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Abstract Background Shiga toxin-producing Escherichia coli (STEC) infections are a significant public health concern as they can cause serious illness and outbreaks. In England, STEC incidence is highest among children and guidance recommends that children under six diagnosed with STEC are excluded from childcare until two consecutive stool cultures are negative. We aimed to describe the barriers and facilitators to implementing exclusion and the impact of exclusion policies on young children and their families. Methods Individual level data was obtained from a wider study focusing on shedding duration among STEC cases aged

Abstract Shigellosis is an enteric infection that transmits through the faecal-oral route, which can occur during sex between men who have sex with men (MSM). Between 2009 and 2014, an epidemic of sexually transmissible Shigella flexneri 3a occurred in England that subsequently declined. However, from 2019 to 2021, despite SARS-CoV-2 restrictions, S. flexneri 3a continued to re-emerge. We explored possible drivers of re-emergence by comparing host demography and pathogen genomics. Cases were primarily among 35–64 year old men in London. Genomic analyses of 502 bacterial isolates showed that the majority (58%) of re-emerging MSM strains were a clonal replacement of the original, with reduced antimicrobial resistance, conservation of plasmid col156_1, and two SNPs with 19 predicted effects. The absence of major changes in the pathogen or host demographics suggest that other factors may have driven the re-emergence of S. flexneri 3a and highlight the need for further work in the area.

Abstract The aim of this study was to compare Illumina and Oxford Nanopore Technology (ONT) sequencing data to quantify genetic variation to assess within-outbreak strain relatedness and characterise microevolutionary events in the accessory genomes of a cluster of 23 genetically and epidemiologically linked isolates related to an outbreak of Shiga toxin-producing Escherichia coli O157:H7 caused by the consumption of raw drinking milk. There were seven discrepant variants called between the two technologies, five were false-negative or false-positive variants in the Illumina data and two were false-negative calls in ONT data. After masking horizontally acquired sequences such as prophages, analysis of both short and long-read sequences revealed the 20 isolates linked to the outbreak in 2017 had a maximum SNP distance of one SNP between each other, and a maximum of five SNPs when including three additional strains identified in 2019. Analysis of the ONT data revealed a 47 kbp deletion event in a terminal compound prophage within one sample relative to the remaining samples, and a 0.65 Mbp large chromosomal rearrangement (inversion), within one sample relative to the remaining samples. Furthermore, we detected two bacteriophages encoding the highly pathogenic Shiga toxin (Stx) subtype, Stx2a. One was typical of Stx2a-phage in this sub-lineage (Ic), the other was atypical and inserted into a site usually occupied by Stx2c-encoding phage. Finally, we observed an increase in the size of the pO157 IncFIB plasmid (1.6 kbp) in isolates from 2019 compared to those from 2017, due to the duplication of insertion elements within the plasmids from the more recently isolated strains. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the genome plasticity and virulence between strains of this zoonotic, foodborne pathogen.

Abstract Shigella sonnei causes shigellosis, a severe gastrointestinal illness that is sexually transmissible among men who have sex with men (MSM). Multidrug resistance in S. sonnei is common including against World Health Organisation recommended treatment options, azithromycin, and ciprofloxacin. Recently, an MSM-associated outbreak of extended-spectrum β-lactamase producing, extensively drug resistant S. sonnei was reported in the United Kingdom. Here, we aimed to identify the genetic basis, evolutionary history, and international dissemination of the outbreak strain. Our genomic epidemiological analyses of 3,304 isolates from the United Kingdom, Australia, Belgium, France, and the United States of America revealed an internationally connected outbreak with a most recent common ancestor in 2018 carrying a low-fitness cost resistance plasmid, previously observed in travel associated sublineages of S. flexneri. Our results highlight the persistent threat of horizontally transmitted antimicrobial resistance and the value of continuing to work towards early and open international sharing of genomic surveillance data.

Escherichia albertii is an emerging gastrointestinal pathogen that causes disease in humans and animals, notably birds. In this genomic epidemiology study, the authors investigate characteristics of isolates sampled from humans and birds in Great Britain and find that they tend to cluster separately.

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal ‘sentinel’ surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions: The consortium’s aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

ABSTRACT Enteroinvasive Escherichia coli (EIEC) and Shigella are closely related agents of bacillary dysentery. It is widely viewed that EIEC and Shigella species evolved from E. coli via independent acquisitions of a large virulence plasmid (pINV) encoding a type 3 secretion system (T3SS). Sequence Type (ST)99 O96:H19 E. coli is a novel clone of EIEC responsible for recent outbreaks in Europe and South America. Here, we use 92 whole genome sequences to reconstruct a dated phylogeny of ST99 E. coli, revealing distinct phylogenomic clusters of pINV-positive and -negative isolates. To study the impact of pINV acquisition on the virulence of this clone, we developed an EIEC-zebrafish infection model showing that virulence of ST99 EIEC is thermoregulated. Strikingly, zebrafish infection using a T3SS-deficient ST99 EIEC strain and the oldest available pINV-negative isolate reveals a separate, temperature-independent mechanism of virulence, indicating that ST99 non-EIEC strains were virulent before pINV acquisition. Taken together, these results suggest that an already pathogenic E. coli acquired pINV and that virulence of ST99 isolates became thermoregulated once pINV was acquired. IMPORTANCE Enteroinvasive Escherichia coli (EIEC) and Shigella are etiological agents of bacillary dysentery. Sequence Type (ST)99 is a clone of EIEC hypothesized to cause human disease by the recent acquisition of pINV, a large plasmid encoding a type 3 secretion system (T3SS) that confers the ability to invade human cells. Using Bayesian analysis and zebrafish larvae infection, we show that the virulence of ST99 EIEC isolates is highly dependent on temperature, while T3SS-deficient isolates encode a separate temperature-independent mechanism of virulence. These results indicate that ST99 non-EIEC isolates may have been virulent before pINV acquisition and highlight an important role of pINV acquisition in the dispersal of ST99 EIEC in humans, allowing wider dissemination across Europe and South America.

