Microbiological Data Program - Annual Summary, Calendar Year 2004 

United States 

Department of 

Agriculture 

Agricultural 

Marketing Service 

Science & 

Technology 

Programs 

Microbiological Data Program 

Progress Update and 2004 Data Summary 

Please Visit Our Website at http://www.ams.usda.gov/science/MPO/MDP.htm 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

January 2006 

To the Reader: 

I am pleased to present the USDA Microbiological Data Program 2004 Data 

Summary. In 2004, MDP continued testing four commodities begun in 2002: 

cantaloupe, tomatoes, leaf lettuce, and romaine lettuce. These items were 

selected because they are high consumption fruit and vegetables in the United 

States. Based on consultations with the U.S. Food and Drug Administration 

(FDA), several commodity changes were implemented midyear in 2004. Green 

onions, cilantro, and parsley were introduced to the program and celery was 

discontinued. Leaf and romaine lettuce were combined as a single commodity 

with each variety being sampled at half the regular sampling rates. 

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

sample collection and analyses. Eleven States participated in 2004: 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. 

This summary is intended to provide the reader with an update on the methods, 

modifications, and refinements made during program development, as well as 

an overview of the data obtained during 2004. 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@usda.gov or visit our Web site at 

http://www.ams.usda.gov/science/MPO/MDP.htm. 

Sincerely, 

Lloyd C. Day 

Administrator 

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 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

Contents

Page No.

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

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

 

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

 

Section II. Sampling........................................................................................................................ 3 

 

Section III. Laboratory Operations.................................................................................................. 8 

 

Section IV. Database Management ................................................................................................. 9 

 

Section V. Summary of 2004 Data.................................................................................................. 9 

References ..................................................................................................................................... 13 

Disclaimer ..................................................................................................................................... 13 

Definitions..................................................................................................................................... 14 

 

iii 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

Figures and Tables

Figures Page No. 

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

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

 3 Commodity Origin ........................................................................................................ 6 

 

4 MDP Data Pathway .................................................................................................... 10 

 

Tables

 1 Distribution of Imported Samples ............................................................................... 5 

 

 2 Samples Collected and Analyzed by State .................................................................. 7 

 3 Summary of Sample Analysis for Pathogenic E. coli .............................................. 11 

 4 Characterization of Pathogenic E. coli Isolates Screened by mPCR......................... 12 

 5 Summary of Analysis for Salmonella........................................................................ 12 

 6 Salmonella Identification, Serogroup, and Genomic Fingerprinting ........................ 13 

v 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

vii 

Data presented in this report were collected and 

processed through the efforts of the following 

organizations: 

State Agencies

California Department of Food and Agriculture 

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

California Department of Food and Agriculture 

Division of Inspection Services 

CDFA Agricultural Microbiology Laboratory 

McClellan Park (McClellan AFB), Bldg. 929 

5431 Arnold Ave. 

Sacramento, CA 95652 

 

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 

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 

Acknowledgements 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

viii 

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 x17, Facsimile (703) 369-0678 

E-mail: amsmpo.data@usda.gov

Web site: http://www.ams.usda.gov/science/

MPO/MDP.htm
Microbiological Data Program - Annual Summary, Calendar Year 2004 

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 Food and Drug Administration (FDA), and the 

USDA National Agricultural Statistics Service 

(NASS). The USDA Agricultural Research 

Service (ARS) and Food Safety and Inspection 

Service (FSIS) provide consultation as independent research authorities on laboratory 

methods. 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 2004, 11 States collected fruit and vegetable 

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

Texas, Washington, and Wisconsin). 

