Incidence and Tracking of Escherichia coli O157:H7 in

a Major Produce Production Region in California

Michael Cooley1*, Diana Carychao1, Leta Crawford-Miksza2, Michele T. Jay2, Carol Myers3, Christopher Rose4, Christine Keys5, Jeff Farrar3,

Robert E. Mandrell1*

1 Produce Safety and Microbiology Research Unit, United States Department of Agriculture-Agricultural Research Service, Western Regional Research

Center, Albany, California, United States of America, 2 California Department of Health Services, Food and Drug Laboratory Branch, Richmond,

California, United States of America, 3 California Department of Health Services, Food and Drug Branch, California, United States of America, 4 Central

Coast Regional Water Quality Control Board, California Environmental Protection Agency, San Louis Obispo, California, United States of America,

5 Food and Drug Administration/Center for Food Safety and Applied Nutrition (CFSAN)/Office of Plant and Dairy Foods (OPDF)/DMS, College Park,

Maryland, United States of America

Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995–2006,

22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California.

Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated

farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once

from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy

rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of.100 individual

samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate

and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis

(PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of

identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport

of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow

creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher

water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where

water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving

multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near

produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human

illness.

Citation: Cooley M, Carychao D, Crawford-Miksza L, Jay MT, Myers C, et al (2007) Incidence and Tracking of Escherichia coli O157:H7 in a Major

Produce Production Region in California. PLoS ONE 2(11): e1159. doi:10.1371/journal.pone.0001159

INTRODUCTION

Escherichia coli O157:H7 (EcO157) is an enteric pathogen that can

cause life threatening hemorrhagic colitis and, in very severe cases,

hemolytic uremic syndrome [1]. It has been estimated that

EcO157 causes approximately 73,000 illnesses in the United States

each year. Between 1982 and 2002, 350 outbreaks caused by

EcO157 were reported; 52% and 9% were caused by foodborne

and waterborne sources, respectively [2]. Ground beef and

produce were associated with 75 and 38 outbreaks, respectively,

but the increased consumption of fresh fruits and vegetables in the

U.S. appears to correlate with increased produce-associated

outbreaks [2]. Since 1995, there have been 22 outbreaks of

EcO157 associated with fresh lettuce or spinach, and 9 of these

outbreaks have been traced to, or near, the Salinas Valley region

of California [3–9]. The Salinas Valley region on the central coast

of California is the major leafy vegetable producer in the US [10].

Contamination of produce can occur in the field by application

of raw (or poorly composted) manure, exposure to contaminated

water (irrigation or flooding) [11–14], or by deposition of feces by

livestock or wild animals. Another possible, but unsubstantiated,

source is contaminated dust from concentrated livestock or other

animal operations. The presence of EcO157 in, or near, a raw

produce production environment must be considered a potential

risk factor for both sporadic- and outbreak-related human illness.

Warm-blooded animals and invertebrates are known to carry

EcO157 [15–24]. Persistence of EcO157 in the environment is

variable and likely dependent upon a variety factors, including

temperature, sunlight (ultraviolet), moisture, nutrients and possibly

unknown factors that enhance or decrease amplification of

EcO157. Recent studies indicate that EcO157 will survive in the

field for four to eight weeks [13,25–27], thus supporting the

National Organic Program recommendation of a minimum of

120 days between manure application and harvest [28]. However,

these and other studies also conclude that survival of EcO157 is

improved at lower temperatures, in clay soil and in close

Academic Editor: Debbie Fox, The Research Institute for Children, United States

of America

Received May 15, 2007; Accepted October 13, 2007; Published November 14,

2007

This is an open-access article distributed under the terms of the Creative

Commons Public Domain declaration which stipulates that, once placed in the

public domain, this work may be freely reproduced, distributed, transmitted,

modified, built upon, or otherwise used by anyone for any lawful purpose.

Funding: A portion of this work was funded by CRIS projects 5325-42000-045-00D

and 5325-42000-044-00D, an interagency transfer of funds provided by FDA

(5325-42000-045-01R), a contract with the CAL-EPA State Water Resources Control

Board (05-221-130-0), and a grant from USDA-CSREES Section 32.1 (project no.

2006-01240).

Competing Interests: The authors have declared that no competing interests

exist.

* To whom correspondence should be addressed. E-mail: mcooley@pw.usda.

gov (MC); mandrell@pw.usda.gov (RM)

PLoS ONE | www.plosone.org 1 November 2007 | Issue 11 | e1159
association with roots [11,25,29], and in soil amended with cattle

feces [30]. These results are consistent with the ability to isolate

EcO157 from carrots 168 days after application of contaminated

manure [12]. EcO157 present in agricultural environments are

expected to be stressed, and possibly non-culturable, increasing the

problem of measuring accurately the amount of pathogen in

samples [31,32]. However, in an unintended ‘‘natural’’ experiment of the virulence and survival of EcO157 for humans, the

same EcO157 strain infecting a child exposed to the family garden

fertilized with raw cow manure was isolated from the soil for

approximately 70 days, and survived much better at ambient

temperatures than at 4uC [27]. Nevertheless, contamination of

ready-to-eat commodities with EcO157 is expected to be present

at very low levels as evidenced by the rarity of outbreaks associated

with raw produce compared to the volume of consumption.

The specific risk factors for in-field contamination, survival, and

spread of EcO157 in the produce production environment remain

unclear. Traceback investigations for one EcO157 outbreak

reported by Washington state in 2002 and two outbreaks in

California in 2003 identified a single farm in the Salinas Valley

common to all three outbreaks [33]. The commodities (lettuce and

spinach) involved, and the processing and distribution of these

commodities varied between the outbreaks. The farm was the only

factor common to all three outbreaks and was considered a likely

source of the pre-harvest contaminated produce. Furthermore, an

outbreak associated with a bagged mixed-produce product, including lettuce, occurred in Minnesota in September 2005 [34] and

a large multi-state outbreak involving bagged spinach occurred in

August/September, 2006 [6]. Traceback investigations for these

outbreaks indicated that the produce was grown in the Salinas

(Monterey County) and San Juan (San Benito County) valleys,

respectively. A critical result in both the 2005 and 2006 bagged

produce traceback investigations was the isolation of strains identical

to the human outbreak strains from bagged produce obtained from

patient’s homes; a single lot number on the bags associated with the

2006 spinach outbreak was associated with a production run with

product obtained from four farms/ranches, one in Monterey and

three in San Benito Counties. Details of the results of the

investigation of this outbreak have been reported [9].

An important tool in outbreak and environmental investigations

is ‘‘fingerprinting’’ of strains to identify relatedness and/or

potential sources of contamination. Pulsed-field gel electrophoresis

(PFGE) has become the method of choice in these investigations

due to the availability of the PulseNet database containing

thousands of profiles submitted by public health labs for isolates

associated with sporadic or outbreak illnesses [35]. Nevertheless,

PFGE is a time- and labor-intensive method, requiring specially

trained individuals to acquire, analyze and submit results

comparable between multiple labs. A newer method, Multi-Locus

Variable-number tandem repeat Analysis (MLVA), has been

shown to be more reproducible than PFGE and better at

discriminating between closely related EcO157 isolates [36–39],

and is being evaluated by CDC as a potential next generation

typing system. Discrimination of strain differences with MLVA

data relies on hyper-mutable tandem repeat (TR) elements located

at various places in the genome. Isolates that are closely related

epidemiologically and/or by source (e.g. human clinical or food

isolates from the same outbreak, animal host), often are identical

or differ by only one or two TR changes at a single locus [36].

We report the results of surveillance studies of EcO157

incidence in multiple environments associated with EcO157

foodborne outbreaks. We report that surface water is a vehicle

of transmission of EcO157 and a potential risk factor for preharvest contamination of leafy vegetables in the Salinas region.

Our results demonstrate the suitability of MLVA for typing

EcO157 strains isolated during complex environmental investigations. Furthermore, we report genotyping data measuring the

relatedness of strains, useful for identifying potential environmental point sources of EcO157.

MATERIALS AND METHODS

Sporadic and outbreak clinical isolates

Human clinical isolates were received from Dr. M. Janda at the

California Department of Health Services (CDHS); S. Chu at the

CDHS-LA County; Dr. K. Smith at the Minnesota Department of

Public Health; D. Green at the Washington State Public Health

Laboratory; C. Ball at the Idaho Bureau of Laboratories; W.