ABSTRACT The gut microbiota constitutes an ideal environment for the selection, exchange, and carriage of antibiotic resistance determinants (ARDs), and international travel has been identified as a risk factor for acquisition of resistant organisms. Here, we present a longitudinal metagenomic analysis of the gut resistome in travellers to “high-risk” countries (Gutback). Fifty volunteers, recruited at a travel clinic in London, United Kingdom, provided stool samples before (pre-travel), immediately after (post-travel), and 6 months after their return (follow-up) from a high-risk destination. Fecal DNA was extracted, metagenomic sequencing performed and the resistome profiled. An increase in abundance and diversity of resistome was observed after travel. Significant increases in abundance were seen in antimicrobial genes conferring resistance to macrolides, third-generation cephalosporins, aminoglycosides, and sulfonamides. There was a significant association with increased resistome abundance if the participant experienced diarrhea during travel or took antibiotics, but these two variables were co-correlated. The resistome abundance returned to pre-travel levels by the 6-month sample point but there was evidence of persistence of several ARDs. The post-travel samples had an increase in abundance Escherichia coli which was positively associated with many acquired resistant determinants. Virulence and phylogenetic profiling revealed pathogenic E. coli significantly contributed to this increase abundance. In summary, in this study, foreign travel remains a significant risk factor for acquisition of microbes conferring resistance to multiple classes of antibiotics, often associated with symptomatic exposure to diarrhoeagenic E. coli. IMPORTANCE A future where antimicrobial therapy is severely compromised by the increase in resistant organisms is of grave concern. Given the variability in prevalence and diversity of antimicrobial resistance determinants in different geographical settings, international travel is a known risk factor for acquisition of resistant organisms into the gut microbiota. In this study, we show the utility of metagenomic approaches to quantify the levels of acquisition and carriage of resistance determinants after travel to a “high-risk” setting. Significant modulation to the resistome was seen after travel that is largely resolved within 6 months, although evidence of persistence of several ARDs was observed. Risk factors for acquisition included experiencing a diarrheal episode and the use of antibiotics. Colonization by pathogenic Escherichia coli was correlated with an increase in acquisition of antimicrobial resistance determinants, and as such established public health guidance to travelers on food and water safety remain an important message to reduce the spread of antibiotic resistance.

Globalization of the food supply chain has created conditions favorable for emergence and spread of multidrug-resistant (MDR) foodborne pathogens. In November 2021, the UK Health Security Agency detected an outbreak of 17 cases infected with the same strain of MDR extended spectrum beta-lactamase (ESBL)-producing Shigella sonnei. Phylogenetic analysis of whole-genome sequencing data revealed the outbreak was closely related to strains of S. sonnei isolated from travelers returning to the UK from Egypt. None of the outbreak cases reported travel and all 17 cases reported eating food from a restaurant/food outlet in the week prior to symptom onset, of which 11/17 (64.7%) ate at branches of the same national restaurant franchise. All 17 cases were adults and 14/17 (82.4%) were female. Ingredient-level analyses of the meals consumed by the cases identified spring onions as the common ingredient. Food chain investigations revealed that the spring onions served at the implicated restaurants could be traced back to a single Egyptian producer. The foodborne transmission of ESBL-producing bacteria is an emerging global health concern, and concerted action from all stakeholders is required to ensure an effective response to mitigate the risks to public health.

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) can give rise to a range of clinical outcomes from diarrhea to the life-threatening systemic condition hemolytic-uremic syndrome (HUS). Although STEC O157:H7 is the serotype most frequently associated with HUS, a major outbreak of HUS occurred in 2011 in Germany and was caused by a rare serotype, STEC O104:H4. Prior to 2011 and since the outbreak, STEC O104:H4 strains have only rarely been associated with human infections. From 2012 to 2020, intensified STEC surveillance was performed in Germany where the subtyping of ~8,000 clinical isolates by molecular methods, including whole-genome sequencing, was carried out. A rare STEC serotype, O181:H4, associated with HUS was identified, and like the STEC O104:H4 outbreak strain, this strain belongs to sequence type 678 (ST678). Genomic and virulence comparisons revealed that the two strains are phylogenetically related and differ principally in the gene cluster encoding their respective lipopolysaccharide O-antigens but exhibit similar virulence phenotypes. In addition, five other serotypes belonging to ST678 from human clinical infection, such as OX13:H4, O127:H4, OgN-RKI9:H4, O131:H4, and O69:H4, were identified from diverse locations worldwide. IMPORTANCE Our data suggest that the high-virulence ensemble of the STEC O104:H4 outbreak strain remains a global threat because genomically similar strains cause disease worldwide but that the horizontal acquisition of O-antigen gene clusters has diversified the O-antigens of strains belonging to ST678. Thus, the identification of these highly pathogenic strains is masked by diverse and rare O-antigens, thereby confounding the interpretation of their potential risk.

Salmonella enterica serovar Enteritidis is one of the most frequent causes of Salmonellosis globally and is commonly transmitted from animals to humans by the consumption of contaminated foodstuffs. In the UK and many other countries in the Global North, a significant proportion of cases are caused by the consumption of imported food products or contracted during foreign travel, therefore, making the rapid identification of the geographical source of new infections a requirement for robust public health outbreak investigations. Herein, we detail the development and application of a hierarchical machine learning model to rapidly identify and trace the geographical source of S. Enteritidis infections from whole genome sequencing data. 2313 S. Enteritidis genomes, collected by the UKHSA between 2014–2019, were used to train a ‘local classifier per node’ hierarchical classifier to attribute isolates to four continents, 11 sub-regions, and 38 countries (53 classes). The highest classification accuracy was achieved at the continental level followed by the sub-regional and country levels (macro F1: 0.954, 0.718, 0.661, respectively). A number of countries commonly visited by UK travelers were predicted with high accuracy (hF1: >0.9). Longitudinal analysis and validation with publicly accessible international samples indicated that predictions were robust to prospective external datasets. The hierarchical machine learning framework provided granular geographical source prediction directly from sequencing reads in

The aim of this study was to describe two foodborne outbreaks caused by contaminated imported melon and make recommendations for future practice. Between March and July 2021, there was an outbreak of 113 cases of Salmonella Braenderup in the UK (62% female, median age 61 years, 33% hospitalized). Analytical epidemiological studies identified Galia melons as the vehicle of infection (OR 671.9, 95% CI 39.0–58,074.0, p

Whole genome sequencing is increasingly being adopted for Shigella sonnei outbreak investigation and surveillance, but there is no global classification standard. Here, the authors develop and validate a genomic framework implemented using open-source software, and demonstrate its application using surveillance data.

