GSE63750
Prolonged growth of a clinical S. aureus strain selects for a stable SCV and Full genomic characterization of the development in a clinical Staphylococcus aureus strain of a stable Small Colony Variant cell-type
We have observed that for a number of S. aureus strains as they switch to a SCV lifestyle there is the formation of an extracellular matrix. We focused our analysis on one strain, WCH-SK2. For bacterial survival in the host, the combination of low nutrients and the prolonged timeframe forms a stress that selects for a specific cell-type from the population. In this context, we used steady-state growth conditions with low nutrients and a controlled low growth rate, for a prolonged time and with methylglyoxal. These conditions induced S. aureus WCH-SK2 into a stable SCV cell-type, they did not revert after sub-culturing. Methods: Transcriptomic profiles of wild-type (WT) and SCV were generated in continuous culture in the presence of stress (high and low level of methylglyoxal). Results: Analysis revealed these cells possessed a metabolic and surface profile that was different from previously described SCVs or biofilm cells. The extracellular matrix was protein and extracellular DNA; but not polysaccharide. The SCV cells induced expression of certain surface proteins (such as Ebh) and lantibiotic synthesis while down-regulating factors that stimulates immune response (leucocidin, capsule, carotenoid). We also studied further their genetic characteristics. They possessed an increased viability in the presence of antibiotics compared to their non-SCV form. Their stability implied there had been genetic changes, we determined the whole genome sequence of WCH-SK2 and its stable SCV forms at a single base resolution, employing Single Molecular Real-Time (SMRT) sequencing that also enables the methylome to be determined. The genetic features of this isolate have been identified; the SCCmec type, the pathogenicity and genetic islands and virulence factors. The comparison has identified a set of genetic changes that occurred in the stable SCV form; most notably to the global regulator MgrA and the phosphoserine phosphatase RsbU (part of the regulatory pathway of the sigma factor SigB). There was a shift in the methylation across the genome. Conclusions: Our data reveal a cell heterogeneity within a S. aureus population and using conditions that resemble long-term survival in the host has identified a previously unnoticed S. aureus cell-type, with a distinctive metabolic and molecular profile. The results from this study represent a unique identification of a suite of epigenetic, genetic and transcriptional factors that are implicated in the switch in S. aureus to its persistent SCV form Overall design: Total RNA of WT (batch culture) and SCV formed in high level of methylglyoxal  (0.031%) at day 55 and SCV formed at low level of methylglyoxal (0.0078%) at day 30 of continuous culture. DNA genomic of WT and stable SCV formed in high level of methylglyoxal (0.031%)