The NaHCO3-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus (MRSA) Is Influenced by mecA Genotype.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of many invasive clinical syndromes, and pose treatment difficulties due to their in vitro resistance to most ß-lactams on standard laboratory testing. A novel phenotype frequently identified in MRSA strains, termed 'NaHCO3-responsiveness', is a property whereby strains are susceptible in vitro to many ß-lactams in the presence of NaHCO3. Specific mecA genotypes, repression of mecA/PBP2a expression and perturbed maturation of PBP2a by NaHCO3 have all been associated with this phenotype. The aim of this study was to define the relationship between specific mecA genotypes and PBP2a substitutions, on the one hand, with NaHCO3-responsiveness in vitro. Mutations were made in the mecA ribosomal binding site (RBS -7) and at amino acid position 246 of its coding region in parental strains MW2 (NaHCO3-responsive) and C36 (NaHCO3- nonresponsive) to generate 'swap' variants, each harboring the other's mecA-RBS/coding region genotypes. Successful swaps were confirmed by both sequencing, as well as predicted swap of in vitro penicillin-clavulanate susceptibility phenotypes. MW2 swap variants harboring the nonresponsive mecA genotypes became NaHCO3-nonresponsive (resistant to the ß-lactam, oxacillin [OXA]), in the presence of NaHCO3. Moreover, these swap variants had lost NaHCO3-mediated repression of mecA/PBP2a expression. In contrast, C36 swap variants harboring the NaHCO3-responsive mecA genotypes remained NaHCO3-nonresponsive phenotypically, and still exhibited nonrepressible mecA/PBP2a expression. These data demonstrate that in addition to the mecA genotype, NaHCO3-responsiveness may also depend on strain-specific genetic backgrounds.

Identification of a staphylococcal complement inhibitor with broad host specificity in equid Staphylococcus aureus strains.

Staphylococcus aureus is a versatile pathogen capable of causing a broad range of diseases in many different hosts. S. aureus can adapt to its host through modification of its genome (e.g. by acquisition and exchange of mobile genetic elements that encode host-specific virulence factors). Recently, the prophage φSaeq1 was discovered in S. aureus strains from six different clonal lineages almost exclusively isolated from equids. Within this phage, we discovered a novel variant of staphylococcal complement inhibitor (SCIN), a secreted protein that interferes with activation of the human complement system, an important line of host defense. We here show that this equine variant of SCIN, eqSCIN, is a potent blocker of equine complement system activation and subsequent phagocytosis of bacteria by phagocytes. Mechanistic studies indicate that eqSCIN blocks equine complement activation by specific inhibition of the C3 convertase enzyme (C3bBb). Whereas SCIN-A from human S. aureus isolates exclusively inhibits human complement, eqSCIN represents the first animal-adapted SCIN variant that functions in a broader range of hosts (horses, humans, and pigs). Binding analyses suggest that the human-specific activity of SCIN-A is related to amino acid differences on both sides of the SCIN-C3b interface. These data suggest that modification of this phage-encoded complement inhibitor plays a role in the host adaptation of S. aureus and are important to understand how this pathogen transfers between different hosts.

Truncation of GdpP mediates ß-lactam resistance in clinical isolates of Staphylococcus aureus.

