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Bioinformatics and Computational Biology analyses were supported by the University of St Andrews Bioinformatics Unit which is funded by a Wellcome Trust ISSF award [grant 097831/Z/11/Z]. The SHAIPI consortium is funded by the Chief Scientist Office through the Scottish Infection Research Network (SIRN10). Outbreaks pose a significant patient safety risk as well as being costly and time consuming to investigate. The implementation of targeted infection prevention and control (IPC) measures relies on infection prevention and control teams (IPCTs) having access to rapid results that accurately detect resistance, and typing results that give clinically useful information on the relatedness of isolates. At present, determining whether transmission has occurred can be a major challenge. Conventional typing results do not always have sufficient granularity or robustness to unequivocally define strains, and sufficient epidemiological data to establish links between patients and the environment is not always available. Whole genome sequencing (WGS) has emerged as the ultimate genotyping tool, but has not yet fully crossed the divide between research method and routine clinical diagnostic microbiology technique. A clinical WGS service was officially established in 2014 as part of the Scottish Healthcare Associated Infection Prevention Institute (SHAIPI) to confirm or refute outbreaks in hospital settings from across Scotland. In this personal view we describe our experiences that we believe provide new insights into the practical application of the use of WGS to investigate healthcare and public health outbreaks. We also propose solutions to overcome barriers to implementation of this technology in a clinical environment. Publisher PDF

This work was supported by the BBSRC Grant BB/N006615/1. Body odour is a characteristic trait of Homo sapiens, however its role in human behaviour and evolution is poorly understood. Remarkably, body odour is linked to the presence of a few species of commensal microbes. Herein we discover a bacterial enzyme, limited to odour-forming staphylococci that are able to cleave odourless precursors of thioalcohols, the most pungent components of body odour. We demonstrated using phylogenetics, biochemistry and structural biology that this cysteine-thiol lyase (C-T lyase) is a PLP-dependent enzyme that moved horizontally into a unique monophyletic group of odour-forming staphylococci about 60 million years ago, and has subsequently tailored its enzymatic function to human-derived thioalcohol precursors. Significantly, transfer of this enzyme alone to non-odour producing staphylococci confers odour production, demonstrating that this C-T lyase is both necessary and sufficient for thioalcohol formation. The structure of the C-T lyase compared to that of other related enzymes reveals how the adaptation to thioalcohol precursors has evolved through changes in the binding site to create a constrained hydrophobic pocket that is selective for branched aliphatic thioalcohol ligands. The ancestral acquisition of this enzyme, and the subsequent evolution of the specificity for thioalcohol precursors implies that body odour production in humans is an ancient process. Publisher PDF

Summary Background BNT162b2 mRNA and ChAdOx1 nCOV-19 adenoviral vector vaccines have been rapidly rolled out in the UK from December, 2020. We aimed to determine the factors associated with vaccine coverage for both vaccines and documented the vaccine effectiveness of the BNT162b2 mRNA vaccine in a cohort of health-care workers undergoing regular asymptomatic testing. Methods The SIREN study is a prospective cohort study among staff (aged ≥18 years) working in publicly-funded hospitals in the UK. Participants were assigned into either the positive cohort (antibody positive or history of infection [indicated by previous positivity of antibody or PCR tests]) or the negative cohort (antibody negative with no previous positive test) at the beginning of the follow-up period. Baseline risk factors were collected at enrolment, symptom status was collected every 2 weeks, and vaccination status was collected through linkage to the National Immunisations Management System and questionnaires. Participants had fortnightly asymptomatic SARS-CoV-2 PCR testing and monthly antibody testing, and all tests (including symptomatic testing) outside SIREN were captured. Data cutoff for this analysis was Feb 5, 2021. The follow-up period was Dec 7, 2020, to Feb 5, 2021. The primary outcomes were vaccinated participants (binary ever vacinated variable; indicated by at least one vaccine dose recorded by at least one of the two vaccination data sources) for the vaccine coverage analysis and SARS-CoV-2 infection confirmed by a PCR test for the vaccine effectiveness analysis. We did a mixed-effect logistic regression analysis to identify factors associated with vaccine coverage. We used a piecewise exponential hazard mixed-effects model (shared frailty-type model) using a Poisson distribution to calculate hazard ratios to compare time-to-infection in unvaccinated and vaccinated participants and estimate the impact of the BNT162b2 vaccine on all PCR-positive infections (asymptomatic and symptomatic). This study is registered with ISRCTN, number ISRCTN11041050, and is ongoing. Findings 23 324 participants from 104 sites (all in England) met the inclusion criteria for this analysis and were enrolled. Included participants had a median age of 46·1 years (IQR 36·0–54·1) and 19 692 (84%) were female; 8203 (35%) were assigned to the positive cohort at the start of the analysis period, and 15 121 (65%) assigned to the negative cohort. Total follow-up time was 2 calendar months and 1 106 905 person-days (396 318 vaccinated and 710 587 unvaccinated). Vaccine coverage was 89% on Feb 5, 2021, 94% of whom had BNT162b2 vaccine. Significantly lower coverage was associated with previous infection, gender, age, ethnicity, job role, and Index of Multiple Deprivation score. During follow-up, there were 977 new infections in the unvaccinated cohort, an incidence density of 14 infections per 10 000 person-days; the vaccinated cohort had 71 new infections 21 days or more after their first dose (incidence density of eight infections per 10 000 person-days) and nine infections 7 days after the second dose (incidence density four infections per 10 000 person-days). In the unvaccinated cohort, 543 (56%) participants had typical COVID-19 symptoms and 140 (14%) were asymptomatic on or 14 days before their PCR positive test date, compared with 29 (36%) with typical COVID-19 symptoms and 15 (19%) asymptomatic in the vaccinated cohort. A single dose of BNT162b2 vaccine showed vaccine effectiveness of 70% (95% CI 55–85) 21 days after first dose and 85% (74–96) 7 days after two doses in the study population. Interpretation Our findings show that the BNT162b2 vaccine can prevent both symptomatic and asymptomatic infection in working-age adults. This cohort was vaccinated when the dominant variant in circulation was B1.1.7 and shows effectiveness against this variant. Funding Public Health England, UK Department of Health and Social Care, and the National Institute for Health Research.

The Holistic Approach to Unravel Antibacterial Resistance in East Africa is a 3-year Global Context Consortia Award (MR/S004785/1) funded by the National Institute for Health Research, Medical Research Council and the Department of Health and Social Care. The award is also part of the EDCTP2 programme supported by the European Union. This work is supported in part by the Makerere University-Uganda Virus Research Institute Centre of Excellence for Infection and Immunity Research and Training (MUII). MUII is supported through the DELTAS Africa Initiative (grant number 107743). The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences and Alliance for Accelerating Excellence in Science in Africa, and is supported by the New Partnership for Africa’s Development Planning and Coordinating Agency with funding from the Wellcome Trust (grant number 107743) and the UK Government. This paper was funded in part by a grant from the National Institutes of Health (grant number U01CA207167). Introduction Antimicrobial resistance (AMR) is a global health threat that requires urgent research using a multidisciplinary approach. The biological drivers of AMR are well understood, but factors related to treatment seeking and the social contexts of antibiotic (AB) use behaviours are less understood. Here we describe the Holistic Approach to Unravel Antibacterial Resistance in East Africa, a multicentre consortium that investigates the diverse drivers of drug resistance in urinary tract infections (UTIs) in East Africa. Methods and analysis This study will take place in Uganda, Kenya and Tanzania. We will conduct geospatial mapping of AB sellers, and conduct mystery client studies and in-depth interviews (IDIs) with drug sellers to investigate AB provision practices. In parallel, we will conduct IDIs with doctors, alongside community focus groups. Clinically diagnosed patients with UTI will be recruited from healthcare centres, provide urine samples and complete a questionnaire capturing retrospective treatment pathways, sociodemographic characteristics, attitudes and knowledge. Bacterial isolates from urine and stool samples will be subject to culture and antibiotic sensitivity testing. Genomic DNA from bacterial isolates will be extracted with a subset being sequenced. A follow-up household interview will be conducted with 1800 UTI-positive patients, where further environmental samples will be collected. A subsample of patients will be interviewed using qualitative tools. Questionnaire data, microbiological analysis and qualitative data will be linked at the individual level. Quantitative data will be analysed using statistical modelling, including Bayesian network analysis, and all forms of qualitative data analysed through iterative thematic content analysis. Ethics and dissemination Approvals have been obtained from all national and local ethical review bodies in East Africa and the UK. Results will be disseminated in communities, with local and global policy stakeholders, and in academic circles. They will have great potential to inform policy, improve clinical practice and build regional pathogen surveillance capacity. Publisher PDF

