| Susceptibility testing,bonding testing,and resistance gene detection were carried out to analyse the epidemic and molecular mechanisms of oqxAB genes in Enterococci; PCR mapping and molecular cloning technology were carried out to analyse Genetic enviroment of oqxAB genes. The epidemiological characteristics of enterococci was analysied by RAPD. The virulence genes were amplified by multiple PCR to analyse the relationship among virulence genes, multidrug-resistant phenotype and phylogenetic type.Broth dilution method was carried out to detect the sensitivity(MIC value)of 83 isolates enterococci against the commonly used antimicrobial agents, PCR amplification was carried out to specificly amplificate the fragment of multi-drug efflux pump genes oqxA, oqxB. The results showed that the drug resistance rate of isolated enterococci against lincomycin, tetracycline, sulfamethoxazole resistance, erythromycin,ceftiofur and olaquindox were 81.36%,78.31%,66.27%,74.58%,72.88%and 72.29%,respectively.And the drug resistance rate against Ceftriaxone, ciprofloxacin and Mequindox were 67.65%,57.83%and 48.64%, respectively. While the drug resistance against Levofloxacin Hydrochloride was at a lower rate of 19.53%. Through the drug sensitivity analysis of Enterococci from different sources we found that the resistance rates of 34 strains of enterococci isolated from feces were higher than the 49 strains of enterococci isolated from pork. Among the 83 isolates, the detection rate of gene-positive of oqxA gene, and oqxB gene was 79.52%,33.73%, respectively. The gene-positive rate of oqxA in the strain isolated from pork was 81.63%(40/49), the gene-positive rate of oqxA in the strain isolated from animal manure was 76.47%(26/34); And The gene-positive rate of oqxB in the strain isolated from pork was 38.78%,the gene-positive rate of oqxB in the strain isolated from animal manure was 26.47%.In this study, the multi-drug resistance efflux pump genes oqxAB were detected in enterococci for the first time in China.The upstream of efflux pump genes OqxAB normally present in the insertion sequence of ISEcpIand IS26,83 strains of enterococci isolated from pork were amplified by positioning PCR,and the results showed that detection rate of IS26 in Enterococcus faecalis was 50%, detection rate of IS26 in enterococci feom feces was 20.69%, while ISEcpIwas not detected in enterococci. Plasmid conjugation test were carried out by using donor(8 Enterococcus from faecalis,8 enterococci from feces)and recipient strains(JH2-2) of bacteria which were cultured overnight,three anti-drug(Rifampin, fusidic acid and olaquindox)screening and verification methods of oqxAB gene, and we compared the resistant phenotype of donor strain, zygote, receptors bacteria.6 Enterococcus conjugant were gained successfully(4 strains from E.faecelis and 2 strains from E.faecium), indicating that oqxAB gene located in plasmid which could be conjugated and transferred. Zygote were drug resistant anginst olaquindox, the sensitivity of drug resistant anainst quinolones, chloramphenicol drugs also decreased, one of the six zygote was drug resistant anginst Mequindox, indicating that the resistance anainst olaquindox can be transferred with oqxAB genes. By digestion, ligation transformation,sequencing the plasmid in enterococci(3EH) which was extracted,we obtained a 8374bp insert fragment,which also carry oqxA,oqxB-like(oqxD), insert IS26 was found in genes of oqxA upstream, and orf68, transposons, hypothetical protein(hpl, hp2), bleomycin resistance gene ble (partial sequence), neomycin or kanamycin resistance gene aph, Tn5 transposon tnpA (partial sequence) were found in preparation.Compared the OqxA gene obtained in this experiment with plasmid pOLA52 from the E.coli,we found that only the 475th bases of G into A,and the 159th amino acids from the Val Val(GTT)becomes isoleucine Ile(ATT); Since insertion of G at 2831 of gene OqxB orf in Escherichia, the misalignment occurs in the followed codon, not only three amino acid mutations, and the orf changed 3153 bp into 2838 nucleotides, and the number of protein amino acids coded changed from 1050 into 945, so the new Oqx genotype gene was named OqxD. The test proved the newly discovered genes OqxD in enterococci located on turn of transposon Tn6010-like, with the full-length of 6837bp, compared with E.coli, Salmonella, Klebsiella pneumoniae Tn6010(6846 bp), the gene short of 9bp nucleotides, and the 13 bp deletion of TAGCACCGCAGAG was found between the spacing of ORF68 and IS26 Tn6010, so the number changed from 49to 36, and inserted four bases in the OqxB. There were 2 more orf in Tn6010-like than that in Tn6010, which encoded two unknown function proteins. By random amplified polymorphic DNA analysis (RAPD)of the enterococci, we found that both clones spread, and horizontal transmission were presencent in the molecular epidemiology of enterococci.Multiplex PCR method was carried out to detected five common virulence gene surface protein(Esp), cell lysis hormone(cylA),gelatinase(gelE), hyaluronic acid(hyl), pheromone-induced protein(asal)in enterococci, and then observed the number and type of portability virulence gene it carried. The results showed that:69 isolates carryed at least one of five virulence genes in a virulence gene, the proportion was 83.13%, the gene-positive in descending order were gelE(54.21%),asal(43.37%),Esp(30.12%),hyl(16.87%),cylA(12.05%). Except gelE,positive rate of Enterococcus faecalis were higher than the Enterococcus faecium in the other four genes.44 of the 49 enterococci isolated from pork carried at least one virulence gene, at the positive rate of 89.80%; 25 of the 34 enterococci isolated from fecal carried at least one virulence gene, at the positive rate of 73.53%. |
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