Antibiotic resistance and biofilm-forming capacity contribute to the success of Staphylococcus aureus as a human pathogen in both healthcare and community settings. These virulence factors do not function independently of each other and the biofilm phenotype expressed by clinical isolates of S. aureus is influenced by acquisition of the methicillin resistance gene mecA. Methicillin-sensitive S. aureus (MSSA) strains commonly produce an icaADBC operon-encoded polysaccharide intercellular adhesin (PIA)-dependent biofilm. In contrast, the release of extracellular DNA (eDNA) and cell surface expression of a number of sortase-anchored proteins, and the major autolysin have been implicated in the biofilm phenotype of methicillin-resistant S. aureus (MRSA) isolates. Expression of high level methicillin resistance in a laboratory MSSA strain resulted in (i) repression of PIA-mediated biofilm production, (ii) down-regulation of the accessory gene regulator (Agr) system, and (iii) attenuation of virulence in murine sepsis and device infection models. Here we review the mechanisms of MSSA and MRSA biofilm production and the relationships between antibiotic resistance, biofilm and virulence gene regulation in S. aureus.
mecA
-dependent methicillin resistance in MRSA is subject to regulation by numerous accessory factors involved in cell wall biosynthesis, nucleotide signaling, and central metabolism. Here, we report that mutations in the TCA cycle gene,
sucC
, increased susceptibility to β-lactam antibiotics and was accompanied by significant accumulation of succinyl-CoA, which in turn perturbed lysine succinylation in the proteome.
Hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) strains typically express high-level, homogeneous (HoR) -lactam resistance, whereas community-associated MRSA (CA-MRSA) more commonly express low-level heterogeneous (HeR) resistance. Expression of the HoR phenotype typically requires both increased expression of the mecA gene, carried on the staphylococcal cassette chromosome mec element (SCCmec), and additional mutational event(s) elsewhere on the chromosome. Here the oxacillin concentration in a chemostat culture of the CA-MRSA strain USA300 was increased from 8 g/ml to 130 g/ml over 13 days to isolate highly oxacillin-resistant derivatives. A stable, small-colony variant, designated HoR34, which had become established in the chemostat culture was found to have acquired mutations in gdpP, clpX, guaA, and camS. Closer inspection of the genome sequence data further revealed that reads covering SCCmec were ϳ10 times overrepresented compared to other parts of the chromosome. Quantitative PCR (qPCR) confirmed Ͼ10-fold-higher levels of mecA DNA on the HoR34 chromosome, and MinION genome sequencing verified the presence of 10 tandem repeats of the SCCmec element. qPCR further demonstrated that subculture of HoR34 in various concentrations of oxacillin (0 to 100 g/ml) was accompanied by accordion-like contraction and amplification of the SCCmec element. Although slower growing than strain USA300, HoR34 outcompeted the parent strain in the presence of subinhibitory oxacillin. These data identify tandem amplification of the SCCmec element as a new mechanism of high-level methicillin resistance in MRSA, which may provide a competitive advantage for MRSA under antibiotic selection.
Prolonging the clinical effectiveness of β-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to β-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between β-lactams and DCS. DCS resensitized MRSA to β-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to β-lactam antibiotics.
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