Increasing levels of antimicrobial resistance (AMR) have been documented in Escherichia coli causing travellers’ diarrhoea, particularly to the third-generation cephalosporins. Diarrhoeagenic E. coli (DEC) can act as a reservoir for the exchange of AMR genes between bacteria residing in the human gut, enabling them to survive and flourish through the selective pressures of antibiotic treatments. Using Oxford Nanopore Technology (ONT), we sequenced eight isolates of DEC from four patients’ specimens who had all recently returned to the United Kingdome from Pakistan. Sequencing yielded two DEC harbouring blaCTX-M-15 per patient, all with different sequence types (ST) and belonging to five different pathotypes. The study aimed to determine whether blaCTX-M-15 was located on the chromosome or plasmid and to characterise the drug-resistant regions to better understand the mechanisms of onward transmission of AMR determinants. Patients A and C both had one isolate where blaCTX-M-15 was located on the plasmid (899037 & 623213, respectively) and one chromosomally encoded (899091 & 623214, respectively). In patient B, blaCTX-M-15 was plasmid-encoded in both DEC isolates (786605 & 7883090), whereas in patient D, blaCTX-M-15 was located on the chromosome in both DEC isolates (542093 & 542099). The two blaCTX-M-15-encoding plasmids associated with patient B were different although the blaCTX-M-15-encoding plasmid isolated from 788309 (IncFIB) exhibited high nucleotide similarity to the blaCTX-M-15-encoding plasmid isolated from 899037 (patient A). In the four isolates where blaCTX-M-15 was chromosomally encoded, two isolates (899091 & 542099) shared the same insertion site. The blaCTX-M-15 insertion site in isolate 623214 was described previously, whereas that of isolate 542093 was unique to this study. Analysis of Nanopore sequencing data enables us to characterise the genomic architecture of mobile genetic elements encoding AMR determinants. These data may contribute to a better understanding of persistence and onward transmission of AMR determinants in multidrug-resistant (MDR) E. coli causing gastrointestinal and extra-intestinal infections.

Signs and symptoms of Shiga toxin–producing Escherichia coli (STEC) serogroup O157:H7 infection range from mild gastrointestinal to bloody diarrhea and hemolytic uremic syndrome (HUS). We assessed the association between Shiga toxin gene (stx) subtype and disease severity for »3,000 patients with STEC O157:H7 in England during 2009–2019. Odds of bloody diarrhea, HUS, or both, were significantly higher for patients infected with STEC O157:H7 possessing stx2a only or stx2a combined with other stx subtypes. Odds of severe signs/symptoms were significantly higher for isolates encoding stx2a only and belonging to sublineage Ic and lineage I/II than for those encoding stx2a only and belonging to sublineage IIb, indicating that stx2a is not the only driver causing HUS. Strains of STEC O157:H7 that had stx1a were also significantly more associated with severe disease than strains with stx2c only. This finding confounds public health risk assessment algorithms based on detection of stx2 as a predictor of severe disease.

Abstract Background Diarrhoeagenic Escherichia coli (DEC) infections are common in children in low-middle income countries (LMICs). However, detecting the various DEC pathotypes is complex as they cannot be differentiated by classical microbiology. We developed four multiplex real-time PCR assays were to detect virulence markers of six DEC pathotypes; specificity was tested using DEC controls and other enteric pathogens. PCR amplicons from the six E. coli pathotypes were purified and amplified to be used to optimize PCR reactions and to calculate reproducibility. After validation, these assays were applied to clinical samples from healthy and diarrhoeal Vietnamese children and associated with clinical data. Results The multiplex real-time PCRs were found to be reproducible, and specific. At least one DEC variant was detected in 34.7% (978/2815) of the faecal samples from diarrhoeal children; EAEC, EIEC and atypical EPEC were most frequent Notably, 41.2% (205/498) of samples from non-diarrhoeal children was positive with a DEC pathotype. In this population, only EIEC, which was detected in 34.3% (99/289) of diarrhoeal samples vs. 0.8% (4/498) non-diarrhoeal samples (p

We aim to provide an evidence-based evaluation of whole genome sequence (WGS) methods, employed at the Salmonella reference laboratory in England, in terms of its impact on public health and whether these methods remain a fit for purpose test under UKAS ISO 15189. The evaluation of the genomic methods were mapped against the value of detecting microbiological clusters to support the investigation of food-borne outbreaks of Salmonella in England between 2012–2020. The analysis of WGS with both SNP- and allelic-based methods provided an unprecedented level of strain discrimination and detection of additional clusters when comparing to all of the previous typing methods. The robustness of the routine genomic sequencing at the reference laboratory ensured confidence in the microbiological identifications, even in large outbreaks with complex international food distribution networks. There was evidence that the phylogeny derived from the WGS data can be used to inform the provenance of strains and support discrimination between domestic and non-domestic transmission events. Further insight on the evolutionary context of the emerging pathogenic strains was enabled with a deep dive of the phylogenetic data, including the detection of nested clusters. The public availability of the WGS data linked to the clinical, epidemiological and environmental context of the sequenced strains has improved the trace-back investigations during outbreaks. The global expansion in the use of WGS-based typing in reference laboratories has shown that the WGS methods are a fit for purpose test in public health as it has ensured the rapid implementation of interventions to protect public health, informed risk assessment and has facilitated the management of national and international food-borne outbreaks of Salmonella.

