Although it is estimated that 20-30% of the general human population are carriers of Staphylococcus aureus, this bacterium is one of the most important etiological agents responsible for healthcare-associated infections. The appearance of methicillin resistant S. aureus (MRSA) strains has created serious therapeutical problems. Detailed understanding of the mechanisms of S. aureus infections seems necessary to develop new effective therapies against this pathogen. In this article, we present an overview of the biochemical and genetic mechanisms of pathogenicity of S. aureus strains. Virulence factors, organization of the genome and regulation of expression of genes involved in virulence, and mechanisms leading to methicilin resistance are presented and briefly discussed.

SCC mec is a mobile genetic element that carries the gene ( mecA ) mediating methicillin resistance in staphylococci. For Staphylococcus aureus , four SCC mec types have been described, one (type IV) of which has been associated with newly identified community-acquired methicillin-resistant S. aureus . However, the distribution of SCC mec types among S. epidermidis is not known. SCC mec typing of a collection of 44 methicillin-resistant Staphylococcus epidermidis (MRSE) isolates recovered between 1973 and 1983 from the blood of patients with prosthetic valve endocarditis (PVE) was performed by PCR amplification of key genetic elements ( mecA , mecI , IS 1272 , and ccrAB ). Of the 44 isolates, 1 (2%) harbored SCC mec type I, 15 (34%) harbored type II, 12 (28%) harbored type III, and 16 (36%) harbored type IV. The complete nucleotide sequence of SCC mec type IV was determined for 16 isolates and found to be identical in size (24 kb) and 98% homologous to DNA sequences published for S. aureus . Type IV SCC mec was also common (5 of 10 isolates) among a geographically dispersed collection of 10 recent (1998 to 2001) S. epidermidis bloodstream isolates. Multilocus sequence typing (MLST) (using the same seven genes presently employed for S. aureus MLST) of these MRSE isolates and of 10 additional recent geographically dispersed methicillin-susceptible isolates demonstrated that all 16 PVE isolates and 2 of 5 recent isolates harboring type IV SCC mec were in three related clonal groups. All three MSSE PVE isolates recovered from patients between 1976 and 1979 were in the same clonal groups as type IV SCC mec MRSE isolates. These data support the hypothesis of intra- and interspecies transfer of type IV SCC mec and suggest that there are clonal associations in S. epidermidis that correlate with SCC mec type.

Ceftaroline is the first member of a novel class of cephalosporins approved for use in the United States. Although prior studies have identified eight ceftaroline-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates in Europe and Asia with MICs ranging from 4 to 8 mg/liter, high-level resistance to ceftaroline (>32 mg/liter) has not been described in MRSA strains isolated in the United States. We isolated a ceftaroline-resistant (MIC > 32 mg/liter) MRSA strain from the blood of a cystic fibrosis patient and five MRSA strains from the respiratory tract of this patient. Whole-genome sequencing identified two amino acid-altering mutations uniquely present in the ceftaroline-binding pocket of the transpeptidase region of penicillin-binding protein 2a (PBP2a) in ceftaroline-resistant isolates. Biochemical analyses and the study of isogenic mutant strains confirmed that these changes caused ceftaroline resistance. Thus, we identified the molecular mechanism of ceftaroline resistance in the first MRSA strain with high-level ceftaroline resistance isolated in the United States.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged to be one of the most important pathogens both in health care and in community-onset infections. Daptomycin (DAP) is a cyclic anionic lipopeptide recommended for treatment of skin infections, bacteremia, and right-sided endocarditis caused by MRSA. Resistance to DAP (DAP ^r ) has been reported in MRSA and is mostly accompanied by a parallel decrease in oxacillin resistance, a process known as the “seesaw effect.” Our study provides evidence that the seesaw effect applies to other β-lactams and carbapenems of clinical use, including nafcillin (NAF), cefotaxime (CTX), amoxicillin-clavulanic (AMC), and imipenem (IMP), in heterogeneous DAP ^r MRSA strains but not in MRSA strains expressing homogeneous β-lactam resistance. The antibacterial efficacy of DAP in combination with β-lactams was evaluated in isogenic DAP-susceptible (DAP ^s )/Dap ^r MRSA strains originally obtained from patients that failed DAP monotherapy. Both in vitro (MIC, synergy-kill curve) and in vivo (wax worm model) approaches were used. In these models, DAP and a β-lactam proved to be highly synergistic against both heterogeneous and homogeneous clinical DAP ^r MRSA strains. Mechanistically, β-lactams induced a reduction in the cell net positive surface charge, reverting the increased repulsion provoked by DAP alone, an effect that may favor the binding of DAP to the cell surface. The ease of in vitro mutant selection was observed when DAP ^s MRSA strains were exposed to DAP. Importantly, the combination of DAP and a β-lactam prevented the selection of DAP ^r variants. In summary, our data show that the DAP–β-lactam combination may significantly enhance both the in vitro and in vivo efficacy of anti-MRSA therapeutic options against DAP ^r MRSA infections and represent an option in preventing DAP ^r selection in persistent or refractory MRSA infections.

Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Ca-dependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR . Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF , vraSR contributes to DAP resistance in the present group of clinical strains.

The transcriptomes of vancomycin intermediate-resistance Staphylococcus aureus (VISA) clinical isolates HIP5827 and Mu50 (MIC = 8 μg/ml) were compared to those of highly vancomycin-resistant S. aureus (VRSA; MIC = 32 μg/ml) passage derivatives by microarray. There were 35 genes with increased transcription and 16 genes with decreased transcription in common between the two VRSAs compared to those of their VISA parents. Of the 35 genes with increased transcription, 15 involved purine biosynthesis or transport, and the regulator ( purR ) of the major purine biosynthetic operon ( purE-purD ) was mutant. We hypothesize that increased energy (ATP) is required to generate the thicker cell walls that characterize resistant mutants.