Antibiotic tolerance and the alternative lifestyles of Staphylococcus aureus.

Staphylococcus aureus has an incredible ability to survive, either by adapting to environmental conditions or defending against exogenous stress. Although there are certainly important genetic traits, in part this ability is provided by the breadth of modes of growth S. aureus can adopt. It has been proposed that while within their host, S. aureus survives host-generated and therapeutic antimicrobial stress via alternative lifestyles: a persister sub-population, through biofilm growth on host tissue or by growing as small colony variants (SCVs). Key to an understanding of chronic and relapsing S. aureus infections is determining the molecular basis for its switch to these quasi-dormant lifestyles. In a multicellular biofilm, the metabolically quiescent bacterial community additionally produces a highly protective extracellular polymeric substance (EPS). Furthermore, there are bacteria within a biofilm community that have an altered physiology potentially equivalent to persister cells. Recent studies have directly linked the cellular ATP production by persister cells as their key feature and the basis for their tolerance of a range of antibiotics. In clinical settings, SCVs of S. aureus have been observed for many years; when cultured, these cells form non-pigmented colonies and are approximately ten times smaller than their counterparts. Various genotypic factors have been identified in attempts to characterize S. aureus SCVs and different environmental stresses have been implicated as important inducers.

A full genomic characterization of the development of a stable Small Colony Variant cell-type by a clinical Staphylococcus aureus strain.

A key to persistent and recurrent Staphylococcus aureus infections is its ability to adapt to diverse and toxic conditions. This ability includes a switch into a biofilm or to the quasi-dormant Small Colony Variant (SCV). The development and molecular attributes of SCVs have been difficult to study due to their rapid reversion to their parental cell-type. We recently described the unique induction of a matrix-embedded and stable SCV cell-type in a clinical S. aureus strain (WCH-SK2) by growing the cells with limiting conditions for a prolonged timeframe. Here we further study their characteristics. They possessed an increased viability in the presence of antibiotics compared to their non-SCV form. Their stability implied that there had been genetic changes; we therefore determined both the genome sequence of WCH-SK2 and its stable SCV form at a single base resolution, employing Single Molecular Real-Time (SMRT) sequencing that enabled the methylome to also be determined. The genetic features of WCH-SK2 have been identified; the SCCmec type, the pathogenicity and genetic islands and virulence factors. The genetic changes that had occurred in the stable SCV form were identified; most notably being in MgrA, a global regulator, and RsbU, a phosphoserine phosphatase within the regulatory pathway of the sigma factor SigB. There was a shift in the methylomes of the non-SCV and stable SCV forms. We have also shown a similar induction of this cell-type in other S. aureus strains and performed a genetic comparison to these and other S. aureus genomes. We additionally map RNAseq data to the WCH-SK2 genome in a transcriptomic analysis of the parental, SCV and stable SCV cells. The results from this study represent the 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.

The induction of Staphylococcus aureus biofilm formation or Small Colony Variants is a strain-specific response to host-generated chemical stresses.

Staphylococcus aureus is extremely versatile. It has a capacity to persist within its host by switching to the alternative lifestyles of biofilm or Small Colony Variants (SCV). The induction of this switch has been presumed to be in response to stressed conditions, however the environmental basis has not been thoroughly investigated. We assessed the response of numerous strains to chemicals that are present in human host. There were some that induced a biofilm or SCV phenotype and indeed some inducing both lifestyles. This result illustrates the diversity within a population and a strain-specific adaptation to the presence of host-generated stresses.

Prolonged growth of a clinical Staphylococcus aureus strain selects for a stable small-colony-variant cell type.

An undetermined feature of Staphylococcus aureus pathogenesis is its persistence and then relapse of disease. This has been explained by its switch to alternative lifestyles, mainly as biofilm or small-colony variants (SCVs). Studying the native characteristics of SCVs has been problematic due to their reversion to the parental lifestyle. We have observed that for a number of S. aureus strains as they switch to an 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 time frame 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; the cells did not revert after subculturing. Analysis revealed these cells possessed a metabolic and surface profile that was different from those of 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 synthesis of lantibiotics while downregulating factors that stimulate the immune response (leucocidin, capsule, and carotenoid). Our data reveal cell heterogeneity within an S. aureus population and under conditions that resemble long-term survival in the host have identified a previously unnoticed S. aureus cell type with a distinctive metabolic and molecular profile.