Funding: Bioinformatics and Computational Biology analyses were supported by the University of St Andrews Bioinformatics Unit that is funded by a Wellcome Trust ISSF award (grant 097831/Z/11/Z). M.T.G.H is supported by the Scottish Infection Research Network and Chief Scientist Office through the Scottish Healthcare Associated Infection Prevention Institute consortium funding (CSO Reference: SIRN10). Objectives Methicillin-resistant Staphylococcus aureus(MRSA) has spread across countries and healthcare settings, with different ecological niches for different clones. It is crucial to understand the comparative epidemiology of MRSA clones between healthcare settings, and independent factors associated with colonization of specific clones. Methods We conducted annual cross-sectional surveillance studies in a network comprising an acute-care hospital and six closely-affiliated intermediate- and long-term care facilities in Singapore, in June-July, 2014-2016. 5,394 patients contributed 16,045 nasal, axillary and groin samples for culture and MRSA isolates for whole genome sequencing. Multivariable multilevel multinomial regression models were constructed to assess for independent factors associated with MRSA colonization. Results MRSA clonal complex(CC) 22 was more prevalent in the acute-care hospital(n=256/493; 51.9%) and intermediate-care(n=348/634; 54.9%) than long-term care(n=88/351; 25.1%) facilities, with clones besides CC22 and CC45 being more prevalent in long-term care facilities(n=144/351; 41.0%) (P

Gene tandem amplifications are thought to drive bacterial evolution, but they are transient in the absence of selection, making their investigation challenging. Here, we analyze genomic sequences of Staphylococcus aureus USA300 isolates from the same geographical area to identify variations in gene copy number, which we confirm by long-read sequencing. We find several hotspots of variation, including the csa1 cluster encoding lipoproteins known to be immunogenic. We also show that the csa1 locus expands and contracts during bacterial growth in vitro and during systemic infection of mice, and recombination creates rapid heterogeneity in initially clonal cultures. Furthermore, csa1 copy number variants differ in their immunostimulatory capacity, revealing a mechanism by which gene copy number variation can modulate the host immune response., Gene tandem amplifications can drive bacterial evolution. Here, Belikova et al. identify copy number variations of lipoprotein-encoding genes in Staphylococcus aureus clinical isolates, and show that the loci expand and contract during bacterial growth in vitro and in mice, leading to changes in immunostimulatory capacity.

Funding: This research was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) (BB/P002757/1). Bacterial superantigens (sAgs) are powerful activators of the immune response that trigger unspecific T cell responses accompanied by the release of pro-inflammatory cytokines. Streptococcus equi (S. equi) and Streptococcus zooepidemicus (S. zooepidemicus) produce sAgs that play an important role in their ability to cause disease. Strangles, caused by S. equi, is one of the most common infectious diseases of horses worldwide. Here, we report the identification of a new sAg of S. zooepidemicus, SpeS, and show that mutation of the putative T cell receptor (TCR)-binding motif (YAY to IAY) abrogated TCR-binding, whilst maintaining interaction with major histocompatibility complex (MHC) class II molecules. The fusion of SpeS and SpeSY39I to six S. equi surface proteins using two different peptide linkers was conducted to determine if MHC class II-binding properties were maintained. Proliferation assays, qPCR and flow cytometry analysis showed that SpeSY39I and its fusion proteins induced less mitogenic activity and interferon gamma expression when compared to SpeS, whilst retaining APC-binding properties. Our data suggest that SpeSY39I-surface protein fusions could be used to direct vaccine antigens towards antigen presenting cells in vivo with the potential to enhance antigen presentation and improve immune responses. Publisher PDF

National networks of laboratory-based surveillance of antimicrobial resistance (AMR) monitor resistance trends and disseminate these data to AMR stakeholders. Whole-genome sequencing (WGS) can support surveillance by pinpointing resistance mechanisms and uncovering transmission patterns. However, genomic surveillance is rare in low- and middle-income countries. Here, we implement WGS within the established Antimicrobial Resistance Surveillance Program of the Philippines via a binational collaboration. In parallel, we characterize bacterial populations of key bug-drug combinations via a retrospective sequencing survey. By linking the resistance phenotypes to genomic data, we reveal the interplay of genetic lineages (strains), AMR mechanisms, and AMR vehicles underlying the expansion of specific resistance phenotypes that coincide with the growing carbapenem resistance rates observed since 2010. Our results enhance our understanding of the drivers of carbapenem resistance in the Philippines, while also serving as the genetic background to contextualize ongoing local prospective surveillance., Whole-genome sequencing (WGS) can support drug resistance surveillance, but is rare in low- and middle-income countries. Here, the authors integrate WGS within the national antimicrobial resistance surveillance program in the Philippines and conduct a retrospective sequencing survey to characterize bacterial populations and dissect resistance phenotypes.

Methicillin Resistant Staphylococcus aureus (MRSA) remains one of the leading causes of both nosocomial and community infections worldwide. In the Philippines, MRSA rates have remained above 50% since 2010, but resistance to other antibiotics, including vancomycin, is low. The MRSA burden can be partially attributed to pathogen-specific characteristics of the circulating clones, but little was known about the S. aureus circulating clones in the Philippines.We sequenced the whole genomes of 116 S. aureus isolates collected in 2013-2014 by the Antimicrobial Resistance Surveillance Program. The multi-locus sequence type, spa type, SCC-mec type, presence of antimicrobial resistance (AMR) determinants and virulence genes, and relatedness between the isolates were all derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined.The MRSA population in the Philippines was composed of a limited number of genetic clones, including several international epidemic clones, such as CC30-spa-t019-SCCmec-IV-PVL+, CC5-SCCmec-typeIV, and ST239-spa-t030-SCCmec-typeIII. The CC30 genomes were related to the South West Pacific clone, but formed a distinct and diverse lineage, with evidence of global dissemination. We showed the independent acquisition of resistance to sulfamethoxazole/trimethoprim across different locations and genetic clones, but mostly in pediatric patients with invasive infections. The concordance between phenotypic and genotypic resistance was 99.68% overall for 8 antibiotics in 7 classes.We produced the first comprehensive genomic survey of S. aureus in the Philippines, which bridges the gap in genomic data from the Western Pacific region and will constitute the genetic background to contextualize ongoing prospective surveillance.

Pseudomonas aeruginosa is an opportunistic pathogen often causing nosocomial infections that are resilient to treatment due to an extensive repertoire of intrinsic and acquired resistance mechanisms. In recent years, increasing resistance rates to antibiotics such as carbapenems and extended-spectrum cephalosporins have been reported, as well as multi-drug resistant and possible extremely drug-resistant rates of approximately 21% and 15%, respectively. However, the molecular epidemiology and AMR mechanisms of this pathogen remains largely uncharacterized.We sequenced the whole genomes of 176 P. aeruginosa isolates collected in 2013-2014 by the Antimicrobial Resistance Surveillance Program. The multi-locus sequence type, presence of antimicrobial resistance (AMR) determinants, and relatedness between the isolates were derived from the sequence data. The concordance between phenotypic and genotypic resistance was also determined.Carbapenem resistance was associated namely with loss-of function of the OprD porin, and acquisition of the metallo-β-lactamase VIM. The concordance between phenotypic and genotypic resistance was 93.27% overall for 6 antibiotics in 3 classes, but varied widely between aminoglycosides. The population of P. aeruginosa in the Philippines was diverse, with clonal expansions of XDR genomes belonging to multi-locus sequence types ST235, ST244, ST309, and ST773. We found evidence of persistence or reintroduction of the predominant clone ST235 in one hospital, as well as transfer between hospitals. Most of the ST235 genomes formed a distinct Philippine lineage when contextualized with international genomes, thus raising the possibility that this is a lineage unique to the Philippines. This was further supported by long-read sequencing of one representative XDR isolate, which revealed the presence of an integron carrying multiple resistance genes, including blaVIM-2, with differences in gene composition and synteny to other P. aeruginosa class 1 integrons described before.We produced the first comprehensive genomic survey of P. aeruginosa in the Philippines, which bridges the gap in genomic data from the Western Pacific region and will constitute the genetic background to contextualize ongoing prospective surveillance. Our results also highlight the importance of infection control interventions aimed to curtail the spread of international epidemic clone ST235 within the country.

