David Robert Greig

Background: Following the global resurgence of cholera in 2022, 51 cholera cases were reported by European countries. We previously characterised 49 Vibrio cholerae O1 isolates from these cases using whole genome sequencing. In 2023 and 2024, 59 additional cholera cases were reported by 11 European countries. Aim: We aimed to confirm that V. cholerae O1 isolates associated with cholera cases reported in Europe in 2023–2024 belonged to the epidemic seventh pandemic El Tor (7PET) lineage rather than to non-epidemic V. cholerae O1 lineages, and to characterise their virulence, antimicrobial resistance (AMR) determinants and phylogenetic relationships. Methods: Fifty V. cholerae O1 isolates were available for whole genome sequencing, of which 49 yielded genomes of sufficient quality for analysis. Genomes were analysed together with more than 1,500 publicly available 7PET genomes to place the European isolates into a global phylogenetic context. Results: All 49 genomes belonged to the 7PET lineage and Wave 3. Four sub-lineages were identified: BD1.2 (2/49), AFR12 (6/49), AFR13 (8/49) and Pre-AFR15 (33/49). The predominance of Pre-AFR15 confirms its continued contribution to the ongoing global cholera resurgence. In contrast to our 2022 survey, eight highly drug-resistant AFR13 isolates were detected, documenting the international spread of this highly drug-resistant clone resistant to multiple first-line antimicrobial agents. Conclusion: Whole genome sequencing should be routinely used by reference laboratories to distinguish epidemic 7PET from non-epidemic V. cholerae O1 lineages and to monitor the emergence and international spread of AMR clones. Continued collaborative genomic surveillance of travel-associated cholera cases can provide early insights into the emergence, international spread and antimicrobial resistance of 7PET sub-lineages.

Shiga toxin-producing Escherichia coli (STEC) are an important public health concern due to their association with foodborne gastroenteritis and severe outcomes including haemolytic uraemic syndrome (HUS), particularly linked to the stx2a subtype of the Shiga toxin. We investigated the temporal dynamics and acquisition of stx2a among STEC isolates submitted to the United Kingdom Health Security Agency (UKHSA) between 2016 and 2024. 12,888 whole genome STEC sequences and associated metadata were analysed. 31.9% of STEC isolates harboured stx2a , spanning 78 O serogroups with a marked shift from STEC O157 to non-O157 serogroups over time. STEC O26:H11 and STEC O145:H28 were the primary drivers of observed increases, most commonly associated with stx2a alone or in combination with stx1a . The widespread and increasing presence of stx2a across the STEC population in England highlights an emerging public health risk and demonstrates the value of routine genomic surveillance in monitoring high-severity Shiga toxin subtypes.

Shiga toxin-producing Escherichia coli (STEC) serotype O157:H7 emerged in the 1980s as a cause of foodborne outbreaks of infectious intestinal disease. Over the last decade, whole-genome sequencing (WGS) has replaced phage typing (PT) as the typing method of choice to detect outbreaks and monitor trends of STEC O157:H7. The aim of this study was to correlate historical PT data with WGS outputs and to describe the continuous trends in STEC O157:H7 notifications in England over a 40-year period from 1985 to 2024. At the UK Health Security Agency (UKHSA), both PT and WGS typing schemes were in use for a 6-year period from 2016 to 2021, providing an opportunity to compare the two typing datasets. We focused on the three major phage types, PT2, PT21/28 and PT8, as the numbers of other phage types were comparatively low and did not make a notable impact on the trends. Lineage and sub-lineage typing data from 2016 to 2024 were superimposed on PT data from 1989 to 2021. Between 2016 and 2021, 3,229 isolates of STEC O157:H7 referred to UKHSA for typing were linked to PT, Shiga toxin ( stx ) subtype and WGS data. For PT21/28 stx2 , there was a good correlation between sub-lineage Ic ( n =495/499, 99.2%). PT8 stx2 isolates were associated with sub-lineage IIb ( n =215/262, 82.1%), and PT8 stx1+2 isolates aligned with sub-lineage IIc ( n =851/857, 99.3%). Notifications of PT2 stx2 were low, and the correlation with lineage I/II during the overlapping time frame was less robust ( n =48/85, 56.5%), although analysis of historical data indicated that the association was stronger when PT2 notifications were at their peak in the 1980s and 1990s. We extrapolated the sub-lineage data for the four major STEC O157:H7 PT and stx profiles in the UK, to bridge the gap across the pre- and post-genomic revolution eras and to facilitate future trend analyses based on sub-lineage alone. We created a window to the past to review the WGS data in the context of the historical PT data. This enabled us to link common exposures, learn from previous outbreak investigations and predict, investigate and prepare for emerging threats to public health.

Introduction. Vibrio cholerae is a diverse species of bacteria that causes watery diarrhoea, vomiting and stomach cramps and is the aetiological agent of cholera. Gap statement. Despite the global upsurge in notifications of cholera and concerns over the impact of climate change, systematic analysis of national and international surveillance data describing the microbiology and epidemiology of V. cholerae is sparse. Aim. We reviewed the microbiology and epidemiology of V. cholerae isolated from travellers returning to the UK. Methodology. All human isolates of V. cholerae detected from 2004 to 2024 were extracted from UK Health Security Agency reference laboratory surveillance systems. Microbiological data were analysed and linked to available epidemiological data and genome sequences for all isolates from 2016 to 2024. Results. There were 984 notifications of V. cholerae from 2004 to 2024 (an average of 51 each year), of which 266 (27.0%) belonged to serogroup O1. There were over 180 different sequence types (STs), of which cholera toxin producing ST69 was the predominant type ( n =99, 28.2%). The highest number of isolates was in 2010 ( n =74), while the lowest was in 2020 ( n =8) and 2021 ( n =4) due to travel restrictions imposed during the COVID-19 pandemic. Children under the age of 10 and the middle-aged and elderly population were most susceptible to infection, and 51.6% of the cases were male. There was a seasonal peak in August and September. Travel was reported by 92.9% of cases, and the most frequently reported travel destinations were India, Pakistan and Kenya. Conclusion. From the UK perspective, to assess the risk to food safety and to more accurately determine the clinical burden of V. cholerae , we recommend (i) widespread molecular testing of shellfish to monitor the emergence of V. cholerae in UK waters due to climate change and (ii) comprehensive testing of faecal specimens from non-travellers with gastrointestinal symptoms. Public health surveillance and information sharing at the global level is essential to assess the impact of investment in water, sanitation and hygiene initiatives for the prevention of cholera.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a globally dispersed zoonotic pathogen capable of causing severe disease outcomes, including bloody diarrhoea and haemolytic uraemic syndrome. While variations in Shiga toxin subtype are well-recognized drivers of disease severity, many unexplained differences remain among strains carrying the same toxin profile. We applied explainable machine learning (ML) approaches – Random Forest and Extreme Gradient Boosting – to whole-genome sequencing data from 1,030 STEC O157:H7 isolates to predict patient clinical outcomes, using data collected over 2 years of routine surveillance in England. A phylogeny-informed cross-validation strategy was implemented to account for population structure and avoid data leakage, ensuring robust model generalizability. Extreme Gradient Boosting outperformed Random Forest in predicting minority classes and correctly predicted high-risk isolates in traditionally low-risk lineages, illustrating its utility for capturing complex genomic signatures beyond known virulence genes. Feature importance analyses highlighted phage-encoded elements, including potentially novel intergenic regulators, alongside established virulence factors. Moreover, key genomic regions linked to small RNAs and stress-response pathways were enriched in isolates causing severe disease. These findings underscore the capacity of explainable ML to refine risk assessments, offering a valuable tool for early detection of high-risk STEC O157:H7 and guiding targeted public health interventions.

