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DEPARTMENT OF HEALTH AND HUMAN SERVICES

FOOD AND DRUG ADMINISTRATION

CENTER FOR FOOD SAFETY AND APPLIED NUTRITION

PUBLIC MEETING:

2005 SPROUT SAFETY

Tuesday, May 17, 2005

8:30 a.m.

Harvey W. Wiley Federal Building Auditorium

5100 Paint Branch Parkway

College Park, Maryland 20740

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C O N T E N T S

PAGE

Opening Remarks

Robert Brackett, Ph.D. 4

Overview

Nega Beru, Ph.D. 8

Government Perspective:

Foodborne Illness Outbreaks

\--Amy Dechet, M.D., Epidemic Intelligence

Officer, Foodborne and Diarrheal Diseases

Branch, Centers for Disease Control (CDC) 12

Sprout Guidance, Background and Next Steps

\--Michelle Smith, Ph.D., Interdisciplinary

Scientist, Office of Plant and Dairy Foods,

CFSAN, FDA 26

Questions and Answers 55

Industry Perspective: 62

State of the Sprout Industry

\--Bob Sanderson, International Sprout

Growers Association (ISGA) 62

Seed Handling and Distribution Systems

\--Bob Rust, International Specialty Supply 81

Consumer Perspective:

\--Caroline Smith DeWaal, Center for

Science in the Public Interest 99

Questions and Answers 113

Scientific Perspective: 129

Microbiological Findings on Sprout

Operations Following FDA Guidance

\--Jed Fahey, D.Sc., Johns Hopkins School

of Medicine 130

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C O N T E N T S (Continued)

PAGE

Scientific Perspective: (Continued)

Interventions

\--William Fett, Ph.D., Eastern Regional

Research Center (ERRC), ARS, USDA 145

\--Kathleen Rajkowski, Ph.D., Eastern

Regional Research Center (ERRC), ARS,

USDA 163

Testing Methodologies and Sampling

\--Mary Lou Tortorello, Ph.D., Research

Microbiologist, Office of Plant and

Dairy Food, CFSAN, FDA 167

\--Tong-Jen Fu, Ph.D., Research Chemical

Engineering, Office of Plant and Dairy

Foods, CFSAN, FDA 182

Alternative Seed Sanitation Methods and the

Results of Practical Field Applications

\--Kean Ashurst, Caudill Seed Company 202

Risk Analysis

\--Don Schaffner, Ph.D., Rutgers 212

Questions and Answers 229

Public Comments 253

Summary of Meeting and Adjourn

Nega Beru, Ph.D. 270

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P R O C E E D I N G S

DR. BERU: Good morning, everyone. I

guess we\"ll get started. We said the program would

start at 8:30. We gave a little time for some of

the s\^D\^Dtragglers, traffic and so on but good

morning and I would like to welcome you to this

public meeting on sprouts.

My name is Nega Beru and I\"m the Associate

Director of the Office of Plant and Dairy Foods in

CFSAn. I also am the moderator for this meeting.

As you know, this meeting is intended to

elicit information on the current science related

to foodborne illness associated with the

consumption of sprouts.

Before I introduce the agenda and the

panelists for our meeting, I would like to invite

Dr. Robert Brackett, Director of the Center for

Food Safety and Applied Nutrition, to make his

opening remarks.

OPENING REMARKS

DR. BRACKETT: Thank you, Nega.

Good morning to all of you and welcome to

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this public meeting on sprout safety. It really is

a pleasure to be with you here today and I had

hoped to be here the entire day with you but I had

a hearing that was changed to today so I will stay

for a little while and head downtown right away

again.

Microbial food safety of fresh produce is

a priority for CFSAN and so is the role of fresh

produce and a healthy diet, which is also important

to us.

In October of 2004, CFSAN finalized an

action plan for fresh produce. This meeting

continues our dialog with stakeholders on how to

ensure that foodborne illnesses associated with

fresh produce, and that includes sprouts, is

minimized to the greatest extent possible.

During the past decade, over 20 percent of

all produce related foodborne illnesses were

associated with the consumption of raw or lightly

cooked sprouts. CDC first brought sprouts to our

attention as a vehicle for foodborne illness back

in 1995. In a 1998 white paper on fresh produce,

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the National Advisory Committee on Microbiological

Criteria for Foods identified raw sprouts as a

special food safety problem. In 1999, the National

Advisory Committee on Microbiological Criteria for

Foods issued a report entitled \"Microbiological

Safety Evaluations and Recommendations on Sprouted

Seeds.\" Since then, FDA has issued several

consumer advisory warnings about health risks

associated with the consumption of raw sprouts.

FDA also released two guidance documents, one on

seed disinfection and the other on testing

irrigation water, concerning practices to minimize

microbial contamination of sprouts. We have worked

collaboratively with other agencies, groups, and

the industry in a variety of different ways.

Although the guidance documents were well received,

and their reissuance and adoption appears to have

resulted in some improvement, outbreaks implicating

sprouts continue to occur.

As a way to address the public health risk

associated with the consumption of raw and lightly

cooked sprouts, FDA is considering the development

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of a regulation. The purpose of this meeting is to

elicit information on the state of the science with

respect to sprout safety and intervention

strategies and also to engage you, the

stakeholders, early on in the rulemaking process.

We believe that the most effective

strategy for reducing foodborne illness from raw

and lightly cooked sprouts is likely to be one that

approaches the problem from several different

angles. For example, we need to look at the seed

producers and distributors as well as the sprout

producers to understand the practices that

contribute to the contamination of sprouts with

human pathogens. It\"s important that we consider

the views and ideas of all of our food-safety

partners. By working together, we think we can

achieve this goal. Consequently, I am looking

forward to hearing your views and seeing them later

on, and your comments, and I hope that you find

that the discussion is worth your while as well.

So with that I would like to again welcome

you to CFSAN and wish that I could stay for the

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whole day. Thanks.

OVERVIEW

DR. BERU: Thank you, Bob.

As you will note from the agenda you

picked up on your way in, we have a pretty fully

day but before I go over the agenda with you let me

just say a few words about housekeeping.

The all important rest rooms\--this is a

full day meeting so I should point that out

first\--are upstairs where you registered as you

came in that hallway. There are also rest rooms

down the hall out this door.

We will provide refreshments during the

breaks but you are on your own for lunch. There is

the Wiley Cafe, which you probably saw as you

walked into the building. And if you drove, there

are a number of places to eat up and down Route 1,

as well as on Kenilworth Avenue.

We have a number of people who have

registered to provide comments at the end of the

formal presentations during the comment portion of

this meeting. If you have not registered but still

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wish to make a comment, please see me some time

during the meeting and we\"ll add you to the list.

And when you ask questions or make

comments, please wait to get the microphones or

step to one of the two microphones on the sides of

the auditorium. We are making a transcript of the

meeting and we want to be sure we capture

everything, including your name and affiliation. The

transcript is going to be made available on

our website in about four weeks time.

Now let me go over the agenda. It is in

your packets if you want to follow along. We\"ve

had the opening remarks by Bob Brackett, our Center

Director.

We\"ll first have a session which we\"ve

termed \"Government Perspective\". Dr. Amy Dechet

from the CDC, who is an Epidemic Intelligence

Service Officer in the Foodborne and Diarrheal

Diseases Branch of the CDC, will first present on

foodborne illness outbreaks.

That will be followed by Dr. Michelle

Smith, Interdisciplinary Scientist in the Office of

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Plant and Dairy Foods, CFSAN. Dr. Smith will

present on the sprout guidance, background and next

steps.

I\"ll ask you to hold questions until both

speakers have gone through their presentations and

they will come to the front and then we\"ll have a

question and answer session.

We\"ll take a break at that point and we\"ll

go into what we term the \"Industry and Consumer

Perspectives.\" First we\"ll have a presentation by

Mr. Bob Sanderson, International Sprout Growers

Association, on the state of the sprout industry.

And then Mr. Bob Rust from the

International Specialty Supply will present on seed

handling and distribution systems.

And we\"ll have a consumer perspective by

Ms. Caroline Smith DeWaal from the Center for

Science in the Public Interest.

When all three have done their

presentations, again we\"ll have a question and

answer session of those three panelists.

Then we\"ll go into what we call the

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\"Scientific Perspective\" section of the meeting.

First microbiological findings on sprout operations

following FDA guidance by Dr. Jed Fahey from Johns

Hopkins School of Medicine.

And we\"ll take a few questions but we\"ll

hold most of the questions for Dr. Fahey until

after the other scientific presentations

have\--we\"ve gone through the other presentations.

We\"ll have lunch at that point. As I say, lunch is

on your own.

And then after lunch the first

presentation will be by Dr. William Fett, Eastern

Regional Research Center, on interventions, and Dr.

Kathleen Rajkowski, Eastern Regional Research

Center again from USDA, ARS.

That will be followed by \"Testing

Methodologies and Sampling\" by two of our own

scientists at the Moffitt Center, Office of Plant

and Dairy Foods, CFSAN. Dr. Mary Lou Tortorello,

who is a research microbiologist at the Moffitt

Center and Dr. T-J Fu, who is a research chemical

engineer, also of the Moffitt Center.

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That will be followed by \"Alternative Seed

Sanitation Methods and the Results of Practical

Field Applications\" by Kean Ashurst\--I hope I\"m

saying that\--pronouncing the name

correctly\--Caudill Seed Company.

And the last of the scientific

presentations will be one on \"Risk Analysis\" by Dr.

Don Schaffner from Rutgers.

There will be a question and answer

session again of the scientific perspective

presentations. We will take a break and we\"ll go

into the public comment section of the meeting. And then I

will summarize the meeting and we will

end that.

With that, I would like to invite Dr. Amy

Dechet from the CDC to present on \"Foodborne

Illness Outbreaks\".

As I said, please hold until both Drs.

Dechet and Smith have made their presentations.

GOVERNMENT PERSPECTIVE

FOODBORNE ILLNESS OUTBREAKS

DR. DECHET: Good morning and thank you

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for the opportunity to speak with you today.

I thought to begin this meeting it would

be helpful to give you an overview of some of the

characteristics of sprouts and their outbreaks, and

put it in the context of the produce-associated

outbreaks as well as foodborne outbreaks in

general.

Each year in the United States we estimate

that there are over 76 million foodborne illnesses,

over 300,000 hospitalizations and over 5,000

deaths. Now these numbers sound large and they are

large but what does this really mean?

This means that one in four Americans will

become ill. So if you look in the room around you

and look at three other people, one of you is

probably going to become ill from some sort of

foodborne illness this year. One in 1,000

Americans are expected to be hospitalized and this

will result in over \$6.5 billion in medical and

other costs such as time lost from work.

The Centers for Disease Control and

Prevention and, in particular, the Foodborne and

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Diarrheal Diseases Branch, where I work, maintains

a Foodborne Outbreak Surveillance System. The

Local and state health departments are a primary

contributor to this system as they are the ones

responsible for detecting, investigating and

controlling outbreaks. At CDC we will participate

in these investigations when we are specifically

requested by the states to do so and we do some

field investigations every year with states and

provide over 100 phone consultations as well.

Once information is gathered from the

states it\"s usually sent to the CDC where we

collect data on the number of cases, the implicated

food and the etiology. To be entered into the

system, we define an outbreak as two or more cases

of a similar illness resulting from the ingestion

of a common food.

I want to point out that this system is

completely voluntary. States are not required to

report, although most do participate and some of

the data we get is incomplete. It could be because

at the state level they were unable to get the

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information or it simply did not make it on to the

form. I\"ll also point out that some of the

information that we have may not match exactly the

same numbers that FDA has and this is because again

our systems are slightly different.

If you look at now at our Foodborne

Outbreak Surveillance System over the past decade

or so, you\"ll see on the X axis the year and on the

Y axis the number of outbreaks per year. You

notice that in 1998 there is a significant jump in

the number of outbreaks. This is probably an

artificial increase because we actually enhanced

surveillance quite a bit during that year and, as

you can see, since that time we\"ve averaged around

13 to 1,400 outbreaks per year.

Looking specifically at produce associated

outbreaks from 1998 to 2002 where we have the most

complete data, we know of 249 outbreaks, and this

represents six percent of outbreaks with a reported

food source and 13 percent of outbreak associated

cases.

If then we turn to the implicated produce,

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many of these produce outbreaks are sort of a

generic or multiple produce item outbreak where

people mention salad or salad was the implicated

item but then we also do have some information on

produce specific outbreaks. And as you can see the

top six there are highlighted in yellow, which

includes lettuce, sprouts, juice, melon, tomato and

berries.

Taking those same top six now I\"ve again

listed here the number of outbreaks. Again this is

up to 2002. And then the percent of population

eating that food in the week prior to interview.

And this is based on our FoodNet population survey

where people are randomly called and asked, \"Did

you eat this food item in the seven days prior to

interview on the phone?\" And as you can see

lettuce, which is the top of our list of outbreaks,

over 70 percent of people reported eating lettuce

in the week prior to interview. The rest of them,

excluding sprouts, range anywhere from 21 to 68

percent but, if you look at sprouts, only eight

percent of people recall eating sprouts in the week

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prior to the interview on the phone and yet it\"s

the second leading cause of produce associated

outbreaks.

Now we fondly refer to sprouts as our

stealth vehicle in foodborne outbreaks because many

people don\"t realize they\"re eating sprouts.

They\"re often in salads or in sandwiches and people

don\"t remember that they ate sprouts. In fact, in

many of the outbreak investigations only 30-40

percent of people will outwardly recall eating

sprouts but then when we look at other implicated

food items and break it down by ingredients we

often do find sprouts then in those food items.

So what is it about sprouts that makes

them so unique? Many of you, I think, probably

already know this information. I\"ll just briefly

review it. There are, of course, multiple

opportunities for contamination along the spectrum

from the farm to the table. At the field level

there are birds flying overhead, there are animals

passing through, and even if animals are grazed on

the field months beforehand we have some evidence

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to show that actually the seeds can be contaminated

when they are grown months later.

During the harvesting process sometimes a

few combines are used for many different fields and

they are also processed in one large central

processing facility and so, therefore, if you have

a contaminated batch it can actually then mix with

other batches and contaminate a larger amount of

sprouts or seeds.

During the scarification process where the

seeds are rubbed between hard surfaces in order to

crack the seed it\"s a perfect time for bacteria to

enter into that seed and then sit there and

actually they can survive there for a several

months period of time.

Then, finally, there\"s multiple times

where during transport it could become

contaminated. From the field to the processing

facility to distribution by the sprouters to the

grocery stores and back home. So you\"ve got many

people handling the sprouts and again bacteria can

amplify throughout the process.

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And then, of course, there\"s the sprouting

process as well. This is a lovely microbiological

environment for bacteria to grow. It\"s warm. It\"s

moist and, depending on the literature that you

read, anywhere from a two to four log increase in

colony forming units per gram can occur.

Unfortunately, it\"s difficult to detect

pathogens. The contamination is usually

non-homogenous so if you sample one part of the

seed or the sprout you may not actually be getting

a representative sample of levels of contamination

elsewhere. Also, there can be low levels of

contamination which are difficult to pick up on

culture methods.

Finally, sprouts are rarely cooked or

washed by the consumer and this is often our final

safety net for many food items that at least you

can hope that the consumer will cook things well

and that will kill a lot of the bacteria but this

usually does not occur with sprouts.

There are a couple of other things that

make sprouts unique. Sprouts is very democratic.

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It likes all sorts of different organisms. We have

many different serotypes of Salmonella that have

been implicated in sprout outbreaks as well as E.

coli. We have some of the serotypes a little bit

unusual here in the United States such as

bovismorbificans, mbandaka and we also have the E.

coli 0157:non-motile. These organisms are more

common in animals overseas and again sort of

suggests that there may be some connection with the

field and contamination at the seed level.

There also are multiple kinds of sprouts

as you very well know. I have just listed a few of

them here. While they have similar

characteristics, they may not all actually be

identical in terms of their likelihood of

transmitting pathogens the way in which they\"re

eaten.

And then, finally, as I\"ve alluded to

already, sprouts\--we have international partners

with sprouts in the seeds and the whole process,

and while it\"s challenging enough to control what

happens here in the United States, it\"s even more

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challenging to control things overseas and on the

individual farms.

So I\"d like now to turn to sprout

outbreaks by year and I\"ve done this in a graphical

form. We have on the X axis again the year and on

the Y axis the number of outbreaks. The peach

colored color is the alfalfa. The dark grey the

mung bean, the orange clover and then green is

mixed, and that generally is clover and alfalfa

combined. As you can see, when we first learned

about sprout associated outbreaks in 1995 with

Salmonella, we had a pretty strong increase up to

1999 which then decreased and again rose in 2003.

I\"m going to take away all but alfalfa

simply to clarify a point here. We know that

alfalfa outbreaks through 2004\--actually to the

current day as far as we\"re aware from 1995 is 26

outbreaks. As you can see there was\--and I think

it was mentioned this morning\--there was growing

concern about the cause of alfalfa sprouts and

other sprouts leading to foodborne illness. So

there was an interim advisory and then guidelines

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passed in 1998 and \"99 by the FDA to advise

chlorination of the seed with 20,000 parts per

million and then also testing of the irrigation

wash water, which I think we\"ll hear a little bit

more about in the next talk.

This looked very hopeful because in 2000,

2001 and 2002 the numbers of outbreaks decreased

but then we had this explosion of outbreaks in 2003

and this raised concern about what might be

happening in the industry.

The next logical question is, well,

perhaps why we have all these outbreaks is that

people weren\"t following the guidelines and that

certainly is a very reasonable question to ask. We

do have some information on who was following the

FDA guidelines. The ones in peach here we don\"t

know whether they were or not. The orange, we have

evidence to suggest that they complied with the

guidelines. And the green suggest they did not

comply. As you can see, we have both orange and

green after the guidelines were passed. So again

what this represents is that perhaps the current

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practices\--current regulations are not sufficient

enough to prevent outbreaks.

I\"d like to turn from that time period of

1998 onwards and just look at the average size of

outbreaks by year. I have again listed the number

of outbreaks, the total number of cases that we\"re

aware of as reported by the states, and then the

mean number of cases per outbreak.

As you can see, particularly looking at

the year 1999 and then looking towards the later

years, 2003-2004, it suggests that perhaps the

outbreaks are getting smaller and this could be a

very positive thing and could be a result of the

guidelines being followed by the sprouters. I

caution, though, in too much interpretation of this

data because we are every year getting better at

detecting outbreaks. We now have a good system of

DNA fingerprinting across the nation and actually

even internationally. So I think we are picking up

outbreaks quicker before they become larger. We

sometimes are picking up outbreaks of two to three

people because states\--they have a lot of people

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working in the state to follow through on outbreaks

to look at some of the DNA fingerprinting measures

and we\"re doing more and more biological testing.

Of course, a remaining question and one

I\"ll end with is, well, perhaps outbreaks are not

the best way to measure the effectiveness of an

intervention. I want to just return very quickly

to a different produce item, to juice, number three

on our list that I showed earlier.

Here we have the number of juice outbreaks

from 1995 to 2005 similar to what I\"ve shown you

with sprouts. Each box represents an outbreak and

the various colors are suggestive of different

kinds of juice. Around the same time period there

was a juice labeling regulation that was passed in

1998-99. As you can see, it certainly rose in \"99

and then actually has decreased and remained low.

There have been various HACCP regulations

throughout.

The last four in red there from 2002 to

2005 actually represent outbreaks that would not

have been included in the HACCP regulations due to

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specific quality and size, for instance, of the

juice facility. So this graph suggests that

actually you can use outbreaks to show that an

intervention is effective or perhaps that it\"s not

as maybe is suggested by the sprout outbreaks.

So to summarize and conclude, sprout

associated outbreaks represent a small proportion

of the foodborne outbreaks in general but they are

one of the most common vehicles identified in

produce associated outbreaks and this is

particularly in the setting of a produce that

actually is not eaten as much as other produce

items in those top six items.

I think we\"re seeing actually with the

organic movement as well as the sort of health

related movements that more and more people are

eating sprouts and again the numbers I suggested

maybe perhaps are not totally accurate because

probably more people are eating sprouts and aren\"t

even aware.

As suggested by the number of outbreaks

most recently, the current practices and

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regulations are not adequate to prevent disease

from sprouts and I think that is why we\"re all here

and looking forward to a good discussion throughout

the day.

Finally, outbreak surveillance does offer

opportunities for tracking effectiveness of

interventions and we certainly at CDC will continue

to collaborate with all parties involved to try to

prevent disease in humans from sprouts.

Thank you very much.

DR. BERU: Thank you, Dr. Dechet.

Our next presenter will be Dr. Michelle

Smith, Interdisciplinary Scientist in the Office of

Plant and Dairy Foods.

By the way, we will have bios copy made

available either during the break or during the

lunch break.

Dr. Smith will present on \"Sprout Guidance

Background and Next Steps.\"

SPROUT GUIDANCE, BACKGROUND AND NEXT STEPS

DR. SMITH: It\"s a pleasure to be here

this morning and I\"d like to thank everyone in the

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room for coming also and I\"m looking forward to

what comes out of this program and, hopefully, we

can make some good progress together.

This is an outline of some of the things

that I intend to cover in my presentation. I don\"t

have a lot of time because mostly we want to hear

from you but I would like to talk a little bit more

about outbreaks, collaborative efforts, consumer

advisories and some of the things that have

happened since we started working together on

sprouts just as food for thought as this effort

goes forward.

Dr. Brackett mentioned the Produce Safety

Action Plan. I\"ll do that also. And Dr. Dechet

brought up some of the questions surrounding the

adequacy of FDA\"s guidance and I\"ll talk a little

bit about them along with next steps. Where do we

go from here and how do we get there?

As has been mentioned, sprouts were first

described as a special problem in the NACMCF

Produce White Paper that became available in 1998.

NACMCF in that paper talked about opportunities for

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contamination for fresh produce items, including

sprouts, and some of these opportunities have been

covered. What makes sprouts special, as everybody

here probably knows, is that the conditions that

foster the germination of the sprouts also promote

the growth of pathogens if they\"re present.

An additional challenge for this group and

for anyone involved in coming up with interventions

is that many of the treatments that have been

looked at to inactivate pathogens may also decrease

germination of the seed, decrease yield or affect

the appearance of sprouts. And treatments that

result in any of these make that treatment option

much less viable.

Now this is my simplified chart for

purposes of this meeting of outbreaks. Our numbers

are just a little bit different from CDC numbers.

According to my total, we have been involved in 27

sprout associated outbreaks since 1996. The first

column is the year, the second column is the total

number of sprout associated outbreaks in

parentheses, followed by columns of different

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sprout types and how that particular outbreak was

attributed to a certain type of sprouts. In a few

cases the numbers don\"t quite add up. Some of that

may be due to the fact that it was a mix of sprout

types and numbers have fallen in more than one

column. For example, in both alfalfa and clover

because we weren\"t able to tease it out. Then

finally the last column is the number of reported

cases of illness associated with the outbreak and

the actual number of illnesses is very likely much

larger than the number of illnesses reported.

Now just a few milestones to put some

context around the different activities. CDC

brought sprouts to our attention in 1995. The

second bullet could run two pages. There have been

many, many, many work groups, collaborative

efforts, other projects started individually and by

agencies and industry in cooperation to try and

address the issue of sprout safety. This is a very

complex issue and we\"re still working on it and

that\"s why we\"re here today.

FDA asked NACMCF to look at sprouts in

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1997 and they published the Sprout White Paper in

1999\. This paper played a very large role in the

guidance that FDA released later in that year.

If you haven\"t seen the Sprout White Paper

there is still a lot of very good information in

that paper looking from soup to nuts at sprouts.

It\"s available at this website and also published

in the International Journal of Food Microbiology.

Some of the findings in the Sprout White

Paper, and it was an extensive paper but some of

the findings I\"d like to highlight here are that

seeds are the most likely source of microbial

contamination. Things can get worse in the

sprouting facility if appropriate practices are not

followed but most, if not all, outbreaks it appears

that the seed brought the contamination in, in the

first place. If the seed is damaged or scarified

that can increase the chances of pathogens becoming

internalized in the seed and it also makes it much

more difficult for any kind of treatment to

disinfect the seed prior to sprouting.

The NACMCF Sprout White Paper specifically

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recommended a five log seed disinfection treatment

to be applied before the initiation of sprouting.

This five log standard was not formally included in

FDA\"s guidance, and I\"ll get to what we said in a

couple of minutes but it has been very widely used

as the target by many of the people looking at

interventions and comparing different

interventions.

We made available two guidance documents

in October of 1999. The first guidance \"Reducing

Microbial Food Safety Hazards for Sprouted Seeds\"

is a very simple, broad guidance document

containing five steps that we feel are important to

minimizing food safety hazards for sprouted seed.

The second guidance document \"Sampling and

Microbial Testing of Spent Irrigation Water During

Sprout Production\" provides additional guidance on

how to implement one of the first five

recommendations. Specifically, the microbial

testing of spent irrigation water. The testing

process is very complex and scientific, and I

brought in our microbiologist to write that. I

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wasn\"t able to write it and if I tried to rewrite

it I would have to share it with them to make sure

I got it right but this issue warranted its own

guidance document just to help people understand

the procedures for the microbiological testing.

Both of these documents are available on our

website which is noted there.

The objectives of the guidance were to

provide recommendations to seed suppliers and

sprout producers about reducing microbial food

safety hazards and sprout associated illnesses and

to ensure that all parties comply with the food

safety provisions of the Food, Drug and Cosmetic

Act.

Our guidance documents are guidance. The

specific recommendations that we feel are important

for microbial food safety for sprouts are just

that. They are recommendations. They are

guidance. The individual recommendations are not

requirements. At the same time the Food, Drug and

Cosmetic Act requires that food be safe and

wholesome and not produced or held under insanitary

33

conditions. That act contains requirements for all

foods.

In the Federal Register notice announcing

availability of the guidance there were several

statements, including \"failure to adopt effective

preventive controls can be considered insanitary

conditions.\" And a notice that FDA will consider

enforcement actions against any party who does not

have effective preventive controls in place. And

we put particular emphasis on microbial testing

because that\"s the last hurdle before the product

is released into the marketplace and gets to the

consumer.

Now in the broad sprout guide there are

five points. Everyone has a responsibility for

this guidance to be effective or as effective as it

can be. Everyone along the way needs to contribute

to improving and maintaining the food safety of

that component over which they have control and it

starts with seed production. We recommended good

agricultural practices for the production

environment for the seed, that seed be conditioned,

34

stored and transported under conditions that would

minimize opportunities for contamination. When you

get into the sprouting facility we are recommending

that producers follow good manufacturing practices

which are set out in 21 CFR Code of Federal

Regulations Part 110 that these GMPs should be

standard operating procedure.

Also, we recommend seed treatment. This

is the FDA language and our guidance. We recommend

applying one or more approved treatments shown to

reduce pathogens prior to sprouting. And as an

example we give the 20,000 parts per million

calcium hypochlorite. This does not say that the

seed treatment has to be 20,000 parts per million

calcium hypochlorite and when we drafted the

guidance back in 1999 we were very hopeful that

treatment alternatives would be discovered and

available because we know there are downsides to

this particular treatment but it at least up until

now has remained pretty much the gold standard.

