[I N D E X]{.underline}

**February 22, 2000**

**[Session 1: Objectives and background information Page]{.underline}**

Welcome and Introduction:

Outline purpose and objectives of workshop

S. Sundlof 4

Antibiotic use in ruminants - An Overview

G. Riddell 12

Antibiotic use in poultry - An Overview

D. Wages 22

Antibiotic use in swine - An Overview

R. Morrison 35

Antibiotic use in aquaculture - An Overview

R. MacMillan 52

Antimicrobial drug discovery and development

J. Watts 70

Antimicrobial new animal drug applications

Review process overview

D. Benz 86

\"558.15\" studies: A historical perspective

J. Cooper 104

Pre-approval assessment of microbial safety

General concepts

B. Flynn 129

Resistance and pathogen load - Factors to consider

D. White 152

Open Comment Period

Facilitator - J. Heslin 190

[I N D E X]{.underline} (Continued)

**[SESSION 2: Study concepts for modeling resistance PAGE]{.underline}**

**[development and/or pathogen load changes]{.underline}**

Introduction

D. White 205

Use of animal studies

K. Ewert 207

Review of an animal study and its relevance

D. Mevius 224

Use of pharmacokinetics/pharmacodynamics

M. Papich 253

Open Comment Period

Facilitator - J. Heslin 280

**KEYNOTE: \"\-\--\" Indicates inaudible in transcript.**

[P R O C E E D I N G S]{.underline}

(8:35 a.m.)

**WELCOME - OUTLINE PURPOSE AND OBJECTIVES OF WORKSHOP**

DR. SUNDLOF: Good morning. If I could ask everybody to please take their
seats, we can begin this morning\'s session.

(Pause.)

DR. SUNDLOF: All right. Good morning, everyone, and welcome to CVM\'s
second workshop on antimicrobial resistance. It is really great to see
the turn out that we have here today. For the next three days we are
going to be discussing some issues that are very important to CVM and to
the industry, and so, I hope everybody is refreshed and ready to go
here.

The purpose of the meeting is to discuss the appropriate design of
pre-approval studies. We talked about pre-approval studies for a long
time in the context of antimicrobial resistance, and we need to consider
in the discussion today both the rate and the extent of resistance
development in the appropriate microbiological organisms and also look
at the issue of pathogen load, which is also a critical factor in
pre-approval testing.

(Slide.)

DR. SUNDLOF: So the meeting\'s objectives are to obtain scientific input
on these issues. These are very complex issues that are meant to give us
some kind of predictive value in assessing what will happen with the
drug after the approval process.

And again, it is a scientific and complex issue, so we need as much as
input as possible, and we would like to hear a lot of different
perspectives and a lot of different alternatives hopefully that will
emerge from this discussion.

(Slide.)

DR. SUNDLOF: Our goals are to get all the ideas out on the table. We
want to hear as many as we can. We want to listen to our experts and ask
them lots of questions. We want to hear from the public. We want to hear
how they view some of the issues we will be dealing with.

We want to discuss these issues in further depth in the breakout
sessions, which will occur tomorrow afternoon and Thursday morning. So
the breakout sessions will give everybody a chance to participate in the
discussion. And we want to do a lot of brainstorming. We want, again,
people to come away from this with a greater understanding and
appreciation than what they came here with. So lots of good ideas,
hopefully, will emerge from this meeting.

I just want to make a statement right now that it is not the intention
to come to a final decision on pre-approval studies at this meeting.
This is not a meeting that is intended to reach a consensus opinion on
what the exact proper study should be.

It is one in which we are going to try to get as many good ideas out as
possible. Those ideas will then form the basis for further comment and
finalization of what we will consider eventually as the proper design
for these

pre-approval studies.

(Slide.)

DR. SUNDLOF: Just to give a little bit of background, in November of
1998 we issued a guidance document, number 78, which says that based on
what we perceive to be the potential public health threat from
antimicrobial resistance development, we believed it is necessary to
consider the potential human health impact of microbial effects
associated with the use of animal drugs.

To do that, we were looking at two different issues. Resistance, and
also, we wanted to consider pathogen load that may increase as the
result of using antimicrobials. So that was guidance document number 78
in November of \'98.

In December of \'98 we issued a paper that we have referred to as the
framework document, and I am sure everybody is familiar with that
document at this time. It states FDA\'s position; that the regulatory
system for antimicrobials for use in food animals should be modified to
address microbial safety. Prior to that, with the exception of a few
cases, we had not considered that in the assessment of safety.

The framework includes a concept of using

pre-approval studies to evaluate the safety of the proposed products,
and so pre-approval studies was a critical element in the framework
document, and that is what this workshop is about; to really focus in on
the pre-approval studies.

(Slide.)

DR. SUNDLOF: Just briefly, we will go through the agenda; what we can
expect in the next three days. First of all, this morning we will have
an overview of antimicrobial use patterns from a number of the producer
organizations. So we will look at poultry versus ruminants versus swine,
et cetera, and how antimicrobials may be used in those practices.

We will also hear a little bit about drug discovery and what is involved
on the industry side on drug development. And finally, just to give
everybody a solid background, we will have some presentations from
people within CVM to explain the regulatory process in general, and
specifically then, how it applies to the regulation of antimicrobial
drugs for food producing animals.

We will also have a presentation based on what we refer to as \"558.15\"
drugs, and those are the sub-therapeutic antimicrobials for which CVM
has, for a number of years, required pre-approval studies. So that can
serve as a kind of background for looking at what we have done in the
past, where we feel the strengths of those studies were and where some
of the weaknesses potentially lie. From there, it may be easier to try
and move forward.

(Slide.)

DR. SUNDLOF: This afternoon we will finish session one. Again, there
will be some talks from CVM people on the regulatory process, and we
will have a discussion of some general concept of microbial safety and
assessment. And we will be listening to some experts, some people that
have got some experience and some ideas on how these studies might best
be performed and the discussion of some of the specific factors to
consider regarding resistance and pathogen load.

And then we will begin session two, which is more conceptual
perspectives. We want to get different perspectives on how we might
approach the issue of

pre-approval studies for microbial safety. So on Wednesday morning then
we will finish up those presentations.

And then, the people who have been talking this afternoon and tomorrow
morning will sit on a panel and will have an open public meeting and a
panel discussion tomorrow morning on some of the things that we have
heard to date.

(Slide.)

DR. SUNDLOF: And then, on Wednesday afternoon, we will begin the
breakout sessions. Those breakout sessions, if you look at your agenda,
are grouped according to the species of interest. So they are species
based breakout groups and people will be allowed to go to whichever of
those breakout sessions that they feel is of most interest to them.

We have provided some questions, and they are in your agenda. The
questions we won\'t go over at this time because a lot of those
questions you really won\'t be able to have a good idea of how to answer
until you have heard the discussions that have led up to the breakout
sessions.

You will have an opportunity during the comment period on Wednesday
afternoon to add additional questions or raise additional concerns for
discussion during the breakout session.

(Slide.)

DR. SUNDLOF: Then, on Thursday, we will finish the breakout sessions and
the chairs or the moderators of those breakout sessions will begin to
prepare the reports. And then, on Thursday afternoon, we will have a
presentation of all of the breakout groups and then further discussion,
and then we will talk about next steps based on what we have heard
during the course of the entire three days.

(Slide.)

DR. SUNDLOF: Okay. But it doesn\'t end there. It doesn\'t end Thursday.
We still think that this is a continuing, ongoing process. It begins
here, but it continues on, and we have opened up a docket and that is
the number of the docket so that additional comments can be sent to FDA.
That is the docket number that you refer to, and we will take all those
comments into account.

Additionally, the transcript of this workshop will be made available. We
will have a full transcript on CVM\'S web site, and there you have our
web site address. And then the final thing I need to do is just a few
little housekeeping details.

Refreshments will be available during the breaks, but because we want to
have as productive a meeting as possible, we would ask everybody to,
please, try to return from the breaks on time so that we can keep on
schedule. Lunches will be on your own. There will be a short reception
on Wednesday at 5:30 in the evening.

And if you have any questions or need anything during the next three
days, the two people that are sitting out there at the table outside of
this room are Alita Sinderlar and Linda Cowatch, and they will be glad
to assist you if you have any problems at all.

So, those are my opening remarks. I would now like to turn the program
over to the moderator of the first session, and that is Dr. Claire
Lathers. Dr. Lathers is relatively new in the Center for Veterinary
Medicine. She is our office director in the Office of New Animal Drug
Evaluation where the studies eventually will be evaluated. So, Claire, I
will turn it over to you.

CHAIRWOMAN LATHERS: Thank you, Steve. Welcome to the Center for
Veterinary Medicine\'s workshop. We will be looking at pre-approval
studies and asking the question: Antimicrobial resistance and pathogen
load; how do we best design our protocols?

On behalf of the Office of the New Animal Drug Evaluation and the
center, I would like to begin by thanking all of those who have
contributed to the effort of making this workshop a success: Bill Flynn,
Dave White, all of the members of the CVM pre-approval protocol group.
They have spent a lot of time discussing possibilities, and now they are
here to share and to listen with you and your ideas.

And Blue has assisted, Linda Towlson, Steve Sundlof, Sharon Thompson,
and indeed, all of the senior management team. And finally, Anita
Sinderlar and Linda Cowatch are the people that are making the actual
workshop happen, if you would, in terms of the mechanics.

So, with the first speech, we will now begin our discussion of the
appropriate designs for the pre-approval studies to evaluate the
microbial effects of antimicrobial drugs intended for use in food
producing animals and ask the question: How do we address the rate and
the extent of resistance development and the changes in the number of
enteric bacteria in the animal\'s intestinal tract that can cause human
illness?

Our first speaker to begin to address these questions is Dr. Gates
Riddell. He is a professor in large animal surgery and medicine at
Auburn University, he is the past president of the American Association
of Bovine Practitioners, he is a member of AVMA\'s drug advisory
committee and a member of the AABP\'s committee on pharmaceutical and
biological issues. Dr. Riddell.

**ANTIBIOTIC USE IN RUMINANTS - AN OVERVIEW**

**By Dr. Gatz Riddell**

DR. RIDDELL: Thank you, Dr. Lathers. I appreciate the opportunity to be
able to bring some perspective from the ruminant species this morning. I
would like to start off my comments by talking about some of the
preparatory steps towards considering the use of antibiotics in
ruminants.

There are numerous tools available to animal agriculture, which can be
implemented to maintain animal health today. These include
well-researched nutritional guidelines, vaccines both old and new to aid
in the prevention of disease, a greater understanding of appropriate
housing designs for various classes of animals and proven protocols for
the integration of these tools in preventive medicine programs.

However, there are also numerous uncontrollable variables which can
impact animal health and which can compromise the effectiveness of
health maintenance protocols. Antibiotics are and will continue to be an
important and necessary tool for the treatment of certain infectious
diseases and prevention of pain and suffering resulting from these
diseases.

It is difficult, if not impossible, to prevent exposure to disease
pathogens. For example, the bacteria, group of bacteria, that have been
associated with causing bovine respiratory disease.

(Slide.)

DR. RIDDELL: Some of these infectious agents are found in regionally
select areas of the United States. Others may require the presence of
specific animal populations, while others may be universally found in
the environment, regardless of the presence or concentration of animal
agriculture.

(Slide.)

DR. RIDDELL: Variables which can place animals at risk for developing
disease subsequent to exposure to these bacterial pathogens include
variation in individual animal susceptibility to disease and response to
effective vaccination protocols. There will be environmental stressors
that are truly uncontrollable, such as weather, drought and other
ambient conditions.

There will be life cycle events that are stressors of themselves, such
as calving in lambing for the yew, and there will be management
stressors, such as diet changes, which are important as we take this
monogastric animal at birth to a ruminant at maturity, and
transportation.

In addition to the potential for bacterial pathogens to cause disease,
there are numerous viral agents which can alter local or systemic immune
system function and open the door for secondary bacterial infection.

(Slide.)

DR. RIDDELL: For those diseases of bacterial origin, the only recognized
therapy may require the use of properly selected, dosed and administered
antibiotics. It is impossible within the scope of this short
presentation to describe all the diseases scenarios for which the use of
parenteral,

(Slide.)

DR. RIDDELL: Invasive surgical procedures will be performed on ruminant
animals under field conditions which can place the animal at risk for
bacterial wound infections. Examples of some of these field procedures
would include exploratory abdominal surgeries and cesarean sections
performed because of obstetrical difficulties encountered in an animal
which cannot be transported to a surgical facility.

Beyond that, even excellent surgical facilities themselves cannot
prevent all bacterial incisional complications. Additionally, ruminant
animals may, by their nature, suffer traumatic injuries which can be
complicated by secondary bacterial wound infections.

The former circumstance, surgical procedures, will require systemic
antibiotics to prevent bacterial infection following contamination, and
the latter, the traumatic injury, may require a full return to health
following the development of a bacterial infection.

(Slide.)

DR. RIDDELL: There are certain conditions under which the potential for
the development of a bacterial infection is increased due to
environmental transport, management, housing or life cycle
circumstances. Respiratory disease in cattle or lambs entering the feed
lot are an example of this.

As with many diseases seen in agriculture, the causes of respiratory
disease outbreaks are considered truly multi-factorial. The stress
associated with transportation and increased exposure risk due to the
commingling of newly introduced and the potentially immuno suppressive
effects of at least one upper respiratory virus all tend to predispose
to bacterial disease.

Now, there are numerous antibiotics on the market today which are
labeled for treatment of bovine respiratory disease, each of which can
be effective against the disease, but none of which will be universally
effective. Therefore, the need for the current armamentarium and
increasing our armamentarium in this area.

For this reason, the wide range of therapeutic options will allow a
practitioner to base treatment upon diagnostic microbiology and previous
experience, with clinical judgment thrown in, and make treatment
adjustments where needed.

(Slide.)

DR. RIDDELL: Another bacterial condition which is life cycle related and
which will respond to antibiotic therapy is a life-threatening uterine
infection, which develops in the first three to 10 days after a cow has
a calf. This condition, known as a toxic or septic metritis, can make an
animal severely ill, may result in her death or render her
reproductively unsound in future years.

In years past, intrauterine antibiotic therapy has been utilized, to a
great degree, to treat this condition. Research on the type and location
of problematic bacteria now suggests that systematic antibiotics, rather
than intrauterine, are markedly more effective.

Monitoring protocols have been developed and implemented on many herds
to develop infections early in the stage of the disease, which involves
something as simple monitoring, daily, the body temperature of the
animal.

These protocols have been able to direct much more specific and limited
antibiotic use because of early intervention. These use of these
protocols allowed treatment to be initiated earlier in the disease in
those animals which are going to develop metritis, thereby enhancing the
therapeutic success rate and minimizing the overall use of antibiotics
because of early intervention.

(Slide.)

DR. RIDDELL: Lameness is a common condition diagnosed in both beef and
dairy cattle. There are specific bacterial conditions, such as
necrotizing pododermatitis, a condition commonly known as foot rot,
which occurs when certain types of anaerobic bacteria gain entry into
the soft tissues of the lower leg and feet of cattle. These infections
respond readily to the use of appropriate systemic antibiotics.

More common causes of lameness are conditions such as sole bruises and
sole ulcers, as you see in the picture here, for which antibiotic
therapy is of little benefit or no benefit. Diagnostic and treatment
protocols for lameness in cattle have been developed which direct
therapy to the specific condition, including antibiotics where necessary
and appropriate.

(Slide.)

DR. RIDDELL: Trained, experienced veterinary practitioners are able to
evaluate disease outbreaks, apply well researched principles and make
predictions as to when disease outbreaks may potentially spread to other
unaffected animals. An excellent example of this type of outbreak is
bovine respiratory disease.

The multi-factorial nature of this disease and the many predisposing
factors have already been outlined. When the predisposing factors are
present and unaffected animals have been placed at risk, the
metaphylactic or prophylactic use of antibiotics in cattle and feeder
lambs at risk for the development of respiratory disease of bacterial
origin can prevent the outright development of disease in large
populations of animals.

Studies have demonstrated that the appropriate application of the
principles of metaphylactic therapy can reduce the overall use of
antibiotics in certain groups of animals.

(Slide.)

DR. RIDDELL: Another example of a preventive strategy which involves the
use of antibiotics would be the use of intramammary antibiotics in the
dairy cow entering the dry period, a time in her life cycle in which
there is a documented increase in risk for the development of bacterial
mastitis.

When a mature lactating cow reaches seven months of pregnancy, she is
dried off and she enter her dry period. This is a time where she is not
milked to allow regeneration of the secretory cells of the mammary gland
before she enters her subsequent lactation with the birth of her next
calf.

The two times that you can see on this graph of greatest risk for the
development of a new intramammary infections during the entire lactation
cycle are the first two weeks and the last two weeks of the dry period.
In addition, lactating dairy cows may enter the dry period with a
subclinical bacterial mastitis.

It has been well established that the infusion of antibiotics into the
mammary gland at a time when the cow will not be milked for 60 days
enhances udder health and promotes the production of higher quality milk
in the subsequent lactation. The high risk period found at the end of
the dry period, on the other hand, is more appropriately mediated by the
use of vaccinations, where appropriate, housing and environmental
upgrades and nutritional programs directed toward maximizing the
performance of the immune system.

(Slide.)

DR. RIDDELL: Very young calves, two to 10 days of age, may encounter
chance overwhelming systemic bacterial infections, typically with Gram
negative organisms. The only treatment which will enable this young
class of animal to overcome the bacterial infection found within the
blood stream will be systemic antibiotics specific to the suspected
organisms.

(Slide.)

DR. RIDDELL: The practice of feeding antimicrobials, such as the
ionophores, which alter ruminant flora populations, to enhance
preferential development of \-- production of volatile fatty acids to
promote efficiency. Bambermycins, tylosin, virginiamycin and
tetracyclines enhance growth promotion, enhance feed efficiency and work
towards disease prevention, resulting in improved animal performance,
productivity and efficiency.

There are numerous label indications for antibiotics in the feed. These
include increased rate of gain, improved feed efficiency, the prevention
or liver abscesses and the control and treatment of anaplasmosis.

The prevention of liver abscesses and the control of anaplasmosis
directly impact animal health and well being. Other methods of control
of these two conditions are limited or non-existent. The well proven
decades old vaccine for anaplasmosis has been off of the market for
several years.

The use of fed antimicrobials to enhance rate of gain and feed
efficiency results in more efficient animal protein production, more
effective use of feed grains and more responsible nutrient utilization
in terms of waste management.

(Slide.)

DR. RIDDELL: As I mentioned in the beginning, it is very difficult to
define, in the limited time available, all potential uses of antibiotics
in ruminants. Hopefully, I have stimulated some thought and provided
some framework for discussion by some of the representative examples I
presented, and hopefully, discussion over the next three days will help
further explore these uses and answer questions pertaining to this
topic.

In summary, antibiotic use in ruminants is necessitated when the
variables involved in animal agriculture, such as environmental
conditions, chance exposure to infectious agents and variations in
individual animal susceptibility predispose to individual animal disease
or outbreaks in herd populations. The proper evaluation, thorough
diagnostic procedures and the implementation of appropriate therapies,
particularly under the auspices of a valid veterinary client patient
relationship, when appropriate, will enhance the efficacy and safety of
the use of antibiotics in ruminants. Thank you.

(Applause.)

CHAIRWOMAN LATHERS: I think we have time for one question, if someone
would like to ask a question.

(No response.)

CHAIRWOMAN LATHERS: Our next speaker will be

Dr. Dennis Wages. Dennis is currently a professor of poultry health
management at the College of Veterinary Medicine at North Carolina State
University. Dennis earned a bachelor of science in poultry science at
the University of Arkansas, a doctorate of veterinary medicine at Kansas
State University, completed a pathology residency at Iowa State
University and is currently at diplomat status with the American College
of Poultry Veterinarians. Dr. Wages.

**ANTIBIOTIC USE IN POULTRY - AN OVERVIEW**

**By Dr. Dennis Wages**

DR. WAGES: Thank you, Dr. Lathers. Good morning. I have been asked to
give an overview of antibiotic use in poultry, probably one of the areas
of most controversy in the use of antibiotics in food animals, and
hopefully, I can give you an idea of why we do what we do and some of
the thinking that is involved in the utilization of antibiotics.

I will touch base on therapeutics in the water, therapeutic feed grade
antibiotics, growth promotion and the use of injectables in the limited
time that I have.

(Slide.)

DR. WAGES: Antibiotic use in the poultry industry has been a fundamental
intervention strategy since the 1960s. Even though preventative disease
management is the primary focus of the industry\'s disease control, and
we maintain much emphasis on vaccination protocols and the study of
immunology, disease outbreaks that do occur, and it is a fact of life
that it requires antibiotic therapy in some cases.

The majority of antibiotic treatment in poultry for acute disease
outbreaks occurs via the water route. When a disease is identified
within a flock, morbidity and mortality are assessed, necropsies are
performed and a diagnostic evaluation is initiated in the diseased
flock.

When culture and antibiotic susceptibility profiling has been performed,
the veterinarian considers farm history, previous diagnostic evaluations
specific to that farm and in that area and initiates appropriate control
measures, which does include environmental and management changes, as
well as, in some cases, the use of antibiotics.

We currently have eight classes of antibiotics used for water
administration, and they represent 15 antibiotics that are approved for
use for the treatment of acute bacterial diseases in poultry. They are
dosed based on milligrams per kilogram of body weight \-- that is, the
pounds of meat in the house \-- at labeled indications or based on the
veterinarian\'s clinical judgment.

Any use of antibiotics not in accordance with the label indications are
to be done within the guidelines outlined by AMDUCA. The antibiotics
commonly chosen for use in appropriate disease outbreaks as intervention
tools for water administration include the tetracyclines, streptomycin,
neomycin, bacitracin and penicillin. These antibiotics represent tools
that we use. We don\'t treat a lot. But when we do, these are the ones
that are used more commonly.

Antibiotics that are less routinely or commonly chosen include
lincomycin, streptomycin, tylosin, erythromycin and sulfonamides. These
antibiotics are used. The latter group are used in the industry;
however, they are used to treat diseases that we don\'t say on a
day-to-day basis. You could take probably eight percent of our treatment
for acute outbreaks and lump them into E. coli bacillosis and falcollera
(sic) in turkeys and chickens, and that is the majority of what we treat
for.

(Slide.)

DR. WAGES: The fluoroquinolones are used at labeled indications. They
are not used in any extra label format, in that it is against federal
law to do so, and they are used sparingly in our industry. They are cost
prohibitive. It is not uncommon to put \$1,500 into a flock of chickens
with the use of the fluoroquinolones, and it is just not cost effective
to utilize such treatments when you look at a cost per pound benefit. It
is a very important drug to our industry.

(Slide.)

DR. WAGES: A survey of the National Chicken Council places
fluoroquinolone use somewhere between one and two percent in the broiler
production. We try to hold it in reserve. The fluoroquinolones currently
represent the only drugs consistently effective for coli bacillosis in
turkeys and chickens, and that is our number one disease.

Because of the economic impact of disease in poultry, disease prevention
through rigid vaccination protocols and management improvements are
emphasized while veterinarians in integrated companies closely regulate
treatments. No, we are not perfect. Companies that have used antibiotics
excessively and inappropriately instead of utilizing stringent disease
prevention programs are simply no longer in the poultry business. They
can\'t afford to be.

The aforementioned antibiotic intervention tools are used in specific
diseased flocks regarding specifically diagnosed bacterial infections,
and we do not use antibiotics in healthy flocks. However, in a house of
25,000, when a disease such as coli bacilloses occurs and we are losing
five to seven birds per thousand, we do have a number of birds that we
call at-risk that are not diseased and appropriate antibiotics are used
in the diseased house. But, in fact, there are birds that aren\'t sick
at that time in a prophylaxis use of antibiotics. Long-term therapy for
chronic infection is not cost effective, nor is it performed in the
poultry industry.

(Slide.)

DR. WAGES: Growth promoting antibiotics are added to the feed primarily
as a control measures for common enteric bacterial diseases,
specifically clostridial infection that result in necrotic enteritis.
History has determined growth promoting antibiotics and their use to be
sub therapeutic, a term that has been used against the poultry industry
and the food animal industry for years and a term that I personally feel
is inappropriate, and I will try to explain.

If one looks at the definition of therapy and treatment, there is, under
all the definitions in Dorland\'s Medical Dictionary, areas where
prophylaxis and prevention are identified as tools in the treatment and
initiation of therapy. The reason sub therapeutic was adopted years ago
to describe growth promotion can be explained in my mind.

In poultry, sub-clinical infections with coccidia in commercially raised
flocks predispose birds to necrotic enteritis. Even though the
anti-coccidial feed additives are routinely and were routinely used in
commercial poultry, antibiotics such as virginiamycin, bacitracin and
lincomycin were added to the feed to prevent necrotic enteritis
infections due to Clostridium perfringes.

These antibiotics were needed because the coccidia preventative feed
additive to control coccidiosis were static and not cidal, thus
preventing clinical coccidiosis in the flock, but not preventing sub
clinical infections and the protozoa proliferation within the intestines
that predisposed the bird to necrotic enteritis infections.

Since the levels used in the feed of these antibiotics to prevent and
control necrotic enteritis were lower than those used to treat active,
acute outbreak of enteric they were coined sub therapeutic; below the
therapeutic dose needed to treat an active infection. Even though we did
control infection. A perfect example is 50 grams per ton of an
antibiotic would control necrotic enteritis. If they broke with the
disease, it would take 400 grams per tone to treat an active infection
within a five to seven-day period.

I don\'t think in today\'s terminology sub therapeutic is appropriate,
although it is very coined and people are very comfortable with it.
Today, we still use virginiamycin, bacitracin, lincomycin and
bambermycin to prevent and control necrotic enteritis and for growth
promotion; however, since the 1980s \-- in fact, about 1982 \-- the
poultry industry has not used the tetracyclines, penicillin,
sulfonamides or erythromycin in feed for growth promotion or at low
levels for disease control.

(Slide.)

DR. WAGES: Besides disease control, antibiotics have other modes of
growth promotion, some known and many unknown. Certain antibiotics used
in poultry increase chilled and hot carcass weight, improve breast yield
and have protein sparing effects in the intestines. Many of these growth
promoting mechanisms and results can be attributed mainly to control of
sub clinical disease, such as necrotic enteritis, and some mechanisms
specifically are unknown.

Some growth promoting antibiotics increase intestinal tensile strength,
the strength of the intestine. This intestinal health and tinsel
strength is important not only for the overall health of the bird, but
also as an advantage at the time of processing to prevent bacterial
contamination.

It has been demonstrated that certain antibiotics increase tinsel
strength and intestinal integrity that prevents the tearing of
intestines during the automated evisceration process. This helps prevent
contamination from intestinal breaking at processing, which decreases
the bacterial load at processing on our carcasses.

(Slide.)

DR. WAGES: Besides overall disease reduction and other cost benefits,
growth promotion increases feed utilization by decreasing the amount of
feed required to produce one pound of gain. To put this in perspective,
if feeding an antibiotic to control necrotic enteritis or to improve
growth promotion and feed efficiency would increase the efficiency 0.01
or 100th of one pound, which would reduce feed conversion from a 2.00 to
1.99. This represents a savings to our industry in the feed utilization
of 375 million pounds.

This reduces the amount of grain required to furnish this feed, reduces
electricity and the milling, or to mill this feed, reduces gasoline to
deliver this feed. It is a snowball and domino effect on reducing the
cost for a chicken.

Enhanced feed utilization also reduces fecal nitrogen and phosphorous
excretion in litter, thus is an environmental advantage when applying
litter to pastures and crops, another point that is of concern in
intensive livestock grazing areas.

Growth promoting antibiotics or any other use of antibiotics are not
used to treat poor management. Antibiotics simply do not replace
deficiencies in management, despite popular press.

(Slide.)

DR. WAGES: Antibiotics added to the feed are rarely used to treat acute
disease outbreaks. Now, this is feed grain antibiotics for acute
outbreaks of disease. Antibiotics that are approved for use in this
manner include the tetracyclines, erythromycin, bacitracin, tylosin and
sulfonamides. And all of my list of antibiotics may not be entirely
complete, but I think they are the ones that are the most common.

These products are difficult to use in the treatment of an acute
outbreak routinely. It requires the removal of coccidiostat from the
feed or requires a cross clearance with the commonly used coccidiostat
during treatment, and either removing the coccidiostat or trying to find
cross clearances and have companies put their money into cross
clearances, none are very palatable to the industry at this time.

There are cases, such as chronic fowl cholera in breeders and some
Mycoplasma species infections where pp. infections where feeding a feed
grade antibiotic at a therapeutic intervention level for 10 to 14 days
may be cost effective and potentially reduce condemnations at
processing, but this procedures is rare.

(Slide.)

DR. WAGES: Injectables. There are antibiotics approved for use as
injections in day old chickens and turkeys to control omphalitis or yolk
sac infections. This procedure has been performed for over 30 years in
the poultry industry.

Now, in the incubation process, at approximately 19 and 27 days of age,
in chicks and poults respectfully is taken from the outside of the chick
or poult and drawn into the body cavity. This period of time is a window
of opportunity for bacteria to enter the developing embryo.

Until 1993, yolk sac infections in chickens were controlled with the
injections of antibiotics such as gentamicin and spectinomycin at 1 day
of age. In 1993, with the approval of Marek\'s disease in ovo
vaccination, which basically vaccinates for Marek\'s from the time that
chickens are transferred from the setting incubators to the hatching
incubators. This process was approved.

This in ovo technique also provided a window of opportunity for an
injection of antibiotic at the time of vaccination for Marek\'s that
would try to and at least potentially more effectively control the yolk
sac infection by placing the antibiotic at the point of contamination
when the yolk sac is withdrawn into the body cavity.

If the bacterial contamination occurred at any point during the egg
collection, storage and incubation of the eggs in ovo antibiotics, in
our mind, felt like there was a benefit to the chick in controlling
bacterial yolk sac infections.

Now, the only antibiotic approved for such injection is sarafloxacin, a
fluoroquinolone. And I will tell you that it is not and has not \-- and
we kind of snubbed sarafloxacin. We do not use it in day old in ovo
injection, nor in any other procedures of injections in poultry.
Sarafloxacin injectable, in the poultry industry\'s mind, is
predominantly a dead issue.

The poultry industry felt that fluoroquinolones were too important to be
used as a day old preventative or control for yolk sac infection or
omphalitis.

The two most commonly used antibiotics for in ovo administration are
ceftiofur and gentamicin. These are not approved for in ovo
administration. They are used under AMDUCA as extra labeled.

Although stringent cleaning and disinfecting of hatcheries and hatchery
equipment are performed daily, these procedures cannot prevent some
bacterial contamination from the egg collection and storage process. The
use of antibiotics in chicks and poults and/or in the developing embryo
provide the poultry veterinarian a useful tool for controlling yolk sac
infections in chicks and poults during the first week of life.

(Slide.)

DR. WAGES: Antibiotic intervention in poultry is a tool. It is one tool
in a total disease prevention program that emphasizes preventative
disease management and vaccination protocols, et cetera. We simply
can\'t afford to have disease in poultry flocks and maintain our current
cost per pound benefit of production.

Our industry does not encourage nor endorse indiscriminate use or
excessive use of antibiotics in our flocks. Currently, the American
Association of Avian Pathologists Committee on Drugs and Therapeutics,
which I am currently the chair, are drafting specific guidelines to
promote judicious use of antimicrobials in poultry to preserve the
efficacy of all antimicrobials in both poultry and human medicine.

And I assure you we are looking at all ways that we use antibiotics and
determine whether we are doing things in the most effective way and what
impact we have. We are convinced that what we do in poultry medicine and
in our food animal species regarding poultry no longer just impacts the
poultry and the growers and the companies. The impacts are global.

This effort supported by the American Veterinary Medical Association and
the AVMA Committee on Judicious Therapeutic Antimicrobial Use. This will
provide the front line poultry veterinarian in poultry to continue to
make informed decisions regarding poultry intervention strategies. Our
antibiotic arsenal is small, but when the need for antibiotic use is
warranted, we need to have access an appropriate therapeutic avenue.

This overview is kind of short. Hopefully sweet and to the point. It
doesn\'t, I am sure, answer all the questions. I hope that it at least
does explain some mechanism and things that we do; why we do. I am sure
it won\'t satisfy everyone, but if there are any questions, I would be
happy to take them now. Thank you very much.

(Applause.)

CHAIRWOMAN LATHERS: Are there any questions for Dr. Wages?

(No response.)

CHAIRWOMAN LATHERS: If not, thank you very much.

DR. WAGES: Thank you.

CHAIRWOMAN LATHERS: The next speaker is Dr. Robert Morrison. Bob is an
associate professor at the College of Veterinary Medicine at the
University of Minnesota. He is director of Pig Champ, a software
business serving the swine industry, he is a co-owner of a 2,000 sow
multiplication herd, he is a board member of Allison Meats, a regional
meat produced and processor, he is vice president of the American
Association of Swine Practitioners, and, as such, he works closely with
veterinarians and has a broad experience which combines both applied
science and business aspects in terms of pork production.

You will now hear his presentation on antibiotic use in swine. Dr.
Morrison.

**ANTIBIOTIC USE IN SWINE - AN OVERVIEW**

**By Dr. Robert Morrison**

DR. MORRISON: Thanks very much. I am going to just giver an overview of
how veterinarians approach the treatment decision within swine
facilities, and I would like to thank the committee for asking the
American Association of Swine Practitioners to be here.

(Slide.)

DR. MORRISON: What I was led to believe at least is that many of you
here maybe weren\'t all that familiar with pork production, and so I put
this slide in just to show you a representative barn. Not, of course, a
barn that all pigs go through like this, but not a atypical barn that a
lot of pigs might go through in terms of their growth process.

And so what you can see here is a barn that might have 1,000 or 1,200
pigs in it, and they would be all relatively similar in age and weight.
They probably came from one sow barn, and they came into this facility
let\'s say around eight or nine weeks of age and they are going to stay
here for three and a half months or so, at which time the barn \--
again, if this was an example barn \-- would be emptied and all of these
pigs would go to market.

The barn would be completely washed down with a power wash and hot water
disinfectant, and basically, the next group of pigs that would come in
would come into the equivalent of a new basically disease-free, if you
think of that way, barn. And that sort of production is what we try to
produce pigs through in a way today so the new group that comes in has a
new chance to do well.

So that is not an atypical barn, as I said. That would be called a
naturally ventilated barn. So you have got curtains on the side, and you
can see the curtain on the left side there is open, letting light in. On
a hot summer day the curtain on both sides will be open, and you will
get nice ventilation going through.

Now, what you can also see there is that those pigs have plenty of
opportunity for touching each other; nose to nose contact, oral fecal
contact, and so there is quite a bit of opportunity for transmission of
infectious agents. And so, when something gets in there, it is very
likely to spread if you don\'t have sort of the set up right to try and
do things right.

(Slide.)

