The Prevalence And Resistance Mechanism Of VanM-positive Vancomycin Variable Enterococci

Vancomycin variable enterococci(VVE)are a group of vancomycin-susceptible enterococci containing vancomycin resistance gene and capable of developing into resistant phenotype under vancomycin pressure.Owing to their potential of escaping from detection,disseminating vancomycin resistance gene and a risk of treatment failure,it is essential to strengthen the surveillance for VVE and to do resistance mechanism research.Recently,several outbreaks and mechanism studies of vanA-positive VVE have been reported in other countries.vanM gene has become a prevalent vancomycin resistance determinant of clinical enterococci in China.In this study,we aim to investigate the prevalence status of vanM-containing VVE in Hangzhou and to further explore the mechanism of resistance conversion.In chapter Ⅰ,we performed the screening of vanM gene among 1284 clinical isolates of enterococci by PCR.Antimicrobial susceptibility testing,ML ST typing and PFGE clustering was conducted on the vanM-positive isolates.S1 nuclease-based plasmid analysis,Southern blot hybridization,whole genome sequencing analysis and Quantitative reverse transcription PCR was conducted to confirm the location and surrounding environment of vanM gene and expression analysis.Our results showed that the prevalence of vanM gene in clinical enterococci was 4.36%(56/1284).These 56 vanM-positive isolates include 55 isolates of E.faecium and one isolate of E.faecalis.Only one E.faecium,SRR22,was found to be glycopeptide-resistant,with vancomycin and teicoplanin MIC>256.Most(54/55)of vanM-positive E.faecium belonged to the main hospital-associated epidemic lineage CC17.vanM was located on plasmid in most of isolates and only in chromosome of one E.faecium ZY11.The vanM transposons in 55 vanM-positive VSE showed diversity because two or three IS1216E elements inserted into different position of the vanM gene cluster leading to partial deletion or interruption of vanR,vanS,vanX or vanH.Quantitative reverse transcription PCR showed that the constitutive expression of vanM in the VRE isolate SRR22 was>5.36 folds higher than that in any of the VSE strains.Our results suggested that the silenced vanM gene cluster in clinical Enterococcus isolates have become a great reservoir of vancomycin resistance gene in Hangzhou.In chapter II,to investigate their capability of converting to vancomycin resistance phenotype,54 VSE strains carrying incomplete vanM gene cluster were induced in vitro by increasing concentrations of vancomycin.Then,susceptibility test and conjugation was performed on the induced resistant isolates.Our results showed that nearly half of(25/54)vanM-positive VSE isolates had the ability of transforming to resistance phenotype,i.e.,they are strictly termed VVE.Besides,the structure integrity of vanM gene cluster appeared to affect the potential of a susceptible VVE strain to turn into resistance.Those carrying vanM gene cluster with partial deletion of vanRS(type ⅢvanM transposon)could be easily induced to resistant cells conferring high level resistance to vancomycin and teicoplanin within two days.However,those having deletion in vanX or deletion in both of vanX and vanRS(type Ⅱ or Ⅰ vanM transposon)required relative long time(3 to 14 days)to turn into VRE cells conferring resistance to vancomycin but susceptible or intermediate resistance to teicoplanin.In addition,we found that the resistant VVE isolates carrying type Ⅲ vanM transposon had high transfer frequency 1.01～5.37×10-3 per donor,except one induced isolate of ZY11 carrying vanM in chromosome.All resistant VVE isolates carrying type Ⅱ and ⅠvanM transposon failed in conjugation.In chapter Ⅲ,to clarify the mechanism of phenotype-switching in vanM-positive VVE,three VVE representative isolates of different vanM transposon types were selected for further investigation.Whole genome sequencing and analysis,quantitative real time PCR and Southern-blotting were performed to explore any changes on DNA,mRNA or cell level between each original isolate(VVE-S)and induced isolate(VVE-R).It showed that compared with VVE-S isolate,the sequence of vanM gene cluster had no change but the copy number of whole cluster increased by tens of folds(45～94 folds)in each VVE-R isolate.The amplification of vanM gene cluster was proven to tandem repeated with flanking IS1216E as joints owing to unassembled reads containing at least five adjoining vanM transposons identified in the raw data of each VVE-R isolate by Nanopore or PacBio.In addition,compared with VVE-S isolate,the expression of vanM increased by tens of folds and the growth rate decreased significantly in VVE-R cells.This study firstly revealed tandem amplification of vanM gene cluster(with flanking IS1216E as joints)as a new mechanism for vancomycin resistance in vanM-positive VVE strains.Besides,genome plasticity,increase of expression level and fitness cost of cells emerged accompanied with this amplification of vanM cluster.