The one that people are most familiar with and the

most data exists for.

35

And then finally because there is no

single treatment that we\"re aware of that can be

counted on to completely eliminate pathogens on

seed, we recommended microbial testing of the spent

irrigation water, the water that flows through the

sprouts during the production practice and we

recommended that this testing be done on each batch

of sprouts before the sprouts enter the food

supply. Salmonella and E. coli O157:H7 are the

pathogens of primary concern in sprouts and these

are the pathogens that we recommend testing for.

Now any guidance, including the sprout

guidance, identifies the most important steps that

we were able to identify at the time the guidance

was released to improve food safety and in this

case we believe that those recommendations should

be implemented immediately to reduce the risk of

sprouts as a vehicle for foodborne illness.

The broad sprout guide did not provide

detailed information on all individual steps that

should be followed to produce seeds and sprouts.

The broad sprout guide just set out the five items.

36

The idea was to have a simple document that could

be pasted to the wall. It was the microbial

testing where we specifically went into details on

how to. However, both documents contain additional

resources and references and we have done

additional things since the guidance to provide

more information.

One of the things that was put together

was an educational video \"Safer Processing of

Sprouts.\" This was a joint California Department

of Health Services-FDA video produced in

cooperation with industry and universities. It was

a fairly large workgroup that got together and came

up with the script. Many sprouting facilities

volunteered their facilities to be filmed as part

of this video.

One of the things I think that is

particularly good about this video is that these

are actual sprout production facilities and if you

look at the video and you listen to what it has to

say it puts a sprout face on many of the very basic

issues like worker health and hygiene and good

37

manufacturing practices. More information about

the video is available both on the California

website and on FDA CFSAN\"s website.

There has been mention of the consumer

advisories that FDA has put out. Our first

advisory issued August 31st, 1998, and that

advisory focused on alfalfa sprouts. When sprouts

first came to our attention alfalfa was the one

that we knew about and so that was the subject of

the advisory.

In 1999, we updated this advisory to cover

all raw sprouts because we were beginning to get

more evidence that at least some of the outbreaks

were not just alfalfa sprouts but maybe alfalfa,

clover mixtures, and at least one, we think, the

clover seed was the source.

The 1999 consumer advisory advised all

persons to be aware of the risks associated with

eating all raw sprouts. We noted, as we do often

in consumer advisories, that people in high risk

categories should not eat sprouts, should not eat

raw sprouts, and there was a new kind of statement

38

in this advisory. Persons wishing to reduce the

risk of foodborne illness from sprouts should not

eat raw sprouts.

There were several driving forces behind

including a statement this strong in the advisory.

One of the driving forces was that the majority of

people experiencing foodborne illness associated

with sprouts were not in the high risk categories.

They were just normal healthy large population

people. The other driving force you can see in

this slide in the red numbers. In 1999, mostly

clustered in the summer time, there were six

outbreaks associated with sprouts. That summer it

felt like sprout outbreaks would never end and I

don\"t think anybody here wants to go back to that

summer again.

Before we released the advisory we did

call in some members of industry and share it with

them and discussed the situation but it was decided

that that\"s the wording that we would go with.

So 1999 was a tough year with the updated

consumer advisory, the six outbreaks. We issued

39

our sprout guidance as a direct final. Usually we

go through two stages, proposed first, ask for

comments, and then go to final incorporating those

comments. Because of the large number of outbreaks

we went straight to final guidance but we did offer

an opportunity for comment on that guidance and

we\"ve collected those comments and considered them.

We also initiated an assignment to inspect

sprout facilities and I\"ll get into that in a

little bit.

Now this was a lot of activity. It wasn\"t

all pleasant but in 2000 there were no outbreaks

associated with alfalfa or clover sprouts so as

painful as this might have been it looked like we

were having a positive effect.

There was one problem. As the alfalfa and

clover situation appeared to be getting better, a

new trend started to emerge and that was outbreaks

involving mung bean sprouts and Salmonella

enteritidis. A couple of things about mung bean

sprout outbreaks. Even though our \"99 advisory was

meant to cover all raw sprouts, we were hearing

40

from some people that maybe mung bean sprout

producers did not see themselves in that guidance.

There are differences in the production practices.

You might look at an alfalfa seed compared to a

mung bean seed and notice that the alfalfa seed has

a much rougher surface and mung bean is very

smooth. There is less opportunity for pathogen

attachment, et cetera. There are still natural

structures on mung bean seeds that pathogens could

very easily attach to so there may have been

somewhat of a false sense of security.

There also was a general assumption at

least on our part that mung bean sprouts were

cooked before consumption so it would have a lower

risk associated with them. When we started

investigating the outbreaks associated with mung

bean seeds and identified the food vehicles

involved, we saw that those food items contained as

an ingredient either raw mung bean sprouts or mung

bean sprouts that had only been very lightly

cooked. Not cooked long enough or at a higher

enough temperature to eliminate any pathogens that

41

might have been present. So the assumption that

mung beans were cooked to the point where microbial

food safety wouldn\"t be a concern didn\"t

necessarily hold true.

So in 2002 we took our previous advisory

and we updated it again to advise all persons to be

aware of the risks associated with eating raw and

lightly cooked sprouts, and we specifically

included mung bean sprouts by name so that there

could no longer be any confusion or misconception

about whether or not bean sprouts were included in

our concerns.

I mentioned that we\"ve done a couple of

field assignments. The first one was in 1998.

This preceded the NACMCF recommendations and it

preceded FDA\"s guidance. So what we looked for in

1998 wasn\"t based on the guidance itself. We

looked at facilities in terms of the current good

manufacturing practice regulations in Part 110. We

targeted 100 firms. We usually draft an assignment

with a goal in mind. We ended up doing inspections

at 83 firms. There was a very long questionnaire.

42

Again largely based on the GMPs and also to gather

demographic information, information about the size

of facilities, products produced, number of

employees, things like that. Eighty firms that

were visited answered questions on this survey.

Finally, we collected samples for

microbial analysis. Samples were collected at

different points in the production process starting

with raw, dry seed and going all the way to

finished product. Samples were tested for the

pathogens, Salmonella, E. Coli 0167:H7 and a number

of other non-pathogenic microorganisms were

enumerated. Samples were collected at 78

firms.

Now this is just for context. I\"m not

going to dwell a lot on the specifics back in \"98

because it\"s not 1998 anymore and I think things

have changed but at that point in time out of the

83 facilities that were inspected 47 of the firms

or a little bit more than half of the firms

received what is called an FDA 483. This is a

report of observations that\"s usually issued when

43

insanitary practices or conditions are observed.

And in some of the more recent assignments I\"ll

give some examples of the things that we see as

insanitary practices or conditions.

In the microbial analysis we found

positives for Salmonella at three firms visited.

At firm C the samples were positive in the

pre-sprouted or pre-soaked seed but prior to

sprouting. Midway between the sprouting process

samples were collected and they were positive.

When firms washed sprouts after harvest, wash water

was collected and this was the case in the third

firm. That was also positive. And finished

product was positive at all three firms.

In no instance in this assignment were any

of the samples of raw seeds that were collected

found to be positive for pathogens and this may

indicate at least using the methodology of the time

some of the difficulties with microbial testing of

seed.

We did a follow-up assignment in 2000.

This time we targeted 150 firms, did a more limited

44

inspection focusing on the recommendations and

FDA\"s guidance, and also used a much shorter

questionnaire. Again looking at the

recommendations in FDA\"s guidance and trying to get

more detail. First of all, were they being applied

and, if so, how were they being applied? We

collected and tested spent irrigation water from

firms that reported that they were doing testing

themselves.

The good news is that we did not find any

samples to be positive for pathogens in the 2000

assignment. The bad news is that FDA 483s or

notice of observations were issued to 99 firms or

72 percent. None of these assignments were

designed in a way where you can do any kind of

quantitative comparison between the numbers. To a

large extent the individual investigators may be

very subjective in what they decide is an

insanitary condition and whether or not they issue

a 483 but there is some qualitative information

that can be gained from this, particularly with

respect to the kinds of observations that were

45

made.

Another thing that we did in 2000 was we

issued a fairly significant number of warning

letters. This is a step above and beyond issuing a

483\. We issued warning letters to 65 firms and we

did this when we found a combination of one or more

insanitary conditions and failure to implement

effective pathogen preventive controls. Again our

emphasis was on microbial testing. There was some

misconception since we did not give the field

authority to issue warning letters based on the

absence of seed disinfection that FDA no longer

thought seed disinfection was important. The

reason we didn\"t give that direct authority was

that seed disinfection has many complexities and it

would have been too difficult, and we still haven\"t

come up with a tool to fully describe to the field

investigators what would or would not constitute an

effective treatment, the variation between

tolerances of different seed types and all those

issues. So when it came to seed treatment, this is

still important, we gathered as much information as

46

we could but we did not make that a criteria for

the warning letter.

Okay. We found that at 54 firms there

were no significant deficiencies noted. We also

found that firms that were inspected in 1998 did

better in 2000 than firms that were being visited

for the first time. So maybe there was some

advantage either to FDA presence or to increased

awareness from having been visited before.

Some of the categories where we saw

deficiencies include personal cleanliness, things

like hand washing facilities, rest room facilities,

their condition, accessibility, even their

existence, insanitary food contact surfaces, how

they were cleaned, what they were made out of, the

presence of pests, including insects and rodents,

and water quality issues. Things like maybe having

a private well as a water source and the private

well had never been tested to ensure water quality.

Now, we said earlier that it looked like

the guidance was doing some good for a little

while. What we have seen in 2001, 2002, a single

47

outbreak associated with alfalfa sprouts, which may

or may not have been mixtures of alfalfa and clover

because we\"re starting to become more aware of that

practice. In 2003 there were five outbreaks. This

is one short of the summer of the \"99. In 2002 or

in 2004\--two outbreaks by mid 2004. So this raises

the question that was mentioned just a little while

ago about the adequacy of the guidance.

Some of the initial reports from the

investigators investigating the outbreak when they

have been able to trace back product to an

individual facility were starting to write in their

report that the sprouter appears to be following

FDA sprout guidance.

Now again our broad guidance is the

equivalent of five bullets so our first question

was does the investigator know enough to assess

accurately whether or not the guidance is being

followed. And we drafted questions here in house

that we\"ve shared with investigators whenever we\"ve

become involved to kind of help them ask the second

and third level of questions to get at whether or

48

not the guidance is being consistently and

appropriately applied by those firms that say

they\"re following the recommended practices.

In all fairness, this also raises the

question about the adequacy of the current

guidance. Is there something different we should

do? Is there more or less or some other way that

the guidance should be revised to be more

effective?

The most recent information we have about

practices within the sprouting industry comes from

a joint California Department of Health Services

Food and Drug Branch and FDA Inspection that was

initiated in February 2004. This was an inspection

of all registered sprout facilities in California

so again the data that comes from this you need to

be careful about extrapolating it to national

numbers but I think that some of the observations

that they made in this assignment are or should be

serious food for thought in what we do next as we

go forward. Again they used the standardized

questionnaire that was largely based on FDA

49

recommendations.

This is the inspection checklist that was

part of the inspection so this is a list of

everything that they looked at.

In this particular assignment 50 percent

of the firms were described as having deficiencies

in the same four areas that were noted before.

Numbers are somewhat different but again this was

not designed for direct comparisons.

When looking at seed treatment the

majority of facilities visited employed some type

of seed disinfection treatment. Seventy percent of

the facilities were using calcium hypochlorite, 25

percent of facilities were using sodium

hypochlorite, ozone and peroxyacetic acid were also

used. However, only two facilities, if you look at

all the steps that are recommended for the seed

disinfection treatment with 20,000 parts per

million calcium hypochlorite, only two facilities

used the correct concentration, the recommended

time and the best method, including agitation and

consideration of the seed solution ratio. So this

50

indicates to me that there is at least a strong

possibility that the folks that are doing seed

disinfection treatments may need a little bit more

help in the steps to follow to do that the most

effective way.

Many firms, 71 percent of firms, collected

spent irrigation water for microbial testing.

Almost all firms tested for Salmonella and E. coli

O157:H7 but the testing method varied greatly and

some of our colleagues here will get into testing

in a lot more detail this afternoon.

When it says \"unapproved test\" we cited

several tests in our guidance document. That

doesn\"t mean that you can\"t use alternative tests

but if you\"re using a different test than the one

that we cited you should have some confidence and

some experience that it really does work on spent

irrigation water.

California also noted that they had some

issues with who does the tests and where the tests

are done, qualified people in a controlled

environment, and also pooling has the potential to

51

dilute pathogens if they\"re present, and holding

samples for an extended period of time. If

pathogens are present they could die off in that

sample and give you a false negative which is a

false sense of confidence, a waste of money, and a

public health concern.

Sixty-five percent were conducting

confirmatory tests. Many firms said that they have

never had an initial positive but would do

confirmatory tests if they saw one. A little more

than a third of the firms were using the enrichment

media for confirmatory testing and this is what

microbiologists would recommend.

Almost half the firms wait to ship until

the results have been received, and again this is

from the confirmatory testing or the follow-up

testing to an initial positive most likely with the

rapid test kit.

One area certainly where more could be

done is the issue of record keeping. One of the

firms visited maintains a record of sampling for

microbial testing but no records of the outcome of

52

that sampling and this would also help us to have

information like this as we look at the incidence

of positives.

Less than 20 percent that conduct

confirmatory testing maintain records of positive

results from that so there\"s even less record

keeping further along the line and most firms do

not contain or do not maintain records of what they

do when they find positives, how was the product

disposed of. Now this may be kind of the

philosophy of don\"t ask, don\"t tell or we don\"t

want to put anything on paper that\"s not good news

but this really would be to the benefit of all

people involved to maintain records of their due

diligence, their appropriate actions, and also

would help us in investigations to know what

happened, and to have documentation to support that

knowledge.

Traceback is another area. A larger

proportion of firms report having the ability to

identify their seed source than firms that reported

having some kind of lot number or other identifying

53

data on finished product. Absence of this

information makes traceback very difficult.

Traceback is even harder when different seedlots

are mixed and the California survey found that for

every production lot of sprouts that they looked at

from one seedlot up to as many as ten were used to

make that lot of sprouts.

Dr. Brackett mentioned the Produce Safety

Action Plan that was finalized in October 2004.

This is the website for that action plan. With

some other commodity groups we\"ve made the

suggestion that they go visit the action plan and

look at it and see if there are ideas about things

that could be done. It covers risk assessment,

education outreach, research components, just a lot

of things that could have application here.

It\"s on our program priority list for this

year to hold a public meeting. We\"ll be able to

check that off after today so thank you all.

As individual tasks are identified they

also make it on to our to do list and one of the

tasks that we have on our priorities list is to

54

evaluate testing protocol for the recovery of

Salmonella in sprout seeds. Now the actions in the

Produce Safety Plan and anything that comes out of

this collaboration might be individual group

actions, they might be collaborative efforts but to

make true progress we really need the work on the

part of all parties.

The Food Code has defined sprouts as a

high risk food. Cut cantaloupe is a high risk

food. There are a number of things on that list.

I think by your presence here and your interest

that\"s an indication that there\"s appreciation that

a rigorous risk reduction strategy is needed. The

question that we\"re working on now with your help

is what should the strategy include to help us

answer the who\"s, how\"s, when\"s and best applied to

get the most appropriate effect of the best

possible solutions.

The Federal Register document announcing

this meeting set out a number of questions to kind

of help focus comment on the areas that we\"re

interested in getting more information on. This is

55

my short list. My personal favorite is how can we

better reach seed producers and distributors.

We\"ve said all along that this is a collaborative

effort. It involves all parties at every step

along the supply chain. Our Produce Safety Action

Plan no longer focuses just on the farm and packing

house. We\"ve extended that to cover the entire

supply chain and that would be an approach that we

would most like to use with sprouts also.

In addition to this public meeting, we

have a comment period that runs until July 18th and

the instructions for submitting additional comments

are in the Federal Register notice for the meeting.

I thank you for your time and attention.

QUESTIONS AND ANSWERS

DR. BERU: Thank you, Michelle. I would

like now to invite you to sit up here together with

Dr. Dechet.

I know we\"re running a little behind time

but we\"ll have a short question and answer session.

As I said before, please use the mikes.

We are recording this and we\"ll be having a

56

transcript made of the meeting which will be posted

on our website so when you have questions either

step to one of the microphones or they can be

handed to you.

Yes, please?

MR. SANDERSON: My name is Bob Sanderson.

I am president of the International Sprout Growers

Association and I would like to point out what\--I

may be hearing things that aren\"t there but

something of an inconsistency in Dr. Dechet\"s

presentation and Dr. Smith\"s.

Dr. Dechet spoke about the chlorine

recommendation of the guidance and discussed it at

some length and then it seemed kind of as an aside

and said, \"And also there is the testing.\" And Dr.

Smith pointed out that when they were evaluating

compliance\--when the FDA was doing this they said,

\"In particular, seed testing or testing

procedures.\"

And I have a feeling that there has been a

mixed message on this emphasis so I think that\"s a

serious problem because growers really are counting

57

every penny and they\"re going to put those pennies

where they really think they\"ll have to and the

power of the inspector is huge. Okay. Thank you.

DR. DECHET: In terms of my reference to

the chlorination process, certainly the sprouters

and the growers would have much more information

about that. I really use that more as a sort of

time table to talk about some of the interventions

that\--or the guidelines that were issued. Probably

Michelle can speak a little bit more about that but

we would, you know, really defer.

I mean, our job at CDC is really to detect

the outbreaks and report that information, and I

think sort of actual guidelines and who is

following those guidelines and what the emphasis is

of those guidelines really is best left to the FDA.

DR. SMITH: And I think maybe another

reason CDC focused on the seed disinfection is that

for a long time it looked as if the firms that were

disinfecting seed with 20,000 parts per million

calcium hypochlorite were not involved in the

outbreaks, whereas firms using the same seedlot

58

that weren\"t consistently or appropriately applying

20,000 were the ones that were having the outbreaks

so that put kind of a spotlight on the seed

disinfection treatment. At the same time there are

issues with the 20,000 parts per million. Not just

whether it\"s being used appropriately but the

impact that it has on seed, whether or not it\"s

consistent with organic production, the fact that

some types of sprouts, not necessarily the major

ones but some of the other types can\"t tolerate

that kind of seed treatment. It\"s very complex.

And then finally there is research about

pathogens surviving that treatment and recovering

during sprouting to the point where they can have a

foodborne\--cause foodborne illness. So our last

hurdle, the final thing that people can do to make

sure that contaminated product is not shipped is

the microbiological testing.

We are looking at a multiple hurdle

approach. I understand people want to do the one

thing that can make the most difference and our

guidance does provide for alternative approaches if

59

they do not violate laws or regulations and if,

hopefully, they get you to the same food safety

place. That second part is really a hard call. I

tend to take calls from the field that observe

something that is just woefully inadequate and I

can say that\"s not an appropriate alternative but

there\"s a huge gray area that I think we need more

information to map out better.

DR. BERU: Yes, please?

MS. SANDERSON: My name is Barbara

Sanderson and I\"m from Jonathan Sprouts also.

Michelle, I really appreciated your reiterating the

guidance where it said seed disinfection such

as\...and I have\--I would like to point out a

situation that happened in the industry. It hasn\"t

to do with your recommendation but how it was used

and I believe it began with an outbreak where a

grower was using a combination of a heat treatment

and 2,000 parts per million calcium hypochlorite

and still had an outbreak. The lawyers used this

information to turn your guidance into a rule that

we had to use 20,000 parts per million calcium

60

hypochlorite and there was no way\--that kind of put

a damper on getting any other kind of treatment

into use or even into the visibility of how

effective it might or might not be with sprouts so

that we really got paralyzed by the legal

profession.

DR. NORTON: I\"m Dick Norton. I\"m a

retired microbiologist from the Food and Drug and

working with different companies and asepsis for

the last 17 years now. Currently I\"m working with

a small company with asepsis and we\"ve sent the

results of Dr. Reiser\"s tests on alfalfa sprouts

for this. It\"s something new to this field. We

have been working off in a little corner somewhere

else and I don\"t think it\"s generally recognized

here but we\"ve been working with this fatty acid

combinations for 14 years now with Tyson\"s, with

various meat, types of meat and fish, and we\"ve

been marketing products for hand anti-asepsis in

hospitals. So we\"re unknown in this field

but we have background and your question as to how

can we all put this together got me interested in

61

finding out how we can help with this and our

interest is in treating seeds. It turns out

there\"s no effect on germination rate. There are

safe compounds that are cheap so if there\"s any

interest in this we would like to work with these

people and unfortunately I have to go to the EPA on

this very same subject at 1:00 o\"clock so I can\"t

stay around for the whole day so if anybody has any

questions.

DR. BERU: Thank you. We are running a

little behind time so let me suggest that we will

have Dr. Dechet and Dr. Smith also join the next

set of panelists during the question and answer

session so if you have any additional questions

let\"s hold them until then and let\"s take a break

until 10 past 10:00 and, as I said, we have

refreshments in the area where we registered.

Thank you.

DR. SMITH: And as Dr. Beru mentioned,

you\"re all on your own for lunch but I have a few

copies of the menu for the cafe that\"s right out

the front door and I\"ll tape the last copy to the

62

table and put a stack out there when I get up.

\[Whereupon, a break was taken.\]

INDUSTRY PERSPECTIVE

DR. BERU: Thank you. Our next presenter

will be Mr. Bob Sanderson and he will present\--he

is president of the International Sprout Growers

Association and he will present on the status of

the sprouting industry.

STATE OF THE SPROUT INDUSTRY

MR. SANDERSON: Thank you, Dr. Beru.

On behalf of the ISGA I would like to

submit what is our current set of sanitary

guidelines, an industry model HACCP and a beginning

to attempt to define good agricultural practices

for seed.

These things weren\"t built for my\--can

people hear me okay if I stand fairly straight?

Dr. Smith suggested that I provide an

overview of the sprout industry\"s, as she put it,

strengths, weaknesses, needs and desires.

My talk is not going to be the ISGA\"s

comment per se. That will be submitted prior to

63

the deadline in July but I\"m going to try to talk

in more general terms about the sprout producer and

his relationship with product safety issues and

their requirements.

In discussing the sprout industry\"s

strengths and weaknesses, there is the product

itself to consider and there are people who are

involved in producing sprouts, and I think any

discussion of the people has got to factor in the

kinds of rules and regulations under which we\"re

operating, whether they\"re clear, consistent and

effective in achieving their intended purpose.

The product itself is remarkable in some

obvious ways. Sprout producers may plant from one

to several crops a week, 52 weeks a year, with an

interval of from four to ten days between beginning

the process and shipping the product to the market.

What happens during this four to ten day period is

a combination of very rapid chemical and biological

processes which transform a mostly unpalatable seed

into a nutritious food.

In addition to being nutritious, it

64

appears that many, if not all, varieties of sprouts

contain high levels of one or more phytochemicals,

which may have significance to human health. The

understanding of these properties is still in the

early stages.

The very rapid growth of sprouts and the

conditions in which they\"re grown present unique

challenges in terms of food safety as well as some

unusual opportunities, which I\"ll come back to in a

moment.

Regarding the people who grow sprouts,

it\"s a very diverse group. The largest in terms of

numbers of producers and amount of sprouts produced

in the U.S. and certainly in the world as well are

those involved in the growing of mung bean sprouts,

which have been a staple food in Asia for thousands

of years. The other main group is companies, which

beginning about 40 years ago, began to introduce a

variety of sprouts, which historically have not

been widely used as a human food. Increasingly,

more and more sprout producers are combining the

growing of the traditional sprouts and the more

65

recently developed varieties so the industry is a

mix of people from very different cultures. Some

carrying on with the family business which may have

been started generations earlier and others who are

relative newcomers.

The increase in concern with sprout safety

seems to have coincided with the appearance on the

market of the newer types of sprouts so, as Dr.

Smith mentioned, there may be some feeling on the

part of the producers of the more traditional

sprouts that they\"ve been caught up in somebody

else\"s problem. After all, these types of sprouts

have been grown for many generations and wouldn\"t

we have known about safety problems long ago?

Although it may be argued that different

types of sprouts, which are used in different ways,

may require different risk reduction strategies,

it\"s not accurate to say that any particular type

of sprout is traditional because the nature of the

market, consumer preferences, production methods

continue to change very rapidly. This is true both

for the producers of bean sprouts and the newer

66

green leaf sprouts since both are functioning in

the context of increasing food safety concerns and

both have to deal with continually increasing

regulatory and third party requirements as well as

changes in the nature of the market and consumer

preferences.

There are some other groups with their

priorities within the sprouting community. The

ISGA being an international organization has

members from many different countries who have

quite different perspectives but since the ISGA

core membership is made up of U.S. growers the

association tends to have a focus on FDA

regulations and food safety which some growers

operating outside the U.S. seem to view as a kind

of negative fixation.

After all, the ISGA was started to promote

the positive aspects of sprouts, not to be talking

about safety all the time and, furthermore, sprouts

seem not to be considered such a high risk in many

other countries. But the issue isn\"t simple

because of differences in types of sprouts, how

67

they\"re usually consumed, total volume sold, as

well as differences in available public health

monitoring and epidemiology.

The FDA\"s recommended 20,000 ppm chlorine

seed treatment is generally considered very

excessive by growers from other countries where in

some cases the use of this treatment wouldn\"t be

allowed. Even among U.S. growers it would be high

on the wish list to have an approved treatment

which did not create such an irritating and

hazardous working environment.

It would also be a good thing for the ISGA

as an international association to have a single

internationally acceptable set of safety practices

or at least an agreement on underlying principles,

which isn\"t to say that sprout growers all around

the world would apply these equally but at least we

would have a common frame of reference.

Another point of debate in the sprout

industry which exists inside the U.S. as well as

between the U.S. and the rest of the world is the

status of organic sprout production and, as I think

68

someone mentioned, sprouts kind of is associated

with organics, which is kind of ironic in a way

nowadays, again primarily relative to the 20,000

ppm chlorine treatment recommendation. Although

some organic certifiers have allowed the use of

this 20,000 ppm treatment, the basis for this

allowance is felt by many to be quite a stretch

with organic production standards and is based on

the need for organic standards to take second place

to FDA safety requirements in any situation where

there may be a perceived conflict.

This is entirely appropriate and necessary

but unfortunately there isn\"t presently a clear way

to evaluate the effectiveness of risk reduction

strategies which might be more consistent with

organic production methods.

So the ISGA\"s wish list might also

include, if possible, the allowance of safety

protocols which were more consistent with organic

standards. This isn\"t to suggest that safety

criteria should be diluted in order to be

acceptable to different groups but that, if

69

possible, safety requirements should be based on

end product criteria rather than on any specific

method of getting there.

