that are more likely to develop that disease are
> also the infants who are going to be receiving this almost exclusively
> through some type of gastric tube feeding, and they\'re not the
> infants who are going to be sucking on a bottle.
>
> So it\'s not going to be possible for the NEC cases that were
> identified to see whether it\'s\--to try and tease out the differences
> between whether it may be related to the plastic tubing, versus the
> powdered formula itself.
>
> With the meningitis cases, I\'m certain that, for instance, that the
> five-week-old infant that I was talking about was most likely a child
> who was normal and healthy and wasn\'t receiving any kind of gavage
> feedings, so that some of those cases of meningitis, if we could find
> more information from\--you know, from the cases themselves, would
> probably indicate that those were children who were on otherwise
> normal feedings, and not related to gavage.
>
> But it\'s not something that\'s clear, that we clearly know at this
> point.
>
> DR. HEUBI: Dr. Fuller.
>
> DR. FULLER: Changing topics a little bit, could you refresh my memory
> or give us a little\--go back over\--what information do we have in E.
> sak in the ingredients used to formulate the powdered formula? Or do
> we have that information?
>
> DR. ANDERSON: I believe information on that topic may be part of the
> talks this afternoon.
>
> DR. HEUBI: Rob?
>
> DR. BAKER: Baker.
>
> I just had two comments. One was about breast feeding. And just sort
> of a priori, you would sort of expect breast feeding or breast milk to
> be protective, in that the information did sort of suggest. So I would
> think that would be something to look at.
>
> The other thing I wanted to mention is that there is another
> population that we haven\'t really talked about that may be at risk,
> and that\'s the graduate of the NICU. And babies are graduating from
> the NICU at earlier and earlier stages, so there are some quite
> premature infants that are being cared for in other parts of the
> hospital or even at home, and they may be at risk for this.
>
> DR. HEUBI: Last question
>
> DR. LEE: Yes, going back to the earlier question\--
>
> DR. HEUBI: Please identify yourself..
>
> DR. LEE: This is Ken Lee. And going back to Dr. Busta\'s question
> about feeding by gavage and establishment of favorable flora, is there
> any attempts in a neonate to try to establish a helpful flora.
> Obviously the line of questioning is that if friendly flora is
> established, then there\'s less opportunity for an opportunistic
> pathogen like E. sak to establish itself. And that kind of thinking
> leads you to the idea that well, maybe, that ought to be put in the
> powder itself to help the patient.
>
> DR. THUREEN: I\'d like to just make a comment on that. This is
> Thureen.
>
> I would have to say that most of these infants that are preterm have
> very altered flora. They\'ve gotten antibiotics staring at
> birth\--often many courses. They\'ve gotten medications that have
> altered the pH of their GI systems. They\'ve undergone many insults.
> So their GI flora is anything but normal.
>
> I think there are a lot of studies that are now underway, looking at
> probiotics as a means of establishing a more normal flora. But I think
> this population is so vulnerable, just because they\'re set up, for
> many, many reasons, for infection from this organism.
>
> DR. LEE: We had a little conversation over here\--the idea, of course,
> is\--that\'s a very good point, in that perhaps there are things
> that\--any growth condition that would lead to an outbreak of the
> pathogen E. sak could also lead to the growth of something that\'s
> non-pathogenic. And if one could find a non-pathogen that would
> inhibit E. sak, then that would also be a helpful thing to be able to
> do.
>
> DR. THUREEN: Thureen\--one more comment.
>
> \[Laughter.\]
>
> DR. THUREEN: You know, I wonder if the E. sakazakii\'s not just sort
> of a marker that\'s given us an interesting epidemiologic chance to
> study these infants, because also my understanding is in any infant
> formula there\'s very low levels of contamination with other
> coliforms, staph aureus, and other organisms, and those are very
> common pathogenic organism for NEC, sepsis, etcetera\--and that we are
> just focusing on E. sak because it\'s such an unusual organism. But,
> in fact, this could be a contributor to a lot of the other infections
> that we see going on.
>
> DR. ALEXANDER: Certainly, it seems like there is\--it\'s easier to
> focus on E. sakazakii because of the fact that there is what appears
> to be a more clearly epidemiologic association between its presence in
> the infant formula and lack of identification in other settings. I
> mean, for these other organisms that you\'re talking about\--the
> coliforms, E. coli, klebsiella, the other enterobacter species\--the
> problems that you run into are those are part of normal human GI
> flora, and so how do you tease out the fact that formula would have
> been what introduced the organism that caused the disease, as opposed
> to just passing through the birth canal, or some other source, whether
> related to what\'s on the health care worker\'s hands, or what would
> be normally the process of sort of GI colonization that, for whatever
> reason in this infant, leads to a more serious infection?
>
> DR. HEUBI: I\'d like to thank the presenters and the committee members
> for the lively discussion. The committee members and the speakers are
> invited to lunch, behind us. The guests are on their own.
>
> \[Laughter.\]
>
> DR. HEUBI: And, I forgot\--if you haven\'t found this out already, the
> restrooms are out here. If you didn\'t know already, I think you\'re
> probably in trouble.
>
> \[Laughter.\]
>
> \[Luncheon recess.\]
>
> [A F T E R N O O N P R O C E E D I N G S]{.underline}
>
> DR. BUSTA: I f you will take a seat we will get started with this
> afternoon\'s presentation.
>
> The committee has received the information requested from Atlanta. You
> see the two graphs that we were sent. This is just for the committee,
> not for public distribution. It\'s unpublished data, not for further
> dissemination . I don\'t know how much more that can be emphasized.
> It\'s right across the top of the slide, but it\'s to help us
> understand the discussion this morning. So, please bear that in
> mind\--not for public distribution.
>
> We also have a table to supplement the presentation this morning, that
> you received.
>
> Thank you for the prompt return to the committee. Brief luncheon
> period, but we will get on our way. We\'ll do our best to stay on time
> this afternoon. It\'s a little tighter. And we had the good fortune of
> having a little extra time this morning. We\'ll have to be tighter
> this afternoon.
>
> The first presentation is on the general microbiology of Enterobacter
> sakazakii. This is from Dr. Maria Nazarowec-White.
>
> DR. HEUBI: As your co-chair, I\'d like to remind everyone to
> speak\--into\-\--the\--microphone.
>
> \[Laughter.\]
>
> **General Microbiology\--Ecology, Pathogenicity, Subtyping, Etc**
>
> DR. NAZAROWEC-WHITE: How to speak into the microphone. Can everybody
> hear me?
>
> First of all, I\'d like to thank you for inviting me to speak about
> Enterobacter sakazakii. This is an organism that I spent a number of
> years studying during my doctoral research. And at that time\--and
> I\'m talking about the early \'90s, because I went back to school
> quite late in life\--there was very little information available. And
> it would have been wonderful to have had a meeting like this where
> different people were presenting different bits of information that
> they have.
>
> I do notice, though, that in the last couple of years, you do hear
> people talking about Enterobacter sakazakii. I work for the Canadian
> Food Inspection Agency in Canada, and we are already in work planning
> meetings talking about maybe putting in a monitoring program. Health
> Canada is planning to include a method for isolation of Enterobacter
> sakazakii in its official analytical methods. CODEX is also talking
> about this organism, through its committees on food hygiene, as well
> as the Committee on Nutrition and Foods for Special Dietary Uses.
>
> Next slide, please.
>
> \[Slide.\]
>
> Now, I\'m just going to briefly touch on a number of things, give you
> a little bit about the history of this organism, and what we actually
> don\'t know bout the ecology or the environment that this organism can
> be found in. I did a bit of a study on the incidence on the Canadian
> retail market. I want to describe some growth studies, looking at
> generation time and lag time of this organism in reconstituted infant
> formula.
>
> I also did a little bit of work on the phenotypic and genotypic
> characterization of the strains that I worked with. And, finally, a
> number of people have asked the question\--a number of people have
> asked a lot of questions this morning\--a little bit about the initial
> work on pathogenicity and virulence factors. The paper is actually
> just out in the [Journal of Food Protection]{.underline}, on the
> pathogenicity.
>
> Next slide.
>
> \[Slide\]
>
> Okay. As we know, until 1980, Enterobacter sakazakii was called
> \"yellow-pigmented cloacae.\" However, there was a proposed name
> change in 1980 based on differences between Enterobacter sakazakii and
> Enterobacter cloacae, using DNA hybridization studies, biochemical
> reactions, and pigment production.
>
> Enterobacter sakazakii has a biochemical profile very similar to
> Enterobacter cloacae, but unlike Enterobacter cloacae, it\'s always
> sorbitol negative, and positive for deoxyribonuclease.
>
> Next.
>
> \[Slide\]
>
> It think it\'s time we saw a picture of this organism that we\'ve been
> talking about all morning. And someone this morning has said that the
> yellow-pigmented colonies were difficult to identify. Well, the yellow
> pigment has a stronger hue when the organism is grown at 15 degrees
> Celsius, rather than at 36, and often growth or isolation is done at
> 36.
>
> The other thing that we found\--and other researchers, as well\--that
> there are two morphologically different colony types. This slide
> illustrates a colony that is mucoid, or dried, and it has a
> slightly\--well, it doesn\'t really show the scalloped edges, but
> it\'s a three-dimensional thing. And when you are trying to touch it
> with the loop on the actual plate, it\'s very rubbery, and it\'s sort
> of almost difficult to get off the plate.
>
> It looks sort of like a raspberry upside down, sitting on the plate.
>
> Next slide, please.
>
> \[Slide.\]
>
> Now, the second colony type is a typical smooth and soft colony. It is
> easily removed with a wire loop. And what also we found is the
> colonies\--the rubbery, scalloped kind\--revert sometimes to this type
> of colony on sub-culturing. At one point in time, when we first saw
> this, we were wondering whether differences in virulence or any other
> phenotypic characteristics would be different between these two
> morphology types.
>
> Next slide, please.
>
> \[Slide.\]
>
> Here\'s a picture\--an electron micrograph\--of an individual
> bacterium. The blue color, of course, has been added. And, as you
> know, it is a motile, gram-negative rod. It grows very easily on
> laboratory medium. And another interesting observation was that after
> 24 hours of growth, all the strains that we had produced a large
> amount of sediment. It appeared that\--it was really quite
> interesting\--and it contained sort of clumped cells and masses that
> were sort of stuck together. I have no idea why that was. But that\'s
> something that could be explored a little further.
>
> Next slide, please.
>
> \[Slide.\]
>
> Ahh\--the environment. Little is known about the environment. There
> was this one study\--or it was mentioned in a letter to one of the
> journals\--and it was\--now I call him \"Mutagens,\" but I guess it\'s
> \"Moyt-yens?\" Am I pronouncing it correctly? He was the one who
> showed that it could not be isolated from surface water, mud rotting
> wood, bird dung, rodents, domestic animals\--I\'m not quite sure which
> ones they mean\--and cows milk.
>
> Now, aside from the infant formula that we have seen the association
> there, there is a paper where sakazakii was isolated from a UHT
> tetrapack box of milk. I have heard that bottled water\--this is
> certainly not a scientific study\--someone mentioned, \"Oh, yes, by
> the way, we did find it there\"\--and raw ground beef. Then there were
> the studies where they were looking at the actual illnesses and
> meningitis, I guess, cases, where they talked about the dish brush and
> the blender that the infant formula was reconstituted in.
>
> So this is certainly an area where research is needed. When we\'re
> looking at the infant formula, I certainly did not look at any of the
> specific ingredients that go into the formula. We know that the
> formula is not sterile, but is the milk? Does it go
> through\--sometimes the milk will go through a pasteurization process
> prior to being put into the formula, and sometimes not. And they
> are\--because it\'s going for further processing\--meaning that it has
> to go through the powder\--to be made into a powder, through the
> drying, so we don\'t know whether it\'s coming from the milk or other
> ingredients. Are they sterile?
>
> Next slide, please.
>
> \[Slide.\]
>
> At the beginning of my research, I contacted children\'s hospitals and
> general hospitals across the country to see if their microbiology
> departments maintained Enterobacter sakazakii strains in their culture
> collections. I managed to obtain nine clinical strains. There was a
> St. Joseph\'s Health Center, affiliated with the University of Western
> Ontario, they sent me two isolates. These isolates are from a
> one-month-old child that had had meningitis with cerebral abscess
> formation. I received another strain from the Montreal Children\'s
> Hospital, and then the Toronto Hospital for Sick Children sent three
> specimens. Two isolates were isolated from cerebrospinal fluid, and
> one strain from a blood culture.
>
> Each of these strains are from different patients, isolated in
> different years. The Laboratory Center for Disease Control in Ottawa
> kindly provided three clinical Enterobacter sakazakii isolates, but
> they did not provide any history as to where these strains came from.
> So these were my clinical strains that I worked with.
>
> There are a number of dried infant formula available on the Canadian
> retail market. We don\'t have very many actual manufacturers in
> Canada. And although there were no incidents reported in the
> literature of E. sakazakii meningitis related to infant formula in
> Canada, in 1990, two incidents of infection were reported to Health
> Canada. In one incident, analysis of two cans of formula obtained from
> the home of the one-month-old infant showed no microbiological
> contamination. However, the original can was discarded, and therefore
> could not be evaluated.
>
> The second incident involved a neonate in a hospital from a different
> city. It appears that in this case, there was a misconception that all
> powdered formula are sterile. And, again, we don\'t know whether the
> less developed intestinal flora or, as someone this morning described,
> the totally different flora in these children that are ill. Even\--it
> sort of emphasizes that we should be very careful in all the
> procedures we use to ensure as low a microbial load as possible.
>
> Samples of dried infant formula were obtained from the manufacturers
> or from retail. And strains of E. sakazakii were isolated. Five cans,
> a retail unit, from five different lots manufactured on five different
> days were used in my study.
>
> Next slide, please.
>
> \[Slide.\]
>
> This slide shows the prevalence of E. sakazakii in the formula we
> evaluated. The incidence varied from zero percent to 12 percent for
> Company A. And we already know that there was the study that was in
> the literature talking about the 141 cans from 35 countries, and they
> found sakazakii in 14 percent of them.
>
> The levels of Enterobacter sakazakii were low, at 0.36
> coliform-forming units per can. However, there was no information on
> the pathogenicity of this organism, and we wanted to see\--learn more
> about the growth of sakazakii.
>
> Next slide, please.
>
> \[Slide.\]
>
> Farmer et al., in his initial paper, tested 57 strains for growth. He
> found that all 57 grew at 25, 36 and 45 degrees Celsius; 50 strains
> grew at 47, but none grew at 4 or 50. Ten Canadian strains\--five
> clinical and five food isolates, were selected, and growth was
> observed over a temperature range of 4 to 50 degrees.
>
> Temperature gradient incubator was used. Laboratory media was spike
> with a thousand cells, and growth was observed over a 15 to 20-day
> period.
>
> Next.
>
> \[Slide\]
>
> This slide shows the minimum growth temperature for both the clinical
> and food isolates. None of the tested strains grew below 5.5 degrees
> Celsius. Now this would indicate that Enterobacter sakazakii would not
> grow at refrigerator temperatures, which we consider 4 degrees
> Celsius. However, in looking at studies of in-home refrigerators, they
> are well above that 4 degrees Celsius. They have shown that the
> temperature range between 7 to 10 is probably what you\'re going to
> find in most in-home refrigerators. And this, in turn, could help
> Enterobacter sakazakii to grow.
>
> And this morning we were hearing how there was a study that was done
> which made up the formula, spiked it, refrigerated overnight, and then
> put it to hang for four hours. Well, depending on what temperature
> that refrigerator was at, if it\'s in there for 24 hours, would allow
> the sakazakii to grow.
>
> In order to evaluate growth in reconstituted dried infant formula,
> three formulae were selected. These three had the highest market share
> on the retail market in Canada at the time. A cocktail of five
> clinical strains and five food isolates were evaluated in three
> different formulae. The formulae were spiked with 10^3^ CFUs, and
> incubated at three different temperatures: 4 degrees, 10 degrees and
> 23 degrees. Four was considered the refrigeration temperature; 10 was
> considered a slightly abusive temperature; and 23, room temperature.
>
> Sampling was done at timed intervals, using direct plating, which
> provided colony counts for determination of generation time and lag
> time. After calculating generation time and lag time, results were
> subjected to analysis of variants to calculate and determine if there
> were any significant differences.
>
> \[Slide\]
>
> This graph shows the generation time. The dark blue is 10 degrees, and
> the light blue is 23 degrees. And there was no statistically
> significant differences found, either among the tested formulae\--in
> other words, formula 1, formula 2\--or between the clinical isolates
> and the food isolates.
>
> At 10 degrees, the generation time varied from 4.18 hours to 5.52
> hours. Food isolates have a slightly lower generation time\--although,
> again, it\'s not statistically significant.
>
> At 23 degrees, the generation time was around 0.67 hours, which
> translates into 40 minutes.
>
> The data for 4 degrees Celsius is not shown here. It was found that it
> remained at the initial levels that were\--that the formula was spiked
> with, or declined over time. And, again, this certainly confirms the
> importance of storing reconstituted infant formula at the correct
> temperature.
>
> When prepared bottles are stored overnight at room temperatures, we
> looked at the data and you could get at least 10^5^ CFU per mil, and
> this is easily obtained.
>
> Next slide, please.
>
> \[Slide.\]
>
> And this is the lag time. Again, there was no statistically
> significant differences among the three formula for both clinical and
> food strains. The lag time varied for from 19 hours\--is it
> backwards?\--19 hours for formula in the food, to 47 hours for formula
> in the clinical. Again, no significant difference.
>
> And, again, you can see the generally shorter lag times for the food
> isolates when compared to the clinical strains. And this is really
> just sort of initial work, and so only using a very few strains.
>
> Next slide, please.
>
> \[Slide.\]
>
> Okay\--typing systems, of course, are based on the premise that
> clonally related isolates share characteristics by which they can be
> differentiated from unrelated isolates. Methods for identification and
> discrimination of bacterial isolates have always been divided into two
> broad categories: phenotype and genotype. The traditional
> micro-techniques, phenotypic typing based on the secondary
> characteristics of bacteria, including biochemical reactions,
> antibiograms, serotyping, bacteriophage typing.
>
> We used the API 20 miniaturized biochemical test strip to identify
> Enterobacter sakazakii. as we isolated it from the infant formula. And
> the 18 strains in our studies were grouped into three biotypes.
>
> Biotype I, included nine strains, and that includes the type strain.
> Six strains out of the 18 were inositol-negative, and three strains
> were VP-positive. Farmer, et al., also found that 98 percent of his 57
> strains tested to be VP-positive, and 25 percent were
> inositol-negative.
>
> Next one.
>
> \[Slide\]
>
> Somebody this morning was talking about antibiotics and antibiotic
> resistence. Again, this is very preliminary work. And, as you know,
> all clinical laboratories do antibiotic susceptibility testing
> routinely. \|We used just the disk diffusion method, using commercial
> antibiotic disks to determine antibiotic resistence in the 18 strains.
> Now, these 11 antibiotics are commonly administered in gram-negative
> infections, and were evaluated using the Enterobacter sakazakii
> isolates that we had.
>
> Included in this list are the gentamicin and ampicillin which, if you
> look through those literature reports that many people were describing
> this morning, if you look at the actual antibiotics that were given to
> some of these infants, the ampicillin/gentamicin combination is often
> considered the gold standard. I think it was Robinson that said that.
>
> Next slide, please.
>
> \[Slide.\]
>
> Now, what I\'m showing here is the resistence to the certain
> antibiotics. We found that there was one\--it was a food strain\--that
> was resistant to ampicillin. The other interesting thing is that there
> were 17 strains that were resistant to cephalothin, and there was only
> one clinical strain that was susceptible to it. The
> sulfisoxazole\--all 18 strains showed resistence to this antibiotic.
>
> So when we looked at the antibiotics, 14 strains fell into Biotype I,
> and this was including the type strain. One strain in Biotype
> II\--antibiogram II\--and two strains that fell into the third
> category, and one strain was antibiogram IV.
>
> Now, antibiograms are not a very discriminatory test, and there have
> been reports in the literature, by Clark, et al., in 1990, that found
> this same thing; that the strains that she tested were categorized
> into\--there were many strains in certain categories.
>
> The other interesting thing was if you took colonies that were
> supposedly clonal on one plate, if you picked five colonies, sometimes
> you would get two or three different antibiotic patterns. So\--don\'t
> know what\'s going on with that, where we\'re assuming they\'re the
> same organism, where the same antibiogram should come up, it doesn\'t.
>
> Next slide, please.
>
> \[Slide.\]
>
> I\'m not going to talk about the actual methodology here, but I do
> want to mention that we did use ribotyping, pulse field gel
> electrophoresis, and random amplification of polymorphic DNA. It\'s
> come a long way since the early \'90s as to some of the work that I
> did and what is being done now.
>
> And the next slide shows the diversity among those 18 strains. It you
> look at the ribotyping, it was grouped into 10 different
> ribotypes\--those 18 strains. In both pulse field electrophoresis, and
> the RAPD, it depending on the restriction enzyme that was used in the
> pulse field, because you can see with XB-A1, we had 18 different
> pulsovars, and with SP-E1, we had 17. And the same thing happened with
> the random application for polymorphic DNA. It depending on which
> primer was used.
>
> Now, the interesting thing\--if you look at the ribotypes\--see these
> three here? All Number III\--these are the three isolates from the one
> hospital\--Toronto sick kids hospital\--but they were isolated in
> three different years. So, with ribotype, they fell into the same
> profile.
>
> And here we have MNW III, IV and V\--these are food isolates. These
> are all the ones from one company. Interestingly, they also fell into
> one category. However, if you go to where we have only 17 different
> profiles, again, these two strains\--MNW III and MNW IV, both in the
> pulse field as well as the RAPD, fell into the same category. So, it
> was interesting. And whereas the third isolate from that particular
> company did not fall into the same category as it did in the
> ribotyping. So, really, what I\'m showing here is the heterogeneity
> among the strains we looked at. There were only 18 strains, but they
> all appear to be genetically heterogeneous.
>
> Okay\--the next slide, please.
>
> \[Slide.\]
>
> Virulence factors. Now, I could find nothing that reported anything
> with respect to Enterobacter sakazakii. There was one study, where it
> was a retrospective in a Danish hospital, that looked at
> enterobacters\--not particularly Enterobacter sakazakii. And it stated
> in that paper that a portal for entry for enterobacter species,
> infection can be the gastrointestinal portal.
>
> So we took that statement and thought, well, we can also find
> Enterobacter sakazakii in infant formula and, of course, it goes
> through the GI tract, and in that paper, again, it said that they did
> find that Enterobacter cloacae produced exotoxins, and E. sakazakii
> and Enterobacter cloacae are kind of related, so it was decided to
> explore a little bit on the pathogenesis or virulence factors of this
> organism.
>
> We used a suckling-mice assay to test for enterotoxin production, and
> then three cell culture lines to look and see what the impact was on
> the morphology of those cells.
>
> Pathogenicity was assessed with the use of suckling mice challenged
> intraperitoneally and orally, with both the clinical and the food
> isolates.
>
> Next slide, please.
>
> \[Slide.\]
>
> For the enterotoxin production, we found of the 18 strains, four
> produced and enterotoxin. Three were clinical, and they were all three
> from three different hospitals, and in three different geographic
> regions in Canada. One food isolate also produced an enterotoxin. And
> the type strain was negative.
>
> Next slide, please.
>
> \[Slide.\]
>
> As far as the tissue culture, we only looked at three isolates. There
> was the type strain, and two clinical strains were used. We used three
> cell lines: Y-1, CHO, and vero cells. Once clinical strain was toxic
> to all three cell lines. On the Y-1 and vero cells, approximately 75
> percent of the cells died. On the CHO cells, there was cell shrinking
> and vacuation.
>
> We also boiled the cells to see if there were any differences there,
> and the boiling produced the same results on the Y-1 and the
> Chinese\--the CHO cells, however the boiling decreased the impact on
> vero cells.
>
> The other clinical strains\--the type strain and one from one of the
> hospitals in Montreal\--had no effect on all three cell lines. Again,
> these are very preliminary and initial work. But we wanted to see
> whether we could, you know, sort of at least see what was going on.
>
> Next slide, please.
>
> \[Slide.\]
>
> I\'m going to talk just a little bit about the infectivity. IP
> injections were lethal at 10^8^ CFU per mouse for all strains tested.
> Death usually occurred within three days of dosing, and the lowest
> level causing death by IP injection was two strains: one food and one
> clinical. The clinical one was at 10^5^\--both of them were actually
> at 10^5^. And the clinical strain also produced an enterotoxin.
>
> By the oral route, only two strains caused death: one clinical and one
> food. These results are the first to be reported with respect to any
> virulence factors in Enterobacter sakazakii. As you can see, much work
> remains to be done.
>
> The paper on the infectivity has just been published in this month\'s
> issue of Journal of Food Protection. So details and tables are in that
> particular paper.
>
> Next slide, please.
>
> \[Slide.\]
>
> Just in summary, incidence in dried infant formula in the Canadian
> market was found to be about 6.7 percent. We\'ve seen values of 14
> percent in the one Dutch study. Minimum growth temperature is between
> 5.5 and 8 degrees, and emphasizes the importance of refrigeration.
>
> Generation time at room at that temperature of 40 minutes. If we
> thinking of putting that bottle at the bedside table so you don\'t
> have to get up in the middle of the night, or taking bottles with you
> when you\'re going shopping and keeping it in the stroller\--looking
> at that time/temperature relationship.
>
> Four of 18 strains produced enterotoxin. The enterotoxin was not
> characterized. Infectious dose\--we have some initial results that
> maybe we could use in further work\--certainly needs to be done in
> this area.
>
> And when we\'re looking at the molecular level, we see that very
> heterogeneous strains.
