Public health investigation of two outbreaks of Shiga toxin-producing Escherichia coli. O157 associated with consumption of watercress

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1 AEM Accepted Manuscript Posted Online 3 April 2015 Appl. Environ. Microbiol. doi: /aem Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 2 Public health investigation of two outbreaks of Shiga toxin-producing Escherichia coli O157 associated with consumption of watercress Claire Jenkins a #, Timothy J. Dallman a, Naomi Launders b, Caroline Willis c, Lisa Byrne b, Frieda Jorgensen c, Mark Eppinger d, Goutam K. Adak b, Heather Aird e, Nicola Elviss f, Kathie A. Grant a, Dilys Morgan b and Jim McLauchlin f Gastrointestinal Bacteria Reference Unit, Public Health England, Colindale, London a ; Gastrointestinal and Emerging Zoonotic Infections Department, Public Health England, Colindale, London b ; Food Water and Environmental Microbiology Laboratory Porton, Public Health England, Porton Down, Salisbury c ; Department of Biology, The University of Texas at San Antonio, One UTSA Circle, San Antonio, TX d ; Food Water and Environmental Microbiology Laboratory York, Public Health England, York e ; Food Water and Environmental Microbiology Laboratory London, Public Health England, Colindale, London f Running title: STEC O157 Watercress outbreaks # Address correspondence to claire.jenkins@phe.gov.uk 1

2 Abstract An increase in the number of cases of Shiga toxin-producing Escherichia coli (STEC) O157 phage type 2 (PT2) in England in September 2013 was epidemiologically linked to watercress consumption. Whole genome sequencing (WGS) identified a phylogenetically related cluster of 22 cases (Outbreak 1). The isolates comprising this cluster were not closely related to any other UK strain in the Public Health England WGS database suggesting a possible imported source. A second outbreak of STEC O157 PT2 (Outbreak 2) was identified epidemiologically following the detection of Outbreak 1. Isolates associated with Outbreak 2 were phylogenetically distinct from those in Outbreak 1. Epidemiologically unrelated isolates on the same branch as the Outbreak 2 cluster included those from human cases in England with domestically-acquired infection and UK domestic cattle. Environmental sampling using PCR resulted in the isolation of STEC O157 PT2 from irrigation water at one implicated watercress farm and WGS showed this isolate belonged to the same phylogenetic cluster as Outbreak 2. Cattle were in close proximity to the watercress bed, and were potentially the source of this second outbreak. Transfer of STEC from the field to the watercress bed may have occurred through wildlife entering the watercress farm or via run-off water. During this complex outbreak investigation, epidemiological studies, comprehensive testing of environmental samples and the use of novel molecular methods proved invaluable in demonstrating that two simultaneous outbreaks of STEC O157 PT2 were both linked to the consumption of watercress but were associated with different sources of contamination. 2

3 Introduction Strains of Shiga toxin-producing Escherichia coli (STEC) serogroup O157 have been associated with outbreaks of severe bloody diarrhoea and haemolytic uraemic syndrome (HUS) for over 30 years (1, 2). In England, outbreaks of STEC are detected through (i) routine investigation of cases by identifying common exposures between cases (ii) detection of the same microbiological subtypes among isolates from cases that are geographically or temporally linked and (iii) detection of an increase in the number of cases in a particular location or associated with a particular subtype. Public Health England (PHE) s Gastrointestinal Bacteria Reference Unit (GBRU) is the National Reference Laboratory and confirms around 900 cases of STEC O157 a year from England & Wales. Approximately 25% of cases are linked to epidemiologically confirmed outbreaks (3, 4). At GBRU, all confirmed STEC O157 isolates are phage typed and typed by multi-locus variable number tandem repeat (VNTR) analysis (MLVA). These typing schemes are instrumental in facilitating both the detection and investigation of outbreaks (4-6). Recently, whole genome sequencing (WGS) has also been evaluated at PHE for its utility in informing outbreak investigation and surveillance of gastrointestinal disease (7-9). On 9 September 2013, the PHE automated outbreak detection system highlighted an increase in the number of cases of STEC O157, phage type (PT) 2, Shiga toxin subtype 2a (stx2a). Ultimately, 19 cases with a distinct MLVA profile ( ) were reported across the UK throughout the duration of the outbreak. Examination of the detailed food histories from the cases rapidly established a link between infection and consumption of watercress purchased from a specific retailer (Retailer A) and this was subsequently demonstrated by an analytical case-case study (6). Retailer A obtained watercress from a single supplier (Supplier A) who processed watercress sourced from ten 3