Chmielecki at the Pennsylvania Department of Health; T. Monson

at the Wisconsin State Laboratory of Hygiene; and the National

Laboratory for Enteric Pathogens (NLEP), National Microbiology

Laboratory (NML), Health Canada. Strains obtained from the

DECA Reference Strain Set are available from the STEC Center,

Michigan State University. A list of all strains used in this study,

including source information, is provided in Supplemental Information, Table S1.

Environmental sampling

Representative samples of soil, sediment, feces, and plants were

collected from the environment into Whirl-Pak bags (Nasco,

Modesto, CA) using either clean latex exam gloves or single-use

sterile spatulas. Pig colon samples were collected by necropsy

performed in the field (Jay MT, Cooley MB, Carychao D, Wiscomb

GW, Sweitzer RA, et al. (2007) Escherichia coli O157:H7 in feral swine

near spinach fields and cattle, central California coast).

Samples of water (100 ml) were collected into sterile, disposable

bottles. The collection points were as close to the centers of

a stream or pond as practical, and collection was accomplished by

attaching bottles to a telescoping pole. GPS data and observations

regarding appearance of the sample or the surroundings were

recorded. Some samples were collected with cotton gauze swabs

(‘‘Moore swabs’’) [40] by anchoring them in a water source to

a monofilament line and retrieving them 5 days later [41]. The

swabs were stored in marked Whirl-Pak bags. All samples were

transported on ice. Sample locations in the Salinas watershed are

shown in Figure 1. Additional details of watershed and 2006

spinach outbreak sample locations have been reported [9,42,43].

Colilert testing

For watershed samples, quantification of total coliforms and E. coli

was determined by the ColilertH QuantiTray 2000 method

according to the manufacturer’s recommendations (Idexx Laboratories, Westbrook, ME). Duplicate samples (111 ml) of surface water

were collected from each site, transported to the laboratory on cold

packs, and stored at 4uC overnight. The following morning, each

sample was diluted 1:10 and 1:100 in Butterfield’s phosphate buffer.

ColilertH reagent containing 4-methylumbelliferyl-beta-D-glucuronide (MUG) was added to 100 ml of each dilution, and samples were

inoculated into 97-well trays and sealed. After 24 hours of

incubation at 35uC, wells were counted for yellow color from

fermentation of O-nitrophenyl-beta-D-galactoside (ONPG), evidence of total coliforms, and blue fluorescence from MU (generic

E. coli), and the Most Probable Number (MPN) were calculated from

the manufacturer’s data tables. Four control strains (Pseudomonas

aeruginosa, ATCC 27853; Klebsiella pneumoniae, ATCC 13882; E. coli,

ATCC 25922; EcO157, ATCC 43888) were cultured and tested

simultaneously for each assay.

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Isolation method

Collected samples were transported and/or stored 12 to 24 hr on

ice until the isolation method was initiated, usually the morning

after collection. Our method was modified from a method

described previously for isolation of EcO157 from cattle feces,

carcass and hide samples [44]. Ten grams of sample, including

soil, sediment, feces or tissue (plant or colon), were removed by

hand using clean exam gloves or with a sterile spatula, and placed

into a 250 ml sterile flask containing 90 ml tryptic soy broth (TSB;

Becton Dickinson, Sparks, MD). Moore swabs were rinsed briefly

in tap water to remove mud and sediment, and were added to

sterile flasks (1 liter) containing 250 ml TSB for enrichment. The

flasks were incubated for two hrs at 25uC with shaking at

200 RPM, then at 42uC for 8 hrs, and held at 4uC without

shaking until the following morning. Water samples were enriched

by adding 11 ml of 106TSB (filter sterilized, 0.45 mm) to 100 ml

of sample, and the mixture was incubated as described above.

One ml of the enrichment broth was tested by adding 20 ml of

anti-O157 antibody bound to magnetic beads (Invitrogen/Dynal,

Carlsbad, CA) and mixing them for 30 min. The beads were

washed twice with PBS containing 0.05% Tween 20 (PBS-T), then

they were re-suspended in 100 ml PBS [45]. Alternately, beads

were incubated and wash using the Dynal BeadRetriever

(Invitrogen/Dynal, Carlsbad, CA) using the EPEC/VTEC Protocol. With either method, 50 ml each of the resuspended beads

were spread on Sorbitol MacConkey agar (Difco Labs; Detroit,

MI) containing cefixime (0.05 mg/ml; Invitrogen/Dynal) and

tellurite (2.5 mg/ml; Invitrogen/Dynal) (CT-SMAC), and on

Figure 1. Map of Salinas, CA region showing sample locations. Farm A is marked as a black dot. The circle marked with ‘‘X’’ is coded by positions

numbered clockwise 1 to 23 and representing the sampling dates show in the list below the circle. The approximate rainfall in cm for the 5 days prior

to sampling is shown in the list. Dates indicated in red text indicate at least one EcO157 strain was isolated on that date. Circles on the map

designated ‘‘A’’ through ‘‘V’’ correspond to the locations where samples were obtained; these are also listed in Table S1. Open circles within the

‘‘Location circle’’ designate that no sample was obtained on that date; blue circles designate that a sample was obtained, but no EcO157 strain was

isolated; and red circles designate that a sample was obtained, and at least one strain of EcO157 was isolated.

doi:10.1371/journal.pone.0001159.g001

E. coli O157:H7 and p\Produce

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Rainbow Agar (Biolog, Hayward, CA) containing novobiocin

(20 mg/ml; Sigma-Aldrich) and tellurite (0.8 mg/ml; Invitrogen/

Dynal) (NT-Rainbow). To aid in identification of authentic

EcO157 colonies, plates of both media were streaked with an

EcO157 strain RM2315 marked with the Green Fluorescent

Protein [46]. Plates were incubated at 37uC overnight (approximately 18 hr).

Suspect colonies and positive control EcO157 strain RM2315,

were patched onto duplicate plates of Luria Broth (LB) agar (Fisher

Scientific, PA) using a numbered grid and incubated at 37uC

overnight. One set of the patched colonies was blotted onto Protran

nitrocellulose membranes (BA 85, Whatman/Schleicher & Schuell;

Sanford, ME). The membrane was washed with 25 mM TRIS

pH 7.4, 0.15 M NaCl, 0.1% Tween 20 (TBS-Tween) and blocked

by immersion in 10 ml of 0.5% casein, 0.01 M TRIS, 0.031 M

sodium azide, 0.15 M NaCl pH 7.4 (blocker) with shaking for

30 minutes at RT. The membrane was incubated in anti-O157

IgG monoclonal antibody (MAb), 13B3 [47], diluted 1:2000 in

10 ml 16 TBS-Tween containing 1% BSA, 0.02% KCl, 0.1%

sodium azide (ELISA Diluting Buffer) and with shaking for

30 minutes at RT. The membrane was washed four times with

100 ml TBS-Tween, then incubated in goat anti-mouse IgG

conjugated with alkaline phosphatase (Invitrogen/Zymed, Carlsbad, CA) diluted 1:2000 in ELISA Diluting Buffer for 30 minutes

with shaking at RT. The membrane was washed four times with

TBS-Tween, then twice in distilled water. The membrane was

developed by the addition of 10 ml of a 0.15 mg/ml and 0.3 mg/

ml solution of 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) and

Nitro Blue Tetrazolium (NBT) Sigma-Aldrich, B5655), respectively. During multiple isolation procedures, we noted that authentic

EcO157 colonies were distinguishable from apparent antibodypositive non-EcO157 bacteria by the rate of the color reaction

compared to EcO157 strain RM2315.

Virulence gene detection via PCR

Putative EcO157 colonies identified by colony blots as described

above were analyzed further by real-time PCR for the presence of

the rfbE gene. Primers were designed based on a 497 bp region of the

rfbE gene reported to be specific for EcO157 [48]. The 20 ml

reaction mix included 10 ml Stratagene Brilliant QPCR master mix,

0.3 mM each rfbE primers (59-TTTCACACTTATTGGATGGTCTCA39 and 59 TGAGTTTATCTGCAAGGTGATTCC-39),

and 0.1 mM of the probe (59 6-FAM-TTCTAACTAGGACCGCAGAGGAAAGAGAGGAATTA-BHQ-1-39, Biosearch Technologies, Inc., San Francisco, CA). Bacteria were transferred from the

duplicate patch plate directly into the PCR tube using a sterile

toothpick. Control tubes containing no template, or EcO157 strain

RM2315 bacteria, were included for amplification in a Stratagene

MX3000P Real-Time PCR machine (95uC for 5 min, then 60 cycles

of 95uC for 15 sec and 60uC for 45 sec).