One aspect of sprouts which many consider

to be their greatest drawback is that they have

proved to be extremely resistant to treatment based

approaches to assuring their safety. This is

because, and this is very much in a nutshell,

sprouts\--with sprouts any sanitizing step is

followed by four or more days of growth at room

temperature. However, the same characteristics

which make sprouts difficult to sanitize may also

make them accessible to more thorough sampling and

testing than is possible with most foods.

The sampling and testing of seedlots prior

to use in sprouting with seed which test positive

for pathogens being diverted to non-food uses is a

common sense preliminary step. The fact that in

several instances sampling and testing of isolated

pathogens from implicated seed following outbreaks

clearly suggests that if the same sampling had been

doing prior to the use of this seedlot for

70

sprouting these outbreaks wouldn\"t have occurred.

Bob Rust is going to go into more detail

on ways to carry out this initial screening of

seedlots.

Since sprouts emerged as a significant

health concern in the late \"90s there have been

dozens of attempts by leading food safety

researchers to develop effective sanitizing

interventions. These investigations continue into

the present and it may be that in the near future a

truly effective seed sanitizing procedure will be

developed which is affordable and which doesn\"t

entail significant risks to sprout growers or have

other serious drawbacks or possibly such a

procedure already exists and, if so, then one can

only hope that it will get a speedy regulatory

approval.

In the event of the allowance of such a

treatment, one question will remain and it will

need to be addressed. It\"s whether the use of such

a treatment would eliminate or significantly reduce

the present need for every batch spent irrigation

71

water testing.

I don\"t know of anyone who presently is

proposing that this should be expected at least in

the near future and this is a very important

question because testing is very, very expensive

and if you introduce the idea of intermittent or

periodic testing in a very competitive market there

will be a significant economic incentive to

minimize it. This may lead to even more confusion

about what constitutes compliance, who is in

compliance and who isn\"t, which will not help the

overall level of confidence in the market.

So while we\"re waiting for improved

treatment interventions or even if we find them, it

seems worthwhile to consider how effective the

recommended testing procedures are. The 1999 FDA

guidance recommends that the spent irrigation water

from all production batches be tested in duplicate

for the two pathogens of concern and that negative

test results be back in house before any product is

shipped from the facility.

In preparing for this meeting I have

72

tended to assume that my company\"s testing program

could be duplicated by other sprout producers but

we are fortunate in being an hour away from a

qualified testing lab which is very responsive to

us and which charges us very reasonable rates for

its services. For other growers I\"ve heard that

lab costs per test can be much greater and some may

have to overnight ship their samples which will add

a full day before they can get their lab results

back. This will add significant problems to

inventory control as well as hold and release

programs which is that you don\"t let anything out

the door until you\"ve got your results back.

So if this every batch testing is going to

be a necessity until some significantly better

treatment is developed, things like cost per test,

how responsive a lab is, how long it takes to get

the sample to the lab, how long it takes to get the

sample results back are very important.

To put this in perspective, if my

competitor and I are both doing all the required

tests but he\"s doing duplicate testing and I am

73

only doing single samples for each test, I will

save enough money on testing alone to offer a price

which will allow me to take my competitor\"s

business and at the same time make more money at

the lower price than I\"m making now. This testing

is very expensive and one thing on many growers\"

wish list is that the required testing could be

significantly reduced.

One bean sprout grower from outside the

U.S. told me that if he tried to do all the kinds

of testing recommended in the FDA guidance he

couldn\"t possibly remain in business.

But I feel it\"s important to come back to

the question do we need to do this testing. It

seems that until some treatment comes along which

clearly indicates that we can cut back on our

testing we need to do it and so we have to focus on

how to make it more effective and more consistent.

High testing costs are frequently

mentioned as a burden to sprout producers but I

think this question needs to be looked at in

context. Are they a burden because the retail

74

price of sprouts is as high as it can be and

consumers will just stop buying them if the price

goes up? Or are they a burden because every grower

is trying to survive in a very competitive market?

And a difference of pennies per package can be a

determining factor in obtaining or keeping an

account with a supermarket customer.

In talking about the costs of the testing

program, I\"m talking about something in the range

of ten cents a package wholesale cost. If you ask

a sprout producer what his bottom line would look

like with an extra dime per package coming in, I

think you\"d see quite a reaction. In my own

company this\--and this is, I think, literally

true\--would finance an entirely new facility and

give everybody a raise at the same time.

So an important question about the burden

of these testing costs is the price sensitivity of

sprouts inherent in the product or is it dependent

on perceived value so that if this value goes up

the customer will readily pay the extra amount.

And I think there is good evidence at least from

75

some markets that customers will readily pay more

for sprouts if they have a reason to believe

they\"re getting a better or safer product.

When the FDA guidance were issued in 1999

sprout producers or sprout products were redefined.

They still looked and tasted the same as they

always had but the costs of producing them to an

adequate standard had increased substantially. At

the same time it was left up to the industry or the

individual producer to convince the customer that

the new improved product which looked and tasted

just like the old one was worth the increase in

price.

Since the supermarket buyer is under

considerable pressure to buy the least expensive

product, it\"s the producer\"s job to justify his

higher production and safety costs but neither the

supermarket buyer nor the consumer has any way to

evaluate the safety claims of the producer. This

can encourage marketing strategies which play upon

customers\" insecurities.

In the present guidelines the recommended

76

treatment and testing protocols are preceded with

the qualifier \"e.g.\" which means \"for example,\"

which suggests that what is recommended is only one

option out of several but in practice these

guidance recommendations are very strictly

interpreted and have acquired the force of

regulations.

Deciding what, if anything, might be

considered \"e.g.\" is a difficult thing to do in

part because it isn\"t clear what the treatment

referred to actually accomplishes. The sprout

industry would like treatment and testing options

to be based on the best available science and we

want, if possible, for them to be flexible so that

\"e.g.\" can allow for a range of approaches provided

they achieve an acceptable end result.

There was an attempt several years ago for

the ISGA to take responsibility for setting safety

standards by way of a seal program. Two problems

were encountered. One was that at the time the

seal program was being developed there had not been

very thorough inquiry into the effectiveness of the

77

safety protocols which were then available. For

example, there was no consideration at that time

that growers could do this every batch sampling. The other

problem was that the ISGA was not in a

position to effectively police compliance with the

requirements of the seal program as it wouldn\"t be

today.

Whether we have a guidance recommendation

or a regulation or whatever the structure, it needs

to be responsive to developments in treatment and

testing technologies and as flexible to the needs

of individual companies as possible. Although the

ISGA can\"t police this, clearly the FDA can\"t

immediately review every new idea that comes along.

And a question, I think, underlying this

is, is it worthwhile to evaluate safety methods for

a very small industry which might be quite

different from what is used with most foods? Of

course, it\"s worth it to the industry to solve its

problems and to prosper but are the sprout

industry\"s challenges relevant to other areas of

food safety or research generally? If we consider

78

the testing challenge in its simplest form to be

the challenge of quickly and accurately telling

friend from foe in the microbial world, this would

seem to connect the sprouting industry\"s needs with

many important research areas. Therefore, the

industry needs to continue to work with people in

government and academic research who have

qualifications and review capabilities necessary to

develop effective sprout safety standards and

procedures. The ISGA technical review

board can provide a framework for this effort.

Allowing for flexibility and safety

protocols has got a number of interrelated parts.

There is the question of how well something works

in a research setting and how well it would

translate into a production setting. This has

repeatedly come up in the case of the 20,000 part

per million chlorine treatment recommendation with

some arguing that lab results using inoculated seed

suggests a very high level of effectiveness in the

real world with others arguing just the opposite,

and I think there\"s good arguments on both sides.

79

Another challenge is how can compliance

with a number of different methods be evaluated by

food safety and third party inspectors. It\"s

certainly simpler for an inspector to evaluate a

single treatment and testing protocol for all

producers in a given category than to evaluate

several which might differ from company to company.

And it becomes even more difficult when,

as in most cases, the sample leaves the building

and is delivered or overnight mailed to a lab. Is

there any way the grower and his food safety

auditor can be assured that the lab is doing the

testing in a way which will provide the best

likelihood of detection of pathogens if they\"re

present in the sample? This may require skills

which are not within the safety inspector\"s usual

job description.

The challenges of developing good sampling

protocols, assuring the best testing methods and

providing adequate inspection criteria is another

area where the ISGA technical review board can

provide a significant contribution by working with

80

growers to develop appropriate inspection

checklists for treating and testing options.

So as long as we have the need for this

testing, we might as well look at it as an

opportunity and make sure it\"s being done as well

as possible.

If the playing field is as level as

possible and the methods are as good as possible,

the added costs may actually benefit the entire

industry by raising the standard of acceptable of

the product in the whole market. In the meantime

we can hope that treatment interventions are

developed which will make the sprout producer\"s

life much simpler. Perhaps some day requiring only

a minimal amount of microbiological monitoring.

Most sprout growers are acutely aware of

the microbial hazards which are possible with their

products, not through any choice. One of the

greatest strengths of the sprouting industry is our

collected awareness of microbial risk and our

growing knowledge of food safety. This may not

only be our best resource for our industry\"s future

81

but may also provide opportunities for learning

which would be relevant to many other areas in the

food industry as well.

I believe in the past calendar year there

haven\"t been any reported outbreaks connected with

sprouts. I don\"t know if the last 12 months\--I\"m

not sure if that is the calendar year but May to

May, which is a very good indicator of significant

improvements in the industry. We need to focus our

energies on how to increase our margins of safety,

restore confidence in sprouts, and make the

sprouting industry a model of food safety.

Thank you.

DR. BERU: Thank you, Mr. Sanderson.

The next presentation will be on Seed

Handling and Distribution Systems and will be made

by Bob Rust, International Specialty Supply.

SEED Handling AND DISTRIBUTION SYSTEMS

MR. RUST: I just wanted to thank Dr.

Michelle Smith and the FDA for inviting me and

giving me an opportunity to discuss a subject that

I\"m passionate about.

82

In order to stay within the allotted time

I recorded the presentation and it will be

available after this meeting in the Seed Safety

Section of our website, www.sproutnet.com.

\[Video presentation.\]

Hello, I\"m Bob Rust, owner of

International Specialty Supply, with divisions of

Prime Seed Company, Prime Packaging, Centrix

Equipment Company and Sun Garden Sprout Company.

We also test sanitation products and

processes for other companies and we have a

pathogen testing laboratory. We do consulting on

such things as GMPs and HACCP. We write a

newsletter called the SproutNet and have an

informational website, www.sproutnet.com, with most

of the research related to sprout safety.

I appreciate the honor of being invited to

speak regarding seed handling and distribution

systems. The world has seen 26 sprout related

outbreaks since 1998, of which 25 involved sprout

growers in the United States and Canada. The FDA

stated that they believed that the source of the

83

contamination in most sprout related outbreaks is

the seed. It\"s impractical for sprout growers to

line up small quantities of seed from farmers all

over the world so seed companies who specialize in

sprouting seed contract the production with farmers

and work with seed processors near the farmer to

process the seed.

It\"s difficult to predict how much seed to

contract and crop failures are common occurrence.

When this happens, seed companies will purchase

seed from other seed companies who may or may not

specialize in sprouting seed. Although there may

be thousands of seed companies throughout the

world, there\"s only a handful that specialize in

sprouting seed.

There are three ways the seed industry can

improve its record of shipping contaminated product

to sprout growers. It can grow, harvest, process,

store and ship seed as though the seed were food

product. It can decontaminate seed destined for

sprouting and it can screen the seed for pathogens

prior to shipping it out.

84

As the owner of a seed company and as a

commercial sprout grower, we\"ve put all three of

these methods to the test. There are so many

farmers and processors involved in so many parts of

the world that our attempts at controlling the

production was unrealistic. There are some good

decontamination possibilities on the horizon but it

may take several years of actual field use to

determine if they are as effective as they appear.

The last one, seed screening, is something

our company has been developing since 2000. There

has been 13 sprout related outbreaks since we

started our sampling protocol and we\"re convinced

that it\"s our seed screening process that has kept

us from being involved in any of these outbreaks.

Seed screening is a simple approach to

risk reduction. The idea is to determine if the

seed is contaminated prior to selling it for

sprouting purposes. The Camden Research Group in

England in their 2004 report titled \"Review of

Microbial Risks Associated with Sprouted Seed\"

concluded that the absence of pathogens in seeds is

85

critical and, consequently, microbial testing of

seeds prior to use for production of sprouts is

essential. Seed sampling is also suggested in the

Codes of Practice of Food Safety in Ireland and the

Ontario Ministry of Agriculture and Food is

adopting its recommendations for seed companies and

sprouters.

In early 2000, Bob Sanderson came up with

the idea that if the source of sprout related

outbreaks is often the seed maybe we should check

to see if the seed is contaminated before we use

it. Hmm. Check to see if the seed is contaminated

before we use it? We thought this was a great idea

so in conjunction with Jonathan Sprouts we

developed a process for screening seed before it

sprouted. When seed arrives it\"s placed in a

quarantined area and inspected for evidence of

mouse droppings on the bags, holes in the bags

where mice or insects may have entered, insect

larvae, bird droppings, that sort of thing. The

bags are black lighted for traces of urine.

In order to find contamination in seed

86

prior to sprouting it, a pathogen must be captured

for identification. Alternatively, one may capture

and identify evidence of pathogen contamination.

Twenty-five grams of sample from each and every

bag. If the sample comes to less than three

kilograms, enough bags are re-sampled in order to

get three kilograms.

The composite sample of seed is very

carefully inspected for indicators of contamination

with both a magnifying glass and a microscope to

determine its fitness for human consumption. The

entire sample is sprouted using commercial sprout

reduction methods, the seed is not sanitized prior

to sprouting. A sample of the runoff water is

collected using FDA procedures recommended for

commercial sprout producers. The water is then

enriched and tested for Salmonella and 0157:H7.

Everything is documented and signed by the person

doing each procedure, their times, such as drop

shipments directly from the country of origin where

sampling and testing isn\"t practical, in these

cases no documentation is provided and the sprout

87

grower needs to perform the sampling and testing

procedures prior to using the seed.

Neither this certificate nor the

procedures we perform is a guarantee that the seed

is not contaminated. It\"s merely a risk reduction

step.

We have an extra step as well. Before we

receive a shipment we bring in a sample and we

inspect it, sprout it out and test it. We stop at

the point of rejection. That is if the seed

doesn\"t pass visual inspection, we reject it

without taking the time to sprout it out.

Recently when I mentioned to a researcher

that we found pathogens four times, she was shocked

that it was only four times. There have been 13

sprout related outbreaks since the year 2000. If

these four lots had not been screened out there

would have been 17 outbreaks instead of the 13.

That\"s 31 percent more outbreaks. I think we would

find more except seed with visible contamination

never makes it past our inspection process and by

doing what we\"re doing we learn whom the safe seed

88

processors are and who to avoid so it\"s also a

supplier screening process.

For over 100 years seed has been sampled

and tested for germination, purity, hard seed,

plant pathogens, and other properties that relate

to the quality of the seed. The statistical

probabilities used to determine these percentages

is well documented and extremely accurate.

Testing seed for human pathogens can be

even far more accurate. When testing for human

pathogens we\"re not looking for percentage but for

any at all. If a single pathogen is detected the

lot is contaminated and can\"t be used for sprouting

purposes.

When it comes to seed sampling for human

pathogens, sample size and total contamination per

lot are the two factors that determine the

probability of capturing a pathogen.

There is a possibility that two or more

pathogens could be lodged on one seed. This makes

our probability charts less reliable so we base

probabilities on contaminated seeds rather than on

89

CFU. Seed while being processed does get mixed

very well, well enough for very accurate sampling

estimates.

For ease of explanation throughout the

rest of this presentation, all samples will be 25

grams and all bags will be 25 kilogram bags so the

sample size is always 1/1,000th of a bag or 1 seed

per 1,000.

Some question the reliability of sampling

because there is a possibility that pathogens can

be in a corner of the bag or in a clump and never

detected. Anything is possible but this scenario

is extremely unlikely. The seed is harvested,

transported and dumped into a silo or bins. It is

then poured or agered into the seed cleaning

equipment, processed and poured into a bag. The

cleaning and grading process doesn\"t allow even two

seeds to clump together or they won\"t fit through

the screens. Seed with pathogens are not going to

stay next to each other throughout this process.

They will be somewhat, if not thoroughly,

distributed.

90

When trying to detect plant pathogens,

similar sample sizes are used and the probabilities

are extremely high as well. When looking for plant

pathogens you are looking for frequency rather than

any at all. In order to determine the percentage

of pathogens in wheat, eight kilograms is sampled

for each 100 tons of seed. Then 300 seeds are

pulled from that eight kilograms for testing.

Distribution of the plant pathogens is good enough

that this method is very accurate. In a same lot

size we would sample and inspect the lot, then 25

million seeds would be tested in order to find a

single human pathogen.

Larger samples increase the probability of

capture at a given contamination rate. Considering

that we sample at least 1/1,000th of a seed, the

larger the lot, the larger the sample. If you have

seed that is contaminated at one seed per kilogram,

pulling one sample from one bag would only get you

a 2.5 percent chance of finding it but, if you

didn\"t find it in the first bag, you have another

shot with identical odds in the second bag. The

91

more bags there are the more 2.5 percent shot you

got at it. You get enough 2.5 shots, say 400, and

you have a 99.99 percent chance of capturing at

least one of those contaminated seeds.

But what if the pathogens are in just a

few bags? This is a problem if you\"re trying to

find a particular level of contamination but it is

not a problem if you are trying to find any at all.

What matters is the total number of pathogens in

the lot, not how many bags they\"re in.

This chart shows what happens if you

sample various size lots in which all lots have

2,000 contaminated seeds. Notice if you have one

contaminated bag, you have 2,000 contaminated seeds

and one pull will capture one of those seeds 86.5

percent of the time. If you divided those 2,000

seeds among two bags, now you have 1,000

contaminated seeds per bag but you have doubled the

number of pulls and the odds even out.

Our charts are actually based on the odds

of capturing clean seed, then we reverse the

numbers in order to predict the number of

92

contaminated seed. If you tried this with small

numbers such as a few marbles in a cup, the odds

will change as you move the marbles from cup to cup

but there are about 12.5 million seeds in a bag.

In this example 1,000 seeds is moved into another

bag with another 12.5 million seeds also. When you

move 1/100,000th of the seed from one bag to

another the number of clean seeds is virtually

unchanged so the probabilities for all practical

purposes remain the same.

This next chart shows a large lot in

which\--except in the bottom row\--is unevenly

distributed among 800 bags. The first row has all

2,000 seeds in one bag. Your odds as in the

previous slide are 86.5 percent but it doesn\"t

matter that you pulled seeds from the 799 bags that

are clean. You pulled one pull from the one bag

that was so contaminated that it gave you an 86.5

percent chance of finding one contaminated seed.

Some say the blending makes seed sampling

unreliable. Blending dilutes contaminated seed per

kilogram but it has no effect on the total number

93

of contaminated seeds and, therefore, no effect on

the probability of capture.

As an example, two bags of seed

contaminated at the rate of four contaminated seed

per kilogram has 200 contaminated seeds. If it\"s

blended in with 800 bags of non-contaminated seed

the new lot still has 200 contaminated seeds. The

probability of detection by pulling 802 samples is

18.5 percent. These are the same odds as if you

had pulled a sample from each of the two

contaminated bags before they were blended in.

However, using our seed sampling protocol

on the two bags, three kilograms would have been

pulled for testing. This increases your sample

size from 1/1,000th to 1/16th. The odds of capture

go up to 99.9994 percent. So blended lots are okay

as long as the seed is screened properly before the

lots are blended.

So why is it hit and miss when health

officials try to find pathogens in seed that they

are certain caused an outbreak? It\"s the total

number of contaminated seeds in the lot that

94

determine the probability of capture. Seed

companies can screen the seed when the entire lot

is intact. This is when the total number of

contaminated seeds is its highest. If just a

portion of that lot is contaminated, the seed in

the contaminated bags are included in the

population that\"s sampled. By the time the

epidemiologists determine that sprouts are the most

probable cause of an outbreak, a good portion of

that lot is gone and a portion, if not all, of the

contaminated seeds was used up in the outbreak.

There\"s something else that should be

pointed out. The effectiveness of seed sampling is

inversely related to the effectiveness of chlorine.

The more contaminated the seed is the less

effective seed sanitizing is. Yet the more

contaminated the seed is the easier it is to detect

a pathogen using seed sampling and testing. Seed

screening along with chlorine or other forms of

decontamination complement each other very well to

reduce the risk that Salmonella or E. coli O157:H7

is in sprouted seeds.

95

Is seed sampling too much of a burden on

the seed industry? Well, first let\"s look at what

sprout growers have to go through in order to

ensure that their sprouts produced from the seed

are safe. An ISS Rototech Rotary Drum uses 50

pounds of seed to produce one batch of sprouts.

The FDA recommends each batch of sprouts be tested

separately and that 12 pounds and 13 ounces of

chlorine be used to sanitize 50 pounds of seed.

At a \$1.55 per pound the chlorine costs

about \$20.00 to sanitize the 50 pound batch.

Runoff water is collected and sent to a lab to be

tested in duplicate for Salmonella and E. coli

O157:H7. The best price we could find shopping

around was \$25.00 for O157:H7 and \$33.00 for

Salmonella. Overnight delivery of the refrigerated

package is \$36.00. Chlorine reduces yields 10 to

30 percent. I just plugged in 10 percent into

these figures. Collection, ice pack, packaging,

shipping, labor and record keeping are at least

\$15.00. I didn\"t put in any time for management,

training, hold and release or false positives. So

96

the minimum it costs a grower who follows the FDA

guidelines is \$195.50 to sanitize and test one 50

pound batch of seed. This is \$3.91 per pound of

seed.

The seed screening by the seed company

consolidates the efforts and the finances. An

experienced sampler can sample 44,000 pounds of

alfalfa in six hours and mung in eight hours. This

is about \$120.00 in labor. The seed and bag

inspection takes a trained technician about three

hours. This is about \$45.00. The seed is about

\$60.00. Growing and disposing of the sprouts is a

couple hours and sending off the sample is \$36.00.

The FDA recommends the tests be done in duplicate

so this costs about \$66.00 for Salmonella and

\$50.00 for O157:H7 tests. Collecting the sample,

packaging it for refrigerated shipping and send it

off is about an hour. This totals \$407.00, which

in 44,000 pounds of seed comes to about a penny a

pound.

So the seed company pays \$407.00 per

44,000 pounds which is less than a penny pound and

97

the sprout growers pay \$3.91 a pound, which in

44,000 pounds comes to about \$172,000.00. That\"s

422 times what it would have cost the seed industry

to screen the seeds. With \$172,000.00 the sprout

grower should have at least got the seed, maybe

even the semi that brought it to him.

Seed screening doesn\"t do the following

things: It doesn\"t cost much. It does not produce

hazardous waste. It doesn\"t put production workers

at risk. It doesn\"t affect germination, vigor,

yield or the quality of the sprouts and it doesn\"t

reduce background flora. It doesn\"t introduce the

possibility of resistance. It doesn\"t

disenfranchise organic sprout growers and it

doesn\"t negatively affect or take away in any way

from other food safety procedures such as

decontamination.

But this is what seed sampling can do: It

can help the seed industry identify practices that

introduce pathogens into foods. It can prevent

contaminated blended lots from entering the market

if sampling is done prior to blending. It can

98

prevent seed with visible contamination from

entering the market, and this includes glass and

other things that don\"t show up on a pathogen test.

It\"s most effective when sanitizers are least

effective. And it can help identify farms and seed

processors lacking good agricultural processes. It

can be used identically to all types of seed and

its application will affect commercial sprout

growers of all sizes with all levels of

sophistication and using all types of equipment.

It can be applied evenly throughout the sprout

industry. It can be used on seed destined for home

sprouters and it may be the only realistic defense

that home sprouters have. Chlorine is not a great

option for home sprouters. It would shift some of

the responsibility of providing safe seed to the

seed industry and it would substantially reduce the

number of foodborne illnesses from sprouts.

In conclusion, I\"d just like to say that

seed needs to be screened for pathogens before it\"s

sold as sprouting seed. It just makes sense.

Thanks for listening.

99

DR. BERU: Our next speaker is Caroline

Smith DeWaal, Center for Science in the Public

Interest.

CONSUMER PERSPECTIVE

MS. DeWAAL: Good morning. My name is

Caroline Smith DeWaal and I don\"t know how many of

you were here\--was it 1998 they had one of these

meetings or \"99\--FDA had another meeting on

sprouts. Probably Bob was here but I was here as

well and I was very interested to realize during

lunch that I was eating with one of the sprout

growers from Vermont, which is my home state. At

that point I think it really hit the sprout

industry by surprise because a lot of the people

who had entered the industry were people who were

really working on producing clean safe food and

suddenly to be hit with a major food safety problem

with their product was something that I know took

the industry by surprise.

The Center for Science in the Public

Interest is a non-profit consumer advocacy

organization and we focus largely on food safety

100

and nutrition issues. We represent consumers both

in Canada and the United States and we\"re funded by

about 900,000 subscribers to our Nutrition Action

Health Letter. As I\"ve just noted, we\"ve really

been involved with the issue of sprout safety for

many years now.

CSPI applauds the Food and Drug

Administration for moving towards the creation of a

regulation to address foodborne illnesses linked to

the consumption of sprouts. The fact that the

number of foodborne illness outbreaks associated

with alfalfa and clover sprouts decreased

immediately after the two sprout guidance documents

were issued by FDA in October 1999 signalled that

those were steps in the right direction. Even at

that time, however, CSPI believed that the guidance

recommendations needed to be mandatory and

advocated for regulatory rulemaking to commence.

The continuing sprout outbreaks over the last five

years are evidence that further FDA action is

needed to ensure the sustained adoption of

effective preventive controls by the seed and

101

sprout industry.

So FDA laid out a series of questions in

preparation of this meeting. I only answer

questions I want to so these are the three that I

decided to focus on: Should the sprout guidance be

expanded or revised? Is the regulation likely to

be effective in reducing foodborne illnesses linked

to sprouts? And how can progress be measured?

Although routine microbial testing is an

essential element in sprout safety, FDA should move

towards the long-term strategy of implementing a

hazard analysis and critical control point system

for sprouts.

However, until an effective pathogen

reduction step is developed and mandated, sprouts

should be labeled as a high risk food.

A regulation is a more effective tool

towards reducing foodborne illnesses associated

with sprout consumption because it will ensure that

all sprout producers are legally required to

institute preventive control measures. It really

puts all sprout growers on a level playing field.

102

One of the sprout guidance documents that

was issued in 1999, \"Sampling and Microbial Testing

of Spent Irrigation Water During Sprout

Production,\" recommended specific techniques for

the routine testing of every production lot or

batch of sprouts for Salmonella and E. coli

O157:H7.

FDA should examine how successful this

microbial testing has been in the past five years,

both in terms of the proportion of the industry

following the recommendations and what their

results were, and I know we\"re hearing some of

those results at this meeting today.