DR. MORRISON: Now, if you think of that barn, I would like you to just
ask yourself which of these three is it most similar to. Is it most
similar to a daycare facility or a residence at a small liberal arts
college or a nursing home? And you could argue which one it is most
similar, but what I am going to propose today is that it is most similar
to a residence where you have got a group of relatively \-- let\'s say
mature individuals, hopefully mature, coming and they are going to stay
there for a while.

And they are not going to come in and go, like in a daycare where you
are going to go home, bringing new infectious agents back every day.
Anybody who has got kids at daycare you know you are sick virtually
every other day.

In a residence, however, you are going to come and you are going to stay
and you are going to stay there for eight months and then you are all
going to go home and the residence is emptied. The other important part
about that residence versus the other two is that they are
immunologically mature.

They are not like a daycare facility where babies don\'t have a well
developed immune system let\'s say or not well exposed. They are not
like a nursing home where you have got perhaps immune compromised
individuals, people who cannot withstand infection. These pigs in this
barn are physiologically and immunologically mature, and they are going
to stay there. They are going to respond well to vaccines, et cetera.

I say there at the bottom, \"We must consider the population when
treating a disease.\" We have those 1,000 pigs in there and maybe three
of them today are sick, but 997 are at risk. So that is very important
to us when we think about the treatment decision for a barn.

When I am teaching veterinary students, I will have 70 students in the
class let\'s say. And on a bad day in February, 10 will be absent from
flu virus, 15 should be absent because they are, you know, sniffling,
they are dripping, they are shedding quantities of virus into the air
that infect the professor, 15 of them are probably feeling kind of
rotten and the other 30 are pretty good.

That is the same situation when we have that barn with 1,000 pigs in it.
If you have something break with it today, a few are going to die. Some
are going to be quite clinically sick, and we want to, hopefully, turn
those around. Maybe half of them are at risk of getting sick, and maybe
30 percent of them are going to do absolutely fine.

(Slide.)

DR. MORRISON: So, when we treat or not, a veterinarian, intuitively, is
going to go through these sort of decisions. What this particular
disease costs in that 1,000 pig barn. What that disease costs. If it
costs nothing, I am very likely not going to do anything.

The impact on the pigs\' well being of that particular disease; how does
it affect their well being. So we may have some diseases that maybe
don\'t have a huge cost, but they may affect the well being of some
individuals, and we may decide to treat on that.

What will likely happen if I ignore it? Will it go away? You know, the
vast majority of infections that take place just go away on their own.
And fortunately, for veterinarians we ride the descending curve, if you
remember your veterinary school of animals and individuals getting
better. Some will stay the same or some will get worse.

The cost of the proposed management changes in treatment. That is one of
the things we are going to always weigh. What is this going to cost, the
scenario that I am treating, versus what is it going to cost for my
recommendation. And hopefully, my recommendation saves money. And the
likelihood of resolving the problem with these changes in treatment. So,
some probability of success.

And by the way, all of these slides are going to be available, I think,
to all of you. So you don\'t need to copy this down.

So, those are the intuitive decisions that veterinarians go through when
he or she is going to recommend treatment. Now, some folks have put
these into very elaborate spreadsheets, and they are very nice tools for
deciding whether to treat or not.

(Slide.)

DR. MORRISON: We diagnose a problem by the following: We are going to
look at records. More and more today pig production is based on very
elaborate and detailed record systems, and so we are going to look at
records. We may have feed consumption, water consumption, weight gain
curves, as well as mortality. Maybe we have a coughing index. Somebody
goes in and measures coughing. So we have got a lot of records to try to
figure out what is going on in that barn of 1,000 pigs.

We will have clinical signs obviously in history. We have got
veterinarians who are trained for many years to figure out what is this
picture telling me. We will have serology done both on cross sections in
some cases and serial bleeding. In other words, we will bleed pigs over
time to figure out are they sero converting to agent \"x.\"

And then we will have postmortem on both pigs that die, and sometimes we
will sacrifice representative pigs and try to figure out what is
happening in the population here. And we will have some pigs that we
will sacrifice and do a postmortem in order to figure out what is going
on in the other 999.

(Slide.)

DR. MORRISON: Now, despite that sort of elaborate protocol that we will
have to try and figure out what is going on, we have some systems that
\-- like here is a farm that has a problem, and you can see each dot
there is a group of 1,000 pigs. And the \"xx\" that is down at the
bottom is time. And the \"y\" axis, by the way, is mortality.

So assume for the moment that every dot is 1,000 pigs, and you can see
that way back in \'96 they had roughly, you know, one percent. Down at
the bottom left you can see we are down around one percent, two percent
mortality. And over time, what you see here is that this mortality is
going up, and, of course, that is very disconcerting to the owners, very
disconcerting to the veterinarians \-- their jobs are on the line \--
and they are spending a lot of money to try to figure this out to make
this curve go down.

But this is a very frustrating case. You can also note, for those of you
who are unfamiliar with finishing, we like to have mortality down two
percent or lower. You are always going to have a few die that just \--
whatever. But we like to have it down in this one, two percent range.
That is a nice, well run barn.

But when you are getting up here in this five, six, seven percent, you
can see that this is also very unpredictable. This is just a nightmare
for these folks to try and figure out what is going on and how to fix
it.

(Slide.)

DR. MORRISON: In that graph what I was showing you was mortality, and
something that we teach and emphasize a lot is that mortality is
probably just the tip of the iceberg and underneath that. So we said we
will tolerate one, two percent mortality. When you get up three, four,
five percent mortality, what you have got is a lot of pigs that are
going to become sick and need to be culled. Or at least they are going
to go to market light than what we would like. Okay? And that is a loss.

And then, furthermore, you have got pigs that are growing slower. This
top group there, some of those will go to different market, like a light
market in town where they consume pigs that are much lighter than most
of those. The greater majority of those are going to go to market light,
and they are going to cost me an enormous amount of money. So mortality
is just the tip of that iceberg when it comes to cost of the producer.

And so, I have got my barn of 1,000 pigs and let\'s say only four
percent are going to die, and I say only in quotation marks. But 96
percent of them are affected, and so that is very, very important when I
make my treatment decision.

(Slide.)

DR. MORRISON: An important point for me is that treatment is a
short-term expense. Every time I add a treatment, it costs money. It
takes money off the bottom line. So I don\'t want to treat any more than
I need to, but you weigh that against management change. If I can go in
and take that last graph where that line was going up, every group of
pigs there is probably going to require some sort of treatment. That is
an expense.

Now, I am going to weigh what can I do in that system or that barn as an
investment to try to change that, to try to turn that curve around. So
management is going to be viewed as a long-term investment, and I am
going to look for some return on the investment.

And you can see here that we might look for changes in housing, we might
change the way by which pigs flow, we might change the health of the
incoming stock and lastly, at the bottom, we might change feed and water
supply. All of those are going to cost, depending on the size of the
farm, hundreds of thousands, perhaps tens of thousands; perhaps millions
of dollars.

I will give you an example, a recent example looking at some numbers
from a farm system. They have looked at groups that go through barns
that have natural ventilation. So the curtains are open; nice summer
breeze coming through. They have looked at those versus groups that go
through with mechanically ventilated barns. So you have got fans
exhausting air and controlled inlets letting the air in.

They have determined that in the groups that have the mechanically
ventilated barn they have roughly about .8 percent lower mortality than
the groups that go through with curtain ventilated barns, and they said,
well, we have to look.

If we have curtain sided barns, we know that we are going to have higher
mortality, we are going to have poor feed efficiency, we are going to
have lower gain, we are going to have more expense for treatment, so
let\'s ante up. And they are actually spending \-- I think it is
\$20,000 per barn to change it over to a mechanically ventilated barn so
that they can reduce the cost and they can improve the performance on
every group that goes through there.

So that is a management change that they will try and impose across all
barns, such that they reduce the treatment expense.

(Slide.)

DR. MORRISON: The treatment program is selected based on these following
criteria: And Dennis gave you a nice summary of actual treatment that
the poultry business uses. I am not going to go through drugs, but they
all will follow this sort of a regime.

What is my diagnosis? And it is a best guess. Hopefully, it is an
educated best guess. Hopefully, it is right. But what is my presumptive
diagnosis? What do I know about this herd historically? What is the
prevalence and incidence of this disease? Do I need to treat at all? Is
it just one pig in the 1,000? And the incidence. How many new pigs are
getting sick every day?

Will the owner do or will the staff do what I ask them to do? A common
cut off let\'s say is in the 10 or 20 percent range of infected pigs or
affected pigs that need treatment. If more than 20 percent are infected
and affected, I probably am not going to get that owner or staff person
to go in and inject pigs. It is just too big a task.

And so, if I say, listen, it is really cheap if you will just go in and
inject these 250 pigs once a day for three days or twice a day for three
days, forget it. You know, the staff person who is getting \$8.00 an
hour isn\'t going to do it.

So I may have to go in and water medicate. Or, in some cases, as we will
see, I may have to go in and feed medicate, because I can\'t get them to
do what I think they ought to do, from a compliance point of view.

Benefit costs of treatment options. I am going to think about that. I am
going to look at my lab results. I am going to weigh, obviously, my
clinical experience and the antibiotic options available. All of those
are the criteria that I am going to consider.

(Slide.)

DR. MORRISON: Now, I just want to \-- there is a very detailed treatment
set of guidelines that the veterinary employs when choosing a treatment.
I just want to go over these very quickly.

(Slide.)

DR. MORRISON: First, the veterinarian is going to decide, well, am I
going to inject or water medicate? Am I going to use a food animal drug
or, in some cases, in the rare case, a non-food animal drug? Am I going
to use it according to label or so-called extra label? If I go in with
an injection or a water medication, am I going to follow up with feed
medication or am I just going to stop? And lastly, if I do switch to a
feed medication, when am I going to stop medication. So that is the
steps that a veterinarian will go through.

(Slide.)

DR. MORRISON: Just very quickly, the first decision. Well, is it three
pigs out of 1,000 or is it 250? So, if it is 250, I am probably going to
have to go in and water medicate because I won\'t get very good
compliance on injection.

(Slide.)

DR. MORRISON: The route of choice is always going to be to choose a drug
labeled for food animals that contains the proper ingredient. It is
always going to be your first choice.

(Slide.)

DR. MORRISON: And then you are going to \-- if you have got this drug in
the proper dosage form, as labeled for the indication, and I believe it
is clinically effective, I am going to use it according to label.

(Slide.)

DR. MORRISON: If not, if not in the proper dosage form, it is not
labeled for indication and I don\'t think it is clinically effective, I
go to extra label. Where I require these criteria I have got to have a
veterinary/client relationship. I have got to know these pigs. In a way,
I have got to be personally responsible and I have got to be available.

I have got to sufficient scientific information to insure an adequate
withdraw, I have got to be able to preserve animal or group ID and I
have got to have records and labels.

(Slide.)

DR. MORRISON: In the rare case where we can\'t go this route, we are
going to use a non-food animal drug where it is not prohibited, and
again, very importantly, where I have got enough evidence to give a
valid withdraw time.

(Slide.)

DR. MORRISON: Dennis covered this briefly. When do we medicate in the
feed? Well, why would we? We would because it gives us the broadest
coverage of the population at risk. I can medicate them all very easily.
It is very labor efficient, it is very simple and it may be the cheapest
program.

But why don\'t we always just go in and medicate with feed then? Well,
it is probably somewhat difficult to achieve therapeutic levels in sick
pigs. Sick people don\'t eat, sick pigs don\'t eat, and so they are not
going to get the medication that we want them to get.

There is the potential for contamination of other feeds. Some pigs that
are not sick will receive the medication. We don\'t want that. They are
at risk, but it is kind of a waste, unless they are going to get sick
without it.

And in some cases, just as it may be the cheapest program, it may be the
most expensive program. So you are always, as a veterinarian, going to
be weighing this. What is my treatment program?

(Slide.)

DR. MORRISON: I did a brief \-- just a little survey in preparation for
this meeting, and I asked some veterinarians, and I was quite impressed
with their awareness and compliance with the guidelines. Some of these
veterinarians have very detailed treatment protocols for clients.

They told me that they choose their product selection based on
effectiveness first and cost second. And remember, this effectiveness is
going to be their clinical perception in many cases and based on
historical experience. For some of them I saw some very elaborate
spreadsheets for comparing drug costs and routes of delivery.

Generally, they will go between 10 and 20 percent as their cut off for
whether they are going to go for injection versus water. Feed medication
was generally used for chronic or preventive situations. And lastly, I
saw a nice spreadsheet for cost effectiveness of growth promotants.

(Slide.)

DR. MORRISON: The last slide. I think it is important to recognize that
within our industry I showed you an example of a 1,000 pig barn. We
could go and I could take you to some barns that are not, in my view,
well run. And I could take you to some other barns that are incredibly
well run.

And so, in our industry I think of health management as being on a
continuum, and you have got some farms out here that don\'t have good
health and you have got some farms out here that have extremely high
health. And when you look at sort of the descriptors of a low health
system or a low health farm, you will find that \-- this is probably
more detail than you need, but they will have multiple sow sources
feeding in. So it is just like that daycare facility.

If you have got 50 kids in a daycare, they are going home every day to
50 different homes and bringing back 200 bugs the next day. Okay? So
multiple sources just creates a wonderful environment for disease to
transmit versus one source.

Again, I won\'t go through any details, but all of these are descriptors
of a poorly run management system. And you come over here and all of
these are nice. We know, from experience, that those are going to be
well run barns if they can do these sorts of things.

(Slide.)

DR. MORRISON: Now, if you could look at the whole industry out there,
you can imagine that there is going to be sort of a bell shaped curve or
normal distribution of health, and the majority of them are going to be
somewhere in the middle. And we will have a few out here that are really
well run, and we will have a few out here that are really poorly run,
and these are the ones that are real challenges.

(Slide.)

DR. MORRISON: The challenge for us as veterinarians \-- this is a fancy
graphic now \-- is to take this and move this curve in the right
direction. So it is a continuum. It is important to recognize that
health management out there is a continuum. We, as veterinarians, are
trying to move everybody over to the right, and it is all sort of
process that we are moving in.

And I am very confident. If I look back where we were 15 years ago \-- I
was in practice at the time \-- I would liken a lot of those farms over
there to low health farms. And I look at where what we work with today,
and a lot of them are moving well over here. It is incredible.

And if I look 15 years from now, I am quite certain that we are going to
be over here on this side of the graph as we continue to move farms off
to the right.

(Slide.)

DR. MORRISON: I would just like to acknowledge that I did do this
survey, and I appreciate the participation of these veterinarians who I
contacted. I also used the AVMA brochure. And also, the AASP has a
pharmaceutical issues task force that I am a part of, and I appreciate
my participation in that group, Tom. Thanks very much for your
attention.

(Applause.)

CHAIRWOMAN LATHERS: Thank you, Dr. Morrison. Are there any questions for
Bob?

(No response.)

CHAIRWOMAN LATHERS: If not, we will now move on to our next discussion
of antibiotic use in aquaculture, presented by Randy MacMillan. Dr.
MacMillan is vice president of research and environmental affairs at
Clear Springs Food, where he is responsible for the research and
development program environment, stewardship and quality assurance.

He is the president of the National Aquaculture Association and the past
president of the U.S. Trout Farmers Association. He is also the past
president of the Idaho Aquaculture Association and the past president of
the American Fisheries Society for Fish Health Session. He is the
current chair of Minor Use/Minor Species Coalition. So, with that, we
will now have a discussion of antibiotic use in aquaculture. Randy.

**ANTIBIOTIC USE IN AQUACULTURE - AN OVERVIEW**

**By Dr. Randy MacMillan**

DR. MacMILLAN: We are having to reboot. I am not promoting Microsoft. It
just happens to be what is on this computer.

(Pause.)

DR. MacMILLAN: I represent a minor animal species group, and we don\'t
have the kinds of resources that other sorts of people have, other sorts
of animal industries have. When you think about minor animal species, it
is important to understand why they are minor animal species. It is
because not many of those animals are eaten.

And in aquaculture, which has been around for 300 or so years in the
world, it is a very young industry in the United States.

(Pause.)

DR. MacMILLAN: So what I will do is go ahead and shut this down
completely, and I will go ahead and start.

CHAIRWOMAN LATHERS: Please do.

DR. MacMILLAN: And then we will go quickly through some of these slides,
once this boots up properly.

I think one of the big questions before this group is to what extent the
antibiotics currently used or potentially used in U.S. aquaculture, and
I really want to emphasize United States aquaculture and emphasize that
throughout this presentation.

But, to what extent the antibiotics or potentially used in U.S.
aquaculture contribute to increased morbidity or mortality, resulting
from a reduction in the efficacy of a specific antimicrobial therapy of
human disease as a consequence of antibiotic resistance by the bacterium
involved in the disease process. And I have a slide that shows this.

But as I understand the purpose of this workshop or the task before us,
it is to identify, with objective methods, how we are going to
quantitate the risk. And I can tell you in United States aquaculture
this is going to be a very formidable task, because there is
considerable evidence, United States evidence, that the risk is so very
low, in spite of some of the rhetoric that has gone on before us, before
me anyway, about how dangerous aquaculture is.

Unfortunately, what has happened is people who have made those claims,
those statements, are using aquaculture practices that are practiced in
third world countries that actually dump human sewage and homothermic
animal waste into those aquaculture facilities as a way to fertilize
those facilities; to provide nutrients for algae that provide food for
zooplankton that then provide food perhaps for the fish.

In the United States, that doesn\'t happen at all, and so, we have
really gotten off the realistic track of what happens in U.S.
aquaculture.

(Pause.)

DR. MacMILLAN: Okay. Here is my opening slide. Again, I wanted to focus
on United States aquaculture, because it is so different than virtually
any place else in the world.

(Slide.)

DR. MacMILLAN: I would like to cover what aquaculture is in the United
States, what our basic culture methods is, because I suspect most people
here have been acquainted with terrestrial animal agriculture. Very few
of you are acquainted with aquaculture, with the growing of animals in
the water.

I want to cover very briefly what we use antibiotics for, how we use
them and, I might add, we only have two, two antibiotics that we can use
in the United States for food fish production.

What are our basic controls? That is, what controls do we use to insure
the judicious application of antibiotics. And then I would like to, with
whatever time I have left, talk about the potential public health risk
and canopy measure in the use of antibiotics in aquaculture.

(Slide.)

DR. MacMILLAN: So, first of all, what is U.S. aquaculture? Well, it is a
very diverse industry. The U.S. Agriculture Department recently
completed a survey of aquaculture in the United States. The very first
one was completed in 1998, and it identified 35 different species of
aquatic animals that are raised in the United States; 35 species that
they could identify or gather enough information on.

There is actually about 50 or so different species that are raised
commercially under aquaculture conditions. Those species are raised in
both fresh water and marine environments, and that becomes a critical
issue in determining where risk might lie.

The species are raised under warm water conditions and cold water
conditions, and that also is a critical factor in identifying where risk
could occur or where potential risk is likely to occur. And as we go
through the next few days, I would suggest to the participants that
temperature and the type of water are going to be key factors that we
need to look at.

We raise vertebrates and invertebrates. The vertebrates are catfish,
trout, salmon, tilapia. The invertebrates are oysters, shrimp and
crawfish, just as examples.

Crawfish don\'t use antibiotics. Nobody in the crawfish enterprises use
antibiotics. Shrimp farmers in the United States should not be using
antibiotics because they are illegal to use. Shrimp farmers in other
countries might be using antibiotics. Catfish, trout and salmon
producers do have two antibiotics that they might elect to use.

(Slide.)

DR. MacMILLAN: We raise food animals and non-food animals. A lot of
non-food animals are imported into the United States in way of the
ornamental fish trade. Sturgeon are raised, a very small industry for
sturgeon, and then tilapia are raised. These are both food animals.

(Slide.)

DR. MacMILLAN: Again, at least 35 minor animal species raised in the
United States. They are raised in various types of cultural practices.
One of the most common is with the ponds. These happen to be catfish
ponds from the Mississippi Delta. Those ponds are generally about three
feet deep. They may be 20 acres in size.

In previous history, they were 50 acres in size. Harvesting upon that
size is really a difficult thing, but 20 acres is more manageable. These
same kinds of ponds may be used to grow shrimp on the coastal areas of
the United States. We have a very, very small shrimp industry in the
United States. There is much more shrimp produced in Ecuador and China
and Thailand than in the United States. Far more.

(Slide.)

DR. MacMILLAN: Another culture method is with flow through systems.
These are typically used for trout culture. These are earthen bottomed.
This particular picture shows an earthen bottomed, earthen sided pond.
They may be cemented ponds. The raceways may go from one raceway to the
next to the next. The water is used repetitively. In this case it is
not.

The water quality requirements for the animals raised in this type of
aquaculture condition are far more stringent than those in the pond
aquaculture conditions, and that is another key factor in identifying
where the risks might come in aquaculture practices.

The water in these systems goes through very rapidly. Frequently the
water right requirements are that you cannot consumptively use the
water. It has to go in and out; in and out. In catfish aquaculture you
can use the water consumptively, and so those ponds are typically static
ponds. Water exchange doesn\'t occur.

Again, those aquatic animals don\'t require the same level of
environment, environmentally stringency, that these colder temperature
animals require.

(Slide.)

DR. MacMILLAN: And then, net pens. Net pens can occur in \-- this is
where they have a netted area for the aquatic animals to be placed and
they are fed there. It can occur in fresh water ponds, in rivers and
most frequently in the ocean.

Much of the salmon production in the United States, and certainly
elsewhere, occurs in net pens in estuarine and in ocean areas.

(Slide.)

DR. MacMILLAN: There is a small type of system that is being looked at.
There is really not any commercial production yet, although people have
been in it for just a few years. But it is with closed recirculating
water systems. Mostly fresh water systems that replace some of the water
daily, and they discharge a small but concentrated eflon (sic.)

(Slide.)

DR. MacMILLAN: So, antibiotics in U.S. aquaculture. In the United Sates
we have two. In other countries there are far more antibiotics
available. In Japan, for example, there are 29 antibiotics available for
aquaculture. In the United Kingdom there are four. In Norway there are
eight. In Chile, anything goes. In Ecuador, anything goes. In China,
anything goes. They don\'t have the same regulatory framework in those
countries that we do in the United States.

(Slide.)

DR. MacMILLAN: So the two drugs that we have available in the United
States for food animals only is oxytetracycline and Romet-30.
Oxytetracycline has been around for, I guess, 30 years or so. Romet-30,
a potentiated sulfonamide, has been around since about the mid \'80s.
No. Mid \'70s to \'80 or so. For very few types of aquatic animals, and
it is only in the feed.

We raise fish in a very intensive way. They are in the water, so they
are not very accessible. The only way we can deliver an antibiotic or
any other kind of drug, in a purposeful way anyway, is in the feed.
There are some water treatments, but those are not antibiotics that are
used for the water treatments.

(Slide.)

DR. MacMILLAN: The NADA is for catfish, salmonids and lobsters only.
They each have a pretty long withdraw time of 21 to 30 days. The lobster
is for the treatment of gaff kemyia. For catfish and salmonids it is for
the treatment of modal \-\-- septicemia, a specific disease of fish
caused by erramonis hydrofla for example. And then for catfish it may
sometimes be used for enteric septicemia of catfish, although that is
not on the label.

(Slide.)

DR. MacMILLAN: The other antibiotic that is available is Romet-30, the
potentiated sulfonamide. For catfish there is a three-day withdrawal
time, and this is specifically for the treatment of a disease called
enteric septicemia of catfish, ESC. There is a three-day withdrawal. And
here, Romet is for the treatment of furunculosis. There is a 42-day
withdrawal time.

The reason for the difference in withdrawal times is that with the
catfish, when they are processed, the skin is removed and this
particular antibiotic can concentrate in the skin. With the salmonids
the skin is left on. So there is a much more protracted withdrawal time.

The interesting thing with Romet 30 is it is hardly used in either
industry anymore. With the salmonids it never was particularly valuable
because of the long withdrawal time. With the catfish they found
alternate ways to manage that particular disease.

(Slide.)

DR. MacMILLAN: One of the things about aquaculture in the United States
is that we don\'t use antibiotics as growth promotants. Never have, and
I can\'t envision ever doing it for two reasons. One, it is very
expensive. But number two, it doesn\'t work. At least not in fish.

We have done some research in my previous history at Mississippi State.
We looked at antibiotics in a research situation to see if we could
promote the growth of catfish. It didn\'t work. I am not aware \-- and I
am fairly familiar with U.S. aquaculture. I am not aware of anybody in
the United States that uses antibiotics as growth promotants.

(Slide.)

DR. MacMILLAN: And there are several reasons why it doesn\'t work. The
most important thing is that the bacterial flora in poikilothermic
animals is itinerant. There is no resident flora. Whatever the fish or
shell fish is eating, that is what will be in the GI tract of that
animal. Or whatever is in the water.

If you take a catfish in cold temperatures and don\'t feed them, their
gut, their GI tract, will essentially go sterile. There is no need for
the bacteria there. It is sterile. If you change the water quality of
the fish, that bacterial flora that you might recover will change.

Poikilothermic animals in aquaculture has what I would call several
natural barriers to the transmission of antibiotic resistance factors or
the occurrence of human pathogens in their system. One is that there are
some basic physiological differences between cold blooded animals and
warm blooded animals.

Those differences become very important when you look at the potential
pathogenicity of bacteria. It is very difficult, for example, to take
salmonella typhimurium that you recover from a catfish or a tilapia and
infect a mammal with that bacteria. There appears to be some sort of
biological adaptation, microbiological adaptation, that has to occur
before that bacteria can cause disease of any kind in a mammal.

(Slide.)

DR. MacMILLAN: There is also some basic temperature differences. Cold
blooded animals are just that, they are cold blooded. So the culture
conditions vary anywhere from say nine or so degrees centigrade, up to
30 degrees or so centigrade.

Most of the bacteria that we are concerned about thrive not at those
colder temperatures, but at the warmer 30 degrees and above type
temperatures.

(Slide.)

DR. MacMILLAN: If you look at \-- and I am not a food scientist or a
food safety expert, but I went through some food safety text and look at
the growth parameters or growth conditions, optimal growth conditions
anyway, of some of the bacteria that we are perhaps looking at here.

Camplyobacter jejuni, no growth at less than 30 degrees centigrade.
Salmonella species, there is a whole complex of salmonella species, the
optimal growth is at 37 degrees. That doesn\'t mean it can\'t happen at
a cooler temperature. It could. But the optimal growth is at 37. E.
coli, 37; shigella, 37; vibrio, 20 to 30 degrees centigrade, but that is
strictly a marine type bacteria.

Forsinia (sic) enterocolitica has a pretty good temperature range, as
does lysteria monocytogenes. The warmer the temperature those, even for
those bacteria, the faster it will grow. None of these bacteria infect
fish. They may occur, but they don\'t cause disease in those fish.

(Slide.)

DR. MacMILLAN: There is also no resident microbial flora on the fish. As
I mentioned earlier, the bacteria that are in the water at the time,
that is what is going to be on or in the fish, the GI tract of the fish.
Okay?

There is also a very, very large water dilution effect. If you think of
raising fish in the ocean, just think how big the ocean is. In most, but
not all, aquaculture situations that are profitable, they have a large
volume of water at their disposal. That is going to cause a tremendous
dilution effect in real life, and that has an impact or potential risk.

There is a limited human aquaculture fish environment interaction where
people don\'t usually get into the water to be with their farmed
animals. It can happen, but it is usually by accident.

(Slide.)

DR. MacMILLAN: There are certain management practices in the United
States that also limit the potential for bacteria to get into humans
from the aquatic environment or for resistance factors, plasmids, for
example, to get transmitted on up the line. One, we use clean water.

The World Health Organization, about 10 years ago, estimated that about
\-- let me see \-- two thirds of the world\'s aquaculture was produced
in environments where human sewage and homothermic animal waste were
purposely put into the ponds or rearing environments for fertilization
purposes. Two thirds.

In China alone, which produces about 65 percent of all the aquaculture
in the world, they still do that. They are changing. They are getting
away from the human waste, but they are still doing the homothermic
animals. The poultry, the pigs and whatever else. That still goes into
aquaculture situations.

In Israel and in England and the UK, the placement of animal manures
into those aquaculture environments goes on. In the United States that
doesn\'t happen. That has a dramatic impact on the types of bacteria
that are present, and hence, a dramatic impact on the relative risk of
an aquaculture practice.

(Slide.)

DR. MacMILLAN: There are very, very few icthyozoonoses associated with
aquacultured fish. Those that have been suspected are of an
international flavor. For example, in Ecuador. There has been a
suggestion that shrimp were the source of an antibiotic resistant vibrio
cholera. Well, Ecuador, in all due respect, their waste management
practices are not nearly as good as we have in the United States. Just
their basic waste management practices.

Another place is in Japan where they \-- in this particular case it was
because they were eating live fish. And then one is in Israel where
somebody got spined from a live fish and they perhaps got exposed to a
vibrio that caused \-- actually caused a mortality.

We have very, very few food-borne pathogens associated with aquaculture
fish. The FDA, in 1998, did a salmonella survey of seafoods, wholesale
seafoods, and seafoods, in general, had about the same cleanliness, if
you will, as red meat products. Red meat products.

About two and a half percent of the seafood they tested, in a global
sense, had salmonella recovered. The catfish, which were all domestic
aquacultured catfish, had about 10 percent salmonella identified.
Tilapia, about six percent. These were imported tilapia, not those
raised in the United States. And then shrimp that were also imported in
the United States, and they had about two and a half or so percent.

The MPNs, the most probable numbers for those, in all those cases was
very low. We are talking .004 to .022, the most probable numbers for
salmonella recovery, meaning that there are very few bacteria present.
The place where they found a lot of bacteria was in wild harvested
shrimp from India.

(Slide.)

DR. MacMILLAN: Another challenge to identifying the risk associated with
aquaculture is that we are so diverse. We are all minor animal species,
so human consumption patterns are going to be very difficult to track.

Another real complicating factor and something that has probably
promoted some misunderstanding about aquaculture\'s role, or potential
role, in the antibiotic resistance issue is that there are bacteria that
grow under aquaculture environments without any antibiotic exposure who
are resistant to the antibiotic, and it really becomes important then to
track and identify the causes of antibiotic resistance.

Is it something that is transferable or not? In this particular case it
was not transferable, but it is a very prevalent finding. It appears to
be associated the \-- in certain aquaculture environments with a highly
nutritious environment, not with antibiotic exposure.

So it is one of the complicating factors that we are going to have to
look at as we move forward in identifying ways to identify risk.

(Slide.)

DR. MacMILLAN: The pathogenic potential of most aquatic bacteria is low.
It is not to say that they can\'t be made pathogenic. They can be made
pathogenic. But it takes several passages through a mammal before they
can become pathogenic.

The microbial flora in the GI tract is itinerant, as well as on the
skin. The measures of resistance that aquaculturists and bacteriologists
that have looked at this in the aquatic environment \-- they have
different measures of resistance internationally, and that is a real
problem in terms of identifying what the real risk is.

It is possible, under laboratory conditions, to demonstrate plasmid
transfer from fish pathogens to potential human pathogens. You can go
the reverse as well. Human pathogens can transfer plasmids to fish
pathogens under laboratory conditions. What we don\'t know is what the
probability of that happening is, and I would suggest to you that 99.9
percent of the time it is a very, very low probability.

(Slide.)

DR. MacMILLAN: So how do we measure? In large respect, the issue, in my
view, for aquaculture comes down to how do you measure the environmental
impact of antibiotic use in aquaculture? How do you measure the
environmental impact? And that is also going to prove to be a very
difficult thing.

(Slide.)

DR. MacMILLAN: There is a cascade of things that has to happen for an
antibiotic that is given to a fish to treat a specific disease; that has
to happen in order for that to eventually have an impact on a human
pathogen. It has got to go through the fish, it has got to be excreted
by the fish, which a large part of antibiotics, the two that we have,
can be excreted by the fish.

It has got to get into the water column, into the sediment, into the
bacteria that are present in the sediment or the water column, and these
are mostly aquatic bacteria that won\'t effect people, and then it has
got to get a plasmid.

For example, it has got to get transferred from the sedimentary type of
bacteria to the terrestrial type of bacteria and then, from there, into
a human and then, from there, to cause disease and then it has to be a
type of bacteria that is resistant to a particular antibiotic that a
person would use. Quite a cascade.

What that means though is that it is going to be very difficult to
quantitate the probability of that happening. I would suggest to you
that the use of antibiotics in U.S. aquaculture has an undetectable
impact on the prevalence of human pathogenic bacteria resistant to
bacteria.

There is an overwhelming bit of qualitative data that supports that
contention. There was a report put out in 1997 by a couple of scientists
from the United Kingdom entitled, \"The Use of Antimicrobial Agents in
Aquaculture.\" It is a report to the Advisory Committee for the
Microbial Safety of Food, the ACMSF working group on antimicrobial drug
resistance.

In this report they shared the same opinion that I do about the relative
risk of aquaculture. It is very, very low. It is not impossible, but it
is very, very low. What they identified as the greatest risk is with the
use of antibiotic in ornamental fish. That is where, from their view,
there is the greatest potential for the transfer of resistance from the
fish to people.

The one last thing is that relative risk is going to be dependent on
water temperature, the species raised and the presence of human or
animal waste. Thank you for your forbearance.

(Applause.)

CHAIRWOMAN LATHERS: Thank you. We will now take a break, and, please, be
back here at 10:30.

(Whereupon, a brief recess was taken.)

CHAIRWOMAN LATHERS: I think it is time to begin. We have now completed
our discussions of antibiotic use in ruminants, poultry, swine and
aquaculture. We will now begin the next session with a discussion of
antimicrobial drug discovery and development by Dr. Jeffrey Watts.

Jeff has a BS in microbiology and a master\'s in microbiology, both
earned at Louisiana Tech University, and he tells me that as of just
February 15th he has completed successfully his Ph.D. dissertation
defense. Congratulations, Jeff.

(Applause.)

CHAIRWOMAN LATHERS: That was in biological sciences, and he has earned
this at Western Michigan University. He is presently a clinical research
scientists too in worldwide product division at Pharmacia and UpJohn
Animal Health in Calamazoo. Jeff.

**ANTIMICROBIAL DRUG DISCOVERY AND DEVELOPMENT**

**By Dr. Jeffrey Watts**

DR. WATTS: Thank you, Dr. Lathers. What I am going to do over the next
few minutes is talk about antimicrobial discovery in animal health, and
particularly, I am going to talk about the impact of the resistance
issues on discovery programs in animal health.

(Slide.)

DR. WATTS: What I am going to do is briefly frame up the resistance
issues, then I am going to talk about the antimicrobial discovery
programs, starting with the human programs and moving into the animal
health programs. It is essential to do it this way because the animal
health programs, as you will learn, very much live at the knee of their
parent.

Then we will talk about the issues that effect antimicrobial discovery
in animal health, what I call the environmental factors, the impetus to
move away from broad spectrum compounds, the impact of the framework
document; should we move toward vaccines, the other things that the
antibacterial support groups do in animal health companies, including
service support activities, and then wrap up with some comments on the
future of discovery in animal health.

