Cell Wall Composition and Decreased Autolytic Activity and Lysostaphin Susceptibility of Glycopeptide-Intermediate Staphylococcus aureus

Abstract

The cell wall composition and autolytic properties of passage-selected glycopeptide-intermediate Staphylococcus aureus (GISA) isolates and their parent strains were studied in order to investigate the mechanism of decreased vancomycin susceptibility. GISA had relatively modest changes in peptidoglycan composition involving peptidoglycan interpeptide bridges and somewhat decreased cross-linking compared to that of parent strains. The cell wall phosphorus content of GISA strains was lower than that of susceptible parent strains, indicating somewhat lower wall teichoic acid levels in the GISA strains. Similar to whole cells, isolated crude cell walls retaining autolytic activity of GISA had drastically reduced autolytic activity compared to that of parent strains, and this arose early in the development of the GISA phenotype. This was due to an alteration in the autolytic enzymes of GISA as revealed by normal susceptibility of GISA-purified cell walls to parental strain autolysin extract and lower activity and altered peptidoglycan hydrolase activity profiles in GISA autolysin extracts compared to those of parent strains. Northern blot analysis indicated that expression of atl, the major autolysin gene, was significantly downregulated in a GISA strain compared to that of its parent strain. In contrast to whole cells, which showed decreased lysostaphin susceptibility, purified cell walls of GISA showed increased susceptibility to lysostaphin. We suggest that in our GISA strains, decreased autolytic activity is involved in the tolerance of vancomycin and the activities of endogenous autolysins are important in conferring sensitivity to lysostaphin on whole cells.

Reduced Vancomycin Susceptibility inStaphylococcus aureus, Including Vancomycin-Intermediate and Heterogeneous Vancomycin-Intermediate Strains: Resistance Mechanisms, Laboratory Detection, and Clinical Implications

“…Reduced autolytic activity is a common feature of hVISA and VISA strains and is a common early phenotypic change in serial isolates obtained during persistent infection (30,31,108,160), although this has not been demonstrated for all isolates (121). Initial studies of Japanese VISA strain Mu50 reported an increase in autolytic activity (362); however, recent data have confirmed that Mu50 demonstrates reduced whole-cell autolytic activity like other VISA strains (362).…”

“…Initial studies of Japanese VISA strain Mu50 reported an increase in autolytic activity (362); however, recent data have confirmed that Mu50 demonstrates reduced whole-cell autolytic activity like other VISA strains (362). Some data suggest a possible role for wall teichoic acids of VISA strains suppressing peptidoglycan degradation by autolytic enzymes (318), while other studies suggested that a reduction in the autolytic activity and altered peptidoglycan hydrolase activity of VISA autolysin extracts are responsible for the reduced autolytic activity (160). It was proposed that vancomycin binding in the staphylococcal cell wall directly blocks the activity of a peptidoglycan hydrolase and explains the reduced autolytic activity demonstrated in the presence of vancomycin (321); however, this does not explain the reduced autolytic activity of hVISA and VISA strains in the absence of vancomycin (121).…”

“…Other common features include a reduced growth rate (62,250) and reduced whole-cell lysostaphin susceptibility (56,62,215). In contrast, purified cell walls of laboratory-induced VISA strains demonstrated increased lysostaphin susceptibility (160).…”

Interconnections between Sigma B, agr , and Proteolytic Activity in Staphylococcus aureus Biofilm Maturation

“…After gels were washed, they were placed in 50 ml of Tris buffer and incubated at 37°C for 15 h. For visualization of protease clearing zones, gels were stained with Coomassie brilliant blue. Autolysin (murein hydrolase) zymography was performed as described previously (42). Cell cultures were grown, and supernatants were prepared and concentrated as described above.…”

Induction of Daptomycin Heterogeneous Susceptibility in Staphylococcus aureus by Exposure to Vancomycin

“…Daptomycin exerts its bactericidal effects by binding to the bacterial cell membrane, resulting in efflux of potassium ions into the cell and subsequent cell death (2,17,18). Vancomycin exerts its activity on the cell wall (6,7). GISA strains demonstrate thicker cell walls with binding sites that sequester the drug (4,6,15).…”