Martin Day

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.

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).

Plasmid-mediated Quinolone Resistance in Salmonella enterica, United Kingdom

To the Editor: Fluoroquinolones are broad-spectrum antimicrobial drugs used to treat many clinical infections. Salmonellosis is treated with fluoroquinolones only in elderly or immunocompromised patients, but these drugs are also used for treating patients with enteric fever, invasive disease, or long-term salmonellae carriage. High-level fluoroquinolone resistance is uncommon, but reduced susceptibility is increasing. Since 1998, plasmid-mediated quinolone resistance encoded by qnr genes A, B, and S that confer low-level resistance to nalidixic acid and reduced susceptibility to ciprofloxacin has been identified in several enterobacterial species, including Salmonella. Their clinical importance is in facilitating resistance to potentially lethal levels of quinolone. Additionally, qnr genes are often associated with strains that produce extended-spectrum β-lactamases. We recently reported identification of qnr genes in Salmonella in the United Kingdom (1). Most isolates were associated with the Far East. Two isolates of S. Virchow were part of an outbreak associated with imported cooked chicken from Thailand. During October 2006–April 2007, we monitored qnr genes in nontyphoidal salmonellae isolated in the United Kingdom that expressed reduced susceptibility to ciprofloxacin (MIC 0.125–1.0 μg/mL) with concomitant susceptibility to nalidixic acid (MIC <16 μg/mL). This resistance phenotype is a useful marker for the qnr gene as the sole quinolone resistance determinant (1). Recent studies showed that isolates of Salmonella spp. and Escherichia coli with decreased susceptibility to ciprofloxacin (MICs >0.06 μg/mL and 0.5 μg/mL, respectively), but with susceptibility or intermediate resistance to nalidixic acid (MIC 8–16 μg/mL and 4–8 μg/mL, respectively), all had qnrA or qnrS genes but lacked mutations in the topoisomerase genes (2,3). Strains with ciprofloxacin MICs >1 μg/mL were also included to monitor involvement of qnr genes in development of high-level ciprofloxacin resistance. Breakpoint concentrations used are based on long-term studies within the Health Protection Agency Laboratory of Enteric Pathogens. Ciprofloxacin Etest (AB Biodisk, Solna, Sweden) results were interpreted according to manufacturer’s procedures. A total of 45 Salmonella spp. strains were tested. Screening for qnr genes by multiplex PCR identified 37 isolates with qnrS and 2 carrying qnrB variants (Table) (4). However, the qnrB primer pair in this multiplex did not fully match all qnrB gene variants. PCR and sequencing using primers FQ1 and FQ2 (5) and qnrS-F and qnrS-R (1), were used to identify specific qnrB and qnrS gene variants. Table Isolates of Salmonella enterica with plasmid-mediated qnr genes, United Kingdom, October 2006–April 2007 The qnrS1-positive salmonellae belong to serotypes Typhimurium (21 isolates), Virchow (10), and Corvallis (6). Most S. Typhimurium isolates were either definitive phage type 120 or 193, and most S. Virchow isolates were phage type 43 (Table). Thirteen qnrS1-positive isolates were from patients who reported recent travel to Egypt, India, Malaysia, Morocco, Thailand, or an undisclosed destination. Twelve isolates from patients who had not traveled abroad were assumed to be from UK-acquired infections. S. Virchow isolates had been associated with cooked chicken from Thailand (1), and qnrS1 has recently been described in S. Corvallis strains from humans in Denmark or isolated in Thailand from humans, chicken, pork, and beef (3). Comparison of pulsed-field gel electrophoresis patterns and resistance phenotypes of qnrS1-positive S. Corvallis strains identified common types, suggesting that some UK patients may have acquired S. Corvallis from chicken from Thailand. Thirteen isolates showed resistance to ceftriaxone, cefotaxime, or ampicillin. Plasmids with qnr genes have been found to co-transfer TEM, SHV, and CTX-M genes (1,5,6). Co-transmission of fluoroquinolone and β-lactamase resistance is clinically important because co-selection of resistance by use of either drug may occur. Twenty-one qnrS1-positive S. Typhimurium were subtyped by variable number tandem repeat (VNTR) analysis to determine whether the increase was caused by spread of >1 distinct strains (7). Twenty isolates produced 1 of 3 related profiles (loci of VNTR profiles are ordered STTR9-STTR5-STTR6-STTR10pl-STTR3): 1–4-0–0-3, 9 isolates; 1–5-0–0-3, 3 isolates; or 1–6-0–0-3, 8 isolates. Alleles 4 and 5, and 5 and 6 at locus STTR5 only differed by an extra 6-bp repeat, which suggests a clonal relationship between the qnrS1-positive S. Typhimurium in this study (Table) (8). S. Typhimurium isolates with the 1–6-0–0-3 profile have been isolated from tourists returning from Asia (7), which suggests that the UK qnrS1-positive S. Typhimurium isolates have originated in the Far East. These findings show increased occurrence of qnr genes, particularly qnrS1, in nontyphoidal salmonellae in the United Kingdom. These data are in contrast to those of recent studies in the United States and France, which show low incidences of qnrS genes in larger strain collections (9,10). The qnr phenotype is in contrast to resistance mediated by mutations in the topoisomerase genes whereby 1 mutation confers low-level resistance to fluoroquinolones and full resistance to nalidixic acid. Our previous study demonstrated that qnrS1 was sufficient to cause decreased susceptibility to ciprofloxacin in the absence of mutations in gyrA (1). In this study, a qnr gene was sufficient to increase the ciprofloxacin MIC to 0.38–0.75 μg/mL. In addition, a qnr gene contributed to high-level ciprofloxacin resistance in 10 isolates, thereby potentially jeopardizing first-line treatment of vulnerable patient groups with ciprofloxacin.