I\"m going to talk briefly about what CSPI

brings to meetings like this. I don\"t just come as

representing consumer advocates. I think Bob

Buchanan at FDA challenged me a number of years ago

to actually bring something to the table so what we

started doing is tracking foodborne illness

outbreaks with our Outbreak Alert Database. It

right now contains about 4,500 outbreaks with both

known food and known etiology which have occurred

103

in the U.S. between 1990 and 2003. It is compiled

from multiple sources, largely especially in

recent years from the Centers for Disease Control

and Prevention, which publishes an outbreak line

listing but they don\"t organize it by food groups

so we take their data and reorganize it so we can

actually track it by the food groups but we also

take data provided by state health departments and

scientific or medical journals.

By linking the outbreaks to specific

foods, CSPI is able to alert consumers of food

safety hazards and provide better information for

food safety resource allocation.

So this is what our data\--I\"m going to go

from the broad dataset to the more narrow issue

that we\"re dealing with today. Between 1990 and

2003 FDA regulated foods were linked to nearly

3,000 outbreaks and over 83,000 cases of illnesses.

These FDA regulated foods constituted 66 percent of

the outbreaks listed in the Outbreak Alert

Database, which again you only get an outbreak

alert if we have known food and known hazard, and

104

it\"s 60 percent of the cases.

And this shows FDA has the lion\"s share of

the outbreaks and the USDA the meat and poultry

products, which we normally associate with

foodborne illnesses, actually are smaller

contributors.

So here is our trends in outbreak

reporting on produce. Produce was the food

category associated with the most foodborne

illnesses linked to outbreaks. This was a surprise

when we first started to find this. We started

publishing our data in 1999 and it took\--we had to

actually FOIA the information from the CDC because

they didn\"t want to give it to us. As you see, the

early years of the database is much less robust

because CDC actually wasn\"t\--they started

electronic reporting of outbreaks to CDC in 1998, I

believe, and so we started getting a much better

dataset. I know you\"ve heard from CDC this morning

but what you see here is produce. Both the

outbreaks and the illnesses from produce are quite

significant.

105

And here we\"ve got the top causes of

produce outbreaks from 1990 to 2003 with norovirus

at the top and the two of greatest concern to the

sprout industry is Salmonella and E. coli.

Okay. This data reflects probably what

you\"ve already seen this morning from CDC that the

principal pathogens of concern here are E. coli,

not just O157:H7 though, there are other types of

pathogenic E. coli strains, and then a large

variety of Salmonella strains.

Okay. I know we\"ve talked a little bit

this morning about this outbreak. This was a 2001

outbreak. In our last meeting actually we looked

at a number of outbreaks and did these kind of

profiles and they were quite interesting. This one

is a little bit different. It was 32 cases of

illness, which is relatively small. Still

significant. It was focused in California,

Arizona, Colorado and New Mexico. And three of the

cases involved or required hospitalization so we

had some high risk individuals who ended up getting

very sick in this outbreak.

106

What was interesting is that it found that

there was a single seed source, a single sprouter

and a single seedlot were associated with the

illnesses but the seeds had been imported from

Australia and had undergone, as we\"ve discussed

earlier, multiple decontamination steps, both heat

and low dose hypochlorite solution.

Now I use this outbreak to illustrate a

couple of points that sprout producers often are

not the source of contamination. Contamination

frequently appears to occur earlier in the

production chain. That seeds intended for human

consumption must be protected from exposure to

manure, animals and contaminated water; that

contamination decontamination treatments need to be

routinely and systematically monitored for

effectiveness; and finally that production

conditions in other countries need to be evaluated

prior to seed import to ensure that the growing

conditions are suitable for seeds intended for

human consumption.

Well, consumers always have an important

107

role to play when it comes to preventing food

safety problems. In this area we can\"t rely on

consumers to make unsafe sprouts safe to eat. We

can\"t ask them to cook their sprouts. I mean

sprouts are natural and fresh and you want to eat

them raw. They were intended to be eaten raw.

It\"s unreasonable to ask people to soak them in

chlorine, for example, once they get them home and

probably wouldn\"t be very good for them anyway so

the onus is on you to really deliver to us a safe

product.

If sprout safety cannot be ensured then

the industry should use labels to alert high risk

consumers to avoid the product.

We recommend that alfalfa seeds should not

be used unless they\"ve been produced under

conditions which are suitable for human

consumption; that FDA should ban the use of mixed

batches to aid in traceback; the FDA should

encourage the development of safe and natural

decontamination methods; the government should

provide greater oversight to the sprout industry;

108

and that consumer warning labels should be required

until effective controls are identified and fully

implemented.

I know this isn\"t what you want to hear or

at least some of it.

So let\"s talk about it. Let\"s talk about

a couple of these recommendations. The practice of

using seeds that have been grown for agricultural

use should stop. I know that this is a radical

suggestion. I know that implementing this

suggestion would have wide implications for the

entire industry. However, the outbreak data

clearly illustrate the contaminated seed are the

primary cause of sprout outbreaks. While farmers

can safely use manure on alfalfa grown for

agricultural purposes it should be strictly banned

in the growth of seeds intended for human

consumption. Farms that supply seeds to sprout

growers should observe strict guidelines for the

growth of these seeds and should exclusively root

their seeds towards the production of human food.

In addition, the practice of growing

109

sprouts from seeds imported from other regions of

the world should stop unless it has been

demonstrated that the seeds have been produced

under similar strict guidelines.

The practice of mixed batches of sprout

seeds makes traceback nearly impossible. Alfalfa

seeds intended for human consumption should be

maintained in intact batches that are carried

through the food production chain from the farm to

the table. The batches of seeds and packages of

sprouts should be clearly labeled or tagged for

ready identification during a recall or traceback.

This will help assure that problem seeds and

sprouts are readily identified and may be quickly

removed from the market.

The methods currently in use, calcium

hypochlorite and chlorine, are primarily reduction

steps and not elimination steps. While these

methods may eliminate some of the harmful

bacterias, others may survive treatment. We

recommend that all decontamination methods should

be challenge tested with seeds positive for E. coli

110

0157:H7 which may be more resistant to some

treatments than other pathogens. Both FDA and the

seed and sprout industry should provide clear and

accurate information to consumers on the

effectiveness of each of the various

decontamination treatment methods.

We think the government should require

that all sprout growers be classified as food

handlers; that unsanitary conditions in the

processing plants can lead to contamination of the

seeds or sprouts, particularly because sprouts are

grown in a warm moist environment which facilitates

bacteria growth along with sprout production.

CSPI recommends regular inspections of

sprout processors by both state and federal food

safety inspectors. In addition, CSPI believes that

implementing a HACCP system for the sprout industry

would be an effective tool and should be

considered.

Although there is no pasteurization step

currently available for sprout processors that

would leave the product fit to eat in its natural

111

form, there are possible barriers or multiple

hurdles that could be incorporated into a HACCP

system.

In response to the foodborne illness

outbreaks associated with sprouts, FDA has issued a

number of consumer advisories over the past few

years so let me tell you where that leaves

consumers. We know that\"s not what\--when it

happens your sales go down dramatically but where

it leaves consumers is this: This relies on

consumers\" memories of what they might have read or

heard in the news to protect themselves. This is a

far less effective technique for transmitting

consumer information than a label on the container.

We believe that labels on sprout containers should

alert consumers that the product may not be safe to

serve to consumers, immunocompromised or elderly

consumers or children.

In previous years FDA has implemented

mandatory labels for several high risk foods,

including unpasteurized apple cider, that pose a

comparable risk to sprouts.

112

So, in conclusion, CSPI urges FDA to move

quickly and expeditiously to start the notice and

comment rulemaking process for sprout safety

regulations. There have been over two dozen

outbreaks linked to sprouts in the past ten years,

representing thousands of consumers who became ill.

Until effective controls are identified and fully

implemented, we believe sprouts should be

prominently labeled to alert consumers to the risk

of eating sprouts.

I want to make one final note. I was very

impressed with the last presentation on actually

putting the burden on the people who distribute the

seeds to actually test them for the safety and it

reminded me of practices in the ground beef

industry. When I was working on the Jack in the

Box outbreak back in the mid 1990s the beef

industry said, \"Oh, we can\"t test the ground beef.

We can\"t test every lot for E. coli 0157:H7. That

would be a terrible burden and it wouldn\"t be

effective.\"

Today many companies in the ground beef

113

industry are testing every lot. They are using

test and hold programs and they are accepting that

responsibility. I thought the presentation that

showed that the people distributing the seeds to

you would be willing to accept that responsibility

by actually sampling the lots is a very important

statement about where we need to be going on this.

Thank you.

QUESTIONS AND ANSWERS

DR. BERU: Thank you, Ms. DeWaal.

I would like now to invite the panelists

who have spoken thus far to the table for a

question and answer session.

If you do have questions, please step up

to the microphones as we are recording this

meeting. The transcript, as I said, will be

available in about four weeks time.

MR. WARRINER: My name is Keith Warriner

from the University of Guelph. This is a question

for Bob R. You mentioned in your talk about

preventing four cases through spent irrigation

water testing. Well, there was two cases where

114

they did do all the testing but they did deliver

contaminated product to the marketplace. So is

this application of no sanitation and just depend

on testing really going to be effective?

MR. RUST: I\"m sorry. I didn\"t understand

the question.

MR. WARRINER: All right. So we\"re

talking about just testing rather than sanitation

and trying to put greater reliance on the testing

of seed batches to determine contamination of that

batch and you said that saved about four cases of

outbreak cases but there was two, wasn\"t there,

that they did all the testing but they still

delivered contaminated product to the marketplace.

MR. RUST: Yes.

MR. WARRINER: So do you think your

application of just relying on testing will really

be adequate?

MR. RUST: Relying on seed sampling and

testing, no, it won\"t be adequate. It\"s just one

step in seed safety, a reduction step.

DR. SMITH: If I can say something, I

115

think the point that you just made is that

screening the seed is an additional step above and

beyond what we specifically laid out in our

guidance and in the instances where contamination

is heavy enough that you can catch it at that

point, you keep that seed out of the food supply

and you avoid some of the outbreaks.

Something else I was thinking of just

before we came to the table, the question earlier

this morning about seemingly mixed messages from

CDC and FDA about the relative importance of seed

disinfection treatment versus testing, I think

there are a lot of examples where the outbreak

investigation shows that seed disinfection had an

impact, either on implicated lots used at

facilities where seed disinfection programs were

not causing illnesses, things like that, or lower

numbers of illnesses at least from firms.

But when it comes to testing, the testing

of the spent irrigation water at the facility, we

have fewer anecdotes from our investigations

possibly because the testing caught contaminated

116

product and avoided the outbreak.

On the other hand, even if you\"re

testing\--if you\"re testing the wrong thing or doing

it the wrong way, you may have a false negative

that lets product through and results in an

outbreak. It may be well intentioned but still an

error in the application of the test itself or an

error in the way that the results from the test are

interpreted, and we have a couple of examples of

situations where that has happened.

MR. WARRINER: Yes. I\"m pretty amazed

that with all this testing going on nobody can

actually give a figure about how effective testing

is and I think people would buy into it more if

they knew it was working.

DR. SMITH: It would be nice to know who

is testing, how often and how often they find a

positive and what they do with product. People are

probably afraid to tell us but we\"d love to know.

MR. WARRINER: Definitely. Thank you.

DR. BERU: Are there any other questions?

Yes, please?

117

MR. LALLEY: Good morning. I\"m Mike

Lalley from Living Foods and a couple of quick

questions. One for Ms. DeWaal. You mentioned in

your presentation that there were 4,500 cases

identified specifically with various food products.

Of which I see a whole 27 have been related to the

various sprout products.

By my poor mathematics, I come out to some

place just less than a half of one percent of all

of these particular issues that you\"ve raised which

are of concern to all of us are related to sprouts.

That seems to me to be a fairly low number and not

one to be ignored obviously but, that being said,

we\"re all well aware of the green onion problem in

the Pennsylvania and Georgia, I believe, where we

had dead bodies littering the streets.

Are we labeling\--is your organization

proposing that we label each package of green

onions with a warning label? It has been shown

that apples which have been defecated upon by birds

are absolutely impossible to decontaminate.

There\"s just no way.

118

Now maybe we should suggest to the public

let us quit consuming raw apples, cook your apples

prior to eating. And these are just the items that

have been identified.

Like I say, if the bird defecates on the

apple, we don\"t have a multi-state situation. He

may go over on to another tree and his cousin and

his brothers, they may be defecating on other

apples and so we may have numerous people ill but

not able to traceback.

So it just seems to me that we\"re a little

over anxious to attribute these terrible situations

to something that is\--to an industry that is in a

position that\"s very difficult to defend ourselves.

DR. BERU: Let\"s give Ms. DeWaal a chance

to respond.

MR. LALLEY: A quick response. I\"d like

to have one other question.

MS. DeWAAL: Thank you very much. I

understand the challenge for the industry of

addressing the problem just because of the

production issues but by my calculation it\"s\--at

119

least in our database it\"s about 29 outbreaks. And

the bottom line is there\--it\"s serious.

If people get E. coli 0157:H7 it can

result in hemolytic uremic syndrome. People

exposed to Salmonella have other serious

complications. So these problems can be addressed

and other industries have faced them.

Now unpasteurized apple cider\--I mean FDA

held meetings just like that to address that issue.

MR. LALLEY: And that contamination came

from\--the unpasteurized apple cider, the

contamination came from apples?

MS. DeWAAL: It\"s apples.

MR. LALLEY: Maybe we should\--are we

warning on apples? I mean, if, in fact, if we drew

some multitude of apples and are able to put E.

coli into the finished product\--well, then it\"s a

no brainer that the apples are contaminated. If

the apples are contaminated at a level to which we

can no longer consume fresh apple cider without

having a warning in our faces, where is the\--maybe

then if I juice my sprouts\--I mean, we\"re\--

120

MS. DeWAAL: This is a process\--

MR. LALLEY: I mean, there\"s just no\--yes,

but you\"re ignoring\--I mean, just\--I mean, is there

any\--well, just answer the question then.

Is there any specific desire on your

organization or FDA or anybody else to warn

consumers of the consumption of raw produce,

fruits, vegetables, et cetera, which we all know

are in a position\--are grown in a warm\--you know,

we have talked about the warm, moist environment in

the sprout operations.

I also do a little dirt production myself

of culinary earth, which just so happens that it\"s

a warm, moist environment that produces all of the

raw fruits and vegetables that we consume. It\"s

not only in our sprout environments so it just

seems to me that we\"re focusing an inadequate or an

over abundant amount of attention on this

particular group.

MS. DeWAAL: Well, as you saw in my

presentation, I did give you the data on all

produce because we do focus our efforts there but

121

more importantly, I think to your question, is that

you are producing a processed food product and the

processing may be something simple and natural that

can be done in my home kitchen but nonetheless if

you bring your products to the market consumers

have an expectation of safety that you need to

address and these outbreaks demonstrate that the

processing needs to be improved.

MR. LALLEY: Absolutely and we\"re in total

agreement but the question is, is there any desire

on your group or the FDA to take this warning label

and apply it to all items that may fall into this

type of a situation? Items that are consumed raw

primarily, fruits and vegetables. Do you have

the\--does your organization have any of that in

process?

MS. DeWAAL: We supported the use of

labels also on another raw process food.

MR. LALLEY: Green onions?

MS. DeWAAL: Excuse me. No.

Raw\--unpasteurized apple cider.

MR. LALLEY: How about green onions? We

122

had dead bodies littering the streets. I mean,

no\--I mean, it\"s\--like I say, by virtue of the fact

that I\"m strong and my position in no way reflects

the fact that I disbelieve that there\"s problems,

potential problems on any food product, with that

being said why the inordinate amount of attention

in the complete looking the other way on these

other products? I mean, it just seems absolutely

ridiculous.

DR. BERU: I think that question has been

asked and\--

MR. LALLEY: And the answer was we are or

we are not considering warning labels on other

fruits and vegetables? Yes or no?

DR. BERU: I can tell you the\--

MR. LALLEY: It\"s a yes or no question.

DR. BERU: \--FDA is not considering

labeling of all produce as you\"re laying that out.

Are there any questions?

While we wait for questions, I had a

question for Dr. Dechet actually. In your

presentation you mentioned that the number of

123

illnesses per outbreak average over the years seems

to have gone down. Can you tell us what you

attribute that to?

DR. DECHET: Certainly one possibility

might be that the guidelines are being followed and

that has had some effect. I think we probably

think it\"s more due to the fact that awareness has

been raised about outbreaks throughout the states.

The Department of Health and Human Services now has

field epidemiologists in all states, EIS officers

in almost all states, so foodborne outbreaks are

very rapidly investigated.

We also have improved our gel

electrophoresis, our DNA fingerprinting program.

It\"s now every\--and this actually touches on one of

the questions that was raised just a minute ago,

you know, are we testing other food products and

how do we know that when Salmonella is due to one

product or another. Depending on the state, for

instance, every tenth sample of Salmonella gets DNA

fingerprinted and those results are sent to the

CDC, and it goes into our national database and

124

then our database staff will compare those isolates

all across the country.

So I think now we\"re able to see that

something in California, oh, look, it\"s the same

exact same DNA fingerprinting as something in

Arkansas, as something in Massachusetts, and then

those states are brought together and then they

actually do an investigation.

We\"ve also included some of the high risk

food items, green onions, unpasteurized cider,

sprouts, on our general\--we call our shotgun

questionnaire when we can\"t find a food item. It

involves over 400 food items and we\"ll start

looking for some of those high risk food items. So

I think it\"s greater awareness. I think it\"s

earlier detection and rapid investigation that

probably has actually reduced the numbers per

outbreak.

DR. BERU: Panelists, feel free to ask

each other questions as well.

Yes?

MR. SANDERSON: This is a question for

125

Caroline. I think all of us agree with you

completely that good agricultural practices for

seed production is absolutely common sense but it\"s

really kind of difficult when you try to figure out

how to bring it about. I wonder if you know of any

industry where good agricultural practices went

from being a good idea to being a regulation that

was consistently followed and how that was done

because maybe we could take some pointers from

that.

MS. DeWAAL: Bob, that\"s a great question

and I\"d be happy if FDA also looked at that issue.

One of the challenges here, and I do work on food

safety issues across both FDA and USDA issues

really from farm to table, but one of the

challenges here is that we don\"t really regulate

farm production and so we are relying on good

agricultural practices at the seed production level

as we do for green onions, as we do for cattle, as

we do for many other items so those are voluntary

standards. So what it means for the sprout growers

as an industry is that you\"re really relying on

126

your source\--on your source material and that\"s the

seed.

I think that we don\"t regulate\--we don\"t

have an agency you can go to that regulates on the

farm. You don\"t have anyone you can go to and say

could you help ensure that the seeds that I\"m being

sold are adequate.

So I think you have to rely on some of the

programs we\"ve heard about today that give you some

level of assurance but, no, I have not seen an on

farm program that\--where good agricultural

practices work. Now, that said, one\--or has moved

to a voluntary program that has been highly

functioning.

Now the exception there\--there is always

an exception\--is eggs. And in Pennsylvania they

developed\--they had a major problem with Salmonella

enteritidis in the flocks and they did develop on

farm practices that showed themselves to be highly

effective at controlling SE in eggs and that is

slowly moving towards a regulation now that will

provide a level playing field for the egg industry.

127

So again it\"s harnessing kind of the kind

of programs that are being field-tested and then

harnessing those into good regulations. That\"s

really where we are. I mean, the sprout issue

isn\"t new. We\"ve been looking at it since the mid

1990s and this is at least the second major meeting

I\"ve attended on this so this is not a new issue.

FDA has not moved quickly to consider regulations

but with the outbreak data today it\"s clear we need

more standards in place.

DR. SMITH: If I can add to that just

briefly. You\"re right that good agricultural

practices are guidance and we don\"t have the

authority to enforce guidance, and that\"s not what

guidance was about but we do have authority over

all FDA regulated foods, including the farm. So we

do\--we are able to get there but we don\"t have the

implementing regulations to lay out what\"s right

and wrong. It has to be an obvious call.

MS. DeWAAL: You can get there after an

outbreak. You cannot implement regulations on the

farm before an outbreak. I mean, you get to the

128

farm after you\--

DR. SMITH: Well, we don\"t have the

implementing regulations and with the exception of

a couple of invited friendly visits we don\"t tend

to go to the farm very often unless there is cause

that brings us.

DR. BERU: Are there any questions?

Yes, please? Again, name and affiliation,

please.

MR. BOYD: My name is Steve Boyd and I\"m

with a company called Hydrose. We do molecular

diagnostics of diseases and plants.

I submit relative to Bob\"s question, Bob

Sanderson, that one model that you might take a

look at is the international seed testing program

that has been slowly developing over the years. In

this case their focus has been primarily on

diseases of plants relative to the plants. They

have no human interest per se but the process and

how they\"ve gone about pulling together other

nations as well as coming up with standardized test

procedures. One of the bigger problems with this

129

whole group is standardized test procedures and

there are a number of different people. In fact,

you just attended one of the meetings recently in

Thailand.

I mean, this is a group that has been

doing this sort of testing. They\"re well aware of

the statistical issues. Their focus is on the

quality of seed both sold into the United States

and globally. The question is what the pathogen is

relative to us so I submit that there is one

organization that if you look at a pathway it might

be of some use.

SCIENTIFIC PERSPECTIVE

DR. BERU: Thank you. We are really

running behind time and so I will stop the

questions and answers at this time and move on to

the next presentation. Thank you, panelists.

We\"ll have one of the science

presentations before we break for lunch and I would

like to invite Dr. Jed Fahey from the Johns Hopkins

School of Medicine to make his presentation on

Microbiological Findings on Sprout Operations

130

Following FDA Guidance.

MICROBIOLOGICAL FINDINGS ON SPROUT

OPERATIONS FOLLOWING FDA GUIDANCE

DR. FAHEY: Good morning. Can you hear

me?

I\"d like to spend the next 15 minutes or

so getting you ready for lunch and talking about

food safety problems of course.

I\"ve got an asterisks next to my name

because I\"ve got my e-mail address there. If there

are slides that you have\--that you\"d like copies of

or if you have questions, I\"d be happy to provide

them to you later.

I didn\"t hear the first couple of

presentations. I apologize for that. I hope I\"m

not re-hashing anything that was already said but

I\"d like to present some data that we developed

actually four years now on hold and release testing

of sprouts, a variety of green sprouts. And we, as

with most of you, were too busy and too overworked

and we just haven\"t had time to try to get

this\--make a serious effort to get this published.

131

We should do that and will do that, especially if

there\"s interest in the data here. So the data has

been around for a few years. We haven\"t been

holding out. We\"ve just been\--just haven\"t gotten

to it.

So by way of overview, I\"d like to tell

you about a one year program of hold and release

testing conducted in concert with strict seed and

facility cleaning procedures by 13 U.S. broccoli

sprout growers. These tests were not all done on

broccoli sprouts but my colleagues and I developed

broccoli sprouts and, in fact, started a company to

promote chemoprotective food products and the

company sells broccoli sprouts. I\"ll show you a

formal disclosure or disclaimer in a second so we

obviously had an interest in working with those

folks that did grow broccoli sprouts.

The microbial contamination tests were

performed on almost 7,000 drums of sprouts,

equivalent to about 5 million consumer packages of

fresh green sprouts, and less than one percent of

those sprout samples gave an initial positive test

132

for E. coli 0157:H7 or for Salmonella. And, when

retested, only three drums again tested positive

and obviously they were destroyed and did not make

it to the marketplace. I\"ll go through some of the

details in a minute.

We also looked at composite testing and

although this is not a commercial message for

composite drum testing, it seemed to be every bit

as good as testing individual drums.

By way of disclosure, we started a company

to sell broccoli sprouts, Braccisca Protection

Products. I\"m an unpaid consultant to the company

now and my stock is under lock down by Johns

Hopkins, who by the way also owns stock in the

company. But clearly we\"ve been working with

broccoli sprouts, we\"ve been working with broccoli

and cruciferous vegetables and have great interest

in their thriving as well as the interest of the

sprout\--all of the green sprouts.

So I present to you just a couple of

slides specifically about broccoli sprouts and then

I won\"t say anything more about them just to remind

133

you that there is considerable or maybe I should

just say significant antibacterial activity in

seeds and sprouts of cruciferous vegetables, which

may indicate that there is some advantage to

growing them in terms of microbial safety. I don\"t

know. But after only a short amount of time when

we measure the antibacterial activity against a lab

strain of E. coli, not the nastier one, we find a

marked impact on bacterial growth from that

leachate or I\"ve called it leachate but it\"s

essentially the rinse water.

And also, and this is published\--the

previous slide wasn\"t but we published a couple of

years ago in the Proceedings of the National

Academy of Sciences there is tremendously potent

antibacterial activity of sulforaphane, one of the

major phytochemical components of broccoli and some

other crucifers against helicobacter pylori, which

is the causative agent of many, if not most, ulcers

and is a risk factor for stomach cancer.

The contrast there between the top four

bars and the bottom one is just to show that

134

there\"s actually two or three logs of difference in

the potency of this particular phytochemical from

broccoli seeds against helicobacter pylori, a human

pathogen, compared to a number of other

environmental bacteria and fungi so I just mention

that in passing. I\"m not going to present any more

data on that but there may be some additional oomph

or punch to be attributed to the cruciferous seeds

and sprouts from them.

So again this may be\--I\"ve heard warm

moist environment a couple of times since I walked

in already. Sprouts, as you know, are seeds that

have been placed in environmentally controlled

hydroponic conditions. They\"re incubated in a warm

moist nutrient rich environment made nutrient rich

by the leachate from the seed itself and they are

ideal environments for microbial growth so that if

E. coli or Salmonella is present it\"s going to

multiply. It\"s almost a guarantee.

So to date I\"m not aware of, and we may

hear data later but I\"m not aware that practical

methods have been developed to check the growth of

135

these contaminants during germination and sprout

growth or processing so you\"ve got to prevent

contaminants from entering the process or if you

find that the sprouts are contaminated obviously

they\"ve got to be destroyed but first you\"ve got to

identify them.

It\"s essential in my mind and our mind in

my group at Johns Hopkins that seed that\"s going to

be used for sprouting undergo some sort of surface

disinfestation or disinfection treatment using a

biocide of some sort. The efficacy of these

agents\--in particular, calcium hypochlorite\--has

been, I think, very well documented by the

laboratories of Dr. Beuchat and others, many

others. When these agents are used correctly the

resulting sprouts are safe to eat and so as you

know and I\"ve seen in earlier presentations there

is a guidance that the FDA issued six years ago on

how to accomplish this surface disinfestation.

So we\--well, this has been said before but

the guidance then indicates that testing the spent

irrigation water is the way to ascertain whether

136

seedlots are contaminated or sprout lots.

The FDA received considerable guidance or

considerable\--not guidance but input from the

sprouting industry and it is continuing to do so

today obviously designed to prevent contaminated

sprouts from ever reaching the public. The FDA, as

far as I\"m aware, followed up the issuance of this

guidance with an inspection of 150 or so sprouters

and determined that only about half of them were

complying with the guidance.

