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1.  Development of Pooled Suppression Subtractive Hybridization to analyze the Pangenome of Staphylococcus aureus 
We describe the development and application of a pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of Staphylococcus aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty nine fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.
doi:10.1016/j.mimet.2010.01.022
PMCID: PMC2843094  PMID: 20138093
Staphylococcus aureus; pangenome; subtractive hybridization
2.  Potential Influence of Staphylococcus aureus Clonal Complex 30 Genotype and Transcriptome on Hematogenous Infections 
Open Forum Infectious Diseases  2015;2(3):ofv093.
Staphylococcus aureus CC30 is distinguished by its ability initiate complicated infections. This ability is due to acquisition of unique genes, SNPs and metabolic changes that attenuate virulence until the conditions become favorable for bacteremia and subsequent hematogenous seeding.
Background. The contemporary Staphylococcus aureus clonal complex (CC) 30 lineage is associated with complicated infections, including endocarditis and osteomyelitis. This lineage diverged from the phage-type 80/81 S aureus clone responsible for a major bacterial epidemic of the 20th century. The genome and transcriptome features that contribute to complicated infections of the CC30 lineage are unknown.
Methods. Twenty-nine clinical methicillin-resistant S aureus (MRSA) strains (8 from CC30 and 21 from other major CCs were evaluated for virulence using murine and Galleria mellonella sepsis models. Genomic features of CC30 were identified by comparative genome sequencing and RNA-Seq transcriptome analysis of the 29 strains and 31 previously sequenced S aureus genomes.
Results. The CC30 isolates displayed lower virulence in the sepsis models compared with other CCs [P < .0001]. Comparisons of orthologous proteins and transcriptome analysis identified genes (eg, nitric oxide reductase) and changes in metabolic pathways (eg, pyrimidine metabolism) that contribute to the distinct CC30 phenotype. Previously reported nonsynonymous single-nucleotide polymorphisms (SNPs) were found in accessory gene regulator C (agrC) and α-hemolysin (hla), molecules important for virulence. Additional nonsynonymous SNPs conserved across clinical CC30 isolates when compared with the first sequenced contemporary CC30 clone, MRSA-16, were identified in multiple genes, suggesting continuing evolutionary divergence in this lineage.
Conclusions. Genomic and transcriptional analyses suggest that the CC30 lineage has acquired metabolic features that contribute to persistent and complicated infections. Absence of sepsis-induced mortality in animal models may be due in part to its unique genomic profile and suggests that specific genotypes of S aureus elicit distinct types of infection types.
doi:10.1093/ofid/ofv093
PMCID: PMC4512144  PMID: 26213692
genome; genotype; hematogenous infections; Staphylococcus aureus; transcriptome
3.  Potential Associations between Severity of Infection and the Presence of Virulence-Associated Genes in Clinical Strains of Staphylococcus aureus 
PLoS ONE  2011;6(4):e18673.
Background
The clinical spectrum of Staphylococcus aureus infection ranges from asymptomatic nasal carriage to osteomyelitis, infective endocarditis (IE) and death. In this study, we evaluate potential association between the presence of specific genes in a collection of prospectively characterized S. aureus clinical isolates and clinical outcome.
Methodology/Principal Findings
Two hundred thirty-nine S. aureus isolates (121 methicillin-resistant S. aureus [MRSA] and 118 methicillin-susceptible S. aureus [MSSA]) were screened by array comparative genomic hybridization (aCGH) to identify genes implicated in complicated infections. After adjustment for multiple tests, 226 genes were significantly associated with severity of infection. Of these 226 genes, 185 were not in the SCCmec element. Within the 185 non-SCCmec genes, 171 were less common and 14 more common in the complicated infection group. Among the 41 genes in the SCCmec element, 37 were more common and 4 were less common in the complicated group. A total of 51 of the 2014 sequences evaluated, 14 non-SCCmec and 37 SCCmec, were identified as genes of interest.
Conclusions/Significance
Of the 171 genes less common in complicated infections, 152 are of unknown function and may contribute to attenuation of virulence. The 14 non-SCCmec genes more common in complicated infections include bacteriophage-encoded genes such as regulatory factors and autolysins with potential roles in tissue adhesion or biofilm formation.
doi:10.1371/journal.pone.0018673
PMCID: PMC3082525  PMID: 21541311

Results 1-3 (3)