Research On The Mutant Prevention Concentration Of Linezolid And Vancomycin Against Methicillin-resistant Staphylococcus Aureus

Objectives: To determine the antibacterial activity and the mutant prevention concentration (MPC) of linezolid and vancomycin to clinical methicilin-resistant Staphylococcus aureus (MRSA) isolates and compare the capacity of two antibiotics to prevent the resistant mutant strains; Furthermore, to investigate the possible molecular mechanism associated with reduced vancomycin susceptibility in the mutant strains by molecular fingerprint techniques.Methods: The minimal inhibitory concentration (MIC) of linezolid and vancomycin against 105 S. aureus were measured by 2-fold agar dilution method according to Clinical and Laboratory Standard Institute (CLSI ) criteria. Quality control was done followed every experiment to sure the accurate; The 56 MRSA clinical isolates were enriched in broth, then the bacterial concentration were adjusted to 1010 colony formation units per milliliter for MPC determine. An approximation was used in which 109 colony-forming units are applied to each agar plate in a series and drug concentration is varied by 2-fold increments. Inoculated plates were incubated at 37℃for 72 h, and colonies were counted at 24h intervals until colony numbers became constant. The drug concentration at which no bacterial mutant is recovered is operationally defined as the MPC.Compared the differences between the wild-type strains and the mutant strains which were selected by vancomycin during the determine of MPC by the Sau-PCR assay and pulsed-field gel electrophoresis fingerprint technique; The graS gene was detected by polymerase chain reaction method; Denaturing gradient gel electrophoresis was used to probe for mutations in the graS gene.Results: 1 The MIC for 90% of the isolates (MIC90) of linezolid and vancomycin to 105 S. aureus clinical isolates were both 2μg/ml, the MIC for 50% of the isolates (MIC50) were 2μg/ml and 1μg/ml,respectively,and the range of MIC were 1-4μg/ml and 0.5-4μg/ml, respectively. 2 The MPC for 90% of the isolates (MPC90) of linezolid and vancomycin for 56 MRSA clinical isolates were 4 and 20.5μg/ml, and the MPC90/MIC90 were 2 and 10.25, respectively. Linezolid selective index(SI=MPC90/ MIC90)with 56 MRSA was lower than vancomycin. Compared to vancomycin, linezolid had a narrower mutant selection window (MSW) against the MRSA. 3 The MIC determination was reperformed for the mutant strains which were selected by vancomycin during the determine of MPC. The MIC for 50% of the isolates (MIC50) and The MIC for 90% of the isolates (MIC90) of vancomycin were 2μg/ml and 4μg/ml,respectively. 4 Analyses by the Sau-PCR assay and pulsed-field gel electrophoresis revealed that the wild-type strains and the mutant strains which were selected by vancomycin during the experimental process of MPC had different DNA profiles.5 Denaturing gradient gel electrophoresis was used to probe for mutations in the graS gene and there were no different banding patterns on the electrophoretograms spectrum.Conclusion: 1 Both linezolid and vancomycin exhibited excellent activity against the S. aureus strains isolated from Shijiazhuang area. We found neither vancomycin-resistant strains nor linezolid-resistant strains. It reveals that the antibacterial activity of vancomycin against S.aureus have been decreasing. 2 The MPC of linezolid and vancomycin with S. aureus were below serum drug peak concentration and the serum drug concentrations were above the MSW. So linezolid and vancomycin are not easy to select resistant mutants of S. aureus. Compared to vancomycin, linezolid has a narrower MSW. The ability of linezolid for restricting the selection of resistant mutants of S. aureus is stronger than that of vancomycin. The possible mechanism are needed to clarify. 3 The MIC determination was reperformed for the mutant strains which were selected by vancomycin during experimental process of MPC. It appeared that the mutant strains had reduced vancomycin susceptibility, which is consistent with the MSW hypothesis. 4 Analyses by the fingerprint technique revealed that the wild-type strains and the mutant strains which were selected by vancomycin during the determing of MPC had different DNA profiles. 5 Denaturing gradient gel electrophoresis was used to probe for mutations in the graS gene and there were no different banding patterns on the electrophoretograms spectrum. It indicates that there may be mutations in other genes or exist other resistant mechanisms.