The work was funded by the Scottish Executive via the Chief Scientists Office through the provision of a grant to establish the Scottish Healthcare Associated Infection Prevention Institute (SHAIPI). The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication. Bacterial type III secretion systems (T3SSs) play an important role in pathogenesis of Gram-negative infections. Enteropathogenic and enterohemorrhagic Escherichia coli contain a well-defined T3SS but in addition a second T3SS termed E. coli T3SS 2 (ETT2) has been described in a number of strains of E. coli. The majority of pathogenic E. coli contain elements of a genetic locus encoding ETT2, but which has undergone significant mutational attrition rendering it without predicted function. Only a very few strains have been reported to contain an intact ETT2 locus. To investigate the occurrence of the ETT2 locus in strains of human pathogenic E. coli, we carried out genomic sequencing of 162 isolates obtained from patient blood cultures in Scotland. We found that 22 of 26 sequence type (ST) 69 isolates from this collection contained an intact ETT2 together with an associated eip locus which encodes putative secreted ETT2 effectors as well as eilA, a gene encoding a putative transcriptional regulator of ETT2 associated genes. Using a reporter gene for eilA activation, we defined conditions under which this gene was differentially activated. Analysis of published E. coli genomes with worldwide representation showed that ST69 contained an intact ETT2 in these strains as well. The conservation of the genes encoding ETT2 in human pathogenic ST69 strains strongly suggests it has importance in infection, although its exact functional role remains obscure. Publisher PDF

ObjectivesTo characterise the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland.MethodsLinezolid-resistant E. faecalis were identified in three Scottish Health Boards and confirmed to carry the optrA gene at the national reference laboratory. WGS was performed with short read (Illumina MiSeq) and long read (Oxford Nanopore MinION) technologies to generate complete genome assemblies. Illumina reads for 94 E. faecalis bloodstream isolates were used to place the optrA-positive isolates in a larger UK phylogeny.ResultsSix optrA-positive linezolid-resistant E. faecalis were isolated from urogenital samples in three Scottish Health Boards (2014-2017). No epidemiological links were identified between the patients, four were community-based, and only one had recent linezolid exposure. Reference-based mapping confirmed the isolates were genetically distinct (>13,900 core SNPs). optrA was located on a plasmid in each isolate and these plasmids showed limited nucleotide similarity. There was variable presence of transposable elements surrounding optrA, (including IS1216, IS3, and Tn3) and not always as a recognisable gene cassette. OptrA amino acid sequences were also divergent, resulting in four protein variants differing in 1-20 residues. One isolate belonged to ST16 and clustered with three other isolates in the UK collection (76-182 SNPs), otherwise the optrA-positive isolates were genetically distinct from the bloodstream isolates (>6,000 SNPs).ConclusionsWe report multiple variants of the linezolid resistance gene optrA in diverse E. faecalis strain and plasmid backgrounds, suggesting multiple introductions of the gene into the E. faecalis population and selection driving recent emergence.

Streptococcus pyogenes is a genetically diverse pathogen, with over 200 different genotypes defined by emm typing, but only a minority of these genotypes are responsible for the majority of human infection in high-income countries. Two prevalent genotypes associated with disease rose to international dominance following recombination of a toxin locus that conferred increased expression. Here, we found that recombination of this locus and promoter has occurred in other diverse genotypes, events that may allow these genotypes to expand in the population. We identified an association between the loss of hyaluronic acid capsule synthesis and high toxin expression, which we propose may be associated with an adaptive advantage. As S. pyogenes pathogenesis depends both on capsule and toxin production, new variants with altered expression may result in abrupt changes in the molecular epidemiology of this pathogen in the human population over time., Gene transfer and homologous recombination in Streptococcus pyogenes has the potential to trigger the emergence of pandemic lineages, as exemplified by lineages of emm1 and emm89 that emerged in the 1980s and 2000s, respectively. Although near-identical replacement gene transfer events in the nga (NADase) and slo (streptolysin O) loci conferring high expression of these toxins underpinned the success of these lineages, extension to other emm genotype lineages is unreported. The emergent emm89 lineage was characterized by five regions of homologous recombination additional to nga-slo, including complete loss of the hyaluronic acid capsule synthesis locus hasABC, a genetic trait replicated in two other leading emm types and recapitulated by other emm types by inactivating mutations. We hypothesized that other leading genotypes may have undergone similar recombination events. We analyzed a longitudinal data set of genomes from 344 clinical invasive disease isolates representative of locations across England, dating from 2001 to 2011, and an international collection of S. pyogenes genomes representing 54 different genotypes and found frequent evidence of recombination events at the nga-slo locus predicted to confer higher toxin genotype. We identified multiple associations between recombination at this locus and inactivating mutations within hasAB, suggesting convergent evolutionary pathways in successful genotypes. This included common genotypes emm28 and emm87. The combination of no or low capsule and high expression of nga and slo may underpin the success of many emergent S. pyogenes lineages of different genotypes, triggering new pandemics, and could change the way S. pyogenes causes disease.

OBJECTIVEWe report the utility of whole-genome sequencing (WGS) conducted in a clinically relevant time frame (ie, sufficient for guiding management decision), in managing a Streptococcus pyogenes outbreak, and present a comparison of its performance with emm typing.SETTINGA 2,000-bed tertiary-care psychiatric hospital.METHODSActive surveillance was conducted to identify new cases of S. pyogenes. WGS guided targeted epidemiological investigations, and infection control measures were implemented. Single-nucleotide polymorphism (SNP)–based genome phylogeny, emm typing, and multilocus sequence typing (MLST) were performed. We compared the ability of WGS and emm typing to correctly identify person-to-person transmission and to guide the management of the outbreak.RESULTSThe study included 204 patients and 152 staff. We identified 35 patients and 2 staff members with S. pyogenes. WGS revealed polyclonal S. pyogenes infections with 3 genetically distinct phylogenetic clusters (C1–C3). Cluster C1 isolates were all emm type 4, sequence type 915 and had pairwise SNP differences of 0–5, which suggested recent person-to-person transmissions. Epidemiological investigation revealed that cluster C1 was mediated by dermal colonization and transmission of S. pyogenes in a male residential ward. Clusters C2 and C3 were genomically diverse, with pairwise SNP differences of 21–45 and 26–58, and emm 11 and mostly emm120, respectively. Clusters C2 and C3, which may have been considered person-to-person transmissions by emm typing, were shown by WGS to be unlikely by integrating pairwise SNP differences with epidemiology.CONCLUSIONSWGS had higher resolution than emm typing in identifying clusters with recent and ongoing person-to-person transmissions, which allowed implementation of targeted intervention to control the outbreak.Infect Control Hosp Epidemiol 2018;852–860

Identifying anti-spike antibodies that exhibit strong neutralizing activity against current dominant circulating variants, and antibodies that are escaped by these variants, has important implications in the development of therapeutic and diagnostic solutions and in improving understanding of the humoral response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We characterized seven anti-SARS-CoV-2 receptor binding domain (RBD) antibodies for binding activity, pairing capability, and neutralization activity to SARS-CoV-2 and three variant RBDs via lateral flow immunoassays. The results allowed us to group these antibodies into three distinct epitope bins. Our studies showed that two antibodies had broadly potent neutralizing activity against SARS-CoV-2 and these variant RBDs and that one antibody did not neutralize the South African (SA) and Brazilian P.1 (BR P.1) RBDs. The antibody escaped by the SA and BR P.1 RBDs retained binding activity to SA and BR P.1 RBDs but was unable to induce neutralization. We demonstrated that lateral flow immunoassay could be a rapid and effective tool for antibody characterization, including epitope classification and antibody neutralization kinetics. The potential contributions of the mutations (N501Y, E484K, and K417N/T) contained in these variants’ RBDs to the antibody pairing capability, neutralization activity, and therapeutic antibody targeting strategy are discussed.

Methicillin-resistant Staphylococcus aureus (MRSA) transmission in the hospital setting has been a frequent subject of investigation using bacterial genomes, but previous approaches have not yet fully utilised the extra deductive power provided when multiple pathogen samples are acquired from each host. Here, we used a large dataset of MRSA sequences from multiply-sampled patients to reconstruct colonisation of individuals in a high-transmission setting in a hospital in Thailand. We reconstructed transmission trees for MRSA. We also investigated transmission between anatomical sites on the same individual, finding that this either occurs repeatedly or involves a wide transmission bottleneck. We examined the between-subject bottleneck, finding considerable variation in the amount of diversity transmitted. Finally, we compared our approach to the simpler method of identifying transmission pairs using single nucleotide polymorphism (SNP) counts. This suggested that the optimum threshold for identifying a pair is 39 SNPs, if sensitivities and specificities are equally weighted.

Funding: This research was supported by the Small Innovative Grant (SIG/15033) and Communicable Diseases – Public Health Research Grant (CDPHRG/0008/2014) awarded by the National Healthcare Group and Ministry of Health Singapore respectively. Objectives With the widespread use of antiseptics in healthcare facilities for the prevention of methicillin-resistant Staphylococcus aureus (MRSA) transmission, there are concerns for antiseptic tolerance and resistance. We sought to understand the use of chlorhexidine and octenidine, qac genes carriage and reduced antiseptic susceptibilities. Methods A serial cross-sectional study was conducted in an acute care hospital and three extended-care facilities of a healthcare network in June-July, 2014-2016. Two of the extended-care facilities were exposed to intranasal octenidine and universal daily chlorhexidine/octenidine bathing. The minimum inhibitory concentration (MIC) levels and qac genes were determined by broth microdilution tests and whole genome sequencing respectively. Multivariable logistic regression was used to assess for the independent associations between antiseptic exposures, qac genes and reduced antiseptic susceptibilities. Results A total of 878 MRSA isolates were obtained. There were associations between qacA/B carriage and chlorhexidine (adjusted odds ratio [aOR]: 7.80; 95% confidence interval [CI]: 3.25-18.71) and octenidine (aOR: 11.79; 95%CI: 5.14-27.04) exposures. Chlorhexidine exposure was associated with reduced chlorhexidine susceptibility (MIC≥4mg/L) (aOR: 3.15; 95%CI: 1.14-8.74). Carriage of qacA/B (aOR: 10.65: 95%CI: 4.14-27.40) or qacC (aOR: 2.55; 95%CI: 1.22-5.32) had an association with reduced chlorhexidine susceptibility; while MRSA sequence type modified the association. However, we found no direct association between (i) antiseptics use and qacC carriage, (ii) octenidine exposure and reduced susceptibility and (iii) reduced octenidine susceptibility and qacA/B or qacC carriage. Conclusions Antiseptic exposures were associated with qac genes carriage. Chlorhexidine exposure was associated with reduced chlorhexidine susceptibility, requiring continued surveillance for the emergence of resistance. Postprint