 

The program tested eight commodities (cantaloupe, celery, leaf and romaine lettuce, tomatoes, 

green onions, cilantro, and parsley) for Escherichia coli (E. coli) with pathogenic potential 

and Salmonella. MDP analyzed a total of 11,214 

samples. Seventy-six percent of the samples were 

from domestic sources, 20 percent were imported, 

and approximately 4 percent were of unspecified 

origin. MDP identified 43 samples carrying 

pathogenic E. coli; however, pathogenic E. coli

strains were isolated from only 9 samples. These 

isolates were sent to Pennsylvania State University for further characterization, including 

serotyping and testing for different virulencespecific genes associated with seven different 

categories of pathogenic E. coli. FDA’s Center 

for Veterinary Medicine (CVM) facility 

conducted tests on antimicrobial resistance and 

genomic fingerprinting on these isolates. MDP 

screening also resulted in five Salmonella isolates: 

one each from cantaloupe, cilantro, green onion, 

lettuce (romaine), and parsley. 

A number of important benefits are expected 

from MDP. Microbiological data obtained from 

this fresh produce screening effort will contribute 

significantly to a national produce microbiological baseline. The data will enhance the 

understanding of the microbial ecology of fresh 

fruit and vegetables in the food supply and permit 

the identification of long-term trends. Such 

baseline data, combined with virulence attributes, 

serotypes, antimicrobial resistance, and genomic 

fingerprints, will help collaborators such as CDC 

and FDA in planning public health initiatives. 

Executive Summary 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

1 

I. Introduction 

Many eminent national scientific organizations 

strongly advocate microbiological monitoring (1, 

2). In 2001, Congress authorized funding for a 

microbiological monitoring program to establish a 

microbial baseline for the domestic food supply. 

The Microbiological Data Program (MDP) was 

established as part of the broader 1997 Presidential 

Food Safety Initiative. 

MDP’s 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 2004 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. This publication is 

available on the Internet at http://www.ams.usda. 

gov/science/MPO/MDP.htm. 

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 Food and 

Drug Administration (FDA). The USDA Agricultural Research Service (ARS) and Food Safety and 

Inspection Service (FSIS) provide consultation as 

independent research authorities on laboratory 

methods. 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, particularly those involving 

technical and quality assurance (QA) issues. 

Figure 1 (b) also depicts MDP program testing 

operations. The participating State laboratories and 

the AMS National Science Laboratory (NSL) 

analyze the MDP samples collected by State 

samplers. FDA’s Center for Veterinary Medicine 

(CVM) and Pennsylvania State University provide additional testing services for isolate 

characterization. Information on MDP data and 

isolates is shared with USDA’s ARS and FSIS, 

CDC, and FDA. 

AMS used USDA consumption surveys to select 

commodities that are highly consumed in the 

United States and can be eaten raw: cantaloupe, 

celery, leaf lettuce and romaine lettuce, tomatoes, 

green onions, cilantro, and parsley. Commodities 

were tested for Escherichia coli (E. coli) strains 

with human pathogenic potential including E. 

coli O157:H7 and Salmonella. Isolates of these 

organisms were sent to specialized laboratories 

for further characterization including multiplex 

polymerase chain reaction (mPCR) screening for 

pathogenic E. coli, serotyping, testing for antimicrobial resistance and virulence attributes, and 

genomic fingerprinting. 

Samples were collected in the 11 participating 

States through cooperative agreements with their 

respective agencies (Figure 2). Together these 

States represent over 50 percent of the Nation’s 

population and all geographic regions of the 

country, with significant rural-to-urban variability. Therefore, MDP samples are a statistically 

defensible representation of the country as a 

whole. Also shown in Figure 2 are the 13 

neighboring 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. 

Microbiology laboratory services were provided 

by nine States (California, Colorado, Florida, 

Michigan, Minnesota, New York, Ohio, Washington, and Wisconsin) and the AMS NSL. 

Microbiological Data Program (MDP) 

Annual Summary, Calendar Year 2004 

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

Operations, (IV.) Database Management, (V.) Summary of 2004 Data 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

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. 

Food and Drug 

Administration Participating 

States 

(a) MDP Planning 

(b) MDP Program Operations 

Agricultural 

Research 

Service 

Food Safety 

and Inspection 

Service 

National 

Agricultural 

Statistics Service 

Centers for 

Disease Control 

and Prevention 

Agricultural 

Research 

Service 

Participating 

States 

National Science 

Laboratory 

Pennsylvania 

State University 

Food Safety 

and Inspection 

Service 

Centers for 

Disease Control 

and Prevention 

Food and Drug 

Administration / 

Center for Veterinary 

Medicine 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

3 

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 FDA/CVM for antimicrobial 

resistance testing. These data will be added to the 

National Antimicrobial Resistance Monitoring 

System (NARMS) database. Additionally, CVM 

performs genomic fingerprinting on MDP isolates 

for inclusion in the PulseNet system. 

