Project description:BACKGROUND:?Strategies to prevent Staphylococcus aureus infection in hospitals focus on patient-to-patient transmission. We used whole-genome sequencing to investigate the role of colonized patients as the source of new S. aureus acquisitions, and the reliability of identifying patient-to-patient transmission using the conventional approach of spa typing and overlapping patient stay. METHODS:Over 14 months, all unselected patients admitted to an adult intensive care unit (ICU) were serially screened for S. aureus. All available isolates (n = 275) were spa typed and underwent whole-genome sequencing to investigate their relatedness at high resolution. RESULTS:Staphylococcus aureus was carried by 185 of 1109 patients sampled within 24 hours of ICU admission (16.7%); 59 (5.3%) patients carried methicillin-resistant S. aureus (MRSA). Forty-four S. aureus (22 MRSA) acquisitions while on ICU were detected. Isolates were available for genetic analysis from 37 acquisitions. Whole-genome sequencing indicated that 7 of these 37 (18.9%) were transmissions from other colonized patients. Conventional methods (spa typing combined with overlapping patient stay) falsely identified 3 patient-to-patient transmissions (all MRSA) and failed to detect 2 acquisitions and 4 transmissions (2 MRSA). CONCLUSIONS:Only a minority of S. aureus acquisitions can be explained by patient-to-patient transmission. Whole-genome sequencing provides the resolution to disprove transmission events indicated by conventional methods and also to reveal otherwise unsuspected transmission events. Whole-genome sequencing should replace conventional methods for detection of nosocomial S. aureus transmission.
Project description:Staphylococcus aureus ST291 has been reported as a homologue recombinant double locus variant of the livestock associated S. aureus ST398. However, whole genome sequencing show that ST291 is a unique genetic lineage with highly variable content within its accessory genome compared to both human and livestock associated genome sequenced CC398s.
Project description:Here, we report the draft genome sequences of Burkholderia pseudomallei and Staphylococcus aureus causing chronic rhinosinusitis. Whole-genome sequencing determined the B. pseudomallei as sequence type (ST) 1381 and the S. aureus as ST8. B. pseudomallei possessed the blaOXA-59 gene. This study illustrates the potential emergence of B. pseudomallei in cases of chronic rhinosinusitis.
Project description:Whole-genome sequencing of Staphylococcus xylosus strain JW2311 from bovine mastitis milk identified the novel 49.3-kb macrolide-lincosamide-streptogramin B (MLSB) resistance plasmid pJW2311. It contained the macrolide resistance gene mph(C), the macrolide-streptogramin B resistance gene msr(A), and the new MLSB resistance gene erm(48) and could be transformed into Staphylococcus aureus by electroporation. Functionality of erm(48) was demonstrated by cloning and expression in S. aureus.
Project description:We describe here the genetic analysis of a vancomycin-susceptible Staphylococcus aureus (VSSA) strain, Mu50Omega, a strain related to vancomycin-intermediate S. aureus (VISA) strain Mu50. Using a combination of Mu50Omega whole-genome sequencing and genome engineering, we observed a stepwise evolution of vancomycin resistance from VSSA to VISA after the mutated vraS and graR genes of Mu50 were engineered into Mu50Omega.
Project description:Successive division events in the spherically shaped bacterium Staphylococcus aureus are oriented in three alternating perpendicular planes. The mechanisms that underlie this relatively unique pattern of division and coordinate it with chromosome segregation remain largely unknown. Thus far, the only known spatial regulator of division in this organism is the nucleoid occlusion protein Noc that inhibits assembly of the cytokinetic ring over the chromosome. However, Noc is not essential in S. aureus, indicating that additional regulators are likely to exist. To search for these factors, we screened for mutants that are synthetic lethal with Noc inactivation. Our characterization of these mutants led to the discovery that S. aureus Noc also controls the initiation of DNA replication. We show that cells lacking Noc over-initiate and mutations in the initiator gene dnaA suppress this defect. Importantly, these dnaA mutations also partially suppress the division problems associated with ∆noc. Reciprocally, we show that over-expression of DnaA enhances the over-initiation and cell division phenotypes of cells lacking Noc. Thus, a single factor both blocks cell division over chromosomes and helps to ensure that new rounds of DNA replication are not initiated prematurely. This degree of economy in coordinating key cell biological processes has not been observed in rod-shaped bacteria and may reflect the challenges posed by the reduced cell volume and complicated division pattern of this spherical pathogen. Overall design: Whole Genome Sequencing experiments were performed on wild type and mutant cells of Staphylococcus aureus RN4220 (Nair et al,PMID: 21378186 ) and HG003 (Herbert et al, 2010 PMID: 20212089) derivatives grown in tryptic soy broth (TSB) at 37°C with aeration.