This work was supported by Medical Research Council partnership grants (G1001787/1 and MR/N002660/1) held between the Department of Veterinary Medicine and School of Clinical Medicine at the University of Cambridge, the Moredun Research Institute and the Wellcome Sanger Institute. This publication presents independent research supported by the Health Innovation Challenge Fund (WT098600 and HICF-T5-342)—a parallel funding partnership between the Department of Health and Wellcome Trust. E.M.H. is supported by a UK Research and Innovation Fellowship (MR/S00291X/1). F.C. is supported by the Wellcome Trust (201344/Z/16/Z). X.B. is supported by a UK–China AMR Partnership Grant (MR/P007201/1). Antibiotic resistance in bacterial pathogens threatens the future of modern medicine. One such resistant pathogen is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to nearly all β-lactam antibiotics, limiting treatment options. Here, we show that a significant proportion of MRSA isolates from different lineages, including the epidemic USA300 lineage, are susceptible to penicillins when used in combination with β-lactamase inhibitors such as clavulanic acid. Susceptibility is mediated by a combination of two different mutations in the mecA promoter region that lowers mecA-encoded penicillin-binding protein 2a (PBP2a) expression, and in the majority of isolates by either one of two substitutions in PBP2a (E246G or M122I) that increase the affinity of PBP2a for penicillin in the presence of clavulanic acid. Treatment of S. aureus infections in wax moth and mouse models shows that penicillin/β-lactamase inhibitor susceptibility can be exploited as an effective therapeutic choice for ‘susceptible’ MRSA infection. Finally, we show that isolates with the PBP2a E246G substitution have a growth advantage in the presence of penicillin but the absence of clavulanic acid, which suggests that penicillin/β-lactamase susceptibility is an example of collateral sensitivity (resistance to one antibiotic increases sensitivity to another). Our findings suggest that widely available and currently disregarded antibiotics could be effective in a significant proportion of MRSA infections. Postprint

Group A Streptococcus (GAS; Streptococcus pyogenes) is a bacterial pathogen for which a commercial vaccine for humans is not available. Employing the advantages of high-throughput DNA sequencing technology to vaccine design, we have analyzed 2,083 globally sampled GAS genomes. The global GAS population structure reveals extensive genomic heterogeneity driven by homologous recombination and overlaid with high levels of accessory gene plasticity. We identified the existence of more than 290 clinically associated genomic phylogroups across 22 countries, highlighting challenges in designing vaccines of global utility. To determine vaccine candidate coverage, we investigated all of the previously described GAS candidate antigens for gene carriage and gene sequence heterogeneity. Only 15 of 28 vaccine antigen candidates were found to have both low naturally occurring sequence variation and high (>99%) coverage across this diverse GAS population. This technological platform for vaccine coverage determination is equally applicable to prospective GAS vaccine antigens identified in future studies., info:eu-repo/semantics/published

The environmental bacterium Burkholderia pseudomallei causes melioidosis, an important endemic human disease in tropical and sub-tropical countries. This bacterium occupies broad ecological niches including soil, contaminated water, single-cell microbes, plants and infection in a range of animal species. Here, we performed genome-wide association studies for genetic determinants of environmental and human adaptation using a combined dataset of 1,010 whole genome sequences of B. pseudomallei from Northeast Thailand and Australia, representing two major disease hotspots. With these data, we identified 47 genes from 26 distinct loci associated with clinical or environmental isolates from Thailand and replicated 12 genes in an independent Australian cohort. We next outlined the selective pressures on the genetic loci (dN/dS) and the frequency at which they had been gained or lost throughout their evolutionary history, reflecting the bacterial adaptability to a wide range of ecological niches. Finally, we highlighted loci likely implicated in human disease., Claire Chewapreecha et al. combine 753 newly sequenced Thai Burkholderia pseudomallei isolates with 258 Australian isolates to identify genes associated with either clinical or environmental strains. They find 47 genes, 12 of which replicate in both locations, that may provide clues to the strategy used by this microbe to adapt to survive in wide range of ecological niches, including human hosts.

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors under evaluation as vaccine antigens. Clinical S. aureus isolates have the biosynthetic capability to express either CP5 or CP8 and an understanding of the relationship between CP genotype/phenotype and S. aureus epidemiology is valuable. Using whole genome sequencing, the clonal relatedness and CP genotype were evaluated for disease-associated S. aureus isolates selected from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T) to represent different geographic regions in the United States (US) during 2004 and 2009–10. Thirteen prominent clonal complexes (CC) were identified, with CC5, 8, 30 and 45 representing >80% of disease isolates. CC5 and CC8 isolates were CP type 5 and, CC30 and CC45 isolates were CP type 8. Representative isolates from prevalent CC were susceptible to in vitro opsonophagocytic killing elicited by anti-CP antibodies, demonstrating that susceptibility to opsonic killing is not linked to the genetic lineage. However, as not all S. aureus isolates may express CP, isolates representing the diversity of disease isolates were assessed for CP production. While approximately 35% of isolates (primarily CC8) did not express CP in vitro, CP expression could be clearly demonstrated in vivo for 77% of a subset of these isolates (n = 20) despite the presence of mutations within the capsule operon. CP expression in vivo was also confirmed indirectly by measuring an increase in CP specific antibodies in mice infected with CP5 or CP8 isolates. Detection of antigen expression in vivo in relevant disease states is important to support the inclusion of these antigens in vaccines. Our findings confirm the validity of CP as vaccine targets and the potential of CP-based vaccines to contribute to S. aureus disease prevention. Publisher PDF

Funding: This work was funded by the John Templeton Foundation Grant 60501, “Putting the Extended Evolutionary Synthesis to the Test”. It also received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland, (https://www.masts.ac.uk)) and their support is gratefully acknowledged. MASTS is funded by the Scottish Funding Council (grant reference HR09011) and contributing institutions. DMP received funding from MASTS, funded by the Scottish Funding Council (grant reference HR09011), and AJW received funding under the MASTS small grant scheme (grant reference SG433). Intertidal systems are complex and dynamic environments with many interacting factors influencing biochemical characteristics and microbial communities. One key factor are the actions of resident fauna, many of which are regarded as ecosystem engineers because of their bioturbation, bioirrigation and sediment stabilising activities. The purpose of this investigation was to elucidate the evolutionary implications of the ecosystem engineering process by identifying, if any, aspects that act as selection pressures upon microbial communities. A mesocosm study was performed using the well characterised intertidal ecosystem engineers Corophium volutator, Hediste diversicolor, and microphytobenthos, in addition to manual turbation of sediments to compare effects of bioturbation, bioirrigation and stabilisation. A range of sediment functions and biogeochemical gradients were measured in conjunction with 16S rRNA sequencing and diatom taxonomy, with downstream bacterial metagenome function prediction, to identify selection pressures that incited change to microbial community composition and function. Bacterial communities were predominantly Proteobacteria, with the relative abundance of Bacteroidetes, Alphaproteobacteria and Verrucomicrobia being partially displaced by Deltaproteobacteria, Acidobacteria and Chloroflexi as dissolved oxygen concentration and redox potential decreased. Bacterial community composition was driven strongly by biogeochemistry; surface communities were affected by a combination of sediment functions and overlying water turbidity, and subsurface communities by biogeochemical gradients driven by sediment reworking. Diatom communities were dominated by Nitzschia laevis and Achnanthes sp., and assemblage composition was influenced by overlying water turbidity (manual or biogenic) rather than direct infaunal influences such as grazing. Publisher PDF