(Slide.)

DR. WATTS: Just to briefly frame this, as you know, the emergence of
resistance organisms in human and veterinarian medicine is of great
concern. The more resistant organisms tend to be predominantly those
nosocomial in humans, with the veterinary contributions primarily
through zoonotic pathogens.

There have been short-term responses to these issues, and these include
things like the development of use guidelines, the development of
formularies and therapeutic guidelines and restricted uses of selected
compounds.

(Slide.)

DR. WATTS: When you talk about discovery, you are looking more than two
to three years out. You are looking usually at seven to 10 years out. So
what are the longer term effects, looking at 2005 and beyond? Will the
antibiotic resistance issues prevent the introduction of new
antimicrobial agents in food animals? That is the key question.

Will companies chose to stay in the food animal markets or in animal
health at all? And will clinicians have therapeutic options for current
pathogens or for new emerging pathogens?

(Slide.)

DR. WATTS: Let\'s talk a little bit about the human discovery programs.
The cost of developing a new compound is very high. The estimates for a
new human use antibiotic are \$125 to \$350 million. I have heard
estimates on some of the newer compounds of more than \$500 million.

The time it takes from the time that compound is initially discovered to
the time it is introduced to market is 10 to 12 years; however, the
markets tend to be quite large, or can be quite large, with markets
easily being \$500 million and several compounds making over a billion
dollars per year.

(Slide.)

DR. WATTS: Over the last few decades there has been several strategies
for countering resistant organisms. These are what I term, for the most
part, incremental improvements. It is improving existing structures.

We have seen this happens with beta-lactams and various generations of
cephlosporins, the fluoroquinolones, the antibiotic inhibitor
combinations, what I call re-trading of older compounds, things like the
amoxocillin clavulanate combinations, which can be quite successful.
Augmentim, at its peak, I believe sold over \$2 billion a year
worldwide.

The problem with these types of strategies is the resistance mechanism.
The basic mechanism is already in place, and all it takes is a minor
modification by the organism to ramp resistance back up. So optimally,
what we should do is screen for compounds with new mechanisms of
actions.

(Slide.)

DR. WATTS: And so, our classic screening program involved a
streptomycetes type of fermentation. We would then screen for inhibitory
activity. We would discard any hits here if there was not activity, or
we would discard if there were no hibs, no activity or any hibs that
turned out to be nuisance antibiotics.

If it was active, if it appeared to be unique, then we would go through
a re-fermentation process. The activity would be confirmed. We would
scale up the chemistry efforts, we would identify the structure, then we
would chemists at it into a synthetic chemistry program to develop new
analogs.

(Slide.)

DR. WATTS: In the \'80s this system collapsed, and the reason it
collapsed was we had over 6,000 antibiotics and the system we used could
not recognize new structures. Also, the antibiotic business was
changing. There were only a few mechanisms of action. The customers were
becoming rather disgruntled. They could only stand so many third
generation cephlosporins being introduced into the marketplace, and
there was also a question of whether or not they even needed new
antibiotics.

(Slide.)

DR. WATTS: So the current paradigm is that now what we are doing is we
are using a molecular target. This is targeted as our mechanism of
action. We clone and express this target, we devise an assay, we now
screen chemical libraries and natural products through this assay, we
then select our lead compounds, and again, we throw chemists at it into
an analog to develop usually thousands of analogs to screen from, and
this is what has been termed a mechanistic screening program.

(Slide.)

DR. WATTS: And through the older techniques of incremental improvements
\-- and we are starting to see some of the \-- at least adding
components of mechanistic screening programs. We are seeing a variety of
compounds come to market in human medicine.

We are seeing the broader community use agents, which include the
extended spectrum fluoroquinolones, the glycosides; we\'re seeing
macrolides, particularly the azolides. The ketolides are in development
and moving through the pipeline.

We are also seeing narrow spectrum compounds primarily focused on the
very resistance organisms, such as the enterococci and resistance \--
staphorius, the improved glycopeptides, synercid, the everninomycins and
the oxazolidinones represented by \-\--

(Slide.)

DR. WATTS: So let\'s talk about the animal health markets. The animal
health markets tend to be much smaller than the human health markets.
Generally, they are about one tenth in size. They are usually split
among various animal groups, and these animal groups have varied use
practices and preferences.

Multiple indications are usually necessary in order for a compounds to
be successful in animal health. And because of these multiple
indications and varied use practices in preferences, there has been a
preference toward broad-spectrum compounds in most areas of veterinary
methods.

However, as I said, animal health companies live at the knee of their
parent. The parent company is relied upon for large scale screening, the
chemistry efforts to expand the template, the initial in vitro toxicity
screen, in vitro activity and toxicity screen, and even if you don\'t
work in a class that your parent is working in, you still rely heavily
upon them for things like path/tox services, formulation,
pharmacokinetics and manufacturing production. You live in their
infrastructure.

(Slide.)

DR. WATTS: So the way the process would work is that we would develop a
target compound profile. We would probably look at a large single market
for the first indication. We would have to define what that market would
look like seven to 10 years in the future. We would have to know what
our current competitors are, and we probably have some ideas of what
other compounds are in the pipeline that will be our future competitors.

And we have to know the compound attributes, particularly those that
give us a competitive advantage. And if you look at BRD as an example
here, in the 1980s we were driven by residues. We saw ceftiofur come to
market with no withhold. In the early 1990s this was changed into a
convenience issue where we saw tilmicosin become a dominant player

And so the question becomes, as we head into the 2000s, will resistance
become a dominant issue and a competitive advantage?

For the most part, the animal health discovery companies obtain their
lead compounds from the human health program. We also look at the
available in vitro activity and toxicity data, usually using a human
organism as our veterinary surrogate.

For example, of course, you look at data for E. coli. What you would
look for, if you were interested, is does this culture have pestoral
activity. I would look at H Flu data. Does it have streposis activity? I
would look at streptococcus pneumonia data. So you are making that
transition from those human pathogens.

You would screen for activity specifically against your veterinary
pathogens. These would be in vitro screen, MIC determinations, you would
then screen through various mouse models, target animal models and you
would like for demonstrated efficacy and safety at this time.

At this time you would transition to development, and this is where the
discovery scientist plays a key role, in that usually the discovery
scientist has to be an advocate for its compound, and they are
responsible for successfully transitioning those compounds from
discovery into development.

(Slide.)

DR. WATTS: If we look at the compounds that are currently available and
the programs they came out of, Tilmicosin came out of a animal health
program. The ceftiofur, pirlimycin, enrofloxacin and chloramphenicol
originally arose out of large corporate screens for compounds to be used
in human medicine.

This was the year of the first publication on these articles, and one of
the things you need to keep in mind is if you see ceftiofur at 1987,
that means that compound was originally looked at in about 1980. If we
look at florfenicol at 1980, that means the screen for that compound was
probably in the mid \'70s.

Pirlimycin, lincosamides, in 1985 first described. I can tell you that
the lincosamides screen for pilimycin was discovered in the mid \'70s.
So, when you start talking about new compounds that you would just
introduce, many times those compounds are 10, 15 or 20 years old.

(Slide.)

DR. WATTS: So the question becomes \-- is as we see the mechanistic
screening programs kick in in human medicine, as we see new antibiotic
and new antimicrobial classes with new mechanisms of action being
introduced hopefully over the next 10 years, then will animal health be
allowed to participate and be able to participate in this revolution.

So one of the things that we need to look at are the environmental
factors. Again, the changes in clinical use patterns, the argument over
whether or not we should be developing narrow versus broad spectrum
compounds, the regulatory environment, particularly the framework
document, and prevention strategies. Should we move to just vaccinations
and that becomes our dominant way of controlling diseases and they
replace antimicrobial agents?

(Slide.)

DR. WATTS: There is an excellent talk at ICAC this year by Dr. Bob
Mollering on the argument of narrow versus broad spectrum compounds in
human medicine. Narrow spectrum compounds target a given class of
organisms. Usually gram positive or gram negatives. They target a
specific genus or species even, while broad \-- the definition for broad
tends to be less defined.

We usually know a broad spectrum compound when we see it, in terms of
the type of spectrum it covers, but most people think of broad spectrum
compounds as those that cover both gram positive and gram negative
organisms.

(Slide.)

DR. WATTS: The advantages to a broad spectrum compound are that if you
have an unknown etiological agent, you can cover it or have a better
chance of covering. You can cover polymicrobial infections, and it
provides peace of mind for the clinician.

The disadvantages are that there is a greater impact on normal flora,
what is called the innocent bystander effect, selection of resistance in
multiple species of organisms and it may impart a false sense of
security to the clinician.

(Slide.)

DR. WATTS: The advantages of narrow spectrum are you have reduced
selection for resistance, it is targeted against selective pathogens,
and you have a reduced innocent bystander effect. The disadvantages are
you need a precise diagnosis, and it cannot be used to manage
polymicrobial infections.

(Slide.)

DR. WATTS: This is the way Dr. Mollering summed up his talk, and I think
it is the best way I have seen of summing up the argument of narrow
versus broad. \"Narrow is good, if you can live with it, and broad is
bad, unless you need it.\"

(Slide.)

DR. WATTS: So, should animal health companies focus only on narrow
spectrum compounds? The thing we have to realize is that it will require
more compounds in the portfolio. That is, a company, instead of living
on one or two compounds, now has to manage two, three, four and five
compounds.

You are going to have to have multiple classes of compounds, and that is
difficult to do if your parent program is heavily invested in one class.
So you are going to have to go outside your company in order to find
additional classes.

You have to provide support for each compound. Support means path/tox,
formulation, manufacturing, marketing. You will have limited indications
for each compound and limited label expansions. The problem you also
have is that marketers will tell you there is difficulty marketing
narrow spectrum compounds, particularly in markets where there are broad
spectrum agents available. In a market where there is a narrow and a
broad, the broad always wins and always dominates the market.

(Slide.)

DR. WATTS: The regulatory climate at this point in time primarily
revolves around the framework document, and I have tried to summarize
the categories here, with category one being the compounds considered
essential for treatment of serious or life-threatening disease in
humans.

Category two is important for treatment of serious disease, but
alternative therapy exists, and category three is limited or no use in
human medicine.

(Slide.)

DR. WATTS: What is the impact of a framework document? Short-term,
category one blocks animal health development for new classes in food
animals. Group two limits development to those indications with low risk
of resistance development, and category three will limit compounds to
those of low potency, toxicity problems or high levels of resistance in
human pathogens.

This links the veterinary use to the human use in terms of both
availability of drugs, particularly availability of drugs in human
medicine to treat specific infections, and the resistance levels in
human pathogens.

(Slide.)

DR. WATTS: Another thing we have been told is we should explore vaccines
as an alternative to antimicrobial agents. I believe that vaccines are
important and they are an important component of disease management
programs. They should be used when and wherever possible. I think
prevention is the key.

However, vaccines may not replace antimicrobial agents, and the reasons
are that effective vaccines are difficult to develop for many bacterial
pathogens. They target only one agent or one, so you have to have a
multivalent vaccine.

One of the things that we have very little information about, but
something that may be important, is that vaccines are a selective
pressure. They may change pathogen distributions and they could change
pathogen distribution to a more resistant pathogen. We just don\'t have
a lot of information on that.

Vaccine market cycles are shorter and the vaccine value tends to be much
lower. That is, because the cost of vaccines tend to be much lower than
it is for antibiotics, those market values tend to be much lower. You
tend to have to manage many more vaccines in your portfolio in order to
get the same value that you would for one single antibiotic. This is
truly on example where an ounce of prevention is not worth a pound of
cure.

And also, one of the things that may be required is surveillance of
effect on pathogen distribution. It may be necessary in order for us to
understand what is going on in these various management systems.

(Slide.)

DR. WATTS: The service support activities. This is what I jokingly refer
to as what your discovery people do in their free time. Most
microbiology expertise in animal health companies reside in the
discovery program. Usually more than 50 percent of their time and
resources are spent in this area each year.

You have to remember that most of these groups are fairly small. A group
with 10 to 15 people would be considered quite large for a dedicated
antimicrobial discovery program in animal health.

And these are sort of the things that they do, the activities that they
may be involved in: Generating MIC data for label expansions and
extensions, conducting MIC studies or in vivo to meet regulatory
requirements, a lot of the resistance monitoring efforts reside in the
discovery group, and also, susceptibility test development to support
those compounds. As resistance needs and monitoring needs have ramped
up, that is taking time away from discovery efforts.

(Slide.)

DR. WATTS: So what is the future of animal health antimicrobial
discovery compounds? The compounds currently in development will
probably be the least effective. They will probably make it to market
with some sort of indication. It may be a limited indication at first.

Many of these programs will be re-focused onto the companion animal
markets because the resistance issues have not been as great a concern
there. The food animal markets will be limited to those with reduced
resistance concerns.

The availability of new compounds and the decreased utility of existing
compounds in human medicine may allow the use of some of the newer
classes of antibiotics in food animals, but that is a longer term
scenario. And the gap in food animal compounds will begin to occur about
2005, unless directed efforts in this area remain in place.

(Slide.)

DR. WATTS: In order for this to happen, one of the things that the
animal health companies have to do is their management has to have the
resolve to stay in the game, and they have to have the resolve to make
sure the programs are adequately resourced.

Discovery programs must build in resistance as part of the target
compound profile. That is essential. And so we would do things like
mutation frequency studies, resistance mechanism determinations,
dose/use patterns that minimize resistance, and I will guarantee you
that as new compounds come to market that are safer in terms of
antibiotic resistance, that this will become a marketing issue once
these compounds to the market.

I believe that wraps me up at this point in time. Questions?

(Applause.)

CHAIRWOMAN LATHERS: Thank you very much. Are there any questions?

(No response.)

CHAIRWOMAN LATHERS: Thank you again. We now move on to our next topic,
antimicrobial new animal drug applications, a review process overview.
Dan Benz will be presenting this.

Dan has earned a BS at the University of Illinois and a master\'s at
Colorado State University. He has a Ph.D. in nutrition from Texas A&M
University, and he is presently an animal scientist in the ruminant drug
team in the division of biometrics and production drugs at the Office of
New Animal Drug Evaluation at the Center for Veterinary Medicine. Dan.

**ANTIMICROBIAL NEW ANIMAL DRUG APPLICATIONS**

**REVIEW PROCESS OVERVIEW**

**By Dr. Dan Benz**

DR. BENZ: Thank you. You may wonder why I am here this morning. Well, I
wondered that too. It is not because one Friday afternoon I was sitting
around my office and somebody came in and said, \"What are you doing the
22nd of February? Are you busy?\" And I said, no. Well, they said, you
can give a speech.

It is not because this was originally scheduled to be between 11:00 and
11:30 you would have lunch and somebody said, well, you can make it
short and go to lunch early. It is not for all those reasons. The actual
reason I am here today was that there was a request made that we tell
John Q. Public just what is required to support an NADA.

You know, there is a lot of talk about putting a lot of additional
requirements on the drug companies, so what is currently required? And
that is what I am going to talk about today, and this is pretty much
going to coffer all new animal drug applications, not just
antimicrobial, and I will show you some differences when we get into
those.

What are the contents of an NADA? Well, what supports an NADA? Well, the
first thing we have is a cover letter from the sponsor. They are going
to tell us what they want; a description of the request. We would like
to get this compound approved for this type of animal, et cetera.

We have a lot of miscellaneous information, patent information,
marketing exclusivity information that we tend to put in there. We have
a FDA 356V. I am not sure what the 356V stands for, other than I assume
it was the 356th numbered form that FDA had. I know the V does stand for
veterinary.

That form is based on the regulations 21 CFR 514. If you want to look
them up, that is where it is. A very important thing that I have bolded,
underlined and italicized is it must be signed by a responsible official
or authorized agent by the company. And if it is a foreign company, they
have to have somebody in this country that has the authority to sign
them.

(Slide.)

DR. BENZ: Now, for the quiz of the day. How many can read this? I know
you can\'t, but we are going to talk about it in my ensuing slides. But
I wanted you to see what an application looked like besides NADA. Drug
product, some information here below, some instructions for use,
Paperwork Reduction, a little spot for a doc unit to use. Also, it was
nice to figure out how to use Adobe Acrobat and get it into PowerPoint.

(Slide.)

DR. BENZ: A little information on the back side. Some fine print. Every
good form has got to have its fine print that you sign and don\'t know
about. And a place for the signature and their title with the date.

(Slide.)

DR. BENZ: What is on the form FDA 356V? Well, one of the first things is
drug product information, the established proprietary names. For example
\-- and I am going to stick away from the animal area so I can\'t get in
trouble by particularly picking anybody\'s product out.

Acetaminophen. Tylenol in the human area, the established proprietary.
Advil, Ibuprofin. So there is a couple of examples.

Dosage form. What form will that be? Will it be an injectable? Will it
be an oral in the feed? So we want to know what type will be used up
front.

Proposed indications for use. Whether it is going to be a production or
increased average daily gain, increased milk production, you can go down
that, or some therapeutic use. The species of the animal that it will be
used in: Cattle, swine, sheep, goats, horses, dogs, cats; whatever. And
its proposed marketing status, whether it is going to be prescription or
over-the-counter.

And I suspect, some time when this is updated, it is going to have three
prescription OTC in for the new class of veterinary feed directives. But
right now we have two on the form.

(Slide.)

DR. BENZ: Some additional information. Applicant\'s name and address. We
want to know where they are doing business. The type of application,
whether it is an original or a supplement. Original means it is the
first time we have ever brought it in. Maybe it is a new chemical
entity. We have never looked at it before. A supplement is something
that would be approved products already on the market and the firm is
trying to make some sort of change to that.

A reason for the submission. What are you trying to do? The good old
Paperwork Reduction Statements. We have to have that in there to be in
compliance with our OMB regulations and Paperwork Reduction Act. And
some instructions for submitting an NADA. How many copies we want, et
cetera, are all on that form.

(Slide.)

DR. BENZ: As I said, that was on the second page. There is appropriate
sections, and these sections are checked as necessary. I will give you a
couple of examples which I will go into later. But you don\'t really
have a need for human food safety in companion animals. So that section
would not be checked.

Also with companion animals the environmental assessment is a lot
easier. Lots of times they get a categorical exclusion. So those types
of things may or may not be checked.

And I said the fine print. That is the legally binding statements. No
one was debarred under the Food Drug and Cosmetic Act will be involved
in any capacity. That came out of the generic scandal. And finally, a
warning. A willingly false statement is a criminal offense. In my mind,
that says FDA does mean business.

(Slide.)

DR. BENZ: We are going to go down the sections; right down the 356V. I
know you couldn\'t read it. That is why we came to these slides. We have
the identification of the compounds, table of contents and summary.
Particularly the summary to describe the chemistry of the proposed drug
so we know what it is.

Its clinical purpose. Again, whether it is therapeutic, a growth
promoter. And the summary of the laboratory and clinical studies to
support that application.

(Slide.)

DR. BENZ: Labeling. We have product labeling. It may be the labeling on
a vial. If you also have any packaging that goes along with it. If a
vial comes in a box and that has labeling, we want to know that.

Package inserts. If you go to CVS and pull out a tube of ointment and
you have got an insert, we would also look at the same type of insert
that would be available for an animal drug. And then, if it is a feed,
we want to look at type A, B and C medicated labeling or the feed
labeling.

The reason that we need the labeling, besides the fact that it will be
put out for public display later on, is we also look at the labeling in
conjunction with the safety and effectiveness to see if the two
coincide, if the labeling is supported by the safety and effectiveness
data.

(Slide.)

DR. BENZ: Okay. The components and composition section, a list of all
articles used as components, the statement of the composition, a
description of the fermentation of the antibiotic drug; some sense of
how the products are made.

(Slide.)

DR. BENZ: Again, more on manufacturing methods, facility controls, the
personnel that are involved, the facility equipment, a description of
the drug synthesis, how it was made, raw material controls,
manufacturing information.

(Slide.)

DR. BENZ: And additionally, finished product controls, stability
program, container packaging, lot control number system. In a nutshell,
how was the product made and can they make it again and come up with the
same consistent product over and over.

And then finally, we have a way, with a lot control number system, et
cetera, to monitor that product. You know, if some product gets out in
the marketplace and it is recalled, you have to know where it came from,
and that is where the lot control number system comes from. So we are
looking at all \-- the complete manufacturing process.

(Slide.)

DR. BENZ: Samples. We hand ask for samples upon request. We seldom do. I
assume there must be a reason some time along the way that we have asked
for samples. Examples that I could think of is if you had a question of
is there an active ingredient in this drug or that type of thing.

And again, here is one that I said earlier that only applied to certain
ones as applicable. Analytical methods for residues, only to food
producing animals. Again, we would not look at looking for residues in
companion animals.

(Slide.)

DR. BENZ: We have to have evidence of safety and effectiveness, and this
includes human food safety, target animal safety, user safety, and
again, effectiveness.

(Slide.)

DR. BENZ: Human food safety. We are looking drug residues in animal
tissues. Those include meat, milk, eggs; you name it, what we could call
as edible tissue. We look at acute toxic response. An example is what
would happen, you know, to children who are allergic to peanuts, get a
peanut and have acute response. They might go into convulsions.

We are also looking at those kinds of things with residues of drugs.
What would be a short-term effect if they got a lot of the drug, and
then a chronic exposure toxicity or a long-term exposure. What happens
to them.

We also look, as part of that, antimicrobial resistance and pathogen
load. Those have been called 558.15 studies, salmonella sheddings; they
have gone by a lot of different names. Dr. Cooper is supposed to give
that presentation next.

(Slide.)

DR. BENZ: Target animal safety. We have to have studies or reports to
demonstrate the cumulative effect of the drug on the animals, such that
the drug does not adversely effect the treated animals. Simply put, does
the drug harm the animal? We are going to look to see if the drug harms
the animal at all.

(Slide.)

DR. BENZ: User safety. We look at hazards associated with manufacturing;
direct. Is there any hazards to the occupational exposure to site when
the drug is manufactured. Indirect, such as manufacturing emissions;
hazards associated with administration to animals.

An example might be that you have a product that is very safe in the
animal, but if the human took and injected themselves by accident, it
could be very toxic. So we want to look at that.

Hazards associated with the use of air, water and solid waste
contaminated by use and disposal of the drug. An example that I could
give you would be that if you have a drug that you give every day, if it
is an injectable and you are giving it to 1,000 animals, what are you
going to do with those 1,000 syringes? We look at those types of
environmental concerns that go along with that.

(Slide.)

DR. BENZ: The effectiveness determined by experts, those experts such as
myself by training experience. Must of us have various degrees. We have
some experience in that field. That is what we are paid for.

We, again, fairly and reasonably concluded that the drug will have the
effective reports or it is represented to have the conditions of use to
prescribe recommended suggested labeling. As I said before, we are
looking at the labeling and effective data and see that the two match
and what is actually in the submission will support that labeling and
will that product be used in the marketplace in a reasonable manner.

(Slide.)

DR. BENZ: The effectiveness is based on substantial evidence, and I
won\'t describe what that is \-- that would be a whole other
presentation \-- consisting of one or more adequate and well controlled
investigations, such as a study in a target species, a study in
laboratory animals, a field investigation, bio equivalent studies and in
vitro studies.

And those would depend on what you were trying to do. You might have a
model that would predict a disease condition. It might be appropriate
with an in vitro study or a laboratory study. Some other types of study
might not be appropriate to do that. So you would have to use what was
appropriate for that condition.

(Slide.)

DR. BENZ: Another section is our GLP good laboratory compliance section.
There is a set of regulations that tell you how you should collect data
in a correct manner, such as data signed, dated, it has gone through a
QA unit, a quality assurance unit. We feel pretty comfortable about it
so that the firm has attested that they have collected the data that
needs to be, such as target animal safety and human food safety under
those conditions and have verified that.

Another section is environmental assessment or EA. The use, manufacture
and disposal of that drug does not propose a significant environmental
impact and an NADA must have an EA or a claim for a categorical
exclusion. The categorical exclusion comes in for such things as
companion animals.

Another example would be that you have a drug that is on the market. You
are going to change the labeling to clarify something, but you are not
going to change the overall exposure of the drug in the population. You
might want to rename or reclassify the genus or a species of some
antimicrobe.

(Slide.)

DR. BENZ: Our Freedom of Information summary. That is the information
that is for public disclosure that we make available. That includes
everything but proprietary information, such as chemistry and
manufacturing; how the product was marketed.

That is available in the dockets manager branch or on our web site for
anybody to look at. Such things that are in there are description of the
effectiveness data that was used to support the application, description
of the target animal safety, human food safety and there is a few other
things. But that is the kind of stuff that we are looking at.

And then there is a section called \"other\" that I have never used, but
every application has to have a good other just to catch everything
else.

(Slide.)

DR. BENZ: Now, I told you what you find that the drug company or the
sponsor submitted, but if you wanted to go in and look at an NADA and
see what CVM has got in there, the agency? Basically, these are the
documents that are in an NADA that we have generated.

Review. I generate animal science reviews. We have got people that
generate veterinary reviews, human food safety. But our interpretation
of the data and how it would support that application. We have gone and
looked and this is my scientific review.

Sometimes we have meeting minutes or memorandum of conference. We may
have had a meeting with the firm. We have sat down around a table and
discussed issues and those issues are of importance, so we want to
document in the field that we have had this meeting. Sometimes we have
internal meetings with our supervisors or other colleagues. We want to
document what happened there.

We have that and it goes into the file, so that if five to 10 years, if
these types of questions come up again, we can look and say, well, this
is what those types of decisions were based upon.

Sometimes we have a document summary, which is kind of a historical
basis of where that submission is moved, what is going on, whether it is
human food safety, effectiveness, target animal safety, but the status
at the time. If the drug is going to be approved, we have a memorandum
recommended approval, which is kind of obvious to the name.

That memorandum has a lot of administrative information. Basically it
tells those in our supervisory chain, which in my case is Dr. Lathers,
that I have dotted all the I\'s and crossed the T\'s and followed
everything along the way and that approval is following our policies and
procedures, and they can feel comfortable in signing off and saying,
yes, we should recommend this approval.

And it is a great place for them to pick up and say, what is going on in
this application; in a four to five page document and say this is all
the history I need to know. Sometimes we have an administrative
memorandum.

That administrative memoranda can be because the data didn\'t quite
address a situation. We had some concerns, but there was a policy
decision that set that aside. It could be policy decisions that came
from above. So that is administrative memoranda.

A draft regulation. CVM drafts a regulation which eventually ends up in
the Federal Register for approved products. That is in there. That is
something that we also send forward. We try to provide what will end up
in the CFR and the Federal Register; how we want it.

And then there are letters, letters to the sponsor that are necessary.
Sometimes there is a really nice letter that says, dear company, your
application is approved and you can begin marketing it. Sometimes we
have letters that says please try again, these are the others that we
would like some additional information and please come back and give us
that information, and we will re-evaluate your request.

And, that is the end of my speech. Any questions?

(Applause.)

CHAIRWOMAN LATHERS: There is a question for Dan? Dan, would you wait
just a moment, please. Would you use the microphone, please.

MS. MELLON: Hi. My name is Margaret Mellon. I am from the Union of
Concerned Scientists. From what I \-- from what you have presented,
there doesn\'t seem to be any information collected by the agency on
usage. I mean, whether approved compounds are actually used, in what
amount and in what animal systems.

Is that true? And if you don\'t get it in this process, do you get it in
any other process?

DR. BENZ: There isn\'t any information collected here because this is
the pre-approval process, and it would be hard for us to estimate how
much would be used, other than there is some estimation in the
environmental assessment because they have to have some idea of what
kind of impact.

We have a whole office, the Office of Surveillance and Compliance, that
looks at products post-marketing; whether they are used in accordance
with label directions, how much there is used, et cetera, and that is
their complete function, is surveillance and compliance.

MS. MELLON: Does the agency make that available in a report, like this
is how much of a particular antibiotic that has been used in a
particular system in 1998 so we could track it over time to see whether
antibiotic use is going up or down in particular systems to get a better
idea of exposure?

DR. BENZ: I really don\'t know the answer, because I am from the Office
of New Animal Drug Evaluation. I do the pre-approval, and I am more on
will the product be safe and effective. If there is anybody here from
Surveillance and Compliance that could answer that \-- or we can leave
it for later today. I really don\'t know.

MR. : \-\-- animal drug experience report.

DR. BENZ: Well, there is a drug experience report that would tell how
much the drug \-- each owner of an NADA that is approved must report to
the agency annually in something called a drug experience report. It
tells us how much the drug is used.

And I don\'t know if it is summarized across companies or anything in
public made available, but we do have some indication \--

MS. MELLON: We have tried to locate such reports and have never been
able to do so. But if they are available, we would like to hear about
it.

CHAIRWOMAN LATHERS: Dr. Thompson.

DR. THOMPSON: I am not from surveillance and compliance, but I will try
to answer your question. We do get information, as you are probably
aware, in the drug experience reports, but that is targeted specifically
to the individual drug, and most of that is considered proprietary
information.

There are some problems, which we have stated in the framework document,
with how we currently collect the information, and we are in the process
of trying to make some changes in terms of changing our regulations to
provide a better basis for tracking drug usage information in the future
and providing a better linkage to the resistance data that we are
collecting through the National Antimicrobial Resistance Monitoring
System.

The questions about how the information will be released publicly, we
don\'t really have an answer for that yet. We are looking at that issue
in terms of providing better public information in the future on that.
But some of it is considered proprietary information and is not
releasable by the agency.

MS. MELLON: Well, I do \-- I am glad that the agency recognizes that it
is a problem, and I guess I would just say that there are ways around
proprietary information, aggregating data and all that, which I know you
have thought about.

But I would just encourage you to go in that direction. It is a real
hard issue to address, either from a health standpoint or a public
policy standpoint, when you don\'t have any idea, really, of how much
antibiotic is being used, where and what the trends are over time.

DR. BENZ: Well, I do know. I can\'t make a plug for them, but there is a
commercial service that collects that data.

MS. MELLON: Well, it isn\'t very satisfactory either. Frankly, you know,
being from the public interest community, I want information that has
the kind of authority and credibility that would come from it coming
from the government. That would certainly be our preference. Thank you.

CHAIRWOMAN LATHERS: Are there any other questions at this moment?

(No response.)

CHAIRWOMAN LATHERS: If not, thank you Dan.

Our next speaker has just arrived, and I think we will need a few
minutes to load her PowerPoint slides. One announcement that I have been
asked to make is that Bill Flynn is obtaining as many copies of the
talks that we have heard this morning as possible, and hopefully, copies
will be available for us this afternoon.

In addition, Bill anticipates that these will be on the home page at a
later date. So you will have hard copies today and electronic copies in
the future. Give us just a moment, please.

(Pause.)

CHAIRWOMAN LATHERS: Our next speaker is Jean Cooper. Jean is a 1987
graduate of the University of Illinois Veterinary College. She does have
a master\'s of science in dairy microbiology and nutrition, and an
undergraduate degree in animal science, both earned at Rutger\'s
University.

She is, at this time, chief of the clinical chemistry and toxicology
branch at the Centers for Devices in Radiological Health at the FDA. In
her current position she did work for the Center for Veterinary Medicine
as an application reviewer. In this capacity she reviewed the studies
supporting the 21 CFR 558.15 Regulation on the sub therapeutic use of
antimicrobial drugs in food products. Jean.

**\"558.15\" STUDIES: A HISTORICAL PERSPECTIVE**

**By Dr. Jean Cooper**

DR. COOPER: Thank you. I had another meeting I had to be at earlier, so
that is why I didn\'t make it here until now.

The Food and Drug Administration is the primary federal agency
responsible for insuring the safety in food supply relative to the
impact of drug use in food animals. The Center for Vet Med approves
animal drugs that are effective and safe for animals and for consumers
of animal products from treated animals.

CVM considers the properties of each drug in determining safety concerns
associated with using any antimicrobial drug in food animal medicine. A
drug\'s biochemical effects on living systems will dictate clinical
utility and toxicity, effect on bacteria, bacterial drug resistance.

These parameters are addressed in considering approving drugs for use in
any animal, but particularly in food animals because their tissues
become human food.

When animals are given antibiotics, bacteria associated with the animals
are exposed to the drug. Fecal bacteria exposed to drugs in live animals
have the potential to contaminate animal tissues at slaughter. The
consequences of contaminated animal tissues can effect large segments of
the public when the contamination includes pathogens, particularly if
the pathogen is resistant the drug normally used to treat the infection
associated with that pathogen.

When FDA assess the safety of a new antimicrobial drug, the impact of
the drug on resistance is part of the process, whether the drug is
intended for sub therapeutic or therapeutic use.

Antibiotic resistance has been a concern since the 1970s. The need to
protect the general public from infection with antimicrobial resistant
pathogens continues to be a concern, as evidenced by you all being here.
The Council for Agricultural Science and Technology, CAST, found the
following in its 1994 report on food-borne pathogens:

As many as 33 million people in the United States get sick annually from
eating food contaminated with enteric pathogens. Various foods are
involved in food-borne diseases, but animal foods, beef, pork, poultry,
seafood, milk and eggs and their products are vehicles of more outbreaks
in the United States than are other foods.

According to CAST, antibiotic use in food animals may be associated with
two concerns. Drug therapy can facilitate intestinal colonization by
pathogens, such as salmonella typhimurium, because such therapy can
disrupt normal gut microbial interactions. Antibiotic therapy may be
ineffective or exacerbate conditions if the pathogen is drug resistant.

The center attempted to satisfy the need for implementation through
implementing the 21 CFR 558.15 studies requirements. The following
discussion will address why CVM reexamined this study supporting the
558.15 studies and the consequences of the analysis.

The discussion will explain CVM\'s objectives in determining microbial
safety requirements to address those objectives, and they were the
results from pre-approval studies intended to address the following
objectives:

(Slide.)

DR. COOPER: The goal is to avoid human exposure to increased pathogen
load in antimicrobial resistant genes resulting from antibiotic use in
animals. As I said before, the concern has been \-- FDA has been
attempting to address this concern since the 1970s, when the FDA
questioned the effect of feeding sub therapeutic levels of antibiotics
to a large number of animals to promote growth.

Many prominent scientists expected drug resistance to develop as a
result of feeding antibiotics to animals and that these resistant
organisms could cause human disease. Scientists were also concerned that
the use of antibiotics in food producing animals promotes an increase in
the animal reservoir of salmonella through promotion of cross
colonization and infection prolongation of the carrier state and relapse
for disease.

The Antibiotic in Feed Task Force from the 1970s was formed to address
the safety and efficacy issues associated with antibiotics administered
in animal feeds. This task force report considered target animal safety,
efficacy and public health, including residue issues.

Safety considerations at that time were established based on a risk
benefit ratio. In the 1970s the Antibiotic in Animal Feed Task Force
concluded that therapeutic use presented a small risk to public health,
and therefore, no study requirements were imposed on products intended
for such use; however, therapeutic antimicrobial drugs must still be
safe.

The assumption that therapeutic use of antimicrobial drugs is safe was
justified by a number of arguments at that time. Antibiotics at that
time were used to relieve animal disease that presented a relatively
small risk because they were used at high doses for short durations in
young animals, and it was believed that the benefit outweighed the
potential risks.