OBJECTIVES: High-level ß-lactam resistance in MRSA is mediated in the majority of strains by a mecA or mecC gene. In this study, we identified 10 mec gene-negative MRSA human isolates from Austria and 11 bovine isolates from the UK showing high levels of ß-lactam resistance and sought to understand the molecular basis of the resistance observed. METHODS: Different antimicrobial resistance testing methods (disc diffusion, Etest and VITEK® 2) were used to establish the ß-lactam resistance profiles for the isolates and the isolates were further investigated by WGS. RESULTS: A number of mutations (including novel ones) in PBPs, AcrB, YjbH and the pbp4 promoter were identified in the resistant isolates, but not in closely related susceptible isolates. Importantly, a truncation in the cyclic diadenosine monophosphate phosphodiesterase enzyme, GdpP, was identified in 7 of the 10 Austrian isolates and 10 of the 11 UK isolates. Complementation of four representative isolates with an intact copy of the gdpP gene restored susceptibility to penicillins and abolished the growth defects caused by the truncation. CONCLUSIONS: This study reports naturally occurring inactivation of GdpP protein in Staphylococcus aureus of both human origin and animal origin, and demonstrates clinical relevance to a previously reported association between this truncation and increased ß-lactam resistance and impaired bacterial growth in laboratory-generated mutants. It also highlights possible limitations of genomic determination of antibiotic susceptibility based on single gene presence or absence when choosing the appropriate antimicrobial treatment for patients.

Prospective genomic surveillance of methicillin-resistant Staphylococcus aureus (MRSA) associated with bloodstream infection, England, 1 October 2012 to 30 September 2013.

BackgroundMandatory reporting of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSI) has occurred in England for over 15years. Epidemiological information is recorded, but routine collection of isolates for characterisation has not been routinely undertaken. Ongoing developments in whole-genome sequencing (WGS) have demonstrated its value in outbreak investigations and for determining the spread of antimicrobial resistance and bacterial population structure. Benefits of adding genomics to routine epidemiological MRSA surveillance are unknown.AimTo determine feasibility and potential utility of adding genomics to epidemiological surveillance of MRSA.MethodsWe conducted an epidemiological and genomic survey of MRSA BSI in England over a 1-year period (1 October 2012--30 September 2013).ResultsDuring the study period, 903 cases of MRSA BSI were reported; 425 isolates were available for sequencing of which, 276 (65%) were clonal complex (CC) 22. Addition of 64 MRSA genomes from published outbreak investigations showed that the study genomes could provide context for outbreak isolates and supported cluster identification. Comparison to other MRSA genome collections demonstrated variation in clonal diversity achieved through different sampling strategies and identified potentially high-risk clones e.g. USA300 and local expansion of CC5 MRSA in South West England.ConclusionsWe demonstrate the potential utility of combined epidemiological and genomic MRSA BSI surveillance to determine the national population structure of MRSA, contextualise previous MRSA outbreaks, and detect potentially high-risk lineages. These findings support the integration of epidemiological and genomic surveillance for MRSA BSI as a step towards a comprehensive surveillance programme in England.

Investigation of a Cluster of Sequence Type 22 Methicillin-Resistant Staphylococcus aureus Transmission in a Community Setting.

BACKGROUND: Whole-genome sequencing (WGS) has typically been used to confirm or refute hospital/ward outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) identified through routine practice. However, appropriately targeted WGS strategies that identify routinely "undetectable" transmission remain the ultimate aim. METHODS: WGS of MRSA isolates sent to a regional microbiological laboratory was performed as part of a 12-month prospective observational study. Phylogenetic analyses identified a genetically related cluster of E-MRSA15 isolated from patients registered to the same general practice (GP) surgery. This led to an investigation to identify epidemiological links, find additional cases, and determine potential for ongoing transmission. RESULTS: We identified 15 MRSA-positive individuals with 27 highly related MRSA isolates who were linked to the GP surgery, 2 of whom died with MRSA bacteremia. Of the 13 cases that were further investigated, 11 had attended a leg ulcer/podiatry clinic. Cases lacked epidemiological links to hospitals, suggesting that transmission occurred elsewhere. Environmental and staff screening at the GP surgery did not identify an ongoing source of infection. CONCLUSIONS: Surveillance in the United Kingdom shows that the proportion of MRSA bacteremias apportioned to hospitals is decreasing, suggesting the need for greater focus on the detection of MRSA outbreaks and transmission in the community. This case study confirms that the typically nosocomial lineage (E-MRSA15) can transmit within community settings. Our study exemplifies the continued importance of WGS in detecting outbreaks, including those which may be missed by routine practice, and suggests that universal WGS of bacteremia isolates may help detect outbreaks in low-surveillance settings.

