Microbiological Data Program - Annual Summary, Calendar Year 2008

United States

Department of

Agriculture

Agricultural

Marketing Service

Science &

Technology

Programs

Microbiological Data Program

Progress Update and 2008 Data Summary

Please Visit Our Website at www.ams.usda.gov/mdp

September 2009
Microbiological Data Program - Annual Summary, Calendar Year 2008

United States

Department of

Agriculture

Marketing and

Regulatory

Programs

Agricultural

Marketing

Service

1400 Independence Ave.

Washington, DC

20250

AMS-Agricultural Marketing Service

An Equal Opportunity Provider and Employer

September 2009

To the Reader:

I am pleased to present the USDA Microbiological Data Program (MDP) 2008

Data Summary. In 2008, MDP tested five commodities (alfalfa sprouts,

cantaloupe, lettuce, spinach, and tomatoes). MDP also performed a special survey

of tomatoes, cilantro, bulb onions, green onions, and hot peppers to assist the

Centers for Disease Control and Prevention and the U.S. Food and Drug

Administration during the Salmonella saintpaul outbreak investigation.

MDP is a partnership with cooperating State agencies that are responsible for

sample collection and analysis. Eleven States participated in the program in 2008:

California, Colorado, Florida, Maryland, Michigan, Minnesota, New York, Ohio,

Texas, Washington, and Wisconsin. Because together these States represent all

regions of the country and more than half the Nation’s population, MDP data can

be used to develop inferences about the national food supply. With a sampling

framework and testing laboratory capability in place, MDP has demonstrated its

ability to quickly mobilize and respond to outbreak situations providing data

rapidly during local and national outbreaks.

This summary is intended to provide the reader with an overview of data collected

in 2008 and summarizes program refinements made during that year. MDP data

are important in developing baseline levels of targeted pathogens in the domestic

food supply. As a continuous data-gathering program, MDP data can be used to

identify microbial trends and to develop risk models.

If you have comments or suggestions on how this summary can be improved,

please send electronic-mail to amsmpo.data@ams.usda.gov or visit our Web site at

www.ams.usda.gov/mdp.

Sincerely,

Rayne Pegg

Administrator

Rayne Pegg
Microbiological Data Program - Annual Summary, Calendar Year 2008

Contents

Page No.

Acknowledgements........................................................................................................................ vii

Executive Summary ........................................................................................................................ ix

Section I. Introduction..................................................................................................................... 1

Section II. Sampling........................................................................................................................ 4

Section III. Laboratory Operations.................................................................................................. 9

Section IV. Database Management ............................................................................................... 10

Section V. Summary of 2008 Data................................................................................................ 12

References..................................................................................................................................... 19

Disclaimer ..................................................................................................................................... 19

Definitions..................................................................................................................................... 19

iii
Microbiological Data Program - Annual Summary, Calendar Year 2008

v

Figures and Tables

Figures Page No.

1 MDP Program Planning and Program Testing Operations............................................2

2 Program Participants .....................................................................................................3

3 Commodity Origin.........................................................................................................7

4 MDP Data Pathway ....................................................................................................11

Tables

1 Number of Samples Tested During Salmonella saintpaul Outbreak ...........................5

2 Distribution of Imported Samples................................................................................6

3 Samples Collected by State .........................................................................................8

4 Number of Samples Analyzed....................................................................................13

5 Number of Samples Tested for E. coli .......................................................................13

6 Summary of Sample Analysis for Pathogenic E. coli ................................................14

7 Characterization of Pathogenic E. coli Isolates Screened by mPCR .........................15

8 Number of Samples Tested for Total Viable Count...................................................16

9 Number of Samples Tested for Coliforms .................................................................16

10 Summary of Analysis for Salmonella ........................................................................16

11 Salmonella Identification and Serogroup...................................................................17

12 Summary of Analysis for E. coli O157:H7 ................................................................18

13 Summary of Analysis for Shigella .............................................................................18
Microbiological Data Program - Annual Summary, Calendar Year 2008

vii

Data presented in this report were collected and processed through the efforts of the following

organizations:

State Agencies

California Department of Pesticide Regulation

Colorado Department of Agriculture

Florida Department of Agriculture and Consumer Services

Maryland Department of Agriculture

Michigan Department of Agriculture

Minnesota Department of Agriculture

New York Department of Agriculture and Markets

Ohio Department of Agriculture

Texas Department of Agriculture

Washington State Department of Agriculture

Wisconsin Department of Agriculture, Trade and Consumer Protection

Laboratories

Colorado Department of Agriculture

Inspection & Consumer Services Division

Laboratory Section

2331 West 31st Ave.

Denver, CO 80211-3859

Florida Department of Agriculture and Consumer Services

Bureau of Food Laboratories, Bldg. 9

3125 Conner Blvd.

Tallahassee, FL 32399-1650

Michigan Department of Agriculture

Laboratory Division

1615 South Harrison Rd.

East Lansing, MI 48823-5224

Minnesota Department of Agriculture

Laboratory Services Division

601 Robert Street North

St. Paul, MN 55155-2531

New York Department of Agriculture and Markets

Food Laboratory

1220 Washington Ave.

State Office Campus, Bldg. 7

Albany, NY 12235

Acknowledgements
Microbiological Data Program - Annual Summary, Calendar Year 2008

Ohio Department of Agriculture

Consumer Analytical Laboratory Bldg. 3

8995 East Main St.

Reynoldsburg, OH 43068

U.S. Department of Agriculture

Agricultural Marketing Service

National Science Laboratory

801 Summit Crossing Pl.

Gastonia, NC 28054

Washington State Department of Agriculture

3939 Cleveland Ave., SE.

Olympia, WA 98501

Wisconsin Department of Agriculture, Trade

and Consumer Protection

Bureau of Laboratory Services

4702 University Ave.

Madison, WI 53705

Program Administration

U.S. Department of Agriculture

Agricultural Marketing Service

Deputy Administrator, Science and Technology Programs:

Robert L. Epstein

1400 Independence Ave., SW., Room 1090-S

Washington, DC 20250

Director:

Martha Lamont

Monitoring Programs Office

8609 Sudley Rd., Ste. 206

Manassas, VA 20110

(703) 330-2300 x 117, Facsimile (703) 369-0678

E-mail: amsmpo.data@ams.usda.gov

Web site: www.ams.usda.gov/mdp

viii
Microbiological Data Program - Annual Summary, Calendar Year 2008

ix

In 2001, the U.S. Department of Agriculture

(USDA) Agricultural Marketing Service (AMS)

was charged with implementing microbiological

testing of fresh fruit and vegetables in the

United States. The program’s mission is to

provide statistically reliable information

regarding targeted foodborne pathogens and

indicator organisms on fresh fruit and

vegetables. The Microbiological Data Program

(MDP) is a voluntary data-gathering program,

not a regulatory enforcement effort.

AMS coordinates MDP planning and program

requirements on a continual basis with the

Centers for Disease Control and Prevention

(CDC), the U.S. Food and Drug Administration

(FDA), and the USDA National Agricultural

Statistics Service. The participating States are

an important component of MDP program

planning activities, particularly those involving

technical and quality assurance (QA) issues.

MDP collects produce samples from terminal

markets and wholesale distribution centers on a

year-round basis. The MDP sampling frame is

designed to take into account population and

consumption on a national scale. In 2008, 11

States collected fruit and vegetable samples

(California, Colorado, Florida, Maryland,

Michigan, Minnesota, New York, Ohio, Texas,

Washington, and Wisconsin). MDP samples

are comprised of both domestic and imported

produce, as well as conventional and

organically produced crops and, for some

commodities, pre-washed, ready-to-eat produce.