>
> Next slide, please.
>
> \[Slide.\]
>
> So it was very interesting working on this organism, because anything
> I did was new at the time. I\'m glad to see that there is more
> interesting in it on the one hand; on the other hand much remains to
> be learned about this organism.
>
> And I look forward to the discussion over the next couple of days.
> Thank you.
>
> DR. BUSTA: Thank you very much
>
> This afternoon, being that we have a fairly tight schedule, I would
> like to restrict this to questions of clarification, and then we\'ll
> have all the speakers up at 3:50 for questions in general.
>
> Are there questions for clarification.
>
> VOICE: \[Off mike.\]
>
> DR. BUSTA: The question is: a definition of lag time?
>
> DR. NAZAROWEC-WHITE: Oh, lag time is the time once we spiked the
> formula\--and, as you know, bacteria grows sort of in that S-curve
> way, well, it\'s that beginning of that S, before it begins to double,
> and the doubling is the generation time.
>
> DR. BUSTA: For my clarification\--this is Frank Busta\--for my
> clarification, what was the shortest lag time? I couldn\'t tell that
> from the\--
>
> DR. NAZAROWEC-WHITE: Oh, I know, because it was very\--there was no
> significant difference.
>
> DR. BUSTA: It looked like one of the food ones was very small column,
> and I was wondering what the shortest lag time is\--for an idea
> of\--now, you were using cocktails in each case, so that you in fact,
> were measuring both generation time and lag time, you\'re measuring
> the fastest strain of the five strains.
>
> DR. NAZAROWEC-WHITE: That\'s right.
>
> DR. BUSTA: While you\'re looking for that, can you handle another
> question and look at the same time?
>
> DR. NAZAROWEC-WHITE: No\--lag time\--let\'s see. I don\'t have the
> numbers, but I will be able to supply them for you. They\'re in the
> paper\--the actual numbers. I don\'t have them.
>
> DR. BUSTA: All right. Dr. Fischer?
>
> DR. FISCHER: What was the dose used to kill animals when given orally?
> The oral dose?
>
> DR. NAZAROWEC-WHITE: Oh, the oral dose? We did\--se started off with,
> you know, 10^3^, 10^4^, 10^7^.
>
> DR. FISCHER: What dose killed the animals. You said it killed two
> strains\--I mean\--killed the animals
>
> DR. NAZAROWEC-WHITE: Oh, I\'m sorry\--10^7^. Yes. Orally. And it was
> the two strains.
>
> DR. BUSTA: Any other questions for clarification?
>
> \[No response.\]
>
> DR. BUSTA: All right. Thank you very much.
>
> The next presentation will be by Dr. Burr; Microbial
> Detection\--Clinical and Food\--from the FDA.
>
> **Microbial Detection\--Clinical and Food**
>
> DR. BURR: I want to thank you for inviting me here today. One of the
> nice things about going at this time of the afternoon is that
> essentially everyone\'s covered just about everything I\'m about to
> say. So it makes it kind of nice. But you\'re not going to learn very
> many new things; hopefully a few pictures will come in. But most of
> the stuff that I\'ll talk about has been briefly touched upon already.
>
> I\'ve been given the assignment of discussing the microbial detection
> of Enterobacter sakazakii, both in food and clinical. And\--
>
> Can I have the next slide?
>
> \[Slide\]
>
> \--what I\'d like to do is provide a summary of the methods that are
> available for isolating and quantifying levels of E. sakazakii in food
> and clinical samples. But I am not planning on covering everything
> that is in the White Papers. I\'ll try to go over the high points of
> the methods. And as you\'ll see in your papers, there\'s a bit more
> information that\'s provided.
>
> Next slide.
>
> \[Slide\]
>
> In terms of presentation, I\'d like to divide it into two aspects:
> one, talk a little bit about the initial isolation reports of E.
> sakazakii, and then turn our attention more to the development of the
> quantitative methods for E. sak.
>
> Initially, the first isolation was in 1980. And, again, you\'ve heard
> about all this from previous speakers. 1983\--I think we\'ll go now to
> just Dr. M\--at this point\--
>
> \[Laughter.\]
>
> \--and in 1984, we had Dr. Postupa and Aldova.
>
> Next slide.
>
> \[Slide\]
>
> The initial isolation, or the initial description of E. sakazakii
> being associated with a can of dried milk came in Farmer\'s initial
> description of E. sak as a new species, and that was in 1980. But like
> the other papers that will talk about the initial ones, there\'s no
> real isolation detailed. Okay? They talk about it\'s just being an
> isolate, but they don\'t describe how they got it, where it really
> came from, or what it took to get it.
>
> The next slide, again, is Dr. Muytjens in 1983, and this is the study
> that Karl and Maria talked about, where eight cases of neonatal
> meningitis associated with E. sakazakii. They isolated the organism
> several times, as has been pointed out, from the prepared formula, but
> never from either the powdered formula itself, or the water used in
> preparing the formula. Again, no information was reported on the
> quantity of powdered formula analyzed. However, as Karl has pointed
> out this morning, we have had several conversations or e-mails, and we
> have been informed that the quantity analyzed in this 1983 paper was
> the 10 gram sample.
>
> Next slide.
>
> \[Slide\]
>
> In 1984, Postupa and Aldova\--again, I apologize if I\'ve done
> something to the names here\--described four strains of E. sakazakii
> from powdered milk, two strains from powdered milk infant formula. The
> only details provided was that it was isolated on deoxycholate-citrate
> agar, incubated at 37 degrees for 48 hours, but no details on anything
> in terms of the quantity analyzed, again.
>
> Okay, and now I\'d like to turn attention to the development of the
> quantitative methods, and essentially we\'ll deal with three methods,
> bringing us up to the current one that the FDA is using. And sort of
> for ease of discussion, we\'ll call the first one the \"European,\"
> then we\'ll move to the \"Canadian\" and then we\'ll move to the FDA
> method.
>
> Each one of these\--the \'97 and the FDA method\--is essentially a
> minor modification of the 1988 paper. Sample size, sensitivity remains
> the same and, for the most part, the change has been in the ease of
> being able to do the method.
>
> As we spoke about, this method was first described in 1988. And in
> referring back to their 1983 paper, they commented that although it
> was not cultured from the formula powder itself, this might have been
> due to an unequal distribution in the powder, or it was present in
> such a low concentration that it escaped detection by conventional
> methods. And for a long time we were sort of hindered by, you know,
> what exactly does \"conventional methods\" mean? And that sort of put
> us on trying to get in touch with Dr. Muytjens as quickly as we could,
> and that\'s when we got the information about the 10 gram sample.
>
> So, based on their early results, in the 1988 work they decided to
> culture a large quantity of breast milk substitutes for the presence
> of all Enterobacteriaceae, including E. sakazakii.
>
> Next slide.
>
> \[Slide\]
>
> So essentially what their method is\--and a method paper is probably
> not the best thing after lunch\--but hopefully we\'ll go through it.
> In triplicate, they mixed 100, 10 and 1 gram samples with 900, 90 and
> 9 mils, respectively, of buffered peptone water, 45 degrees, until
> completely dissolved, and this gets incubated overnight at 36 degrees.
> From these overnight cultures, you innoculate 10 mils from each of the
> flasks into 90 mils of Enterobacteriaceae enrichment broth\--EE
> broth\--and, again, this is incubated overnight at 36 degrees.
>
> This first incubation is non-selective, and this is the first area
> where you get an enrichment broth which has some selectivity to it. In
> duplicate, innoculate from these cultures in duplicate; innoculate 1
> mil from each of the enrichment broths into 20 mils of fluid,
> violet-red bioglucose agar. And again incubate this overnight at 36
> degrees C.
>
> Suspect colonies sub-cultured to sheep blood and eosin-methylene blue
> agar, and the strains were identified via the API 20-E system. And, as
> I said, this initial paper was for all of the Enterobacteriaceae, so
> they did additional testing for E. sakazakii by looking at the
> production of yellow colonies on nutrient agar after 48 hours at 25
> degrees, production of extracellular DN-ase. And a positive
> alkaglucocytase reaction.
>
> Next slide.
>
> \[Slide\]
>
> For those of you that are not familiar with what an API system
> is\--and I know Maria talked a little bit about it\--API system is a
> rapid identification process, where essentially you have plastic
> strips that have 20 small wells consisting of dehydrated media. You
> then add your bacteria suspended in saline to each of the wells, and
> then incubate for 16 to 24 hours, and then look at the color reaction.
> So, essentially, you have each one of these wells is an individual
> biochemical reaction. And so after incubation, you then end up
> with\--and this is a series of four different bacteria\--four
> different examples\--of what you see following 24, or 16 to 24 hour
> incubation.
>
> Next slide.
>
> \[Slide\]
>
> What you then do is, based on the color of each of the reactions, it
> will be scored as either a plus or a minus and so on. So you very
> easily just score.
>
> Next slide.
>
> \[Slide\]
>
> This is just a typical reaction of what E. sakazakii would look like
> in an API strip.
>
> Next slide.
>
> \[Slide\]
>
> So then what happens is that you take your score here, and you
> transform the biochemical reaction; so you had a plus here, a plus
> reaction, a negative reaction. You then transform that into a
> numerical profile. And that numerical profile you then just take to
> either a computer program that they provide, or a book that they
> provide, and that then comes up with an identification of E.
> sakazakii. And you have different levels, based on the variation; you
> have good identification, you have excellent identification; good
> identification, acceptable.
>
> Now, sometimes this numerical profile is not considered sufficient
> enough to be able to identify the organism, so API then recommends
> additional testing. And for E. sakazakii, generally the additional
> test would be testing for the yellow pigmentation, which API has, and
> I think the Journal of Clinical Micro has listed as 98 percent of the
> isolates are yellow\--produce yellow pigmentation.
>
> So this is basically what you end up with after doing an API.
>
> Next slide.
>
> \[Slide\]
>
> Now, in terms of the levels of E. sakazakii in the sample, in this
> method and, again, in all the later ones that we\'re going to talk
> about, the actual level is determined by the most-probable-number
> procedure.
>
> Next slide.
>
> \[Slide\]
>
> And I don\'t want to go into a lot of detail, because I\'ll confuse
> myself quicker than I\'ll confuse you, but it\'s a statistical method,
> and it assumes that the bacteria are separate, and the conditions of
> incubation such that every inoculum that contains even one viable
> organism will produce detectible growth. And it\'s based on the number
> of positive samples from each of the series of triplicate cultures of
> the three inoculation levels. So essentially what you\'re doing is
> you\'re scoring the number of positives in the three 100 gram samples,
> in the three 10 gram, and in the three 1 gram. And the tables that you
> go\--the standardized table\--and I\'ll show that in a
> minute\--provides you an MPN number\--most probable number\--plus a 95
> percent confidence interval.
>
> Next slide.
>
> \[Slide\]
>
> This is a typical MPN table. And I think this is actually taken out of
> the BAM manual, and this is for a three-tube at an inoculation of .1,
> .01 and .001 gram. And what it give you\--as I said, it gives you an
> MPN per gram in the 95 confidence level. So, essentially, you look
> here and you\'re saying how may tubes of the three were inoculated
> with the .1 gram are positive? So here you have one positive, zero
> positive, and this gives you an MPN number of 3.6 MPNs per gram, and
> then here is your 95 confidence level.
>
> Now, down here it describes how we will go from this standardized
> table to go into an E. sak of an MPN per hundred gram. And essentially
> what you do is you take the MPN per gram from this table, you divide
> by 1,000 to adjust for the larger sample size. So that gets you down
> to these sort of values here. And that would be giving you an MPN per
> gram. And then you simply multiply 100 to obtain per 100 gram.
>
> So, in this case, if you had 1.00, you would have .36 MPNs per hundred
> grams.
>
> Next slide.
>
> \[Slide\]
>
> The results of the European survey\--and these, I think, have been
> talked about I think twice already, maybe three times. But
> essentially, from the 35 countries, 141 powdered formula samples were
> analyzed. E. sak was isolated from 20 samples, from 13 of the
> countries. And the levels recovered using the MPN was .36 to 66 colony
> forming units\--or CFUs\--per 100 gram.
>
> And the lowest level of detection reported in this method was 0.36 CFU
> per 100 grams.
>
> Now, on the next slide I want to show a little bit about where that
> comes from here, and I want to distinguish sort of a 0.36 per 100 gram
> detection limit, versus a 0.31 per 100 gram\--actually, the lowest
> possible limit.
>
> \[Slide\]
>
> If you take this chart, in order to get this .36 per 100, that\'s
> essentially you have one tube out of the\--of the largest sample. So
> one of the 100-gram sample is positive, and then the 10 gram and the 1
> gram are both negative.
>
> It\'s theoretically possible that you could have zero out of three in
> the 100 gram, zero out of three in the 10 gram, and one out of three
> in the 1 gram, giving you the lowest possible level of a .3 per 100
> grams. But most of the papers that reported, this is the
> number\--where they\'re getting that one particular number that it\'s
> coming from. Okay?
>
> Next slide.
>
> \[Slide\]
>
> In 1997, Maria\--as she discussed\--and this we\'ll call the Canadian
> method\--made modifications of this initial method. And, essentially,
> they just\--the dried infant formula was suspended in sterile water.
> Suspect colonies from the VRBG plate sub-cultured to TSBYE agar. And,
> again, they\'re just very minor modifications. And as in the previous
> study, the API 20 was used for confirmation, and no additional
> biochemicals were used. The levels were again determined by MPN, and
> again you\'re left with the same sensitivity: .36 per 100 grams.
>
> The results of this study: E. sak was isolated from eight of 120 cans;
> this representing five different manufacturers. And, again, the level
> reported is the .36
>
> Next slide.
>
> \[Slide\]
>
> Now, in 2002, the FDA was charged with developing its method. And
> rather than reinventing anything took from the Canadian method and,
> again, just made some minor modifications. And what we did was went to
> direct spreading or streaking over the overnight EE broth, rather than
> the pour plates. The pour plates are a bit laborious in order to set
> up, so this was a much easier method of getting the isolated colonies.
>
> Five presumptive colonies are sub-cultured to tryptikase soy agar, and
> incubated at 25 degrees C for 48 and 72 hours. And here, again, at the
> lower temperature where we\'re looking for only yellow-pigmented
> colonies from the TSA plates are then further worked up in the API 20
> system. Like the Canadian method, we do not do any additional
> biochemical tests, and we just rely on the results from the API 20.
>
> The level of detection by MPN, again we report as the most likely one
> as the .36 per 100 gram. And we can detect levels of E. sak much lower
> than recommended\--by the recommended FAO level of 3 CFU per gram of
> powdered infant formula.
>
> These are just some pictures that have been provided from Sharon
> Mallow who was c\--author in producing this method. This just shows
> the mixture of the powdered milk in the flask.
>
> Next slide.
>
> \[Slide\]
>
> These are an example of the typical colonies of E. sak on the VBGP.
> And, again, you\'re looking for the purest purple colony, surrounded
> by a purple halo of precipitated bio-acids.
>
> Next slide.
>
> \[Slide\]
>
> And this is just the description of the yellow pigmentation that you
> get at room temperature.
>
> Next slide.
>
> \[Slide\]
>
> Just briefly want to touch on clinical isolation, and this was one of
> the questions was brought up earlier this morning. E. sakazakii is
> isolated from clinical samples using standard methods for the
> isolation of Enterobacteriaceae. There\'s no special media has been
> developed for E. sak, and it grows well on all the standard media that
> is used in clinical laboratories. And for the most part, when you read
> the papers, they don\'t even describe the method. They just say
> \"cultured from blood,\" \"cultured from spinal fluid.\" And in
> talking to several friends who do clinical microbiology, again their
> comment is you just follow the normal routine for blood cultures, and
> that there\'s nothing different, nothing unique.
>
> You can confirm with either API 20, as we\'ve done, or there\'s an
> enterotube 2 system. But, again, it just follows the standard methods
> for identification. And essentially because you\'re dealing for the
> most part with supposedly sterile sites, again much easier
> identification process.
>
> So then, finally, and just in conclusion then, the procedures E.
> sakazakii from powdered formula and clinical samples: follow the
> standard microbiological methods for the isolation of other members of
> the family Enterobacteriaceae. Normally, sterile clinical samples pose
> no major problems for isolating E. sak.
>
> Because of the very low levels of E. sakazakii in powdered formula
> samples and its non-random distribution in the powder, larger
> quantities and sub-samples should be cultured for isolation. And in
> both clinical and food microbiology laboratories, appropriate
> incubation times and temperatures should be applied if the diagnostic
> tests were precise. And, again, this appropriate temperature would be
> going down to the lower temperature for pigment production.
>
> I think that\'s the last slide.
>
> So, hopefully that\--as I said, a lot of the information was covered
> in some of the earlier talks, but hopefully this gives it a bit more
> of a clear picture of what exactly is done to get it out of these food
> samples.
>
> Thank you.
>
> DR. BUSTA: Questions\--preferably for clarification.
>
> Dr. Blumberg.
>
> DR. BLUMBERG: Yes\--Henry Blumberg.
>
> Do you know\--generally in the clinical micro lab they\'re not using
> an API strip to identify gram-negative bacteria, and most of the
> identification of susceptibility is done by automated methods like
> Vitec or Microscan\--things like that.
>
> And do you know how well those identify Enterobacter sakazakii,
> versus, you know, other enterobacter species, like cloacae or things
> like that?
>
> DR. BURR: I don\'t, actually, because the only thing that I got from
> the two friends that I had was just that, \"We just do the standard
> way.\" So I\'m assuming that it\'s easy, and that the automated ones
> do pick it up, but I don\'t have the experience with those.
>
> DR. BUSTA: Other questions?
>
> Dr. Baker.
>
> DR. BAKER: Baker. This morning people talked about difficulty picking
> out these organisms among a whole lot of other organisms. Your slide
> certainly didn\'t show that. Is that a problem or not?
>
> DR. BURR: Well, in this only, hopefully it\'s the combination of the
> EE broth plus the selective plate that actually is cutting down on
> whatever else is in there. So we\'re really trying to do this\--the
> selectivity of the method removes a lot of the other bacteria.
>
> DR. BUSTA: Other questions?
>
> Dr. Fuller.
>
> DR. FULLER: I apologize for what may be a very stupid question. When
> you started with the European method, I think it said triplicate runs
> using 100, 10 and 1 gram. Again\--pardon what may be a pretty stupid
> question.
>
> Are we talking about\--
>
> DR. BURR: If I can\'t answer it, you\'re really setting me up here,
> now.
>
> \[Laughter.\]
>
> DR. FULLER: Well, I\'m just asking\--is this from a single can, or are
> these nine different\--you know, is this for a lot? What are we
> sampling?
>
> DR. BURR: It\'s for\--you take\--out of one can you would take\--
>
> DR. FULLER: Each of those.
>
> DR. BURR: \--for each of those.
>
> DR. FULLER: Okay.
>
> DR. BURR: So you would take a total of 333 grams out of one can\--
>
> DR. FULLER: One can. Okay.
>
> DR. BURR: \--and do it in the separate things. Oh, that was a good
> one.
>
> \[Laughter.\]
>
> DR. BUSTA: Dr. Acholonu.
>
> DR. ACHOLONU: You said that there is no special medium or media for
> isolating E. sak. Is there any special need for developing one, since
> you have indicated that it grows in different kinds of media?
>
> DR. BURR: It doesn\'t appear that there is a need for one. As I said,
> for the clinical, it will grow on any of the media that\'s used for
> enterics. And so there\'s been no need to actually get one that\'s
> just specific for E. sakazakii.
>
> DR. BUSTA: Dr. Tompkin.
>
> DR. TOMPKIN: Yes\--Tompkin.
>
> Due to the low prevalence of this organism in the product\--you make a
> statement\--or a statement is made that it is \"non-randomly
> distributed.\" How can you differentiate low prevalence and just
> failure to detect it, versus whether, in fact, it is non-randomly
> distributed. Does this have to do\--did you actually go into a large
> quantity of a given lot and try to determine random distribution, or
> is this just an assumption.
>
> DR. BURR: It\'s just a generic way of\--
>
> DR. TOMPKIN: It\'s an assumption.
>
> DR. BURR: Right.
>
> DR. TOMPKIN: Okay.
>
> DR. BUSTA: I have a question\--this is Busta.
>
> When you\'re doing a normal Enterobacteriaceae enrichment, and then
> you go on to an Enterobacteriaceae agar, what portion of the colonies
> that you\'re selecting would be sakazakii? What portion of the
> colonies? Do you run all colonies from that enrichment onto the API?
>
> DR. BURR: No, as you\--you\'re just taking\--when you go to that
> second plate\--okay\--when you\'re going from the broth to the VRBG
> plate, then you\'re taking\--you\'re looking for the purple colonies
> with the haze around them. So, again, you\'re picking what you think
> are going to be those colonies. Okay? So you\'re looking for typical
> colonies of E. sak on the selective plate.
>
> DR. BUSTA: Dr. Tarr.
>
> DR. TARR: I have two questions that straddle the question and
> clarification border.
>
> First, when you state your sensitivity is 3 or 3.6 colony-forming
> units per gram\--
>
> DR. BURR: 100 grams.
>
> DR. TARR: \--per 100 gram\--do you ever normalize that to the ambient
> flora? I can imagine one E. sakazakii in a kilogram being detected if
> there\'s nothing else there when you amplify it as if it were coming
> our of a blood culture. And I can imagine many E. saks being swamped
> by ambient flora in the grow-up stage.
>
> That\'s my first question.
>
> DR. BURR: I\'m not sure if I\--again, I\'m not sure if I get that
> question.
>
> DR. TARR: Because, again, you\'re growing it up\--
>
> DR. BURR: Remember, you\'re looking for just growth in that one
> particular tube. So you\'re not looking for any particular level
> within that tube when you\'re doing the MPN procedure.
>
> DR. TARR: But you have a subsequent step when you have a sample\--the
> sampling comes out on your plates, looking for the yellow colonies. If
> you have many additional organisms there, you might miss the low
> frequency E. saks. If you have few other organisms there, you might
> get them.
>
> DR. BURR: Right. But again\--
>
> DR. TARR: It\'s a variable that\'s not\--is that taken into account?
>
> DR. BURR: Right. Remember you\'re doing that\--the hundred grams is
> first going in to a non-selective, and then you\'ve got selective. So
> you\'ve got two, really, grow-outs\--
>
> DR. TARR: Right.
>
> DR. BURR: \--that are occurring before you\'re going to any plates. So
> that\'s supposedly going to bring up the levels of any low levels of
> bacteria, to bring it up to where you\'ve got lots in those cultures.
> So, essentially, you\'ve got 48 hours\' growth is what you\'re
> actually putting on plates.
>
> DR. TARR: But that will be limited by the other organisms in there,
> won\'t it?
>
> DR. BURR: And everything should grow. In at least the first one,
> you\'re essentially allowing everything to grow. That initial one is
> non-selective. Everything is going to grow out there. And then when
> you take the smaller portion of that to the enrichment, then you\'re
> more selective for the E. sak colony.
>
> DR. TARR: Okay. And that laterals into my second question.
>
> Have you or anyone else explored the thermal tolerance of this
> organism, as an initial selective step, where the first incubation is
> perhaps done in 45 degrees\--
>
> DR. BURR: We have not net\--
>
> DR. TARR: \--to give competing flora a penalty/
>
> DR. BURR: Yes\--we have not yet.
>
> DR. BUSTA: Thank you very much.
>
> To continue on, with Dr. Buchanan, talking on resistence\--thermal and
> otherwise.
>
> **Resistence\--Thermal and Other**
>
> DR. BUCHANAN: Thank you. Okay. I\'ve got a microphone and I\'m armed.
>
> \[Laughter.\]
>
> What I\'d like to do today is talk mostly about thermal resistence of
> Enterobacter sakazakii. I have a few additional items that I want to
> mention in passing; one that may help address one of the questions
> that came up earlier today. And in talking about resistence, I\'m
> talking now about resistence to the type of things that this organism
> might encounter during its manufacture or distribution. I\'m not going
> to be talking about antibiotic resistence, which was covered earlier.
>
> Next slide.
>
> \[Slide.\]
>
> And I\'m going to break this up into the\--a quick look at a number of
> studies that have been done, first at a laboratory level, and then at
> a pilot plant level. I am going to be focusing a great deal on the
> latter study, that has been completed. It has been submitted for
> publication, but will be relatively new data to the members of the
> committee.
>
> We have had two studies done\--laboratory trials. They were done by
> Maria, who talked to you earlier. And then Sharon and I did one using
> a somewhat different technique. And just so you have an idea of what
> are covered in these, Maria\'s used a stainless steel tube and a
> constant water bath as the heating system. It used five pooled food
> strains and five pooled clinical strains, in separate trials. And in
> that kind of a study, the most resistant organism is what comes out at
> the end.
>
> We used a submerged coil apparatus .and 12 individual strains. A
> submerged coil apparatus is a fairly sophisticated instrument for
> determining thermal resistence, allowing holding times in factions of
> a second.
>
> Next slide.
>
> \[Slide.\]
>
> Before going on, I\'m going to be mentioning a couple of terms. And,
> knowing that this is a mixed group, for the food microbiologists, I\'m
> sorry to bore you\--the others, two terms that I will be using:
> \"d-value,\" is the time at any given temperature that would be needed
> to reduce a microbial population by 90 percent. And it\'s a common
> term that\'s used for measuring the relative thermal resistance of an
> organism.
>
> A second term that\'s used commonly is referred to as the \"z-value.\"
> This is a value that\--what change in temperature, what increase in
> temperature, you would have to have in the processing in order the
> decrease the d-value by 90 percent.
>
> So there are two ways of describing the time-temperature relationship
> of the organism\'s resistence.
>
> Next slide.