4 68 69 farms in Southern England between August and September Retailer A voluntarily withdrew products containing watercress from their shelves on 12 September 2013 (6) The multidisciplinary outbreak control team continued investigations including prospective case ascertainment, extensive sampling and microbiological testing of food, water and environmental samples using conventional microbiological culture and PCR. In addition, the whole genomes of both environmental and human isolates were sequenced and phylogenetically analysed. In this study we evaluated the use of (i) PCR to improve detection of STEC in food and the environment and (ii) WGS to identify additional outbreak cases linked to the outbreak described above (MLVA profile ), to detect and differentiate a second concurrent STEC O157 PT2 stx2a outbreak (MLVA profile ) and to facilitate investigations into the original source of the two outbreaks. Materials & Methods Case Ascertainment Presumptive isolates of STEC were reported directly to PHE Centres and a standardised STEC Enhanced Surveillance Questionnaire (SESQ) ( VTEC_Questionnaire.pdf) was administered to patients either by local health protection practitioners or environmental health professionals. The SESQ collects demographic details; risk status; clinical condition (including progression to HUS); household or other close contact details; laboratory results; exposures including travel, food and water consumption, contact with animals and environmental factors; epidemiological case classification; and outbreak /cluster status. Data from the questionnaires are included in the National Enhanced Surveillance System for STEC in England (NESSS). During the course of outbreak 4

5 93 94 investigations, NESSS was reviewed regularly for cases reporting watercress consumption, and PHE Centres were asked to be alert for cases reporting watercress consumption Food trace-back investigations and environmental inspections Food chain investigations were undertaken guided by the evidence available from epidemiological investigations. Environmental Health Officers from local district councils undertook inspections and sampling of farms and Supplier A s processing plant and undertook local trace-back of the watercress from the public house implicated in Outbreak 2. Microbiological examination of food, water and environmental sample by culture Sampling: Samples of watercress and irrigation water were collected by Environmental Health Practitioners directly from the point of production from the 10 watercress farms supplying Supplier A. As part of the follow-up investigations, further samples were collected of watercress, irrigation water, empty seed sacks and gravel were collected from four farms where the initial round of microbiological sampling results had indicated elevated levels E. coli in the produce. Samples of seeds, peat and gravel were also collected from the seed propagating business that supplied the farms (Table 1). Watercress samples were placed in sterile plastic bags and transported to the laboratory in cold boxes at a controlled temperature of between 0 and 8 C in the same way as the watercress. Seeds, peat and gravel were placed into sterile plastic bags and transported at ambient temperature. Irrigation water samples were collected in sterile plastic bottles containing sodium thiosulphate and transported in cold boxes. All samples were submitted to PHE Food, Water and Environmental Microbiology laboratories for testing within 24 hours of collection. 5