The program tested five commodities: alfalfa

sprouts; cantaloupe; pre-washed, pre-cut,

bagged lettuce; fresh (unwashed) and prewashed, bagged spinach; and tomatoes.

Samples were screened for Salmonella,

Shigella, and pathogenic Escherichia coli (E.

coli) carrying shiga toxins and enterotoxins,

including E. coli O157:H7. All five commodities were tested for generic E. Coli, and bagged

Executive Summary

lettuce and bagged spinach samples were

enumerated for Total Viable Count (TVC) and

total coliforms. Any pathogens that were

isolated and confirmed were shipped to FDA’s

Center for Veterinary Medicine (CVM) for

additional characterization, including antimicrobial resistance and genomic fingerprinting by pulsed field gel electrophoresis

(PFGE); antimicrobial resistance data were

included in the National Antimicrobial Resistance Monitoring System (NARMS) database

and PFGE patterns were entered into CDC’s

Pulsenet database. In addition, CVM performed serotyping on all Salmonella isolates.

All pathogenic E. coli isolates were shipped to

Pennsylvania State University for serotyping

and testing for 17 virulence-specific genes

associated with different categories of pathogenic E. coli. MDP communicates pathogenic

findings to Federal and State agencies that in

turn, notify vendors. Affected products may be

voluntarily removed from the food distribution

system.

During the Salmonella saintpaul outbreak,

MDP laboratories tested additional samples of

tomatoes (601), cilantro (219), bulb onions

(150), green onions (141), and hot peppers

(225) to aid CDC and FDA in the outbreak

investigation. Samples were collected in

Colorado, Maryland, New York, Texas, and

Washington.

MDP analyzed a total of 11,669 samples during

the 11 months of sampling and testing

operations. Seventy-nine percent of the samples were from domestic sources, 19 percent

were imported, and 2 percent were of

unspecified origin. MDP screening resulted in

16 Salmonella isolates, and MDP laboratories

isolated pathogenic E. coli strains from 11

samples. MDP screening for E. coli 157:H7

and Shigella did not result in any confirmed

isolates.
Microbiological Data Program - Annual Summary, Calendar Year 2008

A number of important benefits are being

derived from MDP. Coordination with public

health agencies has increased, allowing early

intervention by regulatory agencies when

problem areas are identified, and communication among State and Federal agencies for

reporting and sharing data on foodborne

outbreaks has improved. Microbiological data

obtained from MDP’s fresh produce screening

effort can be used to enhance the understanding

of indicators and potential pathogens in fresh

fruit and vegetables in the U.S. food supply,

permit the identification of long-term trends,

and contribute significantly to a national

produce microbiological pathogen prevalence

baseline. MDP data, which in part reflect the

changes in cultivation, irrigation, harvesting

practices, postharvest handling and packaging

of fresh produce to meet changing consumer

life styles, preferences, and demands, will help

refine Good Agricultural Practices (GAP) and

Hazard Analysis and Critical Control Points

(HACCP) plans used by growers, processors,

and food handlers. Furthermore, MDP data

which include antimicrobial resistance, genomic fingerprints, serotypes, and virulence

attributes, will help collaborators such as CDC

and FDA in planning public health initiatives

and responding to produce-related foodborne

outbreaks.

x
Microbiological Data Program - Annual Summary, Calendar Year 2008

1

I. Introduction

Fresh produce is recognized as an important

component of a healthy diet. Because most

produce is grown in a natural environment, it may

be vulnerable to contamination with pathogens. Produce is often consumed raw, without

any type of intervention that would reduce or

eliminate pathogens prior to consumption, which

contributes to its potential as a source of foodborne illness (1, 2). In 2001, Congress authorized

funding for a microbiological monitoring

program to collect data on fresh fruit and

vegetables.

The Microbiological Data Program’s (MDP)

mission is to collect information regarding the

incidence and identification of targeted foodborne

pathogens and indicator organisms on fresh fruit

and vegetables. This publication provides an

overview of data collected in 2008 and summarizes program refinements made during that year.

The Agricultural Marketing Service (AMS)

Monitoring Programs Office (MPO) manages

MDP and is responsible for administrative,

sampling, technical, and database activities.

Figure 1 (a) illustrates MDP program planning

activities. AMS coordinates its planning and program requirements with the Centers for Disease

Control and Prevention (CDC) and the U.S. Food

and Drug Administration (FDA). MDP relies on

the expertise of scientists from FDA, CDC, and

academia. AMS and USDA’s National Agricultural Statistics Service (NASS) statisticians

designed sampling plans based on per capita

consumption, marketplace availability, product

origin, and time in transit and storage. The

participating States are an important component

of MDP program planning activities, including

technical and quality assurance (QA) issues.

Figure 1 (b) depicts MDP program testing

operations. The participating State laboratories and AMS National Science Laboratory

(NSL) analyze MDP samples collected by

trained State sample collectors. FDA’s Center

for Veterinary Medicine (CVM) and Pennsylvania State University (PSU) provide

additional testing services for isolate characterization. Information on MDP data and

isolates is shared with CDC and FDA.

Commodities tested were selected in

consultation with FDA and were chosen

because they are high-consumption fruit and

vegetables in the U.S. diet, are often

consumed raw, and have been implicated in

foodborne outbreaks. Commodities tested in

2008 included: alfalfa sprouts; cantaloupe;

pre-washed, pre-cut, bagged lettuce; fresh

(unwashed) and pre-washed, bagged spinach;

and tomatoes. Commodities were tested for

generic Escherichia coli (E. coli), Salmonella,

Shigella, and E. coli strains with human

pathogenic potential, including E. coli

O157:H7. Isolates of these organisms were

sent to specialized laboratories for further

characterization including testing for antimicrobial resistance, genomic fingerprinting,

serotyping, and virulence attributes. Prewashed, pre-cut, bagged lettuce and prewashed, bagged spinach samples were

enumerated for Total Viable Count (TVC)

and total coliforms. A special survey was

conducted on cilantro, bulb onions, green

onions, hot peppers, and tomatoes in response

to the Salmonella saintpaul outbreak of 2008.

Samples were collected in the 11 participating

States through cooperative agreements with

their respective agencies (Figure 2). Also

shown in Figure 2 are the 13 neighboring

Microbiological Data Program (MDP)

Annual Summary, Calendar Year 2008

This summary consists of the following sections: (I.) Introduction, (II.) Sampling, (III.) Laboratory

Operations, (IV.) Database Management, (V.) Summary of 2008 Data
Microbiological Data Program - Annual Summary, Calendar Year 2008

2

Figure 1. MDP Program Planning and Program Testing Operations. This figure illustrates (a) agencies/

groups that support MDP program policy and planning activities, and (b) agencies/groups that analyze

MDP samples, isolates, or results.

U.S. Food and Drug

Administration Participating

States

(a) MDP Planning

(b) MDP Program Operations

USDA,

Agricultural

Research Service

USDA, Food

Safety and

Inspection

Service

USDA, National

Agricultural

Statistics Service

U.S. Centers for

Disease Control

and Prevention

USDA,

Agricultural

Research

Service

Participating

States

USDA, AMS,

National Science

Laboratory

Pennsylvania

State University

USDA, Food

Safety and

Inspection

Service U.S. Centers for

Disease Control

and Prevention

U.S. Food and Drug

Administration /

Center for Veterinary

Medicine
Microbiological Data Program - Annual Summary, Calendar Year 2008

3

States that are in the direct distribution networks

for the MDP collection States: Alaska, Connecticut, Delaware, Hawaii, Idaho, Massachusetts,

Nevada, New Jersey, New Mexico, Oklahoma,

Vermont, Virginia, and Wyoming. Together,

these States represent more than 50 percent of

the Nation’s population and all geographic

regions of the country, with significant rural-tourban variability. Therefore, MDP samples are a

statistically defensible representation of the

country as a whole.

