Beth Blane

Building a genomic framework for prospective MRSA surveillance in the United Kingdom and the Republic of Ireland.

The correct interpretation of microbial sequencing data applied to surveillance and outbreak investigation depends on accessible genomic databases to provide vital genetic context. Our aim was to construct and describe a United Kingdom MRSA database containing over 1000 methicillin-resistant Staphylococcus aureus (MRSA) genomes drawn from England, Northern Ireland, Wales, Scotland, and the Republic of Ireland over a decade. We sequenced 1013 MRSA submitted to the British Society for Antimicrobial Chemotherapy by 46 laboratories between 2001 and 2010. Each isolate was assigned to a regional healthcare referral network in England and was otherwise grouped based on country of origin. Phylogenetic reconstructions were used to contextualize MRSA outbreak investigations and to detect the spread of resistance. The majority of isolates (n = 783, 77%) belonged to CC22, which contains the dominant United Kingdom epidemic clone (EMRSA-15). There was marked geographic structuring of EMRSA-15, consistent with widespread dissemination prior to the sampling decade followed by local diversification. The addition of MRSA genomes from two outbreaks and one pseudo-outbreak demonstrated the certainty with which outbreaks could be confirmed or refuted. We identified local and regional differences in antibiotic resistance profiles, with examples of local expansion, as well as widespread circulation of mobile genetic elements across the bacterial population. We have generated a resource for the future surveillance and outbreak investigation of MRSA in the United Kingdom and Ireland and have shown the value of this during outbreak investigation and tracking of antimicrobial resistance.

Whole-genome sequencing reveals transmission of vancomycin-resistant Enterococcus faecium in a healthcare network

BackgroundBacterial whole-genome sequencing (WGS) has the potential to identify reservoirs of multidrug-resistant organisms and transmission of these pathogens across healthcare networks. We used WGS to define transmission of vancomycin-resistant enterococci (VRE) within a long-term care facility (LTCF), and between this and an acute hospital in the United Kingdom (UK).MethodsA longitudinal prospective observational study of faecal VRE carriage was conducted in a LTCF in Cambridge, UK. Stool samples were collected at recruitment, and then repeatedly until the end of the study period, discharge or death. Selective culture media were used to isolate VRE, which were subsequently sequenced and analysed. We also analysed the genomes of 45 Enterococcus faecium bloodstream isolates collected at Cambridge University Hospitals NHS Foundation Trust (CUH).ResultsForty-five residents were recruited during a 6-month period in 2014, and 693 stools were collected at a frequency of at least 1 week apart. Fifty-one stool samples from 3/45 participants (7 %) were positive for vancomycin-resistant E. faecium. Two residents carried multiple VRE lineages, and one carried a single VRE lineage. Genome analyses based on single nucleotide polymorphisms (SNPs) in the core genome indicated that VRE carried by each of the three residents were unrelated. Participants had extensive contact with the local healthcare network. We found that VRE genomes from LTCF residents and hospital-associated bloodstream infection were interspersed throughout the phylogenetic tree, with several instances of closely related VRE strains from the two settings.ConclusionsA proportion of LTCF residents are long-term carriers of VRE. Evidence for genetic relatedness between these and VRE associated with bloodstream infection in a nearby acute NHS Trust indicate a shared bacterial population.