ABSTRACT The public health value of whole genome sequencing (WGS) for Shigella spp. in England has been limited by a lack of information on sexual identity and behavior. We combined WGS data with other data sources to better understand Shigella flexneri transmission in men who have sex with men (MSM). WGS data for all S. flexneri isolates referred to the national reference laboratory were linked to i) clinical and behavioral data collected in seven of 21 health regions in England using a standardized exposure questionnaire and, ii) national HIV surveillance data. We included 926 S. flexneri isolates, of which 43.0% (n = 398) fell phylogenetically within two domestically circulating clades associated with genotypic markers of azithromycin resistance. Approximately one third of isolates in these clades were from people living with HIV, primarily acquired through sex between men. 182 (19.7%) isolates had linked questionnaire data; 88% (84/95) of MSM isolates fell phylogenetically within the domestically circulating clades, while 92% (72/78) of isolates from other cases fell within lineages linked with travel to high-risk regions. There was no evidence of sustained transmission between networks of MSM and the wider community. MSM were more likely to be admitted to hospital and receive antimicrobials. Our study emphasizes the importance of sex between men as a major route of transmission for S. flexneri. Combined WGS, epidemiological and clinical data provide unique insights that can inform contact tracing, clinical management and the delivery of targeted prevention activities. Future studies should investigate why MSM experience more severe clinical outcomes. IMPORTANCE Within the last 2 decades there have been an increasing number of Shigella spp. outbreaks among men who have sex with men (MSM) worldwide. In 2015, Public Health England (PHE) introduced routine whole genome sequencing (WGS) for the national surveillance of Shigella spp. However, the lack of information on sexual identity and behavior has hindered interpretation. Our study illustrates the power of linking WGS data with epidemiological, behavioral, and clinical data. We provide unique population-level insights into different transmission networks that can inform the delivery of appropriate public health interventions and patient management. Furthermore, we describe and compare clinical characteristics and outcomes of S. flexneri infection in MSM and other exposure groups. We found that MSM were more likely to be admitted to hospital and receive antimicrobials, indicating that their infections were potentially more severe. The exact reasons for this are unclear and require further exploration.

Background: In August 2020, an outbreak of Shiga toxin-producing Escherichia coli (STEC) O157:H7 occurred in the United Kingdom. Whole genome sequencing revealed that these cases formed a genetically distinct cluster. Methods: Hypotheses generated from case interviews were tested in analytical studies, and results informed environmental sampling and food chain analysis. A case–case study used non-outbreak ‘comparison’ STEC cases; a case–control study used a market research panel to recruit controls. Results: A total of 36 cases were identified; all cases reported symptom onset between August 3 and August 16, 2020. The majority of cases (83%) resided in the Midlands region of England and in Wales. A high proportion of cases reported eating out, with one fast-food restaurant chain mentioned by 64% (n = 23) of cases. Both the case–case study (adjusted odds ratio (aOR) 31.8, 95% confidence interval (CI) 1.6–624.9) and the case–control study (aOR 9.19, 95% CI 1.0–82.8) revealed statistically significant results, showing that the consumption of a specific fast-food product was independently associated with infection. Conclusions: Consumption of a specific fast-food product was a likely cause of this outbreak. The only ingredient specific to the product was cucumbers. The supply of cucumbers was immediately halted, and no further cases have been identified.

In this study, multidrug-resistant (MDR) Escherichia coli isolates from retail food and humans assigned into similar Multilocus Sequence Types (MLST) were analyzed using whole genome sequencing (WGS). In silico analysis of assembled sequences revealed the existence of multiple resistance genes among the examined E. coli isolates. Of the six CTX-M-producing isolates from retail food, blaCTX-M-14 was the prevalent variant identified (83.3%, 5/6). Two plasmid-mediated fosfomycin resistance genes, fosA3, and fosA4, were detected from retail food isolates (one each from chicken and beef), where fosA4 was identified in the chicken isolate 82CH that also carried the colistin resistance gene mcr-1. The blaCTX-M-14 and fosA genes in retail food isolates were located adjacent to insertion sequences ISEcp1 and IS26, respectively. Sequence analysis of the reconstructed mcr-1 plasmid (p82CH) showed 96–97% identity to mcr-1-carrying IncI2 plasmids previously identified in human and food E. coli isolates from Egypt. Hierarchical clustering of core genome MLST (HierCC) revealed clustering of chicken isolate 82CH, co-harboring mcr-1 and fosA4 genes, with a chicken E. coli isolate from China at the HC200 level (≤200 core genome allelic differences). As E. coli co-harboring mcr-1 and fosA4 genes has only been recently reported, this study shows rapid spread of this genotype that shares similar genetic structures with regional and international E. coli lineages originating from both humans and food animals. Adopting WGS-based surveillance system is warranted to facilitate monitoring the international spread of MDR pathogens.

Shigella sonnei is one of the main species causing shigellosis worldwide. Here the authors analyse nearly 400 S. sonnei genome sequences and carry out experimental evolution experiments to shed light into the evolutionary processes underlying the recent emergence of fluoroquinolone resistance in this pathogen.

We used whole-genome sequencing to investigate the evolutionary context of an emerging highly pathogenic strain of Shiga toxin–producing Escherichia coli (STEC) O157:H7 in England and Wales. A timed phylogeny of sublineage IIb revealed that the emerging clone evolved from a STEC O157:H7 stx-negative ancestor ≈10 years ago after acquisition of a bacteriophage encoding Shiga toxin (stx) 2a, which in turn had evolved from a stx2c progenitor ≈20 years ago. Infection with the stx2a clone was a significant risk factor for bloody diarrhea (OR 4.61, 95% CI 2.24–9.48; p

