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.
Project description:The genome of Staphylococcus aureus RKI4, a strain isolated from feces of a patient in a case of staphylococcal food poisoning, was sequenced using combined Illumina and single-molecule real-time sequencing. Hierarchical assembly of the genome resulted in a 2,725,654-bp chromosome and a 17,905-bp mobile genetic element.
Project description:Whole-genome sequencing (WGS) can provide excellent resolution in global and local epidemiological investigations of Staphylococcus aureus outbreaks. A variety of sequencing approaches and analytical tools have been used; it is not clear which is ideal. We compared two WGS strategies and two analytical approaches to the standard method of SmaI restriction digestion pulsed-field gel electrophoresis (PFGE) for typing S. aureus Forty-two S. aureus isolates from three outbreaks and 12 reference isolates were studied. Near-complete genomes, assembled de novo with paired-end and long-mate-pair (8 kb) libraries were first assembled and analyzed utilizing an in-house assembly and analytical informatics pipeline. In addition, paired-end data were assembled and analyzed using a commercial software package. Single nucleotide variant (SNP) analysis was performed using the in-house pipeline. Two assembly strategies were used to generate core genome multilocus sequence typing (cgMLST) data. First, the near-complete genome data generated with the in-house pipeline were imported into the commercial software and used to perform cgMLST analysis. Second, the commercial software was used to assemble paired-end data, and resolved assemblies were used to perform cgMLST. Similar isolate clustering was observed using SNP calling and cgMLST, regardless of data assembly strategy. All methods provided more discrimination between outbreaks than did PFGE. Overall, all of the evaluated WGS strategies yielded statistically similar results for S. aureus typing.
Project description:Staphylococcus aureus ATCC BAA-39 is the reference organism for a multidrug-resistant Staphylococcus aureus (MRSA) strain that was used to study drug-induced resistance reversion by an iodine-containing nanomolecular complex, FS-1. PacBio sequencing was performed on both the experimental and control strains, followed by genome assembly, variant calling, and DNA modification profiling.
Project description:The heterogeneous course, severity, and treatment responses among patients with atopic dermatitis (AD; eczema) highlight the complexity of this multifactorial disease. Prior studies have used traditional typing methods on cultivated isolates or sequenced a bacterial marker gene to study the skin microbial communities of AD patients. Shotgun metagenomic sequence analysis provides much greater resolution, elucidating multiple levels of microbial community assembly ranging from kingdom to species and strain-level diversification. We analyzed microbial temporal dynamics from a cohort of pediatric AD patients sampled throughout the disease course. Species-level investigation of AD flares showed greater Staphylococcus aureus predominance in patients with more severe disease and Staphylococcus epidermidis predominance in patients with less severe disease. At the strain level, metagenomic sequencing analyses demonstrated clonal S. aureus strains in more severe patients and heterogeneous S. epidermidis strain communities in all patients. To investigate strain-level biological effects of S. aureus, we topically colonized mice with human strains isolated from AD patients and controls. This cutaneous colonization model demonstrated S. aureus strain-specific differences in eliciting skin inflammation and immune signatures characteristic of AD patients. Specifically, S. aureus isolates from AD patients with more severe flares induced epidermal thickening and expansion of cutaneous T helper 2 (TH2) and TH17 cells. Integrating high-resolution sequencing, culturing, and animal models demonstrated how functional differences of staphylococcal strains may contribute to the complexity of AD disease.