Clonal differences in Staphylococcus aureus bacteraemia-associated mortality

The bacterium Staphylococcus aureus is a major human pathogen for which the emergence of antibiotic resistance is a global public health concern. Infection severity, and in particular bacteraemia-associated mortality, has been attributed to several host-related factors, such as age and the presence of comorbidities. The role of the bacterium in infection severity is less well understood, as it is complicated by the multifaceted nature of bacterial virulence, which has so far prevented a robust mapping between genotype, phenotype and infection outcome. To investigate the role of bacterial factors in contributing to bacteraemia-associated mortality, we phenotyped a collection of sequenced clinical S. aureus isolates from patients with bloodstream infections, representing two globally important clonal types, CC22 and CC30. By adopting a genome-wide association study approach we identified and functionally verified several genetic loci that affect the expression of cytolytic toxicity and biofilm formation. By analysing the pooled data comprising bacterial genotype and phenotype together with clinical metadata within a machine-learning framework, we found significant clonal differences in the determinants most predictive of poor infection outcome. Whereas elevated cytolytic toxicity in combination with low levels of biofilm formation was predictive of an increased risk of mortality in infections by strains of a CC22 background, these virulence-specific factors had little influence on mortality rates associated with CC30 infections. Our results therefore suggest that different clones may have adopted different strategies to overcome host responses and cause severe pathology. Our study further demonstrates the use of a combined genomics and data analytic approach to enhance our understanding of bacterial pathogenesis at the individual level, which will be an important step towards personalized medicine and infectious disease management.A genome-wide association approach identifies differential biofilm and virulence attributes associated with mortality in two Staphylococcus aureus clonal complexes.

Systematic Surveillance Detects Multiple Silent Introductions and Household Transmission of Methicillin-Resistant Staphylococcus aureus USA300 in the East of England

Background. The spread of USA300 methicillin-resistant Staphylococcus aureus (MRSA) across the United States resulted in an epidemic of infections. In Europe, only sporadic cases or small clusters of USA300 infections are described, and its prevalence in England is unknown. We conducted prospective surveillance for USA300 in the east of England. Methods. We undertook a 12-month prospective observational cohort study of all individuals with MRSA isolated from community and hospital samples submitted to a microbiology laboratory. At least 1 MRSA isolate from each individual underwent whole-genome sequencing. USA300 was identified on the basis of sequence analysis, and phylogenetic comparisons were made between these and USA300 genomes from the United States. Results. Between April 2012 and April 2013, we sequenced 2283 MRSA isolates (detected during carriage screening and in clinical samples) from 1465 individuals. USA300 was isolated from 24 cases (1.6%). Ten cases (42%) had skin and soft tissue infection, and 2 cases had invasive disease. Phylogenetic analyses identified multiple introductions and household transmission of USA300. Conclusions. Use of a diagnostic laboratory as a sentinel for surveillance has identified repeated introductions of USA300 in eastern England in 2012–2013, with evidence for limited transmission. Our results show how systematic surveillance could provide an early warning of strain emergence and dissemination.

Transmission of methicillin-resistant Staphylococcus aureus in long-term care facilities and their related healthcare networks

BackgroundLong-term care facilities (LTCF) are potential reservoirs for methicillin-resistant Staphylococcus aureus (MRSA), control of which may reduce MRSA transmission and infection elsewhere in the healthcare system. Whole-genome sequencing (WGS) has been used successfully to understand MRSA epidemiology and transmission in hospitals and has the potential to identify transmission between these and LTCF.MethodsTwo prospective observational studies of MRSA carriage were conducted in LTCF in England and Ireland. MRSA isolates were whole-genome sequenced and analyzed using established methods. Genomic data were available for MRSA isolated in the local healthcare systems (isolates submitted by hospitals and general practitioners).ResultsWe sequenced a total of 181 MRSA isolates from the two study sites. The majority of MRSA were multilocus sequence type (ST)22. WGS identified one likely transmission event between residents in the English LTCF and three putative transmission events in the Irish LTCF. WGS also identified closely related isolates present in colonized Irish residents and their immediate environment. Based on phylogenetic reconstruction, closely related MRSA clades were identified between the LTCF and their healthcare referral network, together with putative MRSA acquisition by LTCF residents during hospital admission.ConclusionsThese data confirm that MRSA is transmitted between residents of LTCF and is both acquired and transmitted to others in referral hospitals and beyond. Our data present compelling evidence for the importance of environmental contamination in MRSA transmission, reinforcing the importance of environmental cleaning. The use of WGS in this study highlights the need to consider infection control in hospitals and community healthcare facilities as a continuum.