As the program evolves, procedures and methods 

will be modified and refined to provide information necessary for making science-based food 

safety decisions. AMS continues to improve data 

collection systems and to use improved microbial 

detection methods that are quicker, more reliable, 

and more sensitive. AMS implemented DNAbased testing of samples in October 2003 

following program-wide validation studies and 

introduced DNA-based screening for E. coli

O157:H7 in April 2004. In 2004, all E. coli

isolates were screened for potential human 

pathogenicity using mPCR technology. 

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 

Nevada 

Idaho 

Wyoming 

New 

Mexico 

Hawaii 

Alaska 

Virginia

Delaware 

Connecticut 

Massachusetts 

Michigan 

Vermont 

New Jersey 

Figure 2. Program Participants. During 2004, AMS established cooperative agreements with 11 States to 

sample and/or test MDP commodities. Samples collected by Maryland are analyzed by the Ohio 

Laboratory. Samples collected by Texas are analyzed by the National Science Laboratory in Gastonia, 

North Carolina. 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 2004 

4 

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 

MPO. 

The sampling of commodities in commerce is 

conducted at distribution centers and terminal 

(wholesale) markets from which food 

commodities are released to supermarkets and 

grocery stores, including domestic and imported 

commodities (refer to Table 1 and Figure 3 for 

sample origin information). Samples are collected 

weekly 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. These 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 samples per 

commodity. Each site sample consists of three 

sub-samples taken from the same lot in each facility (each sub-sample is treated as a separate 

laboratory sample) and the total number of subsamples collected every month for each 

commodity is 192. 

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. In 2004, 

11,214 samples were collected and analyzed from 

over 700 sites across the country. Table 2 

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 in 

consultation with the NASS (3), 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-ofconsumption 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. 

Based on consultations with FDA, several 

commodity changes were implemented in 2004. 

Green onions, cilantro, and parsley were introduced 

to the program and celery was discontinued. Leaf 

and romaine lettuce were combined as a single 

commodity with each variety being sampled at half 

the regular sampling rates. Cantaloupe and tomatoes remained in the program at 2003 levels. These 

crops were selected because they are highconsumption fruit and vegetables in the U.S. diet, 

are often consumed raw, and have been implicated 

in outbreaks. All samples in a State are collected on 

the same day or within a 2-day interval. Samples 

from a site consist of three individual units of 

produce generally collected from the same 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 produce statistically reliable lot 

estimates. Nevertheless, statistical methods can be 

applied to make whole target-population inferences 

from the data and to compare these inferences over 

time. 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

5 

provides a detailed breakdown of sample numbers 

collected by commodity. As a note, cilantro and 

parsley were treated as a single commodity in that 

each product was collected at a half sampling rate 

(to equal the total collected for one commodity). 

All samples are selected, bagged, and packed 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 will request 

that the particular commodity be re-sampled. All 

samples are shipped on the same day as sample 

collection by overnight delivery so that laboratory 

analysis can begin 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 

Maryland and Texas; these State samples are 

shipped to the Ohio laboratory and the AMS NSL, 

Gastonia, North Carolina, respectively, for analysis. 