6 Enrichment: Initial testing of watercress samples followed PHE Standard Method F17 based on BS EN ISO 16654:2001. Briefly, modified Tryptone Soya Broth (mtsb) was added to 25g of sample to give a ten-fold dilution and homogenised. Subsequently, the watercress samples collected during the follow-up investigations (Table 1) and all watercress samples previously tested under PHE Standard Method F17 as part of the initial investigations were re-tested using a modified protocol, where the entire volume of watercress available (143 to 928 grams) for each sample was homogenised with sufficient mtsb to completely cover the sample. Each sample of peat, gravel or seeds was submerged in mtsb. The entire volume of water provided (~1L), was filtered through a 0.45 µm pore size membrane using negative pressure filtration and the membrane was submerged in 100mL mtsb. Culture: Following incubation at 41.5 o C for 22 hours, cultures were processed using automated immunomagnetic separation (IMS) for enhanced detection of E. coli O157 (Dynal ) followed by culture on Cefixime Tellurite Sorbitol MacConkey (CT-SMAC) Agar plates incubated at 37 C for 24 hours according to PHE Standard Method F17. Suspect colonies were confirmed biochemically as E. coli, serotyped and tested for stx by PCR (see below). Real time PCR: After enrichment DNA was extracted from samples using the 16 Cell DNA Purification Kit Maxwell 16 (Promega Corporation, Madison, WI, USA) with the automated Maxwell 16 Instrument using the Blood and Cells setting according to manufacturer s instructions. Real-time PCR was used to test DNA extracts (from enriched samples and from suspect boiled colony picks from primary isolation plates) using the MicroSEQ STEC Detection Kit (Thermofisher Scientific, Waltham, MA, USA) containing primers and probes targeting the stx1, stx2, eae (intimin) and O157rfbE ( performed on a 7500 Real-Time 6

7 PCR system (Applied Biosystems, Foster City, CA) using the following amplification parameters: 95 o C for 2 min, followed by 95 o C for 15s and 60 o C for 60s. Enrichment broths that were PCR positive for the stx and O157 antigen genes were sub-cultured onto CT-SMAC agar and those PCR positive for stx genes only were sub-cultured on to MacConkey agar. Up to 50 colonies were re-tested using the PCR described above Library preparation and whole genome sequencing To provide context, all STEC O157 PT2 isolates submitted to GBRU between December 2012 and November 2013 were selected for whole genome sequencing. These included isolates from sporadic cases and isolates linked to the outbreak from human cases and environmental samples. DNA was extracted for sequencing from a total of 70 cultures of STEC O157 PT2 using the Wizard kit (Promega UK). Paired-end libraries were generated using the Illumina Nextera XT sample preparation kit. Assessment of fragment sizes was performed on the Perkin Elmer Labchip GX after fragmentation and clean-up. After normalisation, samples were pooled by hand and library quantification was performed using the KAPA library quantification kit for Illumina sequencing, on an ABI Viia7. Paired-end sequencing was performed on the Illumina HiSeq 2500 instrument using the TruSeq Rapid SBS kit (200 cycle) and TruSeq Paired-end rapid cluster kit. The following cycle parameters were used for sequencing: Read 1: 101, Index read 1: 8, Index read 2: 8 and Read 2: 101. RTA version was used for generation of base call files. Phylogenetic analysis Illumina reads were mapped to the reference Sakai using BWA-SW (10). The Sequence Alignment Map output from BWA was sorted and indexed to produce a Binary Alignment Map (BAM) using Samtools (11). GATK2 (12) was used to create a Variant Call Format (VCF) file from each of the BAMs, which were further parsed to extract only single nucleotide 7

8 polymorphism (SNP) positions which were of high quality in all genomes (MQ>30, DP>10, 128 GQ>30, Variant Ratio >0.9). Pseudosequences of polymorphic positions were used to create maximum likelihood trees using RAxML (13) and compared to the sequences of additional strains held in the PHE STEC O157 database. This database comprises genomes from 1,216 cultures of STEC O157 submitted to GBRU between 1982 and The majority of isolates were from human cases in England reporting domestically-acquired infection. Previous studies on WGS of STEC O157 at PHE indicate isolates of STEC O157 with less than 5 SNPs differences within their core genome ( nucleotides) are considered closely related and likely to have an epidemiological link (GBRU in-house data). FASTQ sequences were deposited in the NCBI Short Read Archive under the BioProject PRJNA Clade Typing was performed as originally defined by Manning et al. (14). The 8 definitive polymorphic positions adopted by Yokoyama et al. (15) were used to delineate the strains into the 9 clade groupings and Shiga toxin subtyping was performed as described by Ashton et al. (16). Results and Discussion Investigation of Outbreak 1 The phylogenetic relationship between the 70 isolates of STEC O157 PT2, sequenced as part of the investigation and the isolates in the PHE WGS database was determined by comparing SNPs in the core genome (Figure 1). One phylogenetic cluster of 22 isolates (between 0-3 SNPs apart) included 19 isolates from cases associated with Outbreak 1, epidemiologically linked to watercress consumption purchased from Retailer A (Figure 2) (6). Two additional isolates were from cases occurring earlier in the year (February and March 2013). The SESQs for these two cases indicated consumption of watercress purchased from Retailer B (Figure 8

