Evolution And Molecular Mechanism Of Vancomycin Intermediate-resistant Staphylococcus Aureus Strain In Vitro

Objective Methicillin-resistant Staphylococcus aureus(MRSA）wereinitially described in1961and emerged in the last decade as one of themost important nosocomial pathogens highly prevalent in many countriesand throughout the world.Vancomycin used as the first-line therapy forMRSA infections for long time,vancomycin resistant Staphylococcusaureus(VRSA) and vancomycin intermediate-resistant Staphylococcusaureus(VISA) were reported in many countrys, our previous studies haveshown that a tendency toward decreasing susceptibility to vancomycin inMRSA has emerged in China.The number of clinical failures for patientstreated with vancomycin was rising,so MRSA infection treatment presentsa challenge for clinicians.An in-depth understanding of how MRSAresistant to vancomycin,can help our improve the understanding of theresistance of MRSA.This study, induction vancomycin resistant in MRSAby exposure to vancomycin to get VISA strain,then describe characteristicsof VISA from durg resistant and gene mutation.our aim was to investigatethe vacomycin mechanism of VISA.This research was divided into twoparts:fistly,The phenotypic characteristics and genetic backgroud of laboratory-generated vancomycin intermediate-resistant Staphylococcusaureus; In the one part,we found one VISA loss the methicillin resistant,sothe Second part is the molecular mechanism of Spontaneous Deletion ofStaphylococcal Cassette Chromosome mec Element inlaboratory-generated vancomycin intermediate-resistant Staphylococcusaureus strains.Methods MRSA strains was induced by low dose of vancmycin invitro,The MIC of vancomycin and oxacillin was determined by brothmicrodilution to detect the strains during the passage.PFGE was performedon MRSA strains and its VISA strains.PCR used to detect mecA,MLSTand SCCmec typing was used to analysis the relationship among VISA.Sequences of vraS、graR、WalKR and rpoB were compared susceptibleclinical MRSA strain and VISA.PCR was used to detection and conjuctionthe deletion SCCmec.Results12VISA mutants(vancomycin MICs,4-16g/mL) weregenerated in vitro,two ST239-SCCmecIII MRSA、two ST5-SCCmecIIand one new ST type MRSA were reached8-10ug/ml vancmycin MIC after45days induction.two VISA mutant were stable to20nonselectivepassages,other VISA was The two isolates were the same PFGE type,When removal of vancomycin pressure, most of VISA reverted tosusceptibility. Upon acquisition of vancomycin resistance, all mutantsshowed a concomitant decrease in oxacillin resistance,One VISA strain(3503) loss mecA gene and sensitive to oxallin.The A862Tsubstitution in the rpoB gene occurred in3503VISA strain.3503loss themecA gene when the vancomycin MIC reached6ug/ml(named3503VR6),and then reached the higher vancomycin MIC in10ug/ml(named3503VR10).3503VR10showed genotypic change in PFGEpattern with a deletion infragment size of~80kb,and3503VR6showeddeletion of~40kb.both of the two strains SCCmec excisions in the isolatesappeared to be linked with IS431transposable elements.Conclusion VISA is easy to get in vitro,most of them isunstable.Vancomycin resistant may associated with decrease in oxacillinresistance in VISA.Mutations in rpoB may contribute to vancomycinresistant in VISA, no VraSR、GraRS、WalKR mutation was found in VISA.Spontaneous Deletion SCCmec can occur with high level of VancomycinResistance in Staphylococcus aureus, IS431play a central role in theexcision of DNA.