Colonies producing a positive reaction for the rfbE gene were

streaked from the duplicate patch plate for isolation of single

colonies and were analyzed further for the fliC (H7) gene using the

method of Fratamico et. al. [49]. Each 25 ml reaction contained

16 ThermoPol buffer (New England Biolabs, Ipswich, MA),

200 mM each dNTP, 0.25 mM each primer, 2.5 U TAQ, and 1 ml

template. Thermal cycling parameters were 95uC for 5 min, then

30 cycles of 94uC for 30 sec, 58uC for 30 sec, 72uC for 30 sec, and

a final extension at 72uC for 5 min. DNA template was prepared

by re-suspending approximately 1 ml of cells scraped from a plate

with a 1 ml sterile loop into 100 ml of distilled water with

subsequent boiling for 20 minutes. Debris from boiled cells was

removed by centrifugation at 5000 RPM for 10 min.

Other virulence genes (stx1, stx2, eae, hly) were detected by

a multiplex reaction described previously [50]. Each 15 ml

reaction contained 16 Qiagen multiplex PCR master mix

(Qiagen, Valencia, CA; #206143), 0.25 mM each primer, and

1 ml of template. Thermal cycling parameters were 95uC for

15 min, then 35 cycles of 95uC for 1 min, 65uC for 2 min, and

72uC for 1.5 min. Annealing temperatures decrements from 65uC

to 60uC between cycles 10 and 15, and elongation time increments

from 1.5 min to 2.5 min between cycles 25 and 35. All PCR

ingredients were purchased from New England Biolabs. Reactions

were done on a Tetrad thermal cycler (Bio-Rad/MJ Research,

Hercules, CA) and run on a 2% agarose gel.

PFGE method

Selected isolates were typed using the standard PulseNet procedure

with both XbaI and BlnI restriction enzymes [35]. Dendrograms for

comparing the isolates were constructed by analysis of the profiles

for both digests with bandmatching and phylogenetic clustering

analysis methods of Bionumerics (Applied Maths, Sint-MartensLatem, Belgium). PFGE profiles of the isolates were compared

against those present in the PulseNet database.

MLVA method

MLVA was performed using capillary electrophoresis methods

described previously [37,39]. Essentially, 10 loci are amplified in

three multiplex PCR reactions using fluorescent primers. Reaction

1 contained primers for VIC-Vhec1, NED-Vhec3, FAM-Vhec4,

FAM-Vhec5; reaction 2 contained primers for VIC-Vhec1, NEDVhec2, VIC-Vhec6, FAM-Vhec7 [37]; and reaction 3 contained

primers for FAM-O157-17, NED-O157-19, VIC-O157-37 [39].

All fluorescent primers were obtained from Applied Biosystems

(ABI, Foster City, CA). Each PCR reaction of 10 ml contained 16

multiplex PCR master mix (Qiagen), 0.2 mM of each primer and

1 ml template. Thermal cycling parameters for reactions 1 and 2

were 95uC for 15 min, then 25 cycles of 94uC for 30 sec, 63uC for

90 sec, 72uC for 90 sec, with a final extension at 72uC for 10 min.

Parameters for reaction 3 were 95uC for 15 min, then 35 cycles of

94uC for 20 sec, 65uC for 20 sec, and 72uC for 20 sec, with a final

extension of 72uC for 5 min. The multiplex reactions were pooled

and diluted 1:50 into distilled water. One ml of this dilution was

added to a mixture of 12 ml HiDi formamide (ABI) and 0.08 ml of

a MW standard ladder (ROX-labeled MapMarker 1000, Bioventures, Inc., Murfreesboro, TN), and the mixture was heatdenatured for 5 min at 95uC, cooled on ice for 2 min, loaded

onto the ABI 3130xl Genetic Analyzer, and run for 2100 seconds

using the default settings for fragment analysis with ‘‘Dye Set D’’

(ABI) in a 50 cm array containing POP7 polymer. The sizes of

fragments were determined using GeneMapper software (ABI). If

more than one fragment was detected for a single locus, the

fragment with the highest level of fluorescence was selected for TR

determination. Fragment size was converted to number of TR by

subtraction of the amplified, non-repeat sequences and division by

the repeat size. Fractional repeat numbers were rounded to the

nearest whole repeat number for purposes of comparison. MLVA

genotypes for each isolate tested in this study are listed in

Supplemental Information, Table S1.

Phylogenic, statistical methods and precipitation

data

A minimal spanning tree of the MLVA types was created using the

MST algorithm in BioNumerics regarding TRs as categorical

coefficients. All coefficients were equally weighted. When solutions

with identical calculated distances were obtained, a priority rule

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was applied for linkage in the following order; the highest number

of single-locus variants, the highest number of double-locus

variants and the highest number of entries. Clusters were selected

based on criteria that neighbors within the cluster must be single

locus variants and the resulting cluster must contain at least 3

MLVA types. The allelic diversity was based on Nei9s diversity

[51], which is the calculation 1-S (allele frequency)2

. Wilcoxon

signed rank test or t test was used for pairwise comparisons

between generic E. coli levels and EcO157 incidence (SigmaStat

3.0, SPSS, Inc., Chicago). Precipitation totals were determined

from the average of daily accumulative data collected at four

weather sites as part of the California Irrigation Management

Information System (CIMIS). The sites included data for northern

Salinas, southern Salinas, Castroville and the Salinas airport on

the collection date and four days prior to the collection date. Flow

rates in Gabilan Creek, Salinas River and Reclamation Canal

were available at USGS National Water Information System

(http://waterdata/usgs.gov/ca).

RESULTS

Sampling of a farm associated by traceback with

three separate outbreaks in 2002–2003

From June through December 2004, 178 samples were collected

from a farm (Farm A, Figure 1) that was identified during

traceback investigations as having provided produce associated

with three separate outbreaks (Spokane, WA 2002, San Diego, CA

2003, and San Mateo, CA 2003). Samples included soil, water,

compost, wild animal feces, sediment and plants and were

processed for isolation of EcO157. Further details of the

investigation are published in a CDHS report [5]. The only

sample yielding EcO157 was a creek sediment sample collected

adjacent to Farm A in July 2004. A single strain of EcO157 (Table

S1, RM4403) was isolated from the sample. Strain RM4403 was

fliC (H7)-positive by PCR; virulence genes stx1, stx2, eae and hly

also were positive by PCR (data not shown). However, MLVA and

two-enzyme PFGE (XbaI and BlnI) indicated that RM4403 did not

match closely any of the clinical isolates from the three associated

outbreaks (Table S1: see ‘‘Source’’ OB6, OB7, OB8; data not

shown).

Potential upstream sources of EcO157 surveyed subsequently

included a goat ranch and horse ranch, both located,2.4 km

upstream. The goat ranch, situated on a hill approximately 800

meters from the creek, contained approximately 40 goats, 6

horses/ponies, and, infrequently, a few cows. Drainage from this

property fed into a tributary of the creek. The horse ranch,

situated on the creek approximately 1.6 km upstream, contained

12 horses that had direct access to the creek. Nevertheless, water

and sediment samples taken immediately downstream of these

properties were consistently negative for EcO157.

Sampling of the Salinas Valley watershed, 2005

Several other produce-related outbreaks have been linked to the

Salinas region, some of which may have been due to pre-harvest

contamination. The California Central Coast Regional Water

Quality Control Board (CCRWQCB) reported levels of generic E.

coli above acceptable levels at several locations in the watershed

[52]. Farm A, which floods almost every winter, would be exposed

potentially to these contaminated waters. Indeed, surface water

routinely floods onto fields elsewhere in the Salinas region.

Therefore, selected sites in the Salinas Valley watershed were

surveyed for the presence of EcO157 as a measure of the potential

contribution to pre-harvest contamination. We initiated a comprehensive investigation of the Salinas watershed in January 2005,

collecting 584 water samples from 22 watershed locations

(Figure 1). Samples were collected in collaboration with the

CCRWQCB as part of an on-going investigation. Sample

locations and sample frequency were selected in coordination

with the CCRWQCB and the California Department of Health

Services. Due to the potential influence of flooding on pre-harvest

contamination, additional samples were collected after significant

rainfall during winter months (November to April).