9 ). The third additional case was identified in November 2013 and had consumed watercress from Retailer A after watercress was restocked Food chain investigations identified that Supplier A, wholly supplied watercress to Retailer A prior to and during the period of the outbreak, and sourced product from ten farms in the South of England (Figure 2). Retailer B did not share a common supplier with Retailer A. Rather, the watercress sold by Retailer B during the exposure period for the two cases in February and March was imported from North America and mainland Europe by Supplier B (Figure 2). This indicated that the contamination of the watercress was unlikely to have occurred as a result of environmental contamination at the farms under investigation. It was more likely that components of the watercress cultivation process known to be imported, specifically peat or watercress seeds (grown watercress was not imported during the summer months), may represent a potential source of contamination for Outbreak 1. The peat was of mixed origin from more than one European country and the watercress seeds were predominately supplied by a company based in North America, supplemented by a company based in mainland Europe (Figure 2). Supplier A also stated that Supplier B used the same European seed company but this report could not be verified and further trace back investigations failed to establish any links. This trace back investigation highlights difficulties in determining the source of outbreaks when contamination of the imported product, or imported components of the product, occur in the country of origin. Phylogenetic analysis further supported a foreign source for the outbreak. Isolates associated with Outbreak 1 were not closely related to any other strains in the PHE STEC O157 WGS database. The cluster was 292 SNPS away from the nearest domesticallyacquired strains of STEC O157 PT2 and was located on the same branch of the tree as a number of strains of North American origin. To investigate the potential of a North 9

10 American source of contamination, an assembled genome of an isolate from Outbreak 1 was compared to a large database of US STEC isolates maintained at the University of Texas, USA. The closest isolate identified (designated A0ER01) was incorporated into the UK phylogeny and found to be >200 SNPs different in the core genome. Therefore, no microbiological evidence of a link to North America was identified Detection and investigation of a second outbreak (Outbreak 2) During the investigation of Outbreak 1, two additional cases were reported as having consumed watercress as part of a meal at a public house on 1 October Isolates from these two cases were confirmed as STEC O157 PT2 stx2a. WGS analysis showed these two isolates were 0 SNPs from each other but >313 SNPs apart from the isolates from Outbreak 1, and were designated as belonging to Outbreak 2. A further four cases with between 0-3 SNPs apart from each other and the two cases linked to the public house, were identified with onsets from 12 to 30 September 2013 (Figure 1). Thus Outbreak 2 comprised six cases. Five reported consuming watercress, two of whom purchased watercress from Retailer A prior to the recall. Trace back investigations determined that the supplier of watercress to the public house was Supplier A, who supplied Retailer A. This second outbreak was identified epidemiologically due to increased awareness of watercress as a potential vehicle of STEC infection (the Health Protection Team specifically asked whether the cases had consumed watercress after they stated they had consumed salad leaves in the SESQ) and through analysis of the WGS data. Microbiological examination of food, water and environmental samples Extensive sampling and microbiological testing of watercress, irrigation water, gravel, peat and seeds was undertaken, but the Outbreak 1 strain was not isolated. Whilst STEC was not 10

