Metabolic characteristics and genomic epidemiology of Escherichia coli serogroup O145 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand

Shiga toxin-producing Escherichia coli (STEC) are a global public health concern, and can cause severe human disease. Ruminants are asymptomatic reservoirs of STEC, shedding this pathogen via their faeces. There is ‘zero tolerance’ for the Top 7 STEC serogroups (O26, O45, O103, O111, O121, O145 and O157) in ground beef products exported to the USA. STEC may contaminate carcasses during processing and therefore are a major regulatory concern for New Zealand’s meat industry. A previous study investigating the prevalence of STEC in young calves (n=1508) throughout New Zealand identified STEC O145 as the most prevalent serogroup (43%) at the dairy farm level compared to the other Top 7 serogroups. This high prevalence underlines STEC O145 as a public health concern and an issue for the meat industry. Current culture-based methods for STEC detection are not fully discriminatory due to the lack of consistent differential characteristics between STEC and non-pathogenic E. coli. This study aims to (i) investigate metabolic characteristics of E. coli O145 to facilitate the differential culture of this serogroup and (ii) understand the genomic epidemiology of E. coli O145 using whole genome sequencing (WGS). E. coli O145 strains examined in this study were genetically and metabolically diverse, according to carbon utilisation. The metabolic and genomic analyses were unable to differentiate between stx-positive and stx-negative O145 strains and there was no association with isolation source. However, clustering of O145 strains was observed according to multi-locus sequence type and at the level of eae subtype, a gene encoding the protein intimin which is involved in bacterial attachment to intestinal epithelial cells. Carbon substrates such as D-serine and D-malic acid were identified as candidate metabolites to differentiate defined O145 sequence types and may assist with identification in conjunction with currently available molecular methods. This research has demonstrated the genetic heterogeneity of serogroup O145 and has made significant progress in the identification of metabolites that may prove beneficial in the development of a differential media for certain subsets of serogroup O145. Such a medium would prove a valuable tool for maintaining and monitoring public health and providing food quality and safety assurances that New Zealand meat for export is free of this pathogen

Global and regional source attribution of Shiga toxin-producing Escherichia coli infections using analysis of outbreak surveillance data

Shiga toxin-producing Escherichia coli (STEC) infections pose a substantial health and economic burden worldwide. To target interventions to prevent foodborne infections, it is important to determine the types of foods leading to illness. Our objective was to determine the food sources of STEC globally and for the six World Health Organization regions. We used data from STEC outbreaks that have occurred globally to estimate source attribution fractions. We categorised foods according to their ingredients and applied a probabilistic model that used information on implicated foods for source attribution. Data were received from 27 countries covering the period between 1998 and 2017 and three regions: the Americas (AMR), Europe (EUR) and Western-Pacific (WPR). Results showed that the top foods varied across regions. The most important sources in AMR were beef (40%; 95% Uncertainty Interval 39-41%) and produce (35%; 95% UI 34-36%). In EUR, the ranking was similar though with less marked differences between sources (beef 31%; 95% UI 28-34% and produce 30%; 95% UI 27-33%). In contrast, the most common source of STEC in WPR was produce (43%; 95% UI 36-46%), followed by dairy (27%; 95% UI 27-27%). Possible explanations for regional variability include differences in food consumption and preparation, frequency of STEC contamination, the potential of regionally predominant STEC strains to cause severe illness and differences in outbreak investigation and reporting. Despite data gaps, these results provide important information to inform the development of strategies for lowering the global burden of STEC infections

Shiga toxin production and translocation during microaerobic human colonic infection with Shiga toxin-producing E. coli O157:H7 and O104:H4