Evolution of the Staphylococcus argenteus ST2250 Clone in Northeastern Thailand Is Linked with the Acquisition of Livestock-Associated Staphylococcal Genes

ABSTRACT Staphylococcus argenteus is a newly named species previously described as a divergent lineage of Staphylococcus aureus that has recently been shown to have a global distribution. Despite growing evidence of the clinical importance of this species, knowledge about its population epidemiology and genomic architecture is limited. We used whole-genome sequencing to evaluate and compare S. aureus (n = 251) and S. argenteus (n = 68) isolates from adults with staphylococcal sepsis at several hospitals in northeastern Thailand between 2006 and 2013. The majority (82%) of the S. argenteus isolates were of multilocus sequence type 2250 (ST2250). S. aureus was more diverse, although 43% of the isolates belonged to ST121. Bayesian analysis suggested an S. argenteus ST2250 substitution rate of 4.66 (95% confidence interval [CI], 3.12 to 6.38) mutations per genome per year, which was comparable to the S. aureus ST121 substitution rate of 4.07 (95% CI, 2.61 to 5.55). S. argenteus ST2250 emerged in Thailand an estimated 15 years ago, which contrasts with the S. aureus ST1, ST88, and ST121 clades that emerged around 100 to 150 years ago. Comparison of S. argenteus ST2250 genomes from Thailand and a global collection indicated a single introduction into Thailand, followed by transmission to local and more distant countries in Southeast Asia and further afield. S. argenteus and S. aureus shared around half of their core gene repertoire, indicating a high level of divergence and providing strong support for their classification as separate species. Several gene clusters were present in ST2250 isolates but absent from the other S. argenteus and S. aureus study isolates. These included multiple exotoxins and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus, consistent with a livestock reservoir for S. argenteus. These genes appeared to be associated with plasmids and mobile genetic elements and may have contributed to the biological success of ST2250. IMPORTANCE In this study, we used whole-genome sequencing to understand the genome evolution and population structure of a systematic collection of ST2250 S. argenteus isolates. A newly identified ancestral species of S. aureus, S. argenteus has become increasingly known as a clinically important species that has been reported recently across various countries. Our results indicate that S. argenteus has spread at a relatively rapid pace over the past 2 decades across northeastern Thailand and acquired multiple exotoxin and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus. Our findings highlight the clinical importance and potential pathogenicity of S. argenteus as a recently emerging pathogen. IMPORTANCE In this study, we used whole-genome sequencing to understand the genome evolution and population structure of a systematic collection of ST2250 S. argenteus isolates. A newly identified ancestral species of S. aureus, S. argenteus has become increasingly known as a clinically important species that has been reported recently across various countries. Our results indicate that S. argenteus has spread at a relatively rapid pace over the past 2 decades across northeastern Thailand and acquired multiple exotoxin and antibiotic resistance genes that have been linked previously with livestock-associated S. aureus. Our findings highlight the clinical importance and potential pathogenicity of S. argenteus as a recently emerging pathogen.

PBP2a substitutions linked to ceftaroline resistance in MRSA isolates from the UK