Analytical services were provided by

microbiology laboratories in eight States

(Colorado, Florida, Michigan, Minnesota, New

York, Ohio, Washington, and Wisconsin) and

one USDA AMS facility, NSL. Samples

collected by Maryland were analyzed by the

Ohio laboratory. Samples collected by Texas

were analyzed by AMS NSL. The Colorado

laboratory analyzed spinach collected by

California; the Ohio laboratory analyzed alfalfa

sprouts and lettuce collected by California; and

the AMS NSL analyzed cantaloupes and

tomatoes collected by California.

USDA is a member of the interagency Task

Force on Antimicrobial Resistance established

in 1999 to address antimicrobial resistance,

which has been identified as a priority food

safety and public health issue. As such, isolates

from positive MDP samples were sent to

Nevada

Idaho

Wyoming

New

Mexico

Hawaii

Alaska

Virginia

Delaware

Connecticut

Massachusetts

Michigan

Vermont

New Jersey

Figure 2. Program Participants. During 2008, AMS established cooperative agreements with 11 States to

sample and/or test MDP commodities. Samples collected by California, Maryland and Texas are analyzed

by the Ohio laboratory and the National Science Laboratory in Gastonia, NC. In addition, the Colorado

laboratory analyzed cantaloupes and green onions collected by Florida. States that do not participate in

MDP’s sampling program but are in the direct distribution networks of the participating States are also

shown.

New

York

Texas

California

Oklahoma

Washington

Wisconsin

Florida

Participating State

Minnesota

States where produce is directly marketed from

Participating States

Location of Participating State Laboratories (white stars)

Federal Laboratory (black star)

Maryland

Colorado

Ohio
Microbiological Data Program - Annual Summary, Calendar Year 2008

4

II. Sampling

The goal of the MDP sampling program is to

obtain a statistical representation of selected

commodities in the U.S. food supply by

randomly selecting samples from the national

food distribution system. The MDP sampling

frame is designed to take into account regional

diversity, population, and consumption on a

national scale. The sampling rationale was

developed by MPO in consultation with NASS,

FDA, and CDC.

Collecting data over time from a range of

sources permits statistical statements to be

made about the distribution of targeted

pathogens within the target population. The

target population is all units of a commodity

available at the wholesale level in a

participating State during a defined timeframe

(e.g., 1 year). The extension of statistical statements to the distribution of microorganisms

within the inferential population (the entire

amount of the commodity actually consumed

by the U.S. public during the same timeframe)

requires that strong assumptions be made about

the relationship between the participating States

and the U.S. as a whole, and between the

wholesale and point-of-consumption levels.

Nevertheless, because the States that participate

in MDP fully represent the U.S. inferential

population, and many microorganisms may

enter the food supply at or before the wholesale

level, the MDP is a useful and defensible

baseline survey.

Alfalfa sprouts, cantaloupes, lettuce, and

tomatoes remained in the program at 2007

levels. Based on consultations with FDA,

spinach was introduced in 2008 replacing green

onions. The five commodities were selected

because they are high-consumption fruit and

vegetables in the U.S. diet, are often consumed

raw, and have been implicated in outbreaks.

For 21⁄2 months, mid July through the end of

September, MDP implemented a temporary

FDA/CVM for antimicrobial resistance testing.

These data are being added to the National

Antimicrobial Resistance Monitoring System

(NARMS) database. Additionally, CVM performs genomic fingerprinting on MDP isolates

for inclusion in the PulseNet system.

In 2007, MDP laboratories replaced the

enzyme-based assay for enumeration of generic

E. coli and the Most Probable Number (MPN)

analysis with an automated system. This new

system is based on the same unique enzyme

assay; however, the setup, assay, and the

capture and analysis of data have been

automated. The automated system allowed the

laboratories to reduce staff time by

streamlining this labor-intensive assay while at

the same time removing the bias in subjective

interpretation of results. The automated system

was also used for screening bagged lettuce and

bagged spinach samples for total coliforms;

however, the assay for detecting coliform

bacteria is based on acid production due to

fermentation and is different from the one used

for detecting E. coli. The automated system

was also used to provide Total Viable Count

(TVC) data for bagged lettuce and bagged

spinach samples.

AMS employed DNA-based screening for

Salmonella, Shigella, and pathogenic E. coli,

including E. coli O157:H7. All samples were

screened for the presence of pathogenic E. coli

that harbor shiga toxins (STECs) and enterotoxins (ETECs) using mPCR technology.

STEC and ETEC are two groups of E. coli that

cause the majority of enteric diseases and are

therefore important to human health.

As the program evolves, procedures and

methods are being refined to provide information necessary for making science-based food

safety decisions. AMS continues to use

quicker, more reliable and more sensitive

technologies for improved microbial detection

and improve data collection systems for better

database management.
Microbiological Data Program - Annual Summary, Calendar Year 2008

5

program change to focus on the Salmonella

saintpaul foodborne outbreak. During that

timeframe, five States (Colorado, Maryland, New

York, Texas, and Washington) added cilantro,

bulb onions, green onions, hot peppers, and

additional tomatoes to their normal collection

scheme to aid CDC and FDA in their search for

the source of the Salmonella contamination.

Table 1 shows the number of extra samples

collected and tested for each specially added

commodity. MDP resumed its normal collection

schedule in October.

All samples in a State are collected on the same

day of each week. The samples of cantaloupes,

hot peppers, and tomatoes collected from a site

consist of three individual units or groups of

units of produce collected from the same box/

container. Fresh, pre-sealed bags or clamshells

of alfalfa sprouts, lettuce, and spinach samples

are from the same lot number. Other unbagged

fresh commodities, such as loose spinach or

green onion bunches, are collected in groups of

three as specified weights from the same box/

container. Inferences cannot reasonably be made

from the sample units to the lots from which they

originate because the units do not provide

enough information to generate statistically

reliable lot estimates. Nevertheless, statistical

methods can be applied to make whole-targetpopulation inferences from the data and to

compare these inferences over time.

MDP benefited from the well-established sampling framework of the Pesticide Data Program

(PDP), a program administered by MPO since

1991. States that were already providing

sampling services for PDP also began collecting samples for MDP in 2001 and continue, to

date, through annual cooperative agreements

with AMS. All sample collectors receive

training and are provided with factsheets on the

commodities they collect. The information in

each factsheet includes acceptable and unacceptable products, availability, sample size, and

instructions for data entry, packaging, and

shipping. Additional information is provided

on specific requirements for packaging samples

that are sensitive to ethylene.

The sampling of commodities is conducted at

distribution centers and terminal (wholesale)

markets from which food commodities are

released to supermarkets and grocery stores,

and include domestic and imported commodities (refer to Table 2 and Figure 3 for sample

origin information). Except for national emergency situations, such as special collections in

response to a foodborne outbreak, samples are

collected on a year-round basis and typically

over at least two growing seasons to

accommodate differences in growing conditions. Sampling is apportioned according to

population of the participating State. That is,

the higher the population of the State, the

greater the number of samples taken. The

monthly population-based collection numbers

are as follows: California, 14; Colorado, 2;

Florida, 7; Maryland, 4; Michigan, 6;

Minnesota, 2; New York, 9; Ohio, 6; Texas, 8;

Washington, 4; and Wisconsin, 2. This

schedule results in a monthly target of 64 sites

sampled per commodity. At each site, 3

samples are collected from the same lot in each

facility for a total of 192 samples collected

every month for each commodity.