Methicillin-resistant Staphylococcus aureus (MRSA) poses a substantial threat to human health worldwide and evolves rapidly by acquiring mobile genetic elements, such as plasmids. Here we investigate how the copB-mco copper hypertolerance operon carried on a mobile genetic element contributes to the virulence potential of clinical isolates of MRSA. Copper is a key component of innate immune bactericidal defenses. Here we show that copper hypertolerance genes enhance the survival of S. aureus inside primed macrophages and in whole human blood. The copB and mco genes are carried by clinical isolates responsible for invasive infections across Europe, and more broadly among three successful clonal lineages of S. aureus. Our findings show that a gain of copper hypertolerance genes increases the resistance of MRSA to phagocytic killing by host immune cells and imply that acquisition of this mobile genetic element can contribute to the success of MRSA., Pathogens are exposed to toxic levels of copper during infection, and copper tolerance may be a general virulence mechanism used by bacteria to resist host defenses. In support of this, inactivation of copper exporter genes has been found to reduce the virulence of bacterial pathogens in vivo. Here we investigate the role of copper hypertolerance in methicillin-resistant Staphylococcus aureus (MRSA). We show that a copper hypertolerance operon (copB-mco), carried on a mobile genetic element (MGE), is prevalent in a collection of invasive S. aureus strains and more widely among clonal complex 22, 30, and 398 strains. The copB and mco genes encode a copper efflux pump and a multicopper oxidase, respectively. Isogenic mutants lacking copB or mco had impaired growth in subinhibitory concentrations of copper. Transfer of a copB-mco-carrying plasmid to a naive clinical isolate resulted in a gain of copper hypertolerance and enhanced bacterial survival inside primed macrophages. The copB and mco genes were upregulated within infected macrophages, and their expression was dependent on the copper-sensitive operon repressor CsoR. Isogenic copB and mco mutants were impaired in their ability to persist intracellularly in macrophages and were less resistant to phagocytic killing in human blood than the parent strain. The importance of copper-regulated genes in resistance to phagocytic killing was further elaborated using mutants expressing a copper-insensitive variant of CsoR. Our findings suggest that the gain of mobile genetic elements carrying copper hypertolerance genes contributes to the evolution of virulent strains of S. aureus that are better equipped to resist killing by host immune cells.

ST is an Australian National Health and Medical Research Council Career Development Fellow (#1145033). Sequencing was supported by Wellcome Trust grant 098051. Background : In Australia, community-associated methicillin-resistant Staphylococcus aureus (MRSA) lineage sequence type (ST) 93 has rapidly risen to dominance since being described in the early 1990s. We examined 459 ST93 genome sequences from Australia, New Zealand, Samoa and Europe to investigate the evolutionary history of ST93, its emergence in Australia and subsequent spread overseas. Results : Comparisons with other S. aureus genomes indicate that ST93 is an early diverging and recombinant lineage, comprising of segments from the ST59/ST121 lineage and from a divergent but currently unsampled Staphylococcal population. However, within extant ST93 strains limited genetic diversity was apparent with the most recent common ancestor dated to 1977 (95% highest posterior density [HPD] 1973–1981). An epidemic ST93 population arose from a methicillin-susceptible progenitor in remote northern Australia, which has a proportionally large Indigenous population, with documented overcrowded housing and a high burden of skin infection. Methicillin-resistance was acquired three times in these regions, with a clade harbouring a staphylococcal cassette chromosome mec (SCCmec) IVa expanding and spreading to Australia’s east coast by 2000. We observed sporadic and non-sustained introductions of ST93-MRSA-IVa to the United Kingdom. In contrast, in New Zealand, ST93-MRSA-IVa was sustainably transmitted with clonal expansion within the Pacific Islander population, who experience similar disadvantages as Australian Indigenous populations. Conclusions : ST93 has a highly recombinant genome including portions derived from an early diverging S. aureus population. Our findings highlight the need to understand host population factors in the emergence and spread of antimicrobial resistant community pathogens. Publisher PDF

The work was funded by the Scottish Executive via the Chief Scientist Office through the provision of a grant to establish the Scottish Healthcare Associated Infection Prevention Institute (SHAIPI). Bacteraemia caused by Escherichia coli is a growing problem with a significant mortality. The factors that influence the acquisition and outcome of these infections are not clear. Here, we have linked detailed genetic data from the whole-genome sequencing of 162 bacteraemic isolates collected in Scotland, UK, in 2013-2015, with clinical data in order to delineate bacterial and host factors that influence the acquisition in hospital or the community, outcome and antibiotic resistance. We identified four major sequence types (STs) in these isolates: ST131, ST69, ST73 and ST95. Nearly 50% of the bacteraemic isolates had a urinary origin. ST69 was genetically distinct from the other STs, with significantly less sharing of accessory genes and with a distinct plasmid population. Virulence genes were widespread and diversely distributed between the dominant STs. ST131 was significantly associated with hospital-associated infections (HAIs), and ST69 with those from the community. However, there was no association of ST with outcome, although patients with HAI had a higher immediate mortality compared to those with community-associated infections (CAIs). Genome-wide association studies revealed genes involved in antibiotic persistence as significantly associated with HAIs and those encoding elements of a type VI secretion system with CAIs. Antibiotic resistance was common, and there were networks of correlated resistance genes and phenotypic antibiotic resistance. This study has revealed the complex interactions between the genotype of E. coli and its ability to cause bacteraemia, and some of the determinants influencing hospital or community acquisition. In part, these are shaped by antibiotic usage, but strain-specific factors are also important. Publisher PDF

L. H., L. A., S. W., P. L. and A. S. A. are current employees of Pfizer, and this work was funded by Pfizer Inc. Neisseria meningitidis serogroup B (MnB) was responsible for two independent meningococcal disease outbreaks at universities in the USA during 2013. The first at University A in New Jersey included nine confirmed cases reported between March 2013 and March 2014. The second outbreak occurred at University B in California, with four confirmed cases during November 2013. The public health response to these outbreaks included the approval and deployment of a serogroup B meningococcal vaccine that was not yet licensed in the USA. This study investigated the use of whole-genome sequencing(WGS) to examine the genetic profile of the disease-causing outbreak isolates at each university. Comparative WGS revealed differences in evolutionary patterns between the two disease outbreaks. The University A outbreak isolates were very closely related, with differences primarily attributed to single nucleotide polymorphisms/insertion-deletion (SNP/indel) events. In contrast, the University B outbreak isolates segregated into two phylogenetic clades, differing in large part due to recombination events covering extensive regions (>30 kb) of the genome including virulence factors. This high-resolution comparison of two meningococcal disease outbreaks further demonstrates the genetic complexity of meningococcal bacteria as related to evolution and disease virulence. Publisher PDF

Pathogens are exposed to toxic levels of copper during infection and copper tolerance may be a general virulence mechanism used by bacteria to resist host defences. In support of this, inactivation of copper-exporter genes has been found to reduce the virulence of bacterial pathogensin vivo. Here we investigate the role of copper-hypertolerance in methicillin resistantStaphylococcus aureus. We show that a copper-hypertolerance locus (copB-mco), carried on a mobile genetic element, is prevalent in a collection of invasiveS. aureusstrains and more widely among clonal complex 22, 30 and 398 strains. ThecopBandmcogenes encode a copper efflux pump and a multicopper oxidase, respectively. Isogenic mutants lackingcopBormcohad impaired growth in subinhibitory concentrations of copper. Transfer of acopB-mcoencoding plasmid to a naive clinical isolate resulted in a gain of copper hypertolerance and enhanced bacterial survival inside primed macrophages. ThecopBandmcogenes were upregulated within infected macrophages and their expression was dependent on the copper sensitive operon repressor CsoR. IsogeniccopBandmcomutants were impaired in their ability to persist intracellularly in macrophages and were less resistant to phagocytic killing in human blood than the parent strain. The importance of copper-regulated genes in resistance to phagocytic killing was further elaborated using mutants expressing a copper-insensitive variant of CsoR. Our findings suggest that the gain of mobile genetic elements carrying copper-hypertolerance genes contributes to the evolution of virulent strains ofS. aureus, better equipped to resist killing by host immune cells.

Funding: Wellcome Trust [Grant number 104241/z/14/z] (CPH). Scottish Infection Research Network and Chief Scientist Office through the Scottish Healthcare Associated Infection Prevention Institute consortium funding [CSO Reference: SIRN10] (MTGH, KAP, KO). Bioinformatics and Computational Biology analyses were supported by the University of St Andrews Bioinformatics Unit that is funded by a Wellcome Trust ISSF award [grant 097831/Z/11/Z]. Wellcome Trust grant 098051 (JP, MTGH). Biotechnology and Biological Sciences Research Council grant BB/M014088/1 (AEM). Wellcome Trust Senior Research Fellowship in Clinical Science [106865/Z/15/Z] (SJB). Staphylococcus aureus is an opportunistic pathogen and variable component of the human microbiota. In atopic eczema (AE) a characteristic of the disease is colonization by S. aureus, with exacerbations associated with an increased bacterial burden of the organism. Despite this, the origins and genetic diversity of S. aureus colonizing individual patients during AE disease flares is poorly understood. To examine the micro-evolution of S. aureus colonization we have deep-sequenced S. aureus populations from nine children with moderate to severe AE, and 18 non-atopic children asymptomatically carrying S. aureus nasally. Colonization by clonal S. aureus populations was observed in both AE cases and controls, with all but one of the individuals containing colonies belonging to a single sequence type. Phylogenetic analysis revealed that disease flares were associated with the clonal expansion of the S. aureus population, occurring over a period of weeks to months. There was a significant difference in the genetic backgrounds of S. aureus colonizing AE patients versus controls (Fisher’s Exact test, p=0.03). Examination of intra-host genetic heterogeneity of the colonizing S. aureus populations identified evidence of within-host selection in the AE patients, with AE variants being potentially selectively advantageous for intracellular persistence and treatment resistance. Publisher PDF

