肠球菌GyrA基因突变和多重耐药泵与氟喹诺酮耐药性关系研究

Laboratory study on GyrA gene mutation in enterococci and multi-drug resistance efflux pumps result in enterococcal resistance to fluoroquinolones

目的探究肠球菌GyrA基因突变和多重耐药泵与氟喹诺酮类药物耐药性的关系。方法全部试验菌株使用VITEK 2-Compact仪器和GPI卡进行鉴定。通过PCR和单链构象多态性(SSCP)法检测对左氧氟沙星(Levo)与环丙沙星(Cip)耐药的30株粪肠球菌和10株屎肠球菌GyrA基因有无碱基发生突变,并通过基因测序确定其突变类型。检测了30株粪肠球菌在M-H琼脂中加入终浓度为20μg/mL利血平后Cip MIC值的改变。结果 PCR-SSCP分析结果表明40株对Levo和Cip同时耐药肠球菌均扩增出GyrA基因,其中有24株粪肠球菌和4株屎肠球菌的单链泳动带与粪肠球菌ATCC 29212和敏感菌株的单链泳动带相比较出现异常。其中粪肠球菌在第87位上有密码子改变:GAA变为GGA;屎肠球菌在第83位有密码子改变:AGT变为TAT,这些突变均可引起氨基酸的改变。在M-H琼脂中加入利血平可以提高部分粪肠球菌对环丙沙星的敏感性。结论用PCR-SSCP法检测40株肠球菌的GyrA基因并通过测序分析,显示有70%(28株)肠球菌碱基发生了突变,说明GyrA基因喹诺酮类药物耐药决定区(QRDR)突变是肠球菌对氟喹诺酮类药物耐药的重要原因,此外还有药物泵出等耐药机制的存在。

Objective To study the relations between enterococcal GyrA gene mutation,enterococcal multi-drug resistance efflux pumps and fluoroquinolones resistant in enterococci.Methods All strains were identified using VITEK-2 compact and GPI cards.Mutation of GyrA gene in strains of 30 E.faecalis and 10 E.faecium of levofloxacin-resistant and ciprofloxacin-resistant were detected using PCR and single strand configuration polymorphism（SSCP）,testing results and mutant types were confirmed by sequencing analysis.At the same time,the minimal inhibitory concentration（MIC） of 30 strains of enterococci against ciprofloxacin were tested using M-H agar including reserpine that final concentration was 20 μg/mL.Results All the 40 strains of enterococci resistant to Levo and Cip,were successful to amplify GyrA gene;24 strains of E.faecalis and 4 E.faecium single-strand electrophoresis band appeared abnormal compared with ATCC 29212.The sequencing of 2 strains of E.faecalis had codon alterations at 87：GAA→GGA;2 strains of E.faecium had the codon alteration at 83： AGT →TAT.Alterations of codons lead to the change of amino acids.Parts of E.faecalis＇ MICs were decreased adding reserpine in the M-H agar.Conclusion All 40 strains enterococci GyrA gene were detected by PCR-SSCP and confirmed by sequencing analysis,70%（28 strains） had gene mutation.This result indicated GyrA gene mutations are main causes,which resulted in fluoroquinolones-resistance in enterococci.In addition,Multi-drug resistance efflux pumps were also existent in enterococci.