Evolution of mobile genetic element composition in an epidemic methicillin-resistant Staphylococcus aureus: temporal changes correlated with frequent loss and gain events.

BACKGROUND: Horizontal transfer of mobile genetic elements (MGEs) that carry virulence and antimicrobial resistance genes mediates the evolution of methicillin-resistant Staphylococcus aureus, and the emergence of new MRSA clones. Most MRSA lineages show an association with specific MGEs and the evolution of MGE composition following clonal expansion has not been widely studied. RESULTS: We investigated the genomes of 1193 S. aureus bloodstream isolates, 1169 of which were MRSA, collected in the UK and the Republic of Ireland between 2001 and 2010. The majority of isolates belonged to clonal complex (CC)22 (n = 923), which contained diverse MGEs including elements that were found in other MRSA lineages. Several MGEs showed variable distribution across the CC22 phylogeny, including two antimicrobial resistance plasmids (pWBG751-like and SAP078A-like, carrying erythromycin and heavy metal resistance genes, respectively), a pathogenicity island carrying the enterotoxin C gene and two phage types Sa1int and Sa6int. Multiple gains and losses of these five MGEs were identified in the CC22 phylogeny using ancestral state reconstruction. Analysis of the temporal distribution of the five MGEs between 2001 and 2010 revealed an unexpected reduction in prevalence of the two plasmids and the pathogenicity island, and an increase in the two phage types. This occurred across the lineage and was not correlated with changes in the relative prevalence of CC22, or of any sub-lineages within in. CONCLUSIONS: Ancestral state reconstruction coupled with temporal trend analysis demonstrated that epidemic MRSA CC22 has an evolving MGE composition, and indicates that this important MRSA lineage has continued to adapt to changing selective pressure since its emergence.

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.

Old Drugs To Treat Resistant Bugs: Methicillin-Resistant Staphylococcus aureus Isolates with mecC Are Susceptible to a Combination of Penicillin and Clavulanic Acid.

ß-Lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is mediated by the expression of an alternative penicillin-binding protein 2a (PBP2a) (encoded by mecA) with a low affinity for ß-lactam antibiotics. Recently, a novel variant of mecA, known as mecC, was identified in MRSA isolates from both humans and animals. In this study, we demonstrate that mecC-encoded PBP2c does not mediate resistance to penicillin. Rather, broad-spectrum ß-lactam resistance in MRSA strains carrying mecC (mecC-MRSA strains) is mediated by a combination of both PBP2c and the distinct ß-lactamase encoded by the blaZ gene of strain LGA251 (blaZLGA251), which is part of mecC-encoding staphylococcal cassette chromosome mec (SCCmec) type XI. We further demonstrate that mecC-MRSA strains are susceptible to the combination of penicillin and the ß-lactam inhibitor clavulanic acid in vitro and that the same combination is effective in vivo for the treatment of experimental mecC-MRSA infection in wax moth larvae. Thus, we demonstrate how the distinct biological differences between mecA- and mecC-encoded PBP2a and PBP2c have the potential to be exploited as a novel approach for the treatment of mecC-MRSA infections.

Genomic insights into the rapid emergence and evolution of MDR in Staphylococcus pseudintermedius.

