Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm Shiga toxin-producing Escherichia coli survives storage in wheat flour for two years Alexander Gilla,∗, Tanis McMahona, Forest Dussaultb, Nicholas Petronellab aHealth Canada, Bureau of Microbial Hazards, 251 Sir Frederick Banting Driveway, Ottawa, Ontario K1A 0K9, Canada bHealth Canada, Bureau of Food Surveillance and Science Integration, 251 Sir Frederick Banting Driveway, Ottawa, Ontario K1A 0K9, Canada A R T I C L E I N F O Keywords: Shiga toxin Verotoxin Escherichia coli Flour A B S T R A C T Wheat flour has recently been recognised as an exposure vehicle for the foodborne pathogen Shiga toxin-pro- ducing Escherichia coli (STEC). Wheat flour milled on two sequential production days in October 2016, and implicated in a Canada wide outbreak of STEC O121:H19, was analysed for the presence of STEC in November 2018. Stored in sealed containers at ambient temperature, the water activity of individual flour samples was below 0.5 at 6 months post-milling and remained static or decreased slightly in individual samples during 18 months of additional storage. STEC O121 was isolated, with the same genotype (stx2a, eae, hlyA) and core genome multilocus sequence type as previous flour and clinical isolates associated with the outbreak. The result of this analysis demonstrates the potential for STEC to persist in wheat flour at levels associated with outbreak infections for periods of up to two years. This has implications for the potential for STEC to survive in other foods with low water activity. 1. Introduction Shiga toxin-producing Escherichia coli (STEC, also known as ver- otoxin-producing E. coli) are recognised worldwide as a significant cause of food-borne illness (FAO/WHO, 2018). Milled grains were first confirmed as a source of exposure to STEC following an outbreak of STEC of the serogroups O121 and O26, involving 56 cases between December 2015 and September 2016 in the United States of America (Crowe et al., 2017). Wheat flour from a single mill was identified as the vehicle of exposure by the recovery of STEC O121 isolates, which shared the same pulsed field gel electrophoresis (PFGE) patterns as clinical isolates (Crowe et al., 2017). Since 2016, there have been three additional reports of STEC out- breaks involving wheat flour. The first was a multi-province outbreak of STEC O121:H19 in Canada with 30 cases from November 2016 to April 2017 (Morton et al., 2017). The second outbreak of 6 cases, also of STEC O121, occurred concurrently in British Columbia from February to April 2017, but involved an unrelated product and strain (BCCDC, 2017). The third outbreak involved 21 cases of STEC O26 across 9 US states between December 2018 and May 2019 (CDC, 2019a). These reports indicate that wheat flour is a sporadic, reoccurring vehicle for STEC exposure in Canada and the USA. Previously, our laboratory conducted bacteriological analysis of wheat flour implicated in the first Canadian outbreak of STEC O121:H19 (Morton et al., 2017; Gill et al., 2019). STEC, including STEC O121:H19 with the same PFGE pattern as the clinical isolates, was isolated from wheat flour products milled on three successive days in October 2016. The concentration of the STEC O121 in the flour was estimated at 0.15 to 0.43 MPN/100 g, with no significant difference between samples tested at the end of the outbreak, (5–6 months post milling) and 10–11 months post milling (Gill et al., 2019). Flour sam- ples from this outbreak investigation had been retained and the samples were reanalysed two years post milling (November 2018) to evaluate the potential for STEC to survive prolonged storage in wheat flour. 