So what we\"ve done is examine a subset of

these sprouters in whose facility compliance with

the FDA regulations was verified based upon

inspections by two third party auditors.

This hold and release testing obviously

was done\--obviously based on my disclaimer or

disclosure\--with co-packers who agreed to

participate. Co-packers for the broccosprouts

product line and Brassica Protection Products. It

was done during the calendar year, the entirety of

the calendar year 2001, and there were 13

producers. These companies were all established

137

companies. They hadn\"t just sprung up to grow

broccoli sprouts. They grew and continue to grow

many different kinds of green sprouts and they had

agreed to comply with Brassica Protection Products\"

rigid standards of sanitation. We selected the

plants so that they were geographically distributed

across the U.S.

All of the surveyed growers followed all

of the steps specified in 1999 FDA guidance. They

surface disinfested or disinfected\--I apologize. I

use surface disfestation \[sic\] and disinfection

interchangeably. I\"m sure there are subtle

differences but they are unknown to me. We\--they

rather performed microbial testing of the spent

irrigation water from each batch of green sprouts

and they were subject to, and those of them that

are still working with BPP are still, of course,

subject to both announced and unannounced third

party audits as well as state and federal

inspections of their procedures.

The routine was exposure to 20 parts per

million calcium hypochlorite for 15 minutes

138

followed by extensive rinsing to remove residual

chlorine. They were grown in either trays or drums

and, of course, the only three inputs are light,

heat or constant temperature at least and clean

water. And in the case of the 13 sprouters we

examined they used one of three sources of water.

It was either filtered. It was well water or it

was chlorinated municipal water.

So a sample of the irrigation water,

typically one liter, was collected after two days

of growth of the sprouts. Since they are typically

grown for 72 to 120 hours, the rapid microbial

test, therefore, permits the sprouters to abort

contaminated batches either before packaging or

certainly before shipping and distribution. And in

the cases of these sprouters all of them were held

until the test results were obtained.

The data was not forwarded to me. I\"m not

particularly good at handling large masses of other

people\"s paperwork. I have enough problems with my

own. They were forwarded to a company who

specializes in quality assurance measurement,

139

Quality Associates, Incorporated, in Columbia. And

they also reported some other data, including the

types, the other types of green sprouts that they

were growing, which is listed in the second bullet

there. Some of them actually included wash water

or irrigation water from some non-green sprouts,

some mung or soy bean lots. And to the extent that

that data was combined\--in pooled drum samples\--to

the extent that that data was combined with green

sprout data, we included it. To the extent that it

was exclusively bean sprout, we did not include it.

All but one of the growers used the

services of an external micro-testing company to

which samples could be delivered on the day of

collection so these were obviously in their

geographic area.

One of the growers had set up an in-house

laboratory and we insisted that if we were going to

use their data that we receive all the laboratory

notes from their in-house micro lab.

All of the samples were screened for E.

coli O157:H7 and for Salmonella species. Most of

140

the E. coli testing\--you know, you can\"t force

people to use your favorite micro test certainly in

this kind of a voluntary data submission so most of

those testing for E. coli used a test kit that had

been identified in the guidance. Not most of them

or only a small number of them used such test kits

for Salmonella. They used other tests in other

words. Probably motivated by cost of the tests.

Most of the tests were AOAC official

methods. Most of the kits rather. And I\"m not

even familiar\--I\"m not a microbiologist by

training. I\"m not even familiar with some of the

subtle distinctions here. One was an AOAC

performance tested method. One was an FDA

bacterial analytical method for Salmonella testing

and a couple of them didn\"t specify the types of

tests that they used.

All of the results, however, were entered

in the database and the data entries were

independently verified by an audit process at

Quality\--at QAI.

So I\"ve got a list here which is somewhat

141

hard to read but again I\"ll be happy to e-mail this

table to anybody who wants it. This is a list of

the different test methods that were used along

with an indication of their reported sensitivity

and specificity. Some of them, of course, or I

guess all of them were developed for other products

or the sensitivity and specificity rather was

assessed on other products like milk, apple cider,

orange juice and then some vegetables and poultry.

So the distribution of sampling then

included a majority of single drum samples but as

you can see about a third of the tests were

composite tests on two to four drums done because

of the cost. That was, I heard, mentioned a couple

of times earlier. It is an expensive process and a

number of growers are concerned that they be able

to use pooled drum samples and then my

understanding is if there\"s a positive or a

presumptive positive in a pooled drum sample they

would go back and retest individual drums.

So the results that we found were that

there was again less than one percent presumptive

142

positives, a total of 24 presumptive positives

across all of these tests, no apparent skewing

towards multiple drum tests or single drum tests.

The total number of drums tested was 6,839 and of

those 24 presumptive positives, as I mentioned

earlier, when retested only three gave positive

tests on retest and those lots of sprouts were

destroyed and not sold.

So in most but not all cases the

presumptive positive\--the water sample from the

presumptive positive was reanalyzed. In two of the

cases the follow-up analysis was again positive for

Salmonella and the sprouts were destroyed and in a

single case there was a presumptive detection of E.

coli. It was assumed by the grower to be real and

he or she destroyed the sprouts without a follow-up

test to confirm whether this was, in fact, E. coli

0157:H7.

None of the samples included broccoli

sprouts as a parenthetic statement and all of the

sprouts had been held at growing facilities until

the microbial results were back so that there were

143

no instances in which contaminated sprouts were

released for distribution.

So we feel\--as far as I\"m aware this is

the largest collection of such data that\"s at least

been shared publicly. We\"re\--we at Hopkins since

we developed sprouts and we\"re concerned about and

continue to be concerned about food safety, we were

very eager to do this test and happy to have the

cooperation and collaboration of the sprouters who

did join us.

I think this is a kind of testing program

to be proud of and not to hide. My only apology is

that we have had this data for a while and just

haven\"t sort of gotten it out there. It has just

been a case of entropy, I guess, but I\"m glad to

share it with you now.

This work was supported by a philanthropic

donor to our lab, Lewis B. and Dorothy Cullman

Foundation. And I want to acknowledge the

contributions of a number of colleagues. In

particular, Phil Ourisson at QAI, the Quality

Associates, and Kitty Stevenson, who works with me.

144

Again thanks to the sprout companies. I don\"t know

if any of you are here who agreed to be audited and

share their records and sort of show us their

petticoats.

That\"s the end of my presentation.

DR. BERU: Thank you, Dr. Fahey.

We are going to hold questions until all

of the science presentations have been done. It\"s

now 12:00 o\"clock. We\"ll take a lunch break and

we\"ll start promptly at 1:00 o\"clock.

Thank you.

\[Whereupon, at 12:00 p.m., a lunch break

was taken.\]

\- - -

145

A F T E R N O O N P R O C E E D I N G S

\[1:00 p.m.\]

DR. BERU: Good afternoon and welcome back

from lunch.

We will continue the science presentations

and our next speaker is going to be Dr. William

Fett. He is from the Eastern Regional Research

Center at the USDA ARS and his presentation will be

on interventions.

INTERVENTIONS

DR. FETT: Good afternoon. I know it\"s

good snooze time right now so, hopefully, we can

keep you awake throughout the afternoon with all

the presentations.

My presentation is actually joint with Dr.

Rajkowski, who is going to cut me off after the

first 20 minutes.

What I\"m going to do is give you a very

brief overview in the 20 minutes on the

interventions for use on sprout seed that have been

published. I\"ll only be talking about publishing

information in my part of the talk and Kathleen

146

will be talking a little bit about unpublished

information with her work.

So I\"ll start out on interventions for use

on sprout seed and then if time permits talk a

little bit about interventions during sprouting and

postharvest. There is actually less information on

those two areas and then Kathleen will finish up

with a brief presentation of her ongoing

collaborative study with industry.

So we already heard about the current

guidance and that seed should be treated with one

or more approved antimicrobial treatments such as

the 20,000 ppm calcium hypochlorite and that at

least one treatment should be applied immediately

before sprouting.

So the challenge is to obtain a five log

reduction of human pathogens on sprout seed while

maintaining seed germination, vigor, sprout

appearance and yield at acceptable commercial

levels.

Really what we ultimately would like to

achieve is elimination of all pathogens on the seed

147

due to their potential for outgrowth during the

sprouting process that you\"ve already heard about

in a couple of presentations this morning.

So I thought this was an interesting

graphic. I just pulled out the papers that I had

in my file on interventions for application to

seeds or sprouts or during the sprouting process.

As you can see, after the outbreaks began in \"95

and sprout safety became a much more visible arena

in food safety, you can see that initially \"96-98

there was little in the way of published

information but during that time I think it was

obvious that people were thinking about the

problem, submitting proposals, proposals were

becoming funded and the research was getting

underway. You can see the explosion of papers

until 2003 that have been published in this area.

There was a little bit of a drop off in

2004 and I expect for this year there has been two

papers published so far that I\"m aware of and we\"ll

probably end up with around seven or eight papers.

And, you know, research is continuing in this area

148

and you obtained a couple handouts this morning

concerning unpublished information on potential

interventions so it\"s still an active area of

research. We probably won\"t see the number of

papers that we saw in 2003 again but in the produce

arena it\"s probably the most active area of

research for a single produce item.

So there has been a lot of work done on

chemical interventions on seed primarily with

aqueous sanitizers and they are listed here. They

have been tested at room temperature or at elevated

temperatures, 42 to 50 degrees Centigrade, and with

or without the presence of surfactants up to about

one percent.

If you look over the literature the

elevated temperature during treatment was usually

beneficial but the problem was that, even though

you got a little bit higher log kill, the seed

germination was reduced often to an unacceptable

level.

The effect of the addition of surfactants

has been variable. Even with one percent Tween 80,

149

the most increase that is in the literature is

about one log and often less than that.

There has also been a variety of gas phase

treatments looked at for sprout seed and those are

listed here from acetic acid vapor down to ammonia.

In addition there has been physical

methods tested for interventions on sprout seed,

including dry heat, hot water, irradiation, both

gamma and pulsed UV, hydrostatic pressure and radio

frequency dielectric heating.

And, also, there has been biological

intervention work done on a more limited scale than

chemical or physical interventions. There has been

a little bit of work on the use of lactic acid

bacteria as an antagonist to Salmonella and E. coli

0157\. And, also, there has been some work at our

center on the use of fluorescent pseudomonades as

biological agents or competitive exclusion agents.

And, also, some work on whole bacterial

communities, isolated both from laboratory grown

sprouts and also market purchased sprouts.

And, finally, there was one paper out

150

there and at least two groups interested in the

application of bacteriophage for controlling human

pathogens or eliminating human pathogens on seed.

There has also been a limited amount of

work on combination treatments for sanitizing seed,

both combination of aqueous chemicals such as

lactic acid followed by 2,000 parts per million of

chlorine, combination of physical treatments with

aqueous treatments such as ultrasound and heat in

combination with calcium hydroxide Tween 80,

Tsunami 200 and Fit. The last two are commercial

products. I\"m not sure if Fit is still available

in the marketplace. Finally, a combination of

physical treatments such as a dry heat step

followed by gamma irradiation.

So just briefly to examine what has been

found with chlorine and high levels of chlorine.

Usually 16 to 20,000 ppms has been studied in

several laboratories. There are many papers on

this subject. First of all, the data that has been

obtained has been highly variable as far as actual

log reductions that have been reported ranging from

151

two logs to about seven log reduction. This is

probably due in great part to differences in

methodology that have been used in different

laboratories for this kind of research and some are

listed here but there are several others.

I think this makes it difficult to assess

alternative treatments unless the alternative

treatment is done in the same laboratory with the

same set of conditions and along with the high

level of chlorine treatment so you can directly

compare.

The one consistent finding is that this

treatment does not consistently eliminate pathogens

from inoculated seed in the laboratory. As I

mentioned, if we\"re going to compare alternative

chemical treatments it would be best to run 20,000

ppms chlorine in those experiments to get a direct

comparison using the methodology that you\"re using

in your particular laboratory.

Little has been done with chlorine

treatments on naturally contaminated seed. Trevor

Suslow\"s group and my group have both reported that

152

the use of high levels of chlorine with or without

buffering to a neutral pH did eliminate Salmonella

from two naturally contaminated seedlots that we

looked at.

In Dr. Tortorello\"s lab in Chicago they

found that similar treatment with actually one of

the same lots of naturally contaminated seed was

not effective in eliminating Salmonella. There

were some differences in methodology that the three

groups used that could lead to such a difference in

the results.

It also could be just that the particular

group of seed that were used, even though it was

from the same lot, might have had contaminates

located in different locations on the seed where

she might have seed with contaminates that were

more difficult to reach with a chemical aqueous

sanitizer than the seed that we looked at.

So what are some potential alternatives to

high levels of chlorine for treating alfalfa seed

that had been mentioned in the literature? And

there are really not that many that have been put

153

forth as alternatives. Larry Beuchat\"s lab, he has

proposed calcium hydroxide at one percent, ten

minute treatment at room temperature as an

alternative and also the commercial product Fit

used 15 to 30 minutes at room temperature. In our

lab we have looked at a couple of commercial

products, Citrex and Pangermex at 20,000 ppms, 10

minutes at room temperature, and found similar log

reductions to chlorine. We\"re not usually seeing a

five log reduction on alfalfa seed and we\"re

certainly not eliminating the pathogen from the

seed but the log reductions in these direct

comparisons were similar.

One of the problems, if you will, with the

research as a whole here is that they were tested

only in a single laboratory by a single group and

really it would be nice to see some independent

confirmation of efficacy by other laboratories and

also scale up studies. Usually these were done in

very small scale with small amounts of seed. Also

there is again safety issues primarily with calcium

hydroxide and also possibly cost issues with some

154

of these other commercial products.

As far as biological interventions go, the

most successful intervention so far was the

addition of whole microbial communities isolated

from market sprouts. These are added at the seed

soak stage and at day seven the sprouts were

harvested. Actually days one, three and seven the

sprouts were harvested and the populations of

Salmonella were determined and compared to the

control at day seven the sprouts grown from seed

treated with these whole microbial communities

showed a five log reduction in population of

Salmonella. This was some work done at our center

in collaboration with J. Garland down at the

Kennedy Space Center.

One interesting thing was that when they

tried whole microbial communities from laboratory

grown sprouts they weren\"t as active as from market

sprouts.

What has been published as far as chlorine

treatments, high levels of chlorine for mung bean

seed? We did a little bit of work in my lab a few

155

years ago on this and we did find that it was

pretty effective for reducing Salmonella on mung

bean seed. We got about a five log reduction but

not in elimination and with E. coli 0157:H7 about a

four log reduction but again not elimination.

There was no effect on percent seed germination.

So what are the potential alternatives to

chlorine, high levels of chlorine that have been

proposed for mung bean seed? And actually they are

more numerous than for alfalfa seed. As Michelle

mentioned, I think one of the reasons is that I

think mung bean seed is a little bit easier to

sanitize than alfalfa seed because the seed coat is

often intact, fairly smooth, where as alfalfa seed

often has cracks in the seed coat and also you can

get wrinkled seed because of insect damage, which

makes it much more difficult to sanitize than mung

bean seed.

Bob Rust was telling me last night that

often mung bean seed, which usually comes from

China, is actually hand harvested versus alfalfa

seed, which would be combine, which would lead to

156

probably damage to the seed coat.

Anyway, in 1999 a group from Canada,

Pasqual Delaquis, published this paper on gaseous

acetic acid treatment of mung bean seed and they

were very successful in eliminating Salmonella and

E. coli 0157:H7 from inoculated seed. They did not

eliminate Listeria monocytogenes but did report a

five log reduction. There was a slight loss in

germinability of the seed but at least from a

statistical standpoint it wasn\"t significant.

Unfortunately, the same treatment applied to

alfalfa seed led to much greater loss in seed

germination.

More recently Randy Warbol\"s group at

Cornell reported that a prolonged dry heat

treatment of mung bean seed, four to five days, was

very effective in eliminating Salmonella and E.

coli 0157 without affecting seed germination.

Again, the similar treatment of alfalfa seed was

found to be detrimental to seed germination.

A group in Europe reported that hot water

treatment of inoculated mung bean seed were

157

effective in achieving a five log reduction of

Salmonella in these various temperature time

combinations for this and non of the treatments

were reported to reduce seed germination below 95

percent.

So a very\--apparently a very effective

method for mung bean seed with little effect on

seed germination. Most likely due to the fact that

mung bean has a much thicker seed coat than does

alfalfa seed and that protects it from the

detrimental effects\--thermal effects against the

embryo.

Dean Glyver\"s group published this work

several years ago on use of ammonia gas treatments

for mung bean seed and reported very good activity,

although not elimination of Salmonella or E. coli

0157:H7 and no effect of germination. Again the

similar treatment to alfalfa seed was not as

effective but apparently did not affect

germination.

As far as combination treatments go, Dr.

Bari at the National Food Research Center over in

158

Japan reported that if you expose the seed to dry

heat 50 degrees Centigrade for one hour followed by

hot acetic electrolyzed water along with sonication

you would get between around a four or five, 4.6

log reduction of E. coli 0157:H7. You did have

survivors but no effect on germination and sprout

growth rate.

Alternatively, they tried dry heat

treatment followed by gamma irradiation at two

kilogray and two kilogray has been shown to reduce

populations of pathogens on seed without reducing

the subsequent germination or yield so it\"s

probably an acceptable exposure of the seed. And

they got about a 4.6 log reduction of E. coli

0157:H7 with no survivors and no effect on

germination but they did see a reduced sprout

growth rate.

As far as other seed types there is not

much information out there. One group published on

the use of high pressure for reducing populations

of pathogens on garden cress, sesame, radish and

mustard seed. They had most success with garden

159

cress where they obtained a six log reduction of

Salmonella, generic E. coli and Listeria innocua.

There were survivors after treatment and

germination was delayed by about one day but after

that the sprouts grew just fine.

The other seed types tested in this work,

sesame, radish and mustard seed, were more pressure

sensitive and showed a greater loss in germination

and sprout growth rates.

For radish seed Dr. Bari in Japan again

published that a dry heat treatment, 50 degrees C.

for one hour, followed by gamma irradiation at two

kilogray again gave about a five log reduction of

E. coli 0157:H7. Again there were survivors.

So, in summary for the seed, there is no

method currently available which eliminates

pathogens from artificially inoculated alfalfa seed

while maintaining seed germination and vigor. As

far as effectiveness of high levels of chlorine to

eliminate Salmonella from naturally contaminated

seed the data is variable at this point. I think

the published investigations of outbreaks indicate

160

that the use of high level of chlorine may not

always be 100 percent effective under common

commercial practice and that may be due to the way

it is being applied and, as we heard earlier, I

think there is a lot of variability in how these

treatments are actually being applied by commercial

growers but I think it was also evident that it can

reduce risk.

For mung bean seed especially there are

several potential alternative treatments to high

levels of chlorine. Acetic acid vapor treatments,

ammonia gas, dry heat and wet heat. High pressure

might be applicable to some seed types such as

garden cress.

There is another report out with alfalfa

seed and high pressure where they didn\"t have such

good successes with garden cress. They got a large

reduction in germination of the seed by high

pressure treatment.

Finally biological control with whole

communities appears promising but it\"s a very long

term research and development project which I think

161

would take\--you know, I think it\"s worth doing but

I think it\"s going to take years to come up with a

commercial product.

And then most of these results have been

done\--you know, from investigations in a single

laboratory and they really need independent

confirmation. They need scale up studies and

regulatory approvals may be needed for some of

these alternative treatments.

As far as sprouts go, the challenge is to

significantly reduce or eliminate pathogens on

sprout surfaces during growth or postharvest

without compromising sprout appearance. Not yield,

that shouldn\"t be in here but shelf life and

nutrient content.

And there is substantial laboratory

evidence for a variety of sprout types that

Salmonella and E. coli 0157:H7 can actually get

internalized into the sprout that are grown from

inoculated seed or if you inoculate the roots at an

early stage. Whether this happens under commercial

practice I don\"t think we really know but the

162

potential is definitely there.

So during sprouting as far as addition of

antimicrobials to the irrigation water there has

been quite a few chemicals looked at from chlorine

to acetified sodium chlorite but no intervention

was highly effective.

And then I\"m going to quickly go through

this because Kathleen is giving me the cut off sign

here. As far as postharvest interventions there

has actually been a fair amount of work and I\"m

going to skip over that.

And one of the most effective postharvest

interventions has been irradiation, either gamma or

electron beam, chlorine but at 2,000 ppms rather

than 200 ppms, and chlorous acid at 268 ppms but

regulatory approvals to use these chemicals, I

think, are required.

So, in summary, on sprouts really during

the sprouting process there\"s no effective way of

eliminating Salmonella or E. coli 0157:H7. And

addition of the antimicrobials to the irrigation

water would interfere with the spent irrigation

163

water testing. The test may come back negative but

you still might have viable pathogen cells on or in

sprouts.

And after harvest probably only gamma or

beta irradiation would most likely eliminate both

surface borne and internalized pathogens and

neither is approved right now for use on sprouts

postharvest.

So I\"m going to turn it over to Kathleen

to give you just a brief overview of her

collaborative study that\"s ongoing.

INTERVENTIONS

DR. RAJKOWSKI: Good afternoon.

As a quick means of introduction, I was

asked to revisit two issues. One particular one is

a liquid sanitizer, Tsunami, which was reformulated

by the commercial company. And to investigate what

would be considered a chambered ozone treatment.

The first table are the results obtained

when gaseous ozone with a 65 percent relative

humidity was applied to the seeds. There were six

batches or six separate inoculums done at six

164

different times. The consistency was that they

were all done for 24 hours with the same ozone rate

or ppm which was 30,000 ppm. The average reduction

using this ozone treatment is approximately one

log.

But in many of the studies using ozone

what we did find is that the recovery on a

selective ager showed injury. The results

presented here are on a non-selective ager. Based

on the fact that there was indication of an injury

we took these particular treated alfalfa seeds and

gave them treatment with the Tsunami. Now being a

research group I started using the Tsunami at many

different percent levels. I went from the one

percent, which was the approved, all the way up to

five percent to see what would happen and I found

no difference between three and five percent so the

upper level that I used was three percent.

What we did do is we did verify before I

used any of the liquid sanitizer that the ozone

treatment did not damage the seeds whatsoever so

they were verified through independent

165

laboratories. Two separate sprouters were given

the seeds to germinate them and determine the yield

ratio within their sprouting facility.

Unfortunately, these people could not use the

sprouts for sale but they were able to say, yes,

they were good, we would buy these particular

seeds. And one grower supposedly has been using

the ozone treated seeds.

Using the peracetic hydroperoxide

sanitizer to reduce Salmonella using percentage 0

through 3 percent, the non-treated is the control.

The ozone treated is what I retrieved after using

the sanitizer. At the two percent there was a two

log reduction but at the three percent there was no

growth after recovery using plating media, which

was the tryptic soy ager which is a non-selective,

and in order to prove that there was no growth at

the three percent I did what would be considered

the traditional MPN, most probable number, recovery

and it confirmed the no growth scenario.

As Bill mentioned, when I did use the

peracetic hydrogen peroxide sanitizer I also used

166

the 20,000 ppm on the same seeds. After treatment

you notice there it\"s blank because I got the same

value as the non-treated. I could not find a

calcium hypochlorite reduction and as a result we

are going to be revisiting different methods of

drying the seeds after the wet artificial inoculum.

As Bill mentioned, several laboratories have been

using different treatments. No one has been using

consistently the same method of inoculation and

drying the seeds.

I did my initial work using irradiation.

In order to do that work I dried my seeds down to

almost the same level as before inoculation and it

is possible that that might be the reason why I am

not seeing the reduction with the calcium

hypochlorite because I would have seeds very much

similar to a natural inoculated seed and the fact

that it has the same or comparable percent

moisture.

This is all unpublished data. We are

going to be continuing this in collaboration with

the FDA, Dr. Wu, and we will try to do, what Bill

167

suggested, a split sample where I would take my

inoculated seeds, send them to other laboratories

and have them confirm the reduction using their

technique but the same protocol.

Thank you.

DR. BERU: Thank you, Drs. Fett and

Rajkowski.

Our next set of presentations is going to

be on testing methodologies and sampling, and that

will be by Dr. Mary Lou Tortorello and Dr. T-J Fu

from OPDF, Moffitt Center, Chicago.

TESTING METHODOLOGIES

DR. TORTORELLO: Well, as Nega mentioned,

T-J and I are sharing this overview on testing

methodologies and sampling.

My part of this talk is going to be

primarily a historical perspective providing you

with the background, the rationale for how the FDA

guidance in 1999 came to be for testing

recommendations, that is what was known at the time

based on the best available science that we had.

T-J, however, has done a lot more recent

168

work on sprout research and is going to be giving

you more of a looking forward perspective, that is

what have we learned since that guidance was

published and what are some of the new issues that

should be considered.

So I\"ll begin this overview with a

question. Why are we testing when a lot of the

rest of the food industry has decided that

endproduct testing of foods does not really ensure

their safety for a couple of reasons? First of

all, in a lot of foods there are low levels of

microorganisms present and the type of

contamination that is present could be very

sporadic. So endproduct testing for the most part

has been moved away from the rest of the food

industry and this is why HACCP has come to be such

a focus of food safety for the food industry.

However, sprouts are a unique food. In

sprouts there is a natural microbial growth to high

levels during the sprouting process and depending

on the way the sprouts are produced those

microorganisms can be quite uniformly distributed

169

throughout the batch and so for sprouts product

testing could very well make a lot of sense.

And so the questions that we asked before

the guidance was issued was: What do we want to

test and how and when should that testing be done?

And Caroline\--are you still here\--you

asked if anybody remembered that first FDA public

meeting on sprouts and I remember it. It was in

1998 and I remember it quite distinctly because it

was at that meeting that I first heard of the idea

of testing spent irrigation water to indicate the

microbiological condition of the sprouts and that

idea came from an industry representative. At that

time it was decided that we at the National Center

for Food Safety and Technology at the Moffitt

Center in Chicago would try to verify this idea and

so we went out and bought the big rotary drum

sprouters and we bought a bunch of materials that

would allow us to produce sprouts in our pilot

plant and we attempted to verify this idea of

testing the spent irrigation water as opposed to

the sprouts.

170

Spent irrigation water, of course, being

that water that is used to mist and hydrate the

sprouts during their growth. It trickles through

the batch of sprouts and as it trickles through it

sort of collects the microorganisms that are

present in the sprouts. Therefore, making up a

quite uniform representative sampling of that batch

and this is kind of a unique situation in food

production.

So we tested this first looking at the

total microbial counts in the batch looking at the

difference in total counts between the sprouts and

the water and we found that there is a very good

correlation. The mean difference being between

about half and one log in counts between the

sprouts and the spent irrigation water.

We did the same thing on inoculated

sprouts, that is that we inoculated with E. coli

0157 and found again that there was a pretty good

match between counts of E. coli in the sprouts

versus counts in the water.