At least one strain of EcO157 was isolated from 38 (6.5%) of the

total water samples collected. Two-hundred and thirteen Moore

swab samples were placed at several of the same locations starting

in December 2005; 37 were positive for EcO157 (17.4%). A

combined total of 75 of the water ‘‘grab’’ and Moore swab samples

were positive during the 19 month watershed survey (12.8%).

The majority of the EcO157 positive samples (42 of 75 = 56%)

were collected at sites in the Gabilan Creek (designated A to D in

Figure 1), especially upstream sites closer to grazing. The

concentration of generic E. coli determined for duplicate water

samples collected from these locations varied extensively, with the

highest concentrations occurring during months with the highest

measured water flow (Figure 2). During these months, the flow in

the Gabilan Creek at elevated areas can increase more than 1000-

fold over base flow, resulting frequently in flooding downstream

(data from water flow meter at location D, ‘‘Gabilan Creek’’, see

[53]). The average generic E. coli levels measured for locations A to

D increased by five-fold during these months, and the incidence of

EcO157 increased from undetectable to a high of more than 85%

of the samples being positive. Throughout the watershed, high

concentrations of generic E. coli coincided generally with a high

incidence of EcO157 (P = 0.001, Figure 3). Nevertheless, generic

E. coli levels did not correlate well with the incidence of EcO157

when individual sampling sites were compared, except location G

(P = 0.009). Generic E. coli concentrations were frequently high at

sampling times when EcO157 was not recovered.

Spinach outbreak investigation, 2006

During this 19-month study of the Salinas Valley watershed,

a large, multi-state outbreak occurred with illnesses in August/

September 2006 linked to baby spinach produced in Monterey

and/or San Benito counties. USDA-ARS laboratories processed

222 environmental samples collected at four farms/ranches (F/R)

identified in the traceback investigation (Figure 4, F/R A, B, C,

D). Water at each of the four ranches was sampled and tested due

to the incidence and probable transport of EcO157 shown in the

Salinas watershed. Surface water was located in close proximity

(,0.8 km) to fields on all four ranches. However, unlike Farm A

noted previously, the spinach outbreak-investigated fields (F/R A,

B, C, D) were not subject to routine flooding. Thirty-two water

samples were collected, including well water used for irrigation

and surface water (holding ponds and nearby streams). Water

sample locations were selected based on their proximity either to

fields or cattle. Other samples were collected to investigate other

potential sources of contamination included feces (cattle, feral pig,

deer), soil and sediment. Fenced cattle were located directly

adjacent to fields at F/R A and 2.2 km or 0.6 km from fields at F/

R B and C, respectively. Cattle were not evident at or near F/R D.

From 222 samples, 27 were positive for EcO157, which included

21 positive samples at F/R A and 6 positive samples at F/R B.

Positive samples obtained at F/R A came from water, sediment,

cattle and pig feces, and dust. Water and sediment samples that

were positive for EcO157 were all collected at a location on F/R A

near cattle and approximately 1.5 km from the fields. All EcO157-

positive samples from F/R B were from cattle feces.

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Genotypic comparison of EcO157 isolates

There were 102 samples analyzed during the combined watershed

and outbreak studies that were positive for EcO157. Multiple

isolates were subcultured from one or both of the two different

selective agar plates for each sample. then confirmed as EcO157

by anti-O157 MAb and rfbE analyses, and genotyping by MLVA.

MLVA resulted in identification of 92 different MLVA types for

the 1,301 isolates analyzed (Table S1). Isolates confirmed as

EcO157 were analyzed subsequently by PCR for the presence of

fliC (specific for H7 antigen) and four putative virulence genes. All

of the EcO157 isolates found in this study contained fliC (flagellin

gene), eae (intimin), and hly (hemolysin gene). It is noteworthy that

Figure 2. Correlation of water flow, the concentration of generic E. coli, and the incidence of EcO157 in the Gabilan Creek (Salinas, CA). Linear

regression R2 values are indicated. EcO157 incidence is calculated as the fraction of the number of positive samples compared to the total samples

obtained. The flow rate was obtained from data from the USGS sampling station #11152600 located at position D (see Figure 1). EcO157 incidence

and generic E. coli concentration were determined with duplicate samples.

doi:10.1371/journal.pone.0001159.g002

Figure 3. Concentration of generic E. coli in the Salinas watershed. Sample locations refer to Figure 1. Red and black dots indicate isolation or nonisolation of EcO157 for the corresponding duplicate water samples, respectively.

doi:10.1371/journal.pone.0001159.g003

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all of the isolates described in this study were also positive for stx2,

with the exception of one isolate that was also negative for stx1

(Table S1, MLVA 7). Thirty-eight other MLVA types were

negative for stx1 (Table S1). Isolates of the same MLVA type

always had the same profile of virulence genes by PCR analysis.

Interestingly, most MLVA types were isolated only once during

the watershed and outbreak investigations (61 of 92 types).

However, several MLVA types were isolated multiple times from

samples obtained at different times or locations. For example,

there were 19 cases where the same MLVA type was isolated at

more than one sample location during the watershed survey

(Figure 5A). Most (11 of 19) of the multi-site MLVA types were

isolated at multiple locations on the Gabilan Creek, but not at

other watershed locations. MLVA type 8 strains were isolated only

from multiple locations in the Tembladero Slough and Old Salinas

River, but were not isolated from upstream sites. It is noteworthy

that occasionally, the same MLVA type was isolated on the same

day from locations separated by relatively large distances, but in

the same drainage path. This occurred on three separate sampling

dates. MLVA types 1, 2 and 4 strains were recovered repeatedly

on Feb. 16, 2005 from multiple sites along the Gabilan watershed

and the Tembladero Slough (Figure 5, Table S1). An MLVA type

2 strain was also recovered from a site on the Old Salinas River on

that same day. The total distance via the waterways from the

Gabilan sample site (D) to the Old Salinas River site (I) is

approximately 30 km (Figure 1). Similarly, MLVA type 9 strains

were recovered from sites along both the Gabilan and the Old

Salinas River on March 23, 2005, and MLVA type 100 strains

were recovered from sites on the Alisal Creek, Reclamation Ditch

and Tembladero Slough (maximum separation of 25 km) on April

18, 2006 (Figure 1). It is important to note these three sample dates

were during the winter and that flow in the Gabilan and

Reclamation Ditch was high (568, 852 and 624 liter/s on the

respective sample dates).

Additionally, there were 10 cases where the same MLVA type

was isolated from samples collected on different dates (Figure 5B);

on 8 occasions, the same MLVA type was isolated from samples

collected on subsequent dates at the same location. In contrast,

MLVA 97 strains were isolated from Tembladero Slough nearly

two months after its initial isolation from the Gabilan. Additionally, MLVA 93 strains were isolated initially from the Salinas

River and again, two months later, from both the Gabilan and

Tembladero Slough.

EcO157 was isolated also from a single sample of cattle feces

collected near location A. This sample yielded two MLVA types

(15 and 16), both of which were isolated also from water samples

collected from the Gabilan Creek (Figure 5B). The MLVA 15

strain was isolated from a water sample collected at location C

three weeks prior to the feces sample collection. The MLVA 16

strain was isolated from water samples collected nearly eight

months later at location A and E.

Incidence of EcO157 at an elevated site near

a potential point source

EcO157 was isolated most frequently (22% of all positive samples)

at sample location A (Figure 1), and upstream of location A on

Towne Creek (a tributary to Gabilan Creek). Several potential

sources of contamination were evident. For example, at one site

a small tributary moved through a corral containing approximately eight head of cattle. Soil samples taken near the creek

inside the corral were positive for EcO157 (Table S1: MLVA types

143 and 145). No other sources of contamination were evident up

to 0.8 km downstream, suggesting these cattle as an isolated point

source.