Project description:Staphylococcus aureus subsp. anaerobius is responsible for Morel's disease in animals and a cause of abscess in humans. It is characterized by a microaerophilic growth, contrary to the other strains of S. aureus. The 2,604,446-bp genome (32.7% GC content) of S. anaerobius ST1464 comprises one chromosome and no plasmids. The chromosome contains 2,660 open reading frames (ORFs), 49 tRNAs and three complete rRNAs, forming one complete operon. The size of ORFs ranges between 100 to 4,600 bp except for two ORFs of 6,417 and 7,173 bp encoding segregation ATPase and non-ribosomal peptide synthase, respectively. The chromosome harbors Staphylococcus phage 2638A genome and incomplete Staphylococcus phage genome PT1028, but no detectable CRISPRS. The antibiotic resistance gene for tetracycline was found although Staphylococcus aureus subsp. anaerobius is susceptible to tetracycline in-vitro. Intact oxygen detoxification genes encode superoxide dismutase and cytochrome quinol oxidase whereas the catalase gene is impaired by a stop codon. Based on the genome, in-silico multilocus sequence typing indicates that S. aureus subsp. anaerobius emerged as a clone separated from all other S. aureus strains, illustrating host-adaptation linked to missing functions. Availability of S. aureus subsp. anaerobius genome could prompt the development of post-genomic tools for its rapid discrimination from S. aureus.
Project description:Antibiotic-resistant Staphylococcus aureus is an opportunistic pathogen causing serious human infections worldwide. Here, we report the complete annotated genome of bacteriophage SA75, a member of the Siphoviridae family which could be an alternative to traditional antibiotics for treating Staphylococcus infections. We used a hybrid approach combining MinION and Illumina MiSeq sequencing, which yielded a 43,134-bp genome and 65 open reading frames.
Project description:Staphylococcus aureus is an opportunistic Gram-positive pathogen responsible for a wide range of infections from minor skin abscesses to life-threatening diseases. Here, we report the draft genome assembly and current annotation of the HG001 strain, a derivative of the RN1 (NCT8325) strain with restored rbsU (a positive activator of SigB).
Project description:In addition to being an important human pathogen, Staphylococcus aureus is able to cause a variety of infections in numerous other host species. While the S. aureus strains causing infection in several of these hosts have been well characterised, this is not the case for companion rabbits (Oryctolagus cuniculus), where little data are available on S. aureus strains from this host. To address this deficiency we have performed antimicrobial susceptibility testing and genome sequencing on a collection of S. aureus isolates from companion rabbits. The findings show a diverse S. aureus population is able to cause infection in this host, and while antimicrobial resistance was uncommon, the isolates possess a range of known and putative virulence factors consistent with a diverse clinical presentation in companion rabbits including severe abscesses. We additionally show that companion rabbit isolates carry polymorphisms within dltB as described as underlying host-adaption of S. aureus to farmed rabbits. The availability of S. aureus genome sequences from companion rabbits provides an important aid to understanding the pathogenesis of disease in this host and in the clinical management and surveillance of these infections.
Project description:BACKGROUND: Whole genome (optical) mapping (WGM), a state-of-the-art mapping technology based on the generation of high resolution restriction maps, has so far been used for typing clinical outbreak strains and for mapping de novo sequence contigs in genome sequencing projects. We employed WGM to assess the genomic stability of previously sequenced Staphylococcus aureus strains that are commonly used in laboratories as reference standards. RESULTS: S. aureus strains (n?=?12) were mapped on the Argus™ Optical Mapping System (Opgen Inc, Gaithersburg, USA). Assembly of NcoI-restricted DNA molecules, visualization, and editing of whole genome maps was performed employing MapManager and MapSolver softwares (Opgen Inc). In silico whole genome NcoI-restricted maps were also generated from available sequence data, and compared to the laboratory-generated maps. Strains showing differences between the two maps were resequenced using Nextera XT DNA Sample Preparation Kit and Miseq Reagent Kit V2 (MiSeq, Illumina) and de novo assembled into sequence contigs using the Velvet assembly tool. Sequence data were correlated with corresponding whole genome maps to perform contig mapping and genome assembly using MapSolver. Of the twelve strains tested, one (USA300_FPR3757) showed a 19-kbp deletion on WGM compared to its in silico generated map and reference sequence data. Resequencing of the USA300_FPR3757 identified the deleted fragment to be a 13 kbp-long integrative conjugative element ICE6013. CONCLUSIONS: Frequent subculturing and inter-laboratory transfers can induce genomic and therefore, phenotypic changes that could compromise the utility of standard reference strains. WGM can thus be used as a rapid genome screening method to identify genomic rearrangements whose size and type can be confirmed by sequencing.