The commodities collected and tested in 2004 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 which require classification for 

color and/or size). At the packinghouse, the produce is cleaned, trimmed, sized, sorted, 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 food 

grade 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 forcedCommodity Country 

Number of 

Samples 

Cantaloupe Costa Rica 245 

 Dominican Republic 27 

 Guatemala 432 

 Honduras 231 

 Mexico 30 

 Nicaragua 9 

 Unknown 18 

 992 

 

Celery Canada 3 

 Mexico 24 

 27 

 

Green Onions Canada 12 

 Guatemala 12 

 Mexico 444 

 468 

 

Lettuce Canada 6 

 Mexico 15 

 21 

 

Parsley Mexico 27 

 

Tomatoes Belgium 3 

 Canada 93 

 Mexico 537 

 Netherlands 3 

 636 

Table 1. Distribution of Imported Samples. This 

table details the number of imported samples by 

country of origin and by commodity.
Microbiological Data Program - Annual Summary, Calendar Year 2004 

6 

Figure 3. Commodity Origin. The proportion 

of domestic, imported or unknown origin for 

each commodity is depicted for samples tested 

in 2004. 

Domestic 53.2% 

Unknown 2.4% 

Imported 44.4% 

Cantaloupe 

Domestic 94.3% 

Unknown 3.3% 

Imported 2.4% 

Celery 

Domestic 87.0% 

Unknown 3.6% 

Imported 9.4% 

Cilantro 

Domestic 55.6% 

Unknown 2.9% 

Imported 41.5% 

Green Onions 

Domestic 97.5% 

Unknown 1.9% 

Imported 0.6% 

Lettuce 

Domestic 87.2% 

Unknown 8.2% 

Imported 4.6% 

Parsley 

Domestic 60.3% 

Unknown 11.3% 

Imported 28.4% 

Tomatoes 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

7 

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). To minimize spoilage and bruising, the 

produce is often harvested before reaching full 

ripeness. 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), 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) 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 hand-held computers. 

Sample information is captured in the MDP 

database files on the same day as sample collection. 

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-ofState 

 

Total E. coli 

E. coli 

O157:H7 Salmonella 

California 497 252 135 252 753 141 504 2,534 2,531 1,905 2,534 

Colorado 72 36 18 36 108 18 72 360 360 252 360 

Florida 252 126 69 126 378 66 250 1,267 1,267 88 1,264 

Maryland 144 69 33 72 210 33 144 705 705 492 705 

Michigan 216 108 54 108 324 54 213 1,077 1,077 753 1,077 

Minnesota 12 0 12 12 12 12 12 72 72 72 72 

New York 324 162 81 162 486 81 323 1,619 1,619 1,133 1,619 

Ohio 215 108 51 108 321 54 215 1,072 1,072 749 1,072 

Texas 285 144 71 144 429 72 288 1,433 1,433 997 1,433 

Washington 144 72 33 72 213 39 144 717 717 525 717 

Wisconsin 72 36 18 36 106 18 72 358 358 252 358 

Totals 2,233 1,113 575 1,128 3,340 588 2,237 11,214 11,211 8,018 11,211 

Cantaloupe

Celery

Cilantro

Green Onions

Lettuce (Leaf or Romaine)

Parsley 

Tomatoes

Note: There were three samples that were analyzed for E. coli, but not Salmonella. There were three other 

samples that were analyzed for Salmonella, but not E. coli (explains 11,214 vs. 11,211 total). 

Table 2. Samples Collected and Analyzed by State. This table shows the number of samples collected by 

each State by commodity and the total number of collected samples tested for each organism. 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

8 

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 http://www.ams.usda. 

gov/science/MPO/SOPs.htm. 

III. Laboratory Operations 

Ten microbiology laboratories performed analyses 

for MDP in 2004. The Minnesota Department of 

Agriculture laboratory began analyzing routine 

samples in September 2004 after previously 

performing method development studies for MDP. 

Further testing on positive culture samples and 

isolates was performed by the Florida Department 

of Agriculture and Consumer Services (FDACS), 

the Gastroenteric Disease Center at Pennsylvania 

State University, and FDA/CVM. These additional 

tests included multiplex polymerase chain reaction 

(mPCR) screening for pathogenic E. coli, serotyping, testing for antimicrobial resistance and 

virulence attributes, and genomic fingerprinting. 

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 24 hours to allow for different sample arrival 

times from the various collection sites. Only 

excess soil was removed prior to testing. 

Samples were washed in buffered peptone water 

and all analyses were conducted from this surface 

wash eluent. Refinements to methods were introduced in 2004 to enhance sensitivity and selectivity for target pathogens in MDP commodities. 

