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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 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 4°C [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 pre-harvest 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].

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

Colilert testing

For watershed samples, quantification of total coliforms and E. coli was determined by the Colilert® 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 4°C overnight. The following morning, each sample was diluted 1:10 and 1:100 in Butterfield's phosphate buffer. Colilert® 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 35°C, 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.

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 25°C with shaking at 200 RPM, then at 42°C for 8 hrs, and held at 4°C without shaking until the following morning. Water samples were enriched by adding 11 ml of 10× TSB (filter sterilized, 0.45 µm) to 100 ml of sample, and the mixture was incubated as described above.

One ml of the enrichment broth was tested by adding 20 µl 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 µl 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 µl each of the resuspended beads were spread on Sorbitol MacConkey agar (Difco Labs; Detroit, MI) containing cefixime (0.05 µg/ml; Invitrogen/Dynal) and tellurite (2.5 µg/ml; Invitrogen/Dynal) (CT-SMAC), and on Rainbow Agar (Biolog, Hayward, CA) containing novobiocin (20 µg/ml; Sigma-Aldrich) and tellurite (0.8 µg/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 37°C 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 37°C 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 1× 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 antibody-positive 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 µl reaction mix included 10 µl Stratagene Brilliant QPCR master mix, 0.3 µM each rfbE primers (5′-TTTCACACTTATTGGATGGTCTCA3′ and 5′ TGAGTTTATCTGCAAGGTGATTCC-3′), and 0.1 µM of the probe (5′ 6-FAM-TTCTAACTAGGACCGCAGAGGAAAGAGAGGAATTA-BHQ-1-3′, 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 (95°C for 5 min, then 60 cycles of 95°C for 15 sec and 60°C 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 µl reaction contained 1× ThermoPol buffer (New England Biolabs, Ipswich, MA), 200 µM each dNTP, 0.25 µM each primer, 2.5 U TAQ, and 1 µl template. Thermal cycling parameters were 95°C for 5 min, then 30 cycles of 94°C for 30 sec, 58°C for 30 sec, 72°C for 30 sec, and a final extension at 72°C for 5 min. DNA template was prepared by re-suspending approximately 1 µl of cells scraped from a plate with a 1 µl sterile loop into 100 µl 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 µl reaction contained 1× Qiagen multiplex PCR master mix (Qiagen, Valencia, CA; #206143), 0.25 µM each primer, and 1 µl of template. Thermal cycling parameters were 95°C for 15 min, then 35 cycles of 95°C for 1 min, 65°C for 2 min, and 72°C for 1.5 min. Annealing temperatures decrements from 65°C to 60°C 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-Martens-Latem, 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, NED-Vhec2, 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 µl contained 1× multiplex PCR master mix (Qiagen), 0.2 µM of each primer and 1 µl template. Thermal cycling parameters for reactions 1 and 2 were 95°C for 15 min, then 25 cycles of 94°C for 30 sec, 63°C for 90 sec, 72°C for 90 sec, with a final extension at 72°C for 10 min. Parameters for reaction 3 were 95°C for 15 min, then 35 cycles of 94°C for 20 sec, 65°C for 20 sec, and 72°C for 20 sec, with a final extension of 72°C for 5 min. The multiplex reactions were pooled and diluted 1:50 into distilled water. One µl of this dilution was added to a mixture of 12 µl HiDi formamide (ABI) and 0.08 µl of a MW standard ladder (ROX-labeled MapMarker 1000, Bioventures, Inc., Murfreesboro, TN), and the mixture was heat-denatured for 5 min at 95°C, 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 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 Nei′s diversity [51], which is the calculation 1-Σ (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.

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.

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Figure 3. Concentration of generic E. coli in the Salinas watershed.

Sample locations refer to Figure 1. Red and black dots indicate isolation or non-isolation of EcO157 for the corresponding duplicate water samples, respectively.

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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.

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.

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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 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).

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.

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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).

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: ⧫; 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).

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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 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).

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

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 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).

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

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 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.

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

doi:10.1371/journal.pone.0001159.t001

Table 2. MLVA differences between strains of the common PulseNet PFGE profile, EXHX01.0047-EXHA26.0015^a.

doi:10.1371/journal.pone.0001159.t002

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 fro