To determine whether the incidence of EcO157 correlated

spatially with this potential point source, four duplicate water

samples were collected at each of seven locations, at distances from

10 to 730 meters downstream from the corral. In the five days

prior to sampling, the total rainfall was 0.8 cm and water flow was

estimated at 0.1–0.2 m/s depending on location. Isolates recovered from the corral were of MLVA types indistinguishable or

similar to those recovered from downstream water samples

(Figure 6), such as MLVA types 143 and 145 strains isolated

from all samples collected at 10 and 65 meters downstream, and

MLVA 143 isolated from two of four samples collected at 135

meters downstream. None of the samples collected at 230 meters

and further from the point source were positive for

EcO157(Figure 6). Although isolates representing multiple different MLVA types were obtained from these sites, the isolates

appeared to be related, based on subtle differences in the number

of TR at Vhecs 1, 3 and 4 loci (see inset for Figure 6).

Clustering of MLVA types

Phylogenetic analyses indicated that 70% of the environmental

isolates clustered into eight groups (labeled as A–H in Figure 7). A

cluster was defined as those MLVA types that differ at only a single

locus from its nearest neighbor. Also, the strains within any of the

eight clusters had the same stx1 gene profile. For example, strains

in Clusters A, D, E, F and G all contain stx1, whereas Clusters B,

C and H do not. Furthermore, the strains represented within

Clusters C, D, E and F were related to one another also spatially,

i.e., they were isolated from samples obtained within 8 km of one

another. For example, strains in Clusters D were isolated

Figure 4. Map of Salinas and San Juan valleys showing the positions

(A–D) of F/Rs implicated in the traceback investigations associated

with the baby spinach outbreak 2006. Blue lines on the map are

waterways.

doi:10.1371/journal.pone.0001159.g004

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exclusively from the Gabilan Creek and its tributaries. Cluster F

strains were isolated from Salinas River samples only. Strains in

clusters C and E were isolated only from F/R A and B,

respectively. In contrast, the remaining four clusters (A, B, G

and H) contain strains isolated from divergent locations. Clusters

A and G contain strains from both the Gabilan and the

Tembladero Slough, perhaps reflecting that they are connected

hydrologically (Figure 1). However, Cluster B contains strains from

several locations in the Salinas watershed, some of which are not

connected hydrologically (e.g., locations C, J and N). Similarly,

Cluster H contains strains from both F/R A and B, which have no

watershed connection (Figure 4).

Comparison of MLVA and PFGE data for EcO157

strains

The relationships between selected sets of strains were investigated

further by PFGE analysis using two enzymes XbaI and BlnI.

Strains for comparison were selected primarily to further

characterize strains of identical MLVA types that had been

isolated from different locations and/or at different times.

Additional strains were included in the PFGE analysis to enhance

the comparisons of closely related MLVA types collected during

the 2006 baby spinach outbreak investigation. In many cases,

PFGE data failed to discriminate strains of closely related MLVA

Figure 5. MLVA types compared to (A) Salinas watershed location or (B) sample date. Location designations refer to Figure 1 and are separated by

horizontal lines in (A) to indicate separate waterways. Black and red dots indicate isolation of the MLVA type at the indicated location or date. Red

dots indicate that differences were detected between that isolate and the other isolates of that MLVA type by PFGE.

doi:10.1371/journal.pone.0001159.g005

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types (Figure 8). For example, several isolates in MLVA clusters B,

C and D were indistinguishable by PFGE. Nevertheless, PFGE

analysis distinguished occasionally between identical MLVA types

(Figures 5 and 7). Some of these differences in PFGE patterns

contrasted with the hypothetical transport processes of EcO157, as

interpreted from the MLVA data. For instance, an MLVA type 16

strain was isolated from a water sample collected eight months

after it was isolated from a cattle fecal sample. However, PFGE

data indicated the MLVA type 16 strains isolated from feces and

from water were different (Figure 8, cluster D). Similarly, MLVA

93 type strains were isolated from both the Gabilan and the

Salinas River, sites having no hydrologic connection. However,

PFGE data distinguished between the MLVA 93 strains isolated

from the Salinas River and the multiple MLVA 93 strains from the

Gabilan watershed (Figure 8, clusters B and C). PFGE data were

also inconsistent with MLVA data by distinguishing between

identical MLVA types 15, 89 and 142 from the Salinas watershed,

and also some strains isolated from various samples collected

during the spinach outbreak investigation. For example, MLVA

196 isolates from F/R A pig feces and sediment samples were

distinguishable by PFGE (Figure 8, cluster H). Similarly, MLVA

184 and 187 isolates from different cattle feces from F/R B were

distinguishable by PFGE (Figure 8, clusters E, H).

Comparison of environmental and human clinical

EcO157 strains by MLVA

A set of 105 human clinical strains of EcO157, associated

predominately with sporadic illnesses in California, and outbreak

strains from other states, was typed by MLVA for comparison to

MLVA data of environmental isolates. Strains from outbreaks

associated with apple juice (1996), taco meat (1999), spinach (2003)

and lettuce (1999, 2002, 2003, 2005), were included in this

comparison (Table S1). Comparison of the MLVA data shown in

Table S1 revealed a number of strains linked by identical or very

similar MLVA types, but without an epidemiological or spatial

connection: (a) two environmental isolates from F/R A (RM6103,

RM6155) obtained during the 2006 spinach outbreak investigation

were exact matches to the major MLVA type of.40 clinical

isolates associated with the 2006 spinach outbreak (MLVA 163);

(b) an environmental isolate from feral swine feces collected at F/R

A (RM6146) had an MLVA type identical to a clinical strain from

a sporadic illness occurring in California in 2006 (RM5658,

MLVA type 116, Table S1); and (c) two clinical strains from

sporadic illnesses in California (MLVA types 60 and 86) were

related by MLVA to environmental isolates (Table S1). MLVA

type 86 strains differed from environmental isolates within Cluster

D by two to seven TR at Vhec1. MLVA type 60 varied from

MLVA types of other isolates in Cluster B by one to four TR at

Vhec1 and one to three TR at Vhec4 (Table S1). Clinical isolates

typed as MLVA types 112 and 129 were not associated with any

MLVA clusters, but rather to individual environmental isolates of

MLVA types 90 and 4, respectively.

Comparison of environmental EcO157 strain PFGE

profiles in CDC PulseNet

PFGE patterns determined for selected environmental strains were

compared against the CDC PulseNet database, which contained

during the preparation of this study, XbaI patterns for.22,000

EcO157 isolates (Table 1). Numerous environmental isolates from

this study matched XbaI patterns present in the PulseNet database

(17 of 29 strains analyzed). Ten of the 17 PFGE profiles matching

PulseNet XbaI patterns, also matched BlnI patterns. Thirteen

MLVA types identified for environmental isolates corresponded to

10 different PFGE profiles (XbaI and BlnI) associated in PulseNet

with 10 separate outbreaks; 8 of the outbreaks involved multiple

states (Table 1: ‘‘PulseNet Cluster(s)’’). For example, multiple

MLVA types were identified for EcO157 watershed strains with

XbaI-BlnI profiles EXHX01.0343-EXHA26.0569 (MLVA types

Figure 6. The persistence and transport of EcO157 in a stream in an elevated area near a point source. The change in stream elevation is indicated

as a function of the distance from a corral with cattle. Sampling positions are indicated by: X; different MLVA types isolated from each location are

indicated. The inset shows a phylogenetic tree designating the relationship among the MLVA types isolated in this study (see Figure 7 and Table S1

for more detail).

doi:10.1371/journal.pone.0001159.g006

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89, 99), EXHX01.0200-EXHA26.0015 (MLVA types 101, 102,

103), and EXHX01.3099-EXHA26.0265 (MLVA types 94, 95,

96). The MLVA types corresponding to the EXHX01.3099-

EXHA26.0265, EXHX01.0343-EXHA26.0569 and

EXHX01.0200-EXHA26.0015 profiles were closely related based

on 8/10, 8/10 and 7/10 MLVA loci, respectively. An example of

the value of MLVA can be discerned from a comparison of strains

with the XbaI-BlnI profile EXHX01.0047-EXHA26.0015, a common two-enzyme profile represented by.6% of the submitted

profiles in the PulseNet database. MLVA data were available for

some clinical isolates designated with this common profile and also

for two strains reported previously in a comparison of PFGE and

MLVA [39] (Table 2). MLVA of this limited set of strains with no

apparent linkage epidemiologically (i.e. not outbreak related)

revealed that these ‘‘EXHX01.0047-EXHA26.0015’’ strains in the

PulseNet database are not related closely. Indeed, the closest

similarity was with only 6 of 10 MLVA loci with identical number

of TRs.