Haemolytic uraemic syndrome caused by Shiga toxin-producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream. An understanding of Stx-related events in the human gut is limited due to lack of suitable experimental models. In this study, we have used a vertical diffusion chamber system with polarized human colon carcinoma cells to simulate the microaerobic (MA) environment in the human intestine and investigate its influence on Stx release and translocation during STEC O157:H7 and O104:H4 infection. Stx2 was the major toxin type released during infection. Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions. Increased Stx transport was dependent on STEC infection and occurred via a transcellular pathway other than macropinocytosis. While MA conditions had a similar general effect on Stx release and absorption during infection with STEC O157:H7 and O104:H4, both serotypes showed considerable differences in colonization, Stx production, and Stx translocation which suggest alternative virulence strategies. Taken together, our study suggests that the MA environment in the human colon may modulate Stx-related events and enhance Stx absorption during STEC infection

Whole-genome sequencing for national surveillance of Shiga toxin–producing Escherichia coli O157

Background. National surveillance of gastrointestinal pathogens, such as Shiga toxin–producing Escherichia coli O157 (STEC O157), is key to rapidly identifying linked cases in the distributed food network to facilitate public health interventions. In this study, we used whole-genome sequencing (WGS) as a tool to inform national surveillance of STEC O157 in terms of identifying linked cases and clusters and guiding epidemiological investigation. Methods. We retrospectively analyzed 334 isolates randomly sampled from 1002 strains of STEC O157 received by the Gastrointestinal Bacteria Reference Unit at Public Health England, Colindale, in 2012. The genetic distance between each isolate, as estimated by WGS, was calculated and phylogenetic methods were used to place strains in an evolutionary context. Results. Estimates of linked clusters representing STEC O157 outbreaks in England and Wales increased by 2-fold when WGS was used instead of traditional typing techniques. The previously unidentified clusters were often widely geographically distributed and small in size. Phylogenetic analysis facilitated identification of temporally distinct cases sharing common exposures and delineating those that shared epidemiological and temporal links. Comparison with multi locus variable number tandem repeat analysis (MLVA) showed that although MLVA is as sensitive as WGS, WGS provides a more timely resolution to outbreak clustering. Conclusions. WGS has come of age as a molecular typing tool to inform national surveillance of STEC O157; it can be used in real time to provide the highest strain-level resolution for outbreak investigation. WGS allows linked cases to be identified with unprecedented specificity and sensitivity that will facilitate targeted and appropriate public health investigations

Regional Differences in Presence of Shiga toxin-producing Escherichia coli Virulence-Associated Genes in the Environment in the North West and East Anglian regions of England

Shiga toxin-producing Escherichia coli is carried in the intestine of ruminant animals, and outbreaks have occurred after contact with ruminant animals or their environment. The presence of STEC virulence genes in the environment was investigated along recreational walking paths in the North West and East Anglia regions of England. In all, 720 boot sock samples from walkers’ shoes were collected between April 2013 and July 2014. Multiplex PCR was used to detect E. coli based on the amplification of the uidA gene and investigate STEC-associated virulence genes eaeA, stx1 and stx2. The eaeA virulence gene was detected in 45·5% of the samples, where stx1 and/or stx2 was detected in 12·4% of samples. There was a difference between the two regions sampled, with the North West exhibiting a higher proportion of positive boot socks for stx compared to East Anglia. In univariate analysis, ground conditions, river flow and temperature were associated with positive boot socks. The detection of stx genes in the soil samples suggests that STEC is present in the English countryside and individuals may be at risk for infection after outdoor activities even if there is no direct contact with animals. Significance and Impact of the Study: Several outbreaks within the UK have highlighted the danger of contracting Shiga toxin-producing Escherichia coli from contact with areas recently vacated by livestock. This is more likely to occur for STEC infections compared to other zoonotic bacteria given the low infectious dose required. While studies have determined the prevalence of STEC within farms and petting zoos, determining the risk to individuals enjoying recreational outdoor activities that occur near where livestock may be present is less researched. This study describes the prevalence with which stx genes, indicative of STEC bacteria, were found in the environment in the English countryside

Occurrence and characterization of shiga toxin-producing Escherichia coli in raw meat, raw milk, and street vended juices in Bangladesh

