沙门氏菌中与萘啶酮酸和环丙沙星抗性相关基因及突变的检测分析

Detection and Analysis of Naladixic Acid and Ciprofloxacin Resistance-Associated Genes and Mutations in Salmonella

目的:研究与萘啶酮酸和环丙沙星抗性相关基因在分离于陕西、新疆和广东等9省(市)食源性沙门氏菌中的分布及其与菌株耐药表型间的相关性。方法:使用玻片凝集法鉴定沙门氏菌的血清型,琼脂稀释法测定沙门氏菌的药敏性,聚合酶链式反应法测定9种耐药基因。结果:814株沙门氏菌共涵盖83种血清型,其中鼠伤寒沙门氏菌(Salmonella typhimurium,24.08%)、肠炎沙门氏菌(S.enteritidis,19.41%)、印第安纳沙门氏菌(S.indiana,13.27%)和德比沙门氏菌(S.derby,5.16%)等血清型比较常见。553株(67.94%)和219株(26.90%)沙门氏菌分别对萘啶酮酸和环丙沙星耐药。814株沙门氏菌中,oqxB阳性菌株的平均检出率(31.82%)显著高于qnrA(24.94%)、oqxA(24.57%)、qnrB(24.45%)、qnrS(10.32%)和qepA(3.07%)阳性菌株检出率(P<0.05),与aac(6’)-Ib阳性菌株的检出率(27.52%)间无显著性差异。7种耐药基因在5种最常见血清型、不同采样地来源、不同地域来源以及不同样品来源菌株中的检出率间存在一定差异。gyrA中共检出221个氨基酸突变点,以Ser83Phe/Asp87Gly双突变(21.27%)最为常见,其次分别为Ser83Phe(16.29%)、Asp87Gly(13.57%)、Ser83Tyr(12.22%)、Asp87Tyr(11.31%)、Asp87Asn(10.41%)、Ser83Phe/Asp87Asn双突变(9.95%)、Ser83Tyr/Asp87Gly双突变(2.71%)、Asp87Val(0.90%)、Gly75Phe(0.45%)、Asp87Asn/Ile89Val双突变(0.45%)和Asp87Asn/Val90Gly双突变(0.45%);parC中共检出217个突变点,以Ser80Arg(64.49%)突变最为常见,其次分别为Thr57Ser(35.05%)和Ser80Arg/Gly72Phe双突变(0.47%)。结论:陕西等9省(市)食源性沙门氏菌血清型种类繁多,对萘啶酮酸和环丙沙星耐药较为普遍,oqxA、oqxB、qepA、qnrA、qnrB、qnrS和aac(6’)-Ib基因比较流行,在gyrA和parC中检出多种变异,这些基因的存在和抗生素靶位变异可能是导致沙门氏菌耐药的重要原因。

Objective:In this study,the serotype,antimicrobial susceptibility to nalidixic acid and ciprofloxacin,genes associated with quinolone and fluoroquinolone resistance of the foodborne pathogen Salmonella isolated in nine provinces and municipalities of China,including Shaanxi,Xinjiang and Guangdong,were identified and analyzed,aiming to better understand the development and transmission of antimicrobial resistance,and more broadly,to ensure food safety.Methods:Salmonella serovars were determined by the slide agglutination method,the antimicrobial susceptibility was tested by the agar dilution method,and nine genes associated with resistance to quinolone and fluoroquinolines were determined using PCR.Results:A total of 83 serotypes were identified from 814 Salmonella isolates.The most prevalent serotype was S.typhimurium(24.08%),followed by S.enteritidis(19.41%),S.indiana(13.27%)and S.derby(5.16%).Out of the 814 isolates,553(67.94%)and 219(26.90%)resisted to nalidixic acid and ciprofloxacin,respectively.The average prevalence of oqxB positive isolates(31.82%)was significantly(P<0.05)higher than that of qnrA(24.94%),oqxA(24.57%),qnrB(24.45%),qnrS(10.32%)and qepA(3.07%)positive isolates,and it showed no significant difference from that of aac(6’)-Ib positive isolates(27.52%).A difference could be found among five common serotypes,different sampling sites,different sample types and different geographical areas in the prevalence of the seven antibiotic resistance-associated genes.A total of 221 mutations were detected in gyrA,with the most prevalent one being Ser83Phe/Asp87Gly(21.27%),followed by Ser83Phe(16.29%),Asp87Gly(13.57%),Ser83Tyr(12.22%),Asp87Tyr(11.31%),Asp87Asn(10.41%),Ser83Phe/Asp87Asn(9.95%),Ser83Tyr/Asp87Gly(2.71%),Asp87Val(0.90%),Gly75Phe(0.45%),Asp87Asn/Ile89Val(0.45%),and Asp87Asn/Val90Gly(0.45%).A total of 217 mutations were detected in parC,the most prevalent one being Ser80Arg(64.49%),followed by Thr57Ser(35.05%)and Ser80Arg/Gly72Phe(0.47%).Conclusion:The serovars of Salmonella isolates from the nine provincial regions were diverse,and the resistance to nalidixic acid and ciprofloxacin was highly prevalent.oqxA,oqxB,qepA,qnrA,qnrB,qnrS and aac(6’)-Ib were prevalent in Salmonella,and multiple mutations could be detected in both gyrA and parC.The existence of these antibiotic associated genes and mutations in the quinolone resistancedetermining region(QRDR)may play important roles in nalidixic acid and ciprofloxacin resistance.