>
> \[Slide.\]
>
> And, typically, when we do a thermal resistance curve what we do is we
> do a survival curve. These are two examples. They cover the extremes
> that we saw with 12 individual strains. The organism on that has a d
> of 591 is the most resistant strain that we ran into. And the one on
> the left, with a d of 30.5, is the least resistant strain. And the
> d-value is simply the slope of those inactivation curves. That\'s what
> we\'re really measuring in this case.
>
> So those are what I\'ll be referring to as I go along.
>
> Next slide.
>
> \[Slide.\]
>
> We looked at the 12 individual strains and they fell into two distinct
> groups as we examined them. They fell\--half of them fell in the group
> there on the far left. Those six strains had d-values actually less
> than 35 seconds. So they were very sensitive. The group on the right
> all had d-values greater than 300. They range from 320 up to about
> 590. And so we found two distinct populations\--in itself is very
> interesting.
>
> Next slide.
>
> \[Slide.\]
>
> Trying to put this in perspective so that you can sort of judge this
> against other pathogens of a similar nature, we did have some exact
> data that was generated under the same exact conditions, and so I
> plotted these so you can get a relative idea. The Enterobacter
> sakazakii 607 was the most resistant strain we ran into. And, like I
> said, it had a d-value of around 590. So it bracketed at one end. And
> the Enterobacter sakazakii 51329 was very heat sensitive and it
> bracketed the others. The other strains that are of note here: this is
> E. coli 0157, Klebsiella pneumoniae, Salmonella Hartford, which is a
> relatively heat resistant salmonella; a biotype-1 E. coli, and
> Enterobacter aerogenes. And then this, here, is the pooled strains
> from Maria\'s study. So, again, it covers a wide range and sort of
> behaves like other Enterobacteriaceae do. They, themselves, often have
> a distribution of thermal resistences.
>
> Next slide.
>
> \[Slide.\]
>
> Now, this is the\--when you do a thermal resistence determination, and
> you do it at different temperatures, and then you plot the d-values
> you calculate on a log scale. Typically, if it follows the rules that
> everyone says it\'s supposed to follow, it winds up producing a
> straight line.
>
> Well, Enterobacter sakazakii\--this is strain 607\--did a very nice
> job of following the rules. It\'s just\--I\'ve never\--
>
> \[Laughter.\]
>
> \--I couldn\'t draw a better line if I did it on purpose. Each of
> these points represent at least three independent trials. I mean, it
> just fell out beautifully. So this is\--its d-value\'s over a\--almost
> a 15-degree range.
>
> If you then\--well, you\'re not supposed to\--
>
> Can I get the next one? Next slide? Oh, that\'s this one. Okay.
>
> Just to show you that the gods do shine down on you sometimes when
> you\'re doing experimental work, when Maria did her pooled food
> isolates, and pooled clinical isolates, She found that there were no
> significant differences between the two groups, and that they had an
> overall d-value of 5.8 degrees Celsius. And when we did it by an
> entirely different method four years later we came out with 5.6. I
> mean, you can\'t get much closer than that.
>
> Next slide.
>
> \[Slide.\]
>
> Going back to the z-value\--and while you\'re not supposed to
> extrapolate z-values past your experimental ends, we did anyway as a
> way of predicting how the organism should behave when you go up to
> even higher temperatures. And I might note going above higher
> temperatures in the apparatus we used is virtually impossible, because
> you\'re down at fractions of a second. So we did just simply
> extrapolate this starting at the 70, which was our upper end, and it
> basically suggested that when you get above the temperature of 70,
> inactivation becomes almost instantaneous.
>
> But following the linear relationship, for example, to get a 5-d
> inactivation\--which is something we typically would shoot for with
> these kinds of treatments\--a pasteurization-type treatment\--you can
> see that by the time you\'re up to 80 degrees, to get a 5-d kill you
> should be, at this point, at about .7 seconds. And if you go up even
> higher, you see it becomes literally a small fraction of a second to
> get that kind of inactivation.
>
> So one of the things we wanted to check out was whether or not this
> actually occurs in reality, when you\'re dealing with an infant
> formula, and so we designed a simple experiment where what we did is
> we took a commercial dried infant formula. We inoculated it with a
> concentrated culture of Enterobacter sakazakii 607, which was, again,
> our most thermally resistant isolate. We did it under a condition
> where we maintained the dry nature of the product. We pelleted a
> colony\--basically, we pelleted about 10^15^ cells and then
> distributed it throughout a large volume of formula, so we had a dry
> formula with approximately a million cells per gram\--or would produce
> a million cells per gram when it was rehydrated.
>
> We then distributed that inoculated formula into standard plastic baby
> bottles, following the manufacturer\'s instruction on how to weigh it
> out, with the appropriate volume. We then added preheated water to
> those bottles; capped it, agitated the bottles\--the formula\--as one
> would normally do, shaking it periodically; and we allowed it to stand
> for 10 minutes, and then we sampled the bottle and determined the
> remaining cells to see how much inactivation we got.
>
> Next slide.
>
> \[Slide.\]
>
> Just to show you what the heating looks like, we did a series of
> temperatures in approximately 10 degree Celsius ranges, starting at
> boiling water and then going down to 50 degrees Celsius. And then our
> control was just simply room temperature water that was added to it.
>
> Next slide.
>
> \[Slide.\]
>
> So, basically, we have the control is on the far left. This is room
> temperature water added to the formula. You can see that when we added
> them back we had approximately a million cells per gram. And at 50 we
> got a very small degree of inactivation. At 60 we got approximately a
> little over one log cycle inactivation. And then, as predicted by our
> original studies in model systems, at temperatures at 70 above, we got
> greater than a four log kill. We basically got below the limit of
> detection that we could use. We didn\'t try to go into an NPN at this
> point, but certainly we got a very substantial inactivation.
>
> Next slide.
>
> \[Slide.\]
>
> We also wanted to have some information on what would be the impact of
> this type of heating on nutrient content. And so we did have the same
> procedure done by our nutrition analysis center in Atlanta. They
> basically did the same procedure s we did, except they only used
> boiling water as the single temperature they examined. They did that
> because that would have been the worst case in the scenario we set up.
> We did the analysis in triplicate, using four different commercial
> infant formulas. And I might note that\--I\'m going to have to explain
> how the results are expressed, but they were all normalized against
> units per hundred calories, depending on what nutrient we\'re
> analyzing.
>
> Next slide.
>
> \[Slide.\]
>
> Now, this is a very busy series of slides, but I do want to point out
> what you should be looking for. This is the nutrient being analyzed.
> This was the four commercial formulas being analyzed. Within each of
> the boxes there are four values. This is the value on the label. This
> is the value that was found when the product that was rehydrated with
> room temperature water was analyzed. This is the value that was found
> when it was rehydrated with boiling water. And this is the change
> between this and this.
>
> So you\'re basically looking for two things when you\'re looking;
> you\'re looking for the differential between these two and then
> you\'re looking for whether or not what was remaining, versus what was
> on the label. So, in this case, the addition of boiling water produced
> a 3.7 percent decline in the nutrient, but it\'s still well in excess
> of the reported value on the label.
>
> We\'ve done this now for a series of them, and basically here very
> little change in vitamin A, very little change in vitamin D, E, K,
> thiamin.
>
> Next slide.
>
> \[Slide.\]
>
> Riboflavin\--again, you know, we see some fluctuation plus or minus
> between the two groups, but basically little change; little change
> would be 12. Niacin\--again, little change; folic acid, little change.
>
> Next slide.
>
> \[Slide.\]
>
> Pantothenic acid\--actually, a small increase pretty much across the
> board. Biotin\--little change. The only nutrient we found that was a
> significant decrease, in terms of the concentration across the board
> was with vitamin C. This would be expected. This is the most thermally
> sensitive of the nutrients. In some cases it did fall below the label
> value. In other cases, even though there was a decrease, it was still
> above what was indicated on the label.
>
> So, overall, with the exception of vitamin C, the addition of boiling
> water had really not much impact on nutrient content.
>
> Next slide.
>
> \[Slide.\]
>
> There have been only, as far as we can tell, only one study that was
> done at a pilot plant level on Enterobacter sakazakii. This was done
> by Maria also. It used pooled strains, we think\--and, Maria, if this
> is a question you can help answer that. It was unclear. And it was
> done with an HTST pasteurizer.
>
> Next slide.
>
> \[Slide.\]
>
> It was done in a manner that bracketed the different times and
> temperatures associated with pasteurization. And I\'ve tried to take
> what was a great deal of information and reduce it down to a single
> table. But basically, this value you see here is the percent surviving
> population of the initial inoculum. So you can see here with a
> three-second heating at 63 degrees C you got about a 60 percent
> reduction, down to, you know, several log cycles when you get down
> here\--and that it follows pretty closely what we would expect in
> normal time-temperature relationships. That is, it\'s not a very heat
> sensitive organism, and normal pasteurization would certainly knock it
> out.
>
> Next slide.
>
> \[Slide.\]
>
> Okay. Let\'s switch to other treatments or resistences. And when
> we\'re talking about Enterobacteriaceae as general\--and it\'s
> important to keep in the back of your mind that Enterobacter sakazakii
> is a normal Enterobacteriaceae. It is one of the fecal coliforms that
> we have a great deal of experience with in general terms.
>
> Next slide.
>
> \[Slide.\]
>
> and we know, in general Enterobacteriaceae are not heat resistant.
> They can be moderately acid resistant, particularly if they have been
> pre-adapted. Conversely, they can become moderately alkaline resistant
> if pre-adapted. They have low to moderate chlorine resistence; low to
> moderate irradiation resistence. They will remain viable in
> refrigerated and frozen products for extended periods, particularly if
> the pH is neutral. And then, finally, they have moderate to good
> resistence to drying.
>
> Next slide.
>
> \[Slide.\]
>
> Now, when you get specifically to Enterobacter sakazakii there is
> virtually no information available in the literature specifically
> related, and there\'s very little when you start looking at the
> distributions of resistences the way we did with the 12 individual
> strains.
>
> Just summarizing very quickly what we found, the isolation of it from
> a variety of dried foods indicates that it\'s probably resistant to
> drying. And I\'ll come back to that. We did find one paper where it
> tends to be found after treating seeds that are going to be used for
> sprouting with chlorine. This is one of the organisms that remained on
> the dried seeds, and it would be indicative of at least having
> moderate resistence to chlorine, though this would have to be
> confirmed. And, just an odd thing, it seems to be pretty sensitive to
> kitazans, which is used as an inhibitor.
>
> Next slide.
>
> \[Slide.\]
>
> The one additional piece of experimental information that we do have
> and can share with you is this slide\--when we prepped up the
> samples\--the
>
> dried infant formula samples that we used for the baby bottle
> experiment I showed you\--we did keep the leftover formula, and we
> have been periodically sampling it over the course of almost a year
> now. And this is the survival in that infant formula.
>
> Again, we started with about a million per gram upon rehydration, and
> then we\'ve been following it over the course of nine months. There
> was an initial die-off in the first four months of about two log
> cycles\--two-and-a-half log cycles. Since that time, it has basically
> maintained itself as a steady-state level. We anticipate if its
> behavior is like most other Enterobacteriaceae that this value will
> change little over the next year or two. And it\'s worth noting for
> any of you that follow archeology, they have opened up tombs in Egypt
> that have been basically sealed for 10,000 years, and they have found
> fecal coliforms. So, in a very arid and dry environment, don\'t expect
> that it\'s going to disappear. It will probably hang around for at
> least the shelf life of an infant formula.
>
> And I think that\'s the last slide.
>
> Summary\--this is not a particularly heat resistant organism. There
> seems to be a substantial diversity in the thermal resistence among
> the strains, and this seems to be in keeping with the substantial
> genetic diversity that you\'ve heard discussed in other attributes
> today.
>
> There\'s a good agreement among the studies that have been done.
> There\'s been a limited number of studies but, I mean, they\'ve
> matched up, despite the fact that they were done in different ways,
> the agreement is great.
>
> Inactivation at temperatures above 70 degrees occurs almost
> instantaneously, and certainly within a few seconds. And then specific
> information on the organism\'s resistence to other factors or stresses
> it might see in a food-processing type environment, or food
> preparation environment are generally lacking, though I would say that
> every indication is that it will survive dehydration for extended
> periods.
>
> Thank you.
>
> DR. BUSTA: Thank you, Dr. Buchanan.
>
> Are there questions for clarification?
>
> DR. FULLER: We asked it here, and I just want to make sure. On your
> resistence to dehydration slide, that was stored at room temperature?
>
> DR. BUCHANAN: Yes.
>
> DR. FULLER: Is that a correct assumption?
>
> DR. BUCHANAN: Yes, that\'s sitting on a lab bench in the lab. It\'s
> sitting at room temperature.
>
> DR. FULLER: Right.
>
> DR. BUCHANAN: It would probably show it down if we refrigerated it,
> but this is\--we tried to mimic conditions that would occur in the
> home.
>
> DR. BUSTA: Dr. Moyer-Mileur.
>
> DR. MOYER-MILEUR: Moyer-Mileur.
>
> I have a question concerning your results and how they relate to the
> USDA letter that went out to health professionals where it advised not
> using boiling water to reconstitute powdered formula, one, because of
> loss of heat sensitive nutrients, which your results would say is not
> the case, except for vitamin C, as well as the inability to assure
> adequate destruction of E. sakazakii.
>
> From your findings, would you say that those are no longer concerns?
>
> DR. BUCHANAN: What I would suggest is when that original was put out,
> there was a lack of data upon which we could evaluate the efficacy of
> that treatment. Based on that, these experiments were done. I would
> say that the results of these suggests that one, nutrient loss does
> not appear to be an issue\--or not a substantial issue, and that at
> temperatures above 70, it is a viable means of reducing the level that
> may occur within a dried infant formula.
>
> DR. MOYER-MILEUR: Okay. Thank you.
>
> DR. BUCHANAN: The only issue that was not there is that we still have
> not completed studies on the extent of clumping that might occur.
> Those are being evaluated. Our problem right now is we don\'t have a
> standard for what \"clumping\" means for actually measuring clumping.
> We have empirical observations at this point that it does not appear
> to be a major factor, at least in most formulations, but we don\'t
> have an objective measure of the extent of clumping.
>
> DR. BUSTA: Dr. Acholonu.
>
> DR. ACHOLONU: Just for curiosity\--Alex Acholonu\--I saw a slide\--I
> think it was an electromicroscope slide by one of the previous
> presenters. And then I\'ve seen a slide that you showed of E. sak.
> Some look like they have\--
>
> DR. BUCHANAN: By the way, that wasn\'t E sak. Actually, that was E.
> coli.
>
> DR. ACHOLONU: Oh, I see. Okay.
>
> DR. BUCHANAN: Okay? That was just a general Enterobacteriaceae.
>
> DR. ACHOLONU: Okay. Well, anyway, my question still is: do the E. sak
> organisms have flagella? Are they flagellated?
>
> DR. BUCHANAN: Mm-hmm. Yes.
>
> DR. ACHOLONU: Okay. That\'s what I wanted to find out.
>
> Also, are they supposed to be facultative anaerobes, or just
> anaerobes?
>
> DR. BUCHANAN: They\'re facultative anaerobes. They grow best in the
> presence of oxygen; can grow quite nicely in the absence of it,
> though.
>
> DR. ACHOLONU: Thank you.
>
> DR. BUCHANAN: They have all the normal characteristics of an
> enterobacter.
>
> DR. BUSTA: I have a question, Bob.
>
> How did you inoculate the infant formula with that 10^6^?
>
> DR. BUCHANAN: How did we\--okay. What we did is we grew up a very
> large culture of Enterobacter sakazakii. We spun it down into the
> centrifuge. We took it up into a small volume of water. We then added
> it, dropwise, to the infant formula that was in a\--I think it was a
> four-liter flask, as I remember. After adding a drop, we would then
> shake the material to get it transferred as beset we could. We then
> continued that process until we added the entire inoculum to make sure
> that we had a reasonably distributed material. We had added a small
> amount of a blue dye to the inoculum so that we could see how the
> color was distributed throughout. And while I can\'t say that it was
> homogeneously distributed, it had to be pretty close, because we got a
> nice flat color across the entire lot.
>
> DR. BUSTA: Still could have been clumps, though.
>
> DR. BUCHANAN: There could have been very small clumps, but we got it
> distributed as beset we could. And it\'s interesting to note that as
> we pulled samples out over time, the amount of fluctuation or
> differences between the levels we saw was very, very small.
>
> DR. NEILL: Dr. Fischer?
>
> DR. FISCHER: You did this experiment where you inoculated infant
> formula and then looked at the rate of killing?
>
> DR. BUCHANAN: Mm-hmm.
>
> DR. FISCHER: The strain that you used, was that the most
> resistant\--thermally resistant strain\--or-
>
> DR. BUCHANAN: Yes.
>
> DR. FISCHER: It was.
>
> DR. BUCHANAN: Yes\--607 has been consistently the most resistant
> strain we have in our collection now. And that was the strain we used
> for all subsequent experimentation.
>
> DR. BUSTA: Dr. Tompkin.
>
> DR. TOMPKIN: Yes. Tompkin.
>
> Do you have any idea as to the code date and the freshness of the four
> formulas that were used for the vitamin studies?
>
> DR. BUCHANAN: No, but I can get that information, if it would be
> helpful to the committee. We have the details of all of that. I just
> didn\'t try to put it all on one slide.
>
> DR. BUSTA: Dr. Kuzminski?
>
> DR. KUZMINSKI: Kuzminski.
>
> If you\'d had clumps, though, wouldn\'t your death-time curves have
> reflected the presence of clumps by the changing slopes?
>
> DR. BUCHANAN: The question was posed to me. We tried to get as
> homogeneous of a distribution as possible in adding it to it. All
> indications, by the methods that we used, both using the dye to see
> that it was evenly distributed, by the linear nature of the death-time
> curve, and the fact that we didn\'t get substantial shoulders, and by
> the fact that we were able to take samples throughout this large lot
> and they all pretty much came out within .2 log cycles, are indicative
> of the fact that we did a pretty good job.
>
> DR. KUZMINSKI: I agree.
>
> DR. BUSTA: Other questions?
>
> We have a point in the agenda, now, where I\'d like your feeling. I
> arbitrarily postponed the preliminary subcommittee discussion on
> clinical presentations because I felt these last two had some
> implications on some of the clinical questions this morning. We can
> have that discussion now and then go on to the item on the agenda for
> the production situation, by Dr. Zink, or we can have Dr. Zink\'s
> presentation, take a break and come back for an overall preliminary
> discussion, and then have the public comment, and then have another
> discussion.
>
> What is your preference? The other alternative is you really don\'t
> care.
>
> \[Laughter.\]
>
> Next speaker. As close a consensus as we need.
>
> Is Dr. Zink ready? He\'s ready. Here he comes. And Dr. Zink will give
> us the FDA field survey of powdered formula manufacturing.
>
> **FDA Field Survey of Powdered Formula Manufacturing**
>
> DR. ZINK: Can you hear me? I figure, if nothing else, I\'ll try to
> speak up.
>
> What I want to do is accomplish two things with this presentation. I
> want to take what you\'ve learned this morning and put it into an
> industrial scale, using the information we gained from our field
> survey to talk to you about manufacturing practices; give you a real
> good idea of how formula is made; start you thinking about potential
> opportunities of how the organism could get into formula; and then
> talk to you in some detail about our findings.
>
> Could I have the next slide, please?
>
> \[Slide\]
>
> The objective of our field survey was to describe manufacturing
> processes that were used\--excuse me. This is presentation
> objections\--talk about the manufacturing practices used, summary
> micro-criteria used in various jurisdictions and by various governing
> bodies, and to review the results of our survey.
>
> Next slide.
>
> \[Slide.\]
>
> Powdered infant formula manufacturing , we can roughly talk about two
> kinds of processes. One is dry blending, and the other is where all
> the ingredients are mixed together into a homogeneous liquid, and then
> spray dried to produce a dry powder. Each of these processes has
> different challenges and benefits. I wouldn\'t characterize one as
> being preferable or inferior to the other.
>
> Among dry blending, certainly it minimizes the use of water in the
> processing area. Bacteria need water to grow. Anything that minimizes
> the use of water aids your ability to control the growth of bacteria
> in the environment of a manufacturing plant. However you\'re
> vulnerable to the contamination of ingredients. If your buying dry
> ingredients from various suppliers, and you\'re not, you know, hearing
> those ingredients in any way, pretty much what you get in the finished
> product is whatever you start with in these dry ingredients. Also, if
> you\'re going to use microbiological testing of the incoming raw
> materials to qualify them, that\'s a difficult thing to do because of
> the limitations of lot testing, the potential for non-random
> distribution of organisms, and the difficulty of getting a
> microbiological sampling plan that truly detects a positive organism.
>
> Wet mixing, on the other hand, you have the advantage that you\'re
> hydrating all your ingredients and you can pasteurize them to destroy
> all harmful bacteria, including organisms like sakazakii, but this
> requires frequent wet cleaning of very large equipment items. And also
> this use of water in the plant promotes the growth of bacteria in the
> plant and makes it a little harder to control. So you see some pros
> and cons with these.
>
> Next slide.
>
> \[Slide.\]
>
> This is a typical wet mixing, spray drying type process. This does not
> show any one manufacturer\'s process. It\'s sort of a stylized,
> simplified summary of the types of processes used by manufacturers in
> the survey.
>
> In general, you begin with the protein, mineral oil and
> carbohydrate\--often corn syrup\--components. These all go into a
> large tank. It can be three to five thousand gallons in this tank.
> They\'re mixed to homogeneity, and then they go through the
> pasteurizer. I should add that in some cases, manufacturers homogenize
> before they pasteurize, and some pasteurize before they homogenize.
> But, at any rate, knowing from Bob\'s presentation, you\'re dealing
> with pasteurization temperatures that could be anywhere from 170 to
> 260, this is a process that effectively destroys, completely,
> Enterobacter sakazakii. The holding time on this can range from, oh,
> 15 seconds to more than a minute.
>
> Then the product is usually cooled down to something like 170 degrees
> Fahrenheit\--maybe a little bit less. Some heat-sensitive ingredients,
> such as vitamins and perhaps some mineral component, fatty acid
> component, could be added here. Then it\'s homogenized. This is
> generally done at about 170. Again, remember Bob\'s presentation; 170
> is pretty close to 70 degrees centigrade, so you\'re still doing some
> killing here, even though you\'re not documenting it.
>
> Then it goes to a holding tank\--if it goes to a holding tank. This is
> optional. If it goes to a holding tank, it\'s usually cooled down to
> about 40 degrees centigrade, and may be held for some period of hours
> at that temperature. And then it\'s preheated before it goes into a
> spray dryer.
>
> This spray dryer\--and I\'ll show you some pictures of them
> later\--can be a very large structure; seven, eight, nine stories
> tall. And it\'s sprayed into that structure at the top as a fine mist,
> very small droplets. It can have a temperature anywhere between 160,
> 200 degrees Fahrenheit. When the liquid goes in here the air
> temperature of the spray dryer can run anywhere from maybe 260
> Fahrenheit to over 400 degrees Fahrenheit.
>
> So, up to this point the chances that Enterobacter sakazakii would
> have been in this material, survived this process and be alive and be
> contaminating product at this point\--not very great. The lethality
> steps are considerable up to this point.
>
> However, after this point, that is where, should the organism be
> present in the manufacturing environment it could certainly
> contaminate the powder. As this powder falls through the spray dryer,
> the swirling hot air, it very rapidly dries the droplets into
> particles of dry powder. By the time the powder falls to the bottom of
> this funnel-shaped spray dryer, you have a temperature of maybe 170,
> 180 degrees Fahrenheit; maybe as high as 200 degrees Fahrenheit in the
> powder. And it goes into what\'s called a \"fluidized bed.\" And you
> can think of a fluidized bed as a kind of a churning conveyer of
> powder, if you will, which has air flowing through it. And usually
> these fluidized beds are two stages: one that will pump hot air in
> there to do some additional drying, and then maybe several stages of
> cold air to cool it down.
>
> When the powder comes out of the fluidized bed it goes through a
> sifter. And at this point the powder is about room temperature. The
> sifter is to remove large clumps. And from the sifter it can go
> directly to a can-filling line, where it\'s filled, purged with
> nitrogen and hermetically sealed; or, it can be stored temporarily in
> totes or large bags for, say, quality assurance analysis, and then
> scheduled for filling.
>
> This is fairly typical.
>
> There\'s more than one kind of spray dryer. I\'ve diagramed a kind of
> a vertical funnel-shaped dryer here. You can also have box type dryers
> which are more room-shaped. But the principle of operation is the
> same.
>
> Next slide.
>
> \[Slide.\]
>
> In a dry blending type of process, actually most\--the formula
> manufacturers that use dry blending more heavily, it really should be
> thought of as a combination of wet mixing-dry blending. They\'ll take
> some dry materials, particularly the protein fraction, and the oil
> components, and they will wet mix those, again in a perhaps several
> thousand gallon tank, pasteurize them, cool them, homogenize them as
> before. Homogenization, if you\'re not familiar with it, is simply a
> step where the product is raised to several thousand pounds of
> pressure and forced through a small orifice to make a uniform
> distribution of the components, particularly the oils. The oils are
> reduced to very small micro-droplets so that they stay uniformly in
> suspension and don\'t settle out at the top of the product.
>
> Then, again, the holding tank, the pre-heater, and this protein and
> fat fraction is then spray dried. Now, this is\--percentage-wise, this
> is a smaller amount of the formula, and this process is advantageous
> to manufacturers, because they\'re not having to put water in and take
> water out by expenditure of energy into such a large quantity of
> powder.
