The ability of S. aureus
to cause severe infections is dependent upon a large repertoire of virulence factors, often on mobile genetic elements or MGE (including pathogenicity islands and bacteriophage) that are transferred horizontally through the S. aureus
community. The virulence capacity of individual isolates is largely determined by the function and abundance of these variable virulence factors. In our previous work 
, we demonstrated that S. aureus
CC5 and CC30 are associated with the presence of hematogenous complications, including endocarditis, septic arthritis and vertebral osteomyelitis. We now undertake aCGH analysis to examine the association of 2014 variable S. aureus
genes with severity of infection in a collection of 239 isolates with the goal of identifying genes that contribute to virulence. The 239 S. aureus
isolates were selected from three clinical groups in our previous study 
and included 121 MRSA and 118 MSSA isolates. Of the 226 genes that were identified as significantly associated with severity of infection, 51 were more frequent and 175 less frequent in the complicated infection group (Table S4
We acknowledge that as a result of the microarray probe design being limited to the six genomes available when the microarray was constructed, our analysis is compromised and will not capture those genes present in many other S. aureus lineages and strains. As examples, genes encoding numerous heavy metal resistance genes on the SCCmec type III element as well as genes encoding the lukSF-PV Panton Valentine Leukotoxin and ACME element from Community Acquired MRSA (CA-MRSA) will not be identified in our analysis. However, a significant strength of our study is identification of the core S. aureus ORFs present in all tested clinical strains representing fifteen clonal complexes.
To minimize SCCmec
as a confounding factor, the 226 variable genes were segregated into two groups, based on their presence in the SCCmec
element (). Of the 51 genes more common in complicated infection, 37 were carried on SCCmec
and 14 were not carried on SCCmec
. None of these 14 genes encode known virulence factors previously associated with increased virulence and disease severity. Instead, 11 genes include proteins of unknown function, regulatory proteins and autolysins carried on S. aureus
bacteriophage. These findings support previous work demonstrating the contribution of S. aureus
bacteriophage to pathogenesis of infections 
through genome variation, mobilization of pathogenicity islands and transmission of phage encoded virulence factors.
Yet to be resolved is the contribution of prophage to potentiate S. aureus
virulence by influencing its physiology towards growth as a biofilm, a virulence factor that plays a role in chronic infections, such as native valve endocarditis and osteomyelitis. Evidence of bacteriophage release from S. aureus
biofilms has been reported 
, leading to lysis of cells which in turn promotes the persistence and survival of remaining cells and release of extracellular DNA (eDNA) required for extracellular polymeric substance (EPS) development and maturation of biofilms. A second mechanism for cell lysis and release of eDNA required for biofilm development involves autolysis driven by autolysins and regulated by the cid
. These autolysins are capable of hydrolyzing the amide bond between N-acetylmuramic acid and L-alanine in bacterial cell-wall peptidoglycan resulting in cell lysis and release of eDNA. Interestingly, two of the bacteriophage genes more frequently present in complicated infections are autolysins carried on bacteriophages Sa2MW (MW1380) and L54a (SACOL0389) (). Although, no role has yet to be assigned to these two putative autolysin genes, their presence in cases of complicated infection suggests that they may influence biofilm maturation and disease persistence. An alternative function for the S. aureus
phage encoded autolysins is suggested by recent work on bacteriophage-encoded autolysins in Streptococcus mitis
, which demonstrated their role in binding to human platelets through interaction with fibrinogen 
Of the remaining 9 genes carried on prophage, three are DNA binding proteins with potential regulatory functions (SAS0928, SAR1502 and SAR1555) and six (SACOL0329, SAR1546, SAR1522, MW1385, SACOL0338 and SA0397) are genes of unknown function. The function of three remaining genes (VRA0002, SA1483 and VRA0005) relative to virulence is also not clear. Association of these genes with increased disease severity may be a result of their linkage to unidentified genes not on the microarray and associated with virulence. This study did not start with candidate genes, and as such uncovered a list of genes for which little is known about the function. Detailed functional analysis of these and other S. aureus bacteriophage genes of unknown function will likely lead to a greater understanding of their role in virulence and enabling mutation and adaptation of S. aureus to new environments.
The 37 genes that were more frequently present in complicated infection and carried by SCCmec
span the entire length of the type II SCCmec
element, which is found in the majority (97/120; 81%) of MRSA isolates included in this study. While there is no direct evidence that SCCmec
contributes to S. aureus
virulence, recent work by Queck et. al. 
has shown the type II SCCmec
elements encode a new phenol soluble modulin (PSM) that alters the capacity of S. aureus
to cause disease. Although the arrays used in this study do not contain a probe for this PSM, two genes flanking the PSM encoding region in SCCmec
type II, mecI
(N315-SA0039) and xylR
(N315-SA0041) are associated with severity of infection, suggesting that the new PSM may also be a significant component in our study. However, sequence analysis of this region showed that the PSM is present in all members of a subset of 69 isolates from the study group representing all 3 clinical groups and student controls. Outside of the SCCmec
element, there were 140 genes associated with SCCmec
and severity of infection (Table S4
). This is intriguing as it suggests the possibility of co-evolution of genes in the S. aureus
genome with SCCmec
and the possible preferential insertion of SCCmec
into specific strains.
An unexpected finding in our study was the identification of 171 genes more frequent in strains isolated in subjects with uncomplicated infection. These genes (included in Table S4
) include 152 genes of unknown function, some of which may be involved in attenuation of virulence. Approximately 38% of S. aureus
genes have no assigned function and an additional 10% have a putative function based on functional domain matches 
. Their roles as genes that contribute to S. aureus
specific functions such as pathogenicity or host niche adaptation, remains an unexplored area of this significant bacterial pathogen.
In summary, we have demonstrated a statistical association between the presence/absence of individual genes and clinical outcome. Our data strongly supports the concept that differences in disease potential are rooted in the S. aureus genotype. However, the use of aCGH in our current study limits the results to identification of large-scale genomic changes, such as gain or loss of genes, responsible for phenotypic changes. Additional genotypic factors, including genomic polymorphisms, novel genes and differential gene expression, associated with complicated infections, will be determined by resequencing of representative members of the SABG collection and global transcriptional analysis from in vitro and in vivo environments. Correlation of these genomic and transcriptional differences with clinical outcome will contribute significantly to our understanding of S. aureus virulence.