Shigella

In December 2015, six cases of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 stx2a/stx2c phage type (PT) 24 were identified by the national gastrointestinal disease surveillance system at Public Health England (PHE). Frozen grated coconut imported from India was implicated as the vehicle of infection. Short and long read sequencing data were interrogated for genomic markers to provide evidence that the outbreak strain was from an imported source. The outbreak strain belonged to a sub-lineage (IIa) rare in domestically acquired infection in the United Kingdom, and indicative of an imported strain. Phylogenetic analysis identified the most closely related isolates to the outbreak strain were from cases reporting recent travel not to India, but to Uganda. Phylo-geographical signals based on travel data may be confounded by the failure of local and/or global monitoring systems to capture the full diversity of strains in a given country. This may be due to low prevalence strains circulating in-country under the surveillance radar, or a recent importation event involving the migration of animals and/or people. Comparison of stx2a-encoding prophage harbored by the outbreak strain with publicly available stx2a-encoding prophage sequences revealed that it was most closely related to stx2a-encoding prophage acquired by STEC O157:H7 that caused the first outbreak of STEC-hemolytic uremic syndrome (HUS) in England in 1982–83. Animal and people migration events may facilitate the transfer of stx2a-encoding prophage from indigenous STEC O157:H7 to recently imported strains, or vice versa. Monitoring the global transmission of STEC O157:H7 and tracking the exchange of stx2a-encoding phage between imported and indigenous strains may provide an early warning of emerging threats to public health.

We compared genomes from multiple isolations of Shiga toxin-producing Escherichia coli (STEC) O157:H7 from the same patient, in cases notified to Public Health England (PHE) between 2015 and 2019. There were 261 cases where multiple isolates were sequenced from the same patient comprising 589 isolates. Serial isolates from the same patient fell within five single nucleotide polymorphisms (SNPs) of each other for 260/261 (99.6%) of the cases, indicating that there was little evidence of within host variation. The investigation into the 13 SNP discrepancy between one isolate pair revealed the cause to be a recombination event within a stx2a-encoding prophage resulting in the insertion/deletion of a fragment of the genome. This 50 kbp prophage fragment was homologous to a prophage in the reference genome, and the short reads from the isolate that had the 50 kbp fragment, mapped unambiguously to this region. The discrepant variants in the isolate without the 50 kbp fragment were attributed to ambiguous mapping of the short reads from other prophage regions to the 50 kbp fragment in the reference genome. Identification of such recombination events in this dataset appeared to be rare, most likely because the majority of prophage regions in the Sakai reference genome are masked during the analysis. Identification of SNPs under neutral selection, and masking recombination events, is a requirement for phylogenetic analysis used for public health surveillance, and for the detection of point source outbreaks. However, assaying the accessory genome by combining the use of short and long read technologies for public health surveillance may provide insight into how recombination events impact on the evolutionary course of STEC O157:H7.

Enteroinvasive Escherichia coli (EIEC) cause intestinal illness through the same pathogenic mechanism used by Shigella spp. The latter species can be typed through genomic and phenotypic methods used for E. coli and have been proposed for reclassification within E. coli species. Recently the first appearance of a highly pathogenic EIEC O96:H19 was described in Europe as the causative agent of two large outbreaks that occurred in Italy and in the United Kingdom. In contrast to Shigella spp and to the majority of EIEC strains, EIEC O96:H19 fermented lactose, lacked pathoadaptive mutations, and showed good fitness in extracellular environment, similarly to non-pathogenic E. coli, suggesting they have emerged following acquisition of the invasion plasmid by a non-pathogenic E. coli. Here we describe the whole genome comparison of two EIEC O96:H19 strains isolated from severe cases of diarrhea in Uruguay in 2014 with the sequences of EIEC O96:H19 available in the public domain. The phylogenetic comparison grouped all the O96:H19 strains in a single cluster, while reference EIEC strains branched into different clades with Shigella strains occupying apical positions. The comparison of the virulence plasmids showed the presence of a complete conjugation region in at least one O96:H19 EIEC. Reverse Transcriptase Real Time PCR experiments confirmed in this strain the expression of the pilin-encoding gene and conjugation experiments suggested its ability to mobilize an accessory plasmid in a recipient strain. Noteworthy, the tra region was comprised between two reversely oriented IS600 elements, which were also found as remnants in another EIEC O96:H19 plasmid lacking the tra locus. We hypothesize that an IS-mediated recombination mechanism may have caused the loss of the conjugation region commonly observed in EIEC and Shigella virulence plasmids. The results of this study support the hypothesis of EIEC originating from non-pathogenic E. coli through the acquisition of the virulence plasmid via conjugation. Remarkably, this study showed the ability of a circulating EIEC strain to mobilize plasmids through conjugation, suggesting a mechanism for the emergence of novel EIEC clones.

Abstract The provisional molecular approach, proposed by EFSA in 2013, for the pathogenicity assessment of Shiga toxin‐producing Escherichia coli (STEC) has been reviewed. Analysis of the confirmed reported human STEC infections in the EU/EEA (2012–2017) demonstrated that isolates positive for any of the reported Shiga toxin (Stx) subtypes (and encoding stx gene subtypes) may be associated with severe illness (defined as bloody diarrhoea (BD), haemolytic uraemic syndrome (HUS) and/or hospitalisation). Although strains positive for stx2a gene showed the highest rates, strains with all other stx subtypes, or combinations thereof, were also associated with at least one human case with a severe clinical outcome. Serogroup cannot be used as a predictor of clinical outcome and the presence of the intimin gene (eae) is not essential for severe illness. These findings are supported by the published literature, a review of which suggested there was no single or combination of virulence markers associated exclusively with severe illness. Based on available evidence, it was concluded that all STEC strains are pathogenic in humans, capable of causing at least diarrhoea and that all STEC subtypes may be associated with severe illness. Source attribution analysis, based on ‘strong evidence’ outbreak data in the EU/EEA (2012–2017), suggests that ‘bovine meat and products thereof’, ‘milk and dairy products’, ‘tap water including well water’ and ‘vegetables, fruit and products thereof’ are the main sources of STEC infections in the EU/EEA, but a ranking between these categories cannot be made as the data are insufficient. Other food commodities are also potentially associated with STEC infections but rank lower. Data gaps are identified, and are primarily caused by the lack of harmonisation in sampling strategies, sampling methods, detection and characterisation methods, data collation and reporting within the EU.