11 detected from seeds, gravel or peat by culture, STEC-PCR analysis of the DNA extract from the mtsb enrichment broths amplified the O157rfbE gene targets in seven of 15 seed samples (in two of these eae was also amplified) but the stx genes were not detected in any of these samples (Table 1). The significance of these results is unclear as the detection of O157rfbE gene indicates that E. coli O157 may be present in the sample but the absence of the stx gene suggests that these strains of E. coli O157 strains are unlikely to be linked to either outbreak. Seeds have been epidemiologically implicated as the vehicle of infection in previous outbreaks but detection of the aetiological agent in this sample type is notoriously technically difficult (17, 18). Initial testing of watercress and irrigation water samples by the PHE Standard Method F17 did not reveal any presumptive STEC O157 colonies, but PCR analysis of the mtsb enrichment broth from the irrigation water from Farm 10 amplified the stx, eae and O157rfbE gene targets (Table 1). Following repeat testing of this sample, a single suspect colony was detected and confirmed to be STEC O157 PT2 stx2a. WGS confirmed this isolate was identical to those from Outbreak 2 (Figure 1). All isolates from cases from Outbreak 2 and the irrigation water isolate from Farm 10 were within 3 SNPs of each other but comprised a separate distinct phylogenetic cluster to those from Outbreak 1 (>313 SNPs apart). Isolates from Outbreak 2 closely clustered in the same clade (<39 SNPs) as those from human cases of domestically-acquired infection and those from domestic cattle (Figure 1). The pathogen was detected in the exit irrigation water from the implicated watercress bed only after enhanced testing using PCR, indicating levels of contamination were low. This is consistent with the epidemiological observation that both outbreaks were small in numbers, 11

12 relatively widely dispersed and with a low attack rate for infection. In order to detect STEC in these samples, testing methodologies were amended to increase sensitivity (15- to 20- fold increase in sample volume examined). PCR was useful in identifying samples with low level contamination that can be targeted for further testing. Whilst this approach may not be practical for all routine monitoring by producers and commercial laboratories, it is suggested that such modified procedures are used for future surveillance and outbreak investigations on uncooked salad vegetables in order to gain a better understanding of the risks involved in these product types. The STEC O157 PT2 from Farm 10 was isolated from exit water used to irrigate the watercress beds. Environmental investigations revealed that the watercress beds were adjacent to a field which contained grazing cattle. The field had a steep hill, which ran down to the watercress beds, down which rainwater could run-off into the irrigation channels belonging to the watercress farm. There was also evidence of the presence of wildlife including rodents, birds, deer and badgers, moving freely between the watercress farm and the adjacent field. Samples were taken from two cow pats, one set of pheasant droppings and one set of suspected deer droppings found close to the watercress beds but all were negative for the presence of STEC O157. Despite these negative results, it is possible that the cattle in the field adjacent to the watercress farm were the source of the strain of STEC O157 that caused Outbreak 2 and that the bacteria were transferred from the field to the watercress beds either by wildlife or farm workers entering the watercress farm or by run-off water (19). The reservoir of STEC is animals, most commonly ruminants, and in particular, cattle (20, 21). Transmission from cattle to humans occurs through direct or indirect contact with animals or their environment, or consumption of contaminated food (meat products or vegetable 12

13 produce exposed to animals in the environment) or water (22, 23). Between 1992 and 2006, 2,274 foodborne outbreaks of infectious intestinal disease were reported in England and Wales, of which 82 (4%) were associated with the consumption of prepared salads (24). It is widely recognised that uncooked salad vegetables can be a cause of foodborne infectious disease (25-27). Although there are few reports specifically identifying watercress as being associated with cases of bacterial foodborne disease, a previous review of risk factors for STEC O157 cases in England and Wales demonstrated that consumption of watercress was associated with infection (28). The low levels of contamination described in this study in a product dispersed over a wide geographical region may explain why outbreaks associated with salad vegetables go undetected. While both outbreaks described here were relatively small, the potential exists for much larger outbreaks to occur via the same transmission route. Prevention of outbreaks associated with consumption of salad and raw vegetables must consider all elements of the complex food chain of fresh produce from farm to table. The risk of such outbreaks is likely to be reduced by protecting produce from direct or indirect contact with the faeces from animals during cultivation and harvest (29). Since removal of bacterial pathogens from salad vegetables is technically difficult, prevention of contamination in the field is essential to ensure the bio-safety of all salad vegetables (30). A multidisciplinary approach to outbreak investigations, including descriptive and analytical epidemiological investigation alongside food-chain trace back, environmental investigation, microbiological sampling including PCR and use of WGS, proved invaluable in investigating two simultaneous outbreaks of STEC linked to consumption of watercress but associated with different sources of contamination. This study demonstrated the added insights provided by the use of PCR and WGS in national surveillance of STEC O157. Without PCR, it 13

