Improved characterisation of MRSA transmission using within-host bacterial sequence diversity

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

Genome sequencing defines phylogeny and spread of methicillin-resistant Staphylococcus aureus in a high transmission setting.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infection. Whole-genome sequencing of MRSA has been used to define phylogeny and transmission in well-resourced healthcare settings, yet the greatest burden of nosocomial infection occurs in resource-restricted settings where barriers to transmission are lower. Here, we study the flux and genetic diversity of MRSA on ward and individual patient levels in a hospital where transmission was common. We repeatedly screened all patients on two intensive care units for MRSA carriage over a 3-mo period. All MRSA belonged to multilocus sequence type 239 (ST 239). We defined the population structure and charted the spread of MRSA by sequencing 79 isolates from 46 patients and five members of staff, including the first MRSA-positive screen isolates and up to two repeat isolates where available. Phylogenetic analysis identified a flux of distinct ST 239 clades over time in each intensive care unit. In total, five main clades were identified, which varied in the carriage of plasmids encoding antiseptic and antimicrobial resistance determinants. Sequence data confirmed intra- and interwards transmission events and identified individual patients who were colonized by more than one clade. One patient on each unit was the source of numerous transmission events, and deep sampling of one of these cases demonstrated colonization with a "cloud" of related MRSA variants. The application of whole-genome sequencing and analysis provides novel insights into the transmission of MRSA in under-resourced healthcare settings and has relevance to wider global health.The authors acknowledge financial support from the UKCRC Translational Infection Research (TIR) 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 on behalf of the Department of Health, and the Chief Scientist Office of the Scottish Government Health Directorate (to Professor Peacock); from Wellcome Trust grant number 098051 awarded to the Wellcome Trust Sanger Institute; and the NIHR Cambridge Biomedical Research Centre (to Professor Peacock). S.Y.C.T. is an Australian National Health and Medical Research Council Career Development Fellow (1065736)This is the final version of the article. It first appeared at http://www.genome.org/cgi/doi/10.1101/gr.174730.114

Factors Predicting and Reducing Mortality in Patients with Invasive Staphylococcus aureus Disease in a Developing Country

BACKGROUND: Invasive Staphylococcus aureus infection is increasingly recognised as an important cause of serious sepsis across the developing world, with mortality rates higher than those in the developed world. The factors determining mortality in developing countries have not been identified. METHODS: A prospective, observational study of invasive S. aureus disease was conducted at a provincial hospital in northeast Thailand over a 1-year period. All-cause and S. aureus-attributable mortality rates were determined, and the relationship was assessed between death and patient characteristics, clinical presentations, antibiotic therapy and resistance, drainage of pus and carriage of genes encoding Panton-Valentine Leukocidin (PVL). PRINCIPAL FINDINGS: A total of 270 patients with invasive S. aureus infection were recruited. The range of clinical manifestations was broad and comparable to that described in developed countries. All-cause and S. aureus-attributable mortality rates were 26% and 20%, respectively. Early antibiotic therapy and drainage of pus were associated with a survival advantage (both p<0.001) on univariate analysis. Patients infected by a PVL gene-positive isolate (122/248 tested, 49%) had a strong survival advantage compared with patients infected by a PVL gene-negative isolate (all-cause mortality 11% versus 39% respectively, p<0.001). Multiple logistic regression analysis using all variables significant on univariate analysis revealed that age, underlying cardiac disease and respiratory infection were risk factors for all-cause and S. aureus-attributable mortality, while one or more abscesses as the presenting clinical feature and procedures for infectious source control were associated with survival. CONCLUSIONS: Drainage of pus and timely antibiotic therapy are key to the successful management of S. aureus infection in the developing world. Defining the presence of genes encoding PVL provides no practical bedside information and draws attention away from identifying verified clinical risk factors and those interventions that save lives

Emergence of Community-Associated Methicillin-Resistant Staphylococcus aureus Carriage in Children in Cambodia

We previously described the first reported isolation of methicillin-resistant Staphylococcus aureus (MRSA) (a case series of pediatric community-associated MRSA infections) in Cambodia. We define the rate of pediatric MRSA carriage in the same population and characterize the associated bacterial genotypes by using pulsed-field gel electrophoresis and multilocus sequence typing. A prospective cohort study of MRSA carriage conducted over one month at the Angkor Hospital for Children, Siem Reap, Cambodia, identified MRSA carriage in 87 (3.5%) of 2,485 children who came to the outpatient department, and 6 (4.1%) of 145 inpatients, including at least two with cases of nosocomial acquisition. Genotyping of all 93 MRSA isolates resolved 5 genotypes. Most (91%) isolates were assigned to sequence type 834. Only 28 (32%) of 87 MRSA carriers identified in the outpatient department had no history of recent healthcare contact. The study findings have important implications for healthcare in a setting where diagnostic microbiology and access to antimicrobial drugs with efficacy against MRSA are limited

Feasibility of Modified Surviving Sepsis Campaign Guidelines in a Resource-Restricted Setting Based on a Cohort Study of Severe S. Aureus Sepsis

[This corrects the article on p. e29858 in vol. 7.]