Commodity

Number of Samples Collected

and Analyzed

Cilantro 219

Green Onions 141

Hot Peppers 225

Onions, Bulb 150

Tomatoes 601

TOTAL 1,336

Table 1. Number of Samples Tested during

Salmonella saintpaul Outbreak. This table

shows the number of samples collected and

analyzed in response to the Salmonella saintpaul

outbreak. The 601 tomatoes are in additional to

the regularly scheduled tomatoes.
Microbiological Data Program - Annual Summary, Calendar Year 2008

Distribution centers and terminal markets in

each State are selected at random based on

probability proportional to the site’s distribution volume (i.e., the amount of produce that

moves through the site). Therefore, the larger

the site, the greater the chance it will be

sampled. If the commodity of interest is not

available at the designated primary site, an

alternate site may be chosen. MDP does not

allow samples to be taken from public markets

or retail stores because of the potential for

contamination by the consumer and because

commodity handling practices at this level in the

distribution chain may vary widely. Between

February and December 2008, 11,669 samples

were collected from 376 sites across the country

and analyzed by MDP laboratories. Table 3

provides a detailed breakdown of sample

numbers collected by commodity.

All samples are selected and bagged using

aseptic techniques (i.e., sterile latex gloves and

sterile sample bags). Once bagged, samples

must be properly identified and tamper-proofed

to ensure that chain-of-custody requirements are

met. Sufficient frozen ice packs and the use of

adequate packing materials for cushioning and

insulation are required to maintain refrigerated

temperatures during transport. Sample temperatures and the condition of each sample are

observed and recorded upon receipt at each

laboratory. If the integrity of a sample is in

question, the laboratory requests that the

particular commodity be sampled again. All

samples are shipped on the same day as sample

collection by overnight delivery so that the

laboratory can begin analysis the following day.

Unlike PDP operations, where specific commodities are sent to laboratories specializing in

the analysis of a particular commodity, MDP

laboratory analyses are performed in the same

State from which the sample was collected.

Exceptions include California, Maryland, and

Texas; these State samples are shipped to the

Colorado and Ohio laboratories and AMS NSL

in Gastonia, NC, for analysis.

6

Commodity Country

Number of

Samples

Cilantro Mexico 15

TOTAL 15

Cantaloupe Canada 3

Costa Rica 162

Guatemala 324

Guatemala/Honduras 12

Honduras 171

Mexico 87

Nicaragua 12

TOTAL 771

Green Canada 15

Onions Mexico 63

Mexico/USA 3

TOTAL 81

Hot Peppers Mexico 9

TOTAL 9

Lettuce, Canada 3

Bagged Mexico/USA 21

TOTAL 24

Onions Mexico 12

TOTAL 12

Spinach Canada 24

Mexico 60

Mexico/USA 18

TOTAL 102

Tomatoes Canada 175

Mexico 1,072

Mexico/USA 6

TOTAL 1,253

Table 2. Distribution of Imported Samples. This

table details the number of imported samples by

country of origin and by commodity. None of the

alfalfa sprouts were reported as imported.
Microbiological Data Program - Annual Summary, Calendar Year 2008

Alfalfa sprouts, cantaloupe, lettuce, spinach,

and tomatoes were collected and tested as

routine commodities for 2008. Alfalfa sprouts

are most often grown in drums and packaged in

controlled environments. Cantaloupe and

tomatoes are shipped and sold to distribution

centers and terminal markets as whole,

individual units. For pre-washed, pre-cut

bagged lettuce, all lettuce varieties were

acceptable, whether packaged as a single variety

or mixed. For fresh, unwashed and pre-washed,

bagged spinach, all spinach varieties were

acceptable; pre-washed bagged spinach was

acceptable either as a single variety or mixed.

Bags containing lettuce or spinach mixed with

other greens were not acceptable. Most of these

commodities are harvested primarily by hand

although some mechanical harvesting does

occur.

The produce may be packaged in the field or

taken to a packinghouse (e.g., tomatoes require

7

Hot Peppers (225 Samples)

Green Onions (141 Samples)

Figure 3. Commodity Origin. The proportion of domestic, imported or unknown origin for

each commodity is depicted for samples tested in 2008.

Domestic 60.2%

Unknown 1.9%

Imported 37.9%

Cantaloupe (2,037 Samples)

Domestic 83.6%

Unknown 9.6%

Imported 6.8%

Cilantro (219 Samples)

Domestic 40.4%

Unknown 2.1%

Imported 57.5%

Domestic 82.7%

Unknown 13.3%

Domestic 97.6%

Unknown 1.2%

Imported 1.2%

Lettuce (Bagged) (2,078 Samples)

Imported 4.0%

Onions (150 Samples)

Domestic 92.0%

Imported 8.0%

Spinach (2,078 Samples)

Domestic 94.7%

Unknown 0.4%

Domestic 98.8%

Unknown 1.2%

Sprouts (Alfalfa) (2,039 Samples)

Imported 4.9%

Tomatoes (2,702 Samples)

Domestic 51.8%

Imported 46.4%

Unknown 1.8%
Microbiological Data Program - Annual Summary, Calendar Year 2008

8

classification for color and/or size). At the

packinghouse, some types of produce are

cleaned, trimmed, sized, sorted, chopped into

small pieces for ready-to-eat purposes, bagged,

wrapped, and chilled for preservation until

arrival at distribution centers and terminal

markets. Cleaning is typically accomplished

with chlorinated water, although other

disinfecting agents, such as ozone, may be

used. Some commodities may have a foodgrade wax applied to replace natural waxes

removed during washing to help prevent water

loss. Fungicides may be added to the wax or

applied separately to retard spoilage. Chilling

may be accomplished by various means such as

vacuum cooling, hydrovac cooling, room

chilling, or forced air cooling. After initial

chilling, the produce is stored under chilled

conditions (avoiding freezing) and, depending

on the commodity, under low-oxygen

atmospheric conditions (primarily carbon

dioxide).

Except for leafy greens and alfalfa sprouts, the

produce is often harvested before reaching full

ripeness to minimize spoilage and bruising.

Prior to shipment to distribution centers and

terminal markets, some commodities are often

artificially ripened using techniques such as

ethylene oxide gassing. Some shipping

companies transport produce in refrigerated

trucks or rail cars; others use ice; still others use

no method of cooling, depending on the

commodity. Therefore, MDP data reflect not

only agricultural practices but also handling

practices occurring during harvesting, storage

(including postharvest treatment), bagging, and

shipping operations.

MDP uses Sample Information Forms (SIFs) to

document information required for chain-ofcustody and to capture other information needed

to characterize the sample. Sample collectors use

the forms to record information such as: (1) State

of sample collection, (2) collection date, (3)

State

California 420 455 462 438 423

Colorado 66 66 66 66 314

Florida 231 231 231 231 231

Maryland 132 33 24 36 129 131 123 132

Michigan 198 198 198 195 198

Minnesota 66 66 66 66 65

New York 297 81 81 81 297 297 297 300

Ohio 198 198 198 198 198

Texas 231 72 72 240 150 231 239 643

Washington 132 33 36 36 132 132 120 132

Wisconsin 66 66 66 66 66

Totals 2,037 219 141 225 2,078 150 2,078 2,039 2,702

Total

2,198

578

1,155

740

987

329

1,731

990

1,878

753

330

11,669

Green Onions

Lettuce (Bagged)

Table 3. Samples Collected by State. This table shows the number of samples collected by each State

by commodity.