OBJECTIVES: MDR methicillin-resistant Staphylococcus pseudintermedius (MRSP) strains have emerged rapidly as major canine pathogens and present serious treatment issues and concerns to public health due to their, albeit low, zoonotic potential. A further understanding of the genetics of resistance arising from a broadly susceptible background of S. pseudintermedius is needed. METHODS: We sequenced the genomes of 12 S. pseudintermedius isolates of varied STs and resistance phenotypes. RESULTS: Nine distinct clonal lineages had acquired either staphylococcal cassette chromosome (SCC) mec elements and/or Tn5405-like elements carrying up to five resistance genes [aphA3, sat, aadE, erm(B), dfrG] to generate MRSP, MDR methicillin-susceptible S. pseudintermedius and MDR MRSP populations. The most successful and clinically problematic MDR MRSP clones, ST68 SCCmecV(T) and ST71 SCCmecII-III, have further accumulated mutations in gyrA and grlA conferring resistance to fluoroquinolones. The carriage of additional mobile genetic elements (MGEs) was highly variable, suggesting that horizontal gene transfer is frequent in S. pseudintermedius populations. CONCLUSIONS: Importantly, the data suggest that MDR MRSP evolved rapidly by the acquisition of a very limited number of MGEs and mutations, and that the use of many classes of antimicrobials may co-select for the spread and emergence of MDR and XDR strains. Antimicrobial stewardship will need to be comprehensive, encompassing human medicine and veterinary disciplines to successfully preserve antimicrobial efficacy.

Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is an important global health problem. MRSA resistance to ß-lactam antibiotics is mediated by the mecA or mecC genes, which encode an alternative penicillin-binding protein (PBP) 2a that has a low affinity to ß-lactam antibiotics. Detection of mec genes or PBP2a is regarded as the gold standard for the diagnosis of MRSA. We identified four MRSA isolates that lacked mecA or mecC genes, but were still phenotypically resistant to pencillinase-resistant ß-lactam antibiotics. METHODS: The four human S. aureus isolates were investigated by whole genome sequencing and a range of phenotypic assays. RESULTS: We identified a number of amino acid substitutions present in the endogenous PBPs 1, 2 and 3 that were found in the resistant isolates but were absent in closely related susceptible isolates and which may be the basis of resistance. Of particular interest are three identical amino acid substitutions in PBPs 1, 2 and 3, occurring independently in isolates from at least two separate multilocus sequence types. Two different non-conservative substitutions were also present in the same amino acid of PBP1 in two isolates from two different sequence types. CONCLUSIONS: This work suggests that phenotypically resistant MRSA could be misdiagnosed using molecular methods alone and provides evidence of alternative mechanisms for ß-lactam resistance in MRSA that may need to be considered by diagnostic laboratories.

A novel hybrid SCCmec-mecC region in Staphylococcus sciuri.

OBJECTIVES: Methicillin resistance in Staphylococcus spp. results from the expression of an alternative penicillin-binding protein 2a (encoded by mecA) with a low affinity for ß-lactam antibiotics. Recently, a novel variant of mecA known as mecC (formerly mecALGA251) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified two Staphylococcus sciuri subsp. carnaticus isolates from bovine infections that harbour three different mecA homologues: mecA, mecA1 and mecC. METHODS: We subjected the two isolates to whole-genome sequencing to further understand the genetic context of the mec-containing region. We also used PCR and RT-PCR to investigate the excision and expression of the SCCmec element and mec genes, respectively. RESULTS: Whole-genome sequencing revealed a novel hybrid SCCmec region at the orfX locus consisting of a class E mec complex (mecI-mecR1-mecC1-blaZ) located immediately downstream of a staphylococcal cassette chromosome mec (SCCmec) type VII element. A second SCCmec attL site (attL2), which was imperfect, was present downstream of the mecC region. PCR analysis of stationary-phase cultures showed that both the SCCmec type VII element and a hybrid SCCmec-mecC element were capable of excision from the genome and forming a circular intermediate. Transcriptional analysis showed that mecC and mecA, but not mecA1, were both expressed in liquid culture supplemented with oxacillin. CONCLUSIONS: Overall, this study further highlights that a range of staphylococcal species harbour the mecC gene and furthers the view that coagulase-negative staphylococci associated with animals may act as reservoirs of antibiotic resistance genes for more pathogenic staphylococcal species.

