Molecular Epidemiology Of Vancomycin-resistant Enterococcus And Risk Factors Of Bloodstream Infections In Parts Of China

Background:Enterococcus is conditional pathogens that are associated with nosocomial infections,often causing bloodstream and urinary tract infections.It has inherent resistance and acquired resistance,including vancomycin and daptomycin resistance which were considered as the last line of defense.Vancomycin-resistant Enterococci(VRE)mostly is Enterococcus faecium carrying vanA type in China,but vanM type was reported in several hospitals in Shanghai.Most VRE strains in hospitals are clonal complex 17(CC17).This study retrospectively studied the molecular epidemiology of Vancomycin-Resistant Enterococcus in multiple centers and risk factors of bloodstream infections of VRE strains in China from 2006 to 2017.The aim of this study was to understand the epidemiological characteristics of VRE in China and provide the evidence for the control and prevention of nosocomial infection.Methods:A collection of 245 VRE strains(240 VREfm and 5 VREfs strains)from 16 hospitals of China from January 2006 to December 2007 were selected in this study.Minimal inhibitory concentrations(MICs)of antibiotics were determined by using a microdilution broth method.Vancomycin resistance genes and virulence genes were detected by PCR with specific primers.The horizontal transferability of vancomycin resistance genes and virulence genes of VRE strains was examined by Southern blot and plasmid conjugation assays.The clonal dissemination and genetic relationship of strains were analyzed by pulsed-field gel electrophoresis(PFGE)and multilocus sequence typing(MLST).The VREfin was screened by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry(MALDI-TOF MS)method.Twenty-five VREfm isolates from blood were analyzed by whole genome sequencing,and vancomycin resistance genes and virulence genes were further identified.The phylogenetic tree conducted by whole genome sequence Multilocus sequence typing(wgSNP)and core genome sequence Multilocus sequence typing(cgMLST)was used to presume the genetic relationship of Enterococcus faecium.In total,16 patients with VRE bloodstream infection with complete clinical records were selected.According to the ratio of 1:2,32 patients with concurrent vancomycin-sensitive Enterococcus faecium infection were used as the control group to carry out the risk factor analysis of VER bloodstream infection.Results:VRE isolates from urine,blood and abdominal drainage accounted for 47.5%(116/245),16.7%(41/245)and 16.3%(40/245),respectively.No tetracycline,daptomycin and linezolid non-sensitive strains were found,but the increased trends for MICs for tetracycline and daptomycin were observed.In total,93.3%(224/240)of the strains carried vanA,while the strains carrying vanA and vanM accounted for 6.3%(15/240),which were all from Beijing.Up to 82.9%(199/240)and 55.4%(133/240)of the strains carried esp and hyl,respectively,while the detection rates of asal,gelE,and cylA genes were quite low.94.0%(63/67)of the strains were successfully conjugated,and southern blot results showed that vanA gene of all the strains were located on the plasmid.The majority of ST types of 240 VREfm strains belonged to ST78(45.8%,110/240),ST17(7.9%,19/240)and ST18(7.0%,17/240),which grouped into clone complex 17(CC17).The results of PFGE showed a highly diverse genetic background.Among 25 VREfm strains from blood,10 strains(40%,10/25)were ST78,which belonged to four different cgMLST subtypes,CT1,CT2,CT6,and CT8,showed a long distance on phylogenetic tree.The results of PFGE typing of the 25 VREfin strains were less consistent with cgMLST and wgSNP,while the results of cgMLST and wgSNP were more consistent.Comparing the results of the MALDI-TOF typing with the wgSNP,only two cluster of 10(40%,10/25)VREfm strains are similar.WgSNP of VRE strains revealed highly clonal cluster,while VSE strains were more diverse.The presence of glycopeptides,aminoglycosides,high levels of gentamycin,erythromycin,and tetracycline resistance genes were found in different strains by WGS analysis.Virulence genes efaA,piliB,ebpB,collagen binding protein encoding genes acm and scm,and two cystic related genes(cpsA,cpsB)were found in all the strains,Enterococcal surface protein(Esp)was found in 80%(20/25)strains,while 36%(9/25)strains carried both piliE and piliF genes,and 52%(13/25)strains carried piliA genes.Statistically significant factors in VRE and VSE bloodstream infection patients included more basic diseases(>4),tumors,glycopeptides or carbapenem antibiotics,meropenem usage within six months,and blood transfusion experience.Conclusion:At present,VRE in China is still dominated by vanA-VanA type.In 2008,Enterococcus Faecium strains carrying both vanA and vanM were emerging.Tetracycline,daptomycin,and linezolid still have good activities for VRE in vitro,but the increased trends for MICs for tetracycline and daptomycin were observed.The dominant clone of VRE in China was ST78-CC17.Based on wgSNP typing,a few VREfin strains were epidemic in the same ward or among different wards in the same hospital,and even in different hospitals and cities,sometimes caused outbreaks.ICU and emergency wards are high incidence areas.MALDI-TOF MS is an effective method to identify VRE and VSE.It can play a role in rapid screening during nosocomial infections and outbreak control,but the typing analysis of VREfin by MALDI-TOF MS is less relevant to wgSNP.It is not suitable for VRE and VSE typing analysis.VRE bloodstream infection was associated with more basic diseases(>4),tumors,glycopeptides or carbapenem antibiotics,meropenem usage within six months,and blood transfusion experience.