14 would not have been possible to detect and isolate STEC O157 from the irrigation water. In the UK, foodborne outbreaks of STEC O157 are often small and nationally distributed and, therefore, difficult to resolve. Although the MLVA profiles enabled us to differentiate the two outbreaks, WGS demonstrated unparalleled sensitivity and accuracy in identifying linked cases over time (10 month time-frame) and space (nationally distributed across England). During this investigation, WGS was robust and reliable enough to direct epidemiological investigations with respect to the source of contamination being either domestic or imported, as well as inferring epidemiological context through evolutionary relationships. Sharing of WGS of STEC O157 data across international borders will greatly facilitate evidence-based trace back of isolates to their country of origin. Likewise, sharing WGS data across clinical, agricultural and veterinary disciplines will direct and improve the ascertainment of specific risk factors in the food chain, thus facilitating the targeting of resources and public health interventions in order to have maximum impact on the burden of STEC O157 disease. 14

15 Acknowledgements We would like to acknowledge the contribution of everyone on the Outbreak Control Team, in partiuclar Chris Williams and Rob Smith (Public Health Wales), Mary Locking and Lynda Browning (Public Health Scotland), Neil Irvine (Public Health Agency Northern Ireland), Maree Barnett and Ruth Parry (Department of Health) and Rachel Castles and Darren Chant (Basingstoke and Deane Borough Council). We would especially like to acknowledge the invaluable support and advice from Drazenka Tubin-Delic and her colleagues at the Food Standards Agency. We would also like to thank Marie Chattaway, Neil Perry, Vivienne do Nascimento, Michael Wright and Yoshini Taylor at the Gastrointestinal Bacteria Reference Unit and Kirsten Glen and Natalie Adams at the department of Gastrointestinal and Emerging Zoonotic Infections, Public Health England. Downloaded from on July 5, 2018 by guest 15

16 References 1. Wells JG, Davis BR, Wachsmuth IK, Riley LW, Remis RS, Sokolow R, Morris GK Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J Clin Microbiol 18: Ihekweazu C, Carroll K, Adak B, Smith G, Pritchard GC, Gillespie IA, Verlander NQ, Harvey-Vince L, Reacher M, Edeghere O, Sultan B, Cooper R, Morgan G, Kinross PT, Boxall NS, Iversen A, Bickler G Large outbreak of verocytotoxin-producing Escherichia coli O157 infection in visitors to a petting farm in South East England, Epidemiol Infect 140: Strachan NJ, Dunn GM, Locking ME, Reid TM, Ogden ID Escherichia coli O157: burger bug or environmental pathogen? Int J Food Microbiol 112: Byrne L, Elson R, Dallman TJ, Perry N, Ashton P, Wain J, Adak GK, Grant KA, Jenkins C Evaluating the use of multilocus variable number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 as a routine public health tool in England. PLoS One 9:e Perry N, Cheasty T, Dallman T, Launders N, Willshaw G Application of multilocus variable number tandem repeat analysis to monitor Verocytotoxin-producing Escherichia coli O157 phage type 8 in England and Wales: emergence of a profile associated with a national outbreak. J Appl Microbiol 115: Launders N, Byrne L, Adams N, Glen K, Jenkins C, Tubin-Delic D, Locking M, Williams C, Morgan D; Outbreak Control Team Outbreak of Shiga toxin-producing E. coli 16