M.T.G.H. was supported by the Scottish Infection Research Network and Chief Scientist Office through Scottish Healthcare Associated Infection Prevention Institute consortium funding (CSO reference: SIRN10). The capacity for some pathogens to jump into different host-species populations is a major threat to public health and food security. Staphylococcus aureus is a multi-host bacterial pathogen responsible for important human and livestock diseases. Here, using a population-genomic approach, we identify humans as a major hub for ancient and recent S. aureus host-switching events linked to the emergence of endemic livestock strains, and cows as the main animal reservoir for the emergence of human epidemic clones. Such host-species transitions are associated with horizontal acquisition of genetic elements from host-specific gene pools conferring traits required for survival in the new host-niche. Importantly, genes associated with antimicrobial resistance are unevenly distributed among human and animal hosts, reflecting distinct antibiotic usage practices in medicine and agriculture. In addition to gene acquisition, genetic diversification has occurred in pathways associated with nutrient acquisition, implying metabolic remodelling after a host switch in response to distinct nutrient availability. For example, S. aureus from dairy cattle exhibit enhanced utilization of lactose-a major source of carbohydrate in bovine milk. Overall, our findings highlight the influence of human activities on the multi-host ecology of a major bacterial pathogen, underpinned by horizontal gene transfer and core genome diversification. Postprint

International audience; Whole genome sequencing (WGS) offers the potential to predict antimicrobial susceptibility from a single assay. The European Committee on Antimicrobial Susceptibility Testing established a subcommittee to review the current development status of WGS for bacterial antimicrobial susceptibility testing (AST). The published evidence for using WGS as a tool to infer antimicrobial susceptibility accurately is currently either poor or non-existent and the evidence / knowledge base requires significant expansion. The primary comparators for assessing genotypicephenotypic concordance from WGS data should be changed to epidemiological cut-off values in order to improve differentiation of wild-type from non-wild-type isolates (harbouring an acquired resistance). Clinical breakpoints should be a secondary comparator. This assessment will reveal whether genetic predictions could also be used to guide clinical decision making. Internationally agreed principles and quality control (QC) metrics will facilitate early harmonization of analytical approaches and interpretive criteria for WGS-based predictive AST. Only data sets that pass agreed QC metrics should be used in AST predictions. Minimum performance standards should exist and comparative accuracies across different WGS laboratories and processes should be measured. To facilitate comparisons, a single public database of all known resistance loci should be established, regularly updated and strictly curated using minimum standards for the inclusion of resistance loci. For most bacterial species the major limitations to widespread adoption for WGS-based AST in clinical laboratories remain the current high-cost and limited speed of inferring antimicrobial susceptibility from WGS data as well as the dependency on previous culture because analysis directly on specimens remains challenging. For most bacterial species there is currently insufficient evidence to support the use of WGS-inferred AST to guide clinical decision making. WGS-AST should be a funding priority if it is to become a rival to phenotypic AST. This report will be updated as the available evidence increases. Crown Copyright (C) 2016 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. All rights reserved.

Iceland is free of the major infectious diseases of horses. However, in 2010 an epidemic of respiratory disease of unknown cause spread through the country’s native horse population of 77,000. Microbiological investigations ruled out known viral agents but identified the opportunistic pathogen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) in diseased animals. We sequenced the genomes of 257 isolates of S. zooepidemicus to differentiate epidemic from endemic strains. We found that although multiple endemic clones of S. zooepidemicus were present, one particular clone, sequence type 209 (ST209), was likely to have been responsible for the epidemic. Concurrent with the epidemic, ST209 was also recovered from a human case of septicemia, highlighting the pathogenic potential of this strain. Epidemiological investigation revealed that the incursion of this strain into one training yard during February 2010 provided a nidus for the infection of multiple horses that then transmitted the strain to farms throughout Iceland. This study represents the first time that whole-genome sequencing has been used to investigate an epidemic on a national scale to identify the likely causative agent and the link to an associated zoonotic infection. Our data highlight the importance of national biosecurity to protect vulnerable populations of animals and also demonstrate the potential impact of S. zooepidemicus transmission to other animals, including humans. IMPORTANCE An epidemic of respiratory disease affected almost the entire native Icelandic horse population of 77,000 animals in 2010, resulting in a self-imposed ban on the export of horses and significant economic costs to associated industries. Although the speed of transmission suggested that a viral pathogen was responsible, only the presence of the opportunistic pathogen Streptococcus zooepidemicus was consistent with the observed clinical signs. We applied genomic sequencing to differentiate epidemic from endemic strains and to shed light on the rapid transmission of the epidemic strain throughout Iceland. We further highlight the ability of epidemic and endemic strains of S. zooepidemicus to infect other animals, including humans. This study represents the first time that whole-genome sequencing has been used to elucidate an outbreak on a national scale and identify the likely causative agent., Wellcome Trust 098051

This work was supported by the Animal Health Trust. The zoonotic bacterium Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a diverse, opportunistic pathogen that can cause mastitis in dairy sheep and goats. We used multilocus sequence typing (MLST) to define the genetic diversity of 60 isolates of S. zooepidemicus, which were recovered from sheep and goats in Spain between 2003 and 2010. We identify a novel clonal complex based on sequence type (ST), ST-236, which accounted for 39 of the 60 isolates. A representative ST-236 strain, S. zooepidemicus strain C7 (SzC7), was sequenced and interrogated for the presence of novel nutritional uptake or utilisation systems, the acquisition of which have previously been shown to be important for environmental adaptation in other streptococcal pathogens. A novel phosphoenolpyruvate sugar phosphotransferase system (PTS), which enabled the utilisation of arbutin, was identified. Functionality of the PTS was confirmed following deletion of the PTS from SzC7. Arbutin is found in multiple animal foodstuffs and we propose that the ability to utilise arbutin may have conferred a selective advantage to strains infecting animals, the diet of which contains this sugar. Postprint

This work was supported by grants from the UK Clinical Research Collaboration (UKCRC) Translational Infection Research Initiative and the Medical Research Council (grant number G1000803) with contributions to the grant from the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research (NIHR) on behalf of the Department of Health and the Chief Scientist Office of the Scottish Government Health Directorate (to S. J. P.); and by Wellcome Trust grants numbers 098051 and 089472 awarded to the Wellcome Trust Sanger Institute and B. G. S respectively. Antibiotic resistance forms a serious threat to the health of hospitalised patients, rendering otherwise treatable bacterial infections potentially life-threatening. A thorough understanding of the mechanisms by which resistance spreads between patients in different hospitals is required in order to design effective control strategies. We measured the differences between bacterial populations of 52 hospitals in the United Kingdom and Ireland, using whole-genome sequences from 1085 MRSA clonal complex 22 isolates collected between 1998 and 2012. The genetic differences between bacterial populations were compared with the number of patients transferred between hospitals and their regional structure. The MRSA populations within single hospitals, regions and countries were genetically distinct from the rest of the bacterial population at each of these levels. Hospitals from the same patient referral regions showed more similar MRSA populations, as did hospitals sharing many patients. Furthermore, the bacterial populations from different time-periods within the same hospital were generally more similar to each other than contemporaneous bacterial populations from different hospitals. We conclude that, while a large part of the dispersal and expansion of MRSA takes place among patients seeking care in single hospitals, inter-hospital spread of resistant bacteria is by no means a rare occurrence. Hospitals are exposed to constant introductions of MRSA on a number of levels: (1) most MRSA is received from hospitals that directly transfer large numbers of patients, while (2) fewer introductions happen between regions or (3) across national borders, reflecting lower numbers of transferred patients. A joint coordinated control effort between hospitals, is therefore paramount for the national control of MRSA, antibiotic-resistant bacteria and other hospital-associated pathogens. Publisher PDF

Type VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being a core component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. In addition, fractionation revealed that although an EsaB fusion to yellow fluorescent protein partially localised to the membrane, it was still membrane-localised when the T7SS was absent. Taken together our findings suggest that EsaB has T7SS-dependent and T7SS-independent roles in S. aureus.

This study was funded by National Health and Medical Research Council (Australia) grants 1046812 and 1098337. E. P. P. is in receipt of a University of the Sunshine Coast Research Fellowship. Melioidosis is a tropical disease caused by the bacterium Burkholderia pseudomallei. Outbreaks are uncommon and can generally be attributed to a single point source and strain. We used whole-genome sequencing to analyse B. pseudomallei isolates collected from an historical 2-year long case cluster that occurred in a remote northern Australian indigenous island community, where infections were previously linked to a contaminated communal water supply. We analysed the genome-wide relatedness of the two most common multilocus sequence types (STs) involved in the outbreak, STs 125 and 126. This analysis showed that although these STs were closely related on a whole-genome level, they demonstrated evidence of multiple recombination events that were unlikely to have occurred over the timeframe of the outbreak. Based on epidemiological and genetic data, we also identified two additional patients not previously associated with this outbreak. Our results confirm the previous hypothesis that a single unchlorinated water source harbouring multiple B. pseudomallei strains was linked to the outbreak, and that increased melioidosis risk in this community was associated with Piper methysticum root (kava) consumption. Publisher PDF

TheStaphylococcus aureusType VII protein secretion system (T7SS) plays important roles in virulence and intra-species competition. Here we show that the T7SS in strain RN6390 is activated by supplementing the growth medium with hemoglobin, and its cofactor hemin (heme B). Transcript analysis and secretion assays suggest that activation by hemin occurs at a transcriptional and a post-translational level. Loss of T7 secretion activity by deletion ofessCresults in upregulation of genes required for iron acquisition. Taken together these findings suggest that the T7SS plays a role in iron homeostasis in at least someS. aureusstrains.