ICEberg: a web-based resource for integrative and conjugative elements found in Bacteria.

ICEberg (http://db-mml.sjtu.edu.cn/ICEberg/) is an integrated database that provides comprehensive information about integrative and conjugative elements (ICEs) found in bacteria. ICEs are conjugative self-transmissible elements that can integrate into and excise from a host chromosome. An ICE contains three typical modules, integration and excision, conjugation, and regulation modules, that collectively promote vertical inheritance and periodic lateral gene flow. Many ICEs carry likely virulence determinants, antibiotic-resistant factors and/or genes coding for other beneficial traits. ICEberg offers a unique, highly organized, readily explorable archive of both predicted and experimentally supported ICE-relevant data. It currently contains details of 428 ICEs found in representatives of 124 bacterial species, and a collection of >400 directly related references. A broad range of similarity search, sequence alignment, genome context browser, phylogenetic and other functional analysis tools are readily accessible via ICEberg. We propose that ICEberg will facilitate efficient, multi-disciplinary and innovative exploration of bacterial ICEs and be of particular interest to researchers in the broad fields of prokaryotic evolution, pathogenesis, biotechnology and metabolism. The ICEberg database will be maintained, updated and improved regularly to ensure its ongoing maximum utility to the research community.

Community MRSA outbreaks in areas of low prevalence.

OBJECTIVES: This review aims to explore the characteristics of outbreaks of community-acquired community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) in low prevalence areas, to understand the factors involved in its rise, and to translate this knowledge into public health policy and further research needs. SOURCES: PubMed, EMBASE and Google Scholar were searched using combinations of the terms "transmission", "acquisition", "CA-MRSA" "MRSA", "community-acquired", "low prevalence", "genomic", "outbreak", "colonisation" and "carriage". Wherever evidence was limited, additional articles were sought specifically, via PubMed searches. Papers where materials were not available in English were excluded. CONTENT: Community-acquired, community onset MRSA infection presents a significant public health challenge, even in low prevalence areas; where MRSA rates are historically lower. Despite successes in reducing hospital-onset MRSA (HO-MRSA), CO-MRSA rates are increasing globally, with a need to understand this trend, and the potential risk factors for re-emergence. Challenges in defining low prevalence areas and the significance of exposure to various risk factors for community acquisition, such as healthcare settings, travel, livestock, and environmental factors, are discussed. The importance of genomic surveillance in identifying outbreak strains and understanding the transmission dynamics is highlighted, along with the need for robust public health policies and control measures. IMPLICATIONS: The findings emphasise the complexity of CO-MRSA transmission and the necessity of a multifaceted approach in low prevalence areas. This includes integrated and systematic surveillance of HO-, CO-, and livestock-associated MRSA (LA-MRSA), as has been effective in some Northern European countries. The evolution of CO-MRSA underscores the need for global collaboration, routine genomic surveillance, and comprehensive antimicrobial stewardship to mitigate the rise of CO-MRSA and address the broader challenge of antimicrobial resistance. These efforts are crucial for maintaining low MRSA prevalence and managing the increasing burden of CO-MRSA in both low and higher prevalence regions.

Evaluating the impact of genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) on hospital infection prevention and control decisions.