FDA recognizes that it is necessary to use antibiotics in animals to
relieve pain and suffering. It has also recognized that healthy animals
do make a better food supplies; however, some justifications were more
appropriate in the 1970s than they are today.

In the days when small family farms were the norm, treatment of frank
disease in food animals was generally limited to 10 days and therapy was
directed to individual animals or small groups of animals. It was
thought that relatively few animals would go to slaughter while
receiving antibiotics since few animals are sick at any one time.

Scientists argue that therapeutic use created less potential adverse
effects than non-therapeutic use because of proportion of use and
because of how non-therapeutic drugs is administered. The bulk of food
animals were being fed antibiotics for non-therapeutic uses at levels
which expose bacteria for sub-lethal doses for prolonged periods of
times.

Conversely, it was believed that therapeutic doses are generally high
enough to kill even the less sensitive bacteria. Drug effects on
pathogen load or antibiotic resistance contributed by therapeutic use of
antibiotics were considered a very low probability event.

The conclusion of the Feed Task Force report suggested that the benefit
from therapeutic use of antibiotics outweighed the limited risk of
microbial adverse effects on humans that such use might create;
therefore,

pre-approval studies to support microbial safety of a product were
directed to antibiotics intended for non-therapeutic uses, including
growth promotion and feed efficiency.

(Slide.)

DR. COOPER: The studies that were designed to support the 21 CFR 558.15
regulation were based on the findings of the task force report that I
have been referring to. The actual regulation does not state criteria
for the studies. They were an offshoot of the report that was generated,
and the sponsors were directed toward contacting the center for specific
information on how to do the studies.

The sponsors of antibiotic products were required to submit study
results demonstrating that their product would not promote bacterial
drug resistance if their product is intended to be administered at feed
for non-prescription use in animals.

The study protocol for these studies were highly defined and heavily
controlled to avoid introducing bias. The study design included a
shedding study and a resistance study. Both studies included a negative
control group and a treated group. An environmental control group was
included in the shedding studies. This was a non-treated,

non-salmonella infected group.

The negative control in treated groups were inoculated with a laboratory
strain of salmonella typhimurium that carried an identifying
characteristic, a nalidixic acid resistance, and the laboratory strain
needed to be free of transferrable resistance.

(Slide.)

DR. COOPER: In the acceptable studies, in the coliform study, which was
an endogenous resistance study, there had to be less than 20 percent
resistance in the inclusion criteria for animals in the study. In either
study, the shedding study or the coliform study, 10 to 12 animals per
treatment per study was the norm.

And as I mentioned in the shedding study, there was an inclusion of an
environmental control. The maximum amount of drug that was intended to
be administered needed to be given at least up to the 75 percent level,
and that had to be bolused if the animal was so sick that it couldn\'t
ingest it on its own. And there could be no contaminants in the feed,
other than the test drug itself.

The animals in both studies needed to be free of endogenous salmonella,
and the animals needed to be inoculated with the marked salmonella
before treatment. They needed to have demonstrated a measurable shedding
level before the drug could be administered.

In the 1970s the bacteria that generated the most concern included gram
negative bacilli, such as E. coli and salmonella. At that time
resistance and gram positive bacteria was poorly understood. E. coli and
salmonella frequently carry drug resistance and can transmit this
resistance to other bacteria.

Consequently, E. coli and salmonella were the organisms chosen in the
studies to demonstrate increases in bacterial contamination or genetic
material carrying drug resistance.

(Slide.)

DR. COOPER: These are the drugs that were picked for the studies for the
battery of susceptibility tests for the E. coli and the salmonella:
Aminomycin, ampicillin, carbenicillin, cephaxitin, chloramphenicol,
gentamicin, typhimurium, sulfamethoxazole, streptomycin, sulfadiazine,
tetracycline, kanamycin. Remember, these were the susceptibility panel
that was picked in the 1970s.

The reason was based on concern about public health and what was \--
drugs that were being commonly used for by physicians and that there was
a good literature base to support that E. coli and salmonella would
carry or had potential to carry resistance to these drugs.

The study design required that fecal and tissue salmonella be counted
and susceptibility tested on a weekly basis in the shedding studies and
fecal E. coli were susceptibility tested in the resistance studies. The
studies generally lasted for eight weeks. The test animals were
generally the same species as the intended market for the product, and
the test animals did not need to be near slaughter age.

Drug effect on pathogen quantity, prevalence and duration of infection,
as well as the drug effects on susceptibility in pathogen levels in
tissues were the parameters used to determine whether products increased
consumer exposure to bacterial resistance or pathogen load.

(Slide.)

DR. COOPER: Again, as I mentioned, quantity, prevalence, duration,
increase in resistance and enhanced pathogenicity. The enhanced
pathogenicity was evaluated by looking at the tissue samples to see if
the salmonella were found at higher rates in the treated groups rather
than the controls.

A second study was conducted to determine drug effect on the endogenous
E. coli resistance. All of these parameters were described in the human
safety criteria and animal safety criteria documents. These microbial
safety criteria are again the products of the Antibiotic in Feed Task
Force effort from the 1970s. As of 1998, they were included in the
center\'s web page as guidance line number 18. I don\'t know if they are
still there.

The determination of passing or failing was generally based on
statistical difference between the treated group and the control groups.

(Slide.)

DR. COOPER: If there was a one to two log difference in quantity in the
salmonella shedding studies, the passing was less than a 20 to 30
difference in prevalence in the shedding studies or a less than seven to
10 day difference in shedding, and there was no \-- for the E. coli
studies there was no significant difference in resistance.

Aside from measuring the drug effects defined by shedding, tissue
evasion and resistance, other measurements and/or information were
collected to insure the integrity of the study. In other words, you had
to have a good enough study in order to evaluate these past criteria.

Some of the questions that were asked of the study was did the product
have antimicrobial properties? If it didn\'t have antimicrobial
properties, then it didn\'t fall under the regulation. Did the study
address cross contamination and did it occur? Salmonella is highly
contagious and there were many controls put in place to prevent the
animals exposing each other during the study.

Were there sufficient numbers of animals in the shedding and resistance
studies in order to determine that a difference \-- to have enough power
in the study to determine whether a difference occurred, if there was
one? Did the animals consume enough drug to test the highest intended
dose? Was any other drug, intentionally or unintentionally, given to the
animals during the study?

Was a natural strain of salmonella present in animal water or feed to
confound the test? Did the test strain of salmonella carry a distinct,
stable marker? Was the strain capable of receiving resistance factors
and could it colonize the animal?

One of the components of the study was to evaluate the transfer of
resistance from a endogenous flora into the salmonella. The procedure
was to have clean salmonella that only had the nalidixic marker to be
passed through the animal and collected. If the collected salmonella had
picked up resistance and it was due to a drug, that would be considered
a failure. So the strain had to be capable of being a recipient of
resistance factors.

Was the microbial methodology appropriate and standardized? What tissues
were examined for salmonella nd how often were samples taken? Were the
studies performed long enough to detect an effect, if an effect
occurred? And what level of resistance did the animals in the resistance
study carry in the coliforms prior to starting the study? Remember, they
had to have less than a 20-percent baseline.

These integrity measurements were the basis for study rejection rather
than outright failure. Minor faults in the integrity measurements were
usually justified as not affecting the outcome.

(Slide.)

DR. COOPER: An analysis was done of the accumulated data from the
shedding of resistance studies described. There were 20 years composites
of studies evaluated and there were 44 sets of studies. In 1997 a
retrospective analysis of the data was performed, and the following
discussion summarizes the results:

Eleven of the studies were repeats of previously submitted studies. This
is 11 sets. Nine of the study sets failed to satisfy the safety
criteria. Two of the studies failed because of increased resistance and
the rest failed because of increased shedding.

Of the three studies where the spectrum of drug matched the test
organism\'s gram response, two of them failed because of resistance. One
of the drugs was a broad spectrum product tested in coliforms and the
other drug was a gram positive product tested against a gram positive
organism.

Two of the applications for approval to market products were not
approved based on failing the safety criteria. In other words, they
failed the studies, but remedies were made and the product eventually
made it to the market, with the exception of two products.

Seven succeeded in satisfying the criteria by redeveloping safe
conditions of use. One of the applications was approved after the
duration of treatment was shortened to two weeks. In other words, it was
a 14-day grace period. That was considered a therapeutic drug. If the
drug was intended to be used for more than 14 days, then it would fall
under this regulation. So, by shortening the duration, they no longer
had to meet the regulation.

One of the applications successfully repeated the study at a lower dose,
and the application was approved at the lower dose. It satisfied the
passing criteria. Two of the studies were repeated and the results were
acceptable on the second attempt. Three of the studies were approved
base don further information.

Twelve of the studies were rejected, 12 of the studies sets, for one or
more of the following critical reasons: The salmonella susceptibility
test results were not submitted, that was two studies; coliform
susceptibility test results were not submitted, three studies;
susceptibility test, quality control was out of control, three studies;
shedding lasted too long to measure prevalence or duration, two studies;
environmental control animals were contaminated, four study sets;
environmental controls were not included, one study set; animals used in
the study failed to meet the 20 percent resistance baseline for
inclusion, one set; and the data was too disorganized to interpret two
data sets and the study contained too few animals, one study set.

(Slide.)

DR. COOPER: I will give you a more specific breakdown. The following are
the results from the analysis, according to the following
pharmacological categories:

Since the results of individual products are proprietary information,
the results are summarized according to broad pharmacological classes.
As you can see, in the macrolide lincosamide group there were nine total
data sets. The number of drugs tested, four. The number of animal
species tested, three. You can\'t multiply them because some of the
animals weren\'t tested for each drug. Repeat tests. There were three;
rejects three, fails two; passed four.

(Slide.)

DR. COOPER: Under the ionophore class: 13 tests, data sets; seven
different drugs; three animal species; repeat tests, three; rejects,
four; failed, two; passed, eight.

(Slide.)

DR. COOPER: Unclassified gram positives: 15 total tests; number of drugs
tested, six; number of animal species, three; repeat tests, five;
rejects, four; failed, four; passed, seven.

(Slide.)

DR. COOPER: Streptogramins: Total tests, one; number of drugs, obviously
one; animal species, one and it was a pass.

(Slide.)

DR. COOPER: Glycopeptides: Two total data sets; two different drugs; one
animal species; one rejected; one passed.

(Slide.)

DR. COOPER: Bambermycins: Total tests, two; it as a single drug; two
animals species; one reject; two pass.

(Slide.)

DR. COOPER: Total tests: Two. Or total data sets two. Number of drugs
tested, two; number of animal species, two; failed, one, passed, one.

(Slide.)

DR. COOPER: After 20 years of receiving studies to prevent products
which increase pathogen load or resistance from being marketed the
controversy remains. There are public health concerns associated with
the use of antibiotics in animals.

The original goal is still valid, but the study requirement can and
should be modified to better address the objectives. This is the year
2000. These studies, as designed, are 30 years old. Science has moved
on.

Target animal safety and efficacy were part of the original plan, but
have not historically been evaluated as part of the 21 CFR 558.15
studies since there were better means to address those concerns. So some
of the issues have been changed since the original proposal in the
\'70s.

(Slide.)

DR. COOPER: Problems with the studies that I just talked about: There is
food safety issues. The studies were designed to look at public health
in general, but they were not specifically geared toward food safety.
The studies and the interpretation of the results are not associated
with human consumption of animal products.

There is not tie between a drug effect detected in the 558.15 studies
and human consumption of animal products, because the studies were
primarily done some time during the animal\'s life, not necessarily at
the time they would be going to slaughter.

Shedding does not reflect pathogen load or the level of resistance at
the point of impact on public health. As I said, when animals become
food.

At the time, in the 1970s when the studies were first proposed, it made
sense to test an animal during its life only when a drug was intended to
be administered in feed at low levels for the life time of the animal.
Since animal received drug up to the time they were slaughtered, the
studies to establish microbiological safety could be conducted at any
time.

Drug use practices have changed. Many drugs are now administered at
therapeutic doses for longer than 14 days, but less than a life time to
control disease. A drug effect demonstrated in an animal exposed to a
drug for a short period of time at the middle of its life may not
persist until the animal is slaughtered. A drug effect on microbial
safety is considered the greatest hazard to the consumer when the animal
becomes food rather than exposure through the environment.

Susceptibility test results are another issue. The way the studies were
designed, they provide limited information regarding the drug effect on
the mixed enteric population. Susceptibility testing is performed on
isolates and may not reflect changes in the sensitivity of the majority
of organisms in the gut population.

A drug effect on the transfer of resistance was difficult to detect
under the 558.15 study design. Most studies demonstrated transfer of
resistance to test salmonella from the enteric flora, but the transfer
occurred equally as frequently in the control as the treated group. So
the effect was not drug induced.

None of the studies show transfer of resistance that may \-- the drug
effect may not have been detected because the salmonella were inherently
resistant to the drugs being tested. In other words, 42 out of 44
products were for gram positive spectrum drugs in the organism we were
testing or gram negative organisms.

The effect may not have been detected because salmonella were inherently
resistant and there was no selective advantage for them to acquire the
new resistance. And, as I am alluding to, there was a mismatch between
the drugs and the bugs.

If you recall, seven of the nine studies that failed did so because of
the increased shedding, and that result is predictable. As I said, most
of the drugs were gram positive drugs, and we were using gram negative
organisms in the study.

Another issue was the artificial duration of treatment trigger when the
studies became required. As I mentioned earlier, under the 558.15
studies, the regulation didn\'t kick in until the drug was intended to
be used for more than 14 days.

Direct therapy in modern food animal production is no longer limited to
a few animals or a few days of exposure. Therapy is applied to large
numbers of clinically healthy animals. Farm husbandry emphasis on
prevention means that more animals receive therapy than when treatment
was reserved for salvaging sick animals.

Another issue is a laboratory strain. I\'m sorry. Let me back up one.

The artificial studies may not reflect conditions when marketed, and
that has been a concern for a long time. In order to measure drug
effects in the 558.15, artificially high inoculum doses of salmonella
doses were given. The differences detected in the studies made by
artifacts created by the unusual or unnatural inoculum concentrations.

Typically, the salmonella typhimurium were the animals were inoculated
at an eighth to a tenth to the tenth concentrations after starvation and
other conditions to try to induce the animal\'s flora to allow the
salmonella to colonize.

The laboratory strain, as I mentioned, it had to be a completely
susceptible strain, except for the market. The nalidixic acid resistant
marker is what was typically used. The laboratory strains used in the
study may not be representative of how natural salmonella perform when
challenged by the drug, and the strict research conditions may not
represent the pathogen to drug interaction that would occur under more
normal management conditions.

Another issue was the animal in the studies were unusually stressed
because of isolation to avoid the cross contamination problems. Studies
were very small in animal numbers, particularly in comparison to large
studies that reflect benefit or effectiveness measures.

The studies had too few animals to be sure that the effect seen was
really due to the drug, and the small sample size created environmental
biases. Drug effects may have been masked by cross contamination when
the sample size is small.

Because cross contamination was likely in these studies due to the
contagious nature of salmonella and resistance factors, the animals were
housed separately. Consequently, differences noted between treatment
groups may have reflected environmental effects, as well as drug
effects.

And the last issue I will bring up here is that these studies did not
evaluate effects when the drug was withdrawn. Two sponsors voluntarily
provided a shedding study after the drug was withdrawn. In both cases,
the level of salmonella temporarily increased after the drug was
withdrawn. This preliminary evidence suggests that withdrawing drugs
near slaughter may impact food contamination.

Conversely, some drug effects on resistance or pathogen load may be
reversible. This information may provide management tools to minimize
adverse public health events.

I understand that you are here to develop better microbial safety
studies to demonstrate whether antimicrobial new animals drugs used in
food animals are safe. I hope that my presentation of the center\'s
experience with the 21 558.15 studies help you in that mission. Thank
you.

(Applause.)

CHAIRWOMAN LATHERS: Thank you, Jean. Jean, I would ask you just one
question. Based on your review of the center\'s experience with the
558.15 studies, what suggestions would you offer to all of us at this
time in terms of the design of pre-approval protocols for our studies?

DR. COOPER: I think some of the suggestions I just went through in the
latter part of the speech would be some of the things that I would
suggest that people consider in designing future studies. Things like
try to match the bug with the drug. Try to develop the susceptibility
pattern. That is one thing I didn\'t mention.

A susceptibility pattern that is appropriate for the type of drug being
used. For example, gram positive, panels with gram positive drugs that
are being tested. Match all of those pieces together more closely than
we have in the past. I suggested look at the effects after the drug has
been removed. I think there is a lot of benefits to doing that.

CHAIRWOMAN LATHERS: Are there any other questions for Jean? Yes?

DR. CARNIVALE: Hi. Rich Carnivale, Animal Health Institute. I wonder if
anybody has any information that might look at the drugs that had passed
the 558.15 studies and what predictability those passing results had on
the future resistance or resistance that might have occurred later;
resistance patterns that may have occurred later or not occurred with
those products.

Is there any way that data could be looked at to see whether those
studies were at all predictive? From some of the comments you made, it
sounds like there were so many flaws in the studies. It is a question of
whether they showed any predictability for future resistance patterns.

DR. ANGUELO: This is Fred Anguelo, from the Centers for Disease Control.
It speaks very much to the limitation in the flaws of the studies,
because they didn\'t even test for the antibiotic that they used. So, of
course, you don\'t expect resistance to be detected, and you don\'t test
for gram negative \-- resistance in gram spectrum antibiotics when 42
products are gram positive drugs.

Of course, there is no resistance. So \-- I think the more disconcerting
thing, as a member of public health, is we should criticize ourselves
now 30 years later, and we could ask the industry how much do these
studies cost.

I mean, to be honest, I see little public health utility for these
studies that we have required the industry to do. Not to be too
critical, but \--

(Laughter.)

DR. ANGUELO: Certainly the pathogen load studies have been worthless,
and the resistance studies, when you don\'t even look at the drug in
what you treat for, were

ill-conceived and flawed from the beginning that could have been
predicted.

DR. COOPER: If I could respond to that?

CHAIRWOMAN LATHERS: Yes.

DR. COOPER: I wasn\'t here 30 years ago. Many of us in the room were not
here 30 years ago. I think at the time they used the best science that
they had. There are products, as I indicated, that didn\'t make it to
market or were modified to some extent. I mean, there were nine out of
44. That percentage may not be wonderful, but it did pick up the
absolute worst. It was not completely ineffective.

It just needs to be improved. It has a lot of room for improvement, and
that is what you all are here for.

CHAIRWOMAN LATHERS: A comment?

MR. SHRYOCK: Yes. Tom Shryock, Alenco Animal Health. Just to put this in
context, I have looked at these studies and have done some of the 558
studies myself, as you know Jean.

In the late \'70s, early \'80s the primary concern was the gram negative
infection and human pharmaceutical companies were coming out with the
multi generation sets, et cetera, to combat that. That is why that
particular study panel was in place.

The concept that a gram positive gut flora bug would cause a problem
basically was non-existent at that time. So we need to remember a little
bit of the history as to why these studies were required in the very
first place. Obviously times have changed, resistance has changed and
that has to be factored in.

The other thing that I would like to just bring forward is to speak to
Rich\'s point. If you look at how predictive these studies are or are
not, obviously these products are on the market and have been for 20 or
30 years in many cases.

If one were to look at the actual salmonella prevalence rates, for
example, in swine, as the NARMS program has done, it is characterized as
very low. I think perhaps 17 percent of the pens that were sampled were
indicated as positive for salmonella. Yet, at least 80 plus percent of
the producers had used a feed additive in some capacity.

So that would suggest to me that over the course of time this really has
not resulted in a significant level of salmonella that is present in at
least the pig population from those two little data bits. I would like
to hear some of the responses or comments from some of the producer
groups that are represented here as to what they think the actual field
impact may or may not be. Thank you.

CHAIRWOMAN LATHERS: Additional comments from the audience?

(No response.)

CHAIRWOMAN LATHERS: Jean, would you like to add any concluded words of
wisdom?

DR. COOPER: Good luck and I congratulate you all for coming together to
do this.

CHAIRWOMAN LATHERS: Okay. Thank you very much.

(Applause.)

CHAIRWOMAN LATHERS: Please return promptly at 1:00, and we will begin
our afternoon session.

(Whereupon, at 12:05 p.m., a lunch recess was taken.)

[A F T E R N O O N S E S S I O N]{.underline}

(1:08 p.m.)

CHAIRWOMAN LATHERS: I think we will begin right now, please. Good
afternoon. Welcome back. This is our continuation of the session one,
which deals with the objectives and the background information. We are,
of course, in the approval studies and antimicrobial resistance and
pathogen load discussion in terms of the February workshop.

We are now going to have a presentation by

Dr. William Flynn. Bill earned his BS in animal science at the
University of Delaware, a master\'s in veterinary preventative medicine
at Ohio State University and his DVM also from Ohio State University.

Bill is currently a biologist with the Office of New Animal Drug
Evaluation, serving as a special assistant to the director for the
Division of Human Food Safety. He will now begin to discuss with us the
pre-approval assessment of microbial safety and general concepts. Bill.

**PRE-APPROVAL ASSESSMENT OF MICROBIAL SAFETY**

**GENERAL CONCEPTS**

**By Dr. Bill Flynn**

DR. FLYNN: Thank you. Good afternoon. Before moving into session two
later this afternoon, which is intended to provide a forum for
presenting different ideas about designing pre-approval studies, it
probably would be helpful to first try to describe what we think the
objectives of these studies are and how the data from these studies
might be used.

The purpose of this presentation is, therefore, to attempt to give some
insight into what CVM\'s current thinking is about pre-approval studies
and their role in microbial safety assessment. I guess I would note
right up front there are still a lot of gaps yet to be filled in on this
overall plan, but we hope that some of these gaps can be filled with the
input from this meeting.

(Slide.)

DR. FLYNN: So, my topic basically will start really with the bigger
picture of looking at what has been for now coined as a microbial safety
assessment and then move into looking at some specifics about
pre-approval studies with regard to objectives and design, and then move
back again to the bigger picture view of how these studies may be
interpreted, their role, and also, a few comments about the relationship
between pre-approval studies and the concept of thresholds.

(Slide.)

DR. FLYNN: So first, starting in general with this concept of a
microbial safety assessment, I guess simply it is really a \-- one way
of looking at it is just a form of a qualitative risk assessment. It is
one that incorporates the concepts of the framework document, which most
of you I think are familiar with; what is in that document.

It essentially is a step-wise process or it is intended to provide a
step-wise process for assessing specific uses of antimicrobials in food
producing animals. It also provides a means by which data requirements
can be determined based on the level of risk that is associated with a
specific proposed use.

(Slide.)

DR. FLYNN: As Dr. Sundlof mentioned this morning, really the basis for
this, regulatory basis, it outlined in this guidance 78, which was put
out as a draft in November of \'98 and finalized in December of 1999.

(Slide.)

DR. FLYNN: In the past, FDA/CVM had evaluated the human health impact of
microbial effects on the certain uses of antimicrobial drugs in animal
feeds, and that is where the 558.15 studies comes in.

What guidance 78 did was basically in that document CVM announced that
it was necessary to consider the potential human impact of microbial
effects associated with all uses of all class of antimicrobial drugs,
not just certain drugs in animal feeds.

(Slide.)

DR. FLYNN: Now, looking at this microbial safety assessment, there is
really \-- at least the vision of it right now is of four main
components, the first which is a concept out of the framework document;
is that drugs would be categorized according to its importance to human
medicine.

Second, there would be a characterization of potential human exposure
associated with the proposed drug use. And then third, a
characterization of the risk associated with the drug class and proposed
use. That is really a consideration of the first two steps. And the
fourth component then would be, when necessary, the inclusion of
pre-approval safety studies.

(Slide.)

DR. FLYNN: Now, the first component drug characterization, again, as I
said, was a concept that is outlined in the framework document, which
basically provides three drug categories based on human importance,
category one being most important for treatment of human disease and
category three being on the other end of the scale.

Currently, this is something that is being worked on by FDA in terms of
actually developing a categorized list of approved antimicrobial drugs.
And clearly, since the relative importance of certain drugs would likely
change over time, this categorization would need to be periodically
updated and revised.

(Slide.)

DR. FLYNN: The second component, which is referred to as exposure
potential, essentially refers to the likelihood that a given use would
result in the transfer of resistant zoonotic enteric pathogens and/or
resistance determinants to humans, and this is also described in the
framework document. And in there it suggests a process by which uses
would be ranked as high, medium or low with regard to exposure.

(Slide.)

DR. FLYNN: The framework document also, when talking about this ranking
process of determining exposure potential, talks about considering the
following factors when ranking drugs:

One, drug attributes. That would include such things as the spectrum of
activity, the mechanisms of resistance for that particular class of
drug, the conditions of use, including the dosage form, dose, duration
of the use, the animal species that it is intended to be used in, and
third, the likelihood for exposure to zoonotic enteric pathogen, and
that being just simply whether \-- you know, are there zoonotic
pathogens of concern present? What are the impacts of management
practices for that particular use for that other food processing
impacts?

However, again another point where still further work is needed in terms
of providing more clear definitions of what we mean by a high, medium or
low exposure drug. So a little bit more work needs to go into developing
a system for categorizing based on exposure potential.

(Slide.)

DR. FLYNN: The third component, which is called risk characterization
here, is really just once you have done the first two steps, categorized
the drug and determined its exposure potential, then essentially, based
on those two factors, you are determining what \-- or trying to
characterize the risk associated with that particular proposed use.

So therefore, based on the class and proposed use, you reach a decision
point here to decide, well, is further information needed to
characterize the risk? Or, based on the risk that you have
characterized, do we need further information, such as conducting
pre-approval studies?

And if you have made a decision that, yes, we need pre-approval studies,
then what are the particular areas of concern, given the particular drug
that you are talking about. And here again you would have to start
considering those factors, such as drug attributes and conditions of use
to determine whether a direct or indirect transfer and pathogen load are
all issues that need to be addressed or is it some subset of these, and
that would all depend on the drug and use combination that you are
talking about.

(Slide.)

DR. FLYNN: That would then get us to the fourth major component I guess
of this safety assessment, and that is the actual pre-approval studies
themselves. Essentially, looking at it in the most simplest way, there
are three sort of main issues or areas that may need to be addressed.

One is potential transfer of resistant zoonotic pathogens or the
so-called direct transfer of resistance. Potential transfer of
resistance determinants; therefore, that would be essentially resistance
determinants transferred from \-- for example, an animal bacteria is
then transferred from animal bacteria to human bacteria or what is
referred to as indirect transfer. Then lastly, whether or not there is a
potential impact on pathogen load.

(Slide.)

DR. FLYNN: So, before moving on then to more specifics about
pre-approval studies, just in general, this concept is essentially a
form of a qualitative risk assessment. It is something that would be
conducted by a drug sponsor prior to approval and reviewed by CVM.

The data requirements for input into the safety assessment would be
dependent on the drug class and the proposed use, and then based on
that, there may or may not be a requirement for pre-approval studies as
part of that assessment.

And then basically, the determination of safety would be based on the
overall conclusions of the microbial safety assessment. So basically,
after considering all four main components of the safety assessment.

(Slide.)

DR. FLYNN: And as I said at the beginning, there are still some gaps
that need to be filled. There is work that needs to be done on the
categorization of drugs. What are the category one, two or three drugs?

We need to have a better definition of the exposure ranking in terms of
what is a high, medium or low \-- represents a high, medium or low
exposure potential. Once we have assigned category and ranked the drug,
what criteria are we going to use to then determine when further
information is needed or when pre-approval studies would then be needed.

And once we design these pre-approval studies, that we need to have
interpretive criteria for evaluating these pre-approval studies.

(Slide.)

DR. FLYNN: Again, now focusing on pre-approval studies themselves, the
basis for this goes back to guidance 78, and basically the guidance lays
out that there is the two main areas that may need to be addressed. One
is to evaluate the rate and extent and development of resistant enteric
bacteria in animal\'s intestinal tract following exposure to a drug, and
then the second is evaluate changes in the enteric bacteria in the
animal\'s intestinal tract, and this is the pathogen load question.

(Slide.)

DR. FLYNN: With regard to the actual \-- the framework document then,
which was published in January of 1999, it laid out the concept of using
pre-approval studies for addressing the microbial effects that were
basically outlined in guidance 78.

(Slide.)

DR. FLYNN: So, to look at the objectives of these studies, first, with
regard to the resistance question, again it comes back. The objective
then sort of evolves from guidance 78 with regard to characterizing rate
and extent of resistance development.

(Slide.)

DR. FLYNN: And what do we mean when we say rate and extent? I guess,
looking at each term, with regard to rate it is really trying to answer
the question of how rapidly will resistant enteric bacteria, or maybe
more appropriately bacteria with increased susceptibility, emerge as a
consequence of drug use.

The second component or second word is the extent, and here it is to
answer the question what is the magnitude of change in vitro
susceptibility of enteric bacteria as a consequence of drug use and what
is the prevalence of those bacteria that exhibit that decreased
susceptibility.

(Slide.)

DR. FLYNN: When we think about the objectives of these studies, there
are some questions, I think, that we should be considering. One, is
there a treatment related shift in susceptibility? What is the magnitude
of that shift? What susceptibility changes are detected at certain time
intervals post-treatment? Should we look at expected withdraw time at
expected age and weight of slaughter?

And what is the impact or re-exposure of the pathogen to the drug,
either when that same animal is treated or when another animal is
exposed to that pathogen and then that animal is treated?

(Slide.)

DR. FLYNN: And then you are looking at objectives from the standpoint of
pathogen load. Again, from guidance 78, to evaluate the change in the
number of enteric bacteria in the animal\'s tract that are potential
human pathogens following exposure to the antimicrobial in the animal
drug.

(Slide.)

DR. FLYNN: Again, there are some questions that we should be looking at
or considering. Are there treatment related changes in the number of
enteric bacteria? Are observed changes transient or persistent? Are
pathogen load changes present at expected withdraw time or at the
expected age of weight of slaughter of the animal? And when a change in
pathogen load is present, does that represent a public health risk?

(Slide.)

DR. FLYNN: Some general points to consider regarding the objectives of
these studies: As outlined in the framework, not all uses or classes of
drugs will require that both resistance and pathogen load be addressed
in

pre-approval studies.

Resistance and pathogen load may be addressed in separate studies, or,
if possible, perhaps in a single study. And then, certain uses or
classes of drugs may not require pre-approval studies at all.

(Slide.)

DR. FLYNN: Now, the purpose of this workshop obviously is to talk about
this very issue of pre-approval study design. I think there are some
points that I think we feel are important. First, looking at this
question of what is the organism of concern.

Zoonotic enteric pathogens that have been identified as primary concern
include salmonella, camplyobacter and E. coli, but other bacteria, such
as enterococci, are of particular concern when transfer of resistance
determinants or indirect transfer is a question.

(Slide.)

DR. FLYNN: So these studies we think should \-- whether you are talking
about resistance or pathogen load should focus on enteric zoonotic
pathogens, or pathogen or pathogens of greatest public health concern
associated with the certain target animal species. And when I say target
animal, I refer to just the animal species for which a given product is
intended to be used.

Given the multitude of bacterial species and strains, et cetera, it is
very possible or likely that we will need to be selecting appropriate
sentinel organisms for use in these studies. We simply can\'t look at
everything, so there is going to have to be some decisions made about
what is an appropriate sentinel organism to include.

(Slide.)

DR. FLYNN: Another point regarding study design with regard to the test
article or the drug we are investing. A study should evaluate proposed
conditions of use of the animal drug, including dosage, form of dose and
duration. We are interested in when you use a particular class of drug
under certain conditions, under certain dosage forms. What potential
effects do we see?

One question that does come up is if a dosage range is proposed, what
represents sort of the worst case scenario, so to speak, with regard to
resistance development and/or pathogen load. So, in that case, if you
have a dose range, can we justify selecting a single dosage regimen to
evaluate in such a study? Or is it necessary to look at more than one
dosage regimen?

With regard to the target animal, the study should provide information
pertinent to the use of the drug in the intended species and production
environment. I would like to point out here that due to the nature of
the studies the use of some type of model study is likely to be
necessary. Here I mean that, simply, it would be difficult to mimic
field conditions in such studies.

For example, we may need to be looking at a study that involves a
challenge organism. But, in any case, it is likely that we will
obviously need to be considering a model. And given that we may have a
model, then we have to address the question of does the model have
adequate inferential value.

(Slide.)

DR. FLYNN: Some other factors regarding the target animal. When looking
at these studies, typical age, weight, when exposed to the drug; typical
age, weight when that animal is slaughtered; prevalence of relevant
zoonotic pathogens in that particular species, and also, the particular
management of environmental conditions associated with the particular
animal species.

(Slide.)

DR. FLYNN: In regard to pre-approval studies that are intended to
address the resistance question, it is logical that an end point of such
a study would involve measurement of susceptibility changes in certain
selected bacteria as a consequence of drug use. Therefore, an important
question that would come up is what drug is used in the susceptibility
testing?

To try to answer that, I think that one point first to consider is that
the primary concern, basically the safety question that we are trying to
assess, is the loss of effectiveness of a drug used to treat disease in
humans due to the use of the same or related drug in animals?

Therefore, we think the focus then should be on measuring susceptibility
of pathogens to the pertinent human drug, following exposure to a
related animal drug. One approach would be then to use the NARMS plate,
which are the National Antimicrobial Resistance Monitoring System, which
includes representative organisms from the various classes of drugs, as
indicator drugs to be used in the susceptibility testing in these
studies.

(Slide.)

DR. FLYNN: Now, moving into some more sort of over-arching questions
regarding pre-approval studies, as discussed earlier these studies \--
the vision is of these studies being a component of a microbial safety
assessment, so therefore, a determination in safety will be based on an
overall evaluation of the microbial safety assessment.

That may or may not include pre-approval studies as a component;
however, appropriate interpretive criteria need to be developed to
evaluate such studies with regard to both resistance and pathogen load.
We believe that pre-approval safety determinations can be made based on
microbial safety assessments. That includes studies with appropriate
design and interpretive criteria.

(Slide.)

DR. FLYNN: In regard to interpretation, we basically need interpretive
criteria that can define whether you have a pass/fail when you run these
studies. However, I think we also need to consider whether this study
can identify possible means of mitigating microbial effects that may
occur, such as modification of particular use conditions.

For example, you may have a \-- with certain use conditions have a
failure, but maybe with alternative use conditions you may pass.

Another point about these studies is that the predictive value of the
studies is important, and clearly these studies would need to be
validated, but that may take time to do and may have to occur with use
or with implementation of these studies. So clearly, there is likely to
be a need for refinement of these studies once we \-- given validation
will probably be a difficult task, and so there will clearly need to be
a need for refinement where necessary.

(Slide.)

DR. FLYNN: So, in terms of the overall role of pre-approval studies in
the microbial safety assessment,

pre-approval studies are intended to serve as a tool for screening
specific proposed uses of antimicrobial drugs intended for use in food
producing animals. The studies are then intended to be included, when
necessary, as a component of the microbial safety assessment.