Abstract This Opinion considers the application of whole genome sequencing (WGS) and metagenomics for outbreak investigation, source attribution and risk assessment of food‐borne pathogens. WGS offers the highest level of bacterial strain discrimination for food‐borne outbreak investigation and source‐attribution as well as potential for more precise hazard identification, thereby facilitating more targeted risk assessment and risk management. WGS improves linking of sporadic cases associated with different food products and geographical regions to a point source outbreak and can facilitate epidemiological investigations, allowing also the use of previously sequenced genomes. Source attribution may be favoured by improved identification of transmission pathways, through the integration of spatial‐temporal factors and the detection of multidirectional transmission and pathogen–host interactions. Metagenomics has potential, especially in relation to the detection and characterisation of non‐culturable, difficult‐to‐culture or slow‐growing microorganisms, for tracking of hazard‐related genetic determinants and the dynamic evaluation of the composition and functionality of complex microbial communities. A SWOT analysis is provided on the use of WGS and metagenomics for Salmonella and Shigatoxin‐producing Escherichia coli (STEC) serotyping and the identification of antimicrobial resistance determinants in bacteria. Close agreement between phenotypic and WGS‐based genotyping data has been observed. WGS provides additional information on the nature and localisation of antimicrobial resistance determinants and on their dissemination potential by horizontal gene transfer, as well as on genes relating to virulence and biological fitness. Interoperable data will play a major role in the future use of WGS and metagenomic data. Capacity building based on harmonised, quality controlled operational systems within European laboratories and worldwide is essential for the investigation of cross‐border outbreaks and for the development of international standardised risk assessments of food‐borne microorganisms.

Abstract Shigella are globally important diarrhoeal pathogens that are endemic in low-to-middle income nations and also occur in high income nations, typically in travellers or community-based risk-groups. Shigella phylogenetics reveals population structures that are more reliable than those built with traditional typing methods, and has identified sublineages associated with specific geographical regions or patient groups. Genomic analyses reveal temporal increases in Shigella antimicrobial resistance (AMR) gene content, which is frequently encoded on mobile genetic elements. Here, we whole genome sequenced representative subsamples of S. flexneri 2a and S. sonnei (n = 366) from the United Kingdom from 2008 to 2014, and analysed these alongside publicly available data to make qualitative insights on the genomic epidemiology of shigellosis and its AMR within the broader global context. Combined phylogenetic, epidemiological and genomic anlayses revealed the presence of domestically-circulating sublineages in patient risk-groups and the importation of travel-related sublineages from both Africa and Asia, including ciprofloxacin-resistant sublineages of both species from Asia. Genomic analyses revealed common AMR determinants among travel-related and domestically-acquired isolates, and the evolution of mutations associated with reduced quinolone susceptibility in domestically-circulating sublineages. Collectively, this study provides unprecedented insights on the contribution and mobility of endemic and travel-imported sublineages and AMR determinants responsible for disease in a high-income nation.

Antimicrobial resistance (AMR) in Shigella species is a public health concern. Here, Baker et al. show how horizontal transfer of an AMR plasmid among Shigella species contributes to epidemics in men who have sex with men in England.

In July 2014, an outbreak of Shiga toxin–producing Escherichia coli (STEC) O55:H7 in England involved 31 patients, 13 (42%) of whom had hemolytic uremic syndrome. Isolates were sequenced, and the sequences were compared with publicly available sequences of E. coli O55:H7 and O157:H7. A core-genome phylogeny of the evolutionary history of the STEC O55:H7 outbreak strain revealed that the most parsimonious model was a progenitor enteropathogenic O55:H7 sorbitol-fermenting strain, lysogenized by a Shiga toxin (Stx) 2a–encoding phage, followed by loss of the ability to ferment sorbitol because of a non-sense mutation in srlA. The parallel, convergent evolutionary histories of STEC O157:H7 and STEC O55:H7 may indicate a common driver in the evolutionary process. Because emergence of STEC O157:H7 as a clinically significant pathogen was associated with acquisition of the Stx2a-encoding phage, the emergence of STEC O55:H7 harboring the stx2a gene is of public health concern.

Abstract Background The ST313 sequence type of Salmonella Typhimurium causes invasive non-typhoidal salmonellosis and was thought to be confined to sub-Saharan Africa. Two distinct phylogenetic lineages of African ST313 have been identified. Methods We analysed the whole genome sequences of S. Typhimurium isolates from UK patients that were generated following the introduction of routine whole-genome sequencing (WGS) of Salmonella enterica by Public Health England in 2014. Results We found that 2.7% (84/3147) of S. Typhimurium from patients in England and Wales were ST313 and were associated with gastrointestinal infection. Phylogenetic analysis revealed novel diversity of ST313 that distinguished UK-linked gastrointestinal isolates from African-associated extra-intestinal isolates. The majority of genome degradation of African ST313 lineage 2 was conserved in the UK-ST313, but the African lineages carried a characteristic prophage and antibiotic resistance gene repertoire. These findings suggest that a strong selection pressure exists for certain horizontally acquired genetic elements in the African setting. One UK-isolated lineage 2 strain that probably originated in Kenya carried a chromosomally located bla CTX-M-15, demonstrating the continual evolution of this sequence type in Africa in response to widespread antibiotic usage. Conclusions The discovery of ST313 isolates responsible for gastroenteritis in the UK reveals new diversity in this important sequence type. This study highlights the power of routine WGS by public health agencies to make epidemiologically significant deductions that would be missed by conventional microbiological methods. We speculate that the niche specialisation of sub-Saharan African ST313 lineages is driven in part by the acquisition of accessory genome elements.