Genomic epidemiology enhances the ability to detect and refute methicillin-resistant Staphylococcus aureus (MRSA) outbreaks in healthcare settings, but its routine introduction requires further evidence of benefits for patients and resource utilization. We performed a 12 month prospective study at Cambridge University Hospitals NHS Foundation Trust in the UK to capture its impact on hospital infection prevention and control (IPC) decisions. MRSA-positive samples were identified via the hospital microbiology laboratory between November 2018 and November 2019. We included samples from in-patients, clinic out-patients, people reviewed in the Emergency Department and healthcare workers screened by Occupational Health. We sequenced the first MRSA isolate from 823 consecutive individuals, defined their pairwise genetic relatedness, and sought epidemiological links in the hospital and community. Genomic analysis of 823 MRSA isolates identified 72 genetic clusters of two or more isolates containing 339/823 (41 %) of the cases. Epidemiological links were identified between two or more cases for 190 (23 %) individuals in 34/72 clusters. Weekly genomic epidemiology updates were shared with the IPC team, culminating in 49 face-to-face meetings and 21 written communications. Seventeen clusters were identified that were consistent with hospital MRSA transmission, discussion of which led to additional IPC actions in 14 of these. Two outbreaks were also identified where transmission had occurred in the community prior to hospital presentation; these were escalated to relevant IPC teams. We identified 38 instances where two or more in-patients shared a ward location on overlapping dates but carried unrelated MRSA isolates (pseudo-outbreaks); research data led to de-escalation of investigations in six of these. Our findings provide further support for the routine use of genomic epidemiology to enhance and target IPC resources.

Antibiotic resistance determination using Enterococcus faecium whole-genome sequences: a diagnostic accuracy study using genotypic and phenotypic data.

BACKGROUND: DNA sequencing could become an alternative to in vitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. Here, we aimed to assess and improve the accuracy of antibiotic resistance determination from Enterococcus faecium genomes for diagnosis and surveillance purposes. METHODS: In this retrospective diagnostic accuracy study, we first conducted a literature search in PubMed on Jan 14, 2021, to compile a catalogue of genes and mutations predictive of antibiotic resistance in E faecium. We then evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different, clinically relevant antibiotics using a diverse population of 4382 E faecium isolates with available whole-genome sequences and in vitro culture-based AST phenotypes. Isolates were obtained from various sources in 11 countries worldwide between 2000 and 2018. We included isolates tested with broth microdilution, Vitek 2, and disc diffusion, and antibiotics with at least 50 susceptible and 50 resistant isolates. Phenotypic resistance was derived from raw minimum inhibitory concentrations and measured inhibition diameters, and harmonised primarily using the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing. A bioinformatics pipeline was developed to process raw sequencing reads, identify antibiotic resistance genetic determinants, and report genotypic resistance. We used our curated database, as well as ResFinder, AMRFinderPlus, and LRE-Finder, to assess the accuracy of genotypic predictions against phenotypic resistance. FINDINGS: We curated a catalogue of 228 genetic markers involved in resistance to 12 antibiotics in E faecium. Very accurate genotypic predictions were obtained for ampicillin (sensitivity 99·7% [95% CI 99·5-99·9] and specificity 97·9% [95·8-99·0]), ciprofloxacin (98·0% [96·4-98·9] and 98·8% [95·9-99·7]), vancomycin (98·8% [98·3-99·2] and 98·8% [98·0-99·3]), and linezolid resistance (after re-testing false negatives: 100·0% [90·8-100·0] and 98·3% [97·8-98·7]). High sensitivity was obtained for tetracycline (99·5% [99·1-99·7]), teicoplanin (98·9% [98·4-99·3]), and high-level resistance to aminoglycosides (97·7% [96·6-98·4] for streptomycin and 96·8% [95·8-97·5] for gentamicin), although at lower specificity (60-90%). Sensitivity was expectedly low for daptomycin (73·6% [65·1-80·6]) and tigecycline (38·3% [27·1-51·0]), for which the genetic basis of resistance is not fully characterised. Compared with other antibiotic resistance databases and bioinformatic tools, our curated database was similarly accurate at detecting resistance to ciprofloxacin and linezolid and high-level resistance to streptomycin and gentamicin, but had better sensitivity for detecting resistance to ampicillin, tigecycline, daptomycin, and quinupristin-dalfopristin, and better specificity for ampicillin, vancomycin, teicoplanin, and tetracycline resistance. In a validation dataset of 382 isolates, similar or improved diagnostic accuracies were also achieved. INTERPRETATION: To our knowledge, this work represents the largest published evaluation to date of the accuracy of antibiotic susceptibility predictions from E faecium genomes. The results and resources will facilitate the adoption of whole-genome sequencing as a tool for the diagnosis and surveillance of antimicrobial resistance in E faecium. A complete characterisation of the genetic basis of resistance to last-line antibiotics, and the mechanisms mediating antibiotic resistance silencing, are needed to close the remaining sensitivity and specificity gaps in genotypic predictions. FUNDING: Wellcome Trust, UK Department of Health, British Society for Antimicrobial Chemotherapy, Academy of Medical Sciences and the Health Foundation, Medical Research Council Newton Fund, Vietnamese Ministry of Science and Technology, and European Society of Clinical Microbiology and Infectious Disease.