(Slide.)

DR. FLYNN: Finally, I just want to make a few comments about the
relationship between pre-approval studies and the threshold concept. As
you are aware, again the framework document proposed the concept of
establishing thresholds as part of a new regulatory framework; however,
this threshold concept is still one which is not very well defined.

But I think there are some points we can make at this point with regard
to their potential relationship to pre-approval studies. I think we
acknowledge that threshold concept is still an important component of
the regulatory framework.

In addition to demonstrating safety through

pre-approval studies, it may be necessary for certain classes of drugs
to also establish thresholds prior to approval. We believe that the
concept of establishing thresholds for certain classes of drugs is an
important post-approval tool.

(Slide.)

DR. FLYNN: We don\'t see that pre-approval studies are not intended to
be used to be used to established thresholds. Although interpretive
criteria are needed for these studies, the interpretation of
pre-approval study results does not necessarily require thresholds.

Therefore, I think the design of pre-approval studies not contingent
upon a threshold concept, which I think helps us move forward in this
workshop here. With regard to the threshold concept, I think there is an
intent to obtain further public input on that concept.

(Slide.)

DR. FLYNN: So briefly, in summary, I will just hit some main points here
with regard to microbial safety. Basically, it is a qualitative risk
assessment conducted prior to approval. It includes drug categorization,
exposure ranking and pre-approval studies when they are determined to be
needed. The determination of safety then would be based on the overall
microbial safety assessment.

(Slide.)

DR. FLYNN: Again, the basis goes back to guidance 78 and the framework
document.

(Slide.)

DR. FLYNN: Some points about the objectives with regard to resistance.
How rapidly does decreased susceptibility emerge? That is, rate. What is
the magnitude and prevalence of the susceptibility change with regard to
pathogen load? Does drug use change the number of enteric zoonotic
pathogens in the animal\'s intestinal tract?

Not all uses and classes of drugs will require both resistance and
pathogen load studies necessarily. If both are needed, it is possible
that it can be addressed in a single study. Certain uses and classes may
not require pre-approval studies at all.

(Slide.)

DR. FLYNN: Some specific design points that I made with regard to
organism: The major enteric zoonotic associated with a target animal
species. We need to look at the proposed conditions of the use of the
test article. We need information pertinent to the use of the drug in
the intended species and production environment and the concept of the
use of the related human drug tested by NARMS for susceptibility
testing.

(Slide.)

DR. FLYNN: We think it is a tool for screening specific proposed uses of
antimicrobial drugs and is a component of the microbial safety
assessment.

(Slide.)

DR. FLYNN: With regard to interpretation, it is based on consideration
of the overall assessment and that we still need interpretive criteria
for these studies.

(Slide.)

DR. FLYNN: With regard to the thresholds, the design \-- the design is
not necessarily contingent on threshold concept, these studies are not
intended to establish thresholds and we will be seeking further input on
the threshold concept. And, I think I will stop there.

(Applause.)

CHAIRWOMAN LATHERS: Thank you, Bill. I have one question. You mentioned
that the pre-approval studies are not intended to establish thresholds.
Can you expand your thinking in that area?

DR. FLYNN: Well, obviously we still need more input on how threshold
concept itself would be implemented. But I think one important point
with regard to moving forward on this workshop was being able to answer
the question of whether we thought the end points that we want to get
out of these studies is somehow going to be measured against some
threshold that was established.

If that were the case, it would be hard to move forward on designing
these studies if we don\'t know exactly how we are going to use the data
coming out of it. So I think right now we believe that we don\'t \--
that there doesn\'t necessarily have to be a link between a threshold
and determining safety based on the outcome of a pre-approval study.

But what that means though then is we still need some criteria in order
to evaluate these outcomes. It is just that those criteria are not
exactly the same thing as a thresholds, because, like we said, we may
not always \-- you may not have thresholds for every drug that we want
to conduct a microbial safety assessment for.

CHAIRWOMAN LATHERS: Okay. Thank you. Questions from the floor? Would you
use the microphone, please.

DR. GOOTZ: Tom Gootz, Pfizer. Traditionally you have looked at \-- when
you mention resistance transfer, you are really talking about transfer
of an organism from the zoonotic state to people, creating a resistance
chain.

But in the framework document now, as you alluded to in your talk, you
are beginning to talk about demonstration of resistant determinant
transfer, i.e., specific genes in perhaps the presence of an
antimicrobial in vitro situation.

My question is how do you envision ranking this? For instance, we
already know in the literature that different species of enterococci can
transfer among themselves the determinants that are required for
vancomycin resistance, and that can be in the presence of vancomycin or
another type of derivative.

So my question is how much is there to be learned and how weight do you
intend to put on the demonstration \-- in vitro or even in vivo,
demonstration of specific determinant from one species to another in a
laboratory or in a use animal?

DR. FLYNN: I think that is a very good question, and I think that is
part of the reason we are here at this workshop. I think that does fit
into that exposure potential ranking, and like I said, how that was
described in the framework document is fairly cursory and I think there
is need for a better definition there.

DR. GOOTZ: Just real quick. Thank you. Vancomycin used clearly in the
United States in humans is \-- I think most people would realize is
responsible for the transfer of vancomycin resistance genes among the
enterococci leading to vancomycin resistant enterococci in the mid
\'80s.

The Lanko Lilley group published an AAC note showing the tonnage or
kilograms of vancomycin sold in the United States, which was about 10
times more than the amount of vancomycin used in human health in the
next highest country, which was France, and the other countries in
Europe fall way off.

So I think at least in the United States, which is our provence,
wouldn\'t most people think that, with vancomycin use, for instance,
that was responsible for pressing these resistance genes creating theory
as opposed to the use of animal health products.

MS. : In terms of the of the scope of the risk assessment, I assume that
many, if not most, of the products you envision assessing are going to
be variants of products that are classes of drugs that are already on
the market.

And to the extent that that is the case, is the risk assessment going to
be addressed to the additional increment of risk represented by the new
product? Or is it going to somehow take into account the risk
represented by the product already approved?

DR. FLYNN: That is getting into the question of how the framework is
going to be implemented with regard to currently approved products, and
I am not sure that I can really answer that question. Right now I think
this microbial safety assessment could include potentially any
application that is coming in, whether it be a totally new product or a
new indication for a currently approved product.

And I think the way it is structured right now is to be looking at that
specific \-- the risk associated with that specific proposed use.

MS. : So it would just be the increment, the additional increment of
risk represented by that single new product that is under assessment?

DR. FLYNN: I am not sure that I can answer that completely, because I
think that is \-- the safety assessment itself is to be assessing that
particular application, that particular use. I am not sure how it would
be applied necessarily to account for other uses that are already out
there.

MS. : Okay. Thank you.

CHAIRWOMAN LATHERS: Thank you, Bill. Our final presentation in this
session one will be given by Dr. David White. David earned a BS in
animal science from the University of Vermont, an MS in animal science
in animal science from the University of Kentucky and his Ph.D. at Penn
State University in veterinary science pathobiology.

He has done a postdoctoral fellowship in molecular biology and
microbiology at Tufts University Medical School. He has a research
interest in mechanism of multiple antimicrobial resistance in both human
and veterinary bacterial pathogens.

He is currently a senior research microbiologist at the Office of
Research in the Center for Veterinary Medicine. David will now discuss
resistance and pathogen load factors to be considered. David.

**RESISTANCE AND PATHOGEN LOAD - FACTORS TO CONSIDER**

**By Dr. David White**

DR. WHITE: Thank you very much. I wanted to thank everyone for making
the time to come out to this meeting and staying for the three days so
we can discuss this important issue. Before we get started, could I have
the lights dimmed a little bit. Dan, behind you.

(Pause.)

DR. WHITE: Well, while we are doing that, what I wanted to get across
today was a little bit \-- some of the talks we have seen today talked
about uses in the different animal production environment. A little bit
on risk assessment.

I would like to address a little bit about the science involved in
antibiotic resistance. A quick micro 101 introduction to get us all on
the same page and then segue into some risk factors or questions to
consider when we are designing these pre-approval studies in both
antibiotic resistance and pathogen load.

(Slide.)

DR. WHITE: Now, we talk about antimicrobials. Of course, they have been
around over 50 years now; introduced in clinical medicine in the \'40s.
They were initially hailed as miracle drugs. Here is a poster showing
that penicillin cures gonorrhea in four hours. That is pretty
impressive.

Of course, they were hailed as miracle drugs. They significantly
decreased morbidity and mortality associated with many infectious
diseases. Unfortunately the miracle has weaned a little bit, and we are
now starting to see resistance occur in a lot of different bacterial
pathogens.

So now what we are seeing a lot of covers in both laymen magazines and
scientific journals looking like this, \"Antibiotics, the end of miracle
drugs.\" And if you remember, near the end of New Year\'s last year you
had all these new shows talking about top 10 lists. What were the top 10
greatest achievements? Who were the top 10 most influential people in
the century?

If we thought about the top 10 greatest achievements of humans in the
20th century, I think you have got to put antibiotics in that top 10
because of how many people they have saved, if you think about it. Does
any of us remember the pre-antibiotic era? Probably not.

(Slide.)

DR. WHITE: Now, if we look at the top 10 drug resistant microbes,
remember this is not something you are going to see on America\'s Most
Wanted or posted in your post office, but we do have a top 10 hit list
of the most resistant bacterial pathogens and most of them are human
pathogens and most of them are in the hospitals.

We do have enterobacteriaceae as one of our top 10 drug resistant
microbes, and these include your E. coli; salmonella. They cause a
variety of diseases, and they have become resistant to almost every
antibiotic. Not all at the same time, but there are mechanism identified
for almost every antibiotic that would be used to treat that type of
infection.

Of course, enterococci is getting a lot of attention now. Right now they
are a significant cause of post-surgical wound infections in hospitals,
and, of course, vancomycin resistant enterococci is seen all over.
Luckily, with the introduction of sinersid as an alternative drug of
last resort, we have a new antibiotic in our arsenal.

But as soon as you use an antibiotic, you open up the Jeannie\'s bottle,
so to speak, and resistance will eventually occur.

We also see resistance in agent influenza, MTB, of course, microbacteria
tuberculosis, which causes tuberculosis. We now have strains of MTB that
are resistant to every single microbacteria drug we have. So what
happens now in a hospital \-- you know what they are doing? They are
going back to pre-antibiotic methods. They are creating microbacteria in
isolation rooms. We are facing a crisis when it comes to some of these
bacteria.

(Slide.)

DR. WHITE: It is not kept to bacteria. It also is being observed \--
resistance is being observed parasites. Plasmodium \-- we are starting
to see chloramid (sic) resistance. We now have strains of staphorius
called VIS, which stand for vancomycin intermediate staphorius, that are
showing decreased susceptibility to vancomycin, which is the drug of
last resort.

Luckily, we do have some new drugs in the pipeline, but it always seems
to be a catch-up battle. It goes back to a story, if you remember, from
mythology. Sisyphus. He was \-- his penalty or penance was to push that
rock up the hill. Remember the story anybody? The rock would roll all
the way back down, and he would have to keep doing it.

So are we doomed to repeat this all the time? Are we always waiting for
the next generation of antibiotics to save our butt? That is not what
antibiotics are to be used for.

(Slide.)

DR. WHITE: We talk about resistance. The definition means many different
things to many different people. First, we can have intrinsic. When we
talk about intrinsic resistance, I think it is a naturally occurring
trait arising from the biology of the organism.

For instance, E. coli and salmonella are intrinsically resistance to
vancomycin because the drug cannot get through to the cell membrane. We
also can have acquired resistance, and this happens when a bacterium
that is previously susceptible acquires a gene making it resistant to a
particular antimicrobial.

We can also have chromosomal resistance versus extrachromosomal
resistance, and usually what that means is it is a mutation on the
chromosome somewhere. For instance, with fluoroquinolones, all it is a
base permutation in the DNA gene. For maybe erythromycin macrolides it
is a permutation in the ribazone.

You also can have extra chromosomal resistance where the genes are
coming in as foreign DNA and the bacteria pick up these genes. We also
have microbiological resistance versus clinical resistance. Now, most
microbiologists are interested in the change in the MIC, the minimum
inhibitory concentrations. The clinicians are concerned about whether
their patient or the animal is responding to treatment.

(Slide.)

DR. WHITE: So what are the causes of resistance? First of all, there is
mutation. Just a simple base pair mutation. This is a spontaneous event
that occurs in the absence of a selection pressure of antibiotics. It is
just a random event. There is also adaptive mutations that are being
described which happen in the presence of anoxic agents, and that is a
growing field.

The most common method on how bacteria become resistant is through the
external acquisition of resistance genes, and this has historically been
on plasmids, transposons and some of the newer DNA elements called
integrons where we get gene cassettes that are inserting into this
transposable element. What happens is this integron can line up to six
different antibiotic resistance genes all in proximity to each other.

So in that case what happens is you have these six cassettes all lined
up. Only one antibiotic will select for all six resistances. We can also
have selection of resistance variance from within the animal or the
patient, meaning there is already a pre-existing resistant population
with in the animal.

We use the antimicrobial, we kill off the competing micro flora and all
of a sudden now, the pathogen now that is resistant is able to flourish.
We can have cross infection. This is primarily nosocomial infections
where we get acquisition of entirely new microbes that are resistant.

(Slide.)

DR. WHITE: So the cycle is very complex and that is probably why it has
been discussed for the past 40 years, but if we try to look at this
cycle of antibiotic resistance, initially how we are trying to start off
here \-- we need some way to start, and the idea is that there is a
pre-existing reservoir of resistant genes in the environment somehow,
and this can be from, believe or not, the actual antibiotic producing
organisms.

A lot of our antibiotics come from soil microbes. If they produce an
antibiotic, they need to have the genes to protect it against that
antibiotic. Don\'t you think? Also what happens is a lot of research has
been done that characterizes resistance genes and they are very similar
to those that have been observed in the antibiotic producing organisms.
So that may be the origins of antibiotic resistance.

So what happens over some type of transmissible event? A resistance gene
gets incorporated, say into a plasmid. Some pathogens incorporate this
plasmid into its strain. We get selection pressure administered, the
susceptible die, leaving the resistant bacteria, they grow by cell
division and they again become a reservoir. So it is a cycle.

And I think, when we start talking down the road about intervention and
mitigation strategies, we need to somehow interrupt some of these steps
in the cycle.

(Slide.)

DR. WHITE: Even though we have hundreds of antibiotics available in both
veterinary and human medicine, what is really interesting is that they
really only come down to one of four mechanisms of action. You would
think it would be much more diverse and much more broad acting, but no.
It really comes down to only one of four ways on how these
antimicrobials inhibit or kill bacteria.

One is through inhibiting cell wall synthesis of the bacteria, and this
is your beta-lactams, bacitracin and vancomycin. We also have
antimicrobials that inhibit nucleic acid synthesis or function. This is
your fluoroquinolones.

We have those that somehow competitively inhibit out a substance in the
bacterial cell. This is your sulfa drugs, trimethroprim. They inhibit
the steps in tetrahydro folate synthesis which is essential. And the
majority of our antibiotics are targeted toward inhibited protein
synthesis, and this is a selective toxicity where these antibiotics are
selected for the bacterial ribosome, but not that toxic for the \-\--
and this is where our aminoglycosides, our phenocols, tetracycline
macrolides and lincosamides all act.

(Slide.)

DR. WHITE: So remember that, that most of these antibiotics have only
one of four mechanisms. Now, bacteria have evolved of course. Resistance
mechanisms is very diverse, but that only comes down to one of four
mechanisms as well. They can somehow inactivate the antibiotic, and that
is usually due to the production of enzymes that render the antibiotic
ineffective.

We also can have alteration of the target enzyme. For instance, the
fluoroquinolones. It is a simple base pair mutation and it reduces the
affinity for the fluoroquinolone for the gyrase. We can have alteration
of the target binding sight, and this is particular for those that
affect the ribosome. So we get mutations in the ribosome and
streptomycin and erythromycin no longer have that affinity to bind that
ribosome to inhibit protein synthesis.

And a newer area that is being investigated is reduced cellular uptake
and/or active efflux. What is happening is we are getting permeability
changes in the bacteria where the antibiotic cannot get in as
effectively as it could before. And if it does get in, it has got a pump
that pumps it right back out into the environment just like your sump
pump.

And there is a lot of different genes that have been described. If you
pick up the latest antibiotic resistance journals, you find a new efflux
pump described in every issue. There have been mechanisms described for
tetracyclines, macrolides, fluoroquinolones, and

beta-lactams. So efflux is becoming more prevalent the more we look into
these types of resistance.

(Slide.)

DR. WHITE: So the question that we have been facing for the past 40
years and really haven\'t come to a significant answer with is how does
the use of an antimicrobial in food animals affect the prevalence of
resistance among zoonotic bacterial pathogens and commensals and the
animal and animal production environment?

And I added commensals in there because there is a grassroots effort now
to think about what is the contribution of the commensals in animals as
reservoirs of resistance. Have we totally missed the train on this one?
When we look at normal flora, gee, gosh, they\'re resistant to five or
six antibiotics. So maybe they are the reservoirs of resistance.

(Slide.)

DR. WHITE: So these cascade of events, this potential consequences of
use, this is how it is playing out. The worst case scenario is what
happens when we use antimicrobials in animals is we get an increase in
the prevalence of resistant bacteria in animals. We then get transfer of
resistant microbes, either pathogens or commensals to humans and this is
either via direct contact with animals or through a consumption of
contaminated food or water, i.e., as Dr. Flynn mentioned, the direct
transfer route.

We possibly have transfer resistance genes to other bacterial genera
species. These would be the, i.e., indirect transfer. Do we have
enterococci that have become resistant that colonize us and can transfer
genes to our resident enterococci?

As a result of these transfers we see an increase in incidents of animal
and human infections caused by resistant pathogens and potential
therapeutic failure in animals and humans.

(Slide.)

DR. WHITE: Now, the ecology of antibiotic resistance. There is numerous
figures out there trying to describe the interactions between the
different environments, and I think this is one of the most simplistic,
but gets the point across. There is a couple out there that have arrows
going in 50 million directions, and I think that is important, but when
you look at that you are overwhelmed.

This was a figure used by Johnsonal in the British Medical Journal, and
it shows that there is three areas of concern. There is a human medicine
community and hospital, vet med and agriculture, and they are all
linked. The arrows go in each direction.

So when we start talking about antibiotic resistance, we have to make
sure, if we do intervention or mitigation strategies, that we do it in
all three areas and now just one and ignore the other two.

(Slide.)

DR. WHITE: This transfer of resistance, just to bring you back to basic
micro 101 on this, this is important, because this actually is factors
to consider when we talk about resistance studies. How do resistance
genes transfer between different organisms? Remember all of this from
way back when?

First, there is transformation, which is uptake of naked DNA. And what
happens, of course, is when cells die, they lice. When they lice, they
release their DNA into the environment. Well, bacteria that are in close
proximity can pick up these genes and incorporate them into their
chromosome.

We also have conjugation, which is cell-to-cell contact via sexpalis
(sic) usually plasmid mediated. Plasma replicates itself and is
transferred to another organism. We also now conjugate transposons that
Dr. Abigail Saulur talks about quite a bit. It is a new type of novel
DNA element that can be transferred this way.

And lastly, genes can be transferred via transduction, which is through
a bacterial virus or bacterial phage. This is how penicillin arose in
some staphylococcus \-\-- strains, and also how the shigatoxins arrived
in E. coli 157 from Shigella, was via a virus. So it is important to
keeps these methods in the back of your mind.

(Slide.)

DR. WHITE: And in terms of resistance genes transfers, how do we look at
the effects of antimicrobials in animals and in potential human health
implications. One thing that researchers tends to do is try to find the
same genes in human isolates as we do in animal isolates, and there is
numerous studies that have gone after resistant pheno types and then
gone after the genes involved and then sequenced them. And guess what?
They are 99 percent identical to a gene that has already been seen in
another pathogen.

For instance, the tet genes seen enterococcus, lactococcus have also
been found in strep pneumonin, gonorrhea neisseria and lysteria
monocytogenes. When they have sequenced the genes, they have been
greater than 95 percent DNA similarity, suggesting that they have
transferred. Now, which way they have transferred we have no idea, but
it does suggest gene transfer happens.

We also have cases of erythromycin resistance. The same gene. The same
gene was found in bacillus sphaericus, which is found normally in the
soil and also found in bacteroides, which causes blood born infections.
The same gene. Also, ampicillin resistance in E. coli. They found the
exact gene in heamophilius influenza.

So people may have thought before, yes, when we get gene transfer is
occurring between E. coli and E. coli or salmonella and salmonella, but
look at the slide. We are talking gram positive and gram negative. We
are talking enteric organisms and blood born infections. We are talking
soil organisms. So gene transfer is happening. We just don\'t know which
direction and how often it happens.

(Slide.)

DR. WHITE: Now, in terms of transposable drug resistances or which
antimicrobial resistances have been shown to be movable, it\'s a long
list. It is pretty much everything that we have. Beta-lactams,
chloramphenicol, tetracycline, macrolides, sulfa drugs; they found they
found streptogramins resistance now in plasmids.

One class that you see that is noticeably absence from this \-- anyone
see what I left off? It is an interactive session. You can talk.
Fluoroquinolones. Right?

Two years ago, however, there was a report of a plasmid medicated in
fluoroquinolone resistance in clebciella (sic). They have not identified
the gene yet, but it is a first report of plasmid mediated resistance.
Is that out there in the vet environment? Tough to say?

(Slide.)

DR. WHITE: So, as Dr. Flynn mentioned, and other speakers, why are we
here? Pre-approval studies? The rest of the talk I want to try to get
into factors to consider and questions. And when you sit down and you
think about the design of these studies, a lot of questions pop in your
head.

So hopefully, I can take the last 25 minutes here to throw out these
questions, and you can take them back into your breakout groups tomorrow
and say, oh, yeah, we have to take these into consideration.

Of course, pre-approval studies are intended to predict the likelihood
that antimicrobial resistance will emerge among enteric zoonotic
pathogens upon exposure to an antimicrobial. Also, they are intended to
predict the potential of antimicrobial to increase zoonotic pathogen
carriage and shedding in the target animal.

We have also, of course, other speakers this afternoon and tomorrow
morning that are going to go into more detail regarding these subject
areas, so I am just going to try to touch on some general ideas.

(Slide.)

DR. WHITE: I think some questions jump out at us right away when we talk
about designing pre-approval studies. The first one that jumped in my
mind was is there only one acceptable pre-approval design for all uses
and classes of antimicrobials? Is it going to be apples versus apples?
Or are we going to have apples, oranges, pears? This is my fruit
analogy.

Is there only going to be one acceptable

pre-approval design for all animal species? One study for swine? One
study for cattle? One study for poultry? What pathogen should be the
focus of pre-approval studies? As we saw earlier, historically
salmonella has been the pathogen of concern. Now that camplyobacter has
become the number one enteric pathogen, shouldn\'t we be focusing on
camplyobacter as our pathogen of concern?

How should the appropriate pathogen be selected? Is it based upon its
association with a particular animal species? For instance,
camplyobacter in poultry. We really don\'t know the incidence of
camplyobacter in cattle, so would we think about camplyobacter being the
organism to look at cattle studies?

How about aquaculture? We have no idea. Think about the questions that
\-- if we look at aquatic antibiotic approvals, what bacteria are we
going to pick to be our pathogen to look at? Salmonella? Aeromonus?
(sic) Vibrio? A lot of questions there to think about.

Also, in addition to a specific pathogen, shouldn\'t we be thinking
about looking at surrogate or endogenous bacteria? Those are questions
we need to consider.

(Slide.)

DR. WHITE: Some other questions, of course, and you may see some
redundancy, but I think this is good because others speakers will bring
these up and these are definite subjects that we have to talk about at
this meeting. Of course, what is the target animal? That is going to be
one of the first things.

What is going to be the exposure? Is it going to be something used in
the feed for two weeks? Is it going to be a one-time high dose? That
needs to be taken into consideration. If we do a study and we are going
to look at numerating or evaluating susceptibility of bacteria in the
feces, what are the points in time for sampling? Daily? Weekly? How long
do we go out? As Bill mentioned, what are you going to do? Go out until
the withdrawal time? Are we going to go out to the possibility of when
the animal is going to be slaughtered?

What is the nature of resistance to that particular antimicrobial class?
Is there literature already out there describing resistance mechanisms?
Do we know the prevalence of these genes out there? What is the effect
on pathogen carriage in shedding? Have there been any studies done on
similar antimicrobials of that class? And how are the studies validated?
I think that is something we have to think about in the future. How do
we validate these studies? Can we reproduce them?

(Slide.)

DR. WHITE: So, when we look at pre-approval study validation \-- and the
idea behind this pre-approval study, of course, is the determination of
the study\'s ability to assess microbial safety, and this is via
pathogen load and, of course, hopefully attempting to address direct and
indirect transfer resistance. And one question is, of course, the
reproducability. Are we able to reproduce this experiment?

Do we use standardized microbiological culture conditions and
susceptibility testing methods? What is the gold standard? There is the
BMFDA Manual for Bacterial Culturing. Do we use that as our culture
methods? Because when you talk about salmonella isolation, there are
quite a few different techniques for isolating salmonella.

For susceptibility testing, do we just accept NCCLS approved standards
for susceptibility testing? Or can it be something else that at least it
has proper QAQC, but it is not standardized by NCCLS.

(Slide.)

DR. WHITE: Other questions, of course, what is going to be the challenge
or the sentinel organism to be evaluated in the study? Are we even going
to pick a sentinel strain or are we just going to be happy with a
specific pathogen?

Historically, as we saw this morning, the 558 studies used salmonella as
the challenge organism. Is this appropriate? Are there other organisms
that we should consider, like camplyobacter or E. coli or enterococci?
So, are there better candidate organisms for this study?

Should it be antimicrobial class dependent? As you saw, one of the
weaknesses of that 558 was they were looking at resistance in gram
negative bacteria to gram positive drugs. Hopefully we don\'t repeat
that. But if we are using a gram positive drug, we need to look at
resistance development in gram positive bacteria. Likewise, for gram
negatives.

And should it be animal species dependent? Like I said, is there one
type of organism for swine and one type of organism for cattle and one
type of organism for poultry? You see, a lot of questions. A lot of
factors to consider so far.

(Slide.)

DR. WHITE: Some basic factors to consider maybe for the drug sponsor, of
course, is what is the intended method of administration and dose regime
of the antimicrobial? What is the intended dose, what is the frequency,
what is the duration?

What are the pharmacokinetics and pharmacodynamics of the antimicrobial?
These are things that are pretty much done routinely in the development
of an antimicrobial. What are the serum and tissue concentration at the
label doses? What is the concentration activity at the site of
infection? And maybe we have to start thinking about what is the
concentration and activity in relation to the site of potential human
pathogens, i.e., the intestine?

If the drug sponsor is creating an antimicrobial to target respiratory
infections, do they need to start thinking about the concentration of
that antimicrobial in the intestine as well?

(Slide.)

DR. WHITE: You can also think about actually specific antimicrobial
attributes. So we are getting down to that specific antibiotic that will
be the subject of the

pre-approval study. What is the mechanism of action? Is it cidal? Does
it kill? Or is it static? Does is just inhibit growth?

What is the spectrum of activity? Is it a gram positive? Is it gram
negative? Is it effective only against anaerobes or aerobes? What is the
potential to disrupt the GI flora? That is historically how 558 came to
be, is the gram positive drugs, of course, had effects on gram positive
organisms, decreasing their numbers and perhaps increasing the levels of
enteric gram negative bacteria, such as salmonella.

What is the target activity? As Jeff Watts mentioned earlier, broad
versus narrow spectrum. Does it only have a specific effect on a target
pathogen? Or are we having effects on our innocent bystanders, our
normal flora? And, of course, what is the gastrointestinal distribution?
How much of this drug reaches the intestine where these zoonotic enteric
pathogens may reside?

(Slide.)

DR. WHITE: Now, we talk about the antimicrobial use in these studies.
Are we going to base the study based on label use indications? Or are we
going to dream up a worse case scenario to predict how the drug is going
to be misused? The factors to consider here are the dose, frequency of
dose and duration, route of administration, IV, IM, SC, maybe
intrauterine, intramammary.

How numbers expect to be treated? Are we talking about an antimicrobial
that is going to be used in the water or the feed versus an injectable?
Of course, again, what is the animal species and what is the possible
duration of time between treatment and potential human contact?

If we are talking about an antimicrobial that is designed for the feed
lot where it could be four months to a year before that animal is
slaughtered, versus chicken, which is seven to 10 weeks.

(Slide.)

DR. WHITE: Other considerations, of course, is the likelihood of
resistance developing to that antimicrobial, and this will probably be
based on literature, doing literature searches to see if there is
already literature describing resistance out there, because what if it
is a new novel antimicrobial class? We don\'t know the incidence out
there.

What are the mechanisms of resistance and resistance transfer? Once
again, this would be based on the literature out there, any studies that
have been done on similar antimicrobials that are included in a
particular class. Or maybe they have studies based on that particular
drug.

Is there potential cross resistance to drugs of significant concern in
human medicine to both related and unrelated classes, because some of
these efflux pumps, when activated, give you broad specificity. They
give you multiple resistance to multiple classes of antibiotics. It is
just not fluoroquinolone to fluoroquinolone. In some cases it is
beta-lactam to tetracycline to chloramphenicol to fluoroquinolones.

And when we design these studies, can we detect step-wise decreases in
susceptibility to a particular antimicrobial or is it a night/day shift?
What I mean by this is this may be due to the mechanism of resistance.
For instance, fluoroquinolones, as we mentioned, are step-wise \-- you
do see a step-wise increase in resistance that is due to multiple
mutations in DNA gyrase.

So maybe a susceptible E. coli is at 0.03 micrograms per mil, and you
can start seeing the shifts in susceptibility at 0.25, 0.5, 1, 2, 4. We
can measure these shifts? However, when we talk about gentamicin
resistance, and gentamicin resistance is mostly due to the acquisition
of a gene on a transmissible element.

We have strains of E. coli say that are .05 or one microgram per mil.
The next step they are 16. There is really nothing in between, because
you are talking \-- you have acquired a resistance gene. So there we are
not really predicting a linear trend of resistance. It is one and then
it is 16. So once it is already 16, we have already missed the boat with
those.

(Slide.)

DR. WHITE: Let\'s talk about susceptibility testing. This is going to be
a large part of the

pre-approval studies, and Dr. Shryock will talk about this tomorrow, so
I just want to touch on it.

Of course, AST testing is standardized in vitro testing methods that may
provide qualitative and/or quantitative results. Qualitative results are
historically S, I, R, sensitive, intermediate, resistance, and may
indicate how an organism may respond to a drug in vivo. So it is a
prediction. In vitro, does it equal in vivo?

When we do quanitation, it is reported as the MIC, the minimum
inhibitory concentration. Of course, this is the most basic measurement
of antimicrobial activity against a target organism. The exact
definition of them I see is the lowest antimicrobial concentration that
will inhibit growth of a test organism over a defined rate related to
the organism\'s growth rate.

(Slide.)

DR. WHITE: Now, we try to factor in these considerations for
susceptibility testing. First, of course, we have to isolate that
bacteria from each fecal sample, and this goes back to some earlier
questions. Which bacteria are we going to test? Which pathogen? Are we
also going to look at commensals? If so, which commensals? E. coli?
Protius? Enterococci?

What is the gold standard for isolating this bacteria? We said there is
numerous methodologies out there. Are we going to keep one standard that
is the best? Bacteria then subjected to antimicrobial susceptibility
testing. Do we follow NCCLS guidelines?

(Slide.)

DR. WHITE: As Dr. Flynn mentioned as well, what is the appropriate
antimicrobial for susceptibility testing? Do we pick the relevant
animal/human microbial of the same class and how do we determine the
relevant animal/human antimicrobial? Do we test the animal antimicrobial
being tested or do we test both? And can we correlate susceptibility
data between veterinary and human antimicrobials of the same class.

(Slide.)

DR. WHITE: Other questions. How many isolates will be considered
statistically significant for the study? Say we have a salmonella
challenge study and we administer an antibiotic and that antibiotic
kills of 99 percent of the pathogen and we can only measure small
amounts as we go out six weeks. Is that going to be statistically
significant?

If we have colonies on a plate \-- say we have 3,300 colonies, which is
normally what you want when you want to do quantitation, how many of
those colonies do we pick to do susceptibility testing? The class
question never really been answered. Do we pick one colony? Do we pick
five? Do we pick 10?

Based on some of my earlier work at Penn State, when we looked at
genetic diversity, we found that E. coli has a genetic diversity of .25.
What that means is one out of every two isolates is different in the
normal flora. So if you have 300 colonies on that plate, chances are you
could have 150 different genotypes there. And how many different
susceptibility patterns? Tough question.

Of course, what type of susceptibility testing method can be used? There
is numerous methods out there for determining susceptibility methods.
There is only a few that are NCCLS approved, and I think that we have to
insure that proper quality assurance and quality control methodologies
are used when we do these studies, otherwise we can\'t validate them or
standardize them.

(Slide.)

DR. WHITE: So, just to give you a picture of some of the most commonly
used methods for susceptibility testing, is we have the agar dilution,
which is the gold standard of antimicrobial susceptibility testing. We
incorporate the antibiotic into the standardized media.

We also have agar diffusion or disk diffusion. Kirby-Bauer. A lot of the
veterinary diagnostic labs used Kirby-Bauer. We also have a new item
called the E-test, which is a disk diffusion, so to speak, but it is a
strip that is impregnated with antimicrobial and can give you an MIC.

We have broad dilution methods. We have macro dilutions where usually it
is two mils or more in test tubes, or we have micro broth dilution where
we are using a 96 micro plate. We also have papers that have talked
about replica plating and using the spiral plate readers; spiral
plating. These have been published in the literature; however, they are
not NCCLS approved. So, can these studies be done?

(Slide.)

DR. WHITE: Now, for challenge studies, think about it. If we are going
to go ahead and use a salmonella challenge study or another challenge
organism as part of our study, if we see shifts in antimicrobial
susceptibility, do we want to confirm that it is the challenge stream we
started off with? I think it would be an advantage to do that to make
sure another organism didn\'t slip in the side door. Now, how do we do
this?

Historically there has either been a biochemical marker or a particular
sugar that it ferments or an antibiotic resistance marker. As Jean
Cooper mentioned earlier this morning, the earlier salmonella challenge
strains were nalidixic acid resistance.

There are some new molecular biology techniques out there that perhaps
may help us out in this area, and this is the introduction of
florametric genes into the chromosome or plasmid of a particular strain.
These genes fluoresce, green fluoresce in protein, which stands for GFP,
or luciferace; glows in the dark under UV light.

Can we clone these genes into a particular salmonella and follow the
challenge strain that way? If we do, do these genes that we have
incorporated have any effect on the susceptibility of that organism? And
if we do have strains that exhibit decrease in susceptibility, do we
want to genetically confirm that they are the strain that we started
with? We can do this by either post-field \-\-- or ribo printing. So how
far do we want to take these studies to insure it is the same strain we
started off with?

