Philip M. Ashton

MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island

Short-read, high-throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes. The MinION nanopore sequencer can produce long sequencing reads on a device similar in size to a USB memory stick. Here we apply a MinION sequencer to resolve the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58. Nanopore sequencing data from a single 18-h run was used to create a scaffold for an assembly generated from short-read Illumina data. Our results demonstrate the potential of the MinION device in clinical laboratories to fully characterize the epidemic spread of bacterial pathogens.

Detection of the plasmid-mediated mcr-1 gene conferring colistin resistance in human and food isolates of Salmonella enterica and Escherichia coli in England and Wales

OBJECTIVES In response to the first report of transmissible colistin resistance mediated by the mcr-1 gene in Escherichia coli and Klebsiella spp. from animals and humans in China, we sought to determine its presence in Enterobacteriaceae isolated in the UK. METHODS The PHE archive of whole-genome sequences of isolates from surveillance collections, submissions to reference services and research projects was retrospectively analysed for the presence of mcr-1 using Genefinder. The genetic environment of the gene was also analysed. RESULTS Rapid screening of the genomes of ∼24 000 Salmonella enterica, E. coli, Klebsiella spp., Enterobacter spp., Campylobacter spp. and Shigella spp. isolated from food or humans identified 15 mcr-1-positive isolates. These comprised: 10 human S. enterica isolates submitted between 2012 and 2015 (8 Salmonella Typhimurium, 1 Salmonella Paratyphi B var Java and 1 Salmonella Virchow) from 10 patients; 3 isolates of E. coli from 2 patients; and 2 isolates of Salmonella Paratyphi B var Java from poultry meat imported from the EU. The mcr-1 gene was located on diverse plasmids belonging to the IncHI2, IncI2 and IncX4 replicon types and its association with ISApl1 varied. Six mcr-1-positive S. enterica isolates were from patients who had recently travelled to Asia. CONCLUSIONS Analysis of WGS data allowed rapid confirmation of the presence of the plasmid-mediated colistin resistance gene mcr-1 in diverse genetic environments and plasmids. It has been present in E. coli and Salmonella spp. harboured by humans in England and Wales since at least 2012.

Identification of Salmonella for public health surveillance using whole genome sequencing.

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.

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.

Intercontinental dissemination of azithromycin-resistant shigellosis through sexual transmission: a cross-sectional study

BACKGROUND Shigellosis is an acute, severe bacterial colitis that, in high-income countries, is typically associated with travel to high-risk regions (Africa, Asia, and Latin America). Since the 1970s, shigellosis has also been reported as a sexually transmitted infection in men who have sex with men (MSM), in whom transmission is an important component of shigellosis epidemiology in high-income nations. We aimed to use sophisticated subtyping and international sampling to determine factors driving shigellosis emergence in MSM linked to an outbreak in the UK. METHODS We did a large-scale, cross-sectional genomic epidemiological study of shigellosis cases collected from 29 countries between December, 1995, and June 8, 2014. Focusing on an ongoing epidemic in the UK, we collected and whole-genome sequenced clinical isolates of Shigella flexneri serotype 3a from high-risk and low-risk regions, including cases associated with travel and sex between men. We examined relationships between geographical, demographic, and clinical patient data with the isolate antimicrobial susceptibility, genetic data, and inferred evolutionary relationships. FINDINGS We obtained 331 clinical isolates of S flexneri serotype 3a, including 275 from low-risk regions (44 from individuals who travelled to high-risk regions), 52 from high-risk regions, and four outgroup samples (ie, closely related, but genetically distinct isolates used to determine the root of the phylogenetic tree). We identified a recently emerged lineage of S flexneri 3a that has spread intercontinentally in less than 20 years throughout regions traditionally at low risk for shigellosis via sexual transmission in MSM. The lineage had acquired multiple antimicrobial resistance determinants, and prevailing sublineages were strongly associated with resistance to the macrolide azithromycin. Eight (4%) of 206 isolates from the MSM-associated lineage were obtained from patients who had previously provided an isolate; these serial isolations indicated atypical infection patterns (eg, reinfection). INTERPRETATION We identified transmission-facilitating behaviours and atypical course(s) of infection as precipitating factors in shigellosis-affected MSM. The intercontinental spread of antimicrobial-resistant shigella through established transmission routes emphasises the need for new approaches to tackle the public health challenge of sexually transmitted infections in MSM. FUNDING Wellcome Trust (grant number 098051).