Sir, Ceftaroline is a new cephalosporin antibiotic with activity against MRSA. Binding of this drug to the allosteric domain of PBP2a leads to a conformational change that allows a second molecule of ceftaroline to bind to the active site, blocking its activity.1 Recent reports have described MRSA isolates with low-level (>1–8 mg/L) and high-level (>32 mg/L) resistance to ceftaroline.2–5 These have been isolated in geographically dispersed locations (Greece, Spain, Switzerland, Thailand and the USA) and have occurred in numerous genetic backgrounds: ST5 [clonal complex (CC) 5], ST228 (CC5), ST239 (CC8), ST247 (CC8) and ST764 (CC5).2–5 Low-level resistance has been associated with N146K, E150K and E239K substitutions in the allosteric binding domain of PBP2a.1–3 Recent data have shown that the N146K/E150K substitutions mediate resistance by interrupting the allosteric response of ceftaroline binding, preventing a second molecule of ceftaroline blocking the active site.6 Higher-level resistance (8 to >32 mg/L) is mediated by E447K and Y446N/E447K substitutions in the ceftaroline-binding pocket of the transpeptidase region of PBP2a.2,5 Furthermore, the E447K substitution has been identified in laboratory-generated, ceftaroline-resistant isolates, along with other chromosomal mutations likely to be involved in high-level resistance.7 We sought the presence of these mutations in the whole-genome sequence data of 458 MRSA isolates cultured from humans (n = 397) or animals (n = 61). Isolates from humans were predominantly drawn from the east of England between 1985 and 1987 (n = 180) or between 2006 and 2013 (n = 191), with the remainder drawn from other regions of England and Scotland between 2010 and 2012.8,9 Animal isolates were cultured from dogs (41), cats (3), horses (4) or cattle (13) in the UK.8 We identified three isolates (0.66%) that contained substitutions previously reported to mediate ceftaroline resistance (Table ​(Table1).1). Two isolates (ASARM167 and A38) belonging to ST22 (epidemic MRSA-15) had an E239K substitution in PBP2a. ASARM167 was isolated from a patient with bacteraemia at Cambridge University Hospitals NHS Foundation Trust (CUH) in 2008 and A38 was isolated from a canine wound infection in 2006 treated in Wiltshire, south-west England.8 Phylogenetic analysis of these two isolates based on core genome SNPs placed them in different clades separated by >120 SNPs (data not shown), indicating that the E239K mutation arose independently in these two isolates. The third isolate (ASARM130) had the N146K substitution in PBP2a, belonged to ST241 (CC8) and was isolated from a patient with bacteraemia at CUH in 2007. This isolate was also noted to have an N204K substitution, which has not been reported previously in isolates with the N146K substitution.2,3 Table 1. Characteristics of three MRSA isolates with PBP2a substitutions associated previously with ceftaroline resistance The effect of these PBP2a substitutions on the ceftaroline resistance phenotype was evaluated for these three isolates using the disc diffusion assay based on EUCAST guidelines10 and the Etest (bioMerieux, Lyon, France) according to the manufacturers instructions. Two isolates were susceptible and one was resistant to ceftaroline by disc diffusion, but all three isolates were susceptible by Etest (Table ​(Table1).1). Although isolates with the N146K substitution have been reported previously to have an MIC of 0.5 mg/L (susceptible), all previously reported isolates with E239K had an MIC of ≥2 mg/L (resistant).2,4 The lack of association between a resistant phenotype and the N146K substitution indicates that secondary chromosomal mutations are likely to be involved, as reported previously.4,7 The three study isolates were cultured before the clinical introduction of ceftaroline into clinical practice in the USA in 2010 and Europe in 2012, demonstrating that these are natural variants of PBP2a that occur (albeit at low prevalence) even without pressure from ceftaroline use. All previously reported isolates with PBP2a substitutions mediating ceftaroline resistance belonged to CC5 and CC8, which has led to the suggestion that these two lineages might be more prone to such mutations. Our findings suggest that they probably occur in multiple MRSA lineages including the pandemic CC22 lineage, which is important in many parts of the world including Australia and the Middle East and is the dominant MRSA lineage in the UK and much of Europe.

Longitudinal genomic surveillance of MRSA in the UK reveals transmission patterns in hospitals and the community