(Slide.)

DR. WHITE: Also, there are models out there to predict antimicrobial
resistance. We have pharmacodynamic models that have been developed to
identify factors associated with the probability of developing
resistance, and these are based upon time dependent killing, your
percent time serum above MIC, and this is mostly for beta-lactams, also
studies based on the concentration dependent killing, your ratio of the
serum of the curve or c-max to MIC and this has been done for
fluoroquinolones. I hope Dr. Papich will talk a little bit about this
later this afternoon.

(Slide.)

DR. WHITE: We also have other models looking at the combination of
pharmacokinetics and bacterial population dynamics. These models try to
predict and optimize the effectiveness of particular dosing regimes
against susceptible bacteria. It is only done with hospital isolates so
far. So we haven\'t really introduced this technique into veterinary
medicine.

And there is limited information concerning the problem of resistance,
which is only due to susceptible bacteria, and it has been only relevant
to resistance due in mutations only in particular fluoroquinolones, and
I think Dr. Lipsitch will talk about this tomorrow. I hope.

(Slide.)

DR. WHITE: So, let\'s finish up with pathogen load studies and some
questions to consider here. As you saw, there was quite a bit of
questions to consider for the susceptibility part of the studies.
Tomorrow we have Dr. Cray that is going to talk more about this. So I am
just going to hit you with a couple of slides.

Of course, pathogen load studies are designed to detect increases in the
number of pathogens in the environment. This is usually done by
measuring the numbers of bacteria in the feces. They are designed to
detect increased duration of shedding of the specific pathogen and
increases in the prevalence of animals colonized with that specific
pathogen.

Now, we can\'t make broad inferences sometimes about these studies. It
has to be specific or related to the specific pathogen we are talking
about. It needs to be related to the specific antimicrobial involved,
related to the duration of antimicrobial therapy. And once again, I
think we need to address this concept; what is the time between
cessation of therapy and slaughter of the animal?

(Slide.)

DR. WHITE: When we talk about these pathogen load studies, they should
be able to measure the effect of the antimicrobial on two things. One is
the number of target pathogens shed. For instance, when we do these
types of studies, can mean differences be measured between non-treated
and treated animals? What is the sensitivity of the study?

If we only get 10 colonies on a plate from both animals, is that
statistically significant? What is the duration of shedding of a target
pathogen, and this comes down to what are the time points for fecal
collections. How far out do we take this study? That was brought out
before. Eight weeks was historically for 558. Do we want to go longer
than that?

(Slide.)

DR. WHITE: So historically, as Dr. Cooper mentioned, all animals \--
when we talked about salmonella challenge studies, all animals were
orally challenged with

s\. typhimurium. The first thing the drug sponsor had to do was
establish an infective dose. She said that was relatively high; 10 to
the 8th; 10 to the 10th.

How as it introduced to the animal? Was it put in the feed? Was it put
in the water? Were the animals starved? All questions to consider. There
are at least two test groups. One group treated with the antimicrobial
and an untreated control group.

(Slide.)

DR. WHITE: Fecal samples were then obtained prior to and after dosing in
the animal with the antimicrobial. Fecal samples were then cultured for
salmonella, and we had quanitation of numbers. Once again, we bring up
the idea of sensitivity of that. And bacteria were tested for
susceptibility.

As we saw, to test antimicrobials it is not always the case and that
they may have tested that challenge strain to other antimicrobials and
not even the antibiotic that they were testing.

(Slide.)

DR. WHITE: Now, there is some limitation to this, and Dr. Cooper hit on
these as well. The salmonella shedding studies are only a imitation of a
naturally acquired salmonella infection. We are putting this in a test
barn. Is it real life? Because there are a lot of other factors that
effect shedding in salmonella in real life.

Presence of other viral infections, other bacterial infections, other
stresses involved, nutrition status of the animals, immune functions,
and also, variations in pathogenicity among different salmonella
serotypes. Is the one strain we pick for these historic salmonella
challenge studies the best one to use?

(Slide.)

DR. WHITE: So, two major questions. I think that will \-- hopefully you
can address some of these items in the breakout groups. Considering the
collection of fecal samples prior to and after dosing the animal with
the antimicrobial. How much is collected? Do we collect one gram? Ten
grams? Twenty-five grams? These are simple questions. Are we going to
get 25 grams from a chicken? Probably not.

There have been studies that FDA has funded looking at the incidence of
shigella like toxin producing E. coli and they looked at the difference
between taking one gram of fecal material from a cow versus 25 grams.
The prevalence rate increased 10 fold when you went up to 25 grams.

How many samples are collected? Do we do this once a day? What time
during the day do we do this? Does this standardize it somehow? Once
again, what is the interval samples collected? Daily? Weekly?

Can we culture for these select bacteria? Like I say, we are going to
culture for this pathogen that we are doing as a challenge study. Do we
also want to look at the effect of other commensals? Do we use
enrichment methods? If we use enrichment methods to detect the pathogen,
well, we have changed our quantitation. Right? Because we have taken a
bacterium that is normally low, low numbers, we have

pre-enriched it to detectable numbers, but we really can\'t quantitate
that because we have used a selective enrichment.

What are the isolation methods? Is everyone using the same technique?
Are we using method out of the BMFDA Manual or are we using other
established methods that have been proven? And can we quantitate
bacterial populations?

(Slide.)

DR. WHITE: Now, I think there is some future research needs that need to
be addressed. I said earlier there is this grassroots effort to look at
this as what is the contribution of the normal flora to antimicrobial
resistance. I think we need a lot more focus in this area. And are there
innocent bystanders? Are normal flora packing the bags or holding all
the guns, so to speak, for all the other pathogens when they come in
contact with them?

What factors contribute to the selection of resistant microbes? Can we
optimize PK/PD factors to minimize resistance? And can we optimize dose,
frequency of dose, duration and minimize organism exposure? I think this
is something to look into.

(Slide.)

DR. WHITE: What is the frequency of selection for resistance of specific
antimicrobials. We have a lot of laboratory data and in vitro data, but
we really have limited in vivo data; what is happening in the animal.
Most of our studies are done in the lab bench.

How do resistance mechanisms evolve? Are there already pre-existing
sources of resistance genes or organisms in the environment? You may not
see resistance in your target pathogen, but there is other normal flora
out there that have these resistance genes present.

Are there other selection pressure, such as heavy metals and
disinfectants? As we mentioned earlier, some of these transmissible
elements, such as an integron or transposons, sometimes in addition to
antibiotic resistance genes, they have genes for heavy metal resistance,
such as mercury. They also have resistance genes for antiseptics like
ammonia compounds.

Can the use of heavy metal or clots select for antibiotic resistance? In
that case, yes. So even when you stop using the antibiotic, you have
selection pressure that may keep the antibiotic resistance phenotype
around.

Of course, gene linkage we are seeing more of. This is, once again,
integrons and plasmids and transposons. We are seeing multiple
antibiotic resistance now as the norm and not single resistance.

(Slide.)

DR. WHITE: I also want to let you know that CVM has put out a request
for applications for microbiological hazards associated with the food
production environment. The RFA is out on the table. I think the
deadline is April 30th for applications, and we invite you to submit any
proposals dealing with these types of issues.

In summary, I would like to leave you with some bullets to take home. I
think, given sufficient time and use, antibiotic resistance is going to
emerge. It is a given. It is a natural event of the use of an
antibiotic. There are no antibiotics to which resistance has not
eventually appeared.

Resistance is progressive and evolves from low levels through
intermediate to high levels. Of course, unless it is acquired as a
transmissible genetic element where you may go from that night/day shift
I talked about. Usually resistance occurs in small increases in the MIC,
and this is how we have seen it with fluoroquinolone and E. coli,
penicillin resistance to strep pneumonin, and these VIS, this vancomycin
intermediate staphorius. We have seen small progressions in the MIC.

Now, the VIS may forewarn the appearance of

full-blown vancomycin resistance determinants or full-blown vancomycin
resistance stephorius in the future. Bacteria that are resistant to one
antimicrobial are likely to become resistant to others. They roam in
packs.

(Slide.)

DR. WHITE: Once resistance appears, it is likely to decline slowly, if
it all. So, once the cat is out of the bag, how do we get it to reduce
the incidence of resistance? The use of antimicrobials by one person or
animal effects others in the extended and the immediate environment.

I feel we need to minimize the environmental impact of antibiotics and
we do this by reducing the incident of exposure. The less likely
selection and transfer resistance rates will occur if we reduce the
extent of exposure.

So hopefully you have seen that there are many factors to consider
regarding study concepts for modeling resistance development pathogen
load changes, and you can take a lot of these factors to consider back
to the breakout groups tomorrow.

(Slide.)

DR. WHITE: And I wanted to end on one slide to try to summarize
everything that is going on, and I was lucky yesterday that I had Bob
Walker and Pat McDermott come into work and help me with my talk. We
were trying to figure out some way to end the talk, and he brought up
the analogy of Apollo 13. I am going to take you back to this.

If you remember, Apollo 13 was a routine mission to the moon. Of course,
this was the Tom Hanks movie, for those of you who saw that. And they
had a short circuit and an oxygen module blew up, depriving them of
electricity, oxygen and their engines. So they were in dire straits,
heading to the moon without anything.

Well, what happened was at NASA a diverse team of people, computer
engineers, computer scientists, mathematicians, physicists, all came
together to try to get these guys home. And it was said by one of the
reporters there, \"This may be NASA\'s worst disaster.\"

And one of the mission control specialists responded, \"Or it could be
its shining moment.\" So, can we do the same thing that these people did
at NASA? We have such a diverse group ourselves. Microbiologists,
veterinarians, physicians. Can we all come together to try to address
this issue, this crisis of resistance, and come up with a successful end
point? I thank you for your time.

(Applause.)

CHAIRWOMAN LATHERS: Thank you, Dave. I would add one comment, in terms
of your analogy to Apollo. Apollo 17 is an example. We clearly went back
to the moon and walked on it again, and I think you are right. If we
work together as a team, we will resolve some of the questions before
us.

Are there a few questions for Dave before we begin our formal comment
period? Jim, would you care to come up, please.

MR. : I want to reiterate a point you made, and that is by virtue of
transformation, free DNA can be taken up by other bacteria, so
therefore, in many ways dead bacteria have DNA which is just as usable
as live bacteria. And I think that is a critical point right now because
\-- well, just this morning I saw on the news that we have irradiated
meats on the way to our grocery shelves, and they are going to have
plenty DNA with plenty of antimicrobial resistance genes more than
likely.

DR. WHITE: That is a good point, Rick. I think people thought, in the
past, when an organism is dead, that is the end of the story. And we are
finding more and more now that DNA can hang around and be taken up by
live bacteria.

MR. : David, you gave us a whole list of questions. There may have been
several hundred of them, and they were all valid, and they were all
deserving of answers and those answers are only likely to be obtained
with quite a bit of time, energy and research.

In the meantime, we, you, are presented with the problem of producing
something in the short-term to answer the question of what will the
sponsor be asked to do to register a new compound. You referred to
innocent bystanders, and the question of innocent bystanders came up
before. I think there is one innocent bystander in all of this and that
is the poor drug sponsor. Thank you.

DR. WHITE: and the animals too, Robin. Right. Yes.

CHAIRWOMAN LATHERS: Are there other questions for Dave at this time?

(No response.)

CHAIRWOMAN LATHERS: If not, thank you.

(Pause.)

**OPEN COMMENT PERIOD**

DR. HESLIN: Good afternoon. My name is Jim Heslin. I am with FDA\'s
Office of Human Resources, and I have been asked to sort of moderate the
open comment periods. If you look at your agenda, there are several
spots. A couple today, some more in the next two days, where you will
have the opportunity to make your comments.

This is not really a discussion session or question and answer period,
but rather, an opportunity to kind of enter into the record your
perspectives or comments. If it is extensive, keep in mind that
everything can be submitted to the docket to be included in the write up
of the session.

And what I am going to ask you to do is, for those who want to give a
comment, to come to one of the microphones, introduce yourself and the
organization that you represent and focus on anything that has been
discussed so far here today. You know, going back to the sessions this
morning, keeping in mind that there will be other comment periods later.

So, we have about a half an hour. I have no idea how many people would
like to make comments, but initially, I would like to set a guideline of
three minutes for comments. If there is more time later, we will come
back. But I will let you know when you have about half a minute left so
you can close out your comments. Yes?

DR. ANGUELO: I am Fred Anguelo, from the food-born and viral disease
branch in the National Center for Infectious Diseases. I wanted to
follow up on comments made this morning about the 558 studies and my
judgment of their limited value for public health, although perhaps we
should acknowledge that we don\'t know what antibiotics were not
presented to FDA. So perhaps those studies did prevent aminoglycocides
or cephalosporins being approved sub therapeutically.

But nonetheless, we should also recognize that those 558 studies led to
development of gram positive spectrum antibiotics for growth promotion
or for sub therapeutic and only now we are beginning to see consequences
of those, such as macrolide and lincomycin resistance. And we are yet to
see, but we believe we were on the verge of seeing streptogramin
resistance as a consequence.

But those being our conclusions, I think most of would acknowledge that
the flaws that exist in the 558 studies, and those flaws are that the
antibiotic that was tested for was inappropriate. They tested against a
panel of 12 antibiotics, none of which was the antibiotic on which the
organism was tested against.

The pathogen they tested against was to narrow, only looking at
salmonella. And then finally, it was quite an artificial setting. You
know, it wasn\'t a field setting, not taking into effect the dynamics of
spread in the field.

That being said, I think that I am a little bit frustrated, because what
I have seen so far is I almost get the impression, or the suggestion,
that perhaps, as we went through this new paradigm of drug approvals,
that we should be trying to think how to adapt the 558 studies into this
new paradigm, and I petition that we just scrap that whole paradigm and
actually try to think outside the box.

I think that we really need a new paradigm of what we should be doing.
That being said, at the risk of using up all time, I guess I also want
to express my frustration; that it has been 12 months into the framework
process and I haven\'t heard a concept proposed from FDA on what they
propose to do on the pre-approval studies. I have heard lots of
questions. We could have asked these.

You probably would have given us the same questions 12 months ago, and I
don\'t understand why it took 12 months for you to develop those
questions.

But nonetheless then, let me just comment upon two parts of the 558
studies. First, the pathogen load parts. As I mentioned before, I see no
value of the pathogen load studies, especially for the gram positive
spectrum antibiotics. Perhaps there is some value for gram negative
antibiotics.

But, as many of you know, the food-borne viral diseases branch at CDC
does a lot of work studying the pathogens in the food supply, food-borne
pathogens in the food supply, and it is the conclusion of our branch
that such pathogen load studies are of limited value. There are other
national initiatives undertaken to mitigate pathogens in the food
supply, such as educating consumers on new pathogen reduction of
processing and even irradiation, all of which will reduce pathogen load
much more effectively than trying to screen antibiotics before they are
approved.

It is not clear, in fact, whether antibiotic use in food animals
influences pathogen load at all of susceptible organisms, however, it is
clear that antibiotic use in food animals does influence the
transmission of antibiotic resistant pathogens to that antibiotic to
which they are used.

DR. HESLIN: Could I interrupt for just a second, because you are about
out of time? But I wanted to see whether others had intended to make
comments so I could \--

(Show of hands.)

DR. HESLIN: Okay. You can continue then.

DR. ANGUELO: I apologize for being long winded. So I actually then would
hope that maybe there could be a compromise struck with the industry,
because if the resources that are necessary to meet the criteria of
pathogen load testing under the current 558 studies would \-- maybe
those could be redirected to do something more useful in the resistance
studies and maybe we could agree to scrap the pathogen load testing and
use those resources with some enhanced studies in resistance; so then to
focus on what studies might be useful in resistance studies.

My impression of the problems of resistance studies is it is so much
geared as a pass/fail system. Exactly what David White just said, is
emergence of resistance is inevitable. And if we did good enough
studies, everything would fail. I mean, everything would develop
resistance.

So I am not sure that the paradigm should be a pass/fail paradigm. I
think the paradigm should be gathering information that is of public
health utility in the

pre-approval studies. What would be such information that would be of
value?

Well, I think it would be very useful to know the frequency with which
mutation emerges in a laboratory when you give the antibiotics. It is
very interesting to know how frequently fluoroquinolone resistance \--
or mutations occur that cause resistance to fluoroquinolones. That would
be a valued pre-approval, although I wouldn\'t fail an antibiotic if it
does cause a mutation to occur.

It also would be very interesting to know, before a drug is approved,
the frequency with which resistant elements are transferred. Of course,
that would require the existence of a plasmid that mediates such
resistance. In other words, only unless there is a resistant element
already present will you have a predictive model of whether resistance
will be transferred.

So what else would be interesting besides doing mutation studies?
Studies of movement of the resistant element, if a resistant element is
known to occur. So then, what then would be useful pre-approval? Well,
what would be real useful would be to \-- under a field setting, such as
what occurs in phase three clinical trials, in a very holistic approach
to study the environment around the selective pressure to see if
resistance emerges.

The environment could be the intestinal flora of the animal exposed to
the new antibiotic and also the organisms in the environment. It could
be done in a very focused manner by using selective media instead of
using a whole battery of antibiotics. Just use an impregnated selective
media against the antibiotic you are testing and see if any resistance
emerges under the selective \-- as a consequence of selective pressure
that is used in that field setting.

If a resistance should be detected, then you can study that resistant
gene which you have just \-- you will first determine if resistance is
intrinsic or due to the emergence of a resistance gene, and that would
be of great utility, from a public health perspective, because before a
drug is marketed, we might have a library of resistant genes; so that if
resistance should begin to emerge of consequence, we would have a
library of resistance genes that we could compare the resistance for
detecting the human pathogens to the library of resistant genes that we
had before the product was marketed.

Lastly, perhaps the key fundamental thing that I think pre-approval
studies need to support is we need to categorize the drugs. If we have a
brand new class of antibiotics, what class of drugs would it be? And
unless we do a field study to see what resistance emerged from this new
class of antibiotics and then find out what that resistant gene choosing
cross selection for, for which human drug, it is hard to imagine how we
are going to categorize that drug.

If we have a brand new drug that causes \-- there is no cross resistance
to any human drug, it would be one category. But if we find a resistant
gene and find that it causes cross resistance to a category one human
drug, then, of course, it would be a different approach.

Sorry again for the long winded comments, but in conclusion, I would
encourage thinking outside the box on these pre-approval studies. I
would reconsider pathogen load testing, and I would have a more holistic
approach on the pre-approval studies, looking at the emergence of
resistance in all organisms.

And specifically, to the antibiotic that is being tested for, if that
emergence should emerge, look for cross resistance to human antibiotics.
And the whole strength of the pre-approval hopefully would be to develop
categories; help to strengthen the categorization process to support the
threshold and corrective action concept.

DR. HESLIN: Thank you. Yes. I was looking for key words. And when you
say \"lastly,\" I thought it is almost over. But then you said later,
\"and then in conclusion.\" So, any combination of those words is
helpful for me to see how much more time you need.

If you would introduce yourself and your organization.

DR. GOOTZ: My name is Tom Gootz, from Pfizer. My key word is three
minutes. I don\'t know that much about this to talk longer than that.

I agree with the comments just made. The majority of them. They are
certainly very realistic. And I guess I would add a direct comment which
says \-- when I worked, for 16 years, in the human health area for
Pfizer, for antibiotics, we were also asked, for fluoroquinolones for
instance, to do pre-approval studies.

We would have to dose 30 or so healthy volunteers for 10 days with our
product, the same dose we would use in the clinic, and we had to do a
fairly expensive and extensive colony count studies for all the
different types of floras. The facultative bacteria, anaerobes, gram
positive, enterococci and staph, yeast, and I am probably leaving
something out.

This has been published recently by Carl Knord in 1999 who just
summarized all these such studies in the human area. And what we found
was that for about 12 different quinolones, obviously you decrease the
facultative flora in the gut. You also, in some instances, knock down
the gram positive flora. Very rarely do you even touch the anaerobic
flora of the gut. But in 10 days after you stop dosing, all the flora
comes back.

But more importantly, during the study we were asked by the FDA to do
quantitations of any quinolone resistant organisms, and with only one
study that I can remember from Carl Knord\'s publication, we never found
any. The only study that I am aware of in the human area was in 43
patients who had acute leukemia and received one specific
fluoroquinolone for quite some time.

So my comments is \-- and I am very impressed with all the thinking that
is going into this. It is probably one of the most complicated issues
you ever could possibly deal with. Maybe even worse than gene therapy
itself.

But I am just wondering the real value of

pre-approval studies. Maybe that is the key, is that the pre-approval
studies, even though they are very well intentioned, aren\'t gong to
help that much. Again, based upon my experience with the human area of
new antimicrobial development.

What seems to help is post-approval of good, quality surveillance
studies because that, of course, is what is done in the human area. If
in certain areas you see increase in resistance in certain pathogens,
then you have to take effect. You have to limit how those drugs are used
by rotation or something or stop using them for trivial infections, and
that is how we take care of resistance in the human area.

I will just close by saying that I know I will hear a lot about
different approaches in the next couple of days, but I will bet in the
end that is probably how we are going to have to do it in animal health
as well. Thanks.

DR. HESLIN: Thank you. I think there was someone else in the back there
that wanted to make a comment.

MR. : Hi. Steven \-\--, Union of Concerned Scientists, and the key
phrase is shooting for three minutes. Sorry. This is a written
statement.

USC is an independent, non-profit public interest group of citizens and
scientists working to promote a sustainable food system. We advocate
responsible public policies in areas where technology plays a major
role.

USC is working to encourage healthy and sustainable method for crop and
livestock production and to insure the consumers and citizens have a say
in how food is produced. We are facing a human health crisis as the
result of the emergence of antibiotic resistance diseases.

The rise of resistance bacteria could lead to epidemics and infectious
diseases not seen in the United States since the beginning of the
century. Or actually, the beginning of the last century.

The lives of children, the elderly and many others are threatened.
Antibiotic over-use is a problem in the medical, clinical and
agriculture settings. We recognize that medicine\'s over-use of
antibiotics to treat human ailments is the main contributor is the rise
to the resistance, but agriculture plays an important role also.

Although the actual usage is not known with any precision, agricultural
application, usually said to amount for upwards of 40 percent of the
antibiotics used in the United States, and most of this is directed
towards animal growth promotion.

We commend the Center for Veterinary Medicine for its recent work on
this issue and through the development of disease resistance prevention
framework and for offering these public meetings, but this effort is too
little too late.

The framework targets primarily new antibiotics and new uses and
basically ignores those already registered as livestock growth
promoters. The evaluation scheme is complicated and may not work well
enough to effectively arrest the development of resistance. A growing
body of evidence demonstrates that animal use of antibiotics is
threatening human health today. The proposed framework does not make
sense in light of this information.

USC urges a more aggressive plan for dealing with the problem of
antibiotic resistance. A sound first step now would be for the FDA to
develop a plan, as animal growth promoters, those antibiotics that are
also used in or related to those used in human medicine.

Antibiotics could play a role in modern agriculture, but their use must
be prudent. Use as growth promoters is unnecessary. We simply should not
be giving drugs to healthy animals. To endanger public health by
reducing the effectiveness or availability of antibiotics is
unacceptable.

The Food and Drug Administration, under the Food, Drug and Cosmetic Act,
has the authority to withdraw approval of all antibiotics on the basis
of their risk to human health. In the 1970s the FDA embarked on a bold
initiative to remove at least some human use antibiotics from animal
agriculture. Unfortunately, the agency was unsuccessful.

Studies published over the last 20 years have only strengthened the case
that antibiotic use in animal agriculture is leading to resistance
development, and public health officials, physicians and scientists all
agree that there resistance problem is urgent. The arsenal of drugs
available to fight infectious diseases is dwindling. We cannot count on
newer drugs to fill these gaps in the

short-term.

It is striking that the FDA was willing to cancel antibiotics in the
1970s, but now, with stronger scientific support and recognition of
harm, is proposing only a review of new drug approvals. We urge the FDA
to boldly act now as it did in the past to use its authority to prohibit
human use antibiotics in livestock production.

Such action will help prevent the emergence of untreatable infectious
diseases and help safeguard important antibiotics for future use in
human medicine. Thank you.

DR. HESLIN: Thank you. Yes?

MR. MINERSMAN: My name is Rick Minersman. I\'m from the USDA
agricultural research service, and my key word is realistic. One of the
things about all this is predictability.

And to restate something David said again, most of the antimicrobial
resistances that we have known to date are transmitted from bacteria to
bacteria. Most of these are cases where we are dealing with antibiotics,
which are naturally occurring, and then we have grown them up, amplified
them and modified them. But they started out as naturally occurring
products.

With the new generations of synthetic antimicrobials, bacteria are
finding new ways to circumvent the antimicrobials, and a lot of this has
been unpredictable. It could be something that occurs in less than one
out of every 10 or 13 generations.

One of the classes of antimicrobial resistance is transported across the
cell membrane, and we have a class of genes that are called multiple
resistance genes. It has been impossible to predict ahead of time which
antibiotics these genes effect.

You may have a multiple resistance gene which interferes with mercury,
for instance, that initially doesn\'t interfere with a new
antimicrobial, but that becomes \-- it is changed by mutation and that
could arise later as a new antimicrobial resistance.

Again, this is unpredictable, and if it occurs at a frequency of less
than one out of 10 to the 13th, how much can we expect to achieve in a
pre-approval study to look for these very rare events?

DR. HESLIN: Thank you. We have just a couple of minutes left.

DR. MEVIUS: My name is Dik Mevius. I am from the Netherlands National
Institute for Animal Science and Health, and my key word is optimize
dosage regimens. I would like to comment on the very scientific approach
of Dr. Anguelo, which is very nice: To use pre-approval studies to build
a library of new resistant genes and to use these, the knowledge that
you gain, to study what is happening in the field.

I think it is important to realize that you can optimize a dosage
regimen with respect to their selective property of resistance
selection, and we, in the Netherlands, have a long experience or
tradition with use of fluoroquinolones since 1987. I have been in former
meetings talking about licensing of fluoroquinolones here in the USA.

We saw, after licensing in 1987, a very rapid increase of
fluoroquinolone resistance, because this was the new wonder drug that
was used in an irrational way; in large volumes. Because of that, we
have these kind of meetings as well in the Netherlands and the
professional organizations and the veterinarians themselves build a
veterinary antibiotics policy, and we wrote guidelines.

And these guidelines are very important aspects of training of students
and post-graduate training of veterinarians and these guidelines were
not \-- well, a strict guideline to follow. Always fluoroquinolones as
third choice treatment or never use fluoroquinolones, but use them in an
appropriate way for the right indication, in a right way, and that has
really resulted in a reduction of fluoroquinolone resistance in these
clinical isolates of salmonella and pasteurella and E. coli.

So I think you can use pre-approval studies not only for very
scientific, very important information, but also to, as a licensing
authority, optimize an optimal dosage regimen with respect to resistance
selective properties. Thank you.

DR. HESLIN: Thank you. We are out of time for this open comment period,
and I believe the next thing on the agenda is a break, reconvening at
3:15. That is about 15 minutes from now.

(Whereupon, at 2:58 p.m., a brief recess was taken.)

**SESSION 2 - INTRODUCTION**

**By Dr. David White**

DR. WHITE: If we could start sitting down, we can start the afternoon
session.

(Pause.)

DR. WHITE: Can we take our seats, please. All right. We might as well go
ahead and get started and people can wander into the room when they have
a chance.

This is session two. I want to thank Dr. Claire Lathers for keeping us
on time in session one, and I will try to do the same thing. But, as you
will notice, we are already 10 minutes late. People like that break too
much.

Session two is dealing with study concepts for modeling resistance
development and/or pathogen load changes, and it is a little bit
different than the morning session, in that the morning session dealt
with some of the objective and also kind of background information.

This afternoon we have speakers from the pharmaceutical industry, from
government and European experts to talk about some of the concepts that
we need to take into consideration when we think about these
pre-approval studies.

To start off with, we have Dr. Kathy Ewert from Bayer. Dr. Ewert
received her DVM from the University of Illinois. She then completed a
one-year internship and a two-year residency at Perdue University in
large animal medicine, internal medicine. She then returned to the
University of Illinois and obtained a master\'s degree in large animal
parasitology in internal medicine.

Dr. Ewert has worked for Bayer for seven years as a project manager in
pharmaceutical research and development and in the past few years her
emphasis has been on issues surrounding antimicrobial resistance as they
relate to food animal products. Dr. Ewert.

**USE OF ANIMAL STUDIES**

**By Dr. Ewert**

DR. EWERT: Thank you, Dave. I would just like to take a moment to thank
CVM and Bill Flynn for inviting me to participate in this workshop, and
I look forward to a very fruitful discussion over the next few days.

But before I start my presentation, I have to make one disclaimer and a
couple of comments. First of all, I think this morning Dr. Sundlof
mentioned that this afternoon would be full of experts speaking in
different areas, and I would like to put out the disclaimer that I am
not an expert in designing animal studies to address these issues. I was
just the poor, unfortunate soul that was given the task of trying to put
the framework document into a usable protocol before we had much
guidance.

And so I have been working with the agency there, but I am, by no means,
an expert on the use of animal studies.

And then, a couple of other comments or observations. I found myself
agreeing with everything that Dave said and that Bill said and many of
the things that

Dr. Anguelo said, and so I think that is a red letter day when I find
myself agreeing with both the agency and CDC. And I find that very
encouraging because I think that we have the basis then for moving ahead
and finding a solution to this very difficult problem.

Secondly, a lot of the things that I am going to present to you today
have already been presented. So, if you will indulge me with the
redundancy in what I am going to present, I think you will see that we
are all kind of on the same track about what needs to be done and what
needs to be addressed.

(Slide.)

DR. EWERT: First of all, again guidance document 78. The conclusion of
that document said that drug sponsors of all antimicrobial new animal
drug products must look at the potential for human health impact of
microbial effects of such drugs and that pre-approval studies may be
needed. So that was the basis for looking at pre-approval studies.

(Slide.)

DR. EWERT: FDA\'s objectives, as stated in the framework document and
the guidance document, were to assess the rate and extent of resistance
development in enteric bacteria, to evaluate mitigation measures to
determine their effect on decreasing the rate and extent of development
of resistance, and finally, to asses changes in pathogen load.

One other comment I just wanted to make on pathogen load is that in
reading the framework document there is a number of different references
to pathogen load and different definitions. So this definition was taken
from the guidance document, which seemed to be most encompassing; that
pathogen load is the number of enteric bacteria in the animal\'s
intestinal tract that cause human illness. And I inserted \"have the
potential to,\" because not every single enteric bacteria does cause
human illness.

(Slide.)

DR. EWERT: So, what is the basic design of these animal studies?
Basically, you take your target animal, whether it is cattle, swine or
poultry, and all of their resident enteric bacteria and perhaps some
experimental colonized bacteria, and you expose that unit to the new
animal drug in question and then you measure the effects of that new
drug on the gut flora.

You look at changes in susceptibility or pathogen load. So the basic
design of the animal studies are very similar to the 558.15 studies that
have been discussed earlier today.

(Slide.)

DR. EWERT: In this presentation I would like to have you consider
several areas in the animal studies that we need to look at. First of
all, the design of the study, then the conduct of the study, what is the
target animal, what about this new animal drug, what is this target
bacteria that we are looking at, a few thoughts on microbiological
methodology, and then finally, how do we interpret these results.

(Slide.)

DR. EWERT: And, as Dave went through, I have to tell you there is going
to be a lot of questions ahead, and many of my questions will be the
same questions that Dave raised. That is just because we just plain
don\'t know the answers.

(Slide.)

DR. EWERT: So, for example, in study design, how many studies are we
going to have to do to answer these questions? Are we going to do one
study to be able to characterize resistance and assess pathogen load? Or
are there going to be several studies or more than several studies?

And what are the objectives of these studies going to be? Do we want to
characterize and assess this new animal drug at use rate? Or do we want
to exaggerate the use rate or the dosing frequency to push for the
selection of resistance? And what type of study do we want to do? Do we
want to do it in the laboratory or in a field situation? What is more
meaningful? And how many different study sites do we want to do this at?

If we do a feed lot study, do we want to do it at five or 15 or 25 feed
lots? What about study groups? There has been mention of a control group
and a treated group? Well, if you have flexible label or more than one
dosing regimen, do we need to look at all of those different dosing
regimens with in the study group?

What kind of statistical power are we going to generate in looking at
these studies and how many animals do we have to use? And what about the
statistical analysis? What are we going to analyze? I\'m not sure. What
is the end point of the study?

There has been discussion earlier about pass/fail criteria. These need
to be really established prior to the conduct of the study so we know
what our goal is.

(Slide.)

DR. EWERT: What about the timing for the conduct of these studies? In my
opinion, it should be in the early discovery \-- or it should be in
discovery or early development phase, and that information should be
generated, shared with the agency and accepted early on.

Perhaps we could do these studies and combine them with other early
development or discovery studies, such as pharmacokinetic studies or
residue studies. And then also, we need to keep in mind that we want to
minimize the number of studies that we do, because while we are
stressing to promote public health and safety, we have to look at the
personnel that are conducting these studies, and we could be causing
unnecessary exposure of personnel to potential pathogens.

(Slide.)

DR. EWERT: What about the housing of the animals? Do we want to
individually house these animals so that there is no chance for
transmission? Or do we want to put them in small groups? Or do we want
to put them in production conditions where this is the natural way for
the bacteria to move from animal to animal?

What about the effect of water and feed and environmental contamination
on study outcomes? How many of you have ever looked at what goes into
the feed that you put into these animals that we do these studies on?
And do the microbes that are in the feed then show up in the gut flora
of the animals? And what if these microbes are resistant already? Then
is the drug at fault for that or is it some predisposing cause? So what
should be controlled in these studies?

(Slide.)

DR. EWERT: And again, as Dave alluded to, what should be sampled and
when? Do we do fecal samples at certain time points and how often? And
how long? Do we mimic the stress of hauling and taking these animals off
feed as they experience before they go to slaughter and then

re-test them again to see if we can induce shedding?

In the framework document there is some mention of looking at pathogen
load at slaughter. Well, if we are going to do that, should we look at
fecal samples? Or should we look at the carcass? And at what point in
the processing of this carcass should we take samples to see what kind
of contamination is present?

(Slide.)

DR. EWERT: And the thrust of this presentation, of course, is animal
studies. So we want to look at the target animal and define what this
target animal is. Do we want to do these studies in clinically healthy
animals? Or do we want to do it in a sick animal with the disease for
which the antimicrobial is indicated? That is really my target animal.

From a humane point of view, is it humane to colonize an animal that is
already sick with respiratory disease with some other enteric pathogen
that could cause severe clinical illness in that animal? And what should
be the age of these animals that we use? Should it be for label
indications, say in feeder pigs, that are only 40 pounds of weight and
have another four or five months to go to slaughter? Or should we do
these studies close to slaughter, because this is a public health issue?