17 O157 associated with consumption of watercress, United Kingdom, August to September Euro Surveill 18: Byrne L, Fisher I, Peters T, Maher A, Thomson N, Rosner B, Barnard H, McKeown P, Cormican M, Cowden J, Aiyedun V, Coia J, Brown D, Lane C A Multi-Country Outbreak of Salmonella Newport gastroenteritis in Europe associated with watermelon from Brazil confirmed by whole genome sequencing: October 2011-January Euro Surveill 19: McDonnell J, Dallman T, Atkin S, Turbitt DA, Connor TR, Grant KA, Thomson NR, Jenkins C Retrospective analysis of whole genome sequencing compared to prospective typing data in further informing the epidemiological investigation of an outbreak of Shigella sonnei in the UK. Epidemiol Infect 141: Dallman TJ, Chattaway MA, Cowley LA, Doumith M, Tewolde R, Wooldridge DJ, Underwood A, Ready D, Wain J, Foster K, Grant KA, Jenkins C An investigation of the diversity of strains of enteroaggregative Escherichia coli isolated from cases associated with a large multi-pathogen foodborne outbreak in the UK. PLoS One 9:e Li H, Durbin R Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26: Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project Data Processing Subgroup The Sequence Alignment/Map format and SAMtools. Bioinformatics. 25:

18 McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20: Stamatakis A RAxML Version 8: A tool for Phylogenetic Analysis and Post- Analysis of Large Phylogenies. Bioinformatics : Manning SD, Motiwala AS, Springman AC, Qi W, Lacher DW, Ouellette LM, Mladonicky JM, Somsel P, Rudrik JT, Dietrich SE, Zhang W, Swaminathan B, Alland D, Whittam TS Variation in virulence among clades of Escherichia coli O157:H7 associated with disease outbreaks. Proc Natl Acad Sci 105: Yokoyama E, Hirai S, Hashimoto R, Uchimura M Clade analysis of enterohemorrhagic Escherichia coli serotype O157:H7/H- strains and hierarchy of their phylogenetic relationships. Infect Genet Evol 12: Ashton PM, Perry N, Ellis R, Petrovska L, Wain J, Grant KA, Jenkins C, Dallman TJ Insight into Shiga toxin genes encoded by Escherichia coli O157 from whole genome sequencing. PeerJ 3: e Nathan R. American seeds suspected in Japanese food poisoning epidemic Nat Med 3: Tzschoppe M, Martin A, Beutin L. A rapid procedure for the detection and isolation of enterohaemorrhagic Escherichia coli (EHEC) serogroup O26, O103, O111, O118, O121, 18

19 O145 and O157 strains and the aggregative EHEC O104:H4 strain from ready-to-eat vegetables. Int J Food Microbiol 152: Wachtel MR, Whitehand LC, Mandrell RE Association of Escherichia coli O157:H7 with preharvest leaf lettuce upon exposure to contaminated irrigation water. J Food Prot 65: Borczyk AA, Karmali MA, Lior H, Duncan LM Bovine reservoir for verotoxinproducing Escherichia coli O157:H7. Lancet 1(8524): Rasmussen MA, Cray WC, Casey TA, Whipp SC Rumen contents as a reservoir of enterohemorrhagic Escherichia coli FEMS Microbiol. Lett 114: Locking ME, O'Brien SJ, Reilly WJ, Wright EM, Campbell DM, Coia JE, Browning LM, Ramsay CN Risk factors for sporadic cases of Escherichia coli O157 infection: the importance of contact with animal excreta. Epidemiol Infect 127: Gillespie IA, O'Brien SJ, Adak GK, Cheasty T, Willshaw G Foodborne general outbreaks of Shiga toxin-producing Escherichia coli O157 in England and Wales : where are the risks? Epidemiol Infect 133: Little CL, Gillespie IA Prepared salads and public health. J Appl Microbiol 105: Friesema I, Sigmundsdottir G, van der Zwaluw K, Heuvelink A, Schimmer B, de Jager C, Rump B, Briem H, Hardardottir H, Atladottir A, Gudmundsdottir E, van Pelt W