Staphylococcus aureus Bacteraemia in a Tropical Setting: Patient Outcome and Impact of Antibiotic Resistance

Background: Most information on invasive Staphylococcus aureus infections comes from temperate countries. There are considerable knowledge gaps in epidemiology, treatment, drug resistance and outcome of invasive S. aureus infection in the tropics. Methods: A prospective, observational study of S. aureus bacteraemia was conducted in a 1000-bed regional hospital in northeast Thailand over 1 year. Detailed clinical data were collected and final outcomes determined at 12 weeks, and correlated with antimicrobial susceptibility profiles of infecting isolates. Principal Findings: Ninety-eight patients with S. aureus bacteraemia were recruited. The range of clinical manifestations was similar to that reported from temperate countries. The prevalence of endocarditis was 14%. The disease burden was highest at both extremes of age, whilst mortality increased with age. The all-cause mortality rate was 52%, with a mortality attributable to S. aureus of 44%. Methicillin-resistant S. aureus (MRSA) was responsible for 28% of infections, all of which were healthcare-associated. Mortality rates for MRSA and methicillin-susceptible S. aureus (MSSA) were 67% (18/27) and 46% (33/71), respectively (p = 0.11). MRSA isolates were multidrug resistant. Only vancomycin or fusidic acid would be suitable as empirical treatment options for suspected MRSA infection. Conclusions: S. aureus is a significant pathogen in northeast Thailand, with comparable clinical manifestations and a similar endocarditis prevalence but higher mortality than industrialised countries. S. aureus bacteraemia is frequently associated with exposure to healthcare settings with MRSA causing a considerable burden of disease. Further studies are required to define setting-specific strategies to reduce mortality from S. aureus bacteraemia, prevent MRSA transmission, and to define the burden of S. aureus disease and emergence of drug resistance throughout the developing world. © 2009 Nickerson et al

Panton-Valentine leucocidin is the key determinant of Staphylococcus aureus pyomyositis in a bacterial GWAS

Pyomyositis is a severe bacterial infection of skeletal muscle, commonly affecting children in tropical regions, predominantly caused by . To understand the contribution of bacterial genomic factors to pyomyositis, we conducted a genome-wide association study of cultured from 101 children with pyomyositis and 417 children with asymptomatic nasal carriage attending the Angkor Hospital for Children, Cambodia. We found a strong relationship between bacterial genetic variation and pyomyositis, with estimated heritability 63.8% (95% CI 49.2-78.4%). The presence of the Panton-Valentine leucocidin (PVL) locus increased the odds of pyomyositis 130-fold (=10). The signal of association mapped both to the PVL-coding sequence and the sequence immediately upstream. Together these regions explained over 99.9% of heritability (95% CI 93.5-100%). Our results establish staphylococcal pyomyositis, like tetanus and diphtheria, as critically dependent on a single toxin and demonstrate the potential for association studies to identify specific bacterial genes promoting severe human disease

Platelet-Related Variants Identified by Exomechip Meta-analysis in 157,293 Individuals

Platelet production, maintenance, and clearance are tightly controlled processes indicative of platelets important roles in hemostasis and thrombosis. Platelets are common targets for primary and secondary prevention of several conditions. They are monitored clinically by complete blood counts, specifically with measurements of platelet count (PLT) and mean platelet volume (MPV). Identifying genetic effects on PLT and MPV can provide mechanistic insights into platelet biology and their role in disease. Therefore, we formed the Blood Cell Consortium (BCX) to perform a large-scale meta-analysis of Exomechip association results for PLT and MPV in 157,293 and 57,617 individuals, respectively. Using the low-frequency/rare coding variant-enriched Exomechip genotyping array, we sought to identify genetic variants associated with PLT and MPV. In addition to confirming 47 known PLT and 20 known MPV associations, we identified 32 PLT and 18 MPV associations not previously observed in the literature across the allele frequency spectrum, including rare large effect (FCER1A), low-frequency (IQGAP2, MAP1A, LY75), and common (ZMIZ2, SMG6, PEAR1, ARFGAP3/PACSIN2) variants. Several variants associated with PLT/MPV (PEAR1, MRVI1, PTGES3) were also associated with platelet reactivity. In concurrent BCX analyses, there was overlap of platelet-associated variants with red (MAP1A, TMPRSS6, ZMIZ2) and white (PEAR1, ZMIZ2, LY75) blood cell traits, suggesting common regulatory pathways with shared genetic architecture among these hematopoietic lineages. Our large-scale Exomechip analyses identified previously undocumented associations with platelet traits and further indicate that several complex quantitative hematological, lipid, and cardiovascular traits share genetic factors

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead