Significance: Zoonotic infections with enterohemorrhagic Escherchia coli O157 have emerged as a serious threat to human health. Conventional sequence-based analyses indicate that most human infections originate from particular pathogenic lineages. In this study, we apply a machine-learning approach to complex pangenome information and predict the human infection potential of cattle E. coli O157 isolates. We demonstrate that only a small subset of bovine strains is likely to cause human disease, even within previously defined pathogenic lineages. The approach was tested across isolates from the United Kingdom and United States and verified with food and cattle isolates from outbreak investigations. This finding has important implications for targeting of control strategies in herds.

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>10³ cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a⁺/stx2c⁺ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium. Enterohaemorrhagic E. coli (EHEC) O157 strains are found in cattle where they are asymptomatic, while human exposure can lead to severe symptoms including bloody diarrhoea and kidney damage due to the activity of Shiga toxin (Stx). The most serious symptoms in humans are associated with isolates that encode Stx subtype 2a. The advantage of these toxins in the animal reservoir is still not clear, however there is experimental evidence implicating Stx with increased bacterial adherence, immune modulation and suppression of predatory protozoa. In this study, the hypothesis that Stx2a is important for super-shedding and calf-to-calf transmission was tested by comparing excretion and transmission dynamics of E. coli O157 strains with and without Stx2a. While Stx2a did not alter excretion levels when calfs were orally challenge, it enabled colonisation of more in contact ‘sentinel’ animals in our transmission model. We show that Stx2a is generally induced more rapidly than Stx2c, resulting in increased levels of Stx2a expression. Both Stx2a and Stx2c were able to restrict cellular proliferation of epithelial cells in cultured bovine enteroids. Taken together, we propose that rapid production of Stx2a and its role in establishing E. coli O157 colonisation in the bovine gastrointestinal tract facilitate effective transmission and have led to its expansion in the cattle E. coli O157 population.

The human zoonotic pathogen Escherichia coli O157:H7 is defined by its extensive prophage repertoire including those that encode Shiga toxin, the factor responsible for inducing life-threatening pathology in humans. As well as introducing genes that can contribute to the virulence of a strain, prophage can enable the generation of large-chromosomal rearrangements (LCRs) by homologous recombination. This work examines the types and frequencies of LCRs across the major lineages of the O157:H7 serotype. We demonstrate that LCRs are a major source of genomic variation across all lineages of E. coli O157:H7 and by using both optical mapping and Oxford Nanopore long-read sequencing prove that LCRs are generated in laboratory cultures started from a single colony and that these variants can be recovered from colonized cattle. LCRs are biased towards the terminus region of the genome and are bounded by specific prophages that share large regions of sequence homology associated with the recombinational activity. RNA transcriptional profiling and phenotyping of specific structural variants indicated that important virulence phenotypes such as Shiga-toxin production, type-3 secretion and motility can be affected by LCRs. In summary, E. coli O157:H7 has acquired multiple prophage regions over time that act to continually produce structural variants of the genome. These findings raise important questions about the significance of this prophage-mediated genome contingency to enhance adaptability between environments.

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx_2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx_2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx_2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans.

An antibiotic susceptibility monitoring programme was conducted from 2004 to 2010, resulting in a collection of 143 Escherichia coli cultured from bovine faecal samples (diarrhoea) and milk-aliquots (mastitis). The isolates were subjected to whole-genome sequencing and were distributed in phylogroups A, B1, B2, C, D, E, and G with no correlation for particular genotypes with pathotypes. In fact, the population structure showed that the strains belonging to the different phylogroups matched broadly to ST complexes; however, the isolates are randomly associated with the diseases, highlighting the necessity to investigate the virulence factors more accurately in order to identify the mechanisms by which they cause disease. The antimicrobial resistance was assessed phenotypically, confirming the genomic prediction on three isolates that were resistant to colistin, although one isolate was positive for the presence of the gene mcr-1 but susceptible to colistin. To further characterise the genomic context, the four strains were sequenced by using a single-molecule long read approach. Genetic analyses indicated that these four isolates harboured complex and diverse plasmids encoding not only antibiotic resistant genes (including mcr-1 and bla) but also virulence genes (siderophore, ColV, T4SS). A detailed description of the plasmids of these four E. coli strains, which are linked to bovine mastitis and diarrhoea, is presented for the first time along with the characterisation of the predicted antibiotic resistance genes. The study highlighted the diversity of incompatibility types encoding complex antibiotic resistance elements such as Tn6330, ISEcp1, Tn6029, and IS5075. The mcr-1 resistance determinant was identified in IncHI2 plasmids pCFS3273-1 and pCFS3292-1, thus providing some of the earliest examples of mcr-1 reported in Europe, and these sequences may be a representative of the early mcr-1 plasmidome characterisation in the EU/EEA.

Twenty-nine hippuricase-positive Campylobacter strains were isolated from wild birds and river water. Previous characterization using atpA typing indicated that these strains were related to Campylobacter jejuni and Campylobacter coli but were most similar to three recently described hippuricase-positive Campylobacter species recovered from zebra finches, i.e. C. aviculae, C. estrildidarum and C. taeniopygiae. Phylogenetic analyses using 330 core genes placed the 29 strains into a clade well separated from the other Campylobacter taxa, indicating that these strains represent a novel Campylobacter species. Pairwise digital DNA–DNA hybridization and average nucleotide identity values were below 70 and 95 %, respectively, thus providing further supporting evidence of a novel taxon. Standard phenotypic testing was performed. All strains are microaerobic or anaerobic, motile, Gram-negative, spiral cells that are oxidase, catalase and nitrate reductase positive, but urease negative. Genomic analyses indicate that the 29 strains can potentially synthesize very few amino acids de novo and are auxotrophic for many amino acids and cofactors, similar to the species composing the Campylobacter lari group. In addition, these strains encode complete Entner–Doudoroff and Leloir pathways, suggesting that they may possess the ability to utilize both glucose and galactose; these pathways were also identified in the genomes of the zebra finch-associated taxa. The data presented here show that these strains represent a novel species within Campylobacter, for which the name Campylobacter molothri sp. nov. (type strain RM10537^T=LMG 32306^T=CCUG 75331^T) is proposed.