>
> They then take this dry protein and oil fraction. It goes through the
> same sort of fluidized bed and sifter process, and into drums or totes
> or bags for storage. And then what happens is additional raw
> materials, particularly the carbohydrate fraction, but also
> potentially other kinds of fractions, are added to this pre-mix, if
> you will, in a ribbon blender. Again, it gets sifted and it goes to
> the can-filling line. The purpose of the ribbon blender here is to
> blend all of the materials to homogeneity.
>
> These are basically the two kinds of processes that are used.
>
> Next slide, please.
>
> \[Slide.\]
>
> This is a diagram, a little bit more detail about spray dryers and
> fluidized beds. First, I want to point out to you: this is the bottom
> of a funnel-type vertical spray dryer. That little figure right there
> is a man. Okay? It\'s a large piece of equipment. And this piece of
> equipment has doors\--access doors into it. They have gaskets.
> It\'s\--we\'re not talking about a smooth tube here. So periodically
> these things have to be cleaned. And they have to be wet cleaned. And
> sometimes you have to open an access door, and people have to go into
> them, or people dangle in a harness in them, trying to clean it. This
> is not what we would call a clean-in-place type operations, where you
> just flush some water through there and away you go. It\'s a
> challenging thing to clean, with lots of little nooks and crannies,
> when you think about it.
>
> So what you see here is this portion of a diagram. So here you have a
> large spray dryer. Hot air is generated here. It passes through high
> efficiency filters that filter out any microorganisms, and it goes in
> the top of the spray dryer, along with the heated liquid formula. The
> whole mixture swirls around and dries and falls to the bottom, goes
> into the fluidized bed. This is a two-stage fluidized bed shown here,
> with a hot air stage and cold air state. Again, this air is filtered
> when it goes in there to prevent microbial contamination\--and comes
> out of the fluidized bed through the sifter, as I showed you in the
> diagram.
>
> Things are always more complicated than you typically can draw them
> and show them. And, in fact, you get some very fine particles of
> powder which don\'t readily settle out to the bottom. These can be
> taken out of the spray dryer, and they go through what are called
> \"cyclones,\" which is, in essence, a centrifugal type of centrifuge.
> And these very fine particles can be separately collected. And those
> may be used for infant formula, or they may be\--excuse me. Those may
> go to waste as animal feed, or they may be recycled back into formula
> through a wet-mixing process. It varies.
>
> Next slide.
>
> \[Slide.\]
>
> This is the inside of a box dryer. And a box dryer is just a square
> spray dryer. It\'s hard to see here\--it\'s dimly lit\--but these
> are\--there\'s nozzles and hot air inlet up here. Imagine this room
> was a box dryer. You have hot air and sprayed liquid coming in at this
> end, swirling around and falling to the floor, where it\'s collected.
> This picture showed liquid being sprayed in a very fine mist, and it
> dries. This detail here shows a chain drive. And how do you get the
> powder off the floor? Well, you just have a bar that\'s pulled by a
> chain that scrapes it over to one side, and then you have an
> auger\--if you go to the\--yes, you can see the auger here, I think,
> in this slide\--and then you have an auger in the corner there against
> the wall that carries the powder away. So that\'s the other type of
> spray dryer used.
>
> Next slide, please.
>
> \[Slide.\]
>
> This is a diagram of a fluidized bed\--not a small piece of equipment,
> either. If a person were standing here, they would be about so
> tall\--okay? So if you\'re standing on the floor, certainly the
> apparatus is taller than you are. It gives you some idea of the scale.
> If you look inside of it\--and, of course, this is clean and not
> operating right now\--you see this kind of a porous bed here that
> vibrates and that air can come up through, and the powder just moves
> along like it\'s flowing in a turbulent stream over the bottom of the
> fluidized bed.
>
> And you can see, there\'s some nooks and crannies in here, too, that
> pose cleaning challenges. And it is\--it\'s a large piece of
> equipment. So there\'s opportunity for contamination to occur here if
> the equipment is not perfectly clean.
>
> Next slide.
>
> \[Slide.\]
>
> On to microbiological standards. Before talking about our findings in
> formula, we thought it would be good if we could review with you what
> we knew about the various microbiological standards currently being
> applied to powdered infant formula.
>
> This is the CODEX standards. You heard it referred to earlier in one
> of the presentations, in relation to an outbreak that occurred in
> Belgium. It\'s permissible\--they take five samples of a lot. It\'s
> permissible to have one of those samples with as many as 20 coliforms
> in it, but you should have fewer than 3\--and \"fewer than 3\" in this
> case means no positive tubes in a three-tube MPN\--but you should have
> fewer than 3 in the remaining four samples; n, number of samples, c
> maximum number of samples that can have a number in excess of m, and M
> is the \"no single sample shall exceed\" limit. Okay?
>
> Next slide.
>
> \[Slide.\]
>
> This is the Canadian standards, and I\'m going to focus in here. They
> don\'t specifically have one for coliforms or Enterobacteriaceae, but
> Escherichia coli comes close. Here they\'re taking 10 samples. One of
> those would be permitted to have as many 10 organisms in it, but the
> remaining nine would have to be less than 1.8\--essentially, probably
> a negative results.
>
> Next slide.
>
> \[Slide.\]
>
> This is a standard currently being used in China: aerobic plate count
> less than 30,000 per gram. They have a coliform standard of less than
> 40 per 100 grams.
>
> Next slide.
>
> \[Slide.\]
>
> This is a standards that was proposed\--or, guidelines, really, that
> were proposed I guess in our\--I don\'t know the exact term we would
> use for it\--but it was proposed as criteria that we would use in
> judging the acceptability of a lot of infant formula. And it was less
> than 3.05 MPN per gram coliforms. There were similar separate
> standards for fecal coliforms.
>
> Next slide.
>
> \[Slide.\]
>
> In our survey of infant formula plants we surveyed all of the plants
> that produce powdered formula in the United States. There were 15 such
> plants. Some of these plants produced concentrates and ready-to-feed
> only. Some produced\--I think seven of the plans produced powdered
> only. Some plants produce ready-to-feed and powder, and I think a
> total of 10 plants each produced powdered formula and all were
> included in this assignment. So this is a complete survey, if you
> will, of the U.S. powdered formula market.
>
> Next slide.
>
> \[Slide.\]
>
> What we wanted to do with this study was to get an official sample of
> powdered infant formula and raw materials to assess the prevalence and
> the number of E. sakazakii in the samples. And the sampling protocol
> that we devised was based on prior literature reporting numbers as few
> as .36 per 100 grams, and we wanted to be certain to have a sampling
> protocol that could quantitate down to that limit; also to conduct an
> inspection at each major domestic infant formula producer in order to
> understand any possible relationship between manufacturing practices
> and the prevalence of E. sakazakii.
>
> Next slide.
>
> \[Slide.\]
>
> Here you can see the 10 plants that were studied. The total number of
> samples collected was 92. Of this 92, you\'ll probably be most
> interested in the finished product samples, of which there were 22. We
> also collected separately samples of carbohydrate fraction, fat, if it
> was present as a dry powder, and protein samples.
>
> These samples were collected regardless of the process used. In other
> words, in some cases this carbohydrate was directly dry blended into
> the finished product without further heat treating. And, in some
> cases, this carbohydrate was part of a wet mixing-spray drying
> operation. It went in into a wet mix and then it got pasteurized
> before it was spray dried. But we wanted to look at prevalence in raw
> materials as well.
>
> Next slide.
>
> \[Slide.\]
>
> Looking at finished products by the types of products\--again, there
> were 22 finished product samples. Fourteen of these were formulas for
> full-term infants; four were formulas intended for pre-term infants;
> and two were metabolic formulas, and two were hydrolysates.
>
> Next.
>
> \[Slide.\]
>
> I want to talk to you a little bit about our procedure. If you\'ve
> gotten the impression everybody does their own thing, sort of, when
> they develop these that\'s kind of the nature of microbiology. We have
> essentially, as we said, used the procedure of Nazarowec-White, with
> very little modification. Okay? However, it\'s important to note that
> we started out with 20 cans here. In the study Don talked to you about
> earlier, they were actually looking at 330 grams in a given lot or can
> of sample. We\'re looking at 1,332 grams, and here\'s how we come to
> it.
>
> Twenty cans were collected, and each of these cans had at least eight
> ounces of powder in it, and a composite was constructed by sorting
> these cans into four groups of five; 100 grams came out of each can in
> the group, so we now had four groups of 500 grams. And those four
> groups of 500 grams were each treated as four separate three-tube
> MPNs. So you had 4 times 3 times 100, times 10, times 1, for a
> total\--out of any given lot sampled\--of 1,332 grams. That\'s
> important to note when you consider the prevalence of the organism
> reported by other investigators looking at smaller quantities of
> powder versus our results.
>
> The theoretical limit of detection here would be one in 1,332 grams.
> That could occur, provided you had optimal conditions; you didn\'t
> have competing microorganisms, etcetera. We don\'t know what the
> actual limit of detection is, of course, because we don\'t know what
> the true contamination was. The minimum limit of quantitation\--as Don
> Burr explained to you\--is 0.31 most probable number in 100 grams.
>
> I want tot diverge just a minute so you don\'t get to where you
> misunderstand what\'s meant by \"most probable number method.\" It has
> limitations, and you need to think of those limitations, and you need
> to think about the statistics associated with it. First thing, the
> most probable number, to be a valid method of quantitating something,
> the main assumption you have to meet is that whatever it is you\'re
> measuring has to be uniformly distributed in what you\'re measuring.
> And that is almost certainly not the case here.
>
> If you remember the paper reporting the outbreak in the Belgian
> hospitals, they referred to the fact that they analyzed five samples;
> four of them were negative and one of them had 20 per gram. Well, that
> shows you what I think a lot of microbiologists\--food
> microbiologists\--know, is that this organism is not uniformly
> distributed in the powder. Contamination theoretically could occur
> from sloughing off of a equipment. That could happen sporadically. So,
> in our MPN analysis, we\'re already violating one of the assumptions
> of the MPN, simply because we had no other alternative way to do it
> that was do-able. And you should keep that in mind. The confidence
> interval for this result\--say you get a result of 0.31 MPN in 100
> grams, the confidence interval for that\--95 percent confidence
> interval\--is anywhere from 0.15 per 100 grams, all the way up to 1
> per 100 grams.
>
> Now, the only way to really understand\--I think it\'s important to
> think about what might actually go into a bottle of formula. On
> average, I guess an eight ounce bottle of formula has something
> like\--what?\--34 grams of powder, something like that, in it. And the
> only way we could really find out what might actually be going into a
> serving would be to do an experiment like this where we culture
> everything in little 28 or 34 gram aliquots. But when we report this
> result of 0.31, or 0.36 per 100 grams, you could very well have a gram
> of that powder with 8, 10 or a dozen organisms in it, and then many
> aliquots of that powder with nothing in it. Okay? So this is a
> statistical result, if you will.
>
> Next slide.
>
> \[Slide.\]
>
> What we found, overall, in finished product, we found five positive
> samples, for a total of 22.7 percent of the sample\--finished product
> sampled\--were positive. In every case, when you analyze this by the
> three-tube MPN tables, we found 0.36 per 100 grams\--at or very close
> to the limit of detection\--22 samples tested.
>
> In the carbohydrate fraction, we found only one positive result. Now,
> I should say we only did quantitative MPN test on finished product,
> not the components of it. And we found one protein fraction that was
> positive.
>
> Next slide.
>
> \[Slide.\]
>
> If you break this down by type of formula, four of 14 were full-term
> formulas; one of four was a pre-term formula. The metabolic and
> hydrolysates weren\'t positive. There\'s no significant difference in
> that result.
>
> Next slide.
>
> \[Slide.\]
>
> All positive finished product samples were at the lowest limit of
> quantitation. So we\'re talking about low numbers here. There was no
> correlation between the test results and manufacturing practices. In
> other words, you know, we got positives from people that wet-mixed,
> and we got positives from operations that incorporated dry blending,
> without apparent difference.
>
> And there was no relationship between the results and product type. We
> had soy products that were positive; we had milk-based products that
> were positive . So there\'s no reason to believe that this has got any
> particular type of association.
>
> I believe that\'s it. Thank you.
>
> DR. BUSTA: Are there questions for clarification for Don?
>
> Dr. Fuller. Oh, I\'m sorry. Dr. Moyer-Mileur.
>
> DR. FULLER:
>
> . DR. MOYER-MILEUR: Moyer-Mileur.
>
> Could you just clarify\--for the pre-term formulas, did that include
> your transitional feeding formulas for pre-term babies, or just the
> in-hospital 24 calorie-per-ounce products.
>
> DR. ZINK: I believe that the pre-term did include what you would call
> transitional.
>
> DR. BUSTA: Dr. Briley.
>
> DR. BRILEY: Margaret Briley.
>
> Could you clarify for me the differences\--you said there were no
> differences in cleaning the vats when they were dry or wet milled.
> Would you say that possibly it would be more difficult in one, or that
> one would be a slight bit more toward the possibility of
> contamination, or\--
>
> DR. ZINK: Okay\--talk about equipment cleanability?
>
> DR. BRILEY: Yes.
>
> DR. ZINK: Dry versus wet blending? All right.
>
> In the wet blending and spray drying, since everything has to be spray
> dried, you\'re generally dealing with some very large capacity spray
> drying systems. And those spray dryers, on average, probably have to
> be cleaned once a week by wet cleaning. And that can take a day or
> two, you know. It\'s a difficult process. The fluidized bed has to be
> cleaned, and all of that associated equipment with it has to be
> thoroughly dried out. That\'s a challenge as well.
>
> In a dry blending operation\--like I said, most people still have some
> wet blending and spray drying going on. But they\'re generally not
> having to clean as large a piece of equipment. And on the dry side of
> it, you can go quite a long time between cleaning, or you can use
> strictly dry cleaning methods for your brushing, vacuuming\--that sort
> of thing. So, on a purely dry side of the process, oh you can go
> weeks, perhaps months without using much water. And keeping it dry is
> a huge advantage in controlling these organisms. You can\'t deny them
> food, but you can certainly deny them water, and they need that water
> to grow and without it they don\'t grow.
>
> DR. BUSTA: Dr. Fuller?
>
> DR. FULLER: Given the\--you mentioned the potential for clumping in
> equipment to release chunks of bacteria, if you will. Did you all do
> any looking at\--do any environmental sampling? And, if not, was there
> any reason for not doing that at the same time?
>
> DR. ZINK: We\'ve had limited ability to go back and do those kinds of
> investigative samples. I believe it was done in one case. I believe
> that organisms were recovered from the environment. Perhaps some of
> these investigations will still be ongoing. I don\'t know what the
> status of that is.
>
> But by the time we get a sample, analyze the sample, and interpret the
> results\--this wasn\'t done to be in real time. So a sample simply
> went in and they were enqueued with the labs other work. Oftentimes,
> by the time we got that result, it might be a month or more from the
> time the sample was collected, and by that time the equipment has gone
> through many cleaning cycles, and the manufacturers themselves were
> monitoring equipment and environment. So I think going back in and
> doing a detailed equipment survey in the timeframe we were working in
> here, that was clearly a secondary or tertiary objective.
>
> So I don\'t think we sought anything meaningful out of that or got
> anything meaningful out of it.
>
> DR. MOYER-MILEUR: So let me just\--let me ask my question a little bit
> differently.
>
> The assumption, then, is that the primary source of the E. sak is
> coming from the ingredients, not from equipment contamination. You
> feel that there\'s enough cleaning going on to prevent that? Or\--I
> mean, I was just trying to get a better understanding.
>
> DR. ZINK: I think if I had to offer you my opinion, I would say the
> organism is probably coming from the equipment, post spray drying. I
> think that the organism, by whatever means unknown, gets into the
> plant and can contaminate that equipment. And the difficulties of
> cleaning this equipment, you can never make that equipment completely
> sterile. And so I think that what low levels of contamination do occur
> are probably coming from that handling equipment.
>
> DR. MOYER-MILEUR: Okay. So we\'ve got that assumption, but no data to
> back that up yet.
>
> DR. ZINK: Nope.
>
> DR. MOYER-MILEUR: Okay.
>
> DR. ZINK: I\'m\--that\'s going to have to be the subject for further
> study.
>
> DR. BUSTA: Dr. Blumberg.
>
> DR. BLUMBERG: Henry Blumberg.
>
> Did you look at other organisms besides Enterobacter sakazakii? Or did
> these studies that you were doing just focus on that?
>
> DR. ZINK: We were specifically looking for Enterobacter sakazakii.
> They were specifically picking yellow colonies.
>
> When they first started this study\--and going over the work sheets of
> the labs, I think they were more liberal in picking colonies as they
> were trying to become familiar with this organism. So I think they
> cast a wider net at first. And, certainly, we did see, in some of
> those early samples, some things like Serratia, Panatea\--other
> organisms cropped up. But, for the most part, after the first few
> positive samples, they were zeroing right in and just picking
> sakazakii.
>
> DR. BUSTA: Dr. Tompkin.
>
> DR. TOMPKIN: This is Tompkin.
>
> You didn\'t do MPNs on the carbohydrate and protein fractions, the
> ones that tested positive. What was the amount of product, or
> ingredient?
>
> DR. ZINK: We were looking\--I\'m trying to remember. You would ask me
> that. I believe we looked at 500 grams as one sample. Is that right?
> Don, do you remember? Is Don Burr back there?
>
> DR. BURR: No, I don\'t remember.
>
> DR. ZINK: I\'m pretty close to that. I\'ll check. But I believe it was
> 500 grams.
>
> DR. BUSTA: Dr. Neill?
>
> DR. NEILL: Peggy Neill.
>
> Don, do you recall what month or season of the year the sampling was
> done?
>
> DR. ZINK: I think we began collecting in August and continued through
> January, or maybe even February.
>
> DR. BUSTA: Dr. Tarr.
>
> DR. TARR: Tarr.
>
> What governs the interval between cleanings? Is it tradition, or is it
> driven by colony counts or other data?
>
> DR. ZINK: It\'s driven by need. You begin to see accumulation of
> material on the walls of the spray dryer. Or else they need to switch
> from product type to some other product type. That\'s another thing
> that would drive it\--say, going from milk to soy, or soy to milk.
>
> DR. BUSTA: Dr. Briley?
>
> DR. BRILEY: Margaret Briley.
>
> Did you, in your survey, by any chance make record of any of these
> plants that had HACCP procedures in place?
>
> DR. ZINK: The question was did we, in our, surveys make a record of
> any of the plants that had HACCP procedures in place. No, I don\'t
> recall that we were specifically looking \"Did you or did you not have
> a HACCP plan?\"
>
> The nature of how these plants operate is it\'s HACCP by any other
> name. In other words, all of these manufacturers had detailed
> procedures. Every one of them had SOPs for cleaning the equipment.
> They all identified critical control points in their process. It
> was\--you know, there was a remarkable sameness as you read through
> all the reports about the methodology of controlling the process.
>
> DR. BUSTA: Dr. Stallings.
>
> DR. STALLINGS: Stallings.
>
> We\'ve really been waiting to see some of these data. So, to clarify
> the one the FDA field survey results\--you did have starting
> product\--carbohydrate and protein\--that had positive cultures.
>
> DR. ZINK: Yes.
>
> DR. STALLINGS: And then the finished product. And so you were saying,
> though, that you really felt like the source of the organisms was
> during the manufacture. So the protein and carbohydrate\--I mean, just
> explain that.
>
> DR. ZINK: I can talk about those in a little bit more detail.
>
> DR. STALLINGS: Okay.
>
> DR. ZINK: In the case of the protein fraction, that was used in a
> product which went through a traditional wet mixing-spray drying
> process. So all of the protein was hydrated into water with the other
> ingredients, and then it went through pasteurization. I remember doing
> a calculation on the lethality, taking that processor\'s exact time
> and temperature conditions, and then only at the
> pasteurizer\--okay?\--not considering other heating contributions. And
> I think I came up with a theoretical lethality of around 200 log cycle
> reduction. Very substantial. No way that organism could possibly have
> survived that process. So, I do not believe that that protein
> contributed\--you know, although it was contaminated, it would not
> have been a factor in contaminating the other product.
>
> You always have to consider, when raw materials come into a plant and
> they\'re contaminated, even though you\'re going to mix them wet and
> pasteurize it, if they get opened and tracked around the plant,
> or\--you know, that could be a problem. But in the case of the
> contaminated carbohydrate, it did go into a product through a dry
> blending process. So, most certainly, what was in it would have been
> expected to also show up in the formula.
>
> DR. BUSTA: Dr. Kuzminski.
>
> DR. KUZMINSKI: Thank you. Larry Kuzminski.
>
> A number of questions. In your survey, did you get a chance to
> review\--and it sounds like you did\--the QA practices\-\--the
> standard quality assurance practices\--during the manufacturing
> process?
>
> DR. ZINK: Yes. All of those procedures were documented and recorded,
> and there was nothing remarkable or deficient showing in any of that
> documentation.
>
> Bear in mind, you know, when all of that field survey material comes
> back to us, they were following specific instructions to collect
> certain kinds of information, and collect certain kinds of
> documentation. I think one of the things you have to ask is: do these
> traditional kinds of critical control point documentation systems and
> SOPs\--do they adequately address risk of contamination with an
> organism like this? I mean, certainly, in looking at the data as it
> came in, you know, it seemed to meet, you know, all of the
> requirements one would expect of a manufacturer of that product\--as
> documented on those reports.
>
> If you were standing there in those plants, I think you have the
> ability to assess things, and consider things, you know, that might
> not be part of formal control programs that might be contributing
> factors.
>
> DR. KUZMINSKI: One of the points that you made was the potential for
> contamination post\--in the process flow, past the spray dryer, once
> heat\'s stopped being applied to components of other process, back to
> ribbon blending and to addition of the minor ingredients; the vitamin
> and mineral pre-mixes.
>
> I noted that\--you perhaps omitted this, but did you get a chance to
> take a look at the vitamin components in the pre-mix for
> microbiological quality?
>
> DR. ZINK: They did not sample any of the vitamin components in this
> survey.
>
> DR. KUZMINSKI: There must\--also from the data on the distribution of
> the nutrients that, I think, Dr. Buchanan showed, it looks like the
> efficiency of the ribbon blender is a pretty efficient blender, in
> terms of getting distribution pretty homogeneously throughout a batch,
> even up into a 5,000 pound batch\--quite a size batch.
>
> Would you think, though, that if something if something is sloughed
> off from the equipment\--a chunk of dried material, caked on material,
> from something that\'s been running for awhile, a number of shifts, a
> number of days, especially in the dry process, that the efficiency of
> the ribbon blender would be there to get that distributed through the
> batch also?
>
> DR. ZINK: Well, to the extent that\--I mean, not all formulas go
> through that kind of ribbon blender dry blending process. Some are
> strictly the wet mix-spray dry, more of a continuous stream type of
> thing.
>
> Yes, I would think the ribbon blender would tend to take non-random
> contamination and randomize it to a degree. I\'m not familiar with any
> kinds of studies\--and I don\'t have any experience to say how
> effective in doing that it would be. But I would expect that the
> ribbon blender, on those dry blended products, would tend to randomize
> the contamination a bit\--yes.
>
> But in the other process, you really don\'t have that at work. So you
> would maybe expect more non-randomness in the wet mix-spray dry type
> of process.
>
> DR. KUZMINSKI: And, lastly, there have been a couple of
> recalls\--voluntary recalls\--in the industry due to E. sak presence.
> Very responsible companies are involved. And in the follow-up
> investigation to these recalls, in terms of process analysis, the
> efficacy presence perhaps and the line flow of ingredients, right back
> to the raw materials\--individual raw material supplier\--to the
> discharge end of the packing line\--can you share with us any
> information that has been gained from those recall incidents, as to
> the occurrence?
>
> DR. ZINK: I can say\--you know, did someone\--did an investigator or a
> company come forward with the smoking gun in each of those cases? No.
> Okay. I mean, I am familiar\--some manufacturers shared the fact that,
> indeed, they were finding the organism in the food processing
> environment, and that controlling the organism in the food processing
> environment is a real challenge. And perhaps we\'ll hear more about
> that later.
>
> DR. BUSTA: Dr. Neill
>
> DR. NEILL: Don, can you tell me\--I have two very different question,
> one of which is how analogous is this process for the production of
> the powdered formula to the production of powdered milk? And then,
> kind of a\--
>
> DR. ZINK: Virtually .identical.
>
> DR. NEILL: All right. And then perhaps somewhere in the later batch of
> questions, one of the individuals who spoke can tell us whether any
> similar work has been done trying to analyze, microbiology speaking,
> any powdered milk for this.
>
> DR. ZINK: Her question was how similar is the infant formula
> manufacturing process to, say, what would be used to produce
> spray-dried milk. And my answer is it\'s virtually identical; same
> types of equipment, same types of processes, probably even similar
> temperatures involved.
>
> We certainly haven\'t done a survey of spray-dried milk plants for
> this organism.
>
> DR. NEILL: And then the second question has to do with the derivation
> of the sampling plan. Since, in looking at that I notice that there\'s
> quite a difference in the distribution of the sample types\--finished
> product versus component ingredients, etcetera\--across the 10 plans,
> and was this derived on the basis of volume of product, or what?
>
> DR. ZINK: I think probably others could talk about that sampling plan
> more, but\--correct me if I\'m wrong\--we wanted to get a good
> sampling of the various different kinds of products that were out
> there. I don\'t believe there was aan attempt to bias it in any way.
> It was really what we could get at within the timeframe allocated for
> the study, really. And we wanted to look at some of each thing,
> but\--you know, some of these formulas are produced
> infrequently\--once or twice a year. And that had a lot to do with it,
> particularly some of the speciality formulas and stuff\--our ability
> to get those samples.
>
> DR. BUSTA: I\'ll take three more questions.
>
> Dr. Acholonu.
>
> DR. ACHOLONU: Doctor, you discussed the microbiological standards in
> Canada, in China and as used by FDA. And each of them has aerobic
> plate count. Since we have been told that E.sak is a facultative
> anaerobe, can that method be used?