But the real test of this idea came from

171

using seeds that were naturally contaminated with

Salmonella that we had obtained from outbreaks and

we were fortunate to be able to get three different

lots of seeds that we tested this idea on.

What we found was that every time we found

Salmonella in the sprouts we also found it in the

water for these naturally contaminated seeds and so

this was a real good proof of concept for us that

we could, in fact, use that spent irrigation water

as an indicator of the contamination status of the

sprouts.

Okay. So that answered the question what

should be tested. We then tried to determine how

and when should testing be done.

As you all know, the guidance has some

very specific recommendations on how to test the

spent irrigation water and what the FDA

microbiologists were charged with at the time was

to come up with some kind of test that would be

practical to use by the sprouting industry. That

is we wanted the testing results to be available

within 48 hours of sampling and we also wanted to

172

be able to recommend a test that had been

extensively validated.

At that time there were two AOAC official

methods for Salmonella. One was the Assurance Gold

Enzyme Immunoassay and the other one was the VIP,

the Visual Immunoprecipitate Assay. These had

undergone the extensive collaborative testing that

is prescribed by AOAC International to become an

official method and at the time for E. coli 0157:H7

the VIP was the only official method. However, we

had had some additional experience within the

agency in testing sprouts with the Reveal test and

we were pretty confident that that would also be a

good test to include in our recommendation.

So the requirements for testing that were

in the guidance as a result of all of this

consideration was to test each sprouting batch for

both Salmonella and E. coli 0157:H7. Because of

the methodology restrictions on Salmonella there

were a couple more enrichment steps included. For

E. coli things were a little bit simpler before

getting to that rapid test kit. These were

173

obviously the screening tests that were going

to\--with results available within 48 hours.

And then the guidance recommended what to

do if you got positive results. Well, you had two

alternatives and that was first either to discard

the sprouts and seed and disinfect the sprouting

equipment or you could go through the confirmatory

series of tests to prove whether that initial

screening result was a true positive or not. The

confirmatory tests are quite extensive. They

involve isolation of the organism as well as

biochemical characterization to prove whether that

isolate is, in fact, the pathogen.

A couple of comments that I wanted to make

here, although the guidance did not explicitly say

so, how to run those confirmatory tests should be

done from the original enrichment. I guess there

was at least one sprout outbreak that occurred

because of this incorrect confirmation. The

confirmatory test, I guess, was run not from the

original enrichment but from the original sample

that had been stored in the refrigerator. This was

174

not explicit but should be made very explicit now.

The other comment that I would like to

make is about the disinfection. If you get a

positive result how do you disinfect your equipment

and how do you validate whether that equipment has

been properly disinfected? This was kind of a

personal consideration of mine because it happened

to me in our own laboratories and I wondered what

was the procedure in the sprouting industry when a

positive happened. How do you validate your

disinfection?

We tested the sensitivity of these rapid

test kits for Salmonella and E. coli and found that

they were pretty sensitive. Both the sprouts and

water could be detected down to an inoculation

level of 1 CFU/gram. However, they are not as

sensitive to be able to get away with not doing an

enrichment and this chart shows that for E. coli

0157 tests, the VIP and Reveal, that the detection

limits are pretty high. That is for the VIP you

need seven log CFU/ml sprouts or water to be able

to detect those organisms. Reveal is a little bit

175

more sensitive. You need five to six log CFU/ml to

detect them and so these tests can never be run

without doing an enrichment because the pathogens

don\"t\--they grow to high levels but not that high

during sprouting.

As is shown on this slide and this is the

growth of Salmonella during sprouting of the

naturally contaminated seeds associated with

outbreaks. And this slide actually helps me to

make two points. The first point is about the

maximum population level that is reached during

sprouting at least for these two lots of seeds. In

lot A populations of Salmonella got up to maybe 10

3

MPN per gram and in lot B perhaps between 104th-5th

MPN per gram. So again to reiterate the test kits

must be used in conjunction with an enrichment

because they\"re just not sensitive enough to be

able to detect these levels.

And the second point that I wanted to make

from these slides is to show you that the maximum

population levels are reached at about the second

day of sprouting, that is 48 hours after initiation

176

of sprouting. So that was the rationale for

including in the guidance the recommendation that

the sample be taken at 48 hours of sprouting

because this has been shown not just for the

Salmonella, as seen here in the naturally

contaminated seeds, but also in other types of

microbial populations that we had tested. We saw

the same trends for the total microorganisms, for

the cola forms and for inoculated E. coli as well.

That said, I will also show you this table

which shows the Salmonella rapid test kit results

for each day of sprouting of naturally contaminated

seeds. We are looking at two seedlots here

showing that on day zero seedlot A was detected as

positive. That must have been a very hot batch.

Lot B\--I see lot B was not detected on day zero.

However, on day one both seedlots showed positive

testing by the EIA so this table would imply that

even though the maximum population levels are shown

at 48 hours after beginning of sprouting it\"s

possible that by day one even though those

populations are not at maximum levels they may be

177

high enough for the test kits to detect the

presence of the contaminate after enrichment.

I also wanted to talk a little bit about

the validation of rapid tests. Although we were

working with AOAC official methods which have, as I

said, undergone very extensive collaborative

testing, I believe there are a minimum of eight to

ten laboratories that simultaneously test a kit in

order for AOAC official method recognition as well

as a lot of other testing that must be performed,

including sensitivity, specificity, false positive

rate, false negative rate. It is very, very

extensive testing.

Even though we were working with known

AOAC official methods we wanted to do our own

in-house verification that these methods were

tested\--were okay to use for sprouts and sprout

water because although the AOAC official method

allows use for various categories of foods, sprouts

may not have been included in that testing. So we

wanted to make sure that these tests were okay for

use with sprout\--for sprouts.

178

And after consulting with the FDA

statisticians who gave us their blessing on the

number of reps to use and number of lots to test

and number of serotypes of Salmonella to include in

the testing, we did a total of four seedlots, a

total of 66 replications for each rapid test kit,

comparing them to the BAM, which is the FDA

reference method for Salmonella. We decided that,

in fact, these tests were good to go for including

in the guidance.

And this type of testing was also done for

E. coli 0157:H7 by Steve Wiegant, who is an FDA

colleagues of our\"s out near Seattle.

I just also wanted to show you some

results that we did on rapid test kits that did not

wind up in the guidance. The guidance had

specified rapid test kits that were all

immunoassays. We wondered if there were any

nucleic acid based rapid test kits that could also

be used and at that time the Gene Trak test for

Salmonella was an AOAC performance tested method,

which is a little less stringent testing regime

179

than an official method. I believe the performance

tested method involves only a minimum of one

independent laboratory confirmation of the test as

opposed to the eight to ten labs that are required

in official method.

Anyway, we looked at the BAX, which is a

PCR based method, compared to the Gene Trak, which

is a nucleic acid hybridization method, and

compared that to the Assurance Gold EIA, which was

included in the guidance and which is an

immunoassay. These are the detection limits of the

three assays and we can see right off the bat that

the immunoassay, the Gold, is the least sensitive

when it comes to detection limit requiring 10

6 CFUs

for positive result to occur. The Gene Trak was

next and requiring about 10

5 cells and the BAX,

which is a PCR based method which amplifies the

genetic target, is very sensitive requiring only

100 CFUs for detection. At that time the BAX was

neither an official method nor a performance tested

method.

So that was pretty impressive to us

180

until\--and these were done for Salmonella cells in

buffer, and that was pretty impressive until we

looked at the performance of these tests in

sprouts. In sprouts and water from the naturally

contaminated seeds.

And here we saw that the BAX did not

perform that well at all and that was largely

because its procedure recommended a non-selective

enrichment of the organism and so it detected very

poorly. Only 13 positives out of 60 samples. The

Gene Trak was very good, detecting 36 out of the 60

samples. And the Assurance Gold EIA detected 41.

Whereas, the BAM detected 30.

Now these were not inoculated samples.

These were naturally contaminated samples so some

of them might not have been positive at all and

that\"s why you see the low percentages of positives

among all of the tests.

My point here is that we need to consider

in considering other tests that could be run, we

need to consider the entire method, that is from

the enrichment all the way through the rapid test,

181

and whether or not the test has been validated for

sprouts.

I\"ve put up here in this table just a few

AOAC performance tested methods and I just wanted

to show you if you look at the column all the way

on your right the matrix for which these various

tests have been approved. The Gene Trak

Salmonella, which is the one that we included in

that last slide, has been performance tested for

very general categories for food, animal feed and

environmental samples. Whereas, some of these

tests have been tested for very specific

applications. Look at the Pathogen E. in the very

bottom row has been performance tested just for raw

ground beef. So my point here is that in

considering other tests we need to look at what the

matrix has been that was included in the testing.

This is an interesting slide because it is

the same slide that Bill Fett put up for chemical

interventions but I did the same thing for pathogen

detection methods and publications in those areas.

It\"s interesting that there are a whole

182

lot of publications on different detection methods

for sprouts. The question, however, should be not

whether the test has been published but whether it

has been validated with independent laboratories

testing the method and not just a single

publication.

So, in summary, in considering what

methods to use, we want to ask has the method been

tested specifically for sprouts and for sprout

irrigation water and has the entire method been

tested against a standard method, including the

enrichment, the detection and the confirmation. In

short, has the method been extensively evaluated or

has it been validated?

And that\"s it. Thank you.

TESTING METHODOLOGIES AND SAMPLING

DR. FU: Okay. Good afternoon.

In this part of the talk what I will do is

to review some of the issues that have been raised

since the publication of the last guidance in 1999

and then provide some of the research findings that

have been published or has been accumulating in our

183

lab that may help to address some of those issues.

Okay. I will divide the presentation into

three areas. First is to look at the issue

relating to the sampling and testing during

sprouting and then I will also look at the sampling

and testing method that has been developed and used

for testing seeds and, finally, if time allows, I

will look at the detection methodologies and share

with you the experience we have in validating and

evaluating commercially available methods.

There are several methods and issues

raised regarding the sampling and testing of spent

irrigation water such as when is the best time to

sample, 24 hours versus 48 hours, and how to

sample. The guidance suggests sampling one liter

per production batch. Some people would like to

pool and there are some considerations for mung

bean so I will go through them.

Regarding the sampling and testing of

spent irrigation water: As I said, people would

like to know if they can test the irrigation water

at 24 hours instead of 48 hours as suggested by the

184

guidance. Testing earlier gives the sprouters some

advantages. For example, they can ship the sample

a day earlier while the sprouts are at the freshest

and also they can free up some of their storage

while putting their sprouts in storage waiting for

the testing results.

But is it appropriate to test irrigation

water at 24 hours? To answer that question we need

to know what level of pathogens are present during

sprouting at 24 hours and since 1999 a lot of

growth study has been published in the literature

and the data provided from the study may help us to

answer the question that I just asked.

So what we have done is to collect those

published studies and to summarize the data and put

out this table. What wee have here is we list the

level of pathogens that was measured at the

beginning, at 24 hours and then 48 hours, during

sprouting of the naturally contaminated seeds, as

well as the artificially inoculated seeds among

different growth studies.

What we can see here is almost all of the

185

studies shows that pathogens proliferate during

sprouting. However, the level of the test

population are different. In general, for the

naturally contaminated seeds the level were lower

and the highest level they reach at 48 hours were

lower as opposed to those you find in artificially

inoculated seeds.

Okay. The other thing I want to point out

here is Lou has pointed out those test kits

recommended by the guidance gives you a detection

limit about one CFU/gram, which is about log

zero\--log value is about zero. So you can see most

of the studies, except one, will give a positive

result using the test kit provided by the guidance

either at 24 or at 48 hours.

Now just to give you a better idea of the

relative log increase between the 24 hours and 48

hours what we have done is to just further analyze

this data and put out this table. What I have here

is to list the range of the log increase that was

calculated from the previous study that was at 24

hours and 48 hours for the naturally contaminated

186

seeds and artificially inoculated seeds.

And if you will divide the value of log

increase at 24 hours by the log increase at 48

hours for each of the studies, what we have found

is average ratio obtained is about .85 for the

naturally contaminated seeds and about .88 for the

artificially inoculated seeds. That is on average

90 percent of increase of population during

sprouting occur at the first 24 hours of sprouting.

So I would say that if you have a test kit that

detection limit is way above the test level you can

reach at 48 hours most likely that test kit will be

able to pick up the pathogen at 24 hours on average

but I want to stress the words \"on average\" because

individually the studies show that the level of

pathogen at 24 hours is about maybe\--on a log scale

it\"s only less than 60 percent compared to that you

will see at 48 hours.

Now the other issue that has been raised

is the issue of pooling. In the guidance, as I

said, the guidance recommend the sample from each

production be tested but we all know that testing

187

requires a lot of cost and so some sprouters are

trying to reduce the costs by pooling water from

multiple production lots and then take a

representative sample from that lot and sending it

out for testing. That, in effect, you dilute the

concentration of the sample in the sample that you

take for analysis and because of this concern the

FDA actually advised against sample pooling.

Now let\"s\--an alternative approach that

may help to resolve this problem is to allow sample

concentration so that you can concentrate all the

sample you pool and then test by a single test. So

the sample pre-concentration, we have work for

several years and I will show you the data in later

slides but I want to also mention that sample

pre-concentration may also be helpful to address

another issue in sampling and testing of spent

irrigation water for mung bean sprouts.

It is known that mung bean sprout

irrigation water cycle and the procedure they use

is different. Typically for mung bean sprout they

dump a lot of water during each irrigation cycle.

188

The water can be as much as 100 fold greater than

the water that you use during alfalfa sprouting and

that creates some concern that the microbial count

that you have in the mung bean spent irrigation

water may be much lower than those of bean sprouts.

Therefore, the testing spent irrigation water may

not indicate the positive status of the mung bean

sprouts.

So the sample pre-concentration may again

help to concentrate the level of microbial

population before you go for testing.

So what I would do in the next several

slides is to share with you a system\--a

concentrated system that we have developed in our

lab and some validation work that we have done to

evaluate the potential for this concentrator to be

used as a part of sample preparation for detection

of Salmonella and E. coli 0157:H7 in large volume

of sprout water.

Okay. This is a picture of the

concentrator. What it has is\--it has a pre-filter

that is about ten micron\--with a pour size of ten

189

micron. It also consists of a tangential flow

filtration cartridge. The idea of a tangential

flow filtration is commonly used in the

fermentation industry and I won\"t have time to go

through the details but anyway this process

concentrator will allow processing up to ten liters

of irrigation water within two hours. It can

concentrate the water by 100 fold to 100 mls and

those concentrated samples can then be

used\--analyzed by either culture method, dipstick

test, ELISA or even biosensor.

And to validate the efficacy of this

concentrator as a part of testing there are two

aspects we look at. First is to determine whether

the increase in background microflora in the

concentrated sample affects the enrichment and

subsequent detection of pathogens by conventional

and/or commercially available rapid test. We also

would like to know the lowest recovery rate of

pathogens that can be recovered from the system.

To evaluate the first point what we have

done here is to concentrate a large volume of spent

190

irrigation water by 100 fold and aliquot out in 10

ml aliquots. And for each aliquot we add different

level of pathogens at about one CFU, 10 and 100.

And then we perform 20 replicates for each

inoculation level. Again this is, just like Lu

said, a blessing from a statistician from FDA.

That is their testing recommendation.

So what we have here is for the sample

that were inoculated at 10 CFU and 100 CFU the test

kit are 100 percent efficient to detect the

presence of Salmonella and they were also confirmed

by the culture method.

When the samples were inoculated at one

CFU the test kit was still very good and picked up

the presence of Salmonella. Okay. So that is the

test kit was not really affected by the

concentration. Here the 10 ml concentrated sample

actually correspond to one liter so from here we

can say that the test kit, although it will be able

with the help of the sample pre-concentration we

were able to detect the presence of one CFU or

Salmonella in one liter of spent irrigation water.

191

The same evaluation was done on the

irrigation water collected from sprouting of

seedlot and a result very similar. That is the

Assurance Gold EIA test still performed nicely with

the concentrated water.

Now with respect to recovery what we would

like to know is if we have one cell in this 10

liter of water will we be able to see it in the

concentrated stream. So what we have done here is

to inoculate a 10 liter of water with one cell, 10

cells and 100 cells, and then test the presence of

Salmonella in the concentrated stream and to see

the rate of positive reaction. And what we have

shown here is for the sample that inoculated at 10

cells per 10 liter or higher, the system will be

able to give you a positive result.

The same experiments were performed also

for E. coli 0157:H7. This is a busy slide but what

I want to show you is in the guidance the

enrichment media is special enrichment media

developed by FDA scientists using the modified BPW

plus some antibiotics. When we used that

192

enrichment media the positive rate were not as good

as we would like and not as good or close as what

you see in Salmonella. Okay. So what we have done

is to try a different kind of enrichment media that

was provided by the manufacturer of the \^Rtest and

we were able to boost the positive rate using that

enrichment media.

But while we are we trying to validate the

result of a device, what we have observed is

sometimes the culture methods fails to give you a

positive reaction when the device indicates really

positive results. So it has turned out that when

you are trying to confirm your culture result the

background microflora in sprouts was so high that

actually it prevents good identification of your

typical colonies. So with the help of

immunomagnetic separation we were able to increase

the positive rate of the culture method so overall

for the Salmonella\--E. coli 0157:H7 we have shown

that the test kit will be able to detect at a level

of 10 CFU or higher per liter of water.

And this is again the recovery study that

193

I talked to you about with Salmonella but for E.

coli 0157:H7 the result was better. We were able

to detect the presence of E. coli 0157:H7 one

CFU/10 liter.

Okay. Let\"s move to the seed sampling and

testing. As several speakers have mentioned that

this is recognized that seeds are the major source

of contamination in sprouts. So to prevent the use

of contaminated seeds for sprout production is

critical to ensure sprout safety and I think most

importantly is to protect the home sprout grower

who grow their sprouts at home. Normally they will

not do any microbial testing or seek treatment so

clean seeds to them is really critical.

As far as the sampling and testing method

that\"s out there, what I can find is most of the

methods were used for either of two ways. For the

outbreak investigation and also for screening seeds

that are used for sprout production. What I will

cover in this section is the method that has been

used for determining the presence of pathogens in

seed and also identify some factors that may affect

194

the efficacy of seed testing.

I also will just briefly mention the

protocol that have been developed by the industry

on screening seeds that are destined for sprout

production. Okay.

Inami, et al., which is a group with the

California Department of Health, they have done

many, many outbreak studies and they actually

developed a sampling plan, and it\"s I guess the

only one\--a very comprehensive sampling plan that

you can find in the literature.

What they did is they take 500 grams of

sample from different bags of lots and then they

take 100 grams of sample unit from their 100 sample

for testing and for this paper that they published

what they did is they take actually two 100 gram

samples from each sample. Two 100 gram sample

units from each sample so they can compare the

different testing methodologies. They want to

compare whether testing the seeds is better or

testing the sprouts that are grown from the seeds

is better. And as far as the detection

195

goes they were following the BAM method so I will

show their result here.

You are invited to look at their paper to

look at their specific protocol but what I want to

show there is to compare the positive rate testing

the seed versus the positive rate observed by

testing the sprout and they have done three

seedlots and on average their success rate for

testing seeds is about only 90 percent than if they

were testing the sprouts.

For us we also did a similar study to look

at testing seeds versus testing sprouts and how

efficacious they are. What we have is three

seedlots and they are associated with the outbreak

indicated here.

What we have done instead of testing the

seeds or sprout itself, we test the water that was

collected from the soak water from the sprouts that

has been grown for two days. What we have found

out is for some seeds the testing for day zero

gives you a pretty good prediction of the status of

the sprouts but for some seeds you will miss the

196

positive status of the seeds if you only test the

soak water. So for our experience testing sprouts

will give you better prediction of the

contamination status of the sprouts.

Other factors that may effect the testing

efficacy is the sampling size. What I have shown

here is for sampling for 100 gram seeds\--for some

lots the success rate is pretty good but for other

lots you hardly will be able to determine the

positive status of the contamination.

But if you will increase the sampling from

100 grams to one kilograms, what do our

experience\--

\[Audio difficulties.\]

\--recognized screening seeds is an

important step in multi-approach to prevent the

occurrence of sprout associated outbreak and Bob

Rust and Bob Sanderson has jointly developed this

six step procedure for seed screening. I think Bob

has mentioned this in more detail earlier so I

won\"t discuss this more here but what I want to

point out is that what they have indicated that is

197

this seed screening has prevented at least one

potential outbreak of 0157:H7 and then prevent the

shipment of contaminated seeds to sprouters and

that further protects the consumer who might be

using and might be consuming those sprouts. So it

provides additional protections.

Okay. The last part is the detection

methodology. As Lou mentioned the validation

performed specifically on sprouts is very critical.

What I would do here is to provide you two examples

from our experience in evaluating the performance

of two commercially available methods.

This is an automatic immunomagnetic

separation system. I will not go into the detail

operation because I don\"t have time. It\"s called a

Pathatrix System but what I want to point out is it

has been AOAC performance certified for detection

of E. coli 0157:H7 and Salmonella at a detection

limit of 1 CFU/25 gram of sample.

So we tried to use this for our sprout

irrigation water using their protocol, enrichment

protocol, as certified by AOAC. We found out that

198

the system failed to detect the presence of the

O157:H7 at the level of 100 grams per sample and

also failed on a concentrated sample. In this case

the concentrated sample corresponds to 2.5 liter of

unconcentrated sprout water.

Okay. So what we have done also is to put

the protocol recommended by the guidance test to

see if that would give us a better detection. Yes,

this did for unconcentrated sprout water and we

were able to detect as low as one cell per 25 gram

but this protocol still is not good if we use the

concentrated water.

What we also tried is to look at the media

provided and in this case we were able to increase

the detection substantially and so that we can

actually reach one cell per 25 ml. For the

tangential flow concentrated sample that is

actually one CFU/2.5 liters.

The same thing is done for the Salmonella.

A gain the protocol provided by the AOAC approved

method really is not as good as they would like

to\--as they expected from other food. It\"s only

199

with some modification we were able to reach the

detection limit that was approved by the AOAC. So

again trying to indicate that the validation

specifically done on sprout system is critical.

Biosensor will provide some opportunity

for real time pathogen monitoring and this fiber

optic biosensor that is commercially available in

one format. This format actually would allow flow

through type of operation and this is more of

sample by sample called RAPTOR.

This biosensor has been shown to be able

to detect E. coli 0157:H7 in ground beef at 3-30

CFU/ml levels. It is really, really sensitive

without enrichment. And so when I saw that I was

really, really excited so I decided to purchase one

and test it in sprouts.

More recently variation of that biosensor

are commercially available and has been shown to

detect the presence of Salmonella in spent

irrigation water collected at 67 hours of sprouting

from seeds inoculated with 50 CFU/gram of pathogen.

But I want to caution this statement

200

because if you go to the paper what happened is the

detection limit of this Salmonella is about 10

5

CFU/ml but the way it is written here indicates in

the 50 CFU/gram will give a false sense that\"s very

sensitive but you want to really put the growth

study in mind that at 67 hours post sprouting the

level can be very high. Okay.

So anyway what we have done here is to

evaluate the efficacy of the Analyte 2000

Fiber-Optic Biosensor with multiple replicates and

what we have shown here is, yes, at the level\--the

low level some tests\--some trials will give you

positive status but until you reach a very high

inoculation level you won\"t get a consistent

positive result.

So again a lot of times when people have a

new method they test three and everything is

positive and they say, wow, this test is great for

something but unless we give you a larger number of

trial then you won\"t be able to really say more

confidently what is the efficacy of the test.

So, in conclusion, I want to say there are

201

a lot of information out there since 1999 and some

of the information will help as we engage in

discussion and try to improve the guidance.

And then sample concentration we have

developed that can help to improve the detection

if\--will provide alternative for the pooling of the

sample.

And seed screening does provide additional

control to prevent the sprout associated outbreak

and again I want to emphasize it will provide

protection for consumers who produce sprout at

home.

And then it appears that testing sprouts

to provide\--will provide better detection than

testing seeds.

And, finally, I want to emphasize that

validation of any method should be done in sprouts

so that\--because the sprout is so high a lot of

times those test kits may fail.

Before I leave I want to thank all these

people. Actually we. Lou and me want to thank all

these people who helped us to collect all the data

202

that we presented.

Thank you.

\[Applause.\]

DR. BERU: Thank you, T-J and Mary Lou.

Our next presenter is Kean Ashurst from

Caudill Seed Company and the title of his

presentation is Alternative Seed Sanitation Methods

and the Results of Practical Field Application.

ALTERNATIVE SEED SANITATION METHODS

AND THE RESULTS OF PRACTICAL FIELD APPLICATIONS

MR. ASHURST: Thank you. Hello. My name

is Kean and good afternoon.

I have been preceded by some very

excellent presentations today and I\"m proud to be

here as a spokesman for this industry and for

Caudill Seed.

Caudill Seed Company is dedicated to

making sprout eating a safe and nutritious pursuit

for all in the industry. CSC provides seed and

sprouting equipment to sprout growers and is

attempting to positively impact the sprout industry

in several ways, both directly through the seed

203

that it sells and indirectly through

recommendations and technology development

assistance to sprout growers. We want to provide a

science-based approach for alternate solutions to

all segments of the sprouting industry.

Review methods or the purpose today is

review methods tested to improve seed cleanliness

and recommend one path; review sprouting procedures

and sanitation; and identify risk points and make

recommendations; support the use of existing

sanitation methods, chlorine, and propose an

alternate that may be as or more effective than

chlorine; alternate seed sanitation methods and

results of field applications.

The sprouting industry seed must be

considered a raw agricultural product and must be

dealt with accordingly. I cannot over stress that.

The 10 to 12 years that I spent with Libby, Nestle

and Hunt-Wesson, we washed our pickles, we washed

our tomatoes, we washed all those vegetables before

we processed them. Okay. You have to make that

assumption. All right.

204

There are other sources of contamination

to be considered and controlled for a total quality

management of sprout production. Seed sanitation

includes not only sanitizers but also techniques

and I think what we\"re going to see as we go

through some of our sprouting facilities we\"re

going to see Company A doing this, Company B doing

that, Company C doing this and so on and so forth,

and that is not standardization. I\"m not saying

that what A is doing is incorrect but we need to

have a total agreement and move forward on a level

playing field and everybody is playing the same

ball game.

Other sources of contamination: It is

essential to recognize that not all risks come from

the seed and other areas of contamination need to

be addressed. Proper application of sanitizing

protocol. Equipment cleaning and sanitation.

Some of you have seen this slide as long

as two or three months ago. I want to take some

brief time and go through it because I think it\"s

an important slide. It\"s not all encompassing but

205

it represents my visits to sprout growers, not all

but most.

I want to start out with seed

contamination. Really no firm regulations on it.

Field growing conditions, we need to have

firm regulations.

Procedures for cleaning seed from the

field impacted.

Storage and transportation impacted.