Figure 7. A comparison of the phylogenetic relationships among 92 MLVA types. The size of the circles indicate relative number of isolates of each

MLVA type (numbers in the circles) recovered from the environment. Those MLVA types shown with the smallest circles were isolated only once.

Letters A–H indicate clusters (colored regions) in which types differ at only 1 of the 10 MLVA loci from a neighbor.

doi:10.1371/journal.pone.0001159.g007

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DISCUSSION

We have described the incidence of EcO157 in an important leafy

vegetable production region. Some molecular similarities among

environmental strains raise questions regarding potential spatial

connections and persistence of certain strains in the environment.

These studies were stimulated by the isolation of a single strain of

EcO157 from the sediment in a creek adjacent to a farm identified

in three separate traceback investigations (Table S1, RM4403).

However, the strain was different by PFGE and MLVA from

clinical strains associated with the three outbreaks (Table S1, see

‘‘Source’’: OB6, OB7, OB8). This result only indicated that

EcO157 was in the production environment as a potential source

of contamination, but no definitive mechanism for pre-harvest

contamination could be discerned. The potential for produce

contamination associated with the watershed was investigated

further by assessing the incidence, survival and transport of

EcO157, initially in the Salinas watershed, and subsequently on

farms and ranches implicated by traceback investigation to the

spinach outbreak. The results obtained from both the Salinas

watershed study and the spinach outbreak investigation indicated

that EcO157 was isolated more frequently from samples obtained

near or on grazing land, compared to other locations. These

results are consistent with reports of frequent incidence of EcO157

in numerous surveys of cattle in other locations of the country [54–

62] and incidence in water [63–66].

The incidence of EcO157 was not uniform in the watershed.

EcO157 was frequently isolated from elevated regions of Gabilan

Creek, but much less frequently elsewhere. This would possibly

explain why EcO157 was only found once in 178 samples from

Farm A. Frequent isolation from the Gabilan watershed suggests

there may be a higher concentration of EcO157 in this region

compared to other sites. However, 45% of duplicate samples

collected 30 seconds apart produced inconsistent results, i.e.,

EcO157 was isolated from one sample, but not the other,

suggesting that the incidence and concentration of EcO157 in

the watershed are dynamic events. Alternatively, the incidence of

EcO157 may be uniform, but present at low concentrations.

During the rainy season, it is anticipated that large volumes of runoff water would collect, dilute and disseminate pathogens from

point sources (e.g. animal feces containing high concentrations),

but resulting in concentrations close to or below the level of

sensitivity of our method of detection of EcO157. Our isolation

method was adapted from a sensitive EcO157 isolation procedure

developed for cattle feces and hides [67]. However, water, wildlife

feces, soil and other environmental samples often yielded growth

of many non-target bacterial CFU on the selection plates that

interfered with identification and selection of authentic EcO157

CFU. Background flora can decrease the sensitivity of any

isolation method, thus resulting in an apparent incidence of

EcO157 lower than may exist. Moore swabs for 5-day sampling

improved the recovery of EcO157, but they also do not support

quantification of viable EcO157, since enrichment is required.

Research is ongoing to improve the sensitivity and speed of

methods for isolating both EcO157 and stx-positive non-O157 E.

coli from a variety of samples.

Rainfall and incidence of EcO157

The 19-month period of sampling of the watershed incorporated

two rainy seasons, facilitating measurement of a correlation

between incidence of EcO157 and stream flow (Figure 2). This

correlation could only be shown in the Gabilan where the

incidence is highest. Nevertheless, even in the Gabilan Creek, the

correlation was not very tight (R2= 0.73), primarily due to very

Figure 8. Dendrogram of selected isolates from PFGE data. Taxa are

designated as RMXXXX (##), where RMXXXX is the isolate name and

## is an arbitrary number assigned to that MLVA type [refer to Table

S1]. Taxa in red type are identical MLVA types differentiated by PFGE.

Colored clusters (labeled B–H) are similar to labeled MLVA clusters in

Figure 7.

doi:10.1371/journal.pone.0001159.g008

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few occurrences of high EcO157 incidence. It is not known if

rainfall (and subsequent flooding) releases bacteria already existing

in the creek sediments, or if it washes contamination into the creek

by run-off, although it is probable that both events occur. Studies

with laboratory models indicate that EcO157 is attached predominantly to very small particles (2 mm) [68,69]. These particles

are too small to settle, minimizing interactions with the sediment

and restricting the bulk of the contamination from entering the

sediment. However, a process called hyporheic exchange can lead

to high rates of suspended particle deposition in sediment beds,

even when the suspended particles are very small and have no

appreciable settling velocity [70]. The rate at which hyporheic

exchange occurs is dependent on several conditions, including the

type of sediment and the particle surface chemistry. Indeed, EcO157

has been shown to survive for extended periods in sediment,

becoming undetectable only after 60 days at 24uC [65]. In a separate

Table 1. Comparison of MLVA types of EcO157 environmental strains with PFGE profile designations. ............................................................................................................................... ...................

MLVA typea Strain no.a PulseNet XbaI

b XbaI, % of PulseNetc PulseNet BlnId PulseNet Cluster(s)e

1 RM4859 EXHX01.3536 0.004 EXHA26.1411

2 RM4869 EXHX01.3578 0.004 EXHA26.1411

3 RM4862 EXHX01.0221 0.031 EXHA26.1411

13 RM4888 EXHX01.3537 0.004 EXHA26.1411

15 RM5036 EXHX01.1271 0.366 EXHA26.0569 CA:Oct-03, Multi:Oct-04

15 RM5038f EXHX01.0263 0.374 EXHA26.0257

16 RM5037f EXHX01.0202 0.113 EXHA26.0569 Multi:Jun-05, Nov-05

17 RM4403 EXHX01.0122 0.053 EXHA26.0354

89 RM5450 EXHX01.0343 0.167 EXHA26.0569 Multi:Sep-04

89 RM5621 EXHX01.3535 0.070 EXHA26.0569h

90 RM5603 EXHX01.2216

EXHX01.2216

0.070 EXHA26.0684 NC:Nov-04

91 RM5604 EXHX01.0509 0.031 EXHA26.1412

92 RM5605 EXHX01.3455 0.009 EXHA26.0569

93 RM5606 EXHX01.0047 6.112g EXHA26.0015 Multi state, Mar-03-Aug-06

94 RM5607 EXHX01.3099 0.048 EXHA26.0265 CA:May-03

95 RM5608 EXHX01.3099 0.048 EXHA26.0265 CA:May-03

96 RM5609 EXHX01.3099 0.048 EXHA26.0265h CA:May-03

97 RM5615 EXHX01.2221 0.044 EXHA26.1395

98 RM5616 EXHX01.2221 0.044 EXHA26.1395

99 RM5620 EXHX01.0343 0.167 EXHA26.0569 Multi:Sep-04

100 RM5628 EXHX01.0200 0.959 EXHA26.0332

101 RM5630 EXHX01.0200 0.959 EXHA26.0015 Multi:Aug-05, Jun-06

102 RM5625 EXHX01.0200 0.959 EXHA26.0015 Multi:Aug-05, Jun-06

102 RM5673 EXHX01.0200 0.959 EXHA26.0015 Multi:Aug-05, Jun-06

103 RM5623 EXHX01.0200 0.959 EXHA26.0015 Multi:Aug-05, Jun-06

104 RM5674 EXHX01.1031 0.194 EXHA26.0982 Multi:Jun-06

105 RM5675 EXHX01.0200 0.959 EXHA26.0332

106 RM5676 EXHX01.3535 0.070 EXHA26.0569h

107 RM5677 EXHX01.2838 0.070 EXHA26.0569 h

108 RM5678 EXHX01.3535 0.070 EXHA26.0569 h

109 RM5679 EXHX01.3535 0.070 EXHA26.0569 h

122 RM5718 EXHX01.3535 0.070 EXHA26.0569 h

163i RM6103 EXHX01.0124 ? EXHA26.0015 Multi:Aug/Sep-06

a

See Table S1 for MLVA data and source information.

b

XbaI profile in PulseNet database (last checked Dec, 2006) matching designated strain. XbaI patterns EXHX01.3535, EXHX01.3536, EXHX01.3537 were new to the

PulseNet database at the time of submission.

c

Frequency of this XbaI profile in PulseNet database. d

BlnI profile in PulseNet database (last checked Dec, 2006) matching designated strain. BlnI patterns EXHA26.1395, EXHA26.1411, EXHA26.1412 are new to the PulseNet

database.

e

Clusters of illnesses identified from PFGE profiles submitted to PulseNet as XbaI profile, or both XbaI and BlnI profiles. Designation is by state (2 letter

abbreviation):month-2 digit year; Multi, multiple states.

f

RM5038 and RM5037 were isolated from the same sample of feces; MLVAs 15 and 16 differ by one TR only in locus 4.

g

XbaI-BlnI profile EXHX01.0047-EXHA26.0015 represents.6% of the PulseNet database (see Table 2). h

One band difference between the profile for this strain and the BlnI PulseNet profile designated. I

Major MLVA type for human clinical strains associated with the spinach outbreak.

