| Objective To explore molecular mechanisms of resistance to linezolid and clonal relatedness in Staphyococcus capitis and Staphylococcus epidermidis clinical strains in order to provide evidence for the prevention and control of nosocomial infection and the rational use of antibiotics.Methods Thirty-five strains of Staphylococci spp were collected from inpatients in seven teaching hospitals,including 30 strains of Staphylococcus capitis(28 strains resistant to linezolid,2 strains sensitive to linezolid)and 5 strains of Staphylococcus epidermdis resistant to linezolid.The identification and antimicrobial susceptibility testing of the strains were carried out by the Vitek-2 Compact system.Linezolid resistance cfr and optrA genes in these stains were identified by PCR and sequencing.Clonal relatedness of the strains were detected by pulse field gel electrophoresis(PFGE)and multilocus sequence typing(MLST).The numbers and size of plasmids in Staphylococci spp was detected by S1-PFGE.Based on Illumina and PacBio RS Ⅱ(SMRT)sequencing platforms,the whole genome of some cfr-carrying strains isolated from different hospital inpatients were sequenced,and then the data were analyzed using softwares including ResFinder and ISFinder.Chromosome and plasmid structures were drawn using InkScape 0.48.1 software.Results ①All of 28 linezolid-resistant Staphylococcus capitis strains and 5 linezolid-resistant Staphylococcus epidermidis strains were resistant to penicillin,erythromycin,ciprofloxacin,levofloxacin and moxifloxacin.And the resistance rates to Oxacillin were 96.4%in linezolid-resistant Staphyococcus capitis strains and 80%in linezolid-resistant Staphylococcus epidermidis strains,respectively.②PCR amplification and product sequencing confirmed that the positive rates of cfr gene in 28 linezolid-resistant Staphylococcus capitis strains and 5 linezolid-resistant Staphylococcus epidermidis strains were 82.1%(23/28)and 80%(4/5),respectively,while cfr gene in two linezolid-sensitive Staphylococcus capitis strains was not detected.In addition,all the 35 Staphylococci spp were negative for optrA gene.③PFGE and cluster analysis showed that 30 strains of capitis were divided into 3 groups(A～C),and the Dice similarity coefficient was 74.9%-100.0%and that 5 linezolid-resistant Staphylococcus epidermidis strains belonging to ST22 had mostly identical PFGE profiles and their Dice similarity coefficient was 89.2%-100.0%.The above results suggested that there were clonal spreads of linezolid-resistant Staphylococcus capitis strains and Staphylococcus epidermidis strains in these hospitals.④The complete chromosome and plasmid sequences of three Staphylococcus capitis strains(QD2,QD12 and QD40)and one Staphylococcus epidermidis QD39 strain were obtained by high-throughput sequencing technology.⑤Bioinformatics analysis showed that each of three Staphylococcus capitis strains(QD2,QD12 and QD40)contained one chromosome and three plasmids,and the cfr genes were located on plasmids pHJP338-2,pHJP381-2 and pHJP410-3,respectively,while Staphylococcus epidermidis strain QD39 contained one chromosome and one plasmid and the cfr gene was located on chromosomes.Comparative genomic analysis showed that the cfr region sequences of the cfr-harboring plasmids pHJP338-2,pHJP381-2 and pHJP410-3 in Staphylococcus capitis strains(QD2,QD12 and QD40)and that of the cfr-carrying plasmid pLRSA417 in MRSA strain LRSA417(GenBank No:KJ922127.1)were highly similar(coverage rate up to 100%,nucleic acid consistency 99.17%).In addition,the cfr region in chromosome HJP409-chr of Staphylococcus epidermidis QD39 strain was composed of ISenFA4-ORF1-CFR-ISenFA4,which was also highly similar to the corresponding sequence of plasmid pLRSA417(GenBank No:KJ922127.1)in MRSA strain LRSA417.Conclusion Our results demonstrated that the main molecular mechanisms of resistance to linezolid in staphylococcus capitis and Staphylococcus epidermidis clinical strains is that these strains carry cfr genes located on plasmids or chromosomes.There were clonal outbreaks of linezolid-resistant Staphylococcus capitis and Staphylococcus epidermidis clinical isolates in hospitals and/or between hospitals in this study. |