Let\"s go to sprout production. Air

quality in the growing chamber. Water quality for

irrigation. Again no hard fast regulations in this

area. Harvesting procedures. Seed not washed and

sanitized. Completely avoiding the guidances. Meetings

like today need to be more frequent.

Industry and regulatory cooperation. Washing,

packaging and shipping.

This is one area, I guess, that I get

really excited about when I see water temperature

at 70-80 degrees. It should be below 40. Pour or

no sanitizer used in the washing system. No real

HACCP program in the facility. Temperature abuse

206

of the product in shipping. I have seen a number

of sprouters sell product on vans, 110 degrees and

the van has got a two hour drive to its

destination. Employee practices, again no

regulations. No standards of identity. No QA/QC

programs. I\"m not saying this applies to

everybody. There are good sprouters and there are

marginal ones.

The other area is facility conditions.

About three weeks ago I had an opportunity to go

into a sprout grower in an unknown state, unknown

name, entered the facility and walked in and within

50 feet I counted 18 critical violations and I

didn\"t move. Equipment condition and design.

Product and employee traffic. Cross traffic. Seed

coming in, sprouts going out, and they\"re going

just like this. Sign wave sanitation, and

everybody in this room knows what it is. You\"re

going to have an inspection tomorrow, let\"s get it

right. Construction of the facility. Okay.

Working\--a seed sanitation program.

Working with Dr. Kathleen Rajkowski since the

207

summer of 2004, we have collectively developed an

evaluation program of current and available

processes for reducing seed contamination. Caudill

Seed provided the seed and the USDA would provide

controlled inoculated seed packets for testing.

The initial findings were presented in October 2004

meeting with the FDA.

Evaluation program. We revisited

irradiation, E-beam, heat treatment of seed and

ozone gas. Irradiation and E-beam technology

resulted in poor germination and in some cases as

low as 40 percent. Unacceptable endproduct

quality. The sprouts were rubbery. They were not

crisp in texture. Consumer acceptance issues with

regards to irradiation and they ignored the needs

of a big segment of the industry, the organic

processors.

Heat treatment rapidly aged the seeds and

temperatures between 145 and 147 can only be

achieved consistently in a fluid bed dryer.

Ozone gas treatment showed some promising

results and did not affect the overall flavor or

208

texture of the sprouts but it failed as a

standalone process. Ozone gas treatment was able

to achieve significant reduction. One log kill

with up to two log injury in controlled humidity

conditions.

Dr. Rajkowski has reviewed these slides

with you.

Tsunami 100 was tested in combination with

the ozone gas treatments and the results were

significant. You\"ve seen these slides earlier.

We\"re indicating here that we\"re getting

anywhere from a minimum of four and as high as six

log reduction.

When was the last time, and this is going

to be a sore subject for some of you\--when was the

last time you got 50 pounds of clothes cleaned

stuffed in a nylon bag and submerged for 25 minutes

in water with an astringent detergent dip? Well,

it doesn\"t work on seeds either. I\"m sure that the

above was not the intent of the guidances.

Effective washing implementation.

Effective agitation of the seed is required for

209

adequate wash. We\"re using a 250 CFM blower here,

an oil-less unit that is used in the food industry

for transporting powdered sugar. Water temperature

is ambient.

I want you to take a look at the slide on

the left. It is hard to document this but what

we\"re trying to achieve is an internal rolling

action in that nylon bag and we succeeded in doing

that. You have to follow the procedure though.

You can\"t overload the bag. You\"ve got to place

the bag in the right part of the washing unit. And

if you look to the right this represents only a

small portion of the debris and the material that

came off of this seed. And I say a very small

portion. This is with Tsunami 100.

One sprout processor is currently using

gaseous ozone treated seed in conjunction with

Tsunami 100 with this washing system on a test

basis. Dr. Rajkowski will be receiving and

evaluating a full scale up system in 14 days at the

research facility in Wyndmoor, Pennsylvania.

Caudill Seed will seek an expansion of

210

this program. We will be requesting FDA to

consider usage of Tsunami 100 at three percent

application. Caudill Seed will be recommending a

science-based continuous improvement program on

seed washing and sanitation.

To combat other sources of contamination

we have made available automated CIP systems for

growing chambers. This was tied in with a touch

screen unit. As soon as you finish your growing

process and you wash it out you hit the wash button

and put the doors back on and hit the wash button

and the unit is sanitized internally. And that has

been a result of some modifications that we\"ve made

to the growing chambers.

In addition to that we\"ve made available a

point of use water purification system for each

growing chamber. This additional feature

incorporates a 10 micron spiral wound filter and a

.5 micron carbon block with UV light at 254

nanometers.

I have been through a number of

facilities. I haven\"t seen the back flow

211

preventers. I have seen a hose attached to the

water line and they\"re using the hose to flush out

the drain. What kind of problems do you think that

we\"re going to have?

The next one is probably very important.

It impacts the organic industry and it helps the

industry overall. Air intake systems for the

growing chamber are equipped with dual wave length

UV lights that react and create hydroxyl radicals.

These radicals serve to sanitize the air and

increase phytochemical production and enhance the

immune system of the emerging sprout.

To explain this in detail I\"ve got a lot

of data on this information that\"s true and

factual. We can do it in the Q&A program and I can

also give you a slide. It would take about four

hours if we discussed it today.

There are some of the original references,

J.R. Vig, U.S. Army Electronics Laboratory,

Institute of Molecular Plant Sciences in the

Netherlands, USDA out of New Orleans, and the

Department of Plant Pathology at the University of

212

California, Davis.

It is our goal at Caudill Seed to provide

the industry with the best available technology and

to assist the processor with their food safety

needs. In addition, there are numerous valid ideas

brought forth by other responsible parties in this

forum that need serious consideration.

That\"s the end of the show. Thank you

very much.

DR. BERU: Thank you, Mr. Ashurst.

Our last speaker in this session, this

part of the public meeting, is Dr. Schaffner from

Rutgers, and his presentation is on Risk Analysis.

RISK ANALYSIS

DR. SCHAFFNER: Thanks very much to my

colleagues at the FDA for the invitation to be here

today.

Being the last formal speaker on the

program it is always tough. One possibility might

be that there is nothing left to say. Another

possibility might be that there is no one here to

listen and another possibility might be that you\"re

213

all just already so overloaded with information

there\"s nothing else I can possibly pack into your

brains.

Well, I\"m going to, hopefully, be able to

stand up to that challenge and the opportunity for

me really again as the last formal speaker is to

try to integrate some of the information that

you\"ve heard already or at least present a

framework for how that information can be

integrated. I am told to do that is risk

assessment.

Why are we interested in sprouts at all in

my laboratory? Well, we had an initial discussion

with Bob Sanderson and Bob was very passionate

about his belief in using irrigation water testing.

And I don\"t know how many of you have ever heard

the expression \"if you give a small boy a hammer he

will find that everything he encounters requires

pounding.\" Well, imagine me as that small boy and

instead of a hammer I\"ve been given the tool of

Monte Carlo simulation. Whenever I\"m presented

with a microbiological problem, my initial\--first

214

initial reaction is, \"Well, let\"s use Monte Carlo

simulation to look at this.\" However, in this

particular case, all kidding aside, I think it\"s a

tool that\"s particularly suited for looking at the

trade offs and the complementation of some of these

different issues.

I also want to acknowledge my collaborator

in all this, Rebecca Montville. Rebecca is a

scientist that is currently working at Yale

University and she did a lot of the grunt work to

get this work done and also contributed a lot of

the ideas.

If you\"d like a copy of my presentation I

would be happy to provide it to you. These are two

of\--our two publications on this particular topic.

They\"re also both available on my website. If you

just Google my last name in conjunction with

Rutgers University and food science I\"m sure it

will pop right up. That\"s probably easier than

giving you the URL to our web page. But again most

of what I\"m going to say here, at least the meat of

what I\"m going to say is contained in these two

215

publications so you can read those at your leisure.

The first\--I should say the first

publication\--our first objective before we looked

at building a Monte Carlo simulation of the entire

process was to look at an analysis of the published

studies on seed treatments. And you\"ve already

heard from a number of my colleagues about the

research that has been done in this area. So what

we tried to do was to look at how much had been

done, to look in particular at the sorts of

treatments, and then try to compile them into some

sort of sensible quantitative framework.

This is one of our slides from that or one

of our figures from that publication. What you can

see is that if you look at 20,000 parts per million

chlorine the log reductions are all over the map.

The frequency, which is the Y axis there,

represents the number of times we were able to see

a particular log reduction from a particular study

so we looked at all of these studies. We broke

down the tables and figures and tried to tease this

data out of all of them.

216

What you can see is that this highly

recommended or at least it\"s in people\"s minds this

is a highly recommended procedure, this procedure

gives, if you look at these studies in the

literature, gives most commonly about a 2.5 log

reduction. In some cases in some people\"s hands it

can give upwards of a five log reduction and even

greater than a five log reduction if you look at

that Lang 2000 study.

Now, I should mention that those authors

listed there are the first authors on those

studies. It\"s not the entire author list. So, for

example, the Taormina 1999 study Larry Beuchat is a

co-author on that study as well. I just had to be

able to quickly reference them and fit them all on

this slide.

So what you can see is that there\"s a

great deal of variation in terms of the

effectiveness of the 20,000 parts per million

chlorine disinfection. Now that\"s not a problem to

a risk assessor or to a modeler because what we can

do is to say, well, okay, these data represent the

217

true variability of the system so we\"ll take that

into account when we do our calculations. I\"ll

give you a little bit more on that later but before

we leave this topic of log reduction I want to just

share this figure with you.

If you take those studies that I just

presented and you look at the relationship between

the log reduction that they reported and the

inoculum size, that is the concentration of

organisms that they put on the seeds to begin with,

you can see that there\"s a bit of a relationship.

Okay. And if you look at that Lang 2000 study

which showed the greatest log reduction, what they

did was they took seeds, they immersed them in an

aqueous environment that had a lot of pathogens and

they vacuum dried that system to boost the

concentration.

Now when I look at data like this and I

see a correlation it makes me immediately

suspicious. What these data appear to show is

that\--and this is, you know, not really that

spectacular a finding, it makes sense. The more

218

organisms you put on the seeds, the more you can

take off, the greater log reduction that you can

document. What this analysis says to me is that we

need standardized methods across laboratories for

the way we inoculate seeds and I think I heard at

least two other presenters this morning say that

same thing.

So it\"s not just enough that scientist

Jones publishes a study that shows a certain log

reduction but it needs to be by methods that we in

the scientific community and in the sprouting

community agree are the appropriate methods for

demonstrating the effectiveness of these

technologies so that\"s a really important take home

message.

Now if you take\--if you look at this

slide, what I did here was I\"ve got all this data

but then I\"ve sort of summarized that data with

that bright yellow line and that\"s what risk

assessors would call a triangular distribution.

That shows that you see at least a one log

reduction, most commonly a 2.5 log reduction, and

219

at most about a 6.5 log reduction.

Another figure from our first study, the

one that was published in JFP in 2004, is this

particular one. What this figure does is it

relates that triangular distribution from all the

different datasets that we analyzed to give us a

quick visual way to compare the effectiveness of

20,000 parts per million calcium hypochlorite,

2,000 ppm calcium hypochlorite, 2,000 ppm sodium

hypochlorite, and 200 ppm sodium hypochlorite. Now

what you can see is that there\"s not a heck of a

lot of difference when you go from 2,000 ppm to

20,000 ppm, at least if you\"re looking at calcium

hypochlorite. In fact, the mode, that is the peak

of the triangular distribution, is actually higher

based on our analysis of the literature data for

2,000 ppm calcium hypochlorite.

Now granted the maximum you see is less

and the minimum that you see is less but the most

common number, the one that comes up in more

studies than any other, is actually higher so we

need to be really careful about recommending one

220

particular log reduction over the other. Again you

can use a risk assessment framework, a Monte Carlo

simulation model to compare the effectiveness of

these various disinfection technologies.

This slide represents a screen snapshot

from a piece of software that we developed. This

computer model was developed using a tool called

Analytica. Analytica is basically\--it\"s like an

Excel spreadsheet except that it represents the

data visually instead of in rows and columns. It\"s

a tool that we have used in my lab and my

colleagues at other universities and federal

agencies and international groups have used to do

some of these microbial risk assessments.

And what\--and again I don\"t have time to

go into the details of it but this particular

computer model is available on my website. I have

made some changes as I\"ve been sitting here in the

audience and listening to people and getting

inspired. This represents a relatively recent

picture and once I get back to Rutgers I\"ll be

posting a new version on my website.

221

If you want\--the great thing about this

particular piece of software is it allows you to

hide complexity. So if this were a live version

and I were to click on that button in the middle

that says \"model\" that particular node would

explode and you could look at all the different

factors that go in and the ways that I\"ve combined

them mathematically. It uses a technique

called\--an approach known as \"influence diagram\" so

you can see which inputs influence which outputs.

And you can see on this particular screen

snapshot that a lot of the information that my

colleagues have presented to you today is contained

here. We have the sample size. We have the batch

size. We have the detection limit that you heard

so eloquently discussed just a few minutes ago, the

number of samples taken, the prevalence and the

concentration of the pathogen when it is present on

the seeds. The computer simulation also takes into

account if you want to use a particular type of

disinfection and what the range of effectiveness of

that disinfection technology is. Also if you want

222

to do sampling of seeds prior to using them for

sprouting or whether you want to evaluate

irrigation water during the sprouting process.

So what this piece of software is, is a

mathematical representation to the best of our

ability of the information that\"s out there in the

scientific literature. Since the scientific

literature is not static this shouldn\"t be static

either and so it can be updated and changed as the

science changes.

I should mention that it was kind of

tailored towards alfalfa because there\"s a lot of

the published literature on alfalfa but if you knew

the particulars of a\--it\"s Salmonella on alfalfa in

particular. If you knew the particulars of another

type of seed or another type of pathogen you could

customize this to aid you, and again think of it

not as the magic solution to all of your problems

but as an aid to guiding your thought process in

evaluating one approach over another or the

interaction of different approaches.

So the way the computer model would work

223

again if this was live I could just click on any of

those \"Calc\" buttons and it would calculate based

on Monte Carlo simulation the estimated fraction of

contaminated batches, for example, given the

assumptions that are input into the computer model.

In the interest of time I\"m not going to

go into a great deal of detail on this particular

table. There is a more complete table that\"s in

the published study that\"s available on my website

but I really just want to focus on the three points

that are on the right-hand side of the screen

there. This actually echoes something that Bob

Rust said earlier in the day.

If the prevalence of the pathogen is high,

which means that when you go and you pull a sample

the odds of finding it are high, then using testing

is good. It\"s going to give you the most bang for

your buck.

If the prevalence is low then disinfection

is going to be more effective and that\"s why an

approach of testing, accepting, rejecting, followed

by disinfection is really a great combination and

224

it gives you again the most bang for your buck in

terms of trying to reduce the risk.

Now another key point here is the

assumptions that you make matter and again I don\"t

have time to go into all the assumptions that

underlie this particular table. Okay. You can

look at the article that we published in Applied

and Environmental Micro in 2005 to see some of

those assumptions but if you change the

assumptions\--in other words if you change

assumptions about prevalence, if you change the

assumptions about the concentration of the pathogen

when it is present, if you change your assumptions

about the detection limit for the pathogen of

concern, these numbers may change.

The good news is that a tool like the one

that we\"ve proposed can help you quickly evaluate

changes in those assumptions without going back to

the laboratory, without doing\--spending another

\$200,000.00 and two years of your life to come back

with the answers. Again the answers from the

computer model may not be the gospel truth but they

225

are a guide to future studies and a guide to

decision making.

Another key point, the number of samples

you take matters, and this just shows the

relationship between the percentage of the time

that you\"re able to detect the presence of

pathogens and the number of sub-samples you take.

Again Bob Rust said this much more eloquently than

I can say and I just wanted to echo some of those

thoughts but remember that the particular shape of

this curve and the particular size of those columns

depend upon the assumptions you make about the

prevalence of the pathogens and also the number of

computer simulations you run.

If you\"re dealing with very, very low

prevalence on the order of one out of every 1,000

25 gram samples being positive for the organism and

you only run the computer simulation 1,000 times

then the chances of actually finding that pathogen

in all of those computer iterations is quite low so

you need to be very aware of those particular

sensitivities.

226

Okay. So what are some lessons learned at

least from the research that we\"ve published. Seed

disinfection is a highly variable process even in

the laboratory and you heard a number of other

people allude to this. If it\"s highly variable in

the laboratory or between laboratories, what

happens when we take it out into the actual

production environment? Well, it\"s certainly not

going to be less variable. So seed disinfection is

a highly variable process even in the laboratory.

Irrigation water sampling can be highly

effective. Again I wanted to call your attention

to something that Dr. Tortorello said is that

conventional wisdom in food microbiology is that

testing doesn\"t work. Well, the good news about

sprouts is that they grow pathogens like gang

busters if they happen to be there and you have a

wonderful environment that is the environment of

the irrigation water from which to take samples.

So in the particular case of sprouts testing

actually can be effective and again it will take a

long time before some food microbiologists accept

227

this concept but testing really does work, I think,

quite well in the particular case of sprouts.

But to do that if the prevalence is very

low you have to test many samples, and by many I

mean hundreds of samples or sub-samples prior to

using that seed in production. You also again have

a wonderful opportunity during the production of

sprouts to sample the irrigation water and to test

that irrigation water. And again you heard from a

number of my colleagues today about how important

that is.

And then, finally, remember that

disinfection and sampling at least based upon the

research that we\"ve done and the data that I\"ve

presented here, disinfection in sampling are

complementary tools.

I just want to leave you with a couple of

my favorite quotes. These are from George Cox and

from John Tukey, a couple of statisticians.

George Cox said, \"All models are wrong so

don\"t believe anything a model tells you but

realize that some computer models can be useful.\"

228

Hopefully, today we\"ve shared some of our models

that are useful to you.

And then another quote from John Tukey who

said that, \"An approximate answer\...\" and these

risk assessment models only give approximate

answers \"\...to the right problem is worth a good

deal more than an exact answer to an approximate

problem.\" So again, hopefully, these approximate

answers that we\"ve given will be useful and shed

some light on the subject at hand.

And for those of you who would rather

process pictures I want to leave you with this

slide from Gary Larson which has been amended

slightly. The Sprouts logo didn\"t appear in the

original cartoon. These are clearly microbial risk

assessors who are studying sprouts because they\"ve

got to be microbiologists, you can see there is the

prerequisite microscope. I\"m sure they\"re modelers

and statisticians because they\"ve got a blackboard

full of equations and finally they must really love

sprouts because they\"re all excited now that the

Sprout truck is here.

229

Thank you all for your attention.

QUESTIONS AND ANSWERS

DR. BERU: Thank you, Dr. Schaffner.

I would like now to invite our speakers to

the table for our question and answer session.

I would like to encourage questions not

only from the audience but, panel members, feel

free to ask each other questions as well. I will

take the moderator\"s prerogative and ask the first

question of Dr. Fett.

Of the methods\--you did an extensive

review of the various treatment methods and I

wanted to ask you in terms of applicability to the

widest varieties of seeds in terms of efficacy,

which one holds the most promise do you think?

DR. FETT: Well, I don\"t know if I have a

good answer for that. The problem is that most of

the methods have been looked at only for alfalfa

seed. There has been very little work on other

types of seed. I would say that as I discussed in

the presentation that I think we do have some

viable alternatives for mung bean that look very

230

promising but the work needs to be extended a bit.

There could be cultivar differences, various

differences in methodologies that could be used

that need to be examined but I think with mung bean

there are several potential alternatives that look

very good.

I think with alfalfa seed I think it\"s a

different story. I don\"t see too much out there

right now that has really\--that has been published

I should say\--that is exciting and that I would

consider a viable alternative to the 20,000 ppms or

chlorine right now. There could be work that will

be published soon but that looks more promising for

alfalfa seed but I think alfalfa is a real problem

and I think that relates to the nature of the seed

coat and the cracks and the wrinkles and everywhere

that the pathogens can hide. I don\"t think\--I

don\"t see one method right now that is too exciting

for all the different seed types.

With chlorine we do know that there are

some problems with effects on germination and yield

for some of the seed types so it\"s not universally

231

acceptable either so I think there\"s not one

universal method that can be really applied for all

seed types right now.

DR. BERU: Thank you.

Go ahead, please.

DR. WARRINER: We always talk about a five

log reduction in testing efficacy. I\"ve got doubts

about that for sprout seed studies for the obvious

reason. If you have a low number of survivors in

20 or 38 hours time you can have 10

3 again so I

just wanted to know Professor Fett\"s thoughts on do

you think that needs to be revised as in the log

five criteria?

DR. FETT: Well, I don\"t know if it needs

to be revised. I mean the rationale for that was

in the back of the white paper, I believe, in \"99

that was published by the committee. I think what

we\"re ideally looking for is elimination. I would

agree with you there. Definitely elimination is

preferable to showing a five log reduction because,

as you just said, if we have some survivors there

there\"s a good potential for them to grow out.

232

One thing that I didn\"t talk about that

I\"m really interested in is trying to combine

chemical and biological methods where we would

treat the seed with a chemical method to knock down

the pathogens as low as we can get them, probably

not eliminate them, and then come in with a

competitive exclusion product which would not allow

whatever survivors are there from the chemical or

physical treatment to grow out to high enough

numbers to cause problems.

I think that\"s a possibility for the

future. It\"s not something that\"s going to happen

very soon unfortunately but I think it\"s an

exciting possibility because I think to totally

eliminate pathogens from this whole gamut of seed

types is probably unrealistic. Eliminating the

pathogens and keeping the seed viable and vigorous

and getting a good yield so I think elimination is

ideal but I\"m not sure we\"re going to get there but

elimination would be a lot better than a five log

reduction.

MR. WARRINER: I agree.

233

DR. BERU: Is the speaker on? For the

benefit of the transcriber, please state your name.

MR. RUST: Now it looks like it\"s on.

DR. BERU: Yes.

MR. RUST: The reason that we are all here

is for risk reduction in sprouts and one of the

things to increase the risk reduction or the risk

that there are pathogens in sprouts is to increase

compliance among sprout growers. And I heard some

really good things here today and a lot of it came

from questions that people from industry sent to

the FDA and the FDA responded, and I think very

well.

One of them is talking about a 24 hour

sample or testing after 24 hours instead of 48

hours, and that does wonders for the sprout grower

because of their hold and release. What can happen

in 24 hours is you test it for 24 hours and then

you have a presumptive positive and then you have

to still have that\--at any rate the quicker that it

can come back the easier it is to comply with

because that is quite a complication when you get a

234

false positive which does happen and then it may or

may not be out there and then you have to make a

decision of whether you want to contact your

customers and tell them that you may or may not

have bad product. At any rate, the quicker that

you can get those samples back the better.

Also, the concentrator, that would be

fantastic. That would save sprout growers a

tremendous amount of money.

Don Schaffner was talking about the 2,000

parts per million. That would make a world of

difference in compliance because right now 20,000

parts per million there\"s a lot of sprouts that

won\"t even grow under 20,000 parts per million and

then so sprout growers, \"Well, this whole thing is

just\--doesn\"t really work.\" And so what is real?

Anyhow\--and of course I like seed sampling

or I wouldn\"t have brought it up but I think those

four points would make a tremendous difference in

the sprouting industry and also I appreciate all

the help that you folks have done to do this and I

think the industry does too.

235

DR. BERU: Go ahead, please.

MS. SANDERSON: I have a question for you,

T-J. When you use the concentrator and then you

put up all those tests that you ran for trying to

detect pathogens, I had some trouble understanding

all your charts but I got the feeling that the

quick tests were pretty good at finding it but the

confirmation tests might not have been quite as

good. I was concerned if we were to use a

concentrator and we got a presumptive positive that

when we went to a confirmation test we might get a

false negative.

DR. FU: That\"s why I wanted to bring up

the improved confirmation tests with the

immunomagnetic separation. With usually the

culture method after enrichment you went straight

to a selected place and with so high\--the

background in sprout water is so high that when you

streak it there\"s a lot of things growing on the

selected plate so your ability to identify the

typical colony is affected by the background. But

if you would be able to use the immunomagnetic

236

separation to capture your pathogen before you

streak on a plate and that helped improve the

confirmative test. So that\"s why I wanted to point

it out that immunomagnetic separation is important

for the culture method.

DR. BERU: Yes, Mr. Sanderson?

MR. SANDERSON: This question is also for

T-J. It\"s very good to acknowledge the problems of

bean sprout growers in doing spent irrigation

sampling and you mentioned something like 100:1

difference in water use maybe. Now did you also do

counts? Okay. I point that out because I have

been doing just aerobic counts and coliform counts

and they\"re consistently higher by at least a log

on bean sprouts from alfalfa sprouts after 48 hours

and the bean sprouts are grown at about 90 degrees

whereas our alfalfa sprouts are grown at 70. This

is Fahrenheit. So I\"m mentioning that because in

looking around at ways to do this like tangential

flow concentration it seems to me there\"s a huge

difference in 10:1 reductions from 100:1 in terms

of costs and kind of availability for sprouters.

237

DR. FU: Right. I want to reiterate that,

yes, if you grow the sprout at a higher

temperature, say 30 degree versus 20 degree, you do

see a log increase in the background. But we

didn\"t see\--if we used immunomagnetic separation

that you can clear up the background and we didn\"t

see too much problem in terms of detection. Does

that answer your question?

MR. SANDERSON: It may be more important

if you understood mine because there\"s a lot of

things that I may not understand really clearly but

just that the levels\--I\"m not sure you would get a

one percent organism count in bean sprout water

after 48 hours. You might get ten percent of the

count for the green sprouts after the same length

of growing. In which case you might not need as

much concentration and the concentrator that you\"ve

shown is\--for me as a person in business\--a very

complicated thing and it\"s expensive. Okay.

DR. FU: Okay. I think now I understand

your question more. You are saying if you grow the

sprout, mung bean sprout, at a higher temperature,

238

the rate is higher and so the dilution you see with

water being irrigated on mung bean may not be too

much a problem because the level was higher.

That\"s good but we haven\"t done really experiment

on mung bean yet.

DR. BERU: I had a question for Dr.

Schaffner on the issue of 2,000 for this 20,000 ppm

calcium hypochlorite. You do show the distribution

of high and low reductions in the 2,000 but you

also I think said in your presentation on the need

to standardize how these measurements are done. In

view of that, what can one really say about those

distributions?

DR. SCHAFFNER: Right. That\"s a really

good question and you\"ve got to remember, too, that

the selection of the people who studied 20,000

probably overlapped with the people that studied

2,000 but it might be from different laboratories.

So you\"re absolutely right that you have to be

very, very careful about making those comparisons.

My only point in showing those two particular

distributions is that people somehow seem to have

239

fixated on 20,000 parts per million chlorine and I

just wanted to sort of try to open the door again

to alternatives to 20,000 and to say, hey, maybe

2,000 is almost as good to at least plant that seed

in your minds but not to say that definitively.

Again maybe to encourage my colleagues who are

looking at disinfection techniques to really sit

down and look at this rigorously to see if maybe

2,000 might not be almost just as good.