The major objective of this study was to investigate the prevalence of Shiga toxin (Stx)–producing Escherichia coli (STEC) in different types of food samples and to compare their genetic relatedness with STEC strains previously isolated from animal sources in Bangladesh. We investigated a total of 213 food samples, including 90 raw meat samples collected from retail butcher shops, 20 raw milk samples from domestic cattle, and 103 fresh juice samples from street vendors in Dhaka city. We found that more than 68% (n=62) of the raw meat samples were positive for the stx gene(s); 34% (n=21) of buffalo meats and 66% (n=41) of beef. Approximately 10% (n=2) of the raw milk and 8% (n=8) of the fresh juice samples were positive for stx. We isolated STEC O157 from seven meat samples (7.8%), of which two were from buffalo meats and five from beef; and no other STEC serotypes could be isolated. We could not isolate STEC from any of the stx-positive raw milk and juice samples. The STEC O157 isolates from raw meats were positive for the stx2, eae, katP, etpD, and enterohemorrhagic E. coli hly virulence genes, and they belonged to three different phage types: 8 (14.3%), 31 (42.8%), and 32 (42.8%). Pulsed-field gel electrophoresis (PFGE) typing revealed six distinct patterns among seven isolates of STEC O157, suggesting a heterogeneous clonal diversity. Of the six PFGE patterns, one was identical and the other two were =90% related to PFGE patterns of STEC O157 strains previously isolated from animal feces, indicating that raw meats are readily contaminated with fecal materials. This study represents the first survey of STEC in the food chain in Bangladesh

Identification of the long polar fimbriae gene variants in the locus of enterocyte effacement-negative Shiga toxin-producing Escherichia coli strains isolated from humans and cattle in Argentina

The long polar fimbriae (Lpf) is one of few adhesive factors of Shiga toxin-producing Escherichia coli (STEC) and it is associated with colonization of the intestine. Studies have demonstrated the presence of lpf genes in several pathogenic E. coli strains, and classification of variants based on polymorphisms in the lpfA1 and lpfA2 genes has been adopted. Using a collection of Argentinean locus of enterocyte effacement (LEE)-negative STEC strains, we determined that the different lpfA types were present in a wide variety of serotypes with no apparent association between the types of lpfA1 or lpfA2 genes and the severity of human disease. The lpfA2-1 was the most prevalent variant identified, which was present in 95.8% of the isolates, and lpfA1-3 and lpfA2-2, proposed as specific biomarkers of E. coli O157:H7, were not found in any of the serotypes studied. The prevalence of lpf genes in a large number of strains is useful to understand the genetic diversity of LEE-negative STEC and to define the association of some of these isolates carrying specific lpf-variants with disease.Fil: Galli, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Torres, Alfredo G.. University of Texas Medical Branch; Estados UnidosFil: Rivas, Marta. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentin

Genotypic Characterization of Non-O157 Shiga Toxin–Producing Escherichia coli in Beef Abattoirs of Argentina