Sprouts (Alfalfa)

Cilantro

Cantaloupe

TomatoesHot Peppers

OnionsSpinach
Microbiological Data Program - Annual Summary, Calendar Year 2008

9

commodity code, (4) testing laboratory code,

and (5) sample collector name. Other

information collected includes the country of

origin of the sample, any production claims

(such as organic), and any postharvest

treatments.

An electronic SIF (e-SIF) capturing system was

implemented in 2003 and continues to be used

to record relevant sample information. A

customized software application allows States

to capture SIFs electronically using laptop or

handheld computers. Sample information is

captured in the MDP database files on the same

day as sample collection or early the next day.

MDP sampling operations are conducted with

the use of Standard Operating Procedures

(SOPs) designed to provide consistency across

the program and ensure the integrity of the

analytical data. SOPs also contain specific

instructions for sample selection, shipping and

handling, and chain-of-custody. SOPs are

updated as needed and serve as a technical

reference for conducting program sampling

reviews to ensure that program goals and

objectives are met. All program SOPs are

available on the Internet at www. ams.usda.gov/

mdp.

III. Laboratory Operations

Participating microbiology laboratories tested

MDP samples for generic E. coli and screened

MDP samples for Salmonella, Shigella, and E.

coli strains carrying shiga toxins and

enterotoxins (STECs and ETECs, respectively),

including E. coli O157:H7. MDP laboratories

performed mPCR screening of all samples for

pathogenic E. coli, based on the presence of

genes coding for shiga toxins and enterotoxins.

Isolates of these organisms were sent to FDA’s

CVM and to the Gastroenteric Disease Center at

PSU for further characterization. Tests

performed by FDA/CVM and PSU included

testing for antimicrobial resistance, genomic

fingerprinting, serotyping, and testing for

virulence attributes.

Upon arrival at the testing facility, samples

were logged, visually examined for acceptability, and discarded if determined to be damaged

(decayed, extensively bruised, or spoiled).

Samples were refrigerated until analysis

commenced. Laboratories were permitted to

refrigerate commodities for up to 48 hours from

time of receipt in the laboratory to allow for

different sample arrival times from the various

collection sites. Only excess soil was removed

prior to testing.

All samples were washed in Universal Preenrichment Broth (UPB) with 0.1 percent

Tween®(Polysorbate 80). Cantaloupes, bulb

onions, green onions, lettuce, hot peppers,

spinach, and tomatoes were manually shaken,

followed by overnight soaking. Alfalfa sprouts

were blended using a Stomacher®blender, the

plant material removed, and the remaining wash

incubated overnight. Soaking followed by

overnight incubation enhances recovery of

pathogens that may be trapped in cracks,

crevices, and biofilms.

For E. coli enumeration, TVC, and total

coliform assays, an AOAC®-certified enzymebased automated TEMPO®

system was used for

detection and enumeration (pre-washed, precut, bagged lettuce and pre-washed, bagged

spinach samples only). This system reports

enumeration in colony-forming units per gram

of sample test (cfu/g).

Genomic DNA was extracted from each

enriched sample and purified for use in

detecting pathogens via DNA-based PCR

assays. During 2008, the laboratories assessed

and began employing automated systems for the

extraction and purification of genomic DNA

from bacterial cultures in order to streamline the

labor-intensive preparation of DNA samples for

PCR assays.

All samples were screened by mPCR

procedures for STECs and ETECs. MDP

laboratories used PCR assays and automated

instruments for the detection of Salmonella,
Microbiological Data Program - Annual Summary, Calendar Year 2008

Shigella, and enterohemorrhagic E. coli

O157:H7 in produce samples. Cultural

methods involving selective growth media and

Immunomagnetic Separation (IMS) technology were employed for isolation of target

bacteria. In addition to cultural methods,

automated identifications based on biochemical tests and serotyping of surface antigens

were used in the confirmation of isolates for

the target pathogens.

The main objectives of the QA/Quality

Control (QC) program were to ensure the

reliability of MDP data and to ensure performance equivalency of participating laboratories.

Direction for the MDP QA program was

provided through written SOPs based on

FDA’s 2001 Bacteriological Analytical

Manual (3), AOAC® methods, USDA’s Food

Safety and Inspection Service (FSIS) Microbiological Laboratory Guide, the U.S. Environmental Protection Agency’s Good Laboratory

Practices, and International Standards

Organization (ISO) 17025 guidelines. MDP

analytical methods are published at

www.ams.usda.gov/mdp. SOPs provide uniform administrative, sampling, and laboratory

procedures. MDP laboratories participated in

the proficiency testing of an unknown

pathogen (Salmonella, Shigella, or E. coli

O157:H7) that was spiked into produce

samples. Proficiency testing rounds are

administered by MPO.

Positive and negative controls and a sterile

media blank were required for each sample set.

MDP laboratories use positive control strains

of Salmonella typhimurium and E. coli

O157:H7 that carry a gene coding for Green

Fluorescent Protein (GFP). Expression of the

GFP, detected by exposing the cultures to

ultraviolet light indicates the presence of the

control cultures without the need for

performing lengthy biochemical tests. All

controls and blanks were taken along with the

sample cultures from the preenrichment step to

isolation and identification of target isolates

using cultural, immunological, and serological

methods. MDP laboratories also used

automated instrumentation for confirmation of

isolates based on biochemical reactions.

A Technical Advisory Group, comprised of

microbiologists from each participating

laboratory, provided technical feedback on

program SOP revisions and addressed

technical and QA issues. Additionally, MDP

consulted with scientists from other Federal

agencies [FDA, CDC, USDA’s Agricultural

Research Service (ARS), and FSIS] and

academia on technical issues. For day-to-day

QA oversight, each participating facility was

required to have a Quality Assurance Unit

(QAU) that operated independently from the

laboratory staff. Preliminary QA/QC review

procedures were performed onsite by each

laboratory’s QAU. Final review procedures are

performed by MPO staff responsible for

collating and reviewing data for conformance

with SOPs.

Laboratory performance was monitored

through on-site reviews by MPO staff to

determine compliance with MDP SOPs.

Corrective actions, if necessary, were

performed as a result of on-site reviews.

IV. Database Management

MDP maintains an electronic database that

serves as a central data repository. The central

database resides at MPO in Manassas, VA. The

data captured and stored in the MDP database

include product information and analytical

findings for each sample collected along with

QA/QC results for each set of samples. The

MDP data pathway is depicted in Figure 4.

MDP uses a Web-based Remote Data Entry

(RDE) system to capture and report MDP data.

The RDE system is centralized, with all user

interface software and database files residing

in Washington, DC. The laboratory users need

only a Web browser to interface with the RDE

system. Access to the RDE system is

controlled through separate user login/

10
Microbiological Data Program - Annual Summary, Calendar Year 2008

11

Figure 4. MDP Data Pathway. An illustration of MDP data path from sample collection, through

laboratory analysis and reporting.

-8 State labs + 1 Federal lab for sample testing

-State Public Health Laboratories

-FDA/CVM Laboratory

-Pennsylvania State University E. coli Reference

Laboratory

-Standardized sample preparation

-Test for specific foodborne pathogens

-Confirmation and serology of isolated pathogen

-Genomic fingerprints

-Antimicrobial susceptibility

Laboratory Analysis

Sample Collection

- Collection in 11 States

- Samples taken close to consumer

- Standardized Sample Information Form

- Data entry on handheld/laptop computers

- Web-based data entry software

- Import data from other systems

- Access controlled by user login

- Extensive data cross-checks

Laboratory Remote Data Entry (RDE)

Data Review at HQ

- Scientists review data on-screen

- Upload data to central database

Year-end Review

- Data reconciliation

Internet

Internet

- Standard & ad hoc reporting

- Annual Summary

- eLEXNET

- PulseNet

- NARMS

Data Reporting
Microbiological Data Program - Annual Summary, Calendar Year 2008

12

password accounts and user access rights for

the various system functions based on position

requirements. The RDE system utilizes Secure

Sockets Layer (SSL) technology to encrypt all

data passed between users’ computers and the

central Web server.