2. Materials and methods 2.1. Flour samples Eight samples of bleached wheat flour recovered in an outbreak investigation, had previously been tested and determined to contain STEC (Gill et al., 2019). The samples were stored in the original sam- pling bags inside opaque, air tight, screw capped plastic containers for shipping biohazardous materials (SAF-T-PAK® STP-200, Inmark Life Sciences, Edmonton, AB, Canada) at ambient temperature (20–25 °C). The flour had been produced at a single mill on two consecutive days, designated day A and day B, in October 2016. Two samples were available from day A and five samples from Day B. https://doi.org/10.1016/j.fm.2019.103380 Received 10 October 2019; Received in revised form 12 November 2019; Accepted 13 November 2019 ∗ Corresponding author. E-mail address: alex.gill@canada.ca (A. Gill). Food Microbiology 87 (2020) 103380 Available online 18 November 2019 0740-0020/ Crown Copyright © 2019 Published by Elsevier Ltd. All rights reserved. T http://www.sciencedirect.com/science/journal/07400020 https://www.elsevier.com/locate/fm https://doi.org/10.1016/j.fm.2019.103380 https://doi.org/10.1016/j.fm.2019.103380 mailto:alex.gill@canada.ca https://doi.org/10.1016/j.fm.2019.103380 http://crossmark.crossref.org/dialog/?doi=10.1016/j.fm.2019.103380&domain=pdf 2.2. Water activity The water activity of the flour samples was measured at 24 ± 1 °C with an Aqua Lab Water Activity Meter (Decagon Devices Inc, Pullman, WA) according to the manufacturer's instructions. Water activity was measured on initial receipt of the samples and after two years storage, immediately prior to bacteriological analysis. 2.3. Analysis for STEC O121 Five analytical units of flour weighing 100 g were taken for STEC analysis from each production day. The analytical units for the day A samples were taken from two samples, three from one and two from the other. The five analytical units for day B were taken from five in- dividual samples. A process control consisting of 100 g of wheat flour inoculated with a colony of E. coli O157:H7 ATCC 35150 and a negative control of un-inoculated enrichment media were analysed in parallel. E. coli O157:H7 ATCC 35150 was used as the process control as it carries both stx1a and stx2a and so would provide the expected PCR positive results from both the VT1 and VT2 primer sets used in the screening PCR, and can be differentiated by morphology and serotype from the outbreak STEC O121:H19. The analytical units underwent analysis for STEC O121 as previously described (Gill et al., 2019). Briefly, 100 g of flour was enriched in 300 mL of liquid media and screened for the genes stx and wzxO121. Isolation of STEC O121, was attempted on enrich- ments which were PCR positive for both stx and wzxO121 by plating on Rainbow O157 (BiOLOG, Hayward, CA) and MacConkey agar (Difco, BD, Sparks, MD, USA), aided by E. coli O121 Immunomagnetic Se- paration Beads (Abraxis, Warminster, PA). Isolates which were identi- fied as stx positive were characterised by multiplex PCR for stx1, stx2, eae, and hlyA (Paton and Paton, 2003), for wzxO121, and for stx-sub- type (Scheutz et al., 2012). Species identity was determined by API 20E biochemical tests (bioMérieux, St. Laurent, QC). 2.4. Genome analysis of STEC The genomes of the STEC isolates recovered were sequenced with paired–end (2 × 300 bp) reads sequencing performed on the MiSeq Platform (Illumina, Inc., San Diego, CA) using Reagent Kit v3 and NexteraXT libraries (Illumina, Inc.). Low quality reads were filtered from each sequence data set using tools from the BBMap software suite v38.35, with default parameters (Bushnell, 2014). Genome assemblies were generated from the quality-controlled reads with SKESA v2.1 (Souvorov et al., 2018) and the assemblies were error corrected with Pilon v1.22 (Walker et al., 2014). 2.5. Most probable number calculation (MPN) An estimate of the STEC O121 MPN level (including 95% confidence limits) in the flour for each production day was performed using the US FDA Microsoft Excel spreadsheet (Blodgett, 2015). The analytical re- sults were treated as a 5 tube, 1 dilution (100 g) MPN array for each production day. 3. Results and discussion The water activity of the flour samples in 2018 was the same or slightly lower than it had been on sample receipt in 2017 (Table 1). The conditions of storage, a sealed container, at ambient temperature, un- exposed to light, is similar to the storage of flour practiced by many consumers. For both production days (A and B), one enrichment broth of the five 100 g analytical units tested was found positive for stx, and these two enrichments were also positive for wzxO121. STEC O121 was iso- lated from both presumptive positive enrichments. The isolates were confirmed as E. coli by API 20E, were negative for stx1, positive for stx2, eae and hlyA, and possessed the stx2a subtype. The genome of the two STEC O121 isolates recovered was compared to the genomes of 6 STEC O121:H19 isolates previously recovered from the same flour samples (Gill et al., 2019) and the closed genome of a STEC O121:H19 clinical isolate from the associated outbreak (Robertson et al., 2018) using the Enterobase cgMLST (core genome multilocus sequence typing) (Zhou et al., 2019, comparison using database from February 7, 2019). The genome sequences of the individual strains were compared by BLAST (Madden, 2013) against the Enterobase cgMLST database. Comparison of 2513 core genes between the epidemiologically linked STEC O121:H19 isolates found a minimum number of single nucleotide polymorphisms (SNPs) of 0 and a maximum of 7 between any two isolates, which is consistent with these isolates belonging to a clonal population. An MPN estimate for the STEC O121 level in the flour, calculated for one positive tube out of five 100 g tubes, is 0.22 MPN/100 g with 95% confidence intervals of 0.03 and 1.59 MPN/100 g. This estimate is within the confidence limits of the estimates of 0.15–0.43 MPN/100 g from analysis conducted at the end of the outbreak (5–6 months) or 10–11 months post milling (Gill et al., 2019). Though at a low level, this stability of the STEC O121 population does not suggest any reduction in the potential hazard to consumers during the two years of storage. Studies of STEC survival in artificially inoculated wheat flour have reported the persistence of the inoculated pathogens for at least 40 weeks, with numbers declining steadily from 7 to 8.5 log CFU/g to below 2.5 log CFU/g within 12–16 weeks (Forghani et al., 2018, 2019). This pattern of an initial decline in STEC populations followed by long term persistence has been reported in other studies with low water activity foods artificially inoculated with STEC, including confectionary and nuts (Baylis et al., 2004; Blessington et al., 2013). Since these studies did not attempt to quantify STEC below the limit of quantifi- cation for direct plating (approximately 2 log CFU/g) it is unknown whether the rate at which cell viability was lost was stable, declined or increased during these experiments. The persistence of STEC O121:H19 at a stable level from 5-6 months to 2 years of storage observed in this study does not indicate continuing loss of cell viability. There are three potential explanations for the differences in these observations: 1. The particular STEC O121:H19 strain isolated in this study, is particularly well adapted to survival at low water activity. 2. The artificial in- oculation studies conducted to date do not accurately model natural contamination processes, resulting in significant differences in cell physiological state (Harrand et al., 2019). 3. The survivors are members of a phenotypically heterogeneous portion of the initial population of cells, similar to the persister cells that resist antibiotic treatment (Verstraeten et al., 2016). It is possible that the STEC O121:H19 strain isolated in this study possesses a relatively unusual ability to survive at low water activity compared to other STEC strains. Survival of STEC, Shigella and Salmonella dried on paper disks and on various low moisture foods has Table 1 Results of analysis of wheat flour for Shiga toxin producing E. coli O121 and water activity measurements at 6 months and 2 years of storage post-milling. Production Day Water Activity STEC O121 positive samples MPN/ 100 g 95% Conf. Limit 6 mon. 24 mon. Low High A 0.358 0.338 1/5 0.22 0.03 1.59 0.495 0.430 B 0.352 0.307 1/5 0.22 0.03 1.59 0.363 0.358 0.360 0.360 0.400 0.381 0.381 0.374 MPN: Most probable number. Mon. Months. Conf. Limit: Confidence Limits. A. Gill, et al. Food Microbiology 87 (2020) 103380 2 been report to vary between strains, and to be independent of STEC serotype (Hiramatsu et al., 2005). This study also found that survival was influenced by the food matrix and enhanced in the presence of sucrose, a compatible solute (Hiramatsu et al., 2005). However, there is evidence that the ability to persist in flour or at low water activity is a common trait among E. coli strains, including STEC. Firstly, low num- bers of generic E. coli are a typical component of the microbiota of wheat flour milled in North America (Sperber, 2007). Secondly, two other STEC strains with different genotypes and serotypes were isolated in the original analysis of the samples used in this study (Gill et al., 2019) and surveys of wheat and rye flour samples for STEC conducted in Germany and Switzerland have reported isolation of STEC of diverse serotype and genotype, unrelated to any identified outbreak (Mäde et al., 2017; Boss and Hummerjohn, 2019; Kindle et al., 2019). Finally, survival of E. coli in low water activity environments involves me- chanisms of resistance to desiccation and osmotic stress, such as accu- mulation of compatible solutes and the RpoS stress response, which are common to the species (Burgess et al., 2016). That the inoculation method influences cell survival at low water activity is suggested by comparing two studies which used different inoculation methods (Forghani et al., 2018; Daryaei et al., 2018). For wheat flour (aw 0.47) inoculated with a STEC from liquid culture, an approximate 4 log CFU reduction in cell numbers occurred during the initial 4 weeks of storage, with no significant difference between strains of different serotypes (Forghani et al., 2018). When agar harvested STEC cells were inoculated to confectionary (aw 0.427), savory sea- soning (aw 0.633), meat powder (aw 0.255), or dry pet food (aw 0.547), a 1 log reduction was observed in all four matrices over 4 weeks of storage (Daryaei et al., 2018). Daryaei et al. (2018) also briefly describe an unpublished study in which they observed no significant decline in STEC over 3 weeks storage at 16 °C in wheat flour (aw 0.25 or 0.55). STEC are generally perceived as pathogens associated with foods with high moisture content or water activity and that are consumed fresh. The foods predominantly associated with STEC outbreaks are fresh meats (particularly beef), fresh fruits and vegetables (particularly leafy greens and sprouts), and dairy products (Devleesschauwer et al., 2019; Pires et al., 2019). However, in addition to flour there have been other reports of STEC outbreaks associated with foods with low water activity including, walnuts, hazel nuts, rice cakes, Soy nut butter, jerky, brownie cake, and wheat snacks (CDC, 2019b; Nabae et al., 2013; Keene et al., 1997). These reports and the potential for prolonged survival of STEC observed in this study suggests that other low moisture foods though infrequently contaminated with STEC could be significant vehicles of sporadic exposure to STEC. 4. Conclusions The result of this analysis demonstrates the potential for STEC to persist in wheat flour at levels associated with outbreak infections for periods of up to two years. 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Food Microbiology 87 (2020) 103380 4 http://refhub.elsevier.com/S0740-0020(19)30990-6/sref25 http://refhub.elsevier.com/S0740-0020(19)30990-6/sref25 http://refhub.elsevier.com/S0740-0020(19)30990-6/sref25 http://refhub.elsevier.com/S0740-0020(19)30990-6/sref25 https://doi.org/10.1017/S095026881900116X https://doi.org/10.1017/S095026881900116X https://doi.org/10.1128/genomeA.01561-17 https://doi.org/10.1128/genomeA.01561-17 https://doi.org/10.1128/JCM.00860-12 https://doi.org/10.1186/s13059-018-1540-z https://doi.org/10.1186/s13059-018-1540-z https://doi.org/10.4315/0362-028X-70.4.1041 https://doi.org/10.1007/978-1-4939-2854-5_1 https://doi.org/10.1007/978-1-4939-2854-5_1 https://doi.org/10.1371/journal.pone.0112963 https://www.biorxiv.org/content/10.1101/613554v1 https://www.biorxiv.org/content/10.1101/613554v1 Shiga toxin-producing Escherichia coli survives storage in wheat flour for two years Introduction Materials and methods Flour samples Water activity Analysis for STEC O121 Genome analysis of STEC Most probable number calculation (MPN) Results and discussion Conclusions mk:H1_10 Acknowledgements References