Longitudinal genomic and epidemiological surveillance of methicillin-resistant Staphylococcus aureus in the UK reveals extensive transmission in hospitals and the community. On the trail of MRSA Genome sequencing of methicillin-resistant Staphylococcus aureus (MRSA) has been successfully applied to investigate suspected outbreaks. Coll et al. now extend its application to the genomic surveillance of MRSA in samples from 1465 people identified over a 12-month period by a diagnostic laboratory in the East of England. This analysis identified 173 putative outbreaks involving 598 patients and included hospital outbreaks, those spanning the hospital and community, and community outbreaks among people registered with the same medical practice or living in the same household or long-term care facility. This study illustrates that sequencing is a powerful tool that could be used to identify infectious disease outbreaks as they happen. Genome sequencing has provided snapshots of the transmission of methicillin-resistant Staphylococcus aureus (MRSA) during suspected outbreaks in isolated hospital wards. Scale-up to populations is now required to establish the full potential of this technology for surveillance. We prospectively identified all individuals over a 12-month period who had at least one MRSA-positive sample processed by a routine diagnostic microbiology laboratory in the East of England, which received samples from three hospitals and 75 general practitioner (GP) practices. We sequenced at least 1 MRSA isolate from 1465 individuals (2282 MRSA isolates) and recorded epidemiological data. An integrated epidemiological and phylogenetic analysis revealed 173 transmission clusters containing between 2 and 44 cases and involving 598 people (40.8%). Of these, 118 clusters (371 people) involved hospital contacts alone, 27 clusters (72 people) involved community contacts alone, and 28 clusters (157 people) had both types of contact. Community- and hospital-associated MRSA lineages were equally capable of transmission in the community, with instances of spread in households, long-term care facilities, and GP practices. Our study provides a comprehensive picture of MRSA transmission in a sampled population of 1465 people and suggests the need to review existing infection control policy and practice.

Longitudinal genomic surveillance of multidrug-resistant Escherichia coli carriage in a long-term care facility in the United Kingdom

BackgroundResidents of long-term care facilities (LTCF) may have high carriage rates of multidrug-resistant pathogens, but are not currently included in surveillance programmes for antimicrobial resistance or healthcare-associated infections. Here, we describe the value derived from a longitudinal epidemiological and genomic surveillance study of drug-resistant Escherichia coli in a LTCF in the United Kingdom (UK).MethodsForty-five of 90 (50%) residents were recruited and followed for six months in 2014. Participants were screened weekly for carriage of extended-spectrum beta-lactamase (ESBL) producing E. coli. Participants positive for ESBL E. coli were also screened for ESBL-negative E. coli. Phenotypic antibiotic susceptibility of E. coli was determined using the Vitek2 instrument and isolates were sequenced on an Illumina HiSeq2000 instrument. Information was collected on episodes of clinical infection and antibiotic consumption.ResultsSeventeen of 45 participants (38%) carried ESBL E. coli. Twenty-three of the 45 participants (51%) had 63 documented episodes of clinical infection treated with antibiotics. Treatment with antibiotics was associated with higher risk of carrying ESBL E. coli. ESBL E. coli was mainly sequence type (ST)131 (16/17, 94%). Non-ESBL E. coli from these 17 cases was more genetically diverse, but ST131 was found in eight (47%) cases. Whole-genome analysis of 297 ST131 E. coli from the 17 cases demonstrated highly related strains from six participants, indicating acquisition from a common source or person-to-person transmission. Five participants carried highly related strains of both ESBL-positive and ESBL-negative ST131. Genome-based comparison of ST131 isolates from the LTCF study participants with ST131 associated with bloodstream infection at a nearby acute hospital and in hospitals across England revealed sharing of highly related lineages between the LTCF and a local hospital.ConclusionsThis study demonstrates the power of genomic surveillance to detect multidrug-resistant pathogens and confirm their connectivity within a healthcare network.

Comparison of two chromogenic media for the detection of vancomycin-resistant enterococcal carriage by nursing home residents

We compared ChromID VRE and Brilliance VRE media for the detection of vancomycin-resistant enterococci (VRE). Using a panel of 28 enterococcal isolates, 10 vanA Enterococcus faecium and three vanA Enterococcus faecalis isolates grew as per manufacturers’ instructions whilst growth of two vanC and eight vancomycin-susceptible enterococci was inhibited on both media. Important differences were noted in the selectivity and chromogenic properties of the two media for vanA Enterococcus raffinosus and vanB E. faecium. The two media were further evaluated using 295 stool samples from nursing home residents, 34 of which grew VRE (11.5%). ChromID and Brilliance had comparable sensitivity, which was increased markedly by prolonging incubation to 48 hours (from 29% to 82%, and from 41% to 85%, respectively) and by a pre-enrichment step (to 97% and 100%, respectively). Brilliance VRE agar had higher selectivity at 48 hours, and after pre-enrichment.