Bacterial–host interactions are non-linear and actually threefold, involving significant selection through predatory lytic bacteriophages in the host environment. In studies of human and animal gut microbiome bacteria, it is important to consider phage in all host–pathogen interactions. We use an important zoonotic pathogen, Shiga toxigenic Escherichia coli (STEC) O157:H7, to investigate this. Our study provides evidence that phage resistance profiles are well maintained at the sub-lineage level with variation in profiles within sub-lineages uncommon. This indicates that phage resistance heterogeneity happened early on in the STEC O157:H7 natural history and that occasional “wobbles” do not often outcompete the stable lineage unless combined with a competitive advantage. We discuss an example of this in the acquisition of stx2a that, while an important virulence factor, also conveys increased phage cross-resistance. We also discuss the role of phage resistance in co-occurrence of the three stable lineages worldwide and whether differing phage resistance is maintaining diversity.

In England in 2015, Shigella sonnei isolates from men who have sex with men produced extended-spectrum β-lactamases and exhibited macrolide resistance. Whole-genome sequencing showed a close relationship among the isolates, which harbored a plasmid that was previously identified in a shigellosis outbreak among this population but has acquired a mobile element.

Shigellae are sensitive indicator species for studying trends in the international transmission of antimicrobial-resistant Enterobacteriaceae. Orthodox Jewish communities (OJCs) are a known risk group for shigellosis; Shigella sonnei is cyclically epidemic in OJCs in Israel, and sporadic outbreaks occur in OJCs elsewhere. We generated whole-genome sequences for 437 isolates of S. sonnei from OJCs and non-OJCs collected over 22 years in Europe (the United Kingdom, France, and Belgium), the United States, Canada, and Israel and analyzed these within a known global genomic context. Through phylogenetic and genomic analysis, we showed that strains from outbreaks in OJCs outside of Israel are distinct from strains in the general population and relate to a single multidrug-resistant sublineage of S. sonnei that prevails in Israel. Further Bayesian phylogenetic analysis showed that this strain emerged approximately 30 years ago, demonstrating the speed at which antimicrobial drug–resistant pathogens can spread widely through geographically dispersed, but internationally connected, communities.

We evaluated clinical Shiga toxin–producing Escherichia coli O157 infections in England and Wales during 1983–2012 to describe changes in microbiological and surveillance methods. A strain replacement event was captured; phage type (PT) 2 decreased to account for just 3% of cases by 2012, whereas PT8 and PT21/28 strains concurrently emerged, constituting almost two thirds of cases by 2012. Despite interventions to control and reduce transmission, incidence remained constant. However, sources of infection changed over time; outbreaks caused by contaminated meat and milk declined, suggesting that interventions aimed at reducing meat cross-contamination were effective. Petting farm and school and nursery outbreaks increased, suggesting the emergence of other modes of transmission and potentially contributing to the sustained incidence over time. Studies assessing interventions and consideration of policies and guidance should be undertaken to reduce Shiga toxin–producing E. coli O157 infections in England and Wales in line with the latest epidemiologic findings.

ABSTRACT Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene (sspA) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens. IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication.

Surveillance of antimicrobial resistance (AMR) in non-typhoidal Salmonella enterica (NTS), is essential for monitoring transmission of resistance from the food chain to humans, and for establishing effective treatment protocols. We evaluated the prediction of phenotypic resistance in NTS from genotypic profiles derived from whole genome sequencing (WGS). Genes and chromosomal mutations responsible for phenotypic resistance were sought in WGS data from 3,491 NTS isolates received by Public Health England’s Gastrointestinal Bacteria Reference Unit between April 2014 and March 2015. Inferred genotypic AMR profiles were compared with phenotypic susceptibilities determined for fifteen antimicrobials using EUCAST guidelines. Discrepancies between phenotypic and genotypic profiles for one or more antimicrobials were detected for 76 isolates (2.18%) although only 88/52,365 (0.17%) isolate/antimicrobial combinations were discordant. Of the discrepant results, the largest number were associated with streptomycin (67.05%, n = 59). Pan-susceptibility was observed in 2,190 isolates (62.73%). Overall, resistance to tetracyclines was most common (26.27% of isolates, n = 917) followed by sulphonamides (23.72%, n = 828) and ampicillin (21.43%, n = 748). Multidrug resistance (MDR), i.e., resistance to three or more antimicrobial classes, was detected in 848 isolates (24.29%) with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines being the most common MDR profile (n = 231; 27.24%). For isolates with this profile, all but one were S. Typhimurium and 94.81% (n = 219) had the resistance determinants blaTEM-1,strA-strB, sul2 and tet(A). Extended-spectrum β-lactamase genes were identified in 41 isolates (1.17%) and multiple mutations in chromosomal genes associated with ciprofloxacin resistance in 82 isolates (2.35%). This study showed that WGS is suitable as a rapid means of determining AMR patterns of NTS for public health surveillance.

National surveillance of Shigella flexneri ensures the rapid detection of outbreaks to facilitate public health investigation and intervention strategies. In this study, we used whole-genome sequencing (WGS) to type S. flexneri in order to detect linked cases and support epidemiological investigations. We prospectively analyzed 330 isolates of S. flexneri received at the Gastrointestinal Bacteria Reference Unit at Public Health England between August 2015 and January 2016. Traditional phenotypic and WGS sub-typing methods were compared. PCR was carried out on isolates exhibiting phenotypic/genotypic discrepancies with respect to serotype. Phylogenetic relationships between isolates were analyzed by WGS using single nucleotide polymorphism (SNP) typing to facilitate cluster detection. For 306/330 (93%) isolates there was concordance between serotype derived from the genome and phenotypic serology. Discrepant results between the phenotypic and genotypic tests were attributed to novel O-antigen synthesis/modification gene combinations or indels identified in O-antigen synthesis/modification genes rendering them dysfunctional. SNP typing identified 36 clusters of two isolates or more. WGS provided microbiological evidence of epidemiologically linked clusters and detected novel O-antigen synthesis/modification gene combinations associated with two outbreaks. WGS provided reliable and robust data for monitoring trends in the incidence of different serotypes over time. SNP typing can be used to facilitate outbreak investigations in real-time thereby informing surveillance strategies and providing the opportunities for implementing timely public health interventions.