>
> DR. ZINK: It will certainly grow up as a colony on the aerobic plate
> count. A lot of other things will too, of course. You would have to,
> at that point, pick the colonies and identify them to differentiate it
> and detect it.
>
> To a microbiologist\--the ideal method to a microbiologist is that you
> take a whole lot of the sample, throw it in some magic broth and only
> what you are interested in grows, and it\'s not the least bit
> inhibited by anything in there, but everything else is inhibited\--and
> out comes the bug. We don\'t have that method for this organism,
> unfortunately.
>
> Infant formula is very clean, microbiologically. Although you saw
> aerobic plate count limits up there of 10,000, 30,000\--things like
> that, most manufacturers are producing more in a norm of about 500 per
> gram.
>
> DR. BUSTA: Dr. Beuchat.
>
> DR. BEUCHAT: Beuchat.
>
> My question concerns the physical structure, or the layout, of a
> typical wet mixing-spray drying process. Is the area in which the raw
> ingredients handled separated physically and/or by air flow,
> ventilation and so on, from the post-spray dry area?
>
> DR. ZINK: In the plants I have been in\--I\'ll answer that question by
> saying yes. In some plants, it\'s outstandingly well separated. And in
> somse plants, there\'s room to improve\--okay? But I think all
> manufacturers strive to achieve segregation, and all of the
> manufacturers I know of have very strict physical environmental
> isolation once they\'re dealing with a finished powder. You just
> can\'t stroll from one area of the plan to the other.
>
> DR. BUSTA: Thank you, Don.
>
> We will now take a 20 minute break. Is that long enough for you? A 20
> minute break, so we\'ll aim at 3:25 reconvening, and we\'ll ask any
> questions of the three speakers so far this afternoon first.
>
> \[Break.\]
>
> DR. BUSTA: Will all of you that are asking questions\--now that\'s how
> to make a point\--right? You\'re asked to talk loudly, as well as into
> the microphone. But even with the microphone, talk loudly, because all
> of this is being recorded and someone down the line has to try and
> transcribe it all. And that\'s\--we\'re trying to make their job not
> as terribly difficult as it might be.
>
> At this point we have about, maybe, 20 minutes if we need that to ask
> questions for any of the four speakers from this afternoon. And we\'re
> open to questions.
>
> Dr. Tompkin.
>
> DR. TOMPKIN: Bruce Tompkin. This is for Maria.
>
> I don\'t recall that you mentioned it in your presentation, but in the
> manuscript that you had published, at 4 degrees centigrade, which is
> almost 40 Fahrenheit, you found that E. sakazakii actually died? But I
> don\'t recall that it stated how rapidly. Could you comment on the
> rate of death at refrigerated temperature?
>
> DR. NAZAROWEC-WHITE: At refrigerated temperatures we took samples, I
> think it was every two days. And there was nothing\--in some of them
> it would be 10^3^ and 10^3^, and then after about\--I don\'t
> know\--two weeks, it was down.
>
> DR. BUSTA: Dr. Thureen.
>
> DR. THUREEN: Thureen.
>
> If this committee\--this is a hypothetical question\--if this
> committee can agree that there should be a standard above which there
> should be no higher level of E. sakazakii contamination, and we decide
> that this is an index marker, because it has such virulence, and
> because it\'s easy to detect, and we establish that standard as being
> the limit above\--or at which we feel comfortable of having to be a
> slight contamination rate, do you think then that we will also
> eliminate risk for serious infection from other organisms that are
> found within the powdered infant formulas? Would that be a good
> benchmark to set to provide overall safety, not just against his
> particular organism. And this is for any of you.
>
> DR. BUCHANAN: You\'re asking a really big question. You\'ve posed a
> hypothetical question that\--the best that I could give you is a
> hypothetical answer\--with all the disclaimers, caveats, etcetera.
>
> The suggestion is that if you could have a standard that controlled
> this organism, would you control others? And the real question here
> is: is their mode of action the same? Is their mechanism of
> pathogenicity the same? Is the impact of the interventions or
> standards that you would use the same?
>
> If you could fulfill all those criteria\--probably. And I hesitate to
> say anything more than probability. If their method of control was
> different, then I would say that you\'d have to actually go through
> and make the case. And, gee, if you\'d ask me, you know, are there
> infections due to Enterobacter cloacae, and would controlling
> Enterobacter sakazakii control Enterobacter cloacae? Yes, I think
> you\'d probably have a good chance. Salmonella? I\'d be less sure of.
>
> DR. THUREEN: And so, along that line, do you think it should be E.
> sakazakii, or do you think it should be enterobacter in general that
> should be sort of the target organism for eradication? Or do you think
> it makes a difference.
>
> DR. ZINK: I think I\'d be more comfortable answering if I knew a
> little more than I know right now. I agree with Bob. If you choose to
> pick an organism as the focus of your control, and it happens to be
> part of a family of organisms that have a similar mode of
> contaminating the product, and similar resistance or susceptibility to
> control measures, then I think picking the most onerous of the family
> probably would be a good way to go.
>
> Talking about Enterobacteriaceae, as you\'ve seen, they\'re all pretty
> similar in their susceptibilities and resistences to control measures.
> So that may be an approach you could use. The trouble is that I\'m
> not\--I don\'t think any of us have a lot of data on the diversity of
> organisms in formula and how they get there, you know.
>
> DR. NAZAROWEC-WHITE: That\'s what\--I agree. I think that we don\'t
> know what\'s in there yet. We do know that there are some salmonella,
> maybe, in the dried formula. So you have one option of sort of setting
> that standard is a possibility. On the other hand, are there any
> intervention strategies\--can we do something with that final product
> to inactivate organisms?
>
> DR. BUSTA: Dr. Tompkin.
>
> DR. TOMPKIN: I didn\'t look it up, Don, but maybe you might remember.
> In the \'96 ANPR for FDA for listeria monocytogenes, do you recall
> what the amount of product it\'s\--your slide says it\'s negative, but
> is it negative in a 25 gram, or 50 grams? Do you remember?
>
> DR. ZINK: I don\'t remember.
>
> DR. TOMPKIN: Or was it specified?
>
> DR. ZINK: It would be specified, if by reference to a method. Okay? I
> don\'t remember whether it was actually spelled out in the ANPR. If it
> wasn\'t, it would have been referred back to the method we used. I
> don\'t know\--Ada, do you know? 220? 250? 250 grams?
>
> You know, you hardly ever see listeria in these dry powder plants. It
> just doesn\'t like dry powder plants. I don\'t think I\'ve ever
> recalled it being an issue.
>
> DR. BUSTA: Dr. Fuller.
>
> DR. FULLER: This is Fuller.
>
> Not being particularly knowledgeable of manufacturing practices,
> etcetera, is it possible\--or maybe it\'s cost prohibitive\--but to
> apply a heat step at the\--you know, just prior to filling the cans?
> Is there\--I mean, does it make any sense? Can you use dry heat? Do
> you have to\--you know, is there anything that would\--could be done
> at that step?
>
> DR. BUCHANAN: I\'ll jump in here for a minute, because I looked at dry
> heating for a number of issues.
>
> The big thing about dry heating is whether or not your product is a
> mixture of protein and carbohydrate, which infant formula are. In some
> instances where you have a single entity, and you don\'t have a
> browning reaction taking place, you can actually get away with dry
> heating\--long enough and hot enough that you would inactivate
> microorganisms. A classic example is salmonella control in egg white.
> But egg white is interesting because they have to de-sugar the egg
> white before they can dry it because if not, it turns brown.
>
> The same thing would be expected in infant formula. You\'d get a lot
> of browning reaction products.
>
> DR. ZINK: One of the problems is these bacteria get a lot more heat
> resistant when they dry. I guess this is best studied with salmonella.
> And there are garden variety salmonella strains that can resist 200
> degrees Fahrenheit for hours. And that\'s a real problem\--when
> they\'re dry, when you dehydrate them.
>
> So, I think that applying heat to the dry powder would be\--it would
> be challenging to get significant log reductions.
>
> DR. BUSTA: Dr. Tarr.
>
> DR. TARR: Since looking for low counts of specific pathogens can be
> frustrating and quite variable, let\'s go to the more robust total
> colony counts, or Enterobacteriaceae counts. Are there trends over
> time that you are observing? Are they generally going down year by
> year in industry, and are they associated with any new practices that
> could be either accelerated or amplified in your production line?
>
> You\'re clearly exceeding the CODEX and the FDA guidelines, but
> you\'re still getting some counts coming through. Are there trends in
> those that would be informative for making a safer product?
>
> DR. ZINK: That might be a better question for industry. I don\'t know
> that we have\--at least not in a readily analyzable form\--data that
> would say, \"In 2003 we\'re better than we were in 1985\"\--in terms
> of counts.
>
> I know there are specific instances within industry where\--going back
> to paper on the Belgian outbreak, citing the plant that was the source
> of that powder. I do know there are specific instances where
> improvements in manufacturing process were introduced that had
> dramatic improvements in the microbiological quality of the product.
>
> DR. BUCHANAN: Phil, I\'d also caution you to be a little careful about
> the use of indicator and index organisms. Because there\'s no
> guarantee that the reduction that you see in total aerobic plate
> counts are equivalent reductions taking place in the pathogens of
> concern in the product. In fact, you could, for example, eliminate
> large numbers of vegetative cells from a product, and if the organism
> of concern was a spore former, it would have no meaning in terms of
> reducing the level of risk.
>
> DR. TARR: I agree with that, but at least with Enterobacteriaceae, if
> we got to zero Enterobacteriaceae we would get to zero E. sakazakii.
> And that should be a target. Maybe not this year.
>
> DR. BUSTA: Dr. Stallings.
>
> DR. STALLINGS: Stallings.
>
> One of the overriding principles we\'re still dealing with is that
> this is a rare disease. You know, that if\--that it\'s a rare bacteria
> in the product, and that it\'s rarely pathogenic, even in the setting
> we\'re using.
>
> And I go back to the concern that someone brought up earlier about the
> changes in clinical microbiological methods, in that many things have
> become more automated. And I don\'t know if any of you know, or if
> it\'s something that we can just get for the committee\--just some
> reassurances that the systems that are now being used, where basically
> they\'re put in banks, and heated, and no human eyes see them until,
> you know, there\'s something that says something\'s growing\--that for
> this fairly rare bug, that we would still be picking it up. Because
> if\--we know the neonates are getting smaller and smaller, and sicker
> and sicker, and there would be some reassurance if we really knew that
> we were doing very good clinical microbiological surveillance, and not
> picking this up, and that it was being identified properly when it
> happened, and that sort of thing.
>
> Do any of you have any information in really what\'s going on in
> clinical hospital settings?
>
> DR. BUCHANAN: I think we can try to find that out. But the only
> comment I would offer is that the most important thing for getting a
> clinical sample done is having the physician ask for it.
>
> DR. STALLINGS: Well, to address that, I think any baby\--if we\'re
> talking about the high risk nursery setting, the signs and symptoms
> you\'re going to see were described. It\'s temperature instability,
> it\'s food intolerance if they\'ve been eating, and\--it\'s very soft
> signs. But in a level 3 neonatal nursery, at the drop of a hat you do
> blood, urine and spinal fluid. That\'s just the way we manage the
> highest risk babies. So, in this one rare setting, I think we probably
> do\--we don\'t do stools, but stools would not be the specimen of
> choice for this.
>
> So\--I\'ll defer to my neonatologist, but it\'s usually a pretty\--if
> you are at all worried about these babies, you do the drill and you do
> it comprehensively.
>
> DR. THUREEN: One of the problems is that, in theory, you\'d like to do
> lumbar punctures on all these infants, but they\'re usually too
> unstable to have that done. Anda I think in many nurseries, just signs
> of NEC would not be enough to trigger spinal sampling being done. And
> it varies from nursery to nursery.
>
> While I have a microphone, I have a question.
>
> DR. BUSTA: Dr. Thureen.
>
> DR. THUREEN: Thureen\--for the record. And I\'m sure there\'s a good
> reason, but why isn\'t irradiation a solution to this problem?
>
> DR. BUCHANAN: I\'ll take that one on. And, again, it\'s the
> characteristics of these organisms in a dry environment.
>
> Just as the thermal resistence of a normally sensitive organism goes
> up drastically when you put it in a dry state, so does the irradiation
> resistence. So you would probably exceed the level of irradiation
> that\'s allowed at this point.
>
> Now, I\'d have to say that I don\'t know of any specific information
> on the irradiation tolerance of this organism in an infant formula
> and, for that matter, in just about anything. It certainly is an area
> that could be looked at. But, traditionally, as you decrease the water
> activity of a product its resistence to radiation goes up
> dramatically. Because the way that you kill organisms by
> irradiation\--there\'s two; there\'s direct and indirect effects. The
> indirect effects are due to the formation of free radicals because of,
> actually, the irradiation of the water. And if you eliminate the
> water, you eliminate all of the secondary effects, which is the
> greatest one for these organisms, and you go back to single hit
> targets on DNA. And it gets to be much more difficult.
>
> And I think if I remember\--your Chair knows a lot about that.
>
> \[Laughter.\]
>
> DR. BUSTA: Don, are you just holding the phone?
>
> \[Laughter.\]
>
> Dr. Kuzminski.
>
> DR. KUZMINSKI: Thank you. Larry Kuzminski. Just a brief question to
> Dr. Nazarowec-White.
>
> You mentioned in your introductory comments to your presentation that
> Health Canada was considering a monitoring program. Can you share any
> more details about that, please?
>
> DR. NAZAROWEC-WHITE: I guess we\'ve talked about it for the past
> couple of years, and it\'s the Canadian Food Inspection Agency. At
> this point in time, we are set up very similar to what you have here
> in the U.S. And we do not have any infant formula companies producing
> powdered formula at this point in time. But monitoring the, you know,
> prevalence of E. sakazakii on the formula that we do import, for
> instance, is something that they are considering doing, but looking at
> other programs that right now have a higher priority.
>
> So I think that depending on how the meetings go, and what we learn,
> as far as that importance, we\'ll see what happens with next year\'s
> programming.
>
> DR. BUSTA: Dr. Beuchat.
>
> DR. BEUCHAT: Beuchat. This question is directed to Maria.
>
> You made a comment, almost in passing, about the observation of some
> of your strains clumping or sedimenting. There\'s some evidence to
> make us think that some enterics that are capable of producing
> capsules\--exopolysaccharide-are more heat resistant, are protected
> from the heat assault, are perhaps more resistant to harsh
> environments\--as simple as acid conditions that would be occurring if
> the cells were taken into a body. And perhaps also virulence.
>
> Do you have any indication of those strains that you did observe that
> did have this trait of sedimenting, of being in the category\--or
> among those strains that you\'ve reported on in your paper on
> infectivity and pathogenicity?
>
> DR. NAZAROWEC-WHITE: We found that all of the strains actually
> produced this sediment. And there were only the infectivity\--you know
> was not that\--you know, the strains that cause infectivity also
> produce sediment. But strains that did not cause infectivity also
> produced sediment. So we don\'t know at this point in time. Further
> research, I guess, to look at how many other strains that are in
> various culture collections\--what\'s happening with them, what
> they\'re doing.
>
> DR. BEUCHAT: And, along that line, do we know anything about biofilm
> formation by E. sakazakii? And also, these strains, if you transfer ad
> infinitum, do they lose this capability? This is something we need to
> be aware of if research is conducted in this area.
>
> We\'ve observed some things along that line with listeria.
>
> DR. NAZAROWEC-WHITE: We haven\'t done anything. And, at this point,
> I\'m not sure what the plans are for the work with this organism.
>
> DR. BUSTA: Dr. Heubi:
>
> DR. HEUBI: Heubi.
>
> I had a side conversation with Dr. Buchanan during the break, and I
> actually wanted to raise this for the group as a whole, because I was
> a little confused about this myself.
>
> According to the proposed standards of the FDA for microbiological
> standards, it doesn\'t include any comment about E. sakazakii. And are
> there new standards that are not printed here that specifically relate
> to E. sakazakii? And can you specifically comment on that for the
> group?
>
> DR. BUCHANAN: Yes, there seems to be a little confusion about
> microbiological standards, or the way we work.
>
> For the standards that you\'ve been talking about\--the CACFH,
> etcetera, those are largely good manufacturing, or good hygienic
> standards; this is what we would expect of an industry that\'s
> producing a product under, you know, what would be good conditions.
>
> Our standard for a ready-to-eat food for a pathogen is the absence,
> usually specified by some sampling protocol, some standardized method.
> But here, sakazakii was considered\--this was considered a
> ready-to-eat food. Sakazakii was considered a pathogen, and the level
> of sensitivity of the test determined whether or not we were finding
> it or not.
>
> This is basically the same standard that we have for, essentially, all
> ready-to-eat food: absence based on the detection using a certain
> method.
>
> Does that help clarify?
>
> DR. BUSTA: Okay. Chris.
>
> DR. TAYLOR: Thank you.
>
> Just to add a little bit to what Dr. Buchanan has said, in the 1996
> proposal, there was an effort to update some of the microbial
> standards and reopening the comment period on this rule in order to
> make it a final rule, there will be the opportunity again to talk
> about specific microbial standards for infant formula specifically.
>
> I just wanted to add that.
>
> DR. BUSTA: Thank you.
>
> Dr. Briley.
>
> DR. BRILEY: Margaret Briley. I wanted to clear up something.
>
> It seems that this pathogen is suspected\--
>
> DR. BUSTA: Dr. Briley, could you speak louder? It\'s on, it\'s on, but
> you just have to speak a little louder.
>
> DR. BRILEY: This pathogen, from what I understand\--maybe you need to
> correct me\--is suspected to be in the equipment, possibly, in the
> manufacturing stage. Did I understand that to be a possibility?
>
> Can you make some comment about any improvement in the cleaning of the
> equipment, or in the process of moving it through the equipment that
> industry might take that would allow this to be corrected?
>
> I don\'t know enough about what\'s inside the big funnel. I can see a
> little man with a bonnet and a robe and covered shoes climbing up and
> scrubbing the walls. But I just would like to know if there\'s some
> comment that you could give, based on your observations?
>
> DR. ZINK: I think maybe some from industry could comment on it better
> than I could. In my capacity at FDA, you know\--that was another life,
> a prior life for me. But, you know, you can always design something a
> little better, or clean something a little better than you have in the
> past. Things come along, and we\'re required to raise the bar. I mean,
> were it not for the Titanic, would we have life preservers on cruise
> ships.
>
> I\'m sure that the discovery of this organism and its significance has
> caused a reexamination of cleaning methods and procedures. I think the
> question becomes to what extent, by procedures and equipment-tweaking
> alone can you get to some other level, and what is that other level.
> And that I don\'t know.
>
> DR. BRILEY: Hopefully, zero tolerance maybe.
>
> DR. BUSTA: Dr. Thureen? Okay, Dr. Tompkin.
>
> DR. TOMPKIN: I sensed a perception that the frequency of cleaning
> could be an issue with these facilities. And I\'d like to have some
> comment, probably from Don, on this.
>
> But, in operating a dry operation it\'s better to run it, I think, as
> long as you can. And actually, from my own experience, I\'ve seen
> situations where your problems occur as a result of cleaning, and that
> this non-random distribution of certain bacteria can occur in the
> first product through the system. So sampling the first product
> through the system may, in fact, pick up a contamination that may have
> occurred in the process of cleaning, or while the equipment was
> sitting and still wet, allowing for some growth.
>
> So, in terms of recommendations or strategies, one that the group may
> come up with is clean more frequently, but I don\'t feel comfortable
> with that, and I would like to know what your view is.
>
> DR. ZINK: I think most manufacturers would agree with you, that
> they\'d rather not break and clean very often; that if they could run
> longer and drier, they would. The fact is that if you\'ve got one
> spray dryer, and you make four different kinds of formula, you know,
> and you have to do these changeovers, that will dictate some need to
> clean.
>
> Also, these formulas have a fair bit of carbohydrate or sugar in them,
> and that can tend to make them form a cake or adhere to the walls of
> the spray dryer. There\'s other operational things that you have to go
> in there, such as, maybe, the accumulation of some small number\--you
> know, burnt particles and things like that might dictate a need to get
> in and clean.
>
> But for the spray dryers, I seem to remember that somewhere between
> once every five to 12 days seems to be the usual frequency at which
> they\'re cleaned. And I think cleaning more often\--I agree with you,
> it might cause more trouble, you know, by the additional water it puts
> in the system.
>
> DR. BUCHANAN: Also, I\'d like to also respond or expand your question
> out a little bit, because I have some concern about misinterpreting
> what is meant by \"low levels of contamination,\" versus an
> nonhomogeneous distribution of a sample. And while I have not seen a
> rigorous evaluation\--I haven\'t seen a chi-square test yet\--on
> looking across a large number of cans out of a single lot that\'s
> known to be contaminated, I do have to reflect that we have examined
> numerous cans from several of the recalled lots. And I think we\'re
> more in an issue of the level of contamination, at least with those
> lots, than we are with \"It\'s in the first three cans and it\'s not
> in the next 400 cans, and then it\'s in the next three.\" Because we
> have found it.
>
> You also have to keep in perspective, when we\'re talking about a
> classic one-pound can, and you\'re down in an MPN of .3 per 100 grams,
> that basically means that you\'re dealing with one-and-a-half in every
> can. No, it\'s not even that. It\'s one per every three cans. So
> you\'re already in the type of distribution that you\'re\--it\'s a
> rare event. You\'re not going to find it in every can. But that
> doesn\'t mean that it\'s not homogeneously distributed within the lot.
>
> DR. BUSTA: Dr. Lee.
>
> DR. LEE: Perhaps a generic question\--as I understand it, E. sak
> wouldn\'t be a problem if we are rehydrating with boiling water. And
> certainly, one can formulate, in anticipation of that, by just putting
> in more vitamin C to offset any potential losses.
>
> So I\'d like to know what the contraindications are to formulating
> this product with boiling water?
>
> Well, as far as I can tell, there are two contraindications\--or two
> factors that haven\'t been taken into account. And first I want to
> emphasize\--you don\'t have to use boiling water. That\'s 30 degrees
> too high. It really only has to be at 70. It needs to be hot water.
>
> Second, is we still don\'t have sufficient data on clumping. And while
> we don\'t think it is a large problem, it\'s material that has to get
> through enteral feeding tubes. And so clumping is something that
> certainly needs to be examined.
>
> And then\--and probably the most significant thing\--when you do heat,
> the formula retains its temperature for quite a long time. I showed
> you a cooling curve of the first ten minutes following the heat. And
> there is\--realistically, you have to be careful. You can\'t feed 100
> degree formula to an infant. They\'ll get burned.
>
> So it has to be balanced by the fact that if you use hot water to
> rehydrate the formula, there does have to be adequate cooling.
>
> DR. LEE: Just to follow up on clumping\--if clumping does occur, is
> this formula normally turbid? Is it clear if it\'s fully dissolved?
> No?
>
> DR. ZINK: No. I mean, it looks kind of like, you know, milk. And you
> can get coagulation of protein that\'s fairly significant there
> without it\'s being obvious. You know, I can see it going into an
> enteral feeding tube and clogging that tube up, and outwardly not
> looking like it had a precipitate or clumps in it.
>
> DR. LEE: But it will pass through cheesecloth or some kind of filter,
> if that\'s needed?
>
> DR. ZINK: Well, I think it would\--you know, when it clumps it clumps.
> You would get some of it retained on cheesecloth, probably.
>
> DR. BUSTA: Dr. Fischer.
>
> DR. FISCHER: Fischer.
>
> I was surprised to hear that\--no, I\'m not surprised to hear that
> powdered\--formulation of powdered milk, or the production of powdered
> milk was very similar to the production of infant formula.
>
> If that\'s the case, have you looked in powdered milk for the
> organism? And if it is there\--and if it isn\'t there\--mightn\'t this
> not be clue as to where it might be coming from, in terms of the raw
> ingredients involved?
>
> First of al, have you looked in DR. TARR: ?
>
> DR. ZINK: We haven\'t specifically run a survey of powdered milk. And
> having just recently joined the agency from industry, let me say that
> in this job your capacity to cause mayhem by your actions far exceeds
> that of what it was when I was in industry.
>
> \[Laughter.\]
>
> You know. No, we haven\'t gone out and looked at, you know, producers
> of dry milk for this organism. And while that might seem like a good
> idea at the moment, I think I would want to think about that long and
> hard. You know, what am I going to do when I find it, and what does
> that mean?
>
> I really would expect, if I did do that, to find it.
>
> DR. BURR: Actually, from the 1984, the Postupa and Aldova paper, they
> have four strains coming from powdered milk.
>
> DR. BUCHANAN: Most normal analysis of powdered milk for gram-negative
> Enterobacteriaceae is to determine whether or not there\'s E. coli in
> it. And so you have E. coli, and then you have everything else, and
> you forget about everything else. No one tries to differentiate all
> the\--enterobacter serratia, klebsiella that may show up as fecal
> coliforms. They\'re worried primarily about E. coli, as an indicator
> organism.
>
> So I\'m sure that it\'s been isolated from powdered milk. I just
> don\'t know of anyone that takes the time to try and differentiate all
> those species.
>
> DR. FISCHER: Yes, it seems to me that\'s helpful\--that information is
> helpful, because then it would tell you it\'s not some substance
> you\'ve added, particularly after the drying process, like the
> vitamins, that is the source of the organism.
>
> I\'m just surprised that the source hasn\'t been stumbled on as yet.
> And it seems to me that is a key issue, that would help everybody out,
> if the source could be found.
>
> DR. BUCHANAN: Can I answer this? And I hesitate to do it because this
> is really a question that needs to be addressed to the industry. But I
> know from past experience of looking for the sources of listeria\--and
> there\'s been a lot of work on that, and people have literally torn
> their plants apart to find out where a small micro-colony of listeria
> is harbored and causing problems.
