Because of the rapid and continuing increase of the prevalence of CA USA300 strains at TCH, we initiated the sequencing of two typical isolates from pediatric patients. During this time, Diep et al
] reported the sequence of USA300-FPR3757. USA300-HOU-MR revealed significant differences from USA300-FPR3757. Though USA300-FPR3757 was chosen as a highly resistant isolate, USA300-HOU-MR, a purely community-acquired strain, harbours a large plasmid encoding multiple antibiotic and cadmium resistance. The mosaic composition of plasmid pUSA300-HOU-MR representing three different staphylococcal plasmids points toward significant genetic exchange between staphylococcal isolates and species. Among the numerous antibacterial resistance genes found on the plasmid, the conferred resistance to bacitracin, a common constituent of topical over-the-counter ointments used to treat or prevent cutaneous infections, is most intriguing. In contrast to other MR strains, the cadmium resistance genes were found on a plasmid and not on the chromosome. Genome-wide alignments revealed that not all MRSA are tightly clustered.
Several indels between USA300-HOU-MR and USA300-FPR3757 were identified. The majority of the indels were within repeat regions or rRNAs, though a 13 kb segment was located in different regions of the two genomes. In USA300-FPR3757, the region is inserted between two genes. In USA300-HOU-MR, the segment interrupts an operon (snoABCDEFG
) that has been implicated in susceptibility to thrombin-induced platelet microbicidal protein 1 (tPMP-1). Bayer et al
] have shown that interruption of this operon reduces the susceptibility of S. aureus
to tPMP-1 in vitro
. Resistance to tPMP-1 may allow US300-HOU-MR to cause more serious disease by evasion of platelet-mediated killing in the blood stream. USA300-FPR3757 has an additional repeat within the gene encoding the IgG binding protein A, so it is possible that IgG binding protein is subject to structural variation. We also identified nine SNPs within this gene. This may suggest that the IgG binding protein undergoes significant variation, possibly in response to the host.
Two prophage were identified, one carrying the PVL genes and the other an hemolysin converting phage carrying the staphylokinase, chemotaxis inhibitory protein and staphylococcal complement inhibitor genes. USA300 lacks a copy of prophage L54a. The GehD lipase, whose gene contains the prophage attachment site, is likely to play a role in colonization events. [36
SNP analysis revealed 47 non-synonymous, non-conserved differences between the strains. Some of these may affect protein function. Two of these, one in gyrA and one in parC, are consistent with antibiotic susceptibilities reported in Table . The SNP analysis also revealed a number of sequencing errors in the FPR3757 sequence.
To further our understanding of the evolution of the USA300-HOU-MR strain, we created a draft sequence of an MS isolate from TCH. This revealed two regions of the MR strain not found in the MS strain. It is likely that the progenitor MS strain acquired these regions (along with the SCCmec
IVa and the arginine deiminase region) by recombination to become USA300-HOU-MR. We propose that USA300-HOU-MS first acquired the arginine deiminase region plus the cassette chromosome recombinase genes (ccrA
B) from a strain similar to S. epidermidis
ATCC12228. This is based on the observation that the ccrAB
genes in USA300-HOU-MR are 95–100% identical to those found near the arginine deiminase region in S. epidermidis
ATCC12228. The SCCmec
was likely acquired in a separate event from another MRSA, or from a MR-S. epidermidis strain
, via ccrAB
recombination within the repeats flanking the arginine deiminase region [24
]. US300-HOU-MS lacks a probable nickel ABC transporter that is linked to ACME in USA300-MR. It is possible that increased intracellular stores of nickel in USA300-HOU-MR could contribute to virulence by enhancing the activity of the nickel-dependent urease. [37
]. The MS strain also lacks a copy of the P-ATPase copper transporter gene; such transporters have been shown to be involved in virulence in Listeria monocytogenes
]. Another potential virulence factor revealed by comparison of the MR and MS strains is a possible secreted actin binding protein.
We used a relatively new predictive tool, SecretomeP2.0, to identify non-classically secreted proteins [39
] since it is not possible to predict the function of most of these proteins by BLAST analysis. We found 406 high scoring genes predicted to encode non-classically secreted proteins in USA300-HOU-MR (Additional file 3
). If some of these are indeed secreted then they may be candidate virulence factors for future study.