Project description:The genome of Staphylococcus aureus has rapidly become one the most frequently sequenced among bacteria, with more than 40000 genome sequences uploaded to public databases. Computational resources required for analysis and quality assessment have lagged behind accumulation of sequence data. Improved analytic pipelines, in combination with the development of customized S. aureus reference databases, can be used to inform S. aureus biology and potentially predict clinical outcome. Here, we review the currently available data about S. aureus genome in public databases, and discuss their potential utility for understanding S. aureus evolution. Also discussed are ways to overcome challenges to the application of whole-genome sequencing data for prevention and management of S. aureus disease.
Project description:Current clinical next-generation sequencing is done by using gene panels and exome analysis, both of which involve selective capturing of target regions. However, capturing has limitations in sufficiently covering coding exons, especially GC-rich regions. We compared whole exome sequencing (WES) with the most recent PCR-free whole genome sequencing (WGS), showing that only the latter is able to provide hitherto unprecedented complete coverage of the coding region of the genome. Thus, from a clinical/technical point of view, WGS is the better WES so that capturing is no longer necessary for the most comprehensive genomic testing of Mendelian disorders.
Project description:Methicillin-resistant Staphylococcus aureus USA300, belonging to sequence type (ST) 8, is a rare cause of necrotizing fasciitis in the USA. We herein report a case of monomicrobial Fournier's gangrene caused by an ST8, methicillin-susceptible Staphylococcus aureus (designated ksw1). Whole-genome sequencing and analyses for virulence determinants revealed that, unlike USA300, ksw1 lacked virulence genes, such as Panton-Valentine leukocidin and SCCmec, while harboring the toxic shock syndrome toxin-1 gene. These genomic features correlate with ST8 CA-MRSA/J, which is the major genotype of ST8 in Japan.
Project description:Children with acute hematogenous osteomyelitis (AHO) have a broad spectrum of illness ranging from mild to severe. The purpose of this study is to evaluate the impact of genomic variation of Staphylococcus aureus on clinical phenotype of affected children and determine which virulence genes correlate with severity of illness.De novo whole genome sequencing was conducted for a strain of Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA), using PacBio Hierarchical Genome Assembly Process (HGAP) from 6 Single Molecule Real Time (SMRT) Cells, as a reference for DNA library assembly of 71 Staphylococcus aureus isolates from children with AHO. Virulence gene annotation was based on exhaustive literature review and genomic data in NCBI for Staphylococcus aureus. Clinical phenotype was assessed using a validated severity score. Kruskal-Wallis rank sum test determined association between clinical severity and virulence gene presence using False Discovery Rate (FDR), significance <0.01.PacBio produced an assembled genome of 2,898,306 bp and 2054 Open Reading Frames (ORFs). Annotation confirmed 201 virulence genes. Statistical analysis of gene presence by clinical severity found 40 genes significantly associated with severity of illness (FDR ≤0.009). MRSA isolates encoded a significantly greater number of virulence genes than did MSSA (p < 0.0001). Phylogenetic analysis by maximum likelihood (PAML) demonstrated the relatedness of genomic distance to clinical phenotype.The Staphylococcus aureus genome contains virulence genes which are significantly associated with severity of illness in children with osteomyelitis. This study introduces a novel reference strain and detailed annotation of Staphylococcus aureus virulence genes. While this study does not address bacterial gene expression, a platform is created for future transcriptome investigations to elucidate the complex mechanisms involved in childhood osteomyelitis.