MOST: a modified MLST typing tool based on short read sequencing

Multilocus sequence typing (MLST) is an effective method to describe bacterial populations. Conventionally, MLST involves Polymerase Chain Reaction (PCR) amplification of housekeeping genes followed by Sanger DNA sequencing. Public Health England (PHE) is in the process of replacing the conventional MLST methodology with a method based on short read sequence data derived from Whole Genome Sequencing (WGS). This paper reports the comparison of the reliability of MLST results derived from WGS data, comparing mapping and assembly-based approaches to conventional methods using 323 bacterial genomes of diverse species. The sensitivity of the two WGS based methods were further investigated with 26 mixed and 29 low coverage genomic data sets from Salmonella enteridis and Streptococcus pneumoniae. Of the 323 samples, 92.9% (n = 300), 97.5% (n = 315) and 99.7% (n = 322) full MLST profiles were derived by the conventional method, assembly- and mapping-based approaches, respectively. The concordance between samples that were typed by conventional (92.9%) and both WGS methods was 100%. From the 55 mixed and low coverage genomes, 89.1% (n = 49) and 67.3% (n = 37) full MLST profiles were derived from the mapping and assembly based approaches, respectively. In conclusion, deriving MLST from WGS data is more sensitive than the conventional method. When comparing WGS based methods, the mapping based approach was the most sensitive. In addition, the mapping based approach described here derives quality metrics, which are difficult to determine quantitatively using conventional and WGS-assembly based approaches.

Applying phylogenomics to understand the emergence of Shiga Toxin producing Escherichia coli O157:H7 strains causing severe human disease in the United Kingdom.

Shiga-toxin-producing Escherichia coli (STEC) O157:H7 is a recently emerged zoonotic pathogen with considerable morbidity. Since the emergence of this serotype in the 1980s, research has focussed on unravelling the evolutionary events from the E. coli O55:H7 ancestor to the contemporaneous globally dispersed strains observed today. In this study, the genomes of over 1000 isolates from both human clinical cases and cattle, spanning the history of STEC O157:H7 in the UK, were sequenced. Phylogenetic analysis revealed the ancestry, key acquisition events and global context of the strains. Dated phylogenies estimated the time to evolution of the most recent common ancestor of the current circulating global clone to be 175 years ago. This event was followed by rapid diversification. We show the acquisition of specific virulence determinates has occurred relatively recently and coincides with its recent detection in the human population. We used clinical outcome data from 493 cases of STEC O157:H7 to assess the relative risk of severe disease including haemolytic uraemic syndrome from each of the defined clades in the population and show the dramatic effect Shiga toxin repertoire has on virulence. We describe two strain replacement events that have occurred in the cattle population in the UK over the last 30 years, one resulting in a highly virulent strain that has accounted for the majority of clinical cases in the UK over the last decade. There is a need to understand the selection pressures maintaining Shiga-toxin-encoding bacteriophages in the ruminant reservoir and the study affirms the requirement for close surveillance of this pathogen in both ruminant and human populations.