>
> These plants are really complex places, and all you need is one little
> small niche somewhere that\'s feeding into it. So, it\'s not as simple
> a job as one might think to track down where in a plant environment
> you actually have that organism residing.
>
> DR. BUSTA: Could I take the chair\'s prerogative and expand on that
> just a second?
>
> Did I hear correctly, in two presentations, that it\'s not in the
> environment?
>
> DR. ZINK: Oh no, it\'s in the environment.
>
> DR. BUSTA: Marie?
>
> DR. NAZAROWEC-WHITE: \[Off mike.\].
>
> DR. BUSTA: Marie,.you said in that list-
>
> DR. NAZAROWEC-WHITE: Right.
>
> DR. BUSTA: \--that this was not the source.
>
> DR. NAZAROWEC-WHITE: Correct. That\'s right.
>
> DR. BUSTA: And that as quite an extensive list of all kinds of
> environment.
>
> DR. NAZAROWEC-WHITE: That\'s correct. And it was not found in that
> particular\--in those particular areas. But we have no idea whether
> they took five samples, or whether they actually\--you know, there has
> been no systematic scientific approach to look at that environment.
>
> DR. BUSTA: So chances are where Enterobacteriaceae are, it is also.
>
> DR. NAZAROWEC-WHITE: Well, I would think so.
>
> DR. ZINK: I think this is a question for industry. I believe, as Bob
> alluded to, is what went on with the meats industry and listeria, I
> think this industry has disassembled their plants and done extensive
> testing and has, you know, probably isolated the organism from many
> places in their plant. And I suspect that, you know, while as an
> agency we\'re not in possession of this kind of data and information,
> I suspect that it\'s known, the types of plant environment where the
> organism can be found.
>
> DR. BUSTA: Two more questions, then.
>
> Dr. Acholonu.
>
> DR. ACHOLONU: Alex Acholonu.
>
> In one of the background materials given to us, the title is
> \"Powdered Infant Formula: An Overview of Manufacturing Processes.\"
> It has a statement which I\'m going to read\--this is for Dr. Zink.
>
> \"Typically, finished product is held until it undergoes a final check
> for conformance to specifications\--\"\--and the next statement is
> \"\--including testing for microbiological contaminants.\"
>
> My question is what method would you use to check for microbiological
> contaminants in a finished product?
>
> DR. ZINK: Well, what manufacturers do right now\--what you want to do
> is you don\'t want to produce value-added garbage. Okay. So if you can
> run your test for quality conformance before you add value by putting
> it in a can and labeling it and casing it, only to find out you have
> to throw it out, if they can they\'ll get these tests done before they
> put it in the can. And they\'re typically testing to two kinds of
> standards. Each manufacturers has its own internal standards;
> typically much more stringent than what a regulator would require. So
> they\'re going to test for conformance to their own internal quality
> assurance standards. And that could include things like aerobic plate
> count, salmonella, listeria, coliforms, E. coli. They take the lead,
> it\'s been my experience, from what regulators require. And they\'ll
> be also testing for, you know, vitamin content, homogeneity\--all
> those sorts of things.
>
> Right now, I think most manufacturers test for the kinds of things
> that a regulator would, and they apply the same or more stringent
> criteria.
>
> DR. ACHOLONU: My understanding of a finished product is one that has
> been prepared and may be canned and ready to go. Do you understand\--
>
> DR. ZINK: Well, you get into semantics. Right. From a standpoint of
> microorganisms, if you have that powder sitting in a tote or a big
> bag, it\'s true that there is some potential for further contamination
> by the time it goes through a can-filling line and into the can.
>
> I\'m familiar with several manufacturers that will test product while
> it\'s in bulk. Then they\'ll go ahead and put it in the cans, and then
> they\'ll test it again.
>
> DR. ACHOLONU: They open the can and test it again?
>
> DR. ZINK: Yes.
>
> DR. ACHOLONU: Doesn\'t that give\--make it get contaminated\--
>
> DR. ZINK: Well, they open the can\--usually, they\'ll have a
> biological cabinet to open the can in. It\'s very common in these
> plants to see laminar flow hoods, and\--you know, they\'re able to
> open those cans in a manner that precludes recontamination.
>
> DR. BUSTA: Thank you all very much for an extended question and answer
> session. I appreciate this.
>
> And now we will go on to the public comment. We have three presenters
> from the International Formula Council.
>
> **Public Comment**
>
> DR. BUSTA: The first one that I have on the list is Dr. John
> Vanderhoff. He\'s vice president, Global Medical Affairs, Mead Johnson
> Nutritionals, a Bristol-Meyers Squibb Company, speaking on Overview
> and Risk Characterization.
>
> \[Pause.\]
>
> Will there be a copy of the slides available, that you are using?
>
> DR. VANDERHOFF: If you\'d like. I don\'t know what we\'ve decided to
> do about that. I\'m speaking for the entire group, so I\'ll have to
> defer that to you, whether we want to leave these slides here or not.
> But they\'re\--
>
> VOICE: \[Off mike.\]
>
> DR. BUSTA: All right. Thank you.
>
> Proceed.
>
> DR. VANDERHOFF: Okay. I think we\'re waiting on a little warmth from
> the projector here.
>
> I very much appreciate the opportunity to kick this off and try to
> give you what I would like to call an industry overview of
> Enterobacter sakazakii, and how we see it. And I think it\'s probably
> going to be a rehash of all the different things that we\'ve hear
> today. Hopefully we\'ll be up here in a jiffy.
>
> I think, as I recall, the first thing that we wanted to do was just to
> cover what is Enterobacter sakazakii. And we know that it\'s a
> gram-negative rod. We know that it is\--whoops. Now do we have to wait
> for it to warm up again?
>
> Okay. Tom, do you have the\--that way, if it does come up we won\'t be
> too far off track here.
>
> But this organism we\'ve all said is an opportunist, which it\'s been
> probably in the environment for some time, and has a predilection to
> cause central nervous system infections. I think that\'s important.
> Because of that, these are not infections that we usually miss when we
> treat children with infectious diseases. And so I guess the question
> is: are we dealing with the tip of an iceberg? And I think probably
> not. These things are the sort of things that one usually sees and
> diagnoses.
>
> The epidemiology of this organism: we know that it\'s found in very
> low concentrations in powdered infant formulas. This seems to be a
> worldwide phenomenon. It\'s also been shown to be found in hospital
> environments. I know I reviewed one paper where numerous swabs had
> been taken around hospital rooms, and several of the swabs
> demonstrated the presence of this organism. It\'s been found in
> numerous food products. It\'s even been found in the gastrointestinal
> tract of a number of fruit flies.
>
> So it\'s probably\--we don\'t know how common this thing is in the
> environment, but we know that it\'s there. And we also know that it\'s
> been\--that it can occasionally be found in the stools of infants.
>
> Yeah\--go ahead and bring us up to\--next one.
>
> \[Slide\]
>
> And we know that it\'s been cultured out of the stools of some infants
> who apparently had no symptoms at all, as part of the epidemiological
> evaluation of outbreaks.
>
> Slide, please.
>
> \[Slide.\]
>
> Who\'s at risk for the infection? Well, the primary risk appears to be
> in premature infants, especially small premies that are being treated
> in neonatal intensive care units. And these children are, of course,
> with some degree of impairment of immunological function because of
> their developmental age. We also know that occasional term infants
> have been reported to have become infected with this agent, but if you
> look at the case reports, a fairly high percentage of these\--and
> there were very few of these\--have some underlying condition, either
> immune or barrier function, or some congenital anomalies or
> chromosomal anomalies and so forth.
>
> And we also know that immunocompromised children and immunocompromised
> adults appear to be able to be infected with this organism. So it
> preys on, basically, immunocompromised people, like most opportunistic
> organisms.
>
> Slide, please.
>
> \[Slide.\]
>
> What does it do? Primarily it causes meningitis, ventriculitis, and
> maybe sepsis. We know it has a predilection for the central nervous
> system. And we know that it can be present in the gastrointestinal
> tract in asymptomatic children.
>
> And as a pediatric gastroenterologist, I would discourage you from
> drawing too close of a connection between the reports of necrotizing
> enterocolitis and this organism. Necrotizing enterocolitis seems to be
> a condition that results when you aggressively feed a premature baby,
> causing some degree of ischemia, and the bowel breaks down, and you
> get an invasion with whatever organism happens to be there. And
> there\'s a long list of these. And everyone\'s thought they found the
> cause of NEC, and that\'s never panned out before, and it probably
> won\'t with this organism either. It can certainly cause a problem if
> it\'s there and you have an ischemic bowel, but considering it an
> etiologic agent for necrotizing enterocolitis is probably not correct.
>
> Slide, please.
>
> \[Slide.\]
>
> What\'s the risk involved in this particular infection? There\'s 50 r
> 60 cases reported worldwide. And this is what we\'re basing our
> information on. Since we\'re dealing with case reports, there are
> probably more cases out there than we know about, but it still
> suggests that it\'s a relatively rare condition. Most of these occur
> in premies. It appears to be an extremely rare condition in health
> term infants, despite the fact that there are billions of feedings of
> term infants with powdered infant formula, we seem to have only a
> handful of cases occurring in this age group.
>
> Next slide.
>
> \[Slide.\]
>
> Well, then why is it so uncommon? The organism itself appears to be a
> minimally pathogenic opportunist. The infective dose is probably high.
> It\'s probably much higher than we normally see in fresh reconstituted
> infant formulas. In fact, if you look at the various case reports,
> almost all of them suggest that there has been some mishandling\--I
> would call it mishandling\--of the infant formula from the standpoint
> of hang times, or preparation or use, that has let the formula sit
> around and proliferate, so that there\'s an increase over the
> background number that\'s quite significant in a number of these cases
> in which infection has occurred.
>
> And most of these have also included something like environmental
> contamination or breakdowns, or contamination of blenders and so
> forth.
>
> Slide, please.
>
> \[Slide.\]
>
> What, then, could the infant formula industry do to deal with this
> condition? Well, I think one of the things we can certainly do is work
> with the FDA, the CDC and other organizations to do everything we can
> to help educate users and health care providers. And this would be to
> do things like minimize hang time with enteral feedings, to minimize
> the use of refrigeration to 24 hours, to use liquid formulas in
> hospital setting whenever possible\--especially in neonatal intensive
> care units.
>
> We\'ve hear a little bit about boiling, and the risks associated with
> that. And we an industry have sort of discouraged that because we\'re
> very concerned about mishaps occurring when people might give a
> formula that\'s too hot to a baby and cause burning in the esophagus
> or even an injury to the preparer of the formula. And there\'s also
> now been case reports\--at least one\--of vitamin C deficiency from a
> mishandled infant formula.
>
> So these things can happen and I think probably the major efforts we
> can utilize are on this slide. And then I think we have to do
> everything we possibly can to maximize the capabilities to reduce the
> levels to as low as possible\--which our next speaker will discuss.
>
> Slide, please.
>
> \[Slide.\]
>
> So, in summary, I think Enterobacter sakazakii poses a limited
> threat\--again, because it\'s a rare condition\--to preterm infants
> and other immunocompromised individuals in a hospital setting. And
> because it\'s occasionally spread through mishandled reconstituted
> powdered infant formula, the infant formula industry wants to take an
> active role in supporting the FDA and the CDC efforts to educate
> consumers and health care professionals, and to work on new policies
> and procedures to minimize the exposure of these high risk patients to
> this organism.
>
> So, now I think Les Smoot is going to tell us a little bit more about
> the manufacturing side.
>
> DR. BUSTA: Dr. Les Smoot is Director of Food Safety Quality Management
> at Nestle USA, Nutrition Division.
>
> DR. SMOOT: Good afternoon. Is this on? Can you hear me all right?
>
> Okay. I\'m going to spend just a few moments to, hopefully, follow on
> and answer a fair amount of the questions\--or at least provide some
> further insight on some of the questions that have been asked most of
> the afternoon.
>
> Next slide, please.
>
> \[Slide.\]
>
> First of all, some comments on what is not known about this organism.
> We know genetic diversity is still something we\'re debating and
> learning as we go along. It would be important for us to understand
> the incidence and behavior of the organism throughout the entire food
> chain. Still we\'re gripping, trying to get a hold of where this
> particular organism\--the origin of this; how does it get into the
> food chain? So the specific environmental niches is something that we
> don\'t have a real clear handle on at this point. And then, finally,
> optimum methods for control, detection and recovery. I think we\'re
> still in the very early stages of the science involved with,
> particularly, the detection and recovery phase. And, as an industry,
> we\'re also continuous ly improving the methods for control.
>
> Next slide.
>
> \[Slide.\]
>
> What is known about the organism\--and this is very similar to what
> you\'ve seen for most of the day\--just recently recognized in 1980 as
> Enterobacter sakazakii. As we saw this afternoon, it\'s not unusually
> heat resistant. I think it\'s very fair to say it survives well in
> warm, dry environments. We\'ll talk a little bit more about that in a
> minute.
>
> We also know, in industry, based on our internal studies, that it is
> not overly resistant to typical food sanitizers; and also knowledge
> that it does appear to have a narrower host range of salmonella, one
> of the common food pathogens that we worked against in this particular
> food product category.
>
> What is known about ES ecology? This is getting to some of the
> questions that have bene asked around the table, relative to where we
> find this in our food supply. A recent report by Cordier, et al., has
> shown we\'ve found it in skim milk powder, a lactose, starch, powdered
> banana flakes and powdered orange flakes\--all of these being typical
> ingredients for infant cereals and infant formulas.
>
> Now, Castano, et al., found it in raw pork for a dry-cure bacon. This
> was a Spanish study. Soriano, et al.\--I think this was a Spanish
> study, as well\--found it in raw lettuce.
>
> From and environmental standpoint, Kandhai, et al., in 2003\--this is
> a paper that\'s being submitted to [Lancet]{.underline} and should be
> published this year\--out of the University of Wagenen in the
> Netherlands, they found this organism isolated from the
> environment\--well, there were actually five milk powder factories,
> one chocolate factory, one infant cereal factory, a potato flour
> factory, pasta factory and numerous households. So we\'re starting to
> see that this is not necessarily an unusual organism to be found in
> the environment where food preparation or food ingredients might be
> prepared.
>
> As mentioned earlier, there have been reports of its isolation from
> insects\--this one on a Japanese dairy farm, another one from the gut
> of the Mexican fruit fly, by Kuzina, et al., in 2001. Two isolations
> have been reported in hospital settings; one by Masaki, et al., in
> 2001 in Japanese geriatric hospital environment, and then most
> recently the Block, et a., a pediatric hospital kitchen in Israel. So
> this gives us a little bit of the flavor of the type of environmental
> ecology, as well as food ingredient and food material ecology,
> hopefully answering some of the questions that have been raised this
> afternoon relative to the industrial process.
>
> What I, hopefully, have here for you is a representation of the
> partial steps within a food supply chain for the preparation of infant
> formula. This is kind of a general step-wise progression. What we\'re
> looking at here is potential sources for E. sakazakii, and also roots
> of recontamination of the product. And, as mentioned earlier, raw
> materials\--there needs to be a better understanding of the raw that
> raw materials play as a potential vector, bringing the organism into
> the environment of the factory. I believe Don Zink had mentioned this
> earlier this afternoon. Also, we have been just discussing, and
> throughout the day, the concept of handling practices during
> reconstitution and consumption.
>
> Ut we want to spend a little bit of item, obviously, where there\'s
> some interest is, what type of intervention strategies are applied in
> the manufacturing process, when we look at these various steps
> throughout the process as were detailed by Don Zink in the wet and dry
> blending operations. What types of activities is industry currently
> applying? What kind of good hygienic practices are we currently using
> to reduce the incidence of this particular organism from the factory
> environment.
>
> Next slide.
>
> \[Slide.\]
>
> Well, it\'s not rocket science, really, but it is something that we
> have to do and continue to do, and we are currently doing. Key to this
> is the raw material supply. And, as mentioned earlier, we adhere very
> much to the good manufacturing practices and the HACCP principles. You
> see here the term \"prerequisite programs.\" These are programs that
> are conducted and embraced within the manufacturing arena that are
> prerequisite for a successful hazard analysis, critical control point
> system to be in place, assuring the quality and, most importantly, the
> safety of the food.
>
> So, as a prerequisite program relative to HACCP on raw materials,
> it\'s very important that you have a very rigorous supplier quality
> assurance program; auditing, working with the supplier, improving
> their industrial process to ensure a very clean supply of raw
> materials. Secondly, though you want to verify how effective they are
> in their industrial process. So it\'s very typical a very rigorous raw
> material monitoring program.
>
> All of this supports the use of the HACCP\--the hazard analysis
> critical control point\--principles, particularly for raw materials,
> hazard analysis on the raw material. Is E. sakazakii, a significant
> hazard, likely to occur in the various raw materials that we would use
> within our manufacturing base? And I believe Don Zink had answered
> earlier that from the perspective of the FDA, they have evidence that
> all manufacturers are adhering to these principles in detail.
>
> Secondly, the processing environment. This seems to be a little bit
> of\--there\'s kind of an open question, in terms of understanding our
> ability to control recontamination of the product stream. Prerequisite
> programs here that are very important; obviously they\'re good
> manufacturing practices; the standard sanitation operating
> practices\--this is your cleaning practices that were questioned
> earlier. The microbial surveillance programs. These are the programs
> that are in place to verify the efficacy of those cleaning programs;
> in other words, are we using hygienic indicators, whether it be
> Enterobacteriaceae, whether it be coliforms, so on and so forth.
>
> And very critical to the processing environment, and I think this goes
> a ways towards answering some of the questions\--this \"hygienic
> zoning\" concept. This is something that\'s embraced in great detail
> in many, if not all of the infant formula manufacturers, where we have
> very strict separation of incoming raw material to finished product
> handling and filling; also very strict separation between the dry
> aspects of the factory operations as well as the wet processing areas,
> because we know the Enterobacteriaceae, as well as the salmonella and
> listeria all need water to grow. So, as mentioned earlier, the drier
> we can keep our environments, in and where we have finished product
> handling, it\'s most important to minimize potential regrowth and then
> recontamination of the product stream.
>
> So these are the cornerstones that we are continuously using to combat
> the potential for recontamination. In the process\--and additionally a
> prerequisite program very critical to the ability to exclude organisms
> like E. sakazakii out of the process\--is the hygienic design of
> equipment. And I believe this is where, over the recent years, there
> has been significant improvement. And this is one of the areas that
> the manufacturers themselves, in terms of the design\--as Don was
> showing you the various diagrams, the various pictures of the spray
> dryers, the drying tunnels and so on and so forth. This is where
> technology has started to make a difference in terms of the hygienic
> design.
>
> In terms of the HACCP aspect on the process, as mentioned very
> clearly, the application of thermal related critical control points.
> And, finally, how effective are these intervention strategies?
> Finished product testing is not an intervention strategy. It\'s
> primarily a HACCP verification program, to assure that these
> particular activities and raw materials, process environment control
> and process control, have been effective in assuring the highest
> quality and safety in the finished product. So the finished product
> monitoring that we\'ve been discussing about, in terms of the
> surveillance data, is really there to verify the effectiveness of the
> HACCP program and these prerequisite programs. And as mentioned in the
> White paper on manufacturing, finished product monitoring is used
> before release of products.
>
> Everyone has been talking quite at length today about the Van Acker
> 2001 report. This is a set of unpublished data. This is data from that
> particular factory in the Netherlands. Shortly after the even occurred
> in the summer of 1998, there\'s mentioned in the CODEX risk profile
> and numerous speakers about the enhanced manufacturing practices were
> put in place at this particular facility to assure improved safety and
> quality of the product.
>
> Here we have four consecutive years of monitoring the finished
> product. These were 100 gram samples coming from that particular
> facility. This was a targeted study, above and beyond the ongoing
> monitoring criteria that were utilized\--cited in the publications.
> But we have here the percent of these samples that were positive for
> EB, and then the percent of those samples that were positive for E.
> sakazakii. And through the years, through continuous improvement in
> areas, particularly in hygienic design of the process, as well as
> environmental control, really learning how to manage the use of water
> in these dry processing environments, through a relatively level
> number of samples over those four years we can see a dramatic decrease
> in the percentage of EB-positive finished product and,
> correspondingly, see a dramatic\--or a reasonably significant decrease
> in the presence of E. sakazakii in those finished products.
>
> So this coming from a well-celebrated problematic factory can show how
> enhanced environmental and process controls can make a difference, can
> drive this number very low, but not necessarily to the point of
> eradication.
>
> Next slide.
>
> \[Slide.\]
>
> So, the frequency in finished products has been reduced through
> environmental and process control. And historically we know that CODEX
> standards have been used to assure the safety of infant formula for
> the intended population. And the industry is proposing, at this point,
> a more stringent standard than the CODEX standard that\'s currently
> based on coliforms. Now we\'ve seen those standards cited earlier. I
> think they were shown in Don Zink\'s presentation.
>
> So this next slide is the industry proposed sampling and testing
> program for powdered infant formula. You\'ll not that we have
> expanded\--as has been mentioned in some discussion\--this particular
> standard to the entire Enterobacteriaceae family. It\'s a sampling
> program on per lot basis, where the sample size would be 5 grams\--the
> analytical unit would be 5 grams. The number of units tested would be
> five, and an acceptable marginal quality would equal 1, where if there
> were no samples\--basically, what we have here is a qualitative test,
> where you have five 5-gram samples; a total of 25 grams being tested.
> If all samples are negative, then you would accept the lot
> unconditionally. If there were one sample that were to show positive,
> then you would only accept the lot if that particular positive sample
> was confirmed to be negative for fecal coliform. If it was found to be
> positive for fecal coliform presence, the lot would be rejected. And
> then if there was a chance where there were two of the lots found
> positive for Enterobacteriaceae, then there would be a conduct of an
> intensive investigation looking for root cause, as well as
> confirmation of presence or absence of fecal coliforms, and this could
> lead to either acceptance or rejection of the finished product lot.
>
> Next slide.
>
> \[Slide.\]
>
> So, in summary, really, the industry\'s ability to control this
> organism in finished product really comes to the implementation of a
> phased effort through raw material management, and working with the
> suppliers, continually improving our ability to control the organism
> in the environment; going after, looking at niches in the environment.
> We do know that this organism has the ability to survive in dry, warm
> environments, and in that particular study, performed on the
> Netherlands factory, we did see a higher prevalence, typically at the
> bottom of the spray dryer, in the warmest\--one of the warmer parts of
> the building.
>
> Process control\--really, the key there is the hygienic design for us,
> in terms of improving how the equipment is designed to minimize the
> possibility for micro-niches to be development; and then, ultimately,
> then finished product verification testing.
>
> Thank you.
>
> DR. BUSTA: Thank you, Les.
>
> And the third presenter is Dr. Russell Merritt, Director Medical
> Affairs, Nutritionals, Ross Products Division of Abbot Laboratories,
> who will be speaking on industry\'s response to FDA charge.
>
> DR. MERRITT: Thank you, Dr. Busta, Dr. Heubi. And Thank you for the
> time to present hereto the committee today.
>
> I\'m speaking to you from the perspective of a clinician who worked in
> nutritional support, including neonatal support, for many years, and
> more recently have been very involved in industry and our concerns
> with safety.
>
> You have heard this afternoon a clinical summary of the issues from
> the industry perspective, and also a summary of the manufacturing
> issues and some of the steps that we are proposing to move forward
> with. It\'s my goal to bring this industry perspective into the
> context of the specific charges that FDA gave to this committee. So I
> will comment specifically on the FDA questions, and this commentary is
> available to you as well in written form.
>
> Next slide, please.
>
> \[Slide.\]
>
> The first charge was: given available information on E. sak and
> powdered infant formula, is there a risk? Are there sub-populations at
> risk? And what about immune status, general health, etcetera, in
> regards to the susceptibility to this infection.
>
> Next slide, please.
>
> \[Slide.\]
>
> For preterm and immunocompromised infants, we\'ve heard considerable
> material today, and it indicates that there is a rare risk to such
> infant. Somewhere in the neighborhood of 75 percent of the identified
> cases have a birth weight less than 2,500 grams, or the infants were
> premature. There\'s some suggestion\--particularly in those cases
> where there were issues with formula preparation, storage and
> delivery, that there may be a dose effect. It appears that it requires
> more than simply the presence of an organism\--one organism in two or
> three cans of formula\--but rather, the presence, a susceptible host,
> and then presumably some defect in the environment, by which the
> formula gets from the can to the baby.
>
> The situation appears to be quite different in regards to healthy,
> term infants. The literature does not\--particularly in North
> America\--support the evidence of a clear association between
> consumption of a formula known to possess this organism, and the
> development of serious clinical infection. If any risk exists, it
> needs to be put in the context of the number of feedings of infant
> formula that are used per year, and the extraordinary rarity of this
> infection. If you think about it, a baby probably gets about 2,000
> feedings over the course of a year, and maybe close to half of those
> in the U.S. come from infant formula. Then crudely, that gives you
> something on the order of four billion feedings\--or two billion
> feedings a year of the four million babies who are born. And yet one
> rarely sees this organism. We heard earlier about the CDC screening
> program in 300 hospitals, where a case was identified, to my
> knowledge, not associated with infant formula.
>
> So part of the critical task of the committee is to differentiate the
> possible presence of E. sakazakii on occasion in infant formula from
> the risk of infection in infants.
>
> Next slide.
>
> \[Slide.\]
>
> If there is a risk, what intervention strategies can be used in infant
> formula manufacturing processes and plants?
>
> Well, you just heard from Dr. Smoot that there is an opportunity in
> almost any situation to enhance environmental monitoring and controls,
> using the HACCP principles. And you have just seen an excellent
> example of where not only E. sakazakii, but also enterobacter,
> presence in infant formula was substantially reduced by adherence to
> these principles.