Or, as Dave mentioned, should we follow these animals out for the whole
life time of the animal to see what happens to the dynamics of the gut
flora? And when we choose these target animals, we want to choose
animals that are representative of the animals that are in production,
of the animals that produce protein that we consume.

(Slide.)

DR. EWERT: The new animal drug is a big issue for those of us in the
pharmaceutical industry. Which formulation should we use? Should we use
the active ingredient? Should we use a near final formulation? Or should
we use GMP produced formulations? Each one of these is a step in the
development process of the drug, and represents significant cost and
investment to the company.

For example, the active ingredient could be used early in the discovery
phase. If we go to GMP produced formulations, that is late in the
development and represents a big risk to the company, should the
pre-approval studies not pass and the drug then would no longer then be
pursued for development.

How do we dose these animals? Do we dose them according to the intended
label claim? Or do we test all dosing regimens? Or do we look at the
highest exposure scenario that has been outlined in the framework
document? That need to be better defined so that we know which dose or
which doses we should look at.

(Slide.)

DR. EWERT: The administration of this drug is critical. The frequency,
should that be administered to the intended label or more frequently to
push again for the development or selection of resistant organisms? And
the root of administration is critical as well.

Once again, do we give this according to the intended label? Or do we
try to insure uniform dosing in these control studies? For example,
water mediation in poultry would be very difficult to assess, that every
bird got exactly the same dose. So, do we have to take every individual
animal and do a gavage of that animal before we conduct this study?

And the timing of the administration of the drug is important. We have
developed the drug so that it would be given at the time that the animal
is ill. If we are using clinically healthy animals, the animals are
usually colonized with one or more enteric pathogens, and then, shortly
after that, the drug is given. Well, is that really a realistic
presentation of what happens to the dynamics of those enteric pathogens?

(Slide.)

DR. EWERT: And just a few things on the target bacteria. What is the
target bacteria? In the framework document there is mention of enteric
bacteria of concern. Does that mean all gut flora? If we have a gram
positive spectrum drug, does that mean that we want to look at
enterococci, for example, to see how the susceptibility patterns change?
Or, if we have a gram negative spectrum drug, do we want to look at the
enterics, like salmonella and camplyobacter?

There has been mention of sentinel as an indicator organism. How
realistic is that? Is there any information in the literature that
anybody knows of that shows that that is a valid comparison to make?

And finally, is we used one serotype or one strain of a certain
bacteria, is that representative of the entire population of the target
bacteria? A good example would be DT104. We know that that particular
phage type typhimurium has a propensity to acquire different drug
resistant genes. If you were to use that particular drug in these
studies, the drug \-- excuse me. That bug in the studies, the drug may
get a bad rap.

On the other hand, if you salmonella enteritidis, that particular
salmonella doesn\'t really show the propensity to change the
susceptibility pattern very much.

(Slide.)

DR. EWERT: And then the whole issue of colonization versus natural
infection has to be looked at. Would we use a confirmed food-borne
pathogen from an outbreak? Or would we use a confirmed target species
colonizer? What is the susceptibility profile of that particular
bacteria?

As Dave mentioned and as Dr. Cooper mentioned this morning, the 558.15
studies looked at bugs that were nalidixic acid resistant. That is
certainly not a bacteria that I would like to use in a fluoroquinolone
study. So that is very important to decide.

And as Dave mentioned, these bacteria could be specially identified, and
there is a number of ways. I am not a microbiologist. Dave gave you a
much more inclusive overview of that. But if we incorporate a resistant
gene into this bacteria or they become metabiotically altered, will they
still then be able to colonize the organism that we are trying to
evaluate?

Yes, when they are identified they do facilitate tracking and it will
allow the differentiation of a wild strain that may be endogenous to the
organism. It shows up later in the study from stress and it allows the
differentiation between that and the colonizing strain.

And then finally, for the target bacteria, how many different bacteria
could we use to colonize these laboratory animals or test animals? If we
are looking at a poultry study, do we want to take camplyobacter and
salmonella and put them in the same study? Or would we have to do
separate studies?

(Slide.)

DR. EWERT: This is just one thought for you to take into your breakout
groups. Perhaps there could be a standardized \-- call them battery of
bugs. They could be chosen according to the class of drug to be
evaluated and they could be similar to the ATCC bacteria where there is
predictable colonization and susceptibilities. That way, when we look at
these bacteria and use them in these studies, there would be some sort
of baseline to begin with.

(Slide.)

DR. EWERT: The microbiological methodologies also need to be considered.
We need standardized techniques for culture and isolation. These
techniques need to be specific for the target bacteria and also for the
target animal. And we need to look very closely at the effect of the
media on the recovery of target bacteria.

If we do enrichment, that enhances growth of certain bacteria, but that
introduces bias not only into subsequent susceptibility testing, but
into the populations dynamics that could be described. If we use
selective media, there is inhibitors that prevent overgrowth of certain
bacteria. But, for example, with camplyobacter, there is a variety of
different growth medias and some of those medias will not allow for the
growth of all camplyobacter.

If we use a media that selects for the growth of certain bacteria with
increased MICs, then we are not able to follow any shift. So it would be
a selection for resistant bacteria that developed, but we couldn\'t see
what was going on at the lower end of the MIC spectrum.

(Slide.)

DR. EWERT: Should susceptibility testing board standardized for each
drug? Should we use NCCLS recognized methods? What drugs should be
tested? Do we want to look at the new animal drug or, as has been
suggested, the representative drug from the human side, such as the
NARMS drugs? Or should we have a battery of drugs? For example, all
fluoroquinolones or all cephlosporins that would be tested at the same
time as we do these studies.

(Slide.)

DR. EWERT: The susceptibility testing methods, such as disk diffusion or
agar or broth dilution, have been used historically to identify the
susceptibility of organisms. Do we want to look at full range MICs or do
we want to use a break point technique?

An old method that has been around that has been resurrected and
discussed extensively is replica plating. That estimates the MIC profile
for an entire culture, but it is not a NCCLS recognized technique, it
has not been standardized and that raises a whole issue of questions
about quality control.

(Slide.)

DR. EWERT: And once again, for susceptibility testing, we could use a
selective media that has a certain level of antimicrobial in there that
selects for bacteria that only have increased MICs to that
antimicrobial.

(Slide.)

DR. EWERT: Of paramount importance when we do this microbiological
methodology is to be able to define resistance. It is important to keep
in mind that we have break points that are established to determine if
bacteria are susceptible or resistant.

Break points are established to predict efficacy against a target
pathogen. For bovine respiratory disease, those target pathogens would
be pasteurella or haemopholous. Potential food-borne pathogens are not
target pathogens for many of the animal antimicrobials, therefore, break
points have not been set. If break points have not been set, we cannot
designate enteric bacteria as either susceptible or resistant.

(Slide.)

DR. EWERT: And furthermore, break points may not have been established
for some of the human drugs of concern. An example would be for
camplyobacter and ciprofloxin (sic). We, by convention, are using a
break point of four micrograms for ml of resistance, but that has never
been established. And that raises a question because there has been a
number of reports where a resistant camplyobacter has been successfully
treated with a fluoroquinolone. So we need to be very careful when we
are judging these bacteria and make sure that we have the right criteria
in place.

(Slide.)

DR. EWERT: How do we interpret these results once we have generated the
data? How can we correlate results from model studies to a production
environment? And how do we correlate, if we use healthy animals, the
results from healthy animals studies to animals that are clinically ill?
that is our real target animal. So we know that those animals are
clinically ill and they are stressed, and the dynamics of the drug/bug
relationship may be totally different in the animal.

How do we correlate increased pathogen load to increase carcass
contamination or to increase prevalence of human illness from target
enteric bacteria? I think that is fundamental, to figure that out, if we
can even do it. And if you have noticed, I put decreased in parenthesis
after increased because I have not heard anyone talk about decreased
pathogen load today, but that is a very real possibility. Some of these
antimicrobials actually cause decreased pathogen load, and so what would
be the correlation there?

(Slide.)

DR. EWERT: Bill addressed this earlier, but this was one of the
questions that I had. Will resistance threshold or some kind of criteria
be arbitrarily set by FDA to determine the pass/fail for the new drugs
for these

pre-approval studies? And would any shift in MICs result in a fail? Or
how much of a shift and what proportion of bacteria would the shift have
to occur?

What if there was a shift at the very low end of the MICs in areas that
would never be considered to be a resistant area? Or would we have to
see a shift to resistance for a pass or a fail to be given?

(Slide.)

DR. EWERT: What if we see asyncronous (sic) results between pathogen
loads and resistance studies? What if there is no change in resistance,
but there is an increase in pathogen load or vice-a-versa? How do we
address that type of outcome in one of these pre-approval studies?

(Slide.)

DR. EWERT: Will the data that we generate from these studies be used to
set microbiologically based withdrawal periods rather than residue based
withdrawal periods? And finally, how will the results of the studies be
used to determine the impact on public health?

(Slide.)

DR. EWERT: A few conclusions for you to take with you to the breakout
sessions; that we need clear objectives to enable the proper design of
the studies, and we must have a study design that is established that
incorporates all of the important components into the study. And the
studies must be conducted and the data either accepted or rejected early
in the development process for this to be a feasible approach for
industry.

The target animals must be clearly defined. We have to know what this
animal is that we are going to deal with in these studies.

(Slide.)

DR. EWERT: And the protocol for drug delivery must be defined. We should
consider the uniformity of the test bacteria and appropriate
microbiological techniques must be employed to minimize data generation
biases. It is amazing how much bias you can introduce into a study by
not using the correct microbiological techniques.

And the criteria for interpretation of the results must be established
to determine what impact they may have on public health. With that, I
will take any questions. Thank you very much.

(Applause.)

DR. WHITE: We have time for any questions.

(No response.)

DR. WHITE: I think Dr. Ewert brought up some good points, some that were
brought up in some other presentations, but I think it shows how the
important they are. She also brings up a bunch of points that we did not
discuss in some of the earlier talks and a lot more food for through for
tomorrow\'s breakout session.

Just to let you know, we also have an open comment period at the end of
this session today, and this session is going to continue on to tomorrow
morning as well. We will also have a study concepts panel. The speakers
that are up this afternoon and tomorrow morning will be up here and we
can ask them questions.

Our next speaker is Dr. Mevius. He is from the Netherlands. He is also a
veterinarian bacteriologist. He is head of the Laboratory for Antibiotic
Research at the Institute for Animal Science and Health in the
Netherlands. His laboratory is the reference laboratory for sensitivity
tests and veterinary diagnostic labs in the Netherlands.

He is also chair of the CVMP working group, antimicrobial resistance
working party, and he will operate as chairman of the newly formed VICH
expert working group on antimicrobial resistance. And we are glad you
could make it over from the Netherlands, and welcome.

**REVIEW OF AN ANIMAL STUDY AND ITS RELEVANCE**

**By Dr. Mevius**

DR. MEVIUS: Thank you very much, Mr. Chairman, for the introduction.
First of all, I would like to thank the FDA for inviting me. In fact,
before they invited me, I had already decided to come because of my
interest in the subject. I made all my travel arrangements and the room
was booked, so it was very nice for my budget that the travel expenses
were dealt with by the FDA.

I am not standing here as chairman of the VICH expert working group
because we have not been together. Four of the members are present here
today. In the present \-- in the near future we are going to meet and
discuss this difficult problem.

Today I am going to give you some information of an animal study we did,
and in this animal study we actually made some of the choices that were
mentioned in all the presentations we had. Specifically, the
presentation of

Dr. White, but also in your presentation. And not all of these choices
are validated. You have to be pragmatical here and there, and I have
tried to give the reasons why we did that.

The animal study was focused on in vivo selection of resistant
enterococci in broilers with antimicrobial growth promoters. Well, here
in the United States, of course, although antimicrobial growth promoters
are also an issue, fluoroquinolones is one of the major issues in
Europe.

We had a big discussion about whether to use antimicrobial growth
promoters and one of the big differences, the major differences between
the licensing of antimicrobial growth promoters in Europe and the USA is
that in Europe they are only licensed for feed enhancement, and that is
for a licensing authority. It makes it easier. They are not licensed for
prevention of necrotic enteritis or other bacterial diseases.

They have that side effect, of course, but they are not licensed for
that, and it was \-- I think for that reason \-- easier to ban these
drugs, which actually took place.

We did this study because there has been an ongoing discussion of
whether these low dosages \-- whether these are sub therapeutic or not.
That is also a scientific discussion which is not irrelevant, but it is
low dosages, select resistance. Yes or no? And we wanted to know in a
normal trial whether we could show some of this resistance selection.

(Slide.)

DR. MEVIUS: Okay. The purpose again was to study the effect of four
different antibiotic growth promoters on selection of resistant
enterococci E. faceium, and we focused on enterococci and there has been
a lot of discussion on which bacterial species to use. These
antimicrobial growth promoters have a spectrum focused at the gram
positive flora, so the enterococci are the logical bacterial species to
look at.

And we have looked at, as I will mention later, all enterococci species
we could isolate, but also specifically aimed at E. feacium, because in
the monitoring systems progressing in Europe E. faecium is one of the
target species these monitoring systems focused on.

The question which type of dosage regimen to use was quite easy because
these growth promoters are licensed for use for the entire period these
animals are fattened, the entire fattening period, and we chose a
fattening period of 42 days, six weeks.

So that wasn\'t easy. We didn\'t have to make that choice, and we are
\-- wanted to know whether a specific growth promoting agent would
select for resistance to itself or what the effect of a specific growth
promoting agents would be for other antibiotics. And we didn\'t look at
all antibiotics. This was some sort of a pilot study for us as well.

(Slide.)

DR. MEVIUS: We used commercial animals, Ross 208 broiler chickens. They
were hatched at our own hatchery and just after hatching, at day zero,
they were transported to the institute, to the animal facilities. There
the animals were sexed and five groups were made, consisting of 40 male
and 40 female animals.

We tried to mimic the field situation in that way, and why 80 animals?
Well, that was more or less based on the pens we could building, and we
wanted to have a density in the pen of 15 animals per square meter,
which is more or less the same as is present in practice.

Each group was housed in a pen totally separated from the other groups.
So there was climate control, there was a clothing regimen. So there was
no contact or minimum contact as possible between the groups.

(Slide.)

DR. MEVIUS: The feeding regimen. We had one batch of standard broiler
feed prepared without cocci static agents. They were fed at liberty and
it was daily refreshed, and the animals had free access to drinking
water. This one batch of feed was divided in five portions of 500
kilograms, and feed A was fed to group A and it contained no
antibiotics. And group B in that group added as the control group.

In group B 15 parts per million virginiamycin was added, group C
bacitracin in the mentioned concentration group D avilamycin and in
group E tylosin was added, and we used these antibiotics because these
were licensed for use in poultry. Tylosin was not licensed for use in
poultry, but spiromycin was, but spiromycin has not been used for a long
time in practice in Holland and we were not able to get. It was not
available anymore. It was not used.

So we used Tylosin instead, just as a model antibiotic in the same
dosage as spiromycin was licensed for use.

I have to make a comment. I have discussed this \-- what we did is these
portions of feed were produced under GMP conditions, so they were very
accurate sheets of how many of the pre-mixes were added and how long
they were mixed. But what we failed to do, and it was really very stupid
of us, we didn\'t store a small sample and actually determine that this
concentration was present. If you look at the results, keep that in
mind.

(Slide.)

DR. MEVIUS: The study was the 40-day period, as I mentioned, and at days
zero, three, 27 and 41 of 10 randomly selected animal of each group
fecal samples were taken by taking cloaco swabs. You can\'t take a large
sample from a small chicken. In fact, we did. We sampled them twice a
week, but we ended up with so many isolates that we chose to process
only these isolates from these sampling periods and the results will
give you some sort of a reflection of what the dynamics of resistance
was in the period. So 10 samples, 10 fecal samples, and they were not
pulled. They were treated as individual samples.

(Slide.)

DR. MEVIUS: As was suggested by Dr. Anguelo, maybe the optimum way of
streaming the fecal flora for presence of resistance is using some sort
of a stream plate. Maybe the replica plating method. So what is used for
E. coli, which is very commonly used in the Netherlands and there is a
lot of experience, is enumerate E. coli present in the gut on a
selective medium.

For instance, \-\-- agar or E and V line agar, which is very easy to do.
You can do it as accurate as this method is. And then also, inoculate
the same sample on a plate with a very point concentrate of antibiotic
and that, of course, needs to be defined. That way you can calculate the
proportion of bacteria or E. coli present in the sample, which is
resistance to the specific antibiotic.

For enterococci we have had some experience with that method as well and
enterococci you will end up with a lot of different problems. These
plates \-- we used Slanetz and Bartley agar, but there are other plates
as well, and these plates are not totally selective. So other bacterial
species will also grow on them. That is a problem. So what are you
enumerating.

These plates are not selective, but also elective. There is a color, a
specific color of the enterococci, but this color is only very clear if
you have loose discrete colonies. So, if you have a large number of
colonies on the plate, it is very discriminate \-- it is very difficult
to discriminate atypical colonies from typical enterococci.

So we decided to do it in a different way. We made serial dilutions of
the sample in buffered peptone water, and these dilutions were plated,
just if you would do an enumeration, and we used the spiral plater. But
the only purpose of this inoculation technique was to obtain nice, loose
colonies.

In the highest solution where we had nice, discreet colonies we could
determine the differences in morphology which is present between the
enterococci species as well. We picked up to a maximum of four colonies.
We picked an enterococci species colony type, we picked an enterococci
\-\-- type, which is not always \-\-- we picked a specific colony type,
and often there were other \-- a specific colony type present. So a
maximum of four colony types were picked.

And it will be arbitrary whether there is truly the predominant flora,
but if you have 10 to the 6th or 10 to the 7th, which is one million to
10 million enterococci present in the gut and dilute the sample in a way
that you will end up with something between 30 and 300 colonies on the
plate, this is really the majority population you are measuring there.
And the chance that you are isolating minority populations is really
very small.

But that is an assumption and how valid that is we don\'t know. Nobody
knows. So, from each sample a maximum of four enterococci were randomly
isolated and typed with molecular and biochemical techniques.

So from each sample, a sampling period from one group, a maximum of 40
enterococci were isolated, and generally, the number of enterococci were
around 30. The total number were around 30.

(Slide.)

DR. MEVIUS: These enterococci we determined MICs with agar dilution
according to NCCLS. Well, the method is, of course, according to NCCLS,
but it has not been described for some of these feed additives. For
tylosin, for the macrolides. For the others there are some publications
from groups from Belgium, and we used break points that I mentioned on
there. Eight international units per mil for bacitracin, eight
micrograms per mil for tylosin, four microgram per mil for virginiamycin
and 60 micrograms per mil for avilamycin.

But really, an NCCLS method and a good control mechanism is not present,
is not published, and we referred to data we obtained from the \-\-- in
Belgium or from Denmark and we had similar results, so we assumed that
the data were valid.

And there has been some talk about break points. What is very important
in these kind of studies is that clinical break points shouldn\'t be
used; are irrelevant in my point of view. What should be used is a break
point that discriminates from the normal susceptibility and the normal
susceptible population from shifts in increased resistance, whichever
resistance gene is present at the moment. So you are just looking for
shifts towards resistance.

(Slide.)

DR. MEVIUS: And I am going to show you that the break points we used are
\-- well, aren\'t really doing that. This is MIC distribution of all
enterococci we isolated. I am going to show later which enterococci
species we isolated.

On the left side you have a large \-- this is the MIC distribution of
virginiamycin and the blue indicates the break points, and we determined
MICs larger than the break points as being resistant. This is not just
thought up by us, but there have been some publications on distribution
of MICs, recent publications in enterococci. So he comes up to the same
conclusion.

And you can see, if you use four micrograms per mil, you are more or
less accurately \-- there will be some mistake distinguishing between a
susceptible population and what seems to be one resistant population.
But if you know the resistance genes of \-- the streptogramins are very
complex. We know three resistant genes at the moment and there are
bacteria that are \-\-- resistant.

We don\'t know which genes are present, so this resistant population is,
in fact, more than one population. The assumption that four micrograms
per mil discriminates between susceptible and less susceptible seems to
be valid based just on the pheno type.

(Slide.)

DR. MEVIUS: If you look at Tylosin, it is very easy. You have a
susceptible population with MICs, around two micrograms per mil, and you
have a highly resistant population. So any discriminating break point
concentration in between would be valid.

(Slide.)

DR. MEVIUS: For bacitracin eight units per mil was advised by \-\-- we
used that. Looking at the MIC distribution you could see that in the
methodology we used it is even a small under estimate. If you used the
criterion larger than eight units per mil it is called resistant. The
proportion of bacteria with an MIC of eight units per mil probably also
contain acquired resistant genes.

(Slide.)

DR. MEVIUS: For avilamycin 16 micrograms per mil is used, and again, the
distribution of this population shows you that it is a small under
estimate. If you would use eight micrograms per mil, it would be based
on this phenotypic distribution of MICs. But 16 micrograms per mil seems
to be a very accurate break point.

(Slide.)

DR. MEVIUS: Well, the results. These are the enterococci species we
isolated, and predominant enterococcus in these broilers was
enterococcus faecium. All the enterococcus faecium types of colonies we
isolated were enterococcus faecium. But to our surprise a lot of the

non-specific colonies on the plates were also enterococcus faecium and
very often they had a very different pheno type and very often they were
more resistant.

And so, if you were to just focus on the typical enterococcus faecium
types of colony which we used in our monitoring, that could effect the
final results. Enterococcus faecium was isolated in low proportions.
Here it was isolated mainly on day 30. On day 27 it was very
predominantly present, and later on the numbers went down to very low
numbers.

Enterococcus gallinarium was present, but in variable numbers. You would
only use gallinarium as a species to focus on and in a lot of sampling
periods you would end up with only small proportions of strains. And
enterococcus avium. Well, enterococcus species but not for the type were
also present in low numbers.

(Slide.)

DR. MEVIUS: This slide \-- I am going to give you some slides with
results in the different groups. This is the results of the dynamics of
resistance in all enterococci that we isolated. That is, different
sampling periods; at day three, 13, 27 and 41. The lines indicate
proportion of resistant enterococci.

On day zero, just after they were hatched, we only isolated enterococcus
\-\-- and avium in low numbers. They were not colonized. But somewhere
between day zero and day three these animals were colonized with a
multiple resistant enterococci flora. Many of the species indicated on
the list were already isolated on day three.

And as you can see on day three, the levels of resistance to
virginiamycin, of bacitracin and tylosin were very, very high already.
So this indicates that these resistant determinants are very
predominantly present in animals in the Netherlands.

Avilamycin resistance, this is a different case. In the control group,
we only found, on day 13, a single or maybe two isolates, I am not sure,
which were considered resistant. As a result of the fact there was no
selection pressure, it was clearly demonstrated by the decrease in
resistance again.

So here it shows that although these animals are colonized with a
multiple resistant flora, the levels of resistance will go down, if the
method we used is a valid method. That is important to keep in mind of
course.

(Slide.)

DR. MEVIUS: If you look at faecium, I am going to show you the results
of all enterococci and then the result of only enterococcus faecium as
the target organism being predominantly present. The general picture is
the same although generally there is more variation. The number of
bacteria are smaller so there is more variation.

As you can see, there is no avilamycin resistance, the white line, and
the increase in resistance didn\'t stop on day three, but went on to day
13, to very high levels. But then the decrease in proportions of
resistance, because of the fact \-- the only factor that I can assume is
that there was no selection pressure. It resulted in similar levels as
in the group with all enterococci.

(Slide.)

DR. MEVIUS: These figures are a bit different. This is not the results
of one group. This just focused on virginiamycin resistance and all
pictures that are coming next are built up in the same way. Again, this
shows you proportions of resistance. This time virginiamycin resistant
at the different sampling times.

But the yellow line in the following picture will always state, in this
case, virginiamycin resistance in the control group as is indicated. And
the light blue line is the proportion of virginiamycin resistance in the
group fed virginiamycin. And the red line is the proportion of
virginiamycin \--in the group fed bacitracin. The dark blue line is the
same for the group fed tylosin.

So the control line is similar as \-- this is total enterococci, not
just E. faecium. The control line is similar as we have seen before. It
is the same line, and it is very clear that if you compare the results
of the proportions of virginiamycin resistance there are very clear
differences.

Whether these differences are significant are still under analysis, so I
am just giving you the general picture. Probably the differences are so
big they will be significant, but it depends also on the statistical
analysis method we use.

For instance, for the group of bacitracin you could see a larger peak in
the control group, but the end will lead to the same levels as the
control group, indicated that it is very important \-- as has been
stated before, at which time period are you going to determine the level
of resistance? At the end or during treatment here. It would lead to
quite different results.

Tylosin, the dark blue line. On the first 27 days the line is similar to
the line \-- the proportion of virginiamycin is similar to what is
observed in the control group, but then you can see a very prominent
increase in resistance, because of the relation between tylosin and
streptogramins and their partial cross resistance it is something that
is not so striking.

What is more striking is that avilamycin is the white line, although at
least the first 13 days the levels are in the same order as the control
group. It also has an indication of a selective effect for virginiamycin
resistance. And we don\'t know \-- for avilamycin we don\'t know the
resistance determinants; what kind of genes.

In this kind of study you can only look at the pheno type, and obviously
the enterococcus flora in these animals is not by nature \-- but because
of the use of these drugs for long periods is already at very high
levels. Acquired resistance is present.

And also, in our monitoring we have an imbalance. We see a lot of
strains that are co-resistance to all these antibiotics.

(Slide.)

DR. MEVIUS: If you just look at enterococcus faecium, you can see \--
well, the picture is the same. Bacitracin is the red line and the
control line is the yellow line; are similar. Virginiamycin is the light
blue line. The tylosin line is reluctant in the beginning, but then also
going up to higher levels. But the general picture is the same as in all
enterococci.

And avilamycin resistance, after day 13 there is an increase. The levels
are not very much different from the control group, but there seems to
be some effect present also in enterococcus faecium.

So, if you just look at enterococcus faecium there is more variability.
So it will be more difficult to really determine whether these dosages
really select resistance in a statistically significant way.

(Slide.)

DR. MEVIUS: This is Tylosin resistance. Again, the yellow the control
group. The blue line is the group fed tylosin. So it is very clear that
tylosin and the macrolides directly selects for high levels of tylosin
resistance. All enterococci were resistant from the first day we could
isolate them. It was 30 percent on day three. One hundred percent of all
enterococci were resistant.

In the group fed avilamycin it was not so rapidly increased in tylosin
resistant. But also avilamycin clearly selects for tylosin resistance.
Again, probably that is

co-resistant. It is very probable that they are also tylosin resistant,
and this is shown here in this picture.

And you can show that although the differences are less prominent, also
bacitracin and virginiamycin have an effect on macrolide resistance and
the effect is different than avilamycin or tylosin, but probably also
significant.

And again, in E. faecium the picture is the same control group here.
Both the blue lines are virginiamycin and tylosin.

(Slide.)

DR. MEVIUS: Bacitracin resistance follow. The control lines does not
have an effect. And also here avilamycin resistance. Bacitracin, the
control line levels go up to 50 percent and then go down again. The red
line is the group fed avilamycin \-- sorry. Bacitracin. It is getting
complicated. But clearly, bacitracin selects for itself.

And also here, avilamycin, the line going up again to higher levels than
the control group and very clearly higher levels at the end of the 41
days period selects for bacitracin resistance. Virginiamycin has that
effect. Tylosin effect is more depending of the time period. It seems to
be \-- whether that is really statistically significant I wouldn\'t
know.

(Slide.)

DR. MEVIUS: And the same picture you will see with enterococcus faecium,
and again, with more variability. For tylosin, this is less prominent.

(Slide.)

DR. MEVIUS: Avilamycin is not so much present in the normal enterococci
population. This can be shown in the yellow line here in all
enterococci, which only a few resistant isolates in the control group
were isolated, and you can see that all the other antibiotics, tylosin,
bacitracin and virginiamycin, will have \-- there are some resistant
isolates present, but probably this is not a real difference.

(Slide.)

DR. MEVIUS: And if you just look at E. faecium, the picture is the same.
No resistance in the control group, no resistance in the group fed
bacitracin. There is some effect in the groups fed virginiamycin and
tylosin, and it is very clear in the group fed avilamycin itself.

(Slide.)

DR. MEVIUS: So conclusions. Virginiamycin selects for resistance to
itself, to tylosin and bacitracin, keeping in mind that there is not
statistical analysis done yet. Tylosin selects for resistance to
tylosin, virginiamycin and bacitracin between records. Whether that is
really true we don\'t know. I don\'t know at the moment.

Bacitracin selects for resistance to bacitracin and tylosin and
avilamycin selects for resistance to all the other antibiotics, with
virginiamycin between brackets. It is not so clear.

It is clear that in a population of bacteria which are very \-- a lot of
them are resistant already to bacitracin or tylosin and virginiamycin.
If a new resistance pheno type or gene is selected, it will also select
for the other resistance, which are resistant genes which are
predominantly present, which is shown very nicely by this study, which
will make it much complicated to decide on these kind of studies.

(Slide.)

DR. MEVIUS: So all growth promoters used select for their own resistance
determinants and the second line is not entirely correct. It shouldn\'t
be most growth promoters. It should be all growth models select for
resistance to most other antibiotics.

And in this case we didn\'t include therapeutic antibiotics because in
our monitoring system we know that the enterococci flora has also very
large proportions of tetracycline resistance \-\-- so probably it will
be much more complicated.

(Slide.)

DR. MEVIUS: So, are these data predictive for the field situation? I
think what is very important in this kind of study is to compare it with
data coming from monitoring systems. It was mentioned antimicrobial use
patterns will always result in resistance, and my point of view is you
can design optimum dosage regimens which have an optimum or at least
less effect on antimicrobial resistance.

If you study that in a model situation and compare it, what you can
relate between antimicrobial use factors in the field and what you
observe in the field, then you can really judge the effect of a specific
antibiotic.

(Slide.)

DR. MEVIUS: These are some results of the monitoring program we started
in 1998 as a result of all the discussion of feed additives and
vancomycin resistance. And we didn\'t only look at E. faecium, but also
E. coli, camplyobacter and salmonella, and these data are published now,
and here are presented proportions of \-- in this case we used
erythromycin as the human analog, bacitracin, virginiamycin and
avilamycin.

Keeping in mind that tylosin is only used as a growth promoter. It is
not used a lot therapeutically, but it is used therapeutically. It is
not used in broilers, it is not used in calves, still \-- well, there
may be some there, some indications of differences, but in the field
situation, the proportions of resistance are more or less similar. So
there are probably more factors dealing with resistance in the field
than just the use of these antimicrobial growth promoters.

For bacitracin it is the same phenomenon. The levels of resistance are
very high in pigs where bacitracin or any drug related to bacitracin is
not used. In broilers bacitracin was used in a high amount; large
amounts of bacitracin was used, and you see an indication of higher
levels, but it is not a real difference.

And for virginiamycin it is the same. It is not used in pigs. It was
used in broilers. It was used in calves, but still the levels are more
or less similar. For avilamycin the difference is more prominent.
Avilamycin is only used for feed enhancement in broilers and it is
licensed for use in early weaning pigs. Before weaning.

And you can see the differences in these large numbers of enterococci
faecium strain. You can see 21 percent of them using the same break
points and criteria are considered resistant in the broilers and only
small proportions are considered resistant and in pigs and calves,
indicating also in the field this selective effect could be shown.

(Slide.)

DR. MEVIUS: So could you translate these data to other bacteria? If you
want to study the effect of fluoroquinolones \-- although we have had a
lot of discussion of which bacterial species to use. In my opinion, the
only option is a bacteria which is, by nature, susceptible to the drug,
which is present in all animals which can be isolated without selective
techniques without antibiotics in the media. For instance, for
camplyobacter.

So really, if you want to use a model study, you should use, if
possible, E. coli or enterococci, which is more different, as model
bacteria. However, E. coli, if you look at the monitoring data, is very
often at levels of more than 25 percent multiple resistant. Not always
resistant to all of these antibiotics mentioned, but very often
resistant to these more conventional older antibiotics.

I can remember a couple of years ago I was here in the same room, and we
had a presentation of Paula Cray of the first resistant data and also in
the salmonella. These resistant pheno types are very commonly present,
and this is more or less established. Maybe all around the world. I
wouldn\'t know.

At least in our situation, in normal E. coli, more than 25 percent are
resistant on amoxicillin, tetracyclines, trimethoprim, chloramphenicol
and streptomycin wasn\'t tested. Probably the levels of streptomycin
resistance would be very high as well.

Just to give you an idea of how many bacteria you are talking about, it
is not just 25 percent. Tenth to the 8th, which is 100 million of E.
coli per gram featured is not a very rare assumption. That is a very
common number of E. coli present in the gut of animals. So, if 10th to
8th E. coli is present in a pig or in a broiler, 2.5 times 10 to the 7th
of them are already resistant.

(Slide.)

DR. MEVIUS: If you look at the hypothetical dynamics, for instance,
fluoroquinolone resistance, the blue indicates here the total number of
E. coli present in any animal or hypothetical animal. The yellow
indicates the numbers of fluoroquinolone resistant E. coli. The two red
arrows indicate one week of fluoroquinolone treatment.

And these fluoroquinolone are present in very low numbers, so you will
only detect them if they are at levels of over 100 to 1,000 bacteria per
gram of feces. So the levels are really very high.

So what you will see during fluoroquinolone treatment is the total
numbers will rapidly go down and after approximately one week you will
isolate low numbers, but high proportion of fluoroquinolone resistance,
which after you stop treatment will go down again and how fast it will
go down, that is really a hypothetical.

But if, a couple of weeks later, these bacteria are treated with any of
these other antibiotics, which major proportions of these E. coli are
already resistant to, and I just choose tetracycline as an example, it
will not only select for itself, but at that time also for
fluoroquinolone resistance, keeping in mind that in the beginning, 10th
to the 8th, very large numbers of E. coli were present.

The dot of the resistant coli in this picture would be only very
slightly beneath the 10th to the 8th. So the selection of
fluoroquinolone resistance will take place in bacteria that are also
already tetracycline resistant.

(Slide.)

DR. MEVIUS: This is just a hypothetical situation. The data coming from
the study that we did with the gram positive organisms show you that it
is not entirely hypothetical. This will really take place. The other way
around it will not take place. Tetracycline will select for tetracycline
resistance.

(Slide.)

DR. MEVIUS: The last slide is not very clear. It shows our institute. We
are very proud, of course, to represent our institute over here. It is a
very typical Dutch institute. Why it is so typical Dutch? You can\'t see
it very clearly, but it is built on the bottom of a former lake. Well,
we are known to make these kind of polars, if that is the right word in
English.

In fact, something like 50 years ago this was even sea. It was the sea.
The country is entirely flat, and the land around the institute has been
used to culture tulips and to produce tulip bulbs. And all these bulbs
were not removed, so every year in the spring you can see we still have
a lot of tulips growing in the field, which is very nice. Typically
Dutch.