A separate Windows®-based system allows

sample collectors to electronically capture the

standardized SIF on handheld or laptop

computers. The electronic SIF (e-SIF) system

generates formatted text files containing

sample information that are e-mailed to MDP

headquarters and then imported into the Webbased RDE system.

The RDE data entry screens have extensive

edits and cross-checks built in to ensure that

acceptable values are entered for all critical

data elements. This task is made easier by the

practice of capturing and storing standardized

codes for all critical alphanumeric data

elements rather than their complete names,

meanings, or descriptions. This coding scheme

allows for faster and more accurate data entry,

saves disk storage space, and makes it easy to

perform queries on the database. The data

entry screens also perform edits on numeric

fields, dates, and other character fields to

ensure that entries are within prescribed

boundaries.

At MDP headquarters, the RDE system allows

scientists to review and approve the data for

inclusion in the central database. The central

MDP database is maintained using Microsoft®

Access and SQL Server database tools.

Access to the central MDP database is limited

to MDP headquarters personnel and is

controlled through password protection and

user access rights.

V. Summary of 2008 Data

Table 2 specifies the distribution of imported

samples by commodity and country of origin.

Figure 3 illustrates the proportion of samples

that were domestic, imported, and of unknown

origin for each commodity. Seventy-nine

percent of the samples were from domestic

sources, 19 percent were imported, and 2

percent were of unspecified origin. Table 3

shows the distribution of samples among each

commodity and collection State.

All samples were washed or blended (alfalfa

sprouts) in UPB in order to streamline the

screening process for all target bacteria. E. coli,

TVC, and total coliform enumerations were

performed using the automated TEMPO®

system – TVC and total coliform testing was

performed only on pre-washed, pre-cut, bagged

lettuce and pre-washed, bagged spinach

samples. A soaking step was implemented for

cantaloupe, lettuce, spinach, and tomatoes to

improve pathogen detection. Alfalfa sprouts

were blended, the plant material removed, and

the remaining wash incubated overnight.

Genomic DNA was extracted from each

enriched sample and purified for use in

detecting pathogens. The BAX®instrument, an

automated PCR system, was used for screening

samples for the presence of Salmonella and

enterohemorrhagic E. coli O157:H7. Similarly,

an appropriate aliquot of extracted DNA for

each sample was used in screening for the

presence of pathogenic E. coli by mPCR or

Shigella by conventional PCR. Table 4 shows

the number of samples tested for each

organism.

Positive individual samples were cultured for

isolation and identification of the organism.

Identification of isolates was confirmed using a

conventional biochemical testing system, an

AOAC®performance-tested kit, or a commercial biochemical kit or system, approved by

MDP. In addition to biochemical identification

of an isolate, all MDP participating State

laboratories were required to confirm the

identification by serotyping. Isolates were then

sent to FDA/CVM for expanded serotyping

(except pathogenic E. coli isolates which were
Microbiological Data Program - Annual Summary, Calendar Year 2008

13

sent to PSU for serotyping and virulence

testing), antimicrobial resistance testing, and

genomic fingerprinting.

Enumeration of E. coli

In 2008, a total of 10,330 samples were

enumerated for E. coli using the automated

TEMPO®system, an AOAC-certified method.

As shown in Table 5, only 525 samples

displayed MPNs greater than 10 (approximately 5% of samples).

E. coli is typically found as a commensal

organism in the intestinal tracts of warmblooded animals and humans. As such, it is

often used as an indicator organism and as a

measure of cleanliness for irrigation waters,

produce wash waters, and other surfaces with

which the produce may be in contact. A

detection of E. coli does not mean or imply the

presence of a pathogen but indicates that there

is a possibility of pathogen presence and that

further testing may be warranted if the number

of E. coli cells is above a certain threshold.

Use of E. coli enumeration is most useful for

Hazard Analysis Critical Control Points

(HACCP) analysis.

State E. coli

Pathogenic

E. coli

E. coli

O157:H7

Salmonella

by BAX

Salmonella

by VIDAS Shigella

Total

Coliform

Total

Viable

Count

Total

Number

of Tests

California 2,198 2,198 2,198 2,198 2,198 917 622 12,529

Colorado 330 330 330 578 90 330 132 102 2,222

Florida 1,155 1,155 1,155 1,155 1,155 462 449 6,686

Maryland 647 647 647 740 644 258 260 3,843

Michigan 987 987 987 987 984 382 378 5,692

Minnesota 329 329 329 329 329 132 126 1,903

New York 1,485 1,485 1,485 1,731 1,484 588 488 8,746

Ohio 990 990 990 990 990 395 396 5,741

Texas 1,231 1,231 1,231 1,878 330 1,230 387 365 7,883

Washington 648 648 648 753 648 264 201 3,810

Wisconsin 330 330 330 330 330 117 117 1,884

Totals 10,330 10,330 10,330 11,669 420 10,322 4,034 3,504 60,939

Table 4. Number of Samples Analyzed. This table shows the number collected samples tested for

each organism.

MPN Range Number of Samples

< 10 9,805

10 - 99 389

100 - 999 108

1,000 - 10,000 13

> 10,000 15

Total Number of

Samples Tested 10,330

Table 5. Number of Samples Tested for E.

coli. This table shows the results of E. coli

enumeration.
Microbiological Data Program - Annual Summary, Calendar Year 2008

14

Pathogenic E. coli

In 2008, a total of 10,330 samples were

screened for pathogenic E. coli that harbor

shiga toxins (STECs) and enterotoxins

(ETECs) using an mPCR assay developed by

FDA. Table 6 displays the number of samples

initially screened for E. coli and further tested

for pathogenic E. coli, as well as the number of

samples that tested positive for pathogenic E.

coli.

Thirty-five samples were identified as positive

for pathogenic E. coli and 11 isolates were

obtained. Seven of these isolates were

identified as carrying shiga toxins (STECs) and

four as carrying enterotoxins (ETECs). In

addition to the technological differences

between detection by PCR and isolation by

cultural means, several other factors influence

the rate of successful isolation, including: an

overwhelming amount of background microflora in comparison to the small number of

target bacterial cells, differential growth rates

of various bacteria, and additional growth

requirements.

The 11 STEC and ETEC isolates were sent to

PSU for serotyping and further characterization

and to FDA/CVM for antimicrobial resistance

testing. For each isolate, PSU conducted 17

tests that included identifying virulencespecific genes associated with different classes

of pathogenic E. coli and serotyping for

somatic O antigens and flagellar H antigens.

FDA/CVM conducted tests on resistance to 15

different antibiotics and genomic fingerprinting on these isolates. The results of PSU

and FDA/CVM testing are shown in Table 7.

Two of the STEC strains were isolated from

pre-washed, pre-cut bagged lettuce and five

from spinach (either fresh, unwashed spinach

or pre-washed, pre-bagged spinach); three

ETEC strains were isolated from spinach and

one from alfalfa sprouts. To characterize an

isolate as a human pathogen capable of causing

disease, there must be an interplay of several

proteins, including toxins, encoded by

respective genes. MDP only identified toxin

genes; the additional testing required to

determine the disease-causing potential of

these isolates is not within the scope of MDP.