...............................................................................................................................

doi:10.1371/journal.pone.0001159.t001

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study, however, investigation of the Oldman River Basin in Canada

for watershed contamination with coliforms indicated that most

contamination was coming from a variety of non-point sources and

not from sediment [71]. In contrast, another study of the Oak Creek

in Arizona indicated that seasonal fecal coliform levels were

associated predominantly with sediment agitation during summer

storms and recreational activity [72]. Thus, the contribution made

by EcO157 sequestered in the sediment to the dynamics of stream

contamination appears complicated by a variety of factors, some

specific to the region being studied.

Although EcO157 was at increased incidence during and after

heavy rain events and flooding, EcO157 was also isolated during

periods of drought. For example, EcO157 was isolated from July

2004 samples from Farm A and one year later from Gabilan Creek

samples. In both locations, cattle, horses and other livestock had

direct access to the streams and were within 1.6 km of the sample

site. Similarly, EcO157 was isolated in May and July 2006 in the

Gabilan and in October 2006 from surface water at F/R A during

the spinach outbreak investigation [9]. The samples were again

obtained near obvious animal point sources that could provide

direct exposure of surface water to animal feces. However,

EcO157 was also recovered August 2006 from the Salinas River

(Figure 1, location O), where no obvious source within 8 km was

noted. Furthermore, the flow rate in the Salinas River on the

sample date was a relatively fast 3100 liter/s (average of gage

readings 14 km upstream and 10.5 km downstream). Therefore,

the probability of detecting contamination in such a large volume

of water is low considering the dilution effect. Detection of the

pathogen in large volumes of water will depend upon the

sensitivity of the isolation procedure, the number of point sources,

and/or the concentrations of viable pathogen in the sources.

Nevertheless, these results, in conjunction with MLVA typing data

(see below), suggest possible survival and transport of viable

pathogen for extended periods and over 16–30 km distances, even

during the drought season. However, the potential contribution of

multiple point sources of strains indistinguishable from those at

distant sites can not be eliminated.

Incidence of coliforms and generic E. coli

Water samples were analyzed also for coliforms and generic E. coli

to assess the total maximum daily load (TMDL) in the watershed.

The results confirmed that the Salinas Valley watershed would be

considered ‘‘impaired’’, since.10% of the samples exceeded

coliform levels of 400 MPN/100 ml, and many samples exceeded

E. coli levels of 404 MPN/100 ml [42]. The incidence of multiple

strains of EcO157 at multiple times of the year and at multiple

sites in the watershed is consistent with this designation. Our

analysis of generic E. coli levels from duplicate watershed samples

indicated a correlation with the incidence of EcO157. Samples

showing high levels of generic E. coli were more likely to contain

EcO157 (P = 0.001) when all samples were included in the

analysis. However, the correlation was insignificant at most

individual sample sites due to a very low incidence of EcO157.

Beyond the Gabilan drainage EcO157 was undetectable in many

samples with high levels of generic E. coli. In general, generic E. coli

is a poor indicator of EcO157.

MLVA of EcO157 strains and comparison to PFGE

Multiple strains of EcO157, isolated from samples obtained from

farms and ranches associated with outbreaks and from the Salinas

Valley watershed, were typed by MLVA and PFGE. Our

experience confirmed that MLVA was more efficient than PFGE

for analysis of a large number of strains obtained during complex

investigations. The ability to MLVA type multiple suspect CFU

and to distinguish between closely related strains facilitated a rapid

assessment of strain relatedness for purposes of source-tracking. For

example, during the spinach F/R investigation, the USDA-ARS

laboratory identified 27 environmental samples positive for

EcO157 and subcultured up to 40 suspect CFU per sample for

further analysis. Of greater than 300 isolates confirmed as EcO157

and typed by MLVA, 32 different MLVA types were identified.

Many of the 32 MLVA types were closely related based on single

TR differences (Table S1), but at least two strains, assessed as

different by MLVA, were identified from 8 of the 27 samples (30%).

Furthermore, 15 of the 32 MLVA types, identified as closely related

to the major MLVA type for spinach outbreak clinical strains

(MLVA 163), were all indistinguishable by two enzyme PFGE

(PulseNet profile EXHX01.0124-EXHA26.0015). Although genotyping of multiple isolates from each clinical sample is not necessary

in most cases, environmental samples provide a greater potential

diversity of strains of a target species, due to increased exposure to

multiple potential sources of pathogen contamination (e.g. wildlife,

compost/manure, water, dust, feed). A similar study to assess

multiple suspect isolates by PFGE would be much more labor

intensive, costly, and less discriminatory compared to MLVA.

Of the 92 MLVA types identified during this study, 32 types

(35% of the total) were isolated exclusively from the Gabilan

watershed or its tributaries, and another 32 types were isolated

during the 2006 spinach outbreak investigation, including 23 types

isolated exclusively from F/R A [43]. Similarly, nine MLVA types

were for strains isolated exclusively from F/R B. As noted above,

many of the strains isolated from specific locations or sample dates

(e.g., intra-farm/ranch or watershed) are related closely (Figure 7,

Table S1). For the purpose of this analysis, strains considered

closely related have variations in only one or two MLVA loci with

small variations in the number of TR. This relationship between

MLVA types is based on the assumption that variation in the

number of TR occurs most often with changes of one or two

repeat units [36]. Often PFGE data supported assumptions of

relatedness, in that MLVA clusters were often also represented in

the PFGE trees, although, usually, a single PFGE profile was

associated with multiple MLVA types. Nevertheless, more research is needed in the molecular biology of TR variation,

especially for MLVA typing of strains isolated from environmental

samples and/or clinical strains not connected epidemiologically,

spatially or temporally.

Table 2. MLVA differences between strains of the common

PulseNet PFGE profile, EXHX01.0047-EXHA26.0015a

. ......................................................................

Name Sourceb

Approx.

isolation date MLVA genotypec

RM5652 Human (CA) Feb-06 15-9-11-18-7-9-9-4-7-6

RM5606 Swab (CA) Jan-06 20-9-11-14-7-9-8-4-7-6

RM5187 Human (CA) Sep-05 20-9-16-20-7-9-4-4-7-6

RM5329 Human (CA) Jul-05 20-8-14-16-7-9-8-4-7-8

RM6051 Human (PA) Sep-05 20-9-13-15-7-9-8-4-7-7

VA_01-577cd Human (VA) 2001 29-7-7-15-X-X-8-4-7-7

F7410d Human (IN) 2000 32-7-13-15-X-X-9-4-7-8

RM6053 Human (PA) Aug-06 38-9-14-18-7-9-10-4-7-7

a

PulseNet patterns designated as described previously [35].

b

Source of isolate (state).

c

The number of TR are listed in the order noted in Table S1.

d

Data for strains designated VA_01-577 and F7410 are from a previous study

comparing PFGE and MLVA data [39]. X, not determined for these two strains.

