Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

Abstract

dDaptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. The emergence of daptomycin-nonsusceptible S. aureus isolates 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) in rpoB (RpoB A 477 D) that decreased susceptibility not only to daptomycin but also to vancomycin, ␤-lactams, and rifampin. Furthermore, the rpoB mutant 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 in clpX partly alleviated the growth defect conferred by the rpoB mutation without changing antibiotic susceptibility. The final isolate acquired three additional mutations, including an SNP in mprF (MprF S 295 L) 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 the vraSR (cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selected in vivo contribute to daptomycin nonsusceptibility, growth fitness, and virulence traits. Staphylococcus aureus is a commensal bacterium that can cause a variety of both localized and more invasive infections. Due to its profound ability to acquire resistance to clinically relevant antibiotics, S. aureus remains a major clinical challenge worldwide (1). The most challenging antimicrobial resistance issue in S. aureus has been the evolution and dissemination of methicillinresistant S. aureus (MRSA) strains first in hospitals and later in the general community (1). MRSA infections are typically treated with last-line antibiotics, such as vancomycin and, more recently, daptomycin (DAP), a cyclic lipopeptide derived from Streptomyces roseosporus with bactericidal activity against a broad range of Gram-positive bacteria (2, 3). The mechanism of action of daptomycin has not been fully elucidated, but it is generally accepted that daptomycin inserts into the cytoplasmic membrane of Grampositive bacteria via a calcium-dependent pathway, leading to potassium efflux, membrane depolarization, and, eventually, cell death (2).The development of daptomycin nonsusceptibility (DAP-NS) in S. aureus seems to be a relatively rare phenomenon not involving horizontal gene transfer but, instead, taking place by a stepwise acqu...

In Vivo -Selected Compensatory Mutations Restore the Fitness Cost of Mosaic penA Alleles That Confer Ceftriaxone Resistance in Neisseria gonorrhoeae

“…Here, we report that the mosaic penA alleles from two well-characterized ESC r clinical isolates, H041 (10) and F89 (12), reduce the fitness of N. gonorrhoeae both in vitro and in vivo. We hypothesized that mosaic penA alleles, while increasing ESC resistance, would impart a fitness deficit to a wild-type strain, but in nature, these resistant strains accumulate fitness-increasing mutations that allow the strains to spread (9,21,23,45), as has been shown for other antibiotic-resistant strains (46,47). We showed that FA19 penA41, but not FA19 penA89, acquired spontaneous compensatory mutations during coinfections in the female mouse model that markedly increased fitness.…”

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus

“…Further study showed that the rpoB-A477D allele modulates reduced susceptibility to daptomycin, vancomycin, and ␤-lactam antibiotics and contributes to a variety of pleiotropic effects, including modulation of Spx levels (61). Spx has been linked with both glycopeptide and ␤-lactam resistance in S. aureus in our own laboratory by virtue of the implication in drug resistance of its negative regulator yjbH (35) and an adaptor gene, trfA, that was shown to be Spx regulated (76).…”

Mechanism of Action and Resistance to Daptomycin inStaphylococcus aureusand Enterococci

“…Additional mutations associated with the DAP-R phenotype include genes encoding the RNA polymerase subunits rpoB and rpoC (Friedman et al 2006; Peleg et al 2012). A mutation in rpoB , resulting in the amino acid change A621E, was associated with increased expression of the dlt operon and correlated with an increase in positive cell surface charge, while RpoB mutations A621E and A477D were both linked to activation of cell wall biosynthesis and increased cell wall thickness (Cui et al 2010; Bæk et al 2015). …”