Claire Jenkins

Temporal shedding patterns and virulence factors of Escherichia coli serogroups O26, O103, O111, O145, and O157 in a cohort of beef calves and their dams

ABSTRACT This study investigated the shedding of Escherichia coli O26, O103, O111, O145, and O157 in a cohort of beef calves from birth over a 5-month period and assessed the relationship between shedding in calves and shedding in their dams, the relationship between shedding and scouring in calves, and the effect of housing on shedding in calves. Fecal samples were tested by immunomagnetic separation and by PCR and DNA hybridization assays. E. coli O26 was shed by 94% of calves. Over 90% of E. coli O26 isolates carried the vtx1, eae, and ehl genes, 6.5% carried vtx1 and vtx2, and one isolate carried vtx2 only. Serogroup O26 isolates comprised seven pulsed-field gel electrophoresis (PFGE) patterns but were dominated by one pattern which represented 85.7% of isolates. E. coli O103 was shed by 51% of calves. Forty-eight percent of E. coli O103 isolates carried eae and ehl, 2% carried vtx2, and none carried vtx1. Serogroup O103 isolates comprised 10 PFGE patterns and were dominated by two patterns representing 62.5% of isolates. Shedding of E. coli O145 and O157 was rare. All serogroup O145 isolates carried eae, but none carried vtx1 or vtx2. All but one serogroup O157 isolate carried vtx2, eae, and ehl. E. coli O111 was not detected. In most calves, the temporal pattern of E. coli O26 and O103 shedding was random. E. coli O26 was detected in three times as many samples as E. coli O103, and the rate at which calves began shedding E. coli O26 for the first time was five times greater than that for E. coli O103. For E. coli O26, O103, and O157, there was no association between shedding by calves and shedding by dams within 1 week of birth. For E. coli O26 and O103, there was no association between shedding and scouring, and there was no significant change in shedding following housing.

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).

Subtyping intimin genes from enteropathogenic Escherichia coli associated with outbreaks and sporadic cases in the United Kingdom and Eire

PCR-RFLP methods for subtyping the intimin gene from strains of typical and atypical enteropathogenic Escherichia coli (EPEC) and Verocytotoxin-producing E. coli (VTEC) were compared. A novel HhaI PCR-RFLP method was developed that was rapid, easy to use and amplified an 1852 bp fragment of the intimin gene from all isolates examined. This method was used to investigate the intimin sub-types of EPEC strains associated with 14 outbreaks of diarrhoeal disease between 1967 and 2001, and 20 sporadic cases between January and December 2000, in the UK and Eire. In this study, genes encoding alpha, beta, gamma, delta and zeta-intimin were detected in the EPEC strains associated with outbreaks and beta, gamma, epsilon, theta and zeta-intimin genes were identified in isolates from sporadic cases. The beta-intimin gene was the most frequently detected sub-type in both the outbreak and sporadic strains.

Sex, drugs and smart phone applications: findings from semistructured interviews with men who have sex with men diagnosed with Shigella flexneri 3a in England and Wales

Objectives To inform control strategies undertaken as part of an outbreak of Shigella flexneri 3a among men who have sex with men (MSM). Methods All men aged ≥18 years diagnosed with S flexneri 3a between October 2012 and May 2013 were invited to participate. Semistructured in-depth quantitative interviews were conducted to explore lifestyle and sexual behaviour factors. Results Of 53 men diagnosed, 42 were interviewed of whom 34 were sexually active MSM. High numbers of sexual partners were reported (median=22) within the previous year; most were casual encounters met through social media networking sites (21/34). 63% (20/32) were HIV-positive and actively sought positive partners for condomless sex. 62% (21/34) of men had used chemsex drugs (mephedrone, crystal methamphetamine and γ-butyrolactone/γ-hydroxybutrate), which facilitate sexually disinhibiting behaviour during sexual encounters. 38% (8/21) reported injecting chemsex drugs. Where reported almost half (12/23) had attended or hosted sex parties. All reported oral–anal contact and fisting was common (16/34). Many had had gonorrhoea (23/34) and chlamydia (17/34). HIV-positive serostatus was associated with both insertive anal intercourse with a casual partner and receptive fisting (adjusted OR=15.0, p=0.01; adjusted OR=18.3, p=0.03) as was the use of web applications that promote and facilitate unprotected sex (adjusted OR=19.8, p=0.02). Conclusions HIV-positive MSM infected with S flexneri 3a used social media to meet sexual partners for unprotected sex mainly at sex parties. The potential for the transmission of S flexneri, HIV and other infections is clear. MSM need to be aware of the effect that chemsex drugs have on their health.