In April 2015, Public Health England implemented whole genome sequencing (WGS) as a routine typing tool for public health surveillance of Salmonella, adopting a multilocus sequence typing (MLST) approach as a replacement for traditional serotyping. The WGS derived sequence type (ST) was compared to the phenotypic serotype for 6,887 isolates of S. enterica subspecies I, and of these, 6,616 (96%) were concordant. Of the 4% (n = 271) of isolates of subspecies I exhibiting a mismatch, 119 were due to a process error in the laboratory, 26 were likely caused by the serotype designation in the MLST database being incorrect and 126 occurred when two different serovars belonged to the same ST. The population structure of S. enterica subspecies II–IV differs markedly from that of subspecies I and, based on current data, defining the serovar from the clonal complex may be less appropriate for the classification of this group. Novel sequence types that were not present in the MLST database were identified in 8.6% of the total number of samples tested (including S. enterica subspecies I–IV and S. bongori) and these 654 isolates belonged to 326 novel STs. For S. enterica subspecies I, WGS MLST derived serotyping is a high throughput, accurate, robust, reliable typing method, well suited to routine public health surveillance. The combined output of ST and serovar supports the maintenance of traditional serovar nomenclature while providing additional insight on the true phylogenetic relationship between isolates.

Shigella flexneri is the most common cause of bacterial dysentery in low-income countries. Despite this, S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on serotyping reactions developed over half-a-century ago. Here we combine whole genome sequencing with geographical and temporal data to examine the natural history of the species. Our analysis subdivides S. flexneri into seven phylogenetic groups (PGs); each containing two-or-more serotypes and characterised by distinct virulence gene complement and geographic range. Within the S. flexneri PGs we identify geographically restricted sub-lineages that appear to have persistently colonised regions for many decades to over 100 years. Although we found abundant evidence of antimicrobial resistance (AMR) determinant acquisition, our dataset shows no evidence of subsequent intercontinental spread of antimicrobial resistant strains. The pattern of colonisation and AMR gene acquisition suggest that S. flexneri has a distinct life-cycle involving local persistence.

The ability of Shiga toxin-producing Escherichia coli (STEC) to cause severe illness in humans is determined by multiple host factors and bacterial characteristics, including Shiga toxin (Stx) subtype. Given the link between Stx2a subtype and disease severity, we sought to identify the stx subtypes present in whole genome sequences (WGS) of 444 isolates of STEC O157. Difficulties in assembling the stx genes in some strains were overcome by using two complementary bioinformatics methods: mapping and de novo assembly. We compared the WGS analysis with the results obtained using a PCR approach and investigated the diversity within and between the subtypes. All strains of STEC O157 in this study had stx1a, stx2a or stx2c or a combination of these three genes. There was over 99% (442/444) concordance between PCR and WGS. When common source strains were excluded, 236/349 strains of STEC O157 had multiple copies of different Stx subtypes and 54 had multiple copies of the same Stx subtype. Of those strains harbouring multiple copies of the same Stx subtype, 33 had variants between the alleles while 21 had identical copies. Strains harbouring Stx2a only were most commonly found to have multiple alleles of the same subtype (42%). Both the PCR and WGS approach to stx subtyping provided a good level of sensitivity and specificity. In addition, the WGS data also showed there were a significant proportion of strains harbouring multiple alleles of the same Stx subtype associated with clinical disease in England.

Mycobacterium tuberculosis isolates cultured from 6 patients associated with an isoniazid-resistant M. tuberculosis outbreak acquired rifampicin resistance. The rpoB gene sequence showed that resistance was associated with rare mutations in each isolate. Three isolates had a mutation outside the rifampicin resistance–determining region.

Shiga toxin–producing Escherichia coli serotype O117:K1:H7 is a cause of persistent diarrhea in travelers to tropical locations. Whole genome sequencing identified genetic mechanisms involved in the pathoadaptive phenotype. Sequencing also identified toxin and putative adherence genes flanked by sequences indicating horizontal gene transfer from Shigella dysenteriae and Salmonella spp., respectively.

Enteroaggregative E. coli (EAEC) is an established diarrhoeagenic pathotype. The association with virulence gene content and ability to cause disease has been studied but little is known about the population structure of EAEC and how this pathotype evolved. Analysis by Multi Locus Sequence Typing of 564 EAEC isolates from cases and controls in Bangladesh, Nigeria and the UK spanning the past 29 years, revealed multiple successful lineages of EAEC. The population structure of EAEC indicates some clusters are statistically associated with disease or carriage, further highlighting the heterogeneous nature of this group of organisms. Different clusters have evolved independently as a result of both mutational and recombination events; the EAEC phenotype is distributed throughout the population of E. coli.

Multilocus variable number tandem repeat analysis (MLVA) provides microbiological support for investigations of clusters of cases of infection with Shiga toxin-producing E. coli (STEC) O157. All confirmed STEC O157 isolated in England and submitted to the Gastrointestinal Bacteria Reference Unit (GBRU) during a six month period were typed using MLVA, with the aim of assessing the impact of this approach on epidemiological investigations. Of 539 cases investigated, 341 (76%) had unique (>2 single locus variants) MLVA profiles, 12% of profiles occurred more than once due to known household transmission and 12% of profiles occurred as part of 41 clusters, 21 of which were previously identified through routine public health investigation of cases. The remaining 20 clusters were not previously detected and STEC enhanced surveillance data for associated cases were retrospectively reviewed for epidemiological links including shared exposures, geography and/or time. Additional evidence of a link between cases was found in twelve clusters. Compared to phage typing, the number of sporadic cases was reduced from 69% to 41% and the diversity index for MLVA was 0.996 versus 0.782 for phage typing. Using MLVA generates more data on the spatial and temporal dispersion of cases, better defining the epidemiology of STEC infection than phage typing. The increased detection of clusters through MLVA typing highlights the challenges to health protection practices, providing a forerunner to the advent of whole genome sequencing as a diagnostic tool.

Following a large outbreak of foodborne gastrointestinal (GI) disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75%) of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC) group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx) genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.