As with all method modifications, all program 

quality assurance (QA)/quality control (QC) 

criteria must be met prior to implementation by 

MDP laboratories. 

Prior to February 2004, MDP used the FDA 2001 

Bacteriological Analytical Manual (BAM)-

specified (4) traditional gas-production method for 

detecting thermotolerant fecal E. coli and the Most 

Probable Number (MPN) method for enumeration. 

In February 2004, MDP switched to an AOAC®-

approved method based on a more sensitive 

enzyme-based assay specific for detecting E. coli, 

with enumeration accomplished using the standard 

MPN method. The presumptive E. coli positive 

cultures were sent to the Florida laboratory 

(FDACS) for mPCR screening for shiga-toxin 

producing and enterotoxigenic E. coli. 

MDP used DNA-based polymerase chain reaction 

(PCR) assays and automated instruments for the 

detection of Salmonella and enterohemorrhagic E. 

coli O157:H7 (introduced in April 2004) in 

produce samples. Cultural and Immunomagnetic 

Separation (IMS) technology were employed for 

isolation of target bacteria. Automated biochemical 

tests and cultural methods were used in the 

verification of any preliminary findings. 

The main objectives of the QA/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 BAM methods, AOAC® methods, the FSIS 

Microbiological Laboratory Guide, and the Environmental Protection Agency’s Good Laboratory 

Practices. MDP analytical methods are published at 

http://www.ams.usda.gov/science/MPO/SOPs.htm. 

SOPs provide uniform administrative, sampling, 

and laboratory procedures. 

Positive and negative controls and a sterile media 

blank were required for each sample set. MDP 

laboratories use positive control strains of E. coli

O157:H7 and Salmonella typhimurium 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 having to perform 

lengthy biochemical tests. All controls and blanks 

were taken through the entire analytical procedure. 

MDP laboratories used automated instrumentation 

for confirmation of isolates. 

A Technical Advisory Group, comprised of microbiologists from each participating laboratory, 

provided technical feedback on program SOP 

revisions and addressed technical and QA issues. 

For day-to-day QA oversight, each participating 

facility was required to have a Quality Assurance 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

Unit (QAU) that operated independently from the 

laboratory staff. Preliminary QA/QC review procedures were performed on-site by each laboratory’s 

QAU. Final review procedures are performed by 

MDP staff that are responsible for collating and 

reviewing data for conformance with SOPs. 

Laboratory performance was monitored through onsite reviews by MDP staff to determine compliance 

with MDP SOPs. Corrective actions, if necessary, 

were performed as a result of on-site reviews. 

Performance equivalency of the participating laboratories was monitored by a program-wide proficiency testing program. 

IV. Database Management

 

MDP maintains an electronic database that serves as 

a central data repository. The central database 

resides at MPO, Manassas, Virginia. 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 utilizes 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/password accounts and user access rights for 

the various system functions based on position 

requirements. The RDE system utilizes Secure 

Socket 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 

Sample Information Form on handheld or laptop 

computers. The e-SIF system generates formatted 

text files containing sample information that are emailed to MDP headquarters and then imported into 

the Web-based 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 in a Windows® operating environment. 

Access to the central MDP database is limited to 

MDP headquarters personnel and is controlled 

through password protection and user access 

rights. The system is backed up each night and 

back-up tapes are sent to off-site storage once a 

week. 

V. Summary of 2004 Data 

Table 1 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-six percent of the 

samples were from domestic sources, 20 percent 

were imported, and approximately 4 percent 

were of unspecified origin. Table 2 shows the 

distribution of samples among each commodity 

and collection State. 

In 2004, the third full year of testing, MDP 

collected 11,214 samples. Of these, 11,211 samples were screened for the presence of E. coli and 

Salmonella; and 8,018 samples were screened for 

enterohemorrhagic E. coli O157:H7. Table 2 

shows the number of samples collected and 

analyzed by each State. E. coli has been used as 

an indicator of fecal contamination in food and 

water; pathogenic E. coli and Salmonella are 

frequently implicated in foodborne outbreaks 

where produce was involved (1). Consequently, 

9 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

10 

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

laboratory analysis and reporting. 