The non-O157 Shiga toxin-producing Escherichia coli (STEC) contamination in carcasses and feces of 811 bovines in nine beef abattoirs from Argentina was analyzed during a period of 17 months. The feces of 181 (22.3%) bovines were positive for non-O157 STEC, while 73 (9.0%) of the carcasses showed non-O157 STEC contamination. Non-O157 STEC strains isolated from feces (227) and carcasses (80) were characterized. The main serotypes identified were O178:H19, O8:H19, O130:H11, and O113:H21, all of which have produced sporadic cases of hemolytic-uremic syndrome in Argentina and worldwide. Twenty-two (7.2%) strains carried a fully virulent stx/eae/ehxA genotype. Among them, strains of serotypes O103:[H2], O145:NM, and O111:NM represented 4.8% of the isolates. XbaI pulsed-field gel electrophoresis pattern analysis showed 234 different patterns, with 76 strains grouped in 30 clusters. Nine of the clusters grouped strains isolated from feces and from carcasses of the same or different bovines in a lot, while three clusters were comprised of strains distributed in more than one abattoir. Patterns AREXSX01.0157, AREXBX01.0015, and AREXPX01.0013 were identified as 100% compatible with the patterns of one strain isolated from a hemolytic-uremic syndrome case and two strains previously isolated from beef medallions, included in the Argentine PulseNet Database. In this survey, 4.8% (39 of 811) of the bovine carcasses appeared to be contaminated with non- O157 STEC strains potentially capable of producing sporadic human disease, and a lower proportion (0.25%) with strains able to produce outbreaks of severe disease.Fil: Masana, Marcelo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Agroindustria. Instituto de Tecnología de Alimentos; ArgentinaFil: D´Astek, B. A.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; ArgentinaFil: Palladino, Pablo Martín. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Agroindustria. Instituto de Tecnología de Alimentos; ArgentinaFil: Galli, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: del Castillo, Lourdes Leonor. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Agroindustria. Instituto de Tecnología de Alimentos; ArgentinaFil: Carbonari, Claudia Carolina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Leotta, Gerardo Anibal. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET- La Plata. Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout". Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Instituto de Genética Veterinaria; Argentina. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Vilacoba, Elisabet. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; ArgentinaFil: Irino, K.. Instituto Adolfo Lutz. Seção de Bacteriologia; BrasilFil: Rivas, M.. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas; Argentin

A food chain approach to control of Shiga toxin-producing Escherichia coli in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Science at Massey University, Palmerston North, New Zealand

Part of Chapter 3 has been published as: Browne, A.S., Midwinter, A.C., Withers, H., Cookson, A.L., Biggs, P.J., Marshall J.C., Benschop, J., Hathaway, S., Haack, N., Akhter, R., & French, N.P. (2018). Molecular epidemiology of Shiga toxin-producing Escherichia coli (STEC) on New Zealand dairy farms: Application of a culture-independent assay and whole-genome sequencing. Applied and Environmental Microbiology, 84(14). DOI: 10.1128/AEM.00481-18This thesis describes the prevalence and molecular epidemiology of Shiga toxin-producing Escherichia coli (STEC) in New Zealand using microbiological, genomic, molecular, and statistical methods. STEC are a zoonotic pathogen that can cause bloody diarrhoea and acute kidney failure. Cattle are a well-recognized STEC reservoir, and previous research has identified living near cattle and contact with their faeces as an increased risk for human infection. Seven STEC serogroups (O157, O26, O45, O103, O111, O121, O145), known as the ‘Top 7’ STEC, have been identified as an increased risk to human health, with the New Zealand meat industry undertaking testing to ensure that veal beef exports to some international markets are free of these ‘Top 7’ serogroups. A random stratified cross-sectional study of ‘Top 7’ STEC prevalence of young dairy calves (n=1,508) on New Zealand dairy farms (n=102) found that approximately 20% of calves and 75% of farms were positive for one or more of the ‘Top 7’ STEC. ‘Top 7’ STEC prevalence was positively associated with increased number of calves in a calf pen, and prevalence significantly varied by region. This study utilized a new culture-independent diagnostic test, NeoSEEK (PCR/MALDI-TOF method), and used statistical and microbiological techniques to evaluate the sensitivity and specificity of the method for this and further studies. A longitudinal study evaluating prevalence and transmission of ‘Top 7’ STEC in animals and the dairy farm environment found evidence of calf-to-calf, dam-to-calf, and environment-to-calf transmission. Whole genome sequencing analysis and prevalence data revealed cross-contamination of young veal calf hides occurs during transport and lairage to processing plants. Analysis of New Zealand serogroup O26 bacterial isolates (n=152), in comparison to publicly available genome sequence data (n=252) from other countries (n=14), suggested introduction of STEC and non-STEC O26 into New Zealand during few periods in the 20th and early 21st century. Populations of New Zealand serogroup O26 E. coli are monophyletic, possibly due to minimal live cattle importations into the country. Further research in this area should focus on effective interventions at the farm and meat processing level to decrease the risk of veal beef contamination, while protecting public health