Introduction. Following two large foodborne outbreaks of the gastrointestinal pathogen, enteroaggregative Escherichia coli (EAEC), in Germany in 2011 and the UK in 2014, the UK Health Security Agency (UKHSA) implemented enhanced surveillance strategies for EAEC. Gap Statement. The surveillance of diarrhoeagenic E. coli in England focuses on Shiga toxin-producing E. coli (STEC), and the true clinical and community burden of EAEC is unknown. This gap extends globally, as many countries lack the infrastructure, diagnostic tools and healthcare facilities to resource surveillance programmes for EAEC. Aim. The aim of the study was to review the microbiological typing data and demographic data linked to isolates and cases diagnosed from 2016 to 2023 and to assess the risk to public health. Methodology. Faecal samples that tested positive by PCR for diarrhoeagenic E. coli at local microbiology diagnostic laboratories were referred to the UKHSA for confirmation and culture. Isolates identified as EAEC were sequenced on the Illumina HiSeq and NextSeq platforms. Sequence type, serotype and antimicrobial resistance (AMR) profile were derived from the genome sequence. Age, sex and travel histories were linked to the typing data. Results. There was a total of 1,402 notifications of EAEC, exhibiting a fivefold increase in diagnoses from 93 in 2016 to 524 in 2023. The most common sequence types (STs) were ST34 ( n =202/1,402, 14.4%), ST10 ( n =185/1,402, 13.2%), ST200 ( n =183/1,402, 13.1%) and ST678 ( n =101/1,402, 7.2%), and the most common serotypes were O92:H33 ( n =130/1,402, 9.3%), O175:H31 ( n =78/1,402, 5.6%) and O99:H10 ( n =78/1,402, 5.6%). Most cases were female ( n =748/1,372, 54.5%) and/or were aged n =387/1,372, 28.2%), within which 299 out of 387 (77.3%) were n =810/1,402, 57.8%) and beta-lactam ( n =807/1,402, 57.6%) antibiotics being the most common. Conclusion. Given the burden of disease caused by EAEC in the community, the high proportion of infections in children and travellers, the risk of the emergence of hybrid STEC/EAEC pathotypes and the high proportion of AMR, we recommend that EAEC should be part of the diagnostic algorithm in the UK.

The life cycle of certain bacteriophages involves their maintenance within the bacterial cell as extrachromosomal elements, complete with replication and partitioning systems. These “phage-plasmids” (P-Ps) are distributed widely among bacterial phyla but are not typically included during genomic surveillance studies, and previous reports do not consider the context of their host strain diversity. We recently identified a P1-like P-P carrying the bla _CTX-M-15 resistance gene in Salmonella enterica serovar Typhi, prompting a subsequent investigation into the overall frequency of P-Ps among gastrointestinal bacteria under routine genomic surveillance within England. We expanded our study to include P-P groups known to be associated with Enterobacteriaceae (P1, D6, SSU5, N15) and scanned a collection of 66,856 genomes of diarrhoeagenic Escherichia spp., Shigella spp. and S. enterica . All four P-P groups were detected in our dataset, totalling 9% of E. coli and Shigella genomes and 2% of S. enterica genomes. A small subset harboured two distinct P-P groups. P1-like P-Ps, some of which carried a cytotoxic necrotising factor, were predominantly associated with Shiga toxin-producing E. coli strains of public health concern, including clonal complexes CC11 (O157:H7), CC29 (O26:H11) and CC165. In contrast, SSU5 group P-Ps were linked with multidrug-resistant lineages of both S. Typhi and Shigella sonnei . We found multiple antimicrobial resistance genes inserted into P-Ps via transposons, integrons and insertion sequences, and numerous defence/anti-defence systems. There was evidence of vertical transmission but also many links across species, time and geography, indicating that horizontal transfer is occurring regularly. Phage-plasmids are often described only as cryptic elements or not detected during genomic surveillance. We show that P-Ps are associated with clinically relevant lineages of human pathogens and can acquire accessory genes that may impact on disease severity and therefore should play a more prominent role in pathogen surveillance and epidemiology.

Listeriosis is a predominantly foodborne infection causing severe, invasive disease in the immunocompromised, with the causative agent being Listeria monocytogenes. While L. monocytogenes possesses innate resistance to several classes of antibiotics, acquired antibiotic resistance is low compared with other foodborne pathogens. Conversely, plasmids possessing stress tolerance mechanisms are common and contribute to their ability to persist in food production environments. However, very few have been identified, which also carry antibiotic resistance genes, particularly multidrug resistance regions. We scanned a UK collection of L. monocytogenes genomes and detected an isolate with the dfrD, lnuG and mphB genes, encoding predicted resistance to trimethoprim, lincosamides and macrolides, respectively. This isolate also possessed a Listeria repA family plasmid replication gene, as well as multiple chromosomal and plasmid-associated stress tolerance factors. Long-read sequencing confirmed the presence of a single 120,420 bp plasmid, which notably is a composite of a highly conserved Listeria plasmid backbone with a novel 35 kb resistance region. Comparative genomic analyses revealed that the plasmid-borne resistance region was likely acquired from other Gram-positive species over several events. The island containing the lnuG and mphB genes has links to porcine and bovine origins, while the dfrD gene is located on a Tn3-like transposon associated with multiple resistance genes, and both elements can move to the chromosome of L. monocytogenes strains from food and human disease. This work furthers the understanding of plasmid-mediated antibiotic resistance as well as the wider circulation of mobile genetic elements among L. monocytogenes and other bacterial species within the food chain.