- 9 State labs + 1 Federal lab for sample testing 

- Standardized sample preparation 

- Test for specific foodborne pathogens

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 & adhoc reporting 

- Annual Summary 

Data Reporting 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

these organisms are of public health significance. 

Baseline data-gathering efforts designed to identify 

relevant trends ideally require data generated over 

multiple growing seasons that span several years. 

Although 2004 provided a third year of data for 

MDP, continued data collection is needed before 

multi-year inferences can be made. Additionally, 

MDP began implementing major changes in 

detection technology that will further affect data 

interpretation. 

The 11,211 samples were initially screened for E. 

coli using an AOAC-official method for detection 

and enumeration. Presumptive E. coli-positive 

samples were further screened for pathogenic E. 

coli that harbor shiga-toxins (STEC) and enterotoxins (ETEC) (refer to Table 3) using a multiplex 

polymerase chain reaction (mPCR) assay developed 

by FDA. Toxin genes associated with pathogenic E. 

coli were found in 43 samples; however, 

pathogenic E. coli strains were isolated in only 9 of 

these samples. In addition to the technological 

differences between the 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 a small number of target 

bacterial cells; differential growth rates of various 

bacteria; and additional growth requirements. 

The 9 isolates were sent to Pennsylvania State University for serotyping and further characterization, 

including 13 virulence-specific genes associated 

with different categories of pathogenic E. coli. 

FDA/CVM conducted tests on antimicrobial resistance and genomic fingerprinting on these isolates. 

Eight of the nine isolates carried two or more 

toxins. Four carried toxin genes from both the 

STEC and ETEC pathogenic classes. Two of the 

isolates showed resistance to various antimicrobial 

agents. For an isolate to be characterized as a 

human pathogen and cause disease, there must be 

an interplay of several proteins including toxins, 

encoded by respective genes. MDP only identified 

toxin genes; additional testing required in order to 

determine the actual pathogenicity of these isolates 

is not within the scope of MDP. The results of 

testing conducted by Pennsylvania State University 

and FDA/CVM are shown in Table 4. 

In 2004 the BAX® instrument, an automated 

PCR system, was used for Salmonella screening. 

In April 2004, MDP introduced screening for 

enterohemorrhagic E. coli O157:H7 by BAXbased PCR. For all BAX determinations, pooled 

samples were initially screened. If a positive 

result was obtained, the three individual samples 

were tested. 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 

MDP-approved commercial biochemical kit or 

system. Isolates were then sent to FDA’s CVM 

for serotyping, antimicrobial resistance testing, 

and genomic fingerprinting. 

As depicted in Table 5, a total of 11,211 samples 

were screened for Salmonella by BAX-PCR. 

Seventeen of these individual samples were 

positive and five Salmonella isolates were 

obtained: one each from cantaloupe, cilantro, 

green onion, lettuce (romaine), and parsley. 

These five isolates were sent to FDA’s CVM for 

identification by serotyping, antimicrobial 

resistance, and genomic fingerprinting. Table 6 

identifies each isolate and lists the associated 

serogroup. One isolate, S. poona, belonging to 

11 

Commodity 

Number of 

Samples 

Tested 

Number of 

Pathogenic E. coliPositive Samples 

Cantaloupe 2,233 2 

Celery 1,113 1 

Cilantro 574 8 

Green Onions 1,128 2 

Lettuce 3,339 19 

Parsley 588 10 

Tomatoes 2,236 1 

Total 11,211 43 

Table 3. 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 2004 

serogroup G, is under further investigation. 

Two isolates, S. newport and S. oraninenburg

belong to serogroup C while S. enteritidis and 

S. anatum belong to serogroups D and E, respectively. These four isolates were not 

resistant to any of the antimicrobial agents 

tested. Also shown in Table 6 is an image of 

the PFGE analysis performed for each isolate. 