MR. LALLEY: I have got just a question

for anybody in the audience or the panel. Ms.

Tortorello referred to the uniformity throughout

the crop of the pathogen in the event of its

existence in a crop. I would have a question which

is if someone could explain to me how a 50 pound

bag of seed which produces perhaps eight to ten to

one pounds of sprouts is giving us an equivalent of

1,600 packages per drum or 10,000 servings per

commercial drum, how, in fact, such small numbers

of people can be affected in the outbreak and in

such small geographical areas as some of the

outbreaks which have been identified or supposedly

240

identified?

I would further like to comment on the

elimination versus germination. Elimination is the

key. Germination is a secondary issue. In my mind

if I get 50 percent germination and I\"m assured of

elimination of the pathogen, I couldn\"t care less

what it cost me for that seed as far as additional

costs to eliminate the pathogen.

And one final question is the inoculation,

the artificial inoculation, of seed for scientific

testing. It is very similar in my mind to taking a

hypodermic needle and infecting cows thoroughly

with E. coli and/or Salmonella and then pointing to

the cow and saying, \"Kill the Salmonella and E.

Coli but don\"t touch the cow.\"

DR. BERU: Did anyone\--yes, please.

DR. TORTORELLO: I\"d like to try answering

the first part. That is the relationship between

uniformity of contamination in a batch and the

number of people who become ill. Is that\--was that

your correlation?

MR. LALLEY: \[Not using microphone.\]

241

DR. TORTORELLO: Okay. Well, I guess I\"ll

try answering that with the consideration that

illness results because of at least two factors.

One, it\"s a quantitative issue. That is you have

to\--and perhaps Dr. Dechet from the CDC can also

chime in here. It depends on the number of

organisms that are ingested and it also depends on

who ingests them. So basically it\"s\--you may have

uniform contamination throughout the batch but not

necessarily uniform numbers throughout the batch so

a person who gets part of that batch may not

actually be ingesting the same amount of viable

microorganisms as another person and that person

also may have a higher resistance or better able to

withstand a pathogen being consumed.

Dr. Dechet, would you like to say

something?

DR. DECHET: Yes, it is actually a very

common question that comes up that if there\"s so

much product out there how is it that so few people

are getting sick. We often refer to the disease

pyramid. If you think about like the part of

242

the\--at the top of the pyramid is the number of

people reported to the CDC of getting ill. But if

you think the bottom, the base of the pyramid,

first you have all the people who eat the product,

then you have those who actually let\"s say get

diarrhea, and if you think about when you\"ve gotten

diarrhea how many of you have actually gone to the

doctor. Probably not that many. Most of people

sort of ride it out at home. Then you have the

group that actually goes to the doctor and how many

of those actually get cultured. As a physician

myself, I know that very rarely do physicians

choose to culture diarrhea because usually by the

time the results get back the patient is better.

It\"s really the most severe cases that get

cultured. And then of those that get cultured, how

many actually make it to the lab, get confirmatory

testing and so forth. How many of those then get

reported and how many\--if at the CDC we get every

tenth Salmonella sample, how many actually make the

CDC? So there\"s actually a multiplier effect down

the road where we may only being seeing six. We

243

expect that the number of people that actually got

sick from a product is really much higher.

MR. LALLEY: How many\--

DR. BERU: Excuse me. Please get to the

microphone because this needs to be captured by the

transcriber.

DR. DECHET: I\"ll just repeat the

question. I believe what it is what is about the

geographical distribution? It\"s a good question.

I think, you know, some of the points that were

just mentioned here are ones that might address

some of those issues. You also have to realize

that the degree of reporting behaviors are

different from state to state and so again while

some states DNA fingerprint every sample that comes

in, some only do every tenth, so that may have

something to do with it. I also am not that

familiar, and probably someone in the industry can

help me with this, about are they sent to different

areas or the sort of concentration that some areas

tend to get seed from one place and not from

another place. I don\"t know but also I think the

244

behaviors of the sprouters are probably quite

different from area to area and I would welcome

other people to comment on that.

DR. BERU: Any other? Yes?

DR. SCHAFFNER: Just a follow up on that.

Another explanation that I think we need to

carefully think about is that it may only be a seed

or two in a batch and we are doing some research

that hasn\"t been published yet but that shows that

the organism actually from a single seed or a very

small handful of seeds will spread rather slowly

across a tray during the four day irrigation so it

may well be that in this entire growing chamber

full of sprouts not all of them are positive for

the pathogen. So it may well be that these

outbreaks, the smaller outbreaks, are isolated

because there\"s not that much product implicated.

And then just a quick reaction if I could

to the concept of elimination. I think one of the

things that we need to think about very carefully

is that elimination of a pathogen\--you can\"t talk

about elimination of a pathogen until you know the

245

starting number. If I have 10

4 and I have a five

log reduction there\"s a probability that I\"ll have

pathogens left at the end but if I start with 10

6

and I have a five log reduction I\"ll have one log

of organisms there so I think it\"s very dangerous

to start talking about elimination. We really need

to be focused on a log reduction.

Now I\"m not here to say whether five log

reduction is right or wrong but we need to move

away from the concept of elimination because

elimination assumes a starting concentration. If

we don\"t make that explicit we end up chasing our

tails for a long, long time.

DR. BERU: Dr. Smith has a question or

comment.

DR. SMITH: Along the five log reduction

line, as Dr. Fett said, NACMCF went through a

calculation and came up with five log. I think

they\"ve explained that in the white paper. Five

log is a pretty common performance standard. We

used it in HACCP. FDA did not explicitly adopt

five log in our guidance for a number of reasons.

246

One was that we, ourselves, had not evaluated the

adequacy. If there are six log pathogens present

and you only get rid of five log you\"ve still got a

problem. And the other thing is before we were to

require five log we\"d like to have a way to get

there.

So we\"re between a rock and a hard spot

and as this process goes forward I can almost

guarantee that the people working on the solution

or the regulation or whatever it ends up being will

be asked to come up with a performance standard, a

description of the endpoint that needs to be

achieved. Whenever we can we describe that

endpoint and then give as much flexibility as

possible on how to get there so I want all the

great minds in the room together to start thinking

about that and put it in your comments.

Also, Dr. Fett, you mentioned

internalization. This is an issue certainly with a

lot of fresh produce items where we know if you put

a warm tomato in cold water a suction effect will

pull in water and pathogens if they\"re present. I

247

saw slides of pathogens getting in the root hairs

of sprouts during the production process during

sprouting but I\"m starting to hear more about the

potential internalization of pathogens in sound,

intact seed, not scarified and not damaged. So if

anybody here can comment on their opinion, the

likelihood of that happening.

DR. FETT: Well, I can just comment that

seeds normally aren\"t sterile. You can get

internalization of native bacteria into seeds and

also plant pathogenic bacteria can be in the seed

coat. Unfortunately, as far as I know with

bacterial human pathogens on sprout seed, and

somebody correct me if I\"m wrong, I don\"t think we

have any good data on where exactly they\"re

located. If some are internal and some are

external or they\"re all external, I don\"t think we

have that information.

But I think it\"s very clear from several

lab studies that the potential for bacterial human

pathogens entering through the root system

primarily where the secondary root hairs emerge

248

where you have natural wounding occurring is a

distinct possibility and I think the potential is

there under commercial conditions that that could

happen.

DR. BERU: This gentleman had a question.

You may need to come down here.

MR. MEYEROWITZ: I\"m Steve Meyerowitz from

Sproutman Publications and I guess I\"m here

representing the home sprouter, the kitchen

gardener as it were, of which there is a large

contingent. I have a comment and a question. My

comment is that if we were to make our way down to

2,000 parts per million of chlorine, the public\--it

would solve a lot of public relations problems for

the industry because right now there is a

perception that the 20,000 parts per million\--this

is a public perception\--is overkill as it was and

it actually discredits any other guidance that the

FDA might come out with because of this kind of

heavy handedness so that\"s the perception.

My question is for you, Dr. Fett. When

you talked about whole microbial communities that

249

really interested me because I really think that it

is at the\--the competitive microbial community is

very interesting because essentially that\"s what\"s

going on inside our systems is that we have a

competitive microbial community which is part of

what keeps our immune systems functioning and

protects us from most of the pathogens\--low levels

of pathogens that are commonly found in food\--in

all foods. And, in fact, in our whole

environment\--I think\--don\"t you think\--I didn\"t

even think about that after I washed my hands in

the rest room and I had to walk out and you have to

touch the door that hundreds of other people touch.

So we really need that internal friendly microbial

community helping us.

So my question to you about this

competitive microbial community is how can we get

more research on the schedule in the queue as it

was and is there any downside to using competitive

microbes here? Can it cause other problems?

DR. FETT: As far as more research, talk

to your congressman. Have him send some money to

250

the FDA and the USDA for that kind of work. I mean

we\"re going to\--we\"re in the process of writing up

our next five year project plan right now in ARS

for all of our food safety related projects and in

my group, which is one of really probably

the\--there\"s three produce safety related groups in

the U.S. and ARS. One is my group in Wyndmoor.

There\"s one in Beltsville and there\"s one in

California.

As far as interventions go, it\"s primarily

our group and some work in Beltsville.

As far as competitive exclusion goes it\"s

our group in Wyndmoor and it\"s essentially one

scientist doing the work and it\"s a very

complicated issue. We have been looking at single

strains of fluorescent pseudomonades as well as

whole communities and a little work on mixtures and

biocontrol is a little bit frustrating in that it

seems to work well and quite often sometimes but

then you have an experiment or two where you have a

failure. I\"m not exactly sure why that is.

Biocontrol has been worked on for years

251

and years and years and years in the plant

pathology field. Since the 1920s there has been a

lot of work on that. There continues to be a lot

of work on that and there\"s really not too many

products out there. That\"s why I\"m saying it\"s a

long term research and development program.

And if you\"re talking about one scientist

being responsible for doing that work it\"s a really

long-term project so, you know, the more emphasis,

I think, on that the better but right now we really

only have one scientist working in that area.

It\"s interesting to me why Salmonella and

E. coli can compete so well with the native

microflora to begin with during the sprouting

process. I wouldn\"t have expected that really to

tell you the truth but there is evidence in the

literature if you inoculate Salmonella on to a

sprout that has been grown for 24-48 hours the

pathogen won\"t grow or grow very, very little.

So the key is to have enough competitive

microflora there at the time of germination of a

contaminated seed that you get that competitive

252

exclusion happening but I wish we had more money

and more people to do the work but right now as far

as I know in ARS there\"s only one person that will

be assigned to that kind of study.

DR. BERU: Thank you, Dr. Fett.

We have to save time for the public

comment portion of the meeting so I hate to end the

question and answer but we\"ll end it here, and I

thank the panelists.

We\"ll take a 15 minute break and be back

here about five minutes to 4:00. Thank you.

\[Whereupon, a break was taken.\]

DR. BERU: Welcome back. I think if you

will take your seats we\"ll continue with the last

phase of this meeting.

Before we go into the public comment

period I have a list of four people who are

pre-registered to give comments. Are there any

others that want to be added to the list?

Okay. Then I\"ll just call on them in the

order they are written here.

Mr. Lincoln Neal is our first commenter.

253

No. Okay. So we have three public commenters.

Mr. Keith Warriner is our next. You can

use the microphone.

PUBLIC COMMENTS

MR. WARRINER: Thank you. Really I\"m just

going to speak five minutes on some of the research

we\"ve been doing up in the University of Guelph. I

think if you picked up the handout that really

describes the overheads and the work. Essentially

what it is, is a seed decontamination method based

on a sanitizer which we termed SDH. It has gone

through various names but essentially SDH has been

used in lots of different products, be it

toothpaste, contact lens cleaners, chemotherapy

drug, which has also been considered so essentially

it is a very safe sanitizing product, if you like,

which has got a long history of safety.

The reason why we chose SDH to

decontaminate the seeds was really based on its

phytocompatibility because it doesn\"t damage human

tissue or eukaryotic tissue it obviously preserves

the actual seed integrity and viability but what it

254

does do is inactivates pathogens fairly readily.

We\"ve just done some studies as you can

see in the handout where we have inoculated seeds

with E. coli or Salmonella, a treatment of these

seeds was something in the range of 200 ppm, which

is very low. It eliminates the pathogen. There is

no pathogens on the subsequent sprouts and that\"s a

key point because we don\"t just test the seeds to

see if we get the five log reduction as was

referred to earlier. We actually cultivate the

sprouts for four days afterwards just to ensure

that there is no pathogens there.

So what\"s really different about this

approach is that whereas other approaches tend to

try these strong oxidants before soaking the seed,

we actually put in the sanitizer and join the seed

soaking stage so we\"ve got a higher contact time

with the seed itself and also as the seed starts

germinating obviously the protective sites within

the seed coat start to decrease and the pathogens

emerge where you can inactivate them.

So we have tried this on a little

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different seed types. It does depend on the seed

but the ones we have successfully decontaminated is

alfalfa, mung beans, clover, flax. The ones we

haven\"t been able to decontaminate is radish and a

few others. So it is seed dependent. We\"re not

claiming to be able to decontaminate every seed but

the simple fact is that it\"s very cost effective.

It\"s a very simple solution. There\"s no part where

you couldn\"t do it domestically large scale.

Another part we\"ve done is actually

produce naturally contaminated seed. We did this

by inoculating the flowers of mung bean plants with

a cocktail of Salmonella and E. coli. When we

actually harvested the subsequent beans seven weeks

later we found all were contaminated to Salmonella

and E. coli existed on a few of them.

Significantly, with our treatment with SDH we could

actually effectively decontaminate the seeds.

We\"ve also done some work with microbial

populations through DGGE work, which essentially is

a molecular technique for seeing if there\"s any

different population changes due to SDH and we

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found only really two main bacterial types were

missing from SDH treated seeds but there were no

new bands which means that no new populations have

been added.

I would just like to finish up with some

work we\"ve also been doing with looking at the

distribution of contamination within sprouting mung

bean beds. As you know, sprouting mung bean beds

are basically containers\--performed in containers,

usually 25 kilograms, unlike alfalfa which is

obviously a tray. What we did to look at the

distribution of contamination was to look for

generic E. coli, fecal coliforms and mesophilic

aeromonas because it is better than looking for

pathogens themselves but what we did find at the

end of the day is that contamination was

heterogeneously distributed. So if you try to

collect one liter sample of spent irrigation water

the chances of that representing actual microbial

status of the mung bean bed are very low indeed and

this is why I was interested in your concentration

method.

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We also did some lab trials using colonies

of sprouting mung beans with E. coli 0157 and

Salmonella. What we did here is we had essentially

a 15 centimeter colony of sprouts and we put one

gram of inoculated seed at different locations to

see how the contamination spread and if we could

detect it in spent irrigation water.

The reason why we took these small

concentrations of inoculated seed and put them in

the sprouting colony is because with seed

decontamination it\"s likely that\"s what you would

have. You would have only a proportion of the seed

perhaps which is going to be contaminated where the

rest has been successfully decontaminated.

What we actually found was that

contamination introduced in this way with

localized\--within specific sites, a specific\--more

significantly over 50 percent of the spent

irrigation water samples gave false negative

results. Whether that\"s actually representative of

what is happening in commercial practice is

questionable but it just illustrates the need to

258

validate testing protocols and identify if it

does\--if spent irrigation water testing does

represent what\"s found in the mung bean bed.

So really that\"s just in a nutshell about

the research we have done and, hopefully, we\"re

going to take the SDH treatment further. It is not

going to be easy getting regulatory approval, of

course, and then commercial trial but I was very

interested in the standardization procedures

because I think that\"s what\"s needed. A

standardized procedure to validate and verify seed

decontamination methods so the industry itself

isn\"t confused by it and we have a level playing

field.

Thanks.

DR. BERU: Thank you, Mr. Warriner.

I\"ll next call on Mr. Richard Norton.

MR. NORTON: Hello. We have a small

company that is interested in getting started in

this field. We don\"t know anybody in the field

hardly but we have had some success with it now.

It\"s an interesting new material because it\"s so

259

nontoxic. We would like to supply small samples to

anybody who is treating seeds and different kind of

seeds and who has the ability to sample and measure

the reductions that they get. So if anybody is

interested, let me know and we\"ll see that you get

some advice. We\"ve worked with these chemicals now

for a long time and we have found for different

uses we have to have different mixtures but that a

lot depends on the surface that is being treated

and the ones that we worked out for this was for

the waxy coated alfalfa seeds. So other uses may

have some other mixtures that we are familiar with.

DR. BERU: Thank you, Mr. Norton.

I would next like to call Mr. Steve

Meyerowitz.

MR. MEYEROWITZ: Thank you. I just have

some brief comments that I want to remind everyone

that we have a wonderful product that is very

unique. Sprouts are the only form of agriculture

where you can grow it in any season of the year in

any locality, whether it\"s Atlanta or Alaska, and

we\"re going to need to protect this form of

260

agriculture. We have an economic system which is

seeing fewer and fewer farms, family size farms, in

our local communities. They\"re being sold.

They\"re being turned into strip malls. They\"re

being turned into condominiums. So our local

economies are losing the advantage of local

agriculture.

Sprouts offer something in return for

local agriculture. If we don\"t have enough land

sprouts can be grown in factories. They can be

grown in factories that are reclaimed from old

industry or paper mills that no longer exist.

So it\"s an industry we need to support for

our local economy and for the fact that it\"s a

wonderful source of food. As our population or the

world population doubles every 20 years, we have

less space, more people, less space to grow food,

our food is becoming internationally distributed

long distances, quality and issues of pathogens are

becoming more and more complex. Sprouts are a form

of agriculture that can return us to local sources

of food, whether it\"s a local sprout grower that\"s

261

in your county or whether it\"s you growing it in

your own kitchen. That\"s what is special about it.

But in addition to that these baby plants

offer many wonderful advantages in terms of

personal health and public health. We\"ve

discovered not only that their nutrition is many

times multiplied in the young plant, such as things

like protein in sprouts is approximately four

percent and in iceberg lettuce, the most common

competitor in the salad bar, it\"s .8 percent. So

less than one percent. So more than four times the

protein. Things like the vitamin A of radishes, of

mature radishes that come from your backyard

garden, is only about 10 international units and in

radish sprouts it\"s 391. It\"s 39 times greater.

But more so than just a question of

vitamins, there is also the minerals and trace

minerals because it\"s one of the only forms of

agriculture where we can eat the whole plant,

including the roots and the roots are where we find

a concentration of minerals and trace minerals.

Then we have things like the

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glucosinolates in the whole brassica category of

sprouts where they can actually interrupt the

development of malignant cells. We have things

like sapponins which are very rich in alfalfa and

clover and the sapponins actually have advantages

in reducing osteoporosis and reducing the symptoms

of menopause and hot flashes. And we have

isoflavones. Soy bean sprouts are wonderful

sources of isoflavones. Also for advantages in the

cardiovascular system. You can go on and on. The

antioxidants and bio-flavinoids. It\"s a wonderful

source of nutrition that\"s just really being

discovered.

And it\"s one of the finest sources of raw

food nutrition that we have available to us. The

National Institutes of Health and the National

Cancer Institutes are constantly telling us to

increase the number of portions of raw fruits and

vegetables in our diet. It\"s harder and harder for

Americans to do that. Sprouts are a source of raw

food nutrition that is available at an affordable

price that is locally grown. I think those are

263

some of the reasons why we have to work harder to

protect this industry to find solutions that are

acceptable to the public.

I mentioned earlier that the public has a

certain\--that the FDA has some credibility issues

with the public. Not all of the\--not all consumers

trust the\--what their government\--what our

government regulations and rules are, especially if

they are perceived as being heavy handed.

We have many issues of food safety in our

food supply. It\"s not just a question of

pathogens. We have issues such as pesticide levels

and what levels of pesticides are acceptable. We

have many dangerous things that exist in our food

supply and we need to find ways to improve the

quality of our food supply and sprouts are one

important area that I think can mean a lot for us

as we go forward in the future with prices of oil

increasing, the cost of transporting food

increasing and the quality of our food and the need

for more fresh fruits and vegetables. Sprouts as

an industry needs to be protected so that we can

264

keep the quality of our food available to the

public, and that\"s my reminder for all of us here.

Thank you very much.

DR. BERU: Thank you, Mr. Meyerowitz.

That brings us to the end of the public

comment portion of the meeting.

By way of concluding remarks I would like

to say that\--

MR. LALLEY: Is there time for one more?

DR. BERU: Yes, by all means.

MR. LALLEY: I am Mike Lalley, Living

Foods. I will try to take just a few seconds or

minutes of your time. First of all, I would like

to recognize and make it crystal clear that I

recognize the risk in any food product.

Specifically to quote the good folks from labs,

they say there is absolutely no such thing as zero

risk and that can be evidenced through the green

onion outbreaks, the tomato, the strawberries, the

apples, the apple cider, et cetera, et cetera.

Since 1996 there has been 160 or 1,600 cases and in

the last three years there has actually been about

265

an average of 35 cases a year.

I\"m holding an FDA document here that

talks about an estimated 120,000 illnesses per year

are caused by the consumption of eggs. Relatively

that number is obviously of greater significance.

However, we\"re not here to talk about eggs.

So what I am here to ask is that FDA and

CDC embark on a thorough and critical review of the

epidemiological evidence which is the prime

evidence against sprouts. There have been\--of the

27 cases in the last ten years there has only been

a very small handful, perhaps two or three, where

they have been able to culture pathogens from

either the seed or the finished products. And, as

such, like I say, that is the prime evidence

against our industry.

In 1995 there was a Salmonella outbreak in

Michigan and a few other states and the respondents

in the epidemiological survey, 25 percent of the

respondents who had the genetic fingerprint, the

supposed genetic fingerprint, only 25 percent of

those respondents said that they had actually

266

consumed sprouts.

In 1999, when there was an E. coli 0157:H7

outbreak in Michigan and Virginia, when the good

folks from CDC, Michigan Department of Agriculture

and Michigan Department of Health knocked on my

door, they had not completed their epidemiological

study but at that point they said, \"The reason

we\"re here is vegetarians are ill and it makes

sense that we should come and talk with you.\"

At that moment in time I thought maybe

they had a valid point. Upon further discussion

with other CDC scientists they tell me that if

there\"s an outbreak amongst a particular ethnic

group, say Middle Easterners, and we ask them what

they\"ve consumed and they tell us that they have

consumed humus, babaganosh and other food items

peculiar to their ethnic origin, and then we

compare them with a control group off of the street

of John Blow and his cousin Freddy, we see that

every time John and Freddy consumed no humus and no

babaganosh and as a result there is a statistical

number that arises that implicates the items which

267

are peculiar to that ethnic group.

In this case the vegetarians obviously

were the ethnic group that I\"m referring to and the

control group in no way, shape or form took that

into any consideration whatsoever.

As a matter of fact, Dr. Brewer expressed

a great deal of satisfaction in his written report

on that study where he was showed a great deal of

satisfaction and I would say joy in the fact that

he was able to get a 46 percent positive response

rate from those afflicted consumers that had the

genetic fingerprint. He did this and admits such

in his report by first of all asking multiple

questions regarding sprouts and, secondly, by

preceding his questioning with a leading

description of the potential culprit. As in, for

instance, a small caucasian man, 5\"7\", blue eyes,

balding, was he the guy? And then, in fact, he was

thrilled when 46 percent said, \"Well, it\"s possible

that could have been,\" because he did say that they

could have been hiding underneath the sliced ham or

baked or who knows what. But he got 46 percent

268

positive rate. 54 percent of the people that were

sick in the State of Michigan, never consumed the

sprouts, swore to god. I\"ve got newspaper

articles\--pardon me?

DR. BERU: We are out of time.

MR. MEYEROWITZ: Okay. I will go really

quickly. Anyway, so these vegetarians\--okay. So

let\"s just see here. I\"ll try to skip through some

of this. Anyway, the epidemiology study in that

\"99 case showed a prophylactic effect was\--occurred

by the consumption of ground beef. Well, now I

don\"t know what you know about ground beef, and I

know very little being a vegetarian since 1969, but

I\"m here to tell you that ground beef will not save

you from human pathogens.

When the recall was issued\--a recall was

subsequently issued in Michigan and Virginia and

the outbreak continued for over six weeks in

Virginia. And it just so happens the reason that

happened is that they had secondary contamination.

One person\--secondary transmission from one person

to another because the slobs in Virginia don\"t wash

269

their hands where thank god in Michigan they did.

Well, I\"m here to tell you that is

absolutely ridiculous. It didn\"t\--that isn\"t the

way it happened.

Anyway, to conclude, risk/benefit.

Obviously there\"s a risk in all food items and we

don\"t care what we call them, what they are, what

your preferences are. Risk/benefit, we had Vioxx

and Celebrex. The risk was 25,000 dead bodies

littering the streets of America. The benefit,

however, great though it may have been, that at

least their back didn\"t hurt for a couple of weeks

prior to passing on.

In the case of sprouts, granted we\"ve got

a situation here where human pathogens can be

transmitted to any food form. However, the

benefits are such that the broccoli sprouts, for

instance, are better\--shown to have been more

effective than the prescription drugs in treating

ulcers. They have been shown to prevent and in

some cases perhaps cure cancer.

So what is the benefit? What is the risk?

270

If I take a 50 pound bag and they bring me a 50

pound bag of lettuce, and I take it into my

facility, could I have GMPs, SOPs, HACCP, and I

take 25 pounds of this bag of seed and I grow it in

my sprouting facility, god forbid that anybody\--a

consumer of any sort who wants to avoid

contamination should consume it. Yet if I take

that same 25 pounds, take it into the warm moist

environment of my field out back behind the top\--my

barn, and where animals have defecated for years,

where rodents, insects are flying in from the

livestock farm across the street, then there is no

risk whatsoever there. Eat up, don\"t worry.

And the final conclusion is what they\"re

trying to do is they\"re trying to turn you all into

little Howard Hughes. You\"ll never have his money

but if you have his fear you will be living in his

world.

SUMMARY OF MEETING AND ADJOURN

DR. BERU: Thank you. Thank you very

much.

That now truly brings us to the end of the

271

public comment portion of this meeting.

By way of concluding remarks, I would like

to say that we do believe that a good first step to

improving the safety of sprouts is to engage in

solicited views of other government agencies at the

federal, state and local levels, industry, consumer

groups, academia and the public generally about the

current science relating to preventing or

minimizing foodborne illness associated with the

consumption of sprouts.

In this respect this has been a very

useful meeting. You have heard from the panelists

as well as the commenters on various aspects of the

issue. From the merits of screening and sampling

seeds prior to sprouting to the various studies

conducted on the various seed and sprout treatment

methods.

We hope to also get additional comments

and the comment period is open through July 18th.

With that, I would like to end the meeting

by thanking all involved in making this truly

important meeting happen.

272

A special thanks to our panelists and

thanks to you all very much for taking time from

your busy schedules to participate in this meeting.

Thank you.

\[Applause.\]

\[Whereupon, at 4:31 p.m., the proceedings

were adjourned.\]

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