Introduction. Diarrhoeagenic Escherichia coli (DEC) pathotypes are defined by genes located on mobile genetic elements, and more than one definitive pathogenicity gene may be present in the same strain. In August 2022, UK Health Security Agency (UKHSA) surveillance systems detected an outbreak of hybrid Shiga toxin-producing E. coli/enterotoxigenic E. coli (STEC–ETEC) serotype O101:H33 harbouring both Shiga toxin (stx) and heat-stable toxin (st). Gap statement. These hybrid strains of DEC are a public health concern, as they are often associated with enhanced pathogenicity. However, little is known about their epidemiology, clinical significance and associated public health burden. Aim. The aim of this study was to describe the microbiology, epidemiology and genomic analysis of this novel hybrid serotype in the context of the STEC–ETEC strains in the UKHSA archive. Methodology. From 2014 to 2023, STEC isolated from faecal specimens testing positive for STEC by PCR were sequenced on the NextSeq 1000 short read platform and a subset were selected for long read nanopore sequencing. Genomes were analysed to determine serotype, stx subtype, DEC pathogenicity genes and antimicrobial resistance determinants. Results. There were 162 STEC–ETEC strains isolated between 2014 and 2023, of which 117/162 were human clinical isolates and 45 were of food or animal origin. An average of 16 STEC–ETEC strains were identified each year, exhibiting a range of different stx subtypes, the most common profiles being stx2g,st (n=65, 40%) and stx2a,st (n=48, 30%). The most common sequence types were ST329 and ST200 (n=24 each), and the most frequently detected serotype was O187:H28 (n=25). Nine cases of genetically linked STEC–ETEC O101:H33, stx1a,st were detected between 8 August and 21 September 2022. Although the temporal and geographical distribution of the cases was characteristic of a foodborne outbreak, the contaminated vehicle was not identified. Conclusions. Phylogenetic analysis and long-read sequencing of the outbreak strain provided insight into the stepwise acquisition of st and stx and the evolutionary history of STEC–ETEC pathotypes. The integration of epidemiological data and whole-genome sequencing for routine surveillance of gastrointestinal pathogens is key to understanding the emergence of zoonotic hybrid DEC pathotypes and monitoring foodborne threats to public health.

Background: The number of cholera cases reported to the World Health Organization (WHO) in 2022 was more than double that of 2021. Nine countries of the WHO European Region reported 51 cases of cholera in 2022 vs five reported cases in 2021. Aim: We aimed to confirm that the Vibrio cholerae O1 isolates reported by WHO European Region countries in 2022 belonged to the seventh pandemic El Tor lineage (7PET). We also studied their virulence, antimicrobial resistance (AMR) determinants and phylogenetic relationships. Methods: We used microbial genomics to study the 49 V. cholerae O1 isolates recovered from the 51 European cases. We also used > 1,450 publicly available 7PET genomes to provide a global phylogenetic context for these 49 isolates. Results: All 46 good-quality genomes obtained belonged to the 7PET lineage. All but two isolates belonged to genomic Wave 3 and were grouped within three sub-lineages, one of which, Pre-AFR15, predominated (34/44). This sub-lineage, corresponding to isolates from several countries in Southern Asia, the Middle East and Eastern or Southern Africa, was probably a major contributor to the global upsurge of cholera cases in 2022. No unusual AMR profiles were inferred from analysis of the AMR gene content of the 46 genomes. Conclusion: Reference laboratories in high-income countries should use whole genome sequencing to assign V. cholerae O1 isolates formally to the 7PET or non-epidemic lineages. Periodic collaborative genomic studies based on isolates from travellers can provide useful information on the circulating strains and their evolution, particularly as concerns AMR.