Total Viable Count

Total Viable Count (TVC) provides an

indication of the presence of microorganisms

such as bacteria in a sample, more specifically,

the total number of colony-forming units per

gram (cfu/g) or milliliter of sample. TVC

Commodity

Number of

Samples

Tested

Number of

Samples Screened

by mPCR

Number of

Pathogenic E. coliPositive Samples

Cantaloupe 2,037 2,037 0

Lettuce, Bagged 2,078 2,078 8

Spinach 2,078 2,078 22

Sprouts (Alfalfa) 2,039 2,039 4

Tomatoes 2,098 2,098 1

Total 10,330 10,330 35

Number of

Positive Isolates

Obtained

0

2

8

1

0

11

Table 6. Summary of Sample Analysis for Pathogenic E. coli. This table summarizes the

number of samples initially screened for E. coli and further tested for pathogenic E. coli

and the number of samples that tested positive for pathogenic E. coli.
Microbiological Data Program - Annual Summary, Calendar Year 2008

MPNs were performed via the TEMPO®

system. Table 8 portrays the TVC MPNs for

bagged lettuce and spinach samples, which

were washed prior to commercial packaging.

Coliform Bacteria

Coliform, or enteric, bacterial count is a subset

of TVC and is often used as an indicator of

direct or indirect fecal contamination. Table 9

shows the total coliform MPNs for bagged

lettuce and spinach samples, which were

washed prior to commercial packaging.

Testing was performed via the automated

TEMPO®system. In this assay, bacteria that

produce acid from lactose fermentation are

enumerated.

Salmonella

As depicted in Table 10, all 11,669 samples

were screened for Salmonella by BAX-PCR. Of

these samples, 86 were positive via the

preliminary screening method. Of these 86

samples, 16 Salmonella isolates were

confirmed: 9 from alfalfa sprouts, 3 from

cantaloupe, 1 from cilantro, 1 from bagged

lettuce, 1 from hot peppers, and 1 from spinach.

Serotyping

Table 7. Characterization of Pathogenic E. coli Isolates Screened by mPCR. This table provides data

obtained from additional testing of pathogenic E. coli isolates initially screened by MDP laboratories.

Information includes: pathogenic class, identified toxin genes, and serotyping results.

Cnf-2 = Cytotoxic necrotizing factor 2

HlyA = hemolysin A

LT = heat-labile toxin

Stx-1 and Stx-2 - shiga toxins 1 and 2, respectively

Neg - no serological reaction; did not react with standard antisera

Commodity

Pathogenic

Class

Toxic Genes

Identified

O

Antigen

H

Antigen Pulsed-Field Gel Electrophoresis

Lettuce, Bagged STEC Stx-2, HlyA 8 28

Lettuce, Bagged STEC STa, Stx-1, HlyA 136 16

Spinach ETEC LT, STb 8 15

Spinach ETEC LT 163 19

Spinach ETEC STa, STb 42 37

Spinach STEC Stx-1, Stx-2, HlyA 73 12

Spinach STEC Stx-2, HlyA 130 11/47

Spinach STEC Stx-2 Neg 38

Spinach STEC Stx-1, Cnf-2 Neg 16

Spinach STEC Stx-1, Stx-2 Neg 16

Sprouts (Alfalfa) ETEC STa Neg 4

15
Microbiological Data Program - Annual Summary, Calendar Year 2008

MPN Range

Number of

Lettuce

Samples

Blank (<250) 21

250 - 999 23

1,000 - 9,999 125

10,000 - 99,999 411

100,000 - 999,999 662

>1,000,000 782

Number of

Spinach

Samples

17

2

6

50

233

1,172

Total Number of

Samples Tested 2,024 1,480

Table 8. Number of Samples Tested for Total

Viable Count. This table depicts the most

probable number (MPN) ranges for total viable

count (TVC) in bagged lettuce and spinach.

MPN Range

Number of

Spinach

Samples

< 3 603

3 - 9 263

10 - 99 496

100 - 999 371

1,000 - 9,999 204

Number of

Lettuce

Samples

927

338

378

245

175

10,000 - 99,999 6 27

100,000 - 999,999 0 0

> 1,000,000 0 1

Total Number of

Samples Tested 2,069 1,965

Table 9. Number of Samples Tested for

Coliforms. This table depicts the most probable

number (MPN) ranges for total coliforms in

bagged lettuce and spinach samples.

Commodity

Number of

Samples Tested

by BAX

Number of

Samples Tested

by VIDAS

Number of

Presumptive

Positive Samples

Cilantro 219 0 2

Cantaloupe 2,037 0 13

Green Onions 141 0 0

Hot Peppers 225 0 1

Lettuce, Bagged 2,078 0 25

Onions 150 0 1

Number of

Positive Isolates

Obtained

1

3

0

1

1

0

Spinach 2,078 0 4 1

Sprouts (Alfalfa) 2,039 0 16 9

Tomatoes 2,702 420 24 0

TOTAL 11,669 420 86 16

Table 10. Summary of Analysis for Salmonella. This table shows the number of samples

screened for Salmonella using BAX and VIDAS, the number of positive individual samples,

and the number of isolates obtained.

16
Microbiological Data Program - Annual Summary, Calendar Year 2008

These 16 isolates were sent to FDA/CVM or

State health department laboratories for

identification by serotyping, antimicrobial

resistance for 15 antibiotics, and genomic

fingerprinting. Table 11 identifies each isolate

and the associated serogroup. The isolates

from alfalfa sprouts were all Salmonella

cubana. While all the same species, they were

not all genetically identical based on genomic

fingerprinting and hence originate from

different sources.

E. coli 0157:H7

No enterohemorrhagic E. coli O157:H7 strain

was isolated from the 10,330 samples screened

(refer to Table 12). In this case, as with

pathogenic E. coli analysis, several factors

contribute to successful isolation, including the

level of background microflora versus the

number of target bacterial cells, differential

bacterial growth rates, and additional growth

requirements.

Commodity Genus Species Serogroup Pulsed-Field Gel Electrophoresis

Cantaloupe Salmonella S. Javiana D1

Cantaloupe Salmonella S. Javiana D1

Cantaloupe Salmonella S. Luciana F

Cilantro Salmonella S. Meleagridis E1

Hot Peppers Salmonella S. Cerro K

Lettuce, Bagged Salmonella S. Typhimurium B

Spinach Salmonella S. Newport C2

Sprouts (Alfalfa) Salmonella S. Cubana G2

Sprouts (Alfalfa) Salmonella S. Cubana G2

Sprouts (Alfalfa) Salmonella S. Cubana G2

Sprouts (Alfalfa) Salmonella S. Cubana G2

Sprouts (Alfalfa) Salmonella S. Cubana G2

Sprouts (Alfalfa) Salmonella S. Cubana G2

Sprouts (Alfalfa) Salmonella S. Cubana G2

Sprouts (Alfalfa) Salmonella S. Cubana G2

Sprouts (Alfalfa) Salmonella S. Cubana G2

Serotype/Identification

Table 11. Salmonella Identification and Serogroup. This table summarizes the genus, species, and

serogroup for each of the 16 Salmonella isolates obtained in 2008.

17
Microbiological Data Program - Annual Summary, Calendar Year 2008

Shigella

No Shigella species were isolated from 10,322

samples screened (refer to Table 13).

Foodborne Outbreaks

In late May 2008, reports of a national

foodborne outbreak were in the press headlines.

At the request of CDC and FDA, MDP quickly

initiated sampling and testing of additional

tomatoes in Colorado and Texas – MDP

sampling States where there were initially

human illnesses – as well as in Maryland, New

York, and Washington. The additional tomatoes

sampled were only tested for Salmonella spp.