Chromosomal location of blaCTX-M genes in clinical isolates of Escherichia coli from Germany, The Netherlands and the UK

This study aimed to detect and characterise clinical Escherichia coli isolates suspected of carrying chromosomally encoded CTX-M enzymes. Escherichia coli (n=356) obtained in Germany, The Netherlands and the UK (2005-2009) and resistant to third-generation cephalosporins were analysed for the presence of ESBL-/AmpC-encoding genes within the European SAFEFOODERA-ESBL project. β-Lactamases and their association with IS26 and ISEcp1 were investigated by PCR. Isolates were typed by phylogenetic grouping, MLST and PFGE. Plasmids were visualised by S1 nuclease PFGE, and the location of blaCTX-M genes was determined by Southern hybridisation of XbaI-, S1- and I-CeuI-digested DNA. ESBL enzymes could not be located on plasmids in 17/356 isolates (4.8%). These 17 isolates, from different countries and years, were ascribed to phylogenetic groups D (9), B2 (6) and B1 (2), and to seven sequence types, with ST38 being the most frequent (7 phylogroup D isolates). Eleven isolates produced CTX-M-15. blaCTX-M-15 genes were associated with ISEcp1. The remaining isolates expressed the CTX-M group 9 β-lactamases CTX-M-14 (4), CTX-M-9 (1) and CTX-M-51 (1). blaCTX-M probes hybridised with I-CeuI- and/or XbaI-digested DNA, but not with S1-digested DNA, corroborating their chromosomal location. To summarise, only 4.8% of a large collection of ESBL-producing E. coli isolates harboured chromosomal blaCTX-M genes. These isolates were of human origin and belonged predominantly to ST38 and ST131, which possibly indicates the role of these sequence types in this phenomenon. However, heterogeneity among isolates was found, suggesting that their spread is not only due to the dispersion of successful E. coli clones.