This work was primarily funded by the Ministry of Health, Singapore, awarded through the Singapore Infectious Disease Initiative grant (SIDI/2015/001). We were also supported by the Molecular Biology Laboratory and the Department of Laboratory Medicine, TTSH, Singapore. The culture analysis of fish was primarily funded by the National Environment Agency, Singapore. The statistical work was funded by the Project MODUS grant, while whole-genome sequencing and analysis were conducted at the Genome Institute of Singapore, partially funded by the POLARIS program, Agency for Science, Technology and Research (A*STAR) and the Singapore Ministry of Health’s National Medical Research Council (NMRC/CIRG/1357/2013). Background : Streptococcus agalactiae (group B Streptococcus [GBS]) has not been described as a foodborne pathogen. However, in 2015, a large outbreak of severe invasive sequence type (ST) 283 GBS infections in adults epidemiologically linked to the consumption of raw freshwater fish occurred in Singapore. We attempted to determine the scale of the outbreak, define the clinical spectrum of disease, and link the outbreak to contaminated fish. Methods : Time-series analysis was performed on microbiology laboratory data. Food handlers and fishmongers were screened for enteric carriage of GBS. A retrospective cohort study was conducted to assess differences in demographic and clinical characteristics of patients with invasive ST283 and non-ST283 infections. Whole-genome sequencing was performed on human and fish ST283 isolates from Singapore, Thailand, and Hong Kong. Results : The outbreak was estimated to have started in late January 2015. Within the study cohort of 408 patients, ST283 accounted for 35.8% of cases. Patients with ST283 infection were younger and had fewer comorbidities but were more likely to develop meningoencephalitis, septic arthritis, and spinal infection. Of 82 food handlers and fishmongers screened, none carried ST283. Culture of 43 fish samples yielded 13 ST283-positive samples. Phylogenomic analysis of 161 ST283 isolates from humans and fish revealed they formed a tight clade distinguished by 93 single-nucleotide polymorphisms. Conclusions : ST283 is a zoonotic GBS clone associated with farmed freshwater fish, capable of causing severe disease in humans. It caused a large foodborne outbreak in Singapore and poses both a regional and potentially more widespread threat. Postprint

This work was supported by the Ministry of Health, Singapore (Communicable Diseases–Public Health Research Grant), the Wellcome Trust (Institutional Strategic Support Fund award [grant 097831/Z/11/Z] to the University of St Andrews Bioinformatics Unit), and the Scottish Infection Research Network and Chief Scientist Office (Scottish Healthcare Associated Infection Prevention Institute consortium funding [Chief Scientist Office reference SIRN10] to K. P. and M. T. G. H.). Background: Methicillin-resistant Staphylococcus aureus (MRSA) is the most common healthcare-associated multidrug-resistant organism. Despite the interconnectedness between acute care hospitals (ACHs) and intermediate- and long-term care facilities (ILTCFs), the transmission dynamics of MRSA between healthcare settings is not well understood. Methods: We conducted a cross-sectional study in a network comprising an ACH and 5 closely affiliated ILTCFs in Singapore. A total of 1700 inpatients were screened for MRSA over a 6-week period in 2014. MRSA isolates underwent whole-genome sequencing, with a pairwise single-nucleotide polymorphism (Hamming distance) cutoff of 60 core genome single-nucleotide polymorphisms used to define recent transmission clusters (clades) for the 3 major clones. Results: MRSA prevalence was significantly higher in intermediate-term (29.9%) and long-term (20.4%) care facilities than in the ACH (11.8%) (P < .001). The predominant clones were sequence type [ST] 22 (n = 183; 47.8%), ST45 (n = 129; 33.7%), and ST239 (n = 26; 6.8%), with greater diversity of STs in ILTCFs relative to the ACH. A large proportion of the clades in ST22 (14 of 21 clades; 67%) and ST45 (7 of 13; 54%) included inpatients from the ACH and ILTCFs. The most frequent source of the interfacility transmissions was the ACH (n = 28 transmission events; 36.4%). Conclusions: MRSA transmission dynamics between the ACH and ILTCFs were complex. The greater diversity of STs in ILTCFs suggests that the ecosystem in such settings might be more conducive for intrafacility transmission events. ST22 and ST45 have successfully established themselves in ILTCFs. The importance of interconnected infection prevention and control measures and strategies cannot be overemphasized. Postprint

The spread of drug-resistant bacterial pathogens pose a major threat to global health. It is widely recognised that the widespread use of antibiotics has generated selective pressures that have driven the emergence of resistant strains. Methicillin-resistantStaphylococcus aureus(MRSA) was first observed in 1960, less than one year after the introduction of this second generation β-lactam antibiotic into clinical practice. Epidemiological evidence has always suggested that resistance arose around this period, when themecAgene encoding methicillin resistance carried on an SCCmecelement, was horizontally transferred to an intrinsically sensitive strain ofS. aureus. Whole genome sequencing a collection of the very first MRSA isolates allowed us to reconstruct the evolutionary history of the archetypal MRSA. Bayesian phylogenetic reconstruction was applied to infer the time point at which this early MRSA lineage arose and when SCCmecwas acquired. MRSA emerged in the mid 1940s, following the acquisition of an ancestral type I SCCmecelement, some fourteen years prior to the first therapeutic use of methicillin. Methicillin use was not the original driving factor in the evolution of MRSA as previously thought. Rather it was the widespread use of first generation β-lactams such as penicillin in the years prior to the introduction of methicillin, which selected forS. aureusstrains carrying themecAdeterminant. Crucially this highlights how new drugs, introduced to circumvent known resistance mechanisms, can be rendered ineffective by unrecognised adaptations in the bacterial population due to the historic selective landscape created by the widespread use of other antibiotics.

The environmental bacterium Burkholderia pseudomallei causes an estimated 165,000 cases of human melioidosis per year worldwide and is also classified as a biothreat agent. We used whole genome sequences of 469 B. pseudomallei isolates from 30 countries collected over 79 years to explore its geographic transmission. Our data point to Australia as an early reservoir, with transmission to Southeast Asia followed by onward transmission to South Asia and East Asia. Repeated reintroductions were observed within the Malay Peninsula and between countries bordered by the Mekong River. Our data support an African origin of the Central and South American isolates with introduction of B. pseudomallei into the Americas between 1650 and 1850, providing a temporal link with the slave trade. We also identified geographically distinct genes/variants in Australasian or Southeast Asian isolates alone, with virulence-associated genes being among those over-represented. This provides a potential explanation for clinical manifestations of melioidosis that are geographically restricted. Postprint

Summary Background The emergence of meticillin-resistant Staphylococcus aureus (MRSA) that can persist in the community and replace existing hospital-adapted lineages of MRSA means that it is necessary to understand transmission dynamics in terms of hospitals and the community as one entity. We assessed the use of whole-genome sequencing to enhance detection of MRSA transmission between these settings. Methods We studied a putative MRSA outbreak on a special care baby unit (SCBU) at a National Health Service Foundation Trust in Cambridge, UK. We used whole-genome sequencing to validate and expand findings from an infection-control team who assessed the outbreak through conventional analysis of epidemiological data and antibiogram profiles. We sequenced isolates from all colonised patients in the SCBU, and sequenced MRSA isolates from patients in the hospital or community with the same antibiotic susceptibility profile as the outbreak strain. Findings The hospital infection-control team identified 12 infants colonised with MRSA in a 6 month period in 2011, who were suspected of being linked, but a persistent outbreak could not be confirmed with conventional methods. With whole-genome sequencing, we identified 26 related cases of MRSA carriage, and showed transmission occurred within the SCBU, between mothers on a postnatal ward, and in the community. The outbreak MRSA type was a new sequence type (ST) 2371, which is closely related to ST22, but contains genes encoding Panton-Valentine leucocidin. Whole-genome sequencing data were used to propose and confirm that MRSA carriage by a staff member had allowed the outbreak to persist during periods without known infection on the SCBU and after a deep clean. Interpretation Whole-genome sequencing holds great promise for rapid, accurate, and comprehensive identification of bacterial transmission pathways in hospital and community settings, with concomitant reductions in infections, morbidity, and costs. Funding UK Clinical Research Collaboration Translational Infection Research Initiative, Wellcome Trust, Health Protection Agency, and the National Institute for Health Research Cambridge Biomedical Research Centre.