Whole Genome Sequencing for Public Health Surveillance of Shiga Toxin-Producing Escherichia coli Other than Serogroup O157

Shiga toxin-producing Escherichia coli (STEC) are considered to be a significant threat to public health due to the severity of gastrointestinal symptoms associated with human infection. In England STEC O157 is the most commonly detected STEC serogroup, however, the implementation of PCR at local hospital laboratories has resulted in an increase in the detection of non-O157 STEC. The aim of this study was to evaluate the use of whole genome sequencing (WGS) for routine public health surveillance of non-O157 STEC by comparing this approach to phenotypic serotyping and PCR for subtyping the stx-encoding genes. Of the 102 isolates where phenotypic and genotypic serotyping could be compared, 98 gave fully concordant results. The most common non-O157 STEC serogroups detected were O146 (22) and O26 (18). All but one of the 38 isolates that could not be phenotypically serotyped (designated O unidentifiable or O rough) were serotyped using the WGS data. Of the 73 isolates where a flagella type was available by traditional phenotypic typing, all results matched the H-type derived from the WGS data. Of the 140 sequenced non-O157 isolates, 52 (37.1%) harboured stx1 only, 42 (30.0%) had stx2 only, 46 (32.9%) carried stx1 and stx2. Of these, stx subtyping PCR results were available for 131 isolates and 121 of these had concordant results with the stx subtype derived from the WGS data. Of the 10 discordant results, non-specific primer binding during PCR amplification, due to the similarity of the stx2 subtype gene sequences was the most likely cause. The results of this study showed WGS provided a reliable and robust one-step process for characterization of STEC. Deriving the full serotype from WGS data in real time has enabled us to report a higher level of strain discrimination while stx subtyping provides data on the pathogenic potential of each isolate, enabling us to predict clinical outcome of each case and to monitor the emergence of hyper-virulent strains.

Prospective use of whole genome sequencing (WGS) detected a multi-country outbreak of Salmonella Enteritidis.

Since April 2015, whole genome sequencing (WGS) has been the routine test for Salmonella identification, surveillance and outbreak investigation at the national reference laboratory in England and Wales. In May 2015, an outbreak of Salmonella Enteritidis cases was detected using WGS data and investigated. UK cases were interviewed to obtain a food history and links between suppliers were mapped to produce a food chain network for chicken eggs. The association between the food chain network and the phylogeny was explored using a network comparison approach. Food and environmental samples were taken from premises linked to cases and tested for Salmonella. Within the outbreak single nucleotide polymorphism defined cluster, 136 cases were identified in the UK and 18 in Spain. One isolate from a food containing chicken eggs was within the outbreak cluster. There was a significant association between the chicken egg food chain of UK cases and phylogeny of outbreak isolates. This is the first published Salmonella outbreak to be prospectively detected using WGS. This outbreak in the UK was linked with contemporaneous cases in Spain by WGS. We conclude that UK and Spanish cases were exposed to a common source of Salmonella-contaminated chicken eggs.

Whole Genome Sequencing demonstrates that Geographic Variation of Escherichia coli O157 Genotypes Dominates Host Association

Genetic variation in an infectious disease pathogen can be driven by ecological niche dissimilarities arising from different host species and different geographical locations. Whole genome sequencing was used to compare E. coli O157 isolates from host reservoirs (cattle and sheep) from Scotland and to compare genetic variation of isolates (human, animal, environmental/food) obtained from Scotland, New Zealand, Netherlands, Canada and the USA. Nei’s genetic distance calculated from core genome single nucleotide polymorphisms (SNPs) demonstrated that the animal isolates were from the same population. Investigation of the Shiga toxin bacteriophage and their insertion sites (SBI typing) revealed that cattle and sheep isolates had statistically indistinguishable rarefaction profiles, diversity and genotypes. In contrast, isolates from different countries exhibited significant differences in Nei’s genetic distance and SBI typing. Hence, after successful international transmission, which has occurred on multiple occasions, local genetic variation occurs, resulting in a global patchwork of continental and trans-continental phylogeographic clades. These findings are important for three reasons: first, understanding transmission and evolution of infectious diseases associated with multiple host reservoirs and multi-geographic locations; second, highlighting the relevance of the sheep reservoir when considering farm based interventions; and third, improving our understanding of why human disease incidence varies across the world.