Vrasr Two-Component Regulatory System Adn Vancomycin MIC Creepin Clinical Staphylooccus Aureus Isolates

Objective To assess the trend of vancomycin MIC creep in staphylococcus aureus (S. aureus) isolated from four tertiary teaching hospitals in Chongqing and Guangzhou city over a 3- year period, and to evaluate the role of two-Component regulatory system VraSR in S. aureus with increased vancomycin MICs.Methods The clinical isolates of S. aureus were collected from one hospitals in Chongqing (indicating as A) and three hospitals in Guangzhou (indicating as B to D) during the period from 2007 to 2009. MRSA was confirmed by Kirby-Bauer disk diffusion method using cefoxitin. MIC of vancomycin against S. aureus was determined by broth microdilution according to the CLSI standard,MIC50, MIC90 and geometric-mean-MIC were calculated for all isolates in each year. Moreover, PCR assay was used to detect the prevalence of regulatory gene vraSR in the methicillin-resistant Staphylococcus aureus (MRSA) followed by gene sequencing. Then sequence of N315 genome from genebank and the isolates in this study were aligned. MRSA isolates were induced by low-concentration Vancomycin in-vitro. Minimum inhibitory concentrations (MICs) were determined at each stage,and the vraSR gene sequencing were performed to determine the vraSR gene mutation. Real-time reverse transcription-PCR was used to analyze the expression.Results From 2007 to 2009, we collected 779 MRSA isolates with 264 isolates in 2007, 342 isolates in 2008, and 173 isolates in 2009, respectively. Overall, the vancomycin geometric-mean-MIC was 0.911, 0.954, and 0.976 with a significantly increased trend over years(P<0.001). A significantly increased MIC trend was found in A and B hospitals(P<0.001), but not found in C and D hospitals(P>0.05). Gene sequencing revealed that a MRSA isolate with MIC value of 2μg/mL harbored a mutated vraR presenting an aminoacid substitution (Glu59-Asp). After 4 day induction, MICs of 5 MRSA strains increased form 0.75 to 2μg/mL. Six days later, the MIC value of one of the MRSA isolates reached 4μg/mL. Twelve days later, 3 isolates of MRSA did so while the remaining 1 isolate keeping MIC in 0.75μg/mL.Although vraR gene expression increasd as MIC values elevated, no vraSR gene mutation found in these induced isolates.Conclusion Generally,vancomycin MIC creep occured in the MRSA strains in the study. Due to a possible regional discrepancy, significant vancomycin MIC creep was only found in hospital A and B. Nevertheless, we found a vraR gene mutation in one MRSA strain. In-vitro induction by low-concentration vancomycin indicated that MIC values and vraRS gene expression of the MRSA strains increased over time; however no vraSR mutation found here.