Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation inrpoBand a Compensatory Inactivation of theclpXGene

Abstract

ABSTRACTDaptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistantStaphylococcus aureus(MRSA) infections. The emergence of daptomycin-nonsusceptibleS. aureusisolates during therapy is often associated with multiple genetic changes; however, the relative contributions of these changes to resistance and other phenotypic changes usually remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single-nucleotide polymorphism (SNP) inrpoB(RpoB A477D) that decreased susceptibility not only to daptomycin but also to vancomycin, β-lactams, and rifampin. Furthermore, therpoBmutant exhibited pleiotropic phenotypes, including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator Spx, and slow growth. A subsequently acquired loss-of-function mutation inclpXpartly alleviated the growth defect conferred by therpoBmutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP inmprF(MprF S295L) known to confer daptomycin nonsusceptibility, and accordingly, this isolate was the only daptomycin-nonsusceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as that of the parental strain, while the level of transcription of thevraSR(cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selectedin vivocontribute to daptomycin nonsusceptibility, growth fitness, and virulence traits.