The extant World War 1 dysentery bacillus NCTC1: a genomic analysis

Summary Background Shigellosis (previously bacillary dysentery) was the primary diarrhoeal disease of World War 1, but outbreaks still occur in military operations, and shigellosis causes hundreds of thousands of deaths per year in developing nations. We aimed to generate a high-quality reference genome of the historical Shigella flexneri isolate NCTC1 and to examine the isolate for resistance to antimicrobials. Methods In this genomic analysis, we sequenced the oldest extant Shigella flexneri serotype 2a isolate using single-molecule real-time (SMRT) sequencing technology. Isolated from a soldier with dysentery from the British forces fighting on the Western Front in World War 1, this bacterium, NCTC1, was the first isolate accessioned into the National Collection of Type Cultures. We created a reference sequence for NCTC1, investigated the isolate for antimicrobial resistance, and undertook comparative genetics with S flexneri reference strains isolated during the 100 years since World War 1. Findings We discovered that NCTC1 belonged to a 2a lineage of S flexneri, with which it shares common characteristics and a large core genome. NCTC1 was resistant to penicillin and erythromycin, and contained a complement of chromosomal antimicrobial resistance genes similar to that of more recent isolates. Genomic islands gained in the S flexneri 2a lineage over time were predominately associated with additional antimicrobial resistances, virulence, and serotype conversion. Interpretation This S flexneri 2a lineage is a well adapted pathogen that has continued to respond to selective pressures. We have created a valuable historical benchmark for shigellae in the form of a high-quality reference sequence for a publicly available isolate. Funding The Wellcome Trust.

Revolutionising Public Health Reference Microbiology using Whole Genome Sequencing: Salmonella as an exemplar

Advances in whole genome sequencing (WGS) platforms and DNA library preparation have led to the development of methods for high throughput sequencing of bacterial genomes at a relatively low cost (Loman et al. 2012; Medini et al. 2008). WGS offers unprecedented resolution for determining degrees of relatedness between strains of bacterial pathogens and has proven a powerful tool for microbial population studies and epidemiological investigations (Harris et al. 2010; Lienau et al. 2011; Holt et al. 2009; Ashton, Peters, et al. 2015). The potential utility of WGS to public health microbiology has been highlighted previously (Köser et al. 2012; Kwong et al. 2013; Reuter et al. 2013; Joensen et al. 2014; Nair et al. 2014; Bakker et al. 2014; D’Auria et al. 2014). Here we report, for the first time, the routine use of WGS as the primary test for identification, surveillance and outbreak investigation by a national reference laboratory. We present data on how this has revolutionised public health microbiology for one of the most common bacterial pathogens in the United Kingdom, the Salmonellae. DATA SUMMARY 1. PHE Salmonella sequencing data is deposited in the Sequence Read Archive in BioProject PRJNA248792. IMPACT STATEMENT The first human genome cost around

ESBL-Producing and Macrolide-Resistant Shigella sonnei Infections among Men Who Have Sex with Men, England, 2015

3 billion, and took around 10 years to complete. Advances in DNA sequencing technology (also referred to as whole genome sequencing (WGS)) allow the same feat to be accomplished today for less than

Clinical Isolates of Salmonella enterica Serovar Agona Producing NDM-1 Metallo-β-Lactamase: First Report from Pakistan

10000 and less than 2 weeks. This remarkable improvement in technology has also led to a step change in microbiology, increasing our understanding of the evolution of major human pathogens such as Yersinia pestis, Salmonella Typhi and Mycobacterium tuberculosis. While these kinds of academic studies provide unparalleled context for public health action, until now, this approach has not been routinely employed at the frontline. At Public Health England, WGS has been implemented for routine public health identification, characterisation and typing of an important human pathogen, Salmonella, replacing methods that have changed little over the last 100 years. Analysis of WGS data has identified outbreaks that were previously undetectable and been used to infer rare antimicrobial resistance patterns. This paper will serve as a notification to the community of the methods PHE are using, and will be of great use to other public health labs considering switching to WGS.

Comparison of phenotypic and WGS-derived antimicrobial resistance profiles of Shigella sonnei isolated from cases of diarrhoeal disease in England and Wales, 2015

In England in 2015, Shigella sonnei isolates from men who have sex with men produced extended-spectrum β-lactamases and exhibited macrolide resistance. Whole-genome sequencing showed a close relationship among the isolates, which harbored a plasmid that was previously identified in a shigellosis outbreak among this population but has acquired a mobile element.

Antimicrobial resistance in Shiga toxin-producing Escherichia coli serogroups O157 and O26 isolated from human cases of diarrhoeal disease in England, 2015

ABSTRACT We report two cases of infantile diarrhea due to multidrug-resistant, NDM-1 metallo-β-lactamase-producing Salmonella enterica serovar Agona from Pakistan. This study alerts toward possible risk of NDM-1 transmission to enteric fever pathogens and encourages microbiologists to consider active screening of carbapenem resistance in nontyphoidal Salmonella isolates.