20 An international outbreak of Shiga toxin-producing Escherichia coli O157 infection due to lettuce, September-October Euro Surveill 13(50) Wendel AM, Johnson DH, Sharapov U, Grant J, Archer JR, Monson T, Koschmann C, Davis JP Multistate outbreak of Escherichia coli O157:H7 infection associated with consumption of packaged spinach, August-September 2006: the Wisconsin investigation. Clin Infect Dis. 48: Slayton RB, Turabelidze G, Bennett SD, Schwensohn CA, Yaffee AQ, Khan F, Butler C, Trees E, Ayers TL, Davis ML, Laufer AS, Gladbach S, Williams I, Gieraltowski LB Outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 associated with romaine lettuce consumption, PLoS One 8:e O'Brien SJ, Adak GK, Gilham C Contact with farming environment as a major risk factor for Shiga toxin (Vero cytotoxin)-producing Escherichia coli O157 infection in humans. Emerg Infect Dis 7: Ackers ML, Mahon BE, Leahy E, Goode B, Damrow T, Hayes PS, Bibb WF, Rice DH, Barrett TJ, Hutwagner L, Griffin PM, Slutsker L An outbreak of Escherichia coli O157:H7 infections associated with leaf lettuce consumption. J Infect Dis 177: Berger CN, Sodha SV, Shaw RK, Griffin PM, Pink D, Hand P, Frankel G Fresh fruit and vegetables as vehicles for the transmission of human pathogens. Environ Microbiol 12:

21 Tables and Figures Table 1. Summary of the microbiological examination of food, water and environmental samples from ten farms and the seed propagating business supplying Supplier A. Farm Sample type and number tested Microbiology results Initial investigations 1 Watercress (6), Spinach (1), Negative Rocket (1), Final rinse water (2) 2 Watercress (2), Irrigation water Negative (1) 3 Watercress (5), Irrigation water E. coli 280 cfu/g in 1 sample of watercress (2) 4 Watercress (6), Irrigation water (4) STEC serogroup O74 detected in 1 sample of watercress 5 Watercress (1), Irrigation water Negative (1) 6 Watercress (1), Irrigation water Negative (1) 7 Watercress (5), Irrigation water Negative (5) 8 Watercress (8), Irrigation water (13) E. coli 280 cfu/g in 1 sample and 20 cfu/g in 2 samples of watercress 9 Watercress (4), Irrigation water (10) E. coli 1860 cfu/g in 1 sample and 20 cfu/g in 1 sample of watercress 10 Watercress (7), Irrigation water (11) E. coli 60 cfu/g in 1 sample and 20 cfu/g in 1 sample of watercress; 3 isolates of E. coli O157 detected all stx negative Follow-up investigations 1 Seeds (32), empty seed sacks (5) Seven seed samples were PCR positive for the O157 antigen but negative for the stx genes. E. coli O157 was not cultured. 4 Watercress (5), Irrigation water Negative (5), gravel (1) 9 Watercress (4), Irrigation water Negative (5), gravel (4) 10 Watercress (13), Irrigation water (10), gravel (3) STEC O157 PT2 stx2 isolated from one sample of irrigation water Seed propagating business Seeds (4), peat (10) Negative 21

22 Figure 1. Maximum likelihood phylogeny of 107 STEC O157:H7 genomes, representing a core genome of nucleotides with 3063 polymorphisms. Nineteen isolates were from cases linked to Outbreak 1 and eight isolates associated with Outbreak 2, including seven from cases and one from the watercress irrigation water. Clusters with known epidemiological links are highlighted in red, sporadic UK cases are denoted with *, and reference strains from the United States of America are denoted USA. Two isolates from a single patient in Outbreak 2 are highlighted with a +. Figure 2. Representation of the links food chain between suppliers, retailers and cases belonging to Outbreaks 1 and 2. Note 1 STEC O157 PT2, the cause of Outbreak 2, was detected in the exit irrigation water at Farm 10. Note 2 Seed Company 2 was used by Supplier A to supplement seed supply and was anecdotally linked (not confirmed) to Supplier B Downloaded from on July 5, 2018 by guest 22

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