A Staphylococcus xylosus isolate with a new mecC allotype.

Recently, a novel variant of mecA known as mecC (mecA(LGA251)) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec.

Use of Vitek 2 antimicrobial susceptibility profile to identify mecC in methicillin-resistant Staphylococcus aureus.

The emergence of mecC methicillin-resistant Staphylococcus aureus (MRSA) poses a diagnostic challenge for clinical microbiology laboratories. Using the Vitek 2 system, we tested a panel of 896 Staphylococcus aureus isolates and found that an oxacillin-sensitive/cefoxitin-resistant profile had a sensitivity of 88.7% and a specificity of 99.5% for the identification of mecC MRSA isolates. The presence of the mecC gene, determined by bacterial whole-genome sequencing, was used as the gold standard. This profile could provide a zero-cost screening method for identification of mecC-positive MRSA strains.

TADB: a web-based resource for Type 2 toxin-antitoxin loci in bacteria and archaea.

TADB (http://bioinfo-mml.sjtu.edu.cn/TADB/) is an integrated database that provides comprehensive information about Type 2 toxin-antitoxin (TA) loci, genetic features that are richly distributed throughout bacterial and archaeal genomes. Two-gene and much less frequently three-gene Type 2 TA loci code for cognate partners that have been hypothesized or demonstrated to play key roles in stress response, bacterial physiology and stabilization of horizontally acquired genetic elements. TADB offers a unique compilation of both predicted and experimentally supported Type 2 TA loci-relevant data and currently contains 10,753 Type 2 TA gene pairs identified within 1240 prokaryotic genomes, and details of over 240 directly relevant scientific publications. A broad range of similarity search, sequence alignment, genome context browser and phylogenetic tools are readily accessible via TADB. We propose that TADB will facilitate efficient, multi-disciplinary and innovative exploration of the bacteria and archaea Type 2 TA space, better defining presently recognized TA-related phenomena and potentially even leading to yet-to-be envisaged frontiers. The TADB database, envisaged as a one-stop shop for Type 2 TA-related research, will be maintained, updated and improved regularly to ensure its ongoing maximum utility to the research community.

Colonization and transmission of Staphylococcus aureus in schools: a citizen science project.

Aggregation of children in schools has been established to be a key driver of transmission of infectious diseases. Mathematical models of transmission used to predict the impact of control measures, such as vaccination and testing, commonly depend on self-reported contact data. However, the link between self-reported social contacts and pathogen transmission has not been well described. To address this, we used Staphylococcus aureus as a model organism to track transmission within two secondary schools in England and test for associations between self-reported social contacts, test positivity and the bacterial strain collected from the same students. Students filled out a social contact survey and their S. aureus colonization status was ascertained through self-administered swabs from which isolates were sequenced. Isolates from the local community were also sequenced to assess the representativeness of school isolates. A low frequency of genome-linked transmission precluded a formal analysis of links between genomic and social networks, suggesting that S. aureus transmission within schools is too rare to make it a viable tool for this purpose. Whilst we found no evidence that schools are an important route of transmission, increased colonization rates found within schools imply that school-age children may be an important source of community transmission.