The only thing that is also very typically Dutch is wearing wooden
shoes. Well, we only wear wooden shoes when you, as Americans, come over
to us as tourists just to show how typically Dutch we are. Thank you for
your attention.

(Applause.)

DR. WHITE: We have time for maybe one or two questions. Remember, if you
could identify yourself as well and the organization.

DR. CARNIVALE: Rich Carnivale, Animal Health Institute. Thank you, Dr.
Mevius. I had a question. If the prevalence of cross selection for
resistance among various classes of antibiotics is as you portray, how
could anyone every run a study on a single antibiotic and predict
whether it will or will not select for resistance?

If other antibiotics are going to interfere with that or select for that
antibiotic, it seems to me that it is almost impossible to design a
study to look at a single antibiotic, apart from the other antibiotic
pressures that may be there.

DR. MEVIUS: Yes. Right. Well, that is a very good point, and I showed
you the data to show you how complex it is; that it is not just one
resistant pheno type or resistant determinant you are selecting for. It
depends greatly on the situation that is already present in the
bacterial population.

For fluoroquinolones the level of resistance is generally low or at
least it was low. It depends on how it was used. So then, for
fluoroquinolones, you can more or less accurately study the effect of
fluoroquinolones itself on its selective properties.

For the other antibiotics it will be very, very difficult at this time
period to really \-- to determine which is most important to study.
Tetracycline for itself or erythromycin or tylosin or use another
antibiotic. The results, if methodology is valid and the assumptions we
make at least show at least these antibiotics have the most effect for
their own resistance determinants. Yes?

But it also shows that it is very important in these studies not just to
include the specific antibiotic that you are using, but including other
antibiotics as well and to use a larger spectrum and include \--
relevant human therapeutics would be very relevant.

If you would show that a specific usage of antibiotic would also select
\-- for instance, vancomycin resistance. I didn\'t show you the data. I
wanted to include \-\-- effect as well, but we were not able to do that
anymore because the product was not available. For instance, tylosin
would have selected for \-\-- or vancomycin resistance. That would have
been very important data I think.

If you have higher concentration dosage, an optimum dosage regimen for a
shorter time period used strategically for \-- for instance,
virginiamycin for treatment for chlosidial infection or control of
chlosidial infections, then the effect on the enterococci flora might be
quite different, and that, I think, should be built into these studies.

This dosage regimen shows you that it is more or less the worst case.
Long-term usage of drugs \-- well, presumably low dosages \-- have a
very clear effect on this bacteria populations. If you would use these
drugs in shorter periods at higher dosages, the effect may be quite
different.

DR. WHITE: We have time for one more question.

MR. BYWATER: Robin Bywater, Pfizer. It is very striking to know that the
birds that were not exposed at all rapidly acquired some resistance.
Presumably from their environment. Was there any history of using these
compounds in those buildings and did you look at for enterococci in the
environment to see whether they fitted the presumption?

DR. MEVIUS: No. At our animal facilities where these animals were
hatched no antimicrobial growth promoters were used. We didn\'t take any
environmental samples. What I would do different in the next time, if we
could do a study like this again, I wouldn\'t separate the animals at
day zero. I would keep them together for a week to more or less
stabilize the flora of these animals and then separate them and then
look at the facts.

So we saw enterococcus species. If you look at the numbers, we isolate
from the different groups. Although they were separated at day zero,
they were really very, very similar. So they were colonized during the
processing at day zero. I think when they were sexed and so on.

MR. BYWATER: It is striking, in a hospital environment, how apparently
enterococci are just absolutely everywhere. One suspects that they
probably are in animals houses as well, but I am not sure anyone has
really looked.

DR. MEVIUS: Well, we have looked for other reasons at hatcheries and
enterococci contact in the air, because we think there is a relation
between enterococcus vicalus and \-\-- which is a specific disease in
chickens. And we know that the content of enterococcus vicalus can be
very, very high at the hatcheries, which can be a dusty environment. But
through levels of 10 to the 6th per cubic meter. Very high levels. Not
nice to know as being there as a technician.

MR. BYWATER: Thank you.

DR. MEVIUS: Thank you.

DR. WHITE: Thank you, Dr. Mevius.

(Applause.)

DR. WHITE: We are right back on schedule. It is 4:35. Our next speaker
is Dr. Mark Papich. He is an associate professor of clinical
pharmacology at North Carolina State University. He received his BS from
Montana University in zoology, a DVM from Colorado State University and
a master\'s in clinical pharmacology from the University of Illinois.

He is a diplomat of the American College of Veterinary Clinical
Pharmacology and is currently president as well, and a fellow and
current council member of the American Academy of Veterinary
Pharmacology Therapeutics. Please, welcome Dr. Papich.

**USE OF PHARMACOKINETICS/PHARMACODYNAMICS**

**By Dr. Papich**

DR. PAPICH: Thank you very much. It was a pleasure to be invited here.
When Dr. Flynn called and asked me a few weeks ago to speak at this
meeting, I had to look into the background of it a little bit and then I
got a little scared that I wouldn\'t have much to offer a meeting on
resistance, but there has already been quite a bit of comment and
discussion about some pharmacokinetic/pharmacodynamic concepts.

Already, in fact, even the last speaker made the point that we should
consider the optimization of therapy or doses. And so that is what I am
going to focus on just a little bit, is to try to bring us up to speed
on some of these concepts and talk about what we know in terms of
optimizing antimicrobial drug therapy, using the
pharmacokinetic/pharmacodynamic relationships that we understand.

(Slide.)

DR. PAPICH: As far as the definitions, I think everybody in the room is
familiar with some of the definitions we are talking about. To optimize
antimicrobial drug therapy using pharmacokinetic/pharmacodynamic
relationships. We recognize that pharmacokinetics describes the
disposition of the drug in the body, and we have gathered
pharmacokinetic information in the drug approval process.

And even after many drugs are approved, there is still other studies
that get published that we recognize in the literature that adds to the
body of information on pharmacokinetic points.

I might add that it would \-- and it has been suggested already by some
people that have looked into this area \-- that it would be an added
benefit to have population pharmacokinetic information as well, and if
we look to the future a little bit about how we might best use this
information that I will be discussing, perhaps population
pharmacokinetics would add something to this.

And then, of course, pharmacodynamics is the action of the drug on the
bacteria. So this describes whether it is bacteria cidal, bacteria
static, whether it is time dependent, concentration dependent and so
forth. And hopefully, by using these relationships together and
understanding them, the dose regimens can be estimated and optimized to
meet these end points.

Now, the important point to recognize for the very onset as I go through
this is that up until now all of the work that has been generated to
optimize these regimens have always been geared towards a therapeutic
end point. That is, you know, there is a pathogen in the lungs or in the
skin or wherever and we are optimizing the
pharmacokinetic/pharmacodynamic relationships towards that pathogen.

The concept of using these relationships to minimize resistance has
really been overlooked and that is, of course, why we are here at this
workshop, is to maybe discuss some opportunity for that. But I still
need to make the point that most of the information that I will be
discussing and the information that has been generated by various
investigators has always been aimed at a pathogen and the therapeutic
use of the drug towards a pathogen, not necessarily to prevent
resistance.

(Slide.)

DR. PAPICH: Okay. So some of these concepts and definitions need to be
brought forward. Everybody recognizes what a typical pharmacokinetic
plasmid concentration curve might look like. I will be showing a couple
of these curves as just examples of what these \-- as hypothetical
examples in which we have the plasmid concentration on the \"y\" axis
time; on a 24-hour scale on the \"x\" axis.

And it is sort of terms that we throw around a lot when we have a plasma
concentration profile. First of all, the c-max or the P concentration.
So we refer a lot to the c-max and how that relates to the MIC. And I
have drawn the MIC just as a hypothetical case here, as a dotted line
across here.

And when we are comparing the c-max to MIC, it is a simple ratio, as I
have shown here, of the c-max to MIC. And you will hear that term a lot,
c-max to MIC ratio. It is a term that comes up often in the publications
and by the investigations that have been done.

Another term that you see is time above the MIC, simply shown as the
length of time that the drug concentration remains above the MIC
throughout the 24-hour dosing interval. Now, in this case, I have only
got one \-- the drug has only been administered once. We could conceive
of another situation in which the drug has been administered through
multiple doses or even perhaps by a constant infusion of some sort. In
that case, it is still \-- I am using the term the time above the MIC is
usually taken over a 24-hour period.

And then finally, as we see here, we could measure the area under the
curve. And another term that has been looked at and particularly applies
to the fluoroquinolones is to take this total area under the curve for
the dosing interval and calculate the AUC/MIC. So that is another term
yet that comes up.

(Slide.)

DR. PAPICH: Some of these I won\'t \-- I won\'t reiterate these, but
these are the important terms. Of course, MIC we know. We are all
familiar with MIC. MBC is minimal bacteria cidal concentration.
Routinely that is usually not determined, but in the laboratory
certainly that is something that can be measured. But then the other
terms I have listed as well

I might add that you will hear sometimes the term inhibitory quotient.
That is another term that is used for the c-max to MIC ratio.

(Slide.)

DR. PAPICH: These ideas aren\'t new. It comes up a lot and it has
recently, but if you look through the literature, clear back in the
early days of antibiotics, Eagle, for example, in the early papers on
penicillin asked the question about the dosage schedule on therapeutic
efficacy of penicillin.

And here is another paper also by Eagle as the principal author where
they asked the question of whether or not it is better to give
penicillin as a continuous therapy or intermittently. Those simple
questions at that time weren\'t known, but they were beginning to ask
them, and this goes back quite a few years. In fact, 50 years.

So you can see these ideas of asking these questions of how to optimize
therapy based on a knowledge of the drug\'s pharmacokinetics have been
asked before.

(Slide.)

DR. PAPICH: And since then, of course, and especially recently, there
have been some very good investigations that have helped to try to
define this better, and I have got just a couple here. We can cite some
other more recent ones in which the pharmacodynamic properties of the
antibiotics have been looked at in application to the dosage regimen
design or the role of pharmacokinetics and the outcome of infections.

And we could go on. I think in the notes that are available to you I
have listed some other references that are primarily review articles,
but nevertheless, are good references that help to summarize some of
these concepts.

(Slide.)

DR. PAPICH: So let\'s just look a little bit at what we mean by
optimizing the pharmacokinetic/pharmacodynamic relationship, and to
start off, we can say, well, what is our goal of antimicrobial therapy
in the first place?

In the most simple sense we could say that we need to administer a
sufficient dose to maintain the drug concentration above the MIC or
perhaps some multiple of the MIC at the site of infection.

(Slide.)

DR. PAPICH: So we could draw a real simple curve here and say, well,
here is our drug concentration and here is our MIC, and all we need to
do is get the drug above the MIC throughout a portion of the dose
interval and we are going to achieve therapeutic success. But it is not
that simple, as we know.

(Slide.)

DR. PAPICH: We can complicate this more by saying, well, that is fine,
but here again we have a curve in a couple of different drugs. I have
got the MIC drawn here as a little dotted line. We can say, well, is it
better to give the drug as one big dose? In this case is it a dose with
a relatively short half life, but it reaches a high peak. In fact, it
falls below the MIC throughout the rest of the dosing interval, if this
drug is given once a day.

Is that the best way to give this drug or it is better to have a drug
that releases more slowly or a drug perhaps with a longer half life in
which the peak may not be as high, but it maintains a concentration
above the MIC for a longer period? So which of those is better? We
don\'t know the answer because I haven\'t mentioned what drug we are
talking about.

(Slide.)

DR. PAPICH: Let\'s look at it in another way and say, okay, I have got a
drug and I have the option of giving it as a big, you know, single dose.
In fact, the peak here that I have drawn is 10 times the MIC. I have got
a hypothetical MIC of around two. Is it better to do that, even though
it falls below the MIC throughout the rest of the dosing interval? Or am
I better off to give that drug \-- break it up into smaller doses, give
it more often and keep it up above the MIC for a longer period of time?

Which of those is better? Well, we don\'t know because we haven\'t
discussed what drug we are talking about, but it does matter what drug
we are talking about. And in some cases it makes a difference what the
bacteria is as well.

(Slide.)

DR. PAPICH: So, in order to understand this, they have divided \-- or in
the discussion over the years and trying to understand it better, they
have tried to break drugs down into either being bacteriostatic or
bacteriocidal and those that are bacteriocidal have even been
subdivided.

(Slide.)

DR. PAPICH: And so, we can start out by saying, well, for a drug that is
bacteriostatic, the concept has been \-- and this has been fairly
consistent over the years, that since it doesn\'t \-- backing in a
bacteriostatic way, it is not killing the bacteria. Really, the optimum
therapy would be achieved by a long duration above the MIC.

In other words, the time above the MIC is probably the most important
predictor of efficacy and that has held true pretty much with any
investigations that have looked at these drugs. Drugs that fall into
these categories, and this isn\'t hard and fast because there are
exceptions to this. But the macrolides I have listed, the tetracyclines,
chloramphenicol and some of its derivatives.

And the reason I said that there are some exceptions is that we know
there are some bacteria, some very susceptible bacteria for which
macrolides may be bacteriocidal. Also, fluoroquinolone and its
derivatives. For some bacteria they may be bacteriocidal.

So one of the problems throughout this whole process is that it has been
difficult to define drugs and pigeonhole them into one category versus
another, because sometimes it makes a difference of the type of bacteria
that we are talking about.

(Slide.)

DR. PAPICH: Okay. For the bacteriocidal drugs, they have been divided
into the time dependent or concentration dependent, and even in this
case it has not always been clear as to what group to lump them into.
There have been some attempts, and we have some pretty good ideas for
some of these drugs. But I don\'t want to imply, even for a second, that
we understand this as completely as we should.

(Slide.)

DR. PAPICH: So, let\'s look at some examples. The aminoglycosides,
gentamicin and all the others. As far as we know, the best evidence that
is available would put these into the concentration dependent mode of
action for the aminoglycosides, and that has been fairly consistent.

And that is to say that for the \-- to achieve the best therapeutic
success and \-- you know, the best part of this is what about the
resistance issue? I can\'t answer that. But at least to achieve the best
\-- to achieve therapeutic success, the best regimen seems to be a
regimen that produces a high c-max, that is a high c-max to MIC ratio.

And after that, whether we give the drug more often or not doesn\'t seem
to help and doesn\'t add to the therapeutic efficacy. So, if we achieve
a high c-max in the range of about eight to 10 times the MIC, we can
give this drug even once a day and because of a long post-antibiotic
effect that follows that high peak, we can achieve therapeutic success
with this drug.

(Slide.)

DR. PAPICH: One of the things that \-- this is a bit of a hard slide to
see, but I will just try to illustrate the sort of things you will see
in some of the publications that have examined these features of this
drug. This is an aminoglycoside on the left, tobramycin, and on the
right is ticarcillin, which is beta-lactam.

On the \"y\" axis is basically the bacterial numbers. So, the lower the
number, the better the kill was achieved. And basically, what this slide
is showing is a comparison of an aminoglycoside to a beta-lactam; is
that \-- the symbols represent a higher and higher concentration above
the MIC. By the way, these are in vitro studies.

But what it shows for an aminoglycoside is the level above the MIC
becomes higher and higher. In other words, the ratio higher and higher.
The greater, the steeper the line is here, and this is the very highest
ratio above the MIC, and the bacterial numbers drop off the most rapidly
as the peak concentration or the c-max to MIC ration is lowered the
lines shift up here to the point where you get no kill at all.

Now, in contrast to that, if you look on the right side panel, which is
ticarcillin, a beta-lactam, as you increase the drug concentration above
the MIC higher and higher, you get to a point where you really don\'t
kill anymore bacteria. You reach a bit of a threshold to where there
really isn\'t any advantage of having a higher drug concentration above
a point of about five times the MIC. So that is a contrast of
aminoglycosides and beta-lactams, and I will come back to the
beta-lactams again in a few minutes.

(Slide.)

DR. PAPICH: But to summarize in the way that we use aminoglycosides, in
the clinical sense nowadays \-- and this is true in veterinarian
medicine, as well as human medicine \-- in human medicine this has been
adopted as pretty much a consensus as the way of using aminoglycosides;
is that instead of our older regimens where we used to give
aminoglycosides three times a day, and I have got here just a common
dose might be around 3.3 milligrams per kilograms, we can take that
entire dose and give it once. In other words, 10 milligrams per
kilogram, and that is a regimen that can be given once a day.

This still surprises veterinarians when we talk to them about these
regimens. And I have to admit, 10 years ago, when these regimens
weren\'t being used, if you were to tell me or other veterinarians that
this could be an effective regimen were you have such a long time where
you are below the MIC, we would have thought that was crazy.

But this is what \-- in our clinic, at North Carolina State University,
for small animal patients, horses and so forth, this is the most common
regimen that we use.

(Slide.)

DR. PAPICH: Just to summarize the studies that are available, once daily
has been shown in several studies to be at least as efficacious and some
have suggested more efficacious as other regimens. And once daily
regimens have been shown, which is an added benefit, either as safe or
perhaps more safe because their kidney isn\'t exposed to the drug as
much as other regimens.

Now, wouldn\'t it be nice if we could add a third point to this and say
that these regimens are also less likely to produce resistant bacteria
in the gut and so forth? That would be a nice extra point, but we don\'t
have that point to add yet because we don\'t know.

(Slide.)

DR. PAPICH: Okay. Let\'s look at another group of bacteria. I mentioned
\-- I promised that we would examine the beta-lactams. Beta-lactams can
kill bacteria, but they kill it more slowly. It takes longer exposure of
the bacteria to the drug to kill a bacteria with a beta-lactam.

(Slide.)

DR. PAPICH: So these are time dependent. So, with a time dependent drug,
what is really important is to maximize the length of time above the MIC
so the time above MIC is the most important.

In this case you either have to give the drug more often or give the
drug in a way that is going to last \-- persist longer in the plasmid,
either because it is a sustained release drug or because of protein
binding or some other pharmacokinetic feature that allows the drug to
stay in the plasma for a longer period of time to keep that level above
the MIC.

(Slide.)

DR. PAPICH: So the optimum killing appears to be time dependent for the
beta-lactams. A couple of slides ago you noticed that with ticarcillin,
once you reached a certain concentration, there wasn\'t increased
killing beyond a certain point, and that point, if we were to summarize
most of the studies, that threshold appears to be about five times the
MIC.

So there is no advantage to giving more and more drug or higher and
higher doses. You don\'t get any better kill; however, there might be an
advantage to a drug that lasts longer. That is, giving it more often or
for the half life to be longer.

Now, it is not quite as simple as the picture I just painted, in that
there are some differences between gram positive and gram negative with
respect to the way

beta-lactams kill bacteria.

(Slide.)

DR. PAPICH: And what I have \-- now, this is from isolates of dogs, and
I apologize for shifting to a non-food animal. But this is a good study,
a very interesting way, I think, to illustrate the differences between
gram positive and gram negative bacteria with respect to the way

beta-lactams kill.

This happened to be cephalexin. But what I have shown here \-- and I
have taken this from a study. I took the data and I put it on a graph to
try to attempt to better illustrate it.

The blue bar represents staphylococcus. The red bar represents E. coli.
What the \"y\" axis represents is cumulative percent of susceptibility.
So, on other words, if you take the staphylococcus \-- and across here
is the MIC. Across the \"x\" axis is the MIC.

So as I go along here, at about .5 I reach the MIC 90 for
staphylococcus, and going beyond that really isn\'t going to help much.
On the other hand, if I look at E. coli, in order to get to the MIC 90,
I have got to go clear out to about an MIC of eight.

Now, that happens to be right at the break point, so these are still
susceptible. It is just that there is a big difference in the
susceptibility. Staphylococcus is quite susceptible, and I can kill
staphylococcus with a relatively low MIC or relatively low
concentration, whereas for the E. coli I am way up here at a several
fold difference in concentration before I have an effective level
against the E. coli.

Now, the reason I bring this up is that it presents a problem in
designing some of these studies. What do you target the drug towards in
designing a resistance type study if the pathogen happens to be a gram
positive organism, for example, and you are designing the best
therapeutic regimen for that patient? What happens to the E. coli that
might be in the gut that are inherently more resistant to the drug?

(Slide.)

DR. PAPICH: So I have just illustrated a little bit the differences here
between gram negative and gram positive bacteria with respect to the
pharmacodynamic properties. Gram negative bacteria are just a little
harder to kill. We don\'t have the post-antibiotic effects that we tend
to see for gram positive bacteria.

For gram positive bacteria they are bacteriocidal at lower
concentrations, but we get \-- either it is a

post-antibiotic effect or probably better termed a sub-MIC effect that
has been observed that allows for longer dosing intervals and that is
typical. The way that we use many of our beta-lactams, we can give them
at less frequent intervals when we are treating a gram positive
infection as compared to a gram negative infection.

(Slide.)

DR. PAPICH: Now, to further I guess complicate it, and just to
illustrate that there is a lot that we still need to learn and it is not
as clear cut as we might imagine, this is just \-- it is a very good
summary in the \-- and actually, I would recommend, for anybody that
interested in learning more about pharmacokinetic/pharmacodynamics, the
Clinical Infectious Disease Journal had a summary of different drugs.

There was an article on the beta-lactams, on the macrolides, on the
fluoroquinolones on aminoglycosides, and this is the one taken from the
beta-lactams. The summary of this paper was, as I have already said, the
time above MIC is probably the best predictor of outcome, but how long
above the MIC does that time need to be?

Most of these studies have been in neutropenic animals, mice and rats,
for example, and that maybe gives us a false impression for what is
really needed in the clinical patient. In the neutropenic animal you
need that time above the MIC to be quite long; 90 to 100 percent of the
dosing intervals.

For ones that have \-- where there is a

post-antibiotic effect, in other words, for gram positive bacteria or
for drugs that do kill a little bit better, such as the carbapenems,
time above the MIC can be maybe a little bit less; 50 to 60 percent of
the dosing interval.

However, if you compare that to non-neutropenic animals, animals that
have a competent immune system, you can get by with a time above the MIC
being significantly less, depending on the drug. This is a summary
article, so it has averaged a lot of this together.

But for carbapenems, for penicillin and for cephlosporins it does vary a
little bit. But, as you can see, it is not necessary for the time above
the MIC in at least an immuno competent animal for the time to be above
the MIC for the entire dosing interval.

(Slide.)

DR. PAPICH: Okay. And then we have the fluoroquinolones, probably the
group of drugs that \-- when we talk about resistance issues, it
certainly becomes one of the more controversial. And I have to say, when
we talk about these pharmacokinetic/pharmacodynamic concepts, it has
been one that has caused some controversy as well, because they haven\'t
been able to quite define which category it falls into or what is the
best predictor of therapeutic efficacy.

(Slide.)

DR. PAPICH: There is a consistent amount of information that says that
this is a concentration dependent drug; that the fluoroquinolones act in
a concentration dependent manner and that optimum therapy is done by
achieving the optimum c-max to MIC ration of about eight to 10.

Some of the studies have varied a little bit from that. But if we were
to try to average them, it would be consistently in the eight to 10
range, as far as an optimum ratio here. So that is a bit similar to what
we saw with the aminoglycosides.

(Slide.)

DR. PAPICH: However, you can look at another work that says, no, really
the best measure is the AUC over MIC ratio, looking at taking the AUC
under that dosing interval, the area under the curve, and looking at
that.

Now, where the AUC/MIC ratio has come out the best predictor, I have to
admit, has been in some \-- primarily some studies in human patients in
which the human patients were very ill. Some were immuno compromised.
These were people in an intensive care unit, for example, on a
respirator with very serious illness.

The figure that they arrived at \-- there is on particular study that
has been cited over and over and over; arrived at an optimum AUC/MIC
ratio of at least 125 and more likely 250 or above. Quite a higher
ratio.

And in examining some of our regimens that we use in the animals that we
treat, it seems to me that we rarely achieve ratios that are that high.

(Slide.)

DR. PAPICH: So these values that have been cited for optimum activity
\-- and they have been cited over and over again. The c-max to MIC ratio
of eight to 10 is pretty consistent across the various studies that have
been done. This AUC/MIC ratio of greater than 125, sure, it would be
great if we could always achieve that.

As I said before, I think that is perhaps a little bit unrealistic,
given the current regimens that we use, and also, considering that this
was largely taken from a population from human patients that are
probably a lot more ill than the patients that we deal with. And
probably a lot more immuno compromised than the patients that we deal
with.

(Slide.)

DR. PAPICH: And maybe just to illustrate that, or try to at least, I am
going to use as an example enrofloxacin, and what I did is just to go
through, you know, available published pharmacokinetic studies for a
whole list of species here, and I have got listed the c-max. This would
be the c-max after a standard common dose.

For most of these species the dose is five milligrams per kilogram,
which is pretty average dose across species that is used for
enrofloxacin. And if you look at the c-max level that is achieved after
either \-- depending on how the drug is administered, either oral or the
usual therapeutic regimen that is used, either oral or injectable, if
you look at this, if we could agree that maybe the average along these
species is about one and consider that a susceptible bacteria, a
bacteria susceptible to enrofloxacin, usually has an MIC of .1 or less
most of the time.

I realize that the break point goes clear up to .5 without getting into
that flexible range, but it could be higher. And if we consider that a
lot of the bacteria that we are trying to treat have MICs of about .1 or
less, looking at these c-maxes, I think you could agree that we usually
do achieve, with most of our regimens, a c-max to MIC ratio of at least
eight or 10. Most of the time.

However, if I come back and \-- I will argue again that I think a high
AUC to MIC ratio that has been of 125 or 250 is a little bit
unrealistic, because if we look at the regimens that are usually used in
therapy and consider that if we look at the AUC\'s achieved \-- and I
don\'t know what an average value would be here, but let\'s say
somewhere between five and 10 among these species would maybe be in the
average range here.

And again, consider that a susceptible bacteria to enrofloxacin is going
to have an MIC probably in the range of .1 or less. And again, do a
quick calculation. You can see that we rarely achieve values over 125,
and we surely don\'t get anywhere near 250.

So I think we could probably, if we had the proper studies, revise \--
if we decided to use AUC over MIC, I would argue that 250 is probably
too high and not realistic. We can probably revise that value.

I should maybe apologize to the people at Bayer. These are enrofloxacin
concentration. Probably, to be fair, I should have added ciperofloxacin
to those concentrations because, for example, with c-max, ciperofloxacin
adds \-- because it is the active metabolite; adds at least 10 to 20
percent to these values. Ciperofloxacin will add probably more like 30,
35 percent to the AUC values.

(Slide.)

DR. PAPICH: Nevertheless, the concept that has been used to try to
optimize fluoroquinolone dosing is to use a dose that maximizes c-max to
MIC ratio, because of the arguments that I just made, in trying to \-- I
am not sure we understand what the optimum c-max to AUC ration has been,
so we will optimize c-max to MIC ratio.

This allows us to give the drug once a day, which is usually used, for
therapeutic purposes, for most of the fluoroquinolones available in
veterinary medicine, and one of the advantages \-- and this is the first
drug that I have mentioned today where the issue of resistance has come
up.

(Slide.)

DR. PAPICH: In the studies that are available, the little bit that is
available, suggests that of these two goals, the one that is best at
reducing the emergence of resistant bacteria has been to achieve a good
high peak, a

c-max to MIC ratio of at least eight or 10. So this is the \-- you know,
maybe we are on to something here.

(Slide.)

DR. PAPICH: In the case of the fluoroquinolones, if we could define the
optimum dosing in the species that we treat and get a little bit better
handle on this and come to an agreement that a c-max to MIC ratio of \--
let\'s use a range of eight to 10 \-- would minimize emergence of
resistance, maybe we could use some of this information to some benefit.

And also, what I have also \-- what we can\'t forget is that susceptible
bacteria \-- bacteria that are susceptible to fluoroquinolones can have
quite a range. I used, just as a round number, an MIC of .1 for a
susceptible bacteria in that little table that I was using.

But we recognize that some bacteria are susceptible with an MIC of less
than .03, and the break point can go clear up to .5. Now, that is quite
a range. So we need to be aware that for our therapeutic use for the
drug we utilize the flexible dosing allowing us to use higher doses for
bacteria that have higher MICs.

(Slide.)

DR. PAPICH: Well, there is one other problem or one other issue I wanted
to bring up just briefly in my last couple of slides, and that is any
discussion of the pharmacokinetic/pharmacodynamic concepts and these
terms have always used plasma concentration, and that is just the way
\-- you know, it is the easiest thing to measure. We can find that
pharmacokinetic information relatively easy, even for many of the
species that we treat, but it always uses plasma concentration.

(Slide.)

DR. PAPICH: And one problem that we have that has yet to be really well
defined for some of the drugs is what happens when you have a drug that
happens to concentrate to a high degree in some tissues? What happens?
You know, the plasma concentration in those cases may not be very
helpful.

(Slide.)

DR. PAPICH: For example, I have a little cartoon here to just help us
try to understand it. If we give our drug and it is circulating in the
bloodstream, but a lot of winds up being deposited in the tissues, and
the amount that you measure in the plasma could be relatively small.

A good example of that really is tilomycin. You know, micotil. You know,
that drug is administered to cattle. It concentrates, to a large extent,
in the lungs, and if you measure it in a plasma, you don\'t find a whole
lot. So it is very difficult, almost impossible really, for us to use
\-- in the way that we have been doing it anyway, to use the
pharmacokinetic values taken from plasma and try to make some
assumptions and some predictions from that.

A good example of this in human medicine that has caused problems in
understanding it has been erythromycin, and I would expect that there is
probably other drugs that could fit this category as well. They
concentrate so much in a certain tissue that a pharmacokinetic parameter
that is derived from the plasma isn\'t helping as much. Maybe we need to
go to the tissue to understand what those levels are.

The other thing that this illustrates perhaps a little bit is if this
tissue happens to be the lungs, for example, in treating pneumonia, what
are the concentrations in the gut where resistant bacteria might be
harboring? That is another answer we don\'t know.

So, if we are going to measure concentrations in the tissue, do we have
to then compare lungs with the concentrations that may appear in the
gut? These are questions that I don\'t know the answer to, but I guess I
don\'t have to apologize for adding more questions to your list of
questions today, because you have already got quite a few.

Well, that is all I wanted to bring up. I hope I have done a little bit
to help you understand pharmacokinetics/pharmacodynamics. If there is
any questions, I would be happy to address them.

(Applause.)

DR. GOOTZ: Tom Gootz, from Pfizer. I noticed for fluoroquinolones,
particularly enrofloxacin, you didn\'t have pharmacokinetic data for
poultry, and I am wondering \-- or other quinolones that can be used in
the poultry industry. I am wondering, particularly given a water soluble
product where administration is somewhat variable for some flocks, I
wonder what the AUC to MIC ratio in tissue or even the levels achievable
in stool of poultry for those types of products might be. Would they be
low, high or in between?

DR. PAPICH: That is a good question. I should know the \-- I have seen
the kinetics on enrofloxacin. In fact, we have done some ourselves in
some chickens, and I can\'t remember what \-- you know, in terms of the
c-max and AUC, I can\'t recall what some of those values are, but they
are known.

But I have to clarify that a little bit. Of the stuff that has been
published, you know, that appears in some journal articles and so forth
on the kinetics of enrofloxacin in chickens or poultry has been largely
from an intramuscular injection or a single administration.

What we don\'t know and this kind of raises a bit of a question and a
problem, is what happens when it is in the water and they are drinking
it over a more prolonged period of time? And in that case you are not
hitting \-- you know, it is not a high peak you are hitting. It is more
of a longer exposure over a longer period of time. That does raise some
questions. I have to admit.

DR. SHRYOCK: Tom Shryock, Elanco. I guess I just want to follow up on
that, that point, Mark. We are talking a lot of the enteric bacteria
here as being problematic, and obviously, when we are looking at plasma
concentrations and that sort of thing, we are not talking enteric
concentrations.

For the breakout sessions tomorrow perhaps it would be useful to provide
some considerations of the factors, the issues that one would have to
consider to try to address those kinds of concentration dosing regimens,
et cetera, so we could explore that a little more fully.

DR. PAPICH: I think that would be great too, and to look at
concentration in the gut, if need be, and that brings into consideration
how the drug is eliminated. Some drugs are eliminated by the feces, and
that, of course, is going to be an issue, as compared to other drugs
that are eliminated by kidneys and we find maybe a smaller amount in the
intestine. Those are all issues that I hope we can discuss.

DR. WHITE: Any other questions?

(No response.)

DR. WHITE: Thank you.

DR. PAPICH: Thank you.

(Applause.)

DR. WHITE: Well, we are entering into our open comment period, so I will
turn it over.

**OPEN COMMENT PERIOD**

**By Dr. Heslin**

DR. HESLIN: I am back, and we have got a 15-minute comment period, and I
assume most of you were here earlier, so you kind of know the ground
rules. There is going to be comment periods again tomorrow. But if you
want to take this opportunity, if anyone wants to enter comments, now is
the time to do it. Just introduce yourself and the organization

DR. ANGUELO: Fred Anguelo, from CDC. I was encouraged by several of the
comments. Encouraged first that I didn\'t hear much support for pathogen
load, and therefore, I take that as a need to focus on the resistance
aspects of the pre-approval studies.

I am encouraged by the comments of Dr. Flynn, that the objectives of
these resistance studies are to characterize the rate and extent of
development of resistant enteric bacteria, which I interpret as an
information gathering exercise, not as a pass/fail exercises.

To characterize the rate and extent \-- because we have already heard,
as David White pointed out, all of these agents are going to select for
resistance, and hence, they would all fail if that was the criteria.

I was encouraged by a comment by Dr. Ewert. She mentioned that the
intention of the pre-approval studies was to determine the impact on the
gut flora, but she raised interesting questions about what kind of
animal testing there would be and whether it should be in a laboratory
setting or a field environment.

My impression would be it should obviously be in the field environment
where the agent is intended to be used, and therefore, that would be the
setting in which to determine the rate and extent of emergence of
resistance.

Dr. Ewert also brought up the questions about microbiological
procedures, and I thought that was nicely answered by Dr. Mevius. Sorry.
Because in just the point that there can be used \-- that selective
media can be used and that the use of that selective media doesn\'t
necessarily have to follow NCCLS methods just to have a standard
approach to be adopted.

And then the comment from Dr. Carnivale about concerns about cross
resistance and questioned what the focus should be, and clearly, the
focus should be on the drug which is under consideration in the
pre-approval process, and therefore, what should be searched for is
resistance to that single drug.

And then, once that resistance to that single drug is found through
using selective media for instance, then you can check for cross
resistance to all the human drugs and then we can categorize the drugs.
And once we categorize the drugs, then we can set up the necessary
thresholds and corrective actions.

DR. HESLIN: Thank you. Any other comments?

(No response.)

DR. HESLIN: Okay. Do you have anything else that you want to say?

DR. WHITE: Do we have any other comments for today?

(No response.)

DR. HESLIN: Okay. Well, I guess that is the end for today, and we are
reconvening tomorrow at 8:30.

(Whereupon, at 5:17 p.m., the meeting was recessed, to reconvene
February 23, 2000, at 8:30 a.m.)