• Characterization of biochemical and genetic mechanisms of vancomycin resistance in Staphylococcus haemolyticus

      Verma, Punam; Schwalbe, Richard (1994)
      Vancomycin-resistant subpopulations can be directly selected in all strains of Staphylococcus haemolyticus displaying a double zone of growth around imipenem (imp) agar-diffusion discs (SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar}). The research plan entailed characterizing both biochemical and genetic mechanisms of vancomycin resistance in SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar}. SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar} isolates from 6 geographically distinct hospitals were analyzed by SDS-PAGE protein profiles, plasmid profiles, and pulsed-field (CHEF-DR II) gel electrophoresis patterns. The results suggested that the imp{dollar}\sp{lcub}\rm dz{rcub}{dollar} phenotype is probably not due to clonal dissemination of a single strain. CHEF electrophoresis patterns and plasmid profiles were better than SDS-PAGE protein profiles at discriminating among clinical SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar} isolates. The imp{dollar}\sp{lcub}\rm dz{rcub}{dollar} could not be transferred and expressed in an imp-sensitive strain of S. haemolyticus by utilizing filter mating and mixed culture procedures. This suggests that imp{dollar}\sp{lcub}\rm dz{rcub}{dollar} may arise by either a spontaneous mutation or coded for on a transposable element. Heterogenous expression of vancomycin resistance in coagulase-negative staphylococci was examined using a modified efficiency of plating model (EOP). Survival kinetics of SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar} demonstrated the presence of a heterogeneous subpopulation. Vancomycin-resistant clones from serially selected S. epidermidis and S. haemolyticus (non-SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar}), and from directly selected SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar} strains were also tested in the EOP model. The survival curves were similar to their respective parent strains, except that the vancomycin concentration required for killing was higher. Supplementation of Mueller-Hinton agar with 2% NaCl in the EOP model resulted in an increase in the recovery of vancomycin-resistant subpopulations. These results suggested that vancomycin resistance in SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar} was heterogeneous and expressed in a manner similar to methicillin resistance in staphylococci. The in vitro activity of the investigational glycopeptide antibiotic LY264826 was compared with glycopeptide compounds (vancomycin and teicoplanin), and the lipopeptide antibiotic, daptomycin for 174 clinical isolates of staphylococci. Microbroth dilution testing demonstrated that the MIC{dollar}\sb{lcub}\rm 90{rcub}{dollar} values for LY264826 were {dollar}\le{dollar}2 {dollar}\mu{dollar}g/ml for all species tested except SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar}. MIC{dollar}\sb{lcub}\rm 90\sp\prime s{rcub}{dollar} of twenty SHEimp{dollar}\sp{lcub}\rm dz{rcub}{dollar} strains were 32 {dollar}\mu{dollar}g/ml when 2% NaCl (final concentration) was added to cation supplemented Mueller-Hinton broth (CSMHB). All strains tested were susceptible to daptomycin (MIC{dollar}\sb{lcub}90{rcub}{dollar} = 0.5 {dollar}\mu{dollar}g/ml). Resistant subpopulations could be recovered in all strains of SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar} capable of growth in BHI containing 32 {dollar}\mu{dollar}g/ml of LY264826. Killing curves performed at a concentration of 8 {dollar}\mu{dollar}g/ml in 2% NaCl indicated that LY264826 and other glycopeptide antibiotics were ineffective at a concentration of 8 {dollar}\mu{dollar}g/ml; growth of SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar} strains was noted between 6 and 24 hours. These results suggested that LY264826 and other glycopeptide antibiotics were ineffective in vitro against SHeimp{dollar}\sp{lcub}\rm dz{rcub}{dollar} strains. There was no appreciable difference in the cell wall composition among the sensitive and resistant clones suggesting that modification of cell wall precursors was not responsible for phenotypic expression of vancomycin resistance. (Abstract shortened by UMI.)