A Preliminary Study Of Epidemiology And Mechanism About The Change Of Susceptibility Of Staphylococcus Aureus To Vancomycin In Partial Area Of Shenyang

Objective: The purpose of this study is to analyze the changes in susceptibility tovancomycin of Staphylococcus aureus from some clinical isolates in partial area ofShenyang and the relationship between the susceptibility changes and treatment failure;and to detect vancomycin heterogeneity intermediate resistant heterogeneousvancomycin intermediate resistant staphylococcus aureus（hVISA) Staphylococcusaureus strains. Besides, we will take a preliminary study on the molecular epidemiologyand possible mechanism in hVISA and vancomycin MIC drifting strains(MIC>1.5μg/ml).Materials and Methods:172Staphylococcus aureus clinical isolates wereobtained from some hospitals in Shenyang area from December2010to December2012,including102MRSA strains,70methicillin-susceptible Staphylococcus aureus (MSSA)strains. E-test method was used to detect the vancomycin MIC. We selected51MRSAstrains with MICs between1.5-3.0μg/ml and screened the hVISA strains usingvancomycin-containing4.0μg/ml Brain Heart infusion agar medium, and screened the(vancomycin-resistant Staphylococcus aureus）VRSA strains with MICs greater than8.0μg/ml using vancomycin-containing8.0μg/ml brain heart infusion agar medium. Wesubcultured MRSA strains on blood plates without vancomycin, and detected changesof vancomycin MICs at the same time. We analysis the availability of treatment withvancomycin in70MRSA Clinical isolates of different MICs from General Hospital ofShenyang.42MRSA clinical isolates from General Hospital of Shenyang MilitaryRegion were typed by PFGE method and analysed the correlation between the PFGEtype and vancomycin MICs of MRSA. We selected5MRSA strains (three strains withMICs of2.0μg/ml, two strains with MICs of1.5μg/ml), and one strain of ATCC29213and cultured them for50generations. For every10passages we observed their changes in cell wall thickness by transmission electron microscopy, and meanwhile determinatetheir MICs. Using polymerase chain reaction (polymerase chain reaction, PCR) andsequencing, we analysed the relationship between the changes of4genes relevant to cellwall thickness (graR, rpoB, walK and clpP) and the changes of the cell wall thickness.Results: For the172clinical Staphylococcus aureus isolates in partial area ofShenyang,146strains have vancomycin MICs between1.0~2.0μg/ml, accounting for84.89%. For the102MRSA strains,88strains have vancomycin MICs between1.0μg/ml and2.0ug/ml, accounting for86.27%, of which there are two strains withMICs of2.5and3.0μg/ml respectively. For70MSSA strains,46strains havevancomycin MICs between1.0-1.5μg/ml, accounting for65.71%, and the remainingless than1.0μg/ml. No hVISA strain and the VRSA strain were screened usingvancomycin-containing4.0μg/ml Brain Heart infusion agar medium andvancomycin-containing8.0μg/ml brain heart infusion agar medium. The treatmenteffectiveness of the patients infected with MRSA in different vancomycin MICs hassignificant difference.Compared to the strains which had vancomycin MIC＜1.5μg/ml,the strains which had vancomycin MICs drifting (MIC>1.5μg/ml) had bad effience onintreantment with vancomycin. Using PFGE42MRSA strains isolated from GeneralHospital of Shenyang Military Region were divided into A~E types. There were33strains identified as A type, which is the epidemic type including A1subtype32strains，A2subtype1strain. There were4strains identified as B type, which is the nearestgenetic kinship with A type. A type is mainly distributed in the emergency intensivecare unit, neurosurgery intensive care unit，emergency and neurosurgery. Other typesscattered in various departments including outpatient department. The PFGE typing andtheir MICs in clinical prevalent MRSA strains results indicated that there was noobvious correlation between them. But one strain of A2subtype and4strains of B typeall had high MICs, which had nearer genetic kinship with A1subtype. The MIC tovancomycin significently increased when it subcultured for10generations in diskwithout vancomycin and then it gradually decreased. The MICs above2.0μg/mlreturned back to the early beginning of the culture when passaged50generations in3strains. For two strains with MIC of1.5μg/ml, one returned back to1.5μg/ml and theother back to below1.5μg/ml when passaged50generations. Generally there wasdiffrence in the cell wall thickness among the cell populations with differentvancomycin MICs through transmission electron microscopy. MRSA which had higervancomycin MIC values, were thicker in cell wall than the lower ones. graR, rpoB, Walk and clpP four genes were amplified by PCR and compared with the standardstrains and no gene mutation was detected.Conclusion: No hVISA strain had been screened. Most of the treatment failure inclinical may be irrelevant with hVISA strains. Most of the clinical treatment failure maydue to strains MICs drift. The MICs did not decreased in MIC drift strains whenseprated from antibiotic environment, suggesting that the drift of the MICs may also berelated to stable genetics and mutation. The MIC drift to vancomycin in Staphylococcusaureus was progressed among same clone strains and there may be tendency that it isprevalent in high MICs subclone strains. Though point mutations were not detected ingraR、rpoB、walK and clpP genes which were associated with cell wall metabolism，the thickening of cell wall is easily seen。So the mechanism about the changes ofsusceptibility of staphylococcus aureus needs further study.