................................................................

doi:10.1371/journal.pone.0001159.t002

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Temporal and spatial clustering of related MLVA types

suggested that, in general, movement of the bacteria in the

environment is restricted. For example, identical or closely related

MLVA types were not identified as randomly dispersed in the

region studied. More than 70% of the strains were clustered

spatially (,8 km or further if hydrologically linked). Strains

indistinguishable by MLVA type and isolated from separate

locations (sometimes.16 km), suggest probable transport of

pathogen via linked watersheds. Probable watershed transport of

EcO157 was indicated most clearly by investigation of a point

source involving cattle in a small corral surrounding a portion of

upper Towne Creek (Figures 1 and 6). All of the EcO157 strains

isolated within 135 meters downstream from this potential source

and strains isolated from the corral (MLVA types143 and 145)

were related closely by MLVA (Table S1 and Figure 6), suggesting

the corral as the probable source of contamination. Several of the

MLVA types were isolated from a only one of the sites, and only

from one or two of the four) samples obtained, again reflecting

either limited sensitivity of the isolation procedure and/or the

dynamic nature of the contamination. Nevertheless, the number of

different strains recovered along the stream was proportional

inversely to the distance from the point source, and one strain

(MLVA type 143) was present at all sample sites up to 135 meters

from the corral. Two other strains represented by MLVA types

145 and 147 were isolated only from samples collected up to 64

meters away, possibly because they were at a lower initial

concentration compared to MLVA type 143. Nevertheless,

potential transport by unknown mechanisms, unrelated to water,

with dissemination to multiple sites cannot be eliminated as

a possible explanation for the spatial re-occurrence of strains.

MLVA data suggest also long distance transport of the pathogen

occurs. Identical types (supported by both MLVA and PFGE data)

were found up to 24 km apart and always in the same drainage.

Furthermore, isolates suspected of being closely related were

recovered often from sites greater than 8 km apart, but nearly

always in the same drainage. Other studies on the persistence of

pathogens in a water source, modeled using indicator organisms,

have examined the effects of a number of attenuation parameters

such as temperature, flow rate, particle size, pH, and sediment

type [73,74]. The general conclusions from these studies is that

bacteria are transported in watersheds relatively far (km), limited

primarily by their ability to survive and remain suspended for

movement in a watershed. MLVA types in clusters B and G

isolated during the winter of 2006 and 2005, respectively (Figures 5

and 7), are of particular interest, since nearly all strains re-isolated

more than 16 km apart are contained in these two clusters. The

relatedness of strains isolated from distant sites might reflect an

increased fitness for survival in the watershed compared to other

strains, consistent with a report of STEC strains varying in length

of survival in well water [75]. Alternately, the isolates in clusters B

and G may have been at high concentrations initially in or near

the Gabilan Creek, thereby increasing the probability of isolating

them again downstream even after significant dilution. Increased

fitness of these strains in the GI tracts of wild or domestic animals

may lead to high concentrations in feces for further dissemination

in the environment, increased incidence, and potential increased

importance to epidemiology of outbreaks [59].

Strains related by MLVA type might be introduced into an

environment from multiple sources and independent of watersheds. However, transport of bacteria by a non-watershed

mechanism would be expected to disperse bacteria more randomly

in the watershed than was observed. Identical strains of EcO157

were only isolated where a hydrological connection existed. Our

data support the conclusion that transport of EcO157 in the

Salinas region is due primarily to water. Nevertheless, two clusters

of related MLVA types represented strains isolated from locations

not linked hydrologically (Figure 7). MLVA cluster B contained

isolates from both the Alisal (MLVA types 100, 105) and Gabilan

Creeks (MLVA types 93, 101, 102 103), which are connected only

where water from both enter into the Reclamation Canal

(Figure 1). Additionally, MLVA cluster H strains were isolated

from F/R A (MLVA types 189, 196; several sources) and F/R B

(MLVA 191, 194; cattle feces). These two F/Rs are approximately

55 km apart and are not connected hydrologically (Figure 4).

MLVA types represented by clusters B and H also are clustered

based on PFGE data (98 and 88% similarity, respectively).

Nevertheless, transport of EcO157 outside of the watershed is

probably occurring also. Obvious mechanisms are through wildlife

and livestock movement or transport, and contaminated feed [76].

Prior to the spinach outbreak investigation, MLVA comparisons

between environmental, and a select group of clinical (sporadic

and outbreak) isolates, revealed no identical matches. Traceback

data obtained during the spinach investigation narrowed the

potential F/R sites to four, and intensive sampling at one of the

ranches (F/R A) yielded two isolates identical to the major human

outbreak strain by MLVA, but also.30 additional isolates that

were related closely by MLVA (Figure 7, Table S1). At least 13

different MLVA types were isolated from feral swine feces and 6 of

the MLVA types were closely related to the outbreak MLVA type

(MLVA 163) and had PFGE profiles indistinguishable from the

outbreak strains (Figure 8, cluster C). These results indicate that

roaming feral swine could transport pathogens from multiple point

sources to other locations. The prevalence of feral swine near some

leafy vegetable production regions, and their wide range and lack

of containment, emphasize the need for additional studies to

determine their incidence in other regions, potential for colonization by EcO157 and concentration in feces. A more complete

analysis of the incidence of EcoliO157 and population density of

feral swine related to the 2006 spinach outbreak will be provided

in a separate report (Jay MT, Cooley MB, Carychao D, Wiscomb

GW, Sweitzer RA, et al. (2007) Escherichia coli O157:H7 in feral

swine near spinach fields and cattle, central California coast,

Emerg. Infect. Dis. (Accepted for publication)).

Comparison of MVLA types and PFGE profiles to

data in PulseNet

Comparisons of the PFGE patterns of our environmental isolates

with the PulseNet database revealed several exact matches. It is

especially intriguing that three PFGE profiles were associated in

PulseNet with multiple multi-state outbreaks occurring in 2003,

2004, 2005 and 2006 (Table 1). Nevertheless, a comparison of

MLVA types for eight environmental or clinical isolates with

identical PFGE (XbaI-BlnI) profiles (Table 2), revealed that the

strains were less related by MLVA than indicated by PFGE.

However, it is recognized also that subtle differences in TR may be

determined in different laboratories (Table 2: strains VA-01-577

and F7410) due to differences in reagents, instrumentation and

analysis. Additionally, it is important to note that matching PFGE

profiles for strains not linked epidemiologically (i.e. environmental

strains from this study and PulseNet-related strains), should be

interpreted with caution. This is especially true since digestion

patterns from six or more enzymes are considered necessary for an

accurate estimation of relatedness with PFGE [76]. However,

MLVA should provide a more accurate assessment of strainrelatedness between non-epidemiologically-related strains than

PFGE. A robust MLVA database would assist in making links

between environmental and clinical isolates. Nevertheless, higher

E. coli O157:H7 and p\Produce

PLoS ONE | www.plosone.org 14 November 2007 | Issue 11 | e1159
resolution genotyping methods, such as single nucleotide polymorphism (SNP) arrays [77,78] or partial genome sequencing of

strains, would be most beneficial for accurate source tracking and

improved epidemiology during investigation of outbreaks and

sporadic illness.

The frequent recovery of EcO157 from a variety of distant sites

in the Salinas watershed and the spinach outbreak F/Rs, and also

the isolation of identical, or closely related MLVA types from the

same locations, days or months apart, indicate that contamination

may be more prevalent than assumed previously. The most likely

explanation for these results is re-introduction from one or more

sources, but long-term survival of the pathogen in the environment

is also possible, stimulating important questions regarding the

source and mechanisms of survival, growth, and prevalence of

EcO157 in these environments [14,79–83]. Understanding the

factors related to EcO157 survival and growth in the leafy

vegetable production environments, along with fate and transport

processes, is critical to developing intervention strategies that can

minimize the probability of an outbreak associated with bagged

leafy vegetables.

SUPPORTING INFORMATION

Table S1 Strains used in this study.

Found at: doi:10.1371/journal.pone.0001159.s001 (0.41 MB

DOC)

ACKNOWLEDGMENTS

We thank all of those cited in the Materials and Methods section for their

kind assistance in providing bacterial strains or samples. We also thank J.

Keen, USDA-ARS, for providing anti-O157 MAb, 13B3. We express our

appreciation also to P. Gerner-Smidt and CDC for access to the PulseNet

database. We also thank G. Wiscomb, USDA-APHIS-Wildlife Services, for

assistance in obtaining feral pig samples. Finally, we want to express our

sincere appreciation for the cooperation of specific owners, managers and

growers associated with ranch and farm properties investigated after the

2002, 2003 and 2006 outbreaks.

Author Contributions

Conceived and designed the experiments: RM LC JF MC. Performed the

experiments: LC MJ CM CR MC DC. Analyzed the data: RM LC MJ

CM MC DC CK. Contributed reagents/materials/analysis tools: LC MJ

MC. Wrote the paper: RM MC.

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PLoS ONE | www.plosone.org 16 November 2007 | Issue 11 | e1159