Spontaneous genomic variation as a survival strategy of nosocomial S. haemolyticus

Staphylococcus haemolyticus is one of the most important nosocomial human pathogens frequently isolated in bloodstream and medical devices related infections. This species is notorious for its multidrug resistance and genome plasticity. However, its mechanisms of evolution and adaptation are still poorly explored. In this study we aimed to characterize the strategies of genetic and phenotypic diversity in S. haemolyticus . Here, we analyzed an invasive S. haemolyticus strain, recovered from a bloodstream infection, for genetic and phenotypic stability after serial passage in vitro (>400 generations) in the absence and presence of sub-inhibitory concentrations of a beta-lactam antibiotic. We performed PFGE of the culture and five colonies at seven time points during stability assays were analyzed for beta-lactams susceptibility, hemolysis, mannitol fermentation and biofilm production. We compared their whole genome regarding chromosomal structure, gene content and mutations and preformed phylogenetic analysis based on core SNPs. We observed a high instability in the PFGE profiles at the different time points during serial passage in vitro in the absence of antibiotic. However, no variation was observed in PFGE patterns in the presence of beta-lactams. Analysis of WGS data for individual colonies collected at different time points showed the occurrence of six large-scale genomic deletions within the oriC environ (36 kbp-348 kbp) in the cell populations analyzed, smaller deletions in non-OriC environ region as well as non-synonymous mutations in clinically relevant genes. The regions of deletion and point mutations included genes encoding amino acid and metal transporters, resistance to environmental stress and beta-lactams, virulence, mannitol fermentation, metabolic processes and IS elements. A parallel variation was additionally detected in clinically significant phenotypic traits such as mannitol fermentation, beta-lactams resistance, hemolysis and biofilm formation. All the genetic variants analyzed were closely related in their core genome (13-292 SNPs). Our results suggest that S. haemolyticus populations are composed of subpopulations of genetic and phenotypic variants that might be affected in antibiotic and stress resistance, specific metabolic processes and virulence. The maintenance of subpopulations in different physiological states might be a strategy to adapt rapidly to a stress situation imposed by the host particularly in the hospital environment. ### Competing Interest Statement The authors have declared no competing interest.