As the investigation continued, MDP was

asked to test cilantro, bulb onions, green

onions, and hot peppers. MDP’s existing

sampling framework allows rapid response, so

sampling and testing of these commodities

was initiated within a week of the request.

MDP’s electronic, standardized reporting

system allows for immediate capture of

sample origin information as well as analytical

results and associated QA data. Over the

course of the outbreak, MDP laboratories

tested 1,336 additional samples for

Salmonella, demonstrating the quick response

and adaptability of the program to foodborne

produce-related outbreaks.

Commodity

Number of

Samples

Tested

Number of

Presumptive

Positive Samples

Number of

Positive Isolates

Obtained

Cantaloupe 2,037 0 0

Lettuce, Bagged 2,077 0 0

Spinach 2,072 0 0

Sprouts (Alfalfa) 2,039 0 0

Tomatoes 2,097 0 0

Total 10,322 0 0

Table 13. Summary of Analysis for Shigella. This table shows the number

of samples tested for Shigella and the number of presumptive positives and

isolates obtained.

Commodity

Number of

Samples

Tested

Number of

Presumptive

Positive Samples

Number of

Positive Isolates

Obtained

Cantaloupe 2,037 0 0

Lettuce, Bagged 2,078 0 0

Spinach 2,078 0 0

Sprouts (Alfalfa) 2,039 0 0

Tomatoes 2,098 0 0

Total 10,330 0 0

Table 12. Summary of Analysis for E. coli O157:H7. This table shows the

number of samples tested for E. coli O157:H7 and the number of

presumptive positives and isolates obtained.

18
Microbiological Data Program - Annual Summary, Calendar Year 2008

References:

1. Produce Safety from Production to Consumption: 2004 Action Plan to Minimize Foodborne

Illness Associated with Fresh Produce Consumption (October 2004). U.S. Food and Drug

Administration, Center for Food Safety and Applied Nutrition, Office of Plant and Dairy Foods,

http://www.fda.gov/Food/FoodSafety/Product-SpecificInformation/FruitsVegetablesJuices/

FDAProduceSafetyActivities/ProduceSafetyActionPlan/ucm129487.htm

2. E. coli O157:H7 Outbreaks (September 2006, December 2006). U.S. Food and Drug

Administration, Center for Food Safety and Applied Nutrition, Office of Plant and Dairy Foods,

http://www.cfsan.fda.gov/list.html

3. 4. Bacteriological Analytical Manual (BAM) (2001). U.S. Food and Drug Administration,

http://www.cfsan.fda.gov/~ebam/bam-toc.html

Disclaimer:

The use of trade, firm, or corporation names, references to published work, and analytical methodology

referred to in this 2008 Microbiological Data Program Summary is for the information and convenience

of the reader. Such use does not constitute an official endorsement or approval by the U.S. Department

of Agriculture of any product, service, or analytical method to the exclusion of others that may be

suitable.

Definitions:

Antimicrobial susceptibility: The result of microbes changing in ways that reduce or eliminate the

effectiveness of drugs, chemicals, or other agents to cure or prevent infections.

AOAC®INTERNATIONAL: An internationally recognized organization that validates and approves

analytical methods for foods and agriculture.

Aseptic: Free of microbial contamination.

Cultural Methods: Use of rich or selective media for the growth and identification of target bacteria.

Deoxyribonucleic acid (DNA): The molecule that encodes genetic information required to constitute a

living and reproducing organism. DNA-based technologies exploit the uniqueness in the DNA

sequences of a given organism in detection and identification methods.

eLEXNET: The electronic laboratory exchange network (eLEXNET) is an electronic system

administered by the Food Emergency Response Network (FERN) that allows the exchange of

laboratory analytical data among over 100 public health laboratories at the Federal, State and local

levels. eLEXNET is FERN’s data capture mechanism.

Enterohemorrhagic E. coli (EHEC): Strains of E. coli that are the primary cause of hemorrhagic colitis

or bloody diarrhea, which can progress to the potentially fatal hemolytic uremic syndrome. EHEC are

typified by the production of verotoxin or Shiga toxins (Stx). E. coli O157:H7 is the prototypic EHEC.

19
Microbiological Data Program - Annual Summary, Calendar Year 2008

20

Enterotoxigenic E. coli (ETEC): Strains of E. coli that are the causative agent of travelers’ diarrhea

and illness characterized by watery diarrhea with little or no fever. Pathogenesis of ETEC is due to the

production of any of several enterotoxins, including heat-labile enterotoxin and heat-stable toxin.

Genomic fingerprinting: Techniques used in the identification and/or classification of organisms

exploiting the differences in the DNA sequence.

Green Fluorescent Protein (GFP): Expression of the gene from jelly fish in bacterial control cultures is

used as a marker.

Indicator organism: A microorganism or group of microorganisms whose presence indicates

unsanitary condition or fecal contamination.

Isolate: Target bacterial strain isolated as a pure culture and identified.

Most Probable Number (MPN): Most Probable Number (MPN) is a statistical expression for

estimating the microbial density in a culture or per unit volume of water.

National Antimicrobial Resistance Monitoring System (NARMS): A collaborative effort among the

Centers for Disease Control and Prevention, the Food and Drug Administration, and the U.S.

Department of Agriculture to monitor antimicrobial resistance of human enteric bacteria, including

Campylobacter, Salmonella, Escherichia coli O157:H7, and Shigella.

Pathogen: Specific causative agent (e.g., a bacterium or virus) of disease.

Polymerase Chain Reaction (PCR): A technique used to amplify a specific region of DNA into a large

number of copies in order to produce enough DNA to be adequately tested. PCR can be used to

identify, with a very high probability, disease-causing viruses and/or bacteria.

Multiplex PCR (mPCR) involves simultaneous amplification of more than one specific region of DNA

or specific genes for various analytes.

Proficiency test sample: Any matrix sample prepared for the purpose of determining biases, accuracy,

and/or precision among analysts and/or laboratories or of a single analyst or laboratory.

PulseNet: A national network of local, State, and Federal public health and food laboratories

coordinated by the Centers for Disease Control and Prevention (CDC) to detect foodborne disease case

clusters and outbreaks and facilitate identification of the source by standardized genomic fingerprinting

(molecular subtyping) of various pathogenic bacteria using pulsed-field gel electrophoresis (PFGE)

technology.

Pulsed field gel electrophoresis: (PFGE) is designed to separate DNA too large to be separated by

conventional gel electrophoresis and is a highly discriminatory method for the differentiation of

bacterial isolates based on differences in DNA content.

Serotyping: An antigen and antibody reaction technique that is used to differentiate strains of

microorganisms based on differences in the antigenic composition of a certain structure such as the cell

wall components or flagella.
Microbiological Data Program - Annual Summary, Calendar Year 2008

The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race,

color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion,

sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual’s income is derived

from any public assistance program (Not all prohibited bases apply to all programs.) Persons with disabilities who require

alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’s

TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of

Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-

6382 (TDD). USDA is an equal opportunity provider and employer.

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Shiga toxin: A family of toxins produced by Shigella dysenteriae type I and shiga toxin-producing E.

coli. These toxins have a cytotoxic effect on intestinal epithelial cells that causes characteristic bloody

diarrhea.

Virulence attributes/factors: A bacterial product, usually a protein or carbohydrate (polysaccharide)

that contributes to virulence or pathogenicity.

Virulence: The degree or intensity of pathogenicity of an organism as indicated by case fatality rates

and/or ability to invade host tissues and cause disease.