No enterohemorrhagic E. coli O157:H7 strain 

was isolated from the 8,018 samples screened, 

although there were 6 samples that tested 

positive by BAX-PCR. In this case, as with 

pathogenic E. coli analysis, a number of 

factors can be involved in the rate of isolation, 

including the level of background microflora 

versus the number of target bacterial cells, 

differential bacterial growth rates, and 

additional growth requirements. 

12 

Commodity 

Number of 

Samples 

Tested 

Number of 

Positive 

Isolates 

Cantaloupe 2,233 1 

Celery 1,113 0 

Cilantro 572 1 

Green Onions 1,128 1 

Lettuce 3,340 1 

Parsley 588 1 

Tomatoes 2,237 0 

TOTALS 11,211 5 

Number of 

Positive 

Individual 

Samples 

3 

0 

3 

1 

3 

0 

7 

17 

Table 5. Summary of Analysis for Salmonella. 

This table shows the number of samples screened 

for Salmonella, the number of positive individual 

samples, and the number of isolates obtained. 

Commodity 

Pathogenic 

Class 

Toxin Genes 

Identified O Antigen H Antigen 

Cantaloupe STEC/ETEC STa, Stx-1 NT 52 

Cilantro STEC/ETEC STa, Stx-1 NT 52 

Cilantro ETEC LT, STb 73w 14 

Cilantro STEC/ETEC STb, Stx-2, hlyA NT 19 

Green Onion1

 ETEC LT, STa 19 H+ 

Lettuce STEC/ETEC STb, Stx-2, eae, hlyA 121 19 

Lettuce ETEC STa 116w 28 

Parsley2

 ETEC STa, STb 117 21 

Parsley ETEC STa, STb 5w 10 

Pulsed-Field Gel Electrophoresis 

Table 4. 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. Also shown is an image 

of the pulsed-field gel electrophoresis (PFGE) analysis performed for each isolate.

STEC Shiga-toxin producing E. coli. 

ETEC Enterotoxigenic E. coli. 

Stx-1/2 Shiga-toxin 1 or 2, eae intimin; hlyA-hemolysin. 

NT Non typable. 

1

 Resistance to antimicrobials: Ampicilin, Streptomycin, Sulfasoxazole, Tetracycline, Trimethoprim/sulfamethoxazole 2

 Resistance to antimicrobials: Streptomycin, Sulfasoxazole 

Serotyping 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

13 

Commodity Genus Species 

Cantaloupe Salmonella Newport 

Cilantro Salmonella Anatum 

Green Onion Salmonella Enteritidis 

Lettuce (Romaine) Salmonella Poona* 

Parsley Salmonella Oraninenburg 

* Under further investigation. 

Table 6. Salmonella Identification, Serogroup, and Genomic Fingerprinting. This table summarizes the 

genus, species, and serogroup for each of the five Salmonella isolates obtained in 2004. Also shown is an 

image of the pulsed-field gel electrophoresis (PFGE) analysis performed for each isolate. 

Serotype / Identification 

Serogroup 

C2

E1

D1

G 

C1

References: 

1. Scientific Criteria to Ensure Safe Food (2003). Institute of Medicine, National Research Council of 

the National Academies. The National Academies Press, Washington, DC. 

2. Isaacson, R.E., Torrence, M., and Buckley, M.R. Preharvest Food Safety and Security. American 

Society for Microbiology. 2004. 

3. USDA, Agricultural Marketing Service, Science and Technology Programs, AMS Microbiological 

Data Program Sampling Rationale and Principles, February 2002 (rev. 2), http://

www.ams.usda.gov/science/MPO/SamplingOverview.pdf

4. Bacteriological Analytical Manual (BAM) (2001). US 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 2004 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. 

Pulsed-Field Gel Electrophoresis 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

14 

Definitions: 

Antimicrobial resistance: 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: Refers to 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. 

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. 

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 encoding this protein is used as a marker in 

control cultures. 

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

fecal contamination. 

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

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 (as 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. 
Microbiological Data Program - Annual Summary, Calendar Year 2004 

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. 

●●●●●

15 

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. 

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. 

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 the 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.