>
> Industry is also proposing confirmatory final product testing for
> enterobacter. Again, this is a confirmation of the effectiveness of
> the environmental and the incoming ingredient controls that are placed
> on the manufacturing process, more than the method of controlling the
> product. The testing is not the method, it\'s the indication that
> those methods are working.
>
> Next slide.
>
> \[Slide.\]
>
> Are there other intervention strategies? And I think, clearly, we\'ve
> heard that there are. We can minimize the use of powder in at-risk
> infant populations to the extent possible. We\'ve heard from Dr.
> Thureen\'s experience a little bit about how difficult this would be
> to eliminate completely. And I believe Dr. Bhatia may address this
> issue as well in his comments.
>
> We can promote the published guidelines from the CDC, from the
> American Dietetic Association that emphasize appropriate hygienic
> practices within the hospital. And, again, picking up on Dr.
> Thureen\'s comment, I was impressed that perhaps if neonatologists
> understood more readily that powdered formula is, in fact, not
> sterile, then the motivation no their part to control the use of these
> products, both in terms of quantity and in terms of practice, might in
> fact represent an opportunity for us.
>
> The hot water reconstitution process has been described\--also some of
> its limitations, including possible incomplete bacteriocidal kill,
> vitamin C loss, protein denaturation and resultant clumping, and
> injury that may result to personnel preparing the formula, if we have
> boiling or hot water in the nursery environment.
>
> In addition, there is opportunity, I think, to limit hang time. And
> based on what we\'ve heard today, the four hours seems to make some
> sense, and more consistent labeling and directions for preparation and
> use is another opportunity.
>
> Next slide.
>
> \[Slide.\]
>
> Continuing the second charge: is it possible to identify lower levels
> of E. sakazakii in powdered formula and, in effect, assign allowable
> levels?
>
> Next slide.
>
> \[Slide.\]
>
> For premature or immunocompromised infants, the data are insufficient
> to really specify an allowable level, given that there\'s at least
> some degree of risk there. For healthy term infants a low, non-zero
> level can be established, based on the historical exposure data\--for
> example, 5 to 15 percent of infant formula, based on a number of
> surveys. The extraordinary rarity of this infection in term infants,
> and the data we have heard today about the numbers of organisms that
> appear to be required in neonatal animal inoculation studies.
>
> Next slide.
>
> \[Slide.\]
>
> What are the critical knowledge gaps and research opportunities?
>
> Next slide.
>
> \[Slide.\]
>
> Well, certainly we need to understand more about the circumstances
> that need to opportunistic pathogenicity; whether it\'s susceptibility
> factors in the host, or the virulence factors in the organism itself.
> We need to understand of the incidence and behavior of the organism in
> the food chain. I think Dr. Smoot\'s comments were helpful in giving
> us some direction in that regard. The probability and circumstances
> under which pathogenic levels\--that is, levels capable of causing
> infection in susceptible populations\--occur. And then, lastly, we
> need to focus attention to the methods that may potentially give us
> environmental control, whether that\'s in ingredients a the level of
> plant manufacture, or at the site of use.
>
> So, again, I thank you for the opportunity to provide these industry
> provisional answers to the questions at hand.
>
> DR. BUSTA: Thank you, Dr. Merritt.
>
> The fourth presentation this afternoon is by Dr. Jatinder Bhatia,
> Medical College of Georgia. The title is, \"Use of Powdered Additives
> in Neonatal NICUs.\"
>
> DR. BHATIA: Dr. Busta, Dr. Heubi, Ms. Latham, distinguished members
> and guests. Thank you for the opportunity to share my thoughts with
> you.
>
> I come from the perspective of being trained as a neonatologist and a
> nutritionist who\'s been practicing his trade for a while.
>
> Next slide, please.
>
> \[Slide.\]
>
> The needs have been highlighted because we truly don\'t have a gold
> standard for pre-term babies. And the gold standard the AP has put
> forth is that post-natal growth that approximates the in utero growth
> of a normal fetus. Well, the question that can be raised is: should
> you achieve maximal achievable gain without adverse metabolic and
> other costs?
>
> We know from a whole body of literature that growth of most of these
> infants remains lower that in utero, and that the nutrient intakes
> remain lower than the fetal counterpart during much of the in-hospital
> stay, and possibly much beyond.
>
> Now, what are the consequences? And this is has been known in the
> animal literature and the human literature for a long time.
> Under-nutrition, especially during these vulnerable periods of brain
> growth, can have permanent CNS development, cognition, behavior and
> somatic growth effects. And these effects may be present, even if
> there\'s sub-optimal growth, as shown in animal studies. For example,
> an animal doesn\'t grow, he may have these side effects, but if the
> animal grows suboptimally, he may still have the same effects.
> Therefore premature infants are at high risk.
>
> And I don\'t need to remind this body that the extremely low birth
> weight infant has additional needs for protein, calcium, sodium that
> far exceed those for the term infant. And these, perhaps, may not be
> completely met with the armamentarium that we have at present.
>
> All this information has ben reiterated by the NICHD-sponsored trials
> published recently, which has heightened the awareness of
> practitioners of nutrient intakes and growth curves. These were
> something that we knew, but we did not have. And I want to share with
> you the data on these three cohorts of very small premature
> babies\--24, 25, 26, 27 and 28, 29 weeks. In general, birth weight was
> regained by about two-and-a-half weeks, with the larger babies gaining
> them a little sooner. But the more important point is the next one. By
> 32 weeks post-conceptional age, growth curves of all cohorts fell
> below the 10^th^ percentile\--and I\'ll show that to you in the next
> slide graphically.
>
> The three cohorts that you have here\--the first one being the 24,
> 25\--the largest one\--all three of them, even if you look at 36 weeks
> post conceptional age, all fall below the 10^th^ percentile, and the
> smaller babies may not catch until almost the first year of life. This
> is despite the maximal feeding that we can do currently. So we are
> taking appropriate-for\--age babies, and now making them small for
> gestational babies by the time they leave the hospital.
>
> So, at 32 weeks the average weight of a 24, 25 infant was 50 grams
> below the 50^th^ percentile; 26 to 27, by 780, and the larger cohort
> by 715 grams. And the postulated reasons, at least to practicing
> neonatologists, is the expected post natal weight loss, and our
> inability to feed babies to the maximum potential, and therefore under
> nutrition.
>
> So what are the strategies that we use? Early initiation of parenteral
> nutrition, starting on day one or day two; early initiation of enteral
> feedings, also limited by fear of necrotizing
> enterocolitis\--although, as Dr. Vanderhoff has pointed out, and other
> people have pointed out, that the starting or not staring of enteral
> nutrition early has no association with it. There are other
> associations, which I\'ll come back to later; increasing nutrient
> density while maintaining relative fluid restriction, because we can
> only go so far as far as fluid volume goes, and therefore the need for
> supplements.
>
> What do we have available? Liquid concentrate, powdered
> formula\--either the premature or transitional formulas\--or single
> nutrients. Or we have MCT, vegetable or lipid emulsions.
>
> Here\'s an example of what you would do if you want to increase
> caloric density by either using a single constituent or a balanced
> powdered formula. For example, on the left hand slide, a 24 cal premie
> has 81 calories, and draw your attention to the completely far right
> hand corner, that if you add a proportionate amount of powder to the
> premature infant formula, you can achieve 98 calories per 100 ml; you
> can increase the protein intake and, more importantly, you can also
> increase calcium intake, which is shown on the bottom right-hand
> corner; whereas, if you use corn oil, as an example of vegetable oil,
> or polycose, all you do is increase calories, you do not increase
> protein, and you don\'t affect calcium. So a balanced addition of
> nutrient is far better than using single product.
>
> Now, I\'m going to share briefly some disease-specific concerns, and
> why these are important. For example, bronchopulmonary dysplasia, a
> chronic lung disease, a fairly common entity known to neonatologists.
> Anywhere between 30 to 80 percent, depending on the cohort you study,
> will have continuing needs. And there is need for additional nutrients
> to compensate for the increased work of breathing, and side effects of
> common drugs. And, more importantly, these babies also need the same
> nutritional intervention after they go home, because\--several studies
> have been done. For example, one study has clearly demonstrated that
> afterwards 73 percent of babies, after they leave the hospital, now
> deviate in their growth. And this can be obviated, to a certain
> extent, by using nutrient-rich formulas, which then come from
> supplementation. We also have an issue of fluid restriction. And since
> they are also born prematurely, they also have high needs of calcium,
> phosphorous and electrolytes.
>
> Necrotizing enterocolitis, which we\'ve been hearing all day\--the
> only two factors that I know that have held the test of time have been
> a baby has to be premature, and maybe the feedings have been advanced
> very rapidly, and this has been reevaluated again as of this month.
> More importantly, in the clinical arena, timing of NEC does not
> always\--or usually does not have\--a temporal relation to the
> addition of powdered nutrients. And there\'s a wide range of onset of
> NEC, with less than 40 percent of them having concomitant positive
> blood or other cultures. In other words, you heard this morning as
> well, that there are whole host of organisms that are associated with
> NEC, but there is also a high incidence of NEC without any organism
> identified at all.
>
> So what are the long term consequences? We know that very little catch
> up growth occurs. I\'ve already shown you the growth curves up to
> post-conception age of 36 weeks. We have been creating, and continue
> to create, shorter, lighter infants, with a smaller head circumference
> compared to the fetal counterpart, and now data as long as eight years
> of age, because of the early growth restriction that occurs in the
> NICU.
>
> And the neurological outcome of these babies is not always explained
> by disease process alone. So I believe that nutrition, or under
> nutrition, has to have played a role as well.
>
> And here\'s a good example of that. These data were published in 2001,
> and what they\'re showing, the left hand, at birth, that babies who
> were above the 10^th^ percentile, and by the time they\'re discharged,
> the number of babies who are above the 10^th^ percentile. And you can
> see, between 70 and 85 percent were appropriate or above 10^th^
> percentile at birth, and barely above zero\--way above 10^th^
> percentile, by the time they reached 36 weeks post-conceptual age. And
> this has been shown, not only in England, it\'s been shown in other
> places in the United States as well.
>
> So, post-discharge, which is now becoming a more and more important
> thing\--evidence demonstrates better growth with enriched formulas,
> i.e., the transitional formulas, or using premature formulas at home.
> These infants are predominantly fed powdered infant formulas due to
> cost. And, with earlier discharge from the hospital, calcium and
> phosphorous, and other nutrient needs cannot or may not be met by
> term-infant formulas, and this statement is supported by the American
> Academy of Pediatrics as well.
>
> So in-hospital practices\--given the variety of scenarios in the
> NICU\--and this is my own anecdotal data, and I believe, if you
> surveyed, you\'ll find similar data. And Dr. Thureen can confirm that
> for me\--up to 50 percent of infants in a large NICU will have some
> form of feeding modification\--either addition, thickening due to
> feeding intolerance or concentrating. However concentrating inherently
> has a problem with changing osmolarity, which then limits the feeding
> problems as well.
>
> And rather than asks the question of how have the in-hospital
> guidelines suggested by CDC affect the current practices, what we\'re
> now seeing is while trying to adhere to the CDC, ADA and FDA
> guidelines, that neonatologists and nutritionists are reluctant to use
> appropriate measures to try and achieve the maximal achievable gain
> that I said before. So we\'ve gone backwards, at least in ability to
> provide the nutrition we believe we need to.
>
> So how do neonatologists weigh the risks? We need to establish enteral
> feedings. We all agree with that. We need to establish adequate weight
> gain, because mortality, morbidity and long-term outcomes are clearly
> related to weight and gestational age. And the risk of E. sak\--20
> cases per 10 years, or however you want to count it\--should be put in
> a perspective that during the same period of time, there were more
> than four million premature infants who were fed and delivered during
> that period of time.
>
> Thank you.
>
> DR. BUSTA: Thank you. We have about 10 minutes to ask questions of
> these last four presentations, if there are any questions.
>
> Dr. Stallings.
>
> If the four of you would come up, we\'d appreciate it.
>
> \[Pause.\]
>
> You just have a comment, Dr. Tompkin? All right. Go ahead.
>
> DR. TOMPKIN: Yes. This is Tompkin.
>
> The white paper that was on the table in front of each of us is the
> white paper that the industry has prepared and shared with us. And,
> likewise, the information that Dr. Merritt presented, in terms of the
> industry\'s perspective on the answers that may be the outcome\--that
> also was prepared by industry. So each of you should have those
> documents.
>
> DR. BUSTA: Dr. Stallings.
>
> DR. STALLINGS: Stallings.
>
> I have almost a question\--short question\--for each of us. So,
> starting with John\--if we were starting at the beginning of this
> question\--and in your slide you summarized that you felt that the
> pathogen, it was a minimally pathogenic organism, and that the
> infective dose was high.
>
> And I guess I\'m not convinced of that. And what kind of animal
> models, neonatal animal models\--I mean, there should be some pretty
> straightforward ways of testing that. Do you, or you guys, have any
> ideas? Because I would feel a lot more comfortable if I knew that from
> something other than we\'ve given, you know, four billion feedings and
> 10 babies have gotten sick.
>
> DR. VANDERHOFF: I think at this point in time that is an assumption
> that we\'re making. And it would be nice if we could verify it with
> some animal models. And I\'m probably not the ideal person to tell you
> which ones would be most valid, but I know there are some. But this, I
> think comes from the epidemiological knowledge that there\'s a huge
> amount of feedings\--Jatinder just told us\--of powdered infant
> formula to pre-term babies; half the premies get added powdered. And,
> of course, all the term infants being exposed to low levels of this
> stuff, 14 percent of the formulas appear to have low-level
> contamination, according to the data yet, you know, I\'ve never seen
> one of these. I asked Jatinder if he\'d ever seen one? I don\'t know
> if you\'ve seen one.
>
> But this is a very uncommon infection. And it would certainly suggest
> to us that there must be some other factors, such as mishandling or
> host defense problems or so forth, involved in doing this. I agree, I
> think investigating it with an animal model would be ideal. I\'m not
> sure we can a hundred percent draw the connection between the two, but
> it will certainly give us an idea.
>
> DR. MERRITT: If you think about it, you\'re never going to have the
> dose response in the human, because the event is going to occur
> sufficiently long after the event, that the actual concentration in
> that formula will never be done. So that forces you to a dependence,
> presumably, on an animal model to try and get that answer. And Dr.
> Nazarowec-White\--I mean, we were desperately searching for some of
> that kind of information. And the paper that came out this month I
> think is the first clue in that regard. And, at least, I believe,
> suckling animal model, it indicates that the number of organisms is
> quite high, and if the practices we are suggesting that we all adhere
> to in conjunction with the levels of detection that were described
> were used, we shouldn\'t see those levels.
>
> Now, I don\'t think we have any way of knowing that the mouse is the
> same as the 800 gram baby, but that\'s the best information we have, I
> believe.
>
> DR. STALLINGS: But, really, it\'s not the 800 gram baby that\'s
> getting the formulas. It\'s usually a little bit later. But\--okay.
>
> So, the next question\--for Les. How big is a lot? You know, when you
> were describing what\'s been proposed, it was, I think, 5-gram samples
> times five samples. And I\'m just not familiar enough to know what is
> a lot, and, you know, really what kind of surveillance might that
> represent?
>
> DR. SMOOT: Right. I can only speak from our experience with Nestle.
> Typically, a lot for us might range in about a 20 ton\--
>
> DR. STALLINGS: How many?
>
> DR. SMOOT: 20 tons.
>
> DR. STALLINGS: Okay. That\'s a lot of cans of baby food.
>
> DR. SMOOT: True. Yes.
>
> DR. STALLINGS: 20 tons, and we would be sampling about 25
> grams\--5-grams times five.
>
> DR. SMOOT: For the family Enterobacteriaceae\--correct.
>
> DR. STALLINGS: Okay.
>
> And then my last question is, are the HACCP regulations\--are they
> regulations? Are they required, or are they voluntary at this point?
> Because there was a lot of reference to using that kind of evaluation
> of manufacturing and risk analysis. And I\'m\--
>
> DR. SMOOT: I don\'t believe it\'s mandatory on FDA. It might be infant
> formula regulations, the GMPs\--we see this more of a mandated effort.
> But in our manufacturing base, typically HACCP right now, the use of
> these principles I would say to be voluntary in these types of
> processes.
>
> DR. STALLINGS: So I just wanted to be clear that that might be
> required in the future, but right now it remains voluntary. Thank you.
>
> DR. BUSTA: Dr. Moyer?
>
> DR. MOYER-MILEUR: Moyer Mileur.
>
> I think I want to direct my comment to Dr. Bhatia, but then get
> response from all of you.
>
> I think your comments\--I appreciate your comments, and I think
> you\'re preaching to the choir when it comes to Dr. Thureen and
> myself, as far as the extremely small or extremely premature infants
> not being served well by\--in that growth is minimized during their
> hospitalization, and continues to be minimized.
>
> The concern I have is that I don\'t see this as an\--the issue of
> powdered formula in these babies as an issue until later on in their
> neonatal period. And my personal experience with these babies is that
> we really can\'t even get up to a full volume of enteral feeding until
> at least 21 days of age, at which time we would either be using
> mother\'s milk or a liquid premature infant formula and then go to
> 24-calorie, and then if not able to get them to grow, then start to
> consider adding additives to those products.
>
> So my bigger concern is that I think the message you had is that
> you\'re worried that neonatologists will stop\--will not provide
> sufficient nutrition because of the worry about contamination from E.
> sakazakii. And I don\'t know\--and so I think maybe directing this
> critical need for education to neonatologists, but not only
> neonatologists, but other care providers who do see preterm infants or
> other infants who are immunocompromised, and that includes
> pediatricians, that includes nurse practitioners, both neonatal and
> pediatric nurse practitioners, as well as registered nurses.
>
> I don\'t know if you\--
>
> DR. BHATIA: Well, I would have changed my slides if I\'d known you two
> were going to be here. Anyway\--the point\'s well taken. The approach
> we take is exactly the same. But the point I think I was trying to get
> across is that without these additives available, we will not be able
> to even feed a human-milk fed baby or give the additional needs of
> specific concerns. I can go on and on.
>
> But these are not additives you add in the first two weeks of life.
> These are much later. This is not to the period of time when you see
> this organism affecting babies, so the temporal relationships seem
> different. My issue was: these are the only things we have left now to
> try and do better with what we have. The current formulas for
> premature babies were made when survivors were above 750 to 800 grams.
> They were not designed for 500 to 800 gram babies. So the best we can
> do is use a balanced additive, and that\'s the point I was trying to
> make\--including the human-milk fed preterm baby.
>
> DR. BUSTA: Dr. Heubi.
>
> DR. HEUBI: Heubi.
>
> My question is one that I think both John and Russ commented
> about\--immunocompromised and pre-term infants being potentially at
> risk.
>
> What are your thoughts about, number one, the issue of kids that have
> motility disturbances, short gut, other kinds of problems, who are
> exposed to potential \"specialty formulas\" that tend to be non-liquid
> in composition and be powder instead, and whether you would consider
> them to be a potential risk from exposure because of their underlying
> condition? And then I have another question after you answer that for
> me.
>
> DR. VANDERHOFF: I think I\'ve seen one case report of a short-gut
> getting infected with E. sakazakii, despite the fact that there are a
> lot of these, and they\'ve been fed with this stuff for a long period
> of time. And I\'ve seen them get infected with almost every other
> enteric organism over the years. So I think it\'s relatively low risk.
>
> The motility disturbance kids\--probably same argument. Anything you
> feed to these kids, because they have mucosal breakdown, anything that
> grows in their gut can get into their blood stream. But I don\'t think
> it\'s a big risk for kids with GI tract disease.
>
> Russ?
>
> DR. MERRITT: I think, clearly, there\'s a literature that indicates
> that short-gut is a special situation immunologically, and these
> children are often not well nourished as well. So it\'s hard to deny
> some potential there. They become somewhat more like the premature
> baby, in terms of having impaired gastrointestinal function and
> barrier.
>
> DR. HEUBI: So, my follow-up question is this: what position will
> industry take in terms of trying to educate physicians and health care
> providers\--just like what Laurie was pointing out\--about the fact
> that these formulae that are powders, are not absolutely sterile.
> Because I think, in general, most clinicians don\'t understand this.
> Now, I don\'t know that we should scare people. But I think, on the
> other hand, it should be clear that these preparations are not
> sterile, and that there are some potential minimal risks, or risks
> associated with their use.
>
> Can you comment on that?
>
> DR. MERRITT: I think the point is well taken, and I think what the
> recent experience with this organism has pointed out to all of us is
> that there are differences in products, according to form. And I think
> there probably has been a lack of education in clinicians, even those
> most involved in the care of these kinds of infants, in this regard.
>
> And so I think that does create an opportunity for us, because I
> believe if clinicians understand the risk they\'ll take all steps they
> can to avoid it. And if they really have not fully understood the
> potential risk\--even if small\--up until this point, then I think
> that gives us some cause for optimism.
>
> DR. VANDERHOFF: Yes. One other comment that concerns me a bit is
> that\--I think there\'s an awful lot that you can do to use these
> formulas in a way in which the risk is very low. There\'s also a risk
> of getting people so concerned about it that they utilize products
> that may not be as useful, simply because they\'re in liquid form. And
> I think this was a concern you brought out, Dr. Bhatia.
>
> And so I think the educational thrust needs to be toward how to use
> the products rather than \"don\'t use them\" if they are, in fact, the
> most appropriate product to use.
>
> DR. MERRITT: Clearly, many of the infants we\'re describing are within
> what appears to be the critical period for developmental programming.
> And I think the neonatologist is in a tremendous bind here, because
> he\'s, one, worried about the acute risks that nutrition poses to the
> immune system; secondly, he is trying his best to prevent the
> long-term sequelae, neurologically, of being malnourished for an
> extended period of time. And now there\'s concern that one of the
> tools that may be most effective in helping him deal with these two
> issues has another side to it. And that\'s why I think\--I think
> there\'s such a need to put this risk in perspective.
>
> DR. BUSTA: Dr. Thureen.
>
> DR. THUREEN: Thank you. Thureen.
>
> I have a two-part question about human-mil fortifiers. Are they
> processed in the same way as powdered infant formulas\--breast-milk
> fortifiers?
>
> DR. MERRITT: In general, these are powdered products, and so the basic
> principles would be the same. Their composition is different, and that
> requires some modification of processing.
>
> DR. THUREEN: If you do do an educational process to inform clinicians
> about the downsides of using powdered formulas, and they should be in
> a different way, would that extend to human-milk fortifiers, too, in
> that that may be the most valuable for the infant powdered product
> that you make.
>
> DR. MERRITT: I think that\'s another educational task, because I can
> tell you, in my capacity, since these recommendations came out of
> CDC\--and for a while there was a recommendation to use hot water for
> reconstitution and the like\--we saw some hospitals turning to strange
> practices to achieve the desired result\--in an attempt, you know, to
> be compliant with infection policies within the institution, or to
> develop new ones.
>
> And so I think, again, there is an educational need. That\'s another
> product where there isn\'t a particularly good substitute for the
> powder, because the concept is that the mother\'s milk is available,
> with all of its potential benefits, including immunologic. One likes
> to dilute it as little as possible, and get the nutrient content up.
> And that becomes, then, the advantage of using a powdered as opposed
> to a liquid isocaloric fortifier, which can bring the volume up but
> does not do anything to increase nutrient value\--or less to increase
> nutrient value.
>
> DR. THUREEN: So do you think that human-milk fortifiers should really
> be excluded from this consideration because it has such an exceptional
> role and the volume that\'s used is actually quite small?
>
> DR. MERRITT: \[Nods affirmative.\]
>
> DR. BUSTA: Dr. Fischer.
>
> DR. FISCHER: I\'d like to ask about your recommendation on what to do
> according to\--when you monitor the finished product, what you\'re
> going to do. This has to do with the sample size equals 5, n equals 5,
> c equals 1. And we have\--when c equals 0, then we\'ll accept the lot
> unconditionally. When c equals 1, then accept the lot if
> identification of fecal coliforms is negative; reject lot if
> identification for fecal coliforms is positive.
>
> And then when c equals 2, then conduct\--it says, \"\--then conduct
> additional investigation, up to and including rejection of the lot.\"
>
> I\'d like to know what the criteria would be, after the investigation,
> for rejection of the lot in that case.
>
> DR. SMOOT: For that particular scenario, when we say
> \"investigation\"\--and it would also occur, I would assure and
> assume, even with the c equal 1. Typically, in manufacturing, when you
> have a product that\'s going to be determined marginal, in terms of
> its release criteria, you will typically commence an investigation to
> look at criteria that have been developed relative to the monitoring
> of the environmental hygienic condition. You\'re going to look at
> process controls, in terms of process indicators in the actual line of
> the process. You will look for evidence of manufacturing practices
> where there might have been interventions on the line; product stops,
> starts\--things of this nature.
>
> You\'re looking for a root cause: why would you find something that\'s
> out of compliance? If you find evidence that you can\--as mentioned
> earlier this morning\--a smoking gun where you find that there is
> clear indication that there was an event that occurred that would
> allow the product to go out of compliance on that, you would typically
> do a safety assessment at that point to determine with the level of
> contamination that you have, indicated by this qualitative
> test\--which now you would have two out of five samples positive for
> an Enterobacteriaceae\--you would look at the confirmatory tests that
> are conducted on that, like the fecal coliform, very possibly further
> to E. sakazakii, and from that you would have enough information to
> determine is there a food safety risk associated with that particular
> lot with the c equal 2. And, from that point\--you said up to and
> equal to the rejection of the lot.