Visualization is frequently used to aid our interpretation of complex datasets. Within microbial genomics, visualizing the relationships between multiple genomes as a tree provides a framework onto which associated data (geographical, temporal, phenotypic and epidemiological) are added to generate hypotheses and to explore the dynamics of the system under investigation. Selected static images are then used within publications to highlight the key findings to a wider audience. However, these images are a very inadequate way of exploring and interpreting the richness of the data. There is, therefore, a need for flexible, interactive software that presents the population genomic outputs and associated data in a user-friendly manner for a wide range of end users, from trained bioinformaticians to front-line epidemiologists and health workers. Here, we present Microreact, a web application for the easy visualization of datasets consisting of any combination of trees, geographical, temporal and associated metadata. Data files can be uploaded to Microreact directly via the web browser or by linking to their location (e.g. from Google Drive/Dropbox or via API), and an integrated visualization via trees, maps, timelines and tables provides interactive querying of the data. The visualization can be shared as a permanent web link among collaborators, or embedded within publications to enable readers to explore and download the data. Microreact can act as an end point for any tool or bioinformatic pipeline that ultimately generates a tree, and provides a simple, yet powerful, visualization method that will aid research and discovery and the open sharing of datasets. Publisher PDF

International audience; The evolution of meticillin-resistant Staphylococcus aureus (MRSA) from meticillin-susceptible S. aureus has been a result of the accumulation of genetic elements under selection pressure from antibiotics. The traditional classification of MRSA into healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA) is no longer relevant as there is significant overlap of identical clones between these groups, with an increasing recognition of human infection caused by livestock-associated MRSA (LA-MRSA). Genomic studies have enabled us to model the epidemiology of MRSA along these lines. In this review, we discuss the clinical relevance of genomic studies, particularly whole-genome sequencing, in the investigation of outbreaks. We also discuss the blurring of each of the three epidemiological groups (HA-MRSA, CA-MRSA and LA-MRSA), demonstrating the limited relevance of this classification. © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Staphylococcus aureus is a major human pathogen and an important cause of livestock infections. The first S. aureus genomes to be published, 15 years ago, provided the first view of genome structure and gene content. Since then, thousands of genomes from a wide array of strains from different sources have been sequenced. Comparison of these sequences has resulted in broad insights into population structure, bacterial evolution, clone emergence and expansion, and the molecular basis of niche adaptation. Furthermore, this information is now being applied clinically in outbreak investigations to inform infection control measures and to determine appropriate treatment regimens. In this review, we summarize some of the broad insights into S. aureus biology gained from the analysis of genomes and discuss future directions and opportunities in this dynamic field of research.

This study was funded by the National Health and Medical Research Council via awards 1046812 and 1098337, and the Wellcome Trust Sanger Institute via award 098051. S.J.P. receives funding from the NIHR Cambridge Biomedical Research Centre. Melioidosis, caused by the highly recombinogenic bacterium Burkholderia pseudomallei, is a disease with high mortality. Tracing the origin of melioidosis outbreaks and understanding how the bacterium spreads and persists in the environment are essential to protecting public and veterinary health and reducing mortality associated with outbreaks. We used whole-genome sequencing to compare isolates from a historical quarter-century outbreak that occurred between 1966 and 1991 in the Avon Valley, Western Australia, a region far outside the known range of B. pseudomallei endemicity. All Avon Valley outbreak isolates shared the same multilocus sequence type (ST-284), which has not been identified outside this region. We found substantial genetic diversity among isolates based on a comparison of genome-wide variants, with no clear correlation between genotypes and temporal, geographical or source data. We observed little evidence of recombination in the outbreak strains, indicating that genetic diversity among these isolates has primarily accrued by mutation. Phylogenomic analysis demonstrated that the isolates confidently grouped within the Australian B. pseudomallei clade, thereby ruling out introduction from a melioidosis-endemic region outside Australia. Collectively, our results point to B. pseudomallei ST-284 being present in the Avon Valley for longer than previously recognized, with its persistence and genomic diversity suggesting long-term, low-prevalence endemicity in this temperate region. Our findings provide a concerning demonstration of the potential for environmental persistence of B. pseudomallei far outside the conventional endemic regions. An expected increase in extreme weather events may reactivate latent B. pseudomallei populations in this region. Publisher PDF

The native horse population of Iceland has remained free of major infectious diseases. Between May and July 2010 an epidemic of respiratory disease swept through the population. Initial microbiological investigations ruled out known equine viral agents as the cause of the infections, but identified the opportunistic pathogenStreptococcus zooepidemicusas being frequently isolated from diseased animals. This diverse bacterial species has a broad host range and is usually regarded as a commensal of horses. By genome sequencingS. zooepidemicusrecovered from horses during the epidemic we show that although multiple clones ofS. zooepidemicuswere present in the population, one particular clone, ST209, was responsible for the epidemic. Concurrent with the epidemic, ST209 caused zoonotic infections, highlighting the pathogenic potential of this clone. Phylogenetic analysis suggests that the original ST209 strain entered Iceland in late 2008 or early 2009. Epidemiological investigation revealed that the incursion of this strain into a training yard that utilized a submerged treadmill between the 5thand 19thof February 2010 was a critical trigger for the ensuing epidemic of disease, provided a nidus for the infection of multiple horses, and subsequent distribution of these animals to multiple sites in Iceland.

S. R. H. and J. P. were supported by Wellcome Trust grant 098051. This work was funded by Pfizer Vaccine Research and Development. Staphylococcus aureus USA300 represents the dominant community-associated methicillin-resistant S. aureus lineage in the USA, where it is a major cause of skin and soft tissue infections. Previous comparative genomic studies have described the population structure and evolution of USA300 based on geographically restricted isolate collections. Here, we investigated the USA300 population by sequencing genomes of a geographically distributed panel of 191 clinical S. aureus isolates belonging to clonal complex 8 (CC8), derived from the Tigecycline Evaluation and Surveillance Trial program. Isolates were collected at 12 healthcare centres across nine USA states in 2004, 2009 or 2010. Reconstruction of evolutionary relationships revealed that CC8 was dominated by USA300 isolates (154/191, 81 %), which were heterogeneous and demonstrated limited phylogeographic clustering. Analysis of the USA300 core genomes revealed an increase in median pairwise SNP distance from 62 to 98 between 2004 and 2010, with a stable pattern of above average dN/dS ratios. The phylogeny of the USA300 population indicated that early diversification events led to the formation of nested clades, which arose through cumulative acquisition of predominantly non-synonymous SNPs in various coding sequences. The accessory genome of USA300 was largely homogenous and consisted of elements previously associated with this lineage. We observed an emergence of SCCmec negative and ACME negative USA300 isolates amongst more recent samples, and an increase in the prevalence of ϕSa5 prophage. Together, the analysed S. aureus USA300 collection revealed an evolving pan-genome through increased core genome heterogeneity and temporal variation in the frequency of certain accessory elements. Publisher PDF

The implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasive Staphylococcus aureus isolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http:// www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show that in silico predictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) communityoriented database infrastructure and analysis tools. IMPORTANCE The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine wholegenome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets., 0, SCOPUS: ar.j, info:eu-repo/semantics/published

Background: The recently isolated MRSA LGA251 has low resistance and carries a new mecA homolog. Results: PBP2ALGA, the protein product of the new mecA, showed a “preference” for penicillins and instability at 37 °C. mecALGA251 introduced into susceptible S. aureus allowed expression of high-level resistance. Significance: This study provides insights into the relationship between structure and function of PBP2A-like proteins., Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-to-strain variation in resistance level, in genetic background, and also in the structure of the chromosomal cassette (SCCmec) that carries the resistance gene mecA. In contrast, strain-to-strain variation in the sequence of the mecA determinant was found to be much more limited among MRSA isolates examined so far. The first exception to this came with the recent identification of MRSA strain LGA251, which carries a new homolog of this gene together with regulatory elements mecI/mecR that also have novel, highly divergent structures. After cloning and purification in Escherichia coli, PBP2ALGA, the protein product of the new mecA homolog, showed aberrant mobility in SDS-PAGE, structural instability and loss of activity at 37 °C, and a higher relative affinity for oxacillin as compared with cefoxitin. The mecA homolog free of its regulatory elements was cloned into a plasmid and introduced into the background of the β-lactam-susceptible S. aureus strain COL-S. In this background, the mecA homolog expressed a high-level resistance to cefoxitin (MIC = 400 μg/ml) and a somewhat lower resistance to oxacillin (minimal inhibitory concentration = 200 μg/ml). Similar to PBP2A, the protein homolog PBP2ALGA was able to replace the essential function of the S. aureus PBP2 for growth. In contrast to PBP2A, PBP2ALGA did not depend on the transglycosylase activity of the native PBP2 for expression of high level resistance to oxacillin, suggesting that the PBP2A homolog may preferentially cooperate with a monofunctional transglycosylase as the alternative source of transglycosylase activity.