| Enterococcus,the important opportunistic pathogen,is widely inhabited in the gut of humans and animals.It has already shown that multidrug resistance of strains is widespread and it poses great challenges for clinical anti-infective therapy.The study analyzed the resistant phenotypes of 135 strains of Enterococcus faecium and 230 strains of Enterococcus faecalis isolated from Jiangsu province.The amphenicols/oxazolidinones resistance genes fexA,fexB,floR,cfr and optrA,as well as fosfomycin resistance genes fosA,fosB,fosC were also detected in this study.Conjugation and genetic environment analysis of drug resistance genes were also carried out for the sake of understanding the resistance mechanisms to amphenicols/oxazolidinones and fosfomycin of Enterococcus faecium and Enterococcus faecalis as well as their horizontal transfer among the strains.Moreover,multiple locus sequence typing(MLST)was used to analyze the homology and difference of strains to explore its propagation mechanism.The study has a far-reaching significance in providing a guidance for the optimal use of antimicrobials.1.Analysis of resistant phenotype of Enterococcusfaecium and Enterococcus faecalisThe broth microdilution method was used to detect the antimicrobial sensitivities of 12 antimicrobials against 135 E.faecium strains and 230 E.faecalis strains,respectively,which were isolated from swine in Jiangsu province.The results showed that the resistant rates of erythromycin,tetracycline and florfenicol in 135 strains of E.faecium were the highest with rates of 99.26%,98.52%and 82.22%,respectively.The resistant rates of other drugs from high to low in E.faecium were ampicillin(75.56%),streptomycin(74.07%),gentamicin(71.1 1%),marbofloxacin(69.63%),penicillin G(45.19%),rifampicin(34.07%),fosfomycin(20.00%),and the lowest linezolid(2.96%),respectively.The resistant rates of tetracycline,erythromycin and florfenicol of 230 strains of E.faecalis were the highest with rates of 100%,98.26%amd 96.96%,respectively.The resistant rates of other drugs from high to low in E.faecalis were gentamicin(78.70%),streptomycin(75.22%),marbofloxacin(70.87%),linezolid(54.78%),rifampicin(30.43%),fosfomycin(12.17%)and ampicillin(1.74%),respectively.The strains of E.faecalis had a lowest resistance toward penicillin G and the rate was 0.43%.Vancomycin resistant strains were not found in both E.faecium and E.faecalis.We also found that most of E.faecium and E.faecalis were multiple drug resistant strains.The E.faecium was mainly resistant to 7 kinds of drugs,which contains 39 strains;and 3 strains of which showed the highest resistance to 10 kinds of drugs.The E.faecalis was mainly resistant to 6 kinds of drugs,which contains 64 strains;with 1 strain of which showed the maximum of 10 kinds of drugs.2.Detection of amphenicols/oxazolidinones resistance genes and fosfomycin resistance genes in E.faecium and E.faecalis strainsPCR was used to detect the distribution of the amphenicols/oxazolidinones resistance genes fexA,fexB,floR,cfr,optrA and fosfomycin resistance genes fosA,fosB and fosC in E.faecium and E.faecalis strains.The results revealed that the positive rates of the amphenicols resistance genes fexA,fexB and floR in E.faecium were 22.22%(30),50.37%(68),19.26%(26),respectively;and the positive rates in E.faecalis were 89.13%(205),1.74%(4),and 64.35%(148),respectively.These are basically consistent with the resistant phenotype of florfenicol.The genes cfr and optrA,which give the resistance to both amphenicols and oxazolidinones,were with rates of 0.74%(1)and 17.04%(23)in E.faecium,respectively;however,with rates of 1.30%(3)and 20.87%(48)in E.faecalis,respectively.The strains carrying these two genes were all intermediate or resistant to linezolid.The fosfomycin resistance gene fosB was positive in one strain of E.faecium and two strains of E.faecalis.The MIC values of fosfomycin of the fosB positive strains were eaqual to or more than 1024 ?g/mL.However,fosfomycin resistance genes fosA and fosC were not found in both E.faecium and E.faecalis.3.Analysis of horizontal transmission mechanism of antibiotic resistance genes cfr,optrA and fosBMolecular typing,conjugation and sequencing analysis were applied to explore the drug resistance mechanism and the possibility of transmission of the amphenicols/oxazolidinones resistance genes cfr,optrA and fosfomycin resistance gene fosB in florfenicol and linezolid resistant strains carrying cfr or optrA and fosfomycin resistant strains harboring fosB.The results showed that the optrA positive florfenicol and linezolid resistant E.faecalis strains mainly belong to ST690,and ST32,ST69,ST86,ST22 and the newly discovered ST910-ST914 existed in partial strains.Two strains of cfr positive florfenicol and linezolid resistant E.faecalis had only one ST type known as ST21 and the other strain belongs to newly discovered ST915.Two strains of fosB positive fosfomycin resistant E.faecalis were newly discovered ST916,and the fosB positive fosfomycin resistant E.faecium was ST908,belonging to CC17.Two strains of fosB positive fosfomycin resistant E.faecalis,two strains of cfr positive florfenicol and linezolid resistant E.faecalis and 18 strains of optrA positive florfenicol and linezolid resistant E.faecalis could successfully conjugated.In contrast,three strains of optrA positive florfenicol and linezolid resistant E.faecalis and fosB positive fosfomycin resistant E.faecium failed to conjugate.The sequencing showed that fosB positive strain ZKB11 also carried a macrolides resistance gene ermB,a conjugated transposon Tn916-like structure and some fragments encoding conjugation protein or mobile element protein.The optrA and fexA positive strains ZKB11 and GM57 also carried Tn554 transposon.The strain GM5 7 was found to have ISEnfa5 composite element carrying cfr,which widly distributs in different species of humans and animals-derived bacteria.In summary,both E.faecium and E.faecalis isolated from swine in Jiangsu province presented serious multi-drug resistance phenotypes.Especially,the resistance rates to florfenicol were higher than the previous studies indicating the resistance increasing year by year and either of fexA,fexB,floR,cfr,optrA genes contributing to the resistance.Linezolid and fosfomycin resistant strains containing plasmid-carried cfr or optrA and fosB were also found in this study.MLST indicated that the strains may have clonal spread between humans and pigs.The fosB gene was first reported in E.faecalis,which carries a Tn916-like structure that facilitates its transfer and spread.The oxazolidinones resistance genes optrA and cfr are also associated with the transferable structures Tn554 and ISENfa5,respectively,indicating a potential for widespread of oxazolidinones drug resistance. |
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