Public Health Investigation of Two Outbreaks of Shiga Toxin-Producing Escherichia coli O157 Associated with Consumption of Watercress

ABSTRACT An increase in the number of cases of Shiga toxin-producing Escherichia coli (STEC) O157 phage type 2 (PT2) in England in September 2013 was epidemiologically linked to watercress consumption. Whole-genome sequencing (WGS) identified a phylogenetically related cluster of 22 cases (outbreak 1). The isolates comprising this cluster were not closely related to any other United Kingdom strain in the Public Health England WGS database, suggesting a possible imported source. A second outbreak of STEC O157 PT2 (outbreak 2) was identified epidemiologically following the detection of outbreak 1. Isolates associated with outbreak 2 were phylogenetically distinct from those in outbreak 1. Epidemiologically unrelated isolates on the same branch as the outbreak 2 cluster included those from human cases in England with domestically acquired infection and United Kingdom domestic cattle. Environmental sampling using PCR resulted in the isolation of STEC O157 PT2 from irrigation water at one implicated watercress farm, and WGS showed this isolate belonged to the same phylogenetic cluster as outbreak 2 isolates. Cattle were in close proximity to the watercress bed and were potentially the source of the second outbreak. Transfer of STEC from the field to the watercress bed may have occurred through wildlife entering the watercress farm or via runoff water. During this complex outbreak investigation, epidemiological studies, comprehensive testing of environmental samples, and the use of novel molecular methods proved invaluable in demonstrating that two simultaneous outbreaks of STEC O157 PT2 were both linked to the consumption of watercress but were associated with different sources of contamination.

Assessment of a real-time PCR for the detection and characterization of verocytotoxigenic Escherichia coli

The European Union Reference Laboratory (EU-RL) has produced guidelines for a real-time PCR for the detection of verocytotoxigenic Escherichia coli (VTEC). In this study, we validated the EU-RL assay on 545 strains of VTEC and evaluated the utility of the assay for the detection of VTEC from stool specimens. The validation study on cultures showed that the EU-RL VTEC PCR was 99.3% sensitive for the detection of vtx genes; only strains harbouring vtx2f genes were not detected. The assay was 100% sensitive and 100% specific for the detection of both the eae and O157 rfbE genes. In a prospective study involving 500 stool samples, the EU-RL VTEC PCR detected vtx genes in 12.4% of specimens, compared to 3.8% specimens found to be culture-positive for E. coli O157 using the Health Protection Agency national standard culture method. This study showed that the EU-RL VTEC assay was reliable and robust, and an effective rapid screening method for the detection of VTEC from stool specimens.

Characterization of a Verocytotoxin-Producing Enteroaggregative Escherichia coli Serogroup O111:H21 Strain Associated with a Household Outbreak in Northern Ireland

ABSTRACT A strain of Escherichia coli O111:H21 recently isolated in the United Kingdom harbored the phage-encoded vtx2c gene and the aggregative adherence plasmid. Although exhibiting the same pathogenic profile as the E. coli O104:H4 strain linked to the outbreak in Germany, there were important differences in strain characteristics and in the epidemiological setting.

Comparison of the Distal Gut Microbiota from People and Animals in Africa

The gut microbiota plays a key role in the maintenance of healthy gut function as well as many other aspects of health. High-throughput sequence analyses have revealed the composition of the gut microbiota, showing that there is a core signature to the human gut microbiota, as well as variation in its composition between people. The gut microbiota of animals is also being investigated. We are interested in the relationship between bacterial taxa of the human gut microbiota and those in the gut microbiota of domestic and semi-wild animals. While it is clear that some human gut bacterial pathogens come from animals (showing that human – animal transmission occurs), the extent to which the usually non-pathogenic commensal taxa are shared between humans and animals has not been explored. To investigate this we compared the distal gut microbiota of humans, cattle and semi-captive chimpanzees in communities that are geographically sympatric in Uganda. The gut microbiotas of these three host species could be distinguished by the different proportions of bacterial taxa present. We defined multiple operational taxonomic units (OTUs) by sequence similarity and found evidence that some OTUs were common between human, cattle and chimpanzees, with the largest number of shared OTUs occurring between chimpanzees and humans, as might be expected with their close physiological similarity. These results show the potential for the sharing of usually commensal bacterial taxa between humans and other animals. This suggests that further investigation of this phenomenon is needed to fully understand how it drives the composition of human and animal gut microbiotas.