Comparison of Shiga toxin-encoding bacteriophages in highly pathogenic strains of Shiga toxin-producing Escherichia coli O157:H7 in the UK

Over the last 35 years in the UK, the burden of Shiga toxin-producing Escherichia coli (STEC) O157:H7 infection has, during different periods of time, been associated with five different sub-lineages (1983-1995, Ia, I/IIa and I/IIb; 1996-2014, Ic; and 2015-2018, IIb). The acquisition of a stx2a-encoding bacteriophage by these five sub-lineages appears to have coincided with their respective emergences. The Oxford Nanopore Technologies (ONT) system was used to sequence, characterize and compare the stx-encoding prophages harboured by each sub-lineage to investigate the integration of this key virulence factor. The stx2a-encoding prophages from each of the lineages causing clinical disease in the UK were all different, including the two UK sub-lineages (Ia and I/IIa) circulating concurrently and causing severe disease in the early 1980s. Comparisons between the stx2a-encoding prophage in sub-lineages I/IIb and IIb revealed similarity to the prophage commonly found to encode stx2c, and the same site of bacteriophage integration (sbcB) as stx2c-encoding prophage. These data suggest independent acquisition of previously unobserved stx2a-encoding phage is more likely to have contributed to the emergence of STEC O157:H7 sub-lineages in the UK than intra-UK lineage to lineage phage transmission. In contrast, the stx2c-encoding prophage showed a high level of similarity across lineages and time, consistent with the model of stx2c being present in the common ancestor to extant STEC O157:H7 and maintained by vertical inheritance in the majority of the population. Studying the nature of the stx-encoding bacteriophage contributes to our understanding of the emergence of highly pathogenic strains of STEC O157:H7

Comparative analysis of core genome MLST and SNP typing within a European Salmonella serovar Enteritidis outbreak

Multi-country outbreaks of foodborne bacterial disease present challenges in their detection, tracking, and notification. As food is increasingly distributed across borders, such outbreaks are becoming more common. This increases the need for high-resolution, accessible, and replicable isolate typing schemes. Here we evaluate a core genome multilocus typing (cgMLST) scheme for the high-resolution reproducible typing of Salmonella enterica (S. enterica) isolates, by its application to a large European outbreak of S. enterica serovar Enteritidis. This outbreak had been extensively characterised using single nucleotide polymorphism (SNP)-based approaches. The cgMLST analysis was congruent with the original SNP-based analysis, the epidemiological data, and whole genome MLST (wgMLST) analysis. Combination of the cgMLST and epidemiological data confirmed that the genetic diversity among the isolates predated the outbreak, and was likely present at the infection source. There was consequently no link between country of isolation and genetic diversity, but the cgMLST clusters were congruent with date of isolation. Furthermore, comparison with publicly available Enteritidis isolate data demonstrated that the cgMLST scheme presented is highly scalable, enabling outbreaks to be contextualised within the Salmonella genus. The cgMLST scheme is therefore shown to be a standardised and scalable typing method, which allows Salmonella outbreaks to be analysed and compared across laboratories and jurisdictions. [Abstract copyright: Copyright © 2018. Published by Elsevier B.V.

Genomic surveillance detects Salmonella enterica serovar Paratyphi A harbouring blaCTX-M-15 from a traveller returning from Bangladesh

Whole genome sequencing (WGS) has been used routinely by Public Health England (PHE) for identification, surveillance and monitoring of resistance determinants in referred Salmonella isolates since 2015. We report the first identified case of extended-spectrum-β-lactamase (ESBL) Salmonella enterica serovar Paratyphi A (S. Paratyphi A) isolated from a traveller returning to England from Bangladesh in November 2017. The isolate (440915) was resistant to ciprofloxacin and harboured both the mobile element ISEcp9 -blaCTX-M-15-hp-tnpA and blaTEM-191, associated with ESBL production. Phenotypic resistance was subsequently confirmed by Antimicrobial Susceptibility Testing (AST). S. Paratyphi A 440915 harboured an IncI1 plasmid previously reported to encode ESBL elements in Enterobacteriaceae and recently described in a S. Typhi isolate from Bangladesh. Results from this study indicate the importance of monitoring imported drug resistance for typhoidal salmonellae as ceftriaxone is the first line antibiotic treatment for complicated enteric fever in England. We conclude that WGS provides a rapid, accurate method for surveillance of drug resistance genes in Salmonella, leading to the first reported case of ESBL producing S. Paratyphi A and continues to inform the national treatment guidelines for management of enteric fever

Analysis of a small outbreak of Shiga toxin-producing Escherichia coli O157:H7 using long-read sequencing

Compared to short-read sequencing data, long-read sequencing facilitates single contiguous de novo assemblies and characterization of the prophage region of the genome. Here, we describe our methodological approach to using Oxford Nanopore Technology (ONT) sequencing data to quantify genetic relatedness and to look for microevolutionary events in the core and accessory genomes to assess the within-outbreak variation of four genetically and epidemiologically linked isolates. Analysis of both Illumina and ONT sequencing data detected one SNP between the four sequences of the outbreak isolates. The variant calling procedure highlighted the importance of masking homologous sequences in the reference genome regardless of the sequencing technology used. Variant calling also highlighted the systemic errors in ONT base-calling and ambiguous mapping of Illumina reads that results in variations in the genetic distance when comparing one technology to the other. The prophage component of the outbreak strain was analysed, and nine of the 16 prophages showed some similarity to the prophage in the Sakai reference genome, including the stx2a-encoding phage. Prophage comparison between the outbreak isolates identified minor genome rearrangements in one of the isolates, including an inversion and a deletion event. 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 evolutionary history, virulence and potentially the likely source and transmission of this zoonotic, foodborne pathogen

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

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

Evolution of a zoonotic pathogen:investigating prophage diversity in enterohaemorrhagic Escherichia coli O157 by long-read sequencing

Enterohaemorrhagic Escherichia coli (EHEC) O157 is a zoonotic pathogen for which colonization of cattle and virulence in humans is associated with multiple horizontally acquired genes, the majority present in active or cryptic prophages. Our understanding of the evolution and phylogeny of EHEC O157 continues to develop primarily based on core genome analyses; however, such short-read sequences have limited value for the analysis of prophage content and its chromosomal location. In this study, we applied Single Molecule Real Time (SMRT) sequencing, using the Pacific Biosciences long-read sequencing platform, to isolates selected from the main sub-clusters of this clonal group. Prophage regions were extracted from these sequences and from published reference strains. Genome position and prophage diversity were analysed along with genetic content. Prophages could be assigned to clusters, with smaller prophages generally exhibiting less diversity and preferential loss of structural genes. Prophages encoding Shiga toxin (Stx) 2a and Stx1a were the most diverse, and more variable compared to prophages encoding Stx2c, further supporting the hypothesis that Stx2c-prophage integration was ancestral to acquisition of other Stx types. The concept that phage type (PT) 21/28 (Stx2a+, Stx2c+) strains evolved from PT32 (Stx2c+) was supported by analysis of strains with excised Stx-encoding prophages. Insertion sequence elements were over-represented in prophage sequences compared to the rest of the genome, showing integration in key genes such as stx and an excisionase, the latter potentially acting to capture the bacteriophage into the genome. Prophage profiling should allow more accurate prediction of the pathogenic potential of isolates