江苏部分地区猪源结肠弯曲菌耐药性分析及多位点序列分型

Drug resistant and multilocus sequence typing of Campylobacter coli from pig source in Jiangsu province

摘要:

【背景】弯曲菌(Campylobacter)是重要的人畜共患肠道病原菌，可通过食物链传播，引起人类腹泻性肠炎。【目的】了解猪源弯曲菌耐药特征和分子遗传特征，对江苏省10个规模化猪场进行弯曲菌分离和耐药性检测，并研究分离株的分子分型。【方法】采用琼脂平板稀释法进行最低抑菌浓度(minimal inhibitory concentration，MIC)测定，PCR方法扩增耐药基因，以弯曲菌7个管家基因(aspA、glnA、gltA、glyA、pgm、tkt和uncA)为目的基因进行多位点序列分型(multilocus sequence typing，MLST)研究。【结果】100份样品共分离出结肠弯曲菌22株，分离率为22%，弯曲菌检出情况与养殖规模和日龄无关(P>0.05)。耐药性试验结果显示，20株分离株为多重耐药菌株(81.82%，20/22)，猪源结肠弯曲菌分离株对10种抗生素耐药程度不一，分别为：庆大霉素36.36%，链霉素50%，克林霉素27.27%，氯霉素13.64%，四环素40.91%，环丙沙星18.18%，萘啶酸63.63%，泰利霉素59.09%，红霉素100%，阿奇霉素81.82%；共检出了4种耐药基因[cfr、adE-Sat4-aphA、ermB和Tet(O)]，检出率分别为4.5%、59.1%、9.1%和100%；多位点序列分型结果显示共获得11个ST型，主要流行CC-828克隆系(100%)；进化树结果显示，所有分离株聚集归为2个大群，分别为2个分支和9个分支。χ2检验和Logistic回归模型表明共有3个序列型(sequence type，ST)与其相对应的抗菌药物有相关性。【结论】猪源结肠弯曲菌对大环内酯类抗生素具有较高的耐药性，Tet(O)基因检出率最高，分离株的序列型呈多样性。

Abstract:

[Background] Campylobacter is one of the most common zoonotic pathogens and is a leading cause of human gastroenteritis through food chain. [Objective] To study the phenotypic resistance and molecular type of Campylobacter isolated from pig source in ten intensive pig farms of Jiangsu province. [Methods] The minimal inhibitory concentrations (MICs) was determined by agar dilution method and the resistance genes were detected by polymerase chain reaction (PCR). Seven Campylobacter housekeeping genes including aspA, glnA, gltA, glyA, pgm, tkt and uncA were amplified and sequenced, then the sequences of genes were analysed through multilocus sequence typing (MLST). [Results] A total of 22 isolates were identified from 100 samples (22%), the detection of Campylobacter was independent of breeding scale and age (P>0.05). 81.82% of the isolates were resistant to three or more than three drugs, resistance rates to ten drugs were under different degrees, gentamicin (36.36%), streptomycin (50%), clindamycin (27.27%), chloramphenicol (13.64%), tetracycline (40.91%), ciprofloxacin (18.18%), nalidixic acid (63.63%), telithromycin (59.09%), erythromycin (100%), azithromycin (81.82%). The resistance-genes detection rates to cfr, adE-Sat4-aphA, ermB and Tet(O) were 4.5%, 59.1%, 9.1% and 100%, MLST results showed that 11 sequence types were found and CC-828 was the prevalent clonal complex (100%), Evolutionary tree results showed that all strains were belonged to two groups, which had two branches and nine branches, respectively. χ2 test and Logistic regression analysis showed that three sequence types (STs) were associated with their corresponding antimicrobial agents. [Conclusion] The high resistance to macrolides antibiotics and the high detection of Tet(O) were found in Campylobacter coli from swine feces, C. coli population showed diverse genetic nature.