Characterization of Staphylococcus aureus Isolates with Decreased Susceptibility to Vancomycin and Teicoplanin: Isolation and Purification of a Constitutively Produced Protein Associated with Decreased Susceptibility

Abstract

"Derivative isolates" with 4- to 8-fold and 8- to 16-fold increases in MICs of vancomycin and teicoplanin, respectively, were selected from 2 susceptible clinical isolates of Staphylococcus aureus by serial incubation in low-level vancomycin. A protein of approximately 39 kDa was demonstrable in the cytoplasmic fraction and occasionally in the membrane fraction by SDS-PAGE of both derivatives. This protein was purified by DEAE chromatography, preparative SDS-PAGE, and electroelution. Derivative bacteria were larger on transmission electron microscopy, had thicker cell walls, and had changes in colony morphology on solid media. Further evidence for cell wall reorganization included loss of phage and capsular typing, decreased susceptibility to lysostaphin/lysozyme killing, and changes in condition for detection of optimal coagulase activity. The mechanism of decreased susceptibility to glycopeptide antibiotics among S. aureus derivative isolates is uncertain. The production of the approximately 39-kDa cytoplasmic protein and cell wall reorganization may mediate changed affinity of glycopeptide-peptidoglycan binding or impairment of glycopeptide access to its cell wall target.

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

“…Cell wall thickening is a consistent feature and was recognized well before the first description of clinical VISA isolates ( Fig. 5) (30,59,62,80,108,121,187,229,267). It may be associated with activated cell wall synthesis (108), and the cell wall thickening is reduced when isolates are serially passaged and resistance levels drop (58).…”

“…For some VISA isolates a small increase in peptidoglycan cross-linking was found (30,32,267). 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).…”

“…For example, 6 laboratory-derived VISA strains demonstrated a stable phenotype after 20 passages on nonselective medium (250). Many of the earlier studies of the features of VISA were performed with laboratory-derived strains after stepwise selection on glycopeptide-containing medium (62,215,250,315,319,320,322); however, more recent studies have utilized clinical isolates (32,56,121,151,314,318), and in many cases, the phenotypic features have been similar. The ability to induce the selection of VISA from vancomycin-susceptible parent isolates varies between strains in terms of both the level of resistance attained and the rate at which resistance develops (58,250,267).…”

“…The ability to induce the selection of VISA from vancomycin-susceptible parent isolates varies between strains in terms of both the level of resistance attained and the rate at which resistance develops (58,250,267). After early studies of the effects of glycopeptides on S. aureus and investigation of laboratory-derived vancomycin-resistant strains of S. aureus and a clinical isolate of teicoplanin-resistant S. aureus revealed an increased level of production of PBP2 and cell wall thickening (62,80,313), much of the early investigation of hVISA and VISA was focused on cell wall changes.…”

Staphylococcus epidermidis device-related infections: pathogenesis and clinical management

“…Most of the reports regarding the mechanism of glycopeptide resistance have focused on S. aureus and it appears that it is intrinsic and deriving from the accumulation of mutations and not due to genetic exchange (Pfeltz et al 2000). Cell-wall thickening associated with vancomycin resistance in S. aureus has been reported by a number of groups and it is thought to be a pre-requisite for vancomycin resistance in staphylococci (Daum et al 1992;Hanaki et al 1998;Pfeltz et al 2000). Nunes et al (2006) recently characterised the glycopeptide susceptibility profiles and cell-wall ultrastructure of three clinical strains of CONS with reduced susceptibility to glycopeptides, including S. epidermidis.…”