>
> DR. FISCHER: Let me see if I have this straight. I assume that you
> would do the analysis again, wouldn\'t you? Wouldn\'t you start out by
> making sure that the c equals 2 is really correct? Or not. You\'re
> saying\--
>
> DR. SMOOT: Well, not provided in this particular monitoring
> specification. I think it might be very safe to say individual
> manufacturers might have a secondary, more detailed examination that
> you would go and subject the lot to. So, you could very well go into a
> secondary examination, where there might be a more extensive test, and
> that might be a part of the investigation that\'s referred to\--yes.
>
> DR. FISCHER: So, you\--
>
> DR. SMOOT: But we don\'t try to test the product into compliance. I
> mean, that\'s not the intent.
>
> DR. FISCHER: I assume you would write down, for the FDA I suppose, the
> criteria you would use to ferret out\--
>
> DR. SMOOT: That would definitely be spelled out. Correct. Yes. Just in
> an abbreviated form, in terms of sample size, number of units, \"this
> is what we\'re proposing at this point.\" But, yes, there would have
> to be more detail relative to the types of decision criteria on that c
> equals 2.
>
> DR. BUSTA: I\'m going to limit this to two more: Dr. Lee, and then Dr.
> Tarr. Dr. Lee first.
>
> DR. LEE: Yes, this is Ken Lee.
>
> I think it was Dr. Merritt, but I\'m not quite so sure\--somebody made
> a casual mention of the protein denaturation. If you\'re using
> 70-degree or even 100-degree reconstitution, are you really changing
> the quality of the proteins? Is there any change in digestibility?
>
> DR. MERRITT: Well, certainly with some of these formulas, one gets
> substantial clumping in the context of what we\'re describing. And
> therefore I think you introduce some uncertainty regarding nutrient
> delivery. You may also have the issue, as described, relative to
> passing a given feeding through a nasogastric tube, or a nasojejunal
> tube, which is something one likes to change as infrequently as
> possible.
>
> DR. BUSTA: Dr. Tarr.
>
> DR. LEE: Just to continue that thread, though\--those proteins, if
> they are thermally professed by a wet preparation process, they would
> have been denatured, then, at the factory, would they not?
>
> DR. MERRITT: Well, I can tell you that our empiric data of utilizing
> the suggested procedure results in some clumping.
>
> DR. BUSTA: Dr. Tarr.
>
> DR. TARR: Tarr.
>
> I\'ve heard three reasons today to continue to have powder available
> for, presumably, the high-risk premature infant: first, expense;
> second, bulk and storage; and, now, most recently, the probable or
> possible nutrient value of being able to deliver more assets to the
> child per unit volume.
>
> I\'m concerned that that third approach will run the risk of
> administering inappropriate amounts of solute\--solute load to
> premature infants, in the goal of attaining growth. And powdered
> formula may not be medically the most appropriate way to address
> this\--the growth issue.
>
> I do not mean to minimize the growth issue, but I am worried about
> adding another problem to that without definite benefit, and also
> retaining the small risk of an infection.
>
> DR. BHATIA: Maybe I should restate that given the constraints of time,
> I did not go into renal solute load, and all those calculations have
> been done.
>
> If you take the example Dr. Thureen about a purely human-milk fed
> baby, that human-milk fed baby, under current guidelines, or any
> guidelines, will have to have an additive added. The type of additives
> I\'m talking about is on the same order of magnitude. We\'re not
> talking about substituting an entire proportion of formula, we\'re
> talking maybe to fine-tune the formula, take it from 24 calories to 27
> calories. So you\'re talking about one-eight of a teaspoon. And we\'ve
> actually done osmolarity and renal solute load calculations. They are
> well done by other places as well.
>
> It just is heightening the awareness\--we need one step higher; just
> like heightening the need for premature babies. We know we cannot feed
> premature babies plain human milk at all.
>
> So, the nutrition risks, if you will, are far less than any other
> risks at this point in time, infection notwithstanding.
>
> DR. MERRITT: If you take the example of the post-discharge formulas,
> which have only a 10 percent difference in caloric concentration at
> recommended dilution, and maybe a 20, 25 percent difference in protein
> content, there are at least three studies now which show better growth
> in these infants when given such formulas.
>
> I think what you\'ll find in many nurseries is a common practice of
> taking the feeding up to 27 calories, more rarely 30. And I think most
> clinicians are hesitant to go beyond that level, both because of free
> water concerns, and also because of the osmotic concerns. So the usual
> modification is modest. But in light of the data that Dr. Bhatia
> showed on the growth history of infants below 1,500 grams. In light of
> the data, for example, that Alan Lucas has presented, that as little
> as three weeks of feeding intervention\--that is, the difference
> between a term and a pre-term formula\--will make a substantial IQ
> points different\--or IQ substantially different, perhaps as late as
> eight years, or now longer.
>
> I think there is a great deal of pressure on the health care
> provider\--and probably appropriately so\--to try to assure better
> growth of these infants than we are currently seeing.
>
> DR. TARR: As a follow on\--can we not come up with a panel of liquid
> sterilized formulas to meet those needs in the majority of children?
>
> DR. VANDERHOFF: Russ, maybe you can say more about this than I can,
> but there are technological considerations\--I hope I\'m saying this
> correctly\--that exist in making a liquid infant formula that don\'t
> exist in making a powdered infant formula. And when you push the
> nutrient concentrations up to a certain level with a liquid formula it
> just doesn\'t work. I mean, you can\'t make it.
>
> So, that\'s almost kind of a limiting step there. And I think this is
> probably why this hasn\'t been done. I mean, I\'m relatively new to
> this industry, but I think that\'s\--
>
> DR. MERRITT: You know, it\'s clearly an interesting challenge, from
> our perspective, in this issue. To date, the technology hasn\'t been
> there to do that, and if it were I think one would have sen such
> products. But continuing to push that is certainly a challenge on the
> industry side.
>
> DR. BUSTA: We need to start our preliminary discussion, but I\'m
> getting all kinds of pressure from this side of the table with some
> urgent questions.
>
> \[Laughter.\]
>
> So if\--there are two of you that have been pushing me, and if you do
> them real fast, we will take three more minutes.
>
> And Dr. Blumberg was first on waving me down. And then Dr. Kuzminski
> will have the last word\--but it\'s real quick, please.
>
> DR. BLUMBERG: I had a quick question on the per-lot testing. Why is
> the sample size five grams? What we heard earlier today was much
> larger sample size, 100 grams in the CDC investigation of the
> Tennessee outbreak. They used 100 grams, and in some of the other
> things. So why are you proposing five grams for the per-lot testing?
>
> DR. SMOOT: Well, currently, as you\'ve seen, the 1986 A&PR criteria
> for infant formula production with coliforms less than 3 per gram as
> manufacturing criteria that they\'re looking to regulate the industry
> at, as well as the CODEX coliform criteria of 3 per gram.
>
> We are looking to increase that 3 from 3 to 25 grams, in terms of
> monitoring the production verification of our HACCP principles, as an
> indicator that the allowable\--not the allowable levels, but the
> levels of this particular bacterium would be at a very low level in
> the infant formula.
>
> When you look at and reason\--in comparison, looking at other
> bacteria\--Dr. Buchanan indicated as contaminants or pathogens and
> zero tolerance\--and relevance, looking at listeria monocytogenes,
> even though mentioned, it\'s not found frequently in manufactured
> infant formula. The requirement there is on the order of only 250
> gram-negative in 250 grams. And in light of the unknown opportunistic
> pathogenicity of this particular organism, the fact that we have
> epidemiological data that there are billions of feedings over the
> years, we felt as a process-control verification, that a 25 gram
> sample\--absence in 25 grams would be adequate to indicate the safety
> and quality\--
>
> DR. BLUMBERG: Do you have data looking at whether, you know, you can
> pick it up with 25 versus 100 grams?
>
> DR. SMOOT: There is industry data that has not been shared with you
> today that would indicate\--the data set that I provided on that one
> particular Dutch factory, where there was absence in 100 grams, that
> was the data there.
>
> There is a data set that that particular factory\'s been operating
> since that time frame under the monitoring criteria of less than .3
> coliforms per gram, as indicated in the risk profile for CODEX. And so
> there shows\--there\'s good correlation, that whether it\'s
> less\--operating at less than .3 coliforms per gram, or operating at
> negative in 100 grams, per gram, for Enterobacteriaceae, there has not
> been evidence that that particular manufactured powder has been
> implicated or presents a risk for infant nutrition.
>
> I think we\'re just trying to resist the issue of going\--keeping the
> risk of the organism in relation to the sample size that\'s being
> monitored on the production-lot basis.
>
> DR. BUSTA: Dr. Kuzminski.
>
> DR. KUZMINSKI: Kuzminski. Thank you. A quick question.
>
> Did I hear you correctly when you mentioned that the lot size was 20
> tons?
>
> DR. SMOOT: Typically, an average, around 20,000 pounds\--correct.
>
> DR. KUZMINSKI: And that\--I guess\--you must have a high degree of
> confidence\--statistical confidence, that the 25 gram sample is truly
> representative of 20 tons.
>
> DR. SMOOT: Well, at this point what we\'re doing is verifying the
> HACCP principles, the HACCP activities and the prerequisite programs
> relative to the family of Enterobacteriaceae, and that representing
> the E. sakazakii. We feel that that gives us a very good
> representation of the microbiological quality, relative to that group
> of organisms.
>
> DR. BUSTA: Thank you very much.
>
> We have 20 minutes to have our preliminary discussion. Time goes fast
> when you\'re having fun.
>
> \[Laughter.\]
>
> First\--well, thank you all from the industry group.
>
> I must say, the bus will leave at 6:00 p.m. Abe\'s shuttle leaves at
> 6:00 p.m. So we\'ve got to keep that in mind. We won\'t want to dawdle
> when we conclude here.
>
> Tomorrow morning, the bus will arrive at 7:15, in case you really
> don\'t know what else to do. But it will leave no later than 7:45. So,
> at 7:45, the bus pulls out, and most of you, I would guess, will want
> to be checked out and have your bag with you. I know I intend to do
> so. Because when we conclude here, there will be opportunities to go
> directly to the various locations\--airports, etcetera.
>
> There will be, at eight o\'clock there will be a continental breakfast
> when we get here in the morning. So much for the mechanics there.
>
> **Preliminary Subcommittee Discussion on Clinical Presentations**
>
> The reason I want to start the preliminary discussion now is to let
> you start thinking about tomorrow. Because tomorrow we will have
> extensive subcommittee discussion in the morning, and then in the
> afternoon we will attempt to have concluding deliberations and
> recommendations from the subcommittee.
>
> One of the approaches that we have used in the past, on other
> committees, is to go and ask each one of the representatives, the
> voting members on the committee, to make concluding comments on what
> they see are the conclusions and responses to the charges. We probably
> will round\--do Charge 1, and then we will do charge two. So you can
> think about this evening, being that the President is not speaking
> tonight.
>
> VOICE: \[Off mike.\]\--starting right away in the morning?
>
> DR. BUSTA: Well, first we will have discussions. So we will start
> discussing. And those concluding remarks will presumably be in the
> afternoon, after lunch.
>
> DR. KUZMINSKI: \[Off mike.\]\--addressing each of the charged
> questions, or\--
>
> DR. BUSTA: The question from Dr. Kuzminski was: will the concluding
> comments discuss each of the charges? And I would say we would go
> Charge 1, and everybody make a comment. And then we would go Charge 2.
> And I haven\'t thought whether we do Charge 2(1), Charge 2(2), or look
> at them as a unit. But, in part, I think our discussion in the
> morning, as we give our opinions and attitudes, will give us an idea
> of how we will give the concluding remarks in the afternoon.
>
> Would someone like to start some comments on their thoughts on Charge
> 1: Characterize the infants at risk?
>
> Please note that you had a new charge today, different from the one in
> your book\--slightly different. Charge 1 is slightly different. So if
> you just look at the one that was handed out this morning in your blue
> packet, that is the current charge.
>
> DR. THUREEN: The distinguishing features?
>
> DR. BUSTA: The distinguishing features in number 1 are that\--in the
> new one, it now says \"consideration of factors\" in the middle line.
> The old one said \"facts.\"
>
> After \"status,\" age has been added. So it\'s \"immune status, age,
> and general health status, etc.\" has been added. And then it \"may
> impact,\" rather than \"impact.\" So there are a couple of
> modifications\--expansions and modifications on the intensity of the
> recommendation.
>
> So would someone with a strong opinion like to say, given the
> available information on E. sak and powdered infant formula, is there
> a risk?
>
> Dr. Thureen.
>
> DR. THUREEN: Thureen. I\'ll start.
>
> Is there a risk? Yes, I think we\'ve identified today that there is a
> risk. But is it an acceptable risk? It seems like that the number of
> infections with E. sak are very low, considered to other neonatal
> infections. And we haven\'t put it in the context of other infections.
> There was some context with the number of doses of infant formula
> given that makes the risk seem quite low. But just as a clinician,
> without having any data here in front of me, I think, that as an
> infection source and risk, it\'s very low, considering the actually
> worldwide number of infants that we have that are in the pre-term
> infant group.
>
> I think the highest risk group\--oh, do you have a question after
> that?
>
> DR. BUSTA: Well, the comment right after that, which you are probably
> going into immediately, is identifying the populations of infants at
> risk. Maybe that\'s a second comment, but\--
>
> DR. THUREEN: Right. And that second comment is: I think the highest
> risk group, by far, are pre-term infants. And, as a group, they\'re at
> risk for so many things, this one is really a drop in the bucket,
> compared to everything else that they\'re exposed to. But it is a risk
> to them.
>
> I\'m not convinced by the data presented that term infants are at
> significant risk, when you look at the risk and the association with
> infant formulas, and the data of all term infants\--not pre-term, but
> term infants\--those cases that have been identified, I think that
> that risk is exceedingly low, and that any limitation of powdered
> formula should probably not extend to term infants, based on the data
> that\'s there, unless more data becomes available. And I think it has
> to be an ongoing data collection, with better surveillance.
>
> But I think the pre-term infant group is at risk, but is it an
> acceptable risk, or an understandable risk, or a risk that can\'t be
> eliminated, looking at the downsides of the alternatives of not using
> powdered infant formula
>
> DR. BUSTA: Is there\--I\'m trying to keep this balanced. Is there
> someone who has a counter comment to that?
>
> Dr. Stallings?
>
> DR. STALLINGS: The only concern that I have to that is I guess I\'m
> still not convinced that term infants that are truly sick enough to be
> in a level 3 nursery, may not still be at some risk. Because if\--I
> mean, we don\'t understand the pathogenesis, so we don\'t know if
> it\'s really a dose effect yet, because we don\'t know if this
> is\--we\'ve never had the opportunity to measure the dose in the
> product that actually got the children sick. We\'ve done retrospective
> analyses.
>
> So, I still am concerned about term infants with various surgical and
> stressed\--
>
> DR. THUREEN: Right. I agree with you completely, Virginia.
>
> DR. STALLINGS: Okay.
>
> DR. THUREEN: I think this should be to the non-NICU population.
>
> DR. STALLINGS: Okay. Good. So that was my only\--I don\'t think we
> know enough the other stuff, and that environment refers\--
>
> DR. THUREEN: Right. And I agree with you. If they\'re sick enough
> there, they should be included, because they would be
> immunocompromised\--presumable, based on some underlying illness.
>
> DR. BUSTA: Dr. Heubi.
>
> DR. HEUBI: I have a question, I think that I still need to have
> clarified from Patti, and maybe from Laurie. And that is, if we simply
> look at the risk-benefit, in terms of growth and neurological
> development for the pre-term infant, is it appropriate to state that
> using powdered fortifiers fall into the category of the risk of E.
> sakazakii is so low that the benefit is so much greater, that this is
> not an issue. And then I think if you follow that, is it\--are the
> other formulas that we\'re talking about, in terms of constituted
> formulas, such as any of the names that you want to name\--are they a
> different category?
>
> DR. THUREEN: Thureen. Do you mean the constituted, ready-to-feed
> formulas?
>
> DR. HEUBI: No. I mean powdered formulas.
>
> DR. THUREEN: Powdered formulas.
>
> DR. HEUBI: E.g., the amino acid or protein hydrolysate formulas, etc.
>
> DR. MOYER-MILEUR: For the pre-term infant, I think that\--
>
> DR. BUSTA: Laurie, would you identify yourself?
>
> DR. MOYER-MILEUR: Moyer-Mileur\--sorry.
>
> I think that when you have a baby who is receiving mother\'s milk and
> that requires fortification, that the fortifier itself is given in
> very small amounts, and that the benefit from having mother\'s milk
> far outweighs the risk from the powdered supplement.
>
> The concern I have in this high risk population is the use of other
> powdered products in these infants. And I would\--you know, there are
> ready-to-feed liquid products available that should be, for a
> hospital, readily available, that that could be substituted. Unless
> they have a metabolic\--inborn error of metabolism.
>
> DR. STALLINGS: Stallings.
>
> Do we have any data on culturing the breast-milk fortifier? I haven\'t
> heard anything that\'s been presented. Because you\'re hearing from
> all of us that that\'s a very unique and important product to the care
> of these kids. So are there any data from the powdered products,
> versus the liquid, sterile products?
>
> DR. BUSTA: I see a negative from the FDA presenter. Negative from
> Canada. Any positives in the\--
>
> DR. STALLINGS: Well, those would be interesting data for us to learn
> about, because that is something that almost didn\'t exist in the
> \'80s when, you know, all of this was first starting.
>
> The other thing, for clarification\--on this list that we got at the
> table today, of \"powdered form only,\" it said \"amino acid based
> formulas.\" And I just\--those really are the metabolic disease
> ones\--I\'m assuming. Because the protein hydrolysates are under the
> powdered and liquid form. And, again, it would be very difficult to do
> the work without the protein hydrolysates, but they are available in a
> sterile liquid form.
>
> So I think if we\'re starting to focus on the highest risk is the
> premies, there really are only a couple of places\--and we eliminate
> the metabolic diseases, because that\'s a different setting, it seems
> to be headed toward the breast-milk fortifiers.
>
> DR. BUSTA: All right.
>
> DR. STALLINGS: Other comments?
>
> DR. BUSTA: Other discussions. Someone else would like to\--Dr. Neill.
>
> DR. NEILL: Neill.
>
> Maybe before we leave this particular aspect that we\'re talking
> about, I think one of the things that has made me a little uneasy
> today si the issue of risk and trying to categorize it along a
> spectrum of quantitation, when you\'re calling it \"low,\" and \"very
> low,\" and words like \"negligible.\"
>
> I think we know that it\'s somewhere in the vicinity of low, but I am
> personally very unsettled that we really have a good box that we can
> draw around this. I guess, maybe, kind of two points that I would make
> to that issue would be that the NNIS hospitals\--which have come up a
> couple of times\--and, Henry, help me out here if you know the answer.
> That acronym is NNIS, for the National Nosocomial Infections
> Surveillance System. And it is, as you know, the 300 hospitals. The
> extent to which those participating hospitals have obstetrics, NICU or
> pediatric services is unknown to me. I think it\'s a natural, in terms
> of a question to ask: whether the NNIS system has any data on E. sak,
> but it may not at all be the mechanism that could give you the answer.
>
> DR. BLUMBERG: I think that\'s a good point, because\--Henry
> Blumberg\--not all the NNIS hospitals may have a neonatal ICU. And if
> they do, the neonatal ICU may not be contributing data to NNIS. It\'s
> like a menu, and there\'s a lot of different surveillance projects.
>
> For instance, we\'re in this hospital, but our NICU, we\'re not
> reporting NICU data, we\'re reporting surgical site infections or
> other things. So you could be in this hospital and not contributing
> anything from, say, the neonatal ICU.
>
> DR. NEILL: The second half of the point that I was going to make on
> that\--because, again, it gets to this idea of trying to quantitate
> \"low,\" because of where I think we\'re going to end up going, where
> we have this terrible clinical conundrum of trying to weigh the
> benefit against the risk\--and that has to do with the fact that the
> data we\'ve talked about today are published cases of diagnosed
> infections, which everyone in this room recognizes is inherently
> biased. And at this point I would not know how many are diagnosed but
> have eluded formal surveillance mechanism, or how many\--to Dr.
> Tarr\'s point from this morning\--of sepsis of unknown etiology, or
> the patients got the antibiotics on board beforehand, so the CSF
> cultures are negative. Then how many of all this other category\--a
> clearly very ill but un-microbiologically diagnosable illness in fact
> could be due to E. sak? And I don\'t know.
>
> But I thought we probably ought to at least get that one out on the
> table.
>
> DR. BUSTA: Dr. Blumberg?
>
> DR. BLUMBERG: Three\'s two things I wanted to mention.
>
> One is, I think it would be useful to query directors of clinical
> microbiology labs to find out do the automated systems that they use
> pick up Enterobacter sakazakii. Because I think that\'s one\--I just
> don\'t know the answer. Because most of the work\'s been done with API
> strips, and what they\'re using in the clinical micro lab is a
> different system for, you know, most of these things.
>
> The other issue is whether there\'s other enterobacter species in the
> formula that could be contributing to illness in the neonatal ICU
> setting. Because from Muytjens\' study that\'s been mentioned, 14
> percent of the samples cultured positive for E. sakazakii, but in that
> same study, 25 percent cultured positive for Enterobacter glomerans,
> and 21 percent for Enterobacter cloacae. And those are very common,
> hospital acquired infections. And they don\'t jump out. Like E.
> sakazakii is unusual, it jumps out at people. But every day hospitals
> are having Enterobacter cloacae or glomerans infections, and those
> things would not jump out. And I\'m just wondering if, in the NICU
> setting, the infant formula could contribute to those infections.
>
> Maybe that\'s beyond our charge.
>
> DR. BUSTA: Are we\--should I look around and see if we\'ve got an
> answer? Does anybody in FDA have any data on those? On the other
> enterobacters that might be coming in in the infant formula\--other
> enterobacters\--glomerans, cloacae\--that may be coming in in infant
> formula that possible could result in a hospital infection?
>
> DR. ALEXANDER: Dr. Alexander.
>
> As I noted during my presentation on neonatal meningitis, I mean,
> there\'s certainly data that speaks to the fact that other
> enterobacter species also cause neonatal meningitis. They don\'t
> necessarily have the same predisposition to developing brain abscesses
> that Enterobacter sakazakii does, but it is a cause of many different
> types of neonatal infection.
>
> And the problem that you get into when you try and figure out is it
> coming from the formula, or is it coming from someplace else, is that
> those organisms\--the glomerans, the Enterobacter cloacae\--are
> organisms that have been identified as part of normal enteric flora
> for adult humans that are interacting with these infants. And also
> it\'s been identified as part of the hospital environment, so it\'s
> difficult to tease out whether the organism in formula would be the
> source of infection.
>
> With E. sakazakii, since the only place that we really seem to be
> finding it consistently seems to be from the powdered formula, it
> seems to more of a clearer direct association. Although, even that,
> has some difficulties; whether there\'s the possibility that one child
> gets contaminated and then hospital practices, or, you know, health
> care workers carrying the organism to multiple people on their hands
> would be another source that we just haven\'t been able to sort out.
>
> DR. BUSTA: Thank you. Dr. Baker? Did I see you\--you have the
> microphone in your hand, and I thought you\--
>
> DR. BAKER: Baker. I had a couple of comments.
>
> I think one is that I think in this list of formulas that they\'re
> talking about\--the amino acid formula\--they\'re really referring to
> NeoKate as available as a powder only.
>
> The other comment I had was about your comments about risk and unease
> about risk. Just to sort of think about that from a philosophical
> point of view, risk is a measurable thing. That is something that you
> can put a number on in science. Meaningful risk, or safety, you can\'t
> put a number on it. That\'s a societal decision. And that\'s what
> we\'re being asked actually to do, is to tell FDA how meaningful this
> risk that has been identified is, and it\'s not easy. Because there\'s
> no scientific way of doing it. You just have to come up with it.
>
> But anyway, talking about the first charge, I felt that there
> definitely is a risk, and the risk to the normal neonate is probably
> negligible, and not a meaningful risk. But the risk to the premature
> or the NICU baby is probably a significant risk that we need to at
> least address. And I wonder about other populations, like the
> immunocompromised normal baby, or the graduate of the NICU\--should be
> worrying about them as well?
>
> I don\'t think I have an answer for what we should say about that yet,
> though.
>
> DR. BUSTA: Dr. Thureen.
>
> DR. THUREEN: I suspect that any recommendations we come up with are
> going to have a lot of provisions based on getting further data, and
> that we might not have final conclusions and recommendations by the
> time we leave this meeting because there are so many questions; and
> that perhaps part of what we should be thinking about tonight is not
> only what recommendations we have based on the information we received
> today, but what kind of further information we might like to collect
> before final rule\--we could at least set up provisional guidelines,
> but that it may be changed based on important information that we feel
> needs to be collected before we can come to definite conclusions.
>
> And your insights into how data is collected, and what some of the
> presentations are may not reflect reality. That\'s important
> information, and there are ways we could suggest gathering that kind
> of information to at least ultimately come up with a good final
> solution, and we may have to settle for provisional recommendations in
> the meantime.
>
> DR. BUSTA: Would someone like to have the last word of today?
>
> \[No response.\]
>
> It\'s me? My last word of the day is we have 10 minutes, according to
> my watch, to meet Abe\'s shuttle in front of the building, which
> leaves at six o\'clock.
>
> Let me thank each and every one of you, all the presenters, all the
> support staff, and everyone else for an outstanding, intense, long and
> good day. Tremendous involvement by everyone on the committee, and we
> appreciate all your efforts. Think about this tonight. Come back
> vigorous and ready to address the questions tomorrow morning.
>
> Thank you all. We\'ll see you on the bus.
>
> \[Whereupon, at 5:50 p.m. the meeting was adjourned, to reconvene at
> 8:00 a.m. on March 19, 2003.\]

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