七彩山鸡养殖场铜绿假单胞菌的耐药性、多位点序列分型及遗传进化分析

Analyses of drug resistance,multi-locus sequence typing and genetic evolution of Peudomonas aeruginosa in pheasant farms

【目的】探究广东省七彩山鸡养殖场中铜绿假单胞菌Pseudomonas aeruginosa的流行特点、耐药性、多位点序列分型(Multi-locus sequence typing,MLST)及遗传进化背景,为临床合理用药提供参考。【方法】从广东省3个规模化七彩山鸡养殖场收集孵化死胚及周围环境样本进行铜绿假单胞菌的分离鉴定,采用K-B纸片扩散法测试其对22种抗菌药物的敏感性,利用MLST分析铜绿假单胞菌分离株的分子流行特点。将各ST型的7个管家基因序列按顺序拼接,用MEGA7软件对拼接好的序列进行遗传进化分析。【结果】在采集的514份样本(死胚样本405份、环境样本109份)中共分离到铜绿假单胞菌145株(分离率28.2%),其中,24株来源于环境样本(分离率22.0%,24/109),121株来源于死胚样本(分离率29.9%,121/405)。145株铜绿假单胞菌除对氨苄西林、卡那霉素和萘啶酸天然耐药外,对复方新诺明、氯霉素和四环素的耐药性较强,耐药率分别为100%、80.0%和77.2%,其次为头孢噻肟(耐药率23.4%)。除天然耐药外,多重耐药的铜绿假单胞菌占比达73.1%(106/145),且对亚胺培南也显现出耐药性。MLST结果显示,89株耐药谱较广的铜绿假单胞菌可分为18个ST型,其中,6个ST型是本研究新发现的。铜绿假单胞菌死胚分离株的优势ST型是ST-260,环境分离株以ST-2100和ST-3202为主。遗传进化分析表明,环境和死胚样品中的铜绿假单胞菌具有较高的相似性。【结论】本研究调查的3个七彩山鸡养殖场的死胚及周围环境中均存在不同程度的铜绿假单胞菌感染或污染,且分离株具有较强的耐药性;建议在养殖过程中不仅要加强饲养管理、提高生物安全意识,还应根据药敏试验结果合理选择抗菌药。

【Objective】To investigate the epidemic characteristics,drug resistance,multi-locus sequence typ-ing(MLST)and genetic evolution background of Pseudomonas aeruginosa in Guangdong Province,and provide a reference for clinical rational drug use.【Method】Samples from dead embryos of pheasant and their surrounding environment were collected for separation and identification of P.aeruginosa.The K-B paper disk diffusion method was used to analyze P.aeruginosa sensitivity to 22 kinds of antimicrobials.The MLST method was applied to analyze the molecular epidemiology of P.aeruginosa strains.Seven house-keeping genes of each ST type were spliced sequentially,and we used MEGA7 software to conduct genetic evolution analysis to the spliced sequence.【Result】A total of 145 P.aeruginosa strains(isolation rate 28.2%)were isolated from the collected 514 samples(405 dead embryo samples and 109 environment samples),including 24 strains from environmental samples(isolation rate 22.0%,24/109)and 121 strains from dead embryos(isolation rate 29.9%,121/405).Antibiotic drug sensitivity test showed that 145 strains of P.aeruginosa were naturally resistant to ampicillin,kanamycin and nalidixic acid,strongly resistant to complex sulfamethoxazole,chloramphenicol,tetracycline,followed by cefotaxime,with the drug resistance rates of 100%,80.0%,77.2%and 23.4%respectively.Except the natural drug resistance,the proportion of multiple drug resistant P.aeruginosa was up to 73.1%(106/145),and a certain proportion of P.aeruginosa strains which were resistant to imipenem appeared.MLST analysis showed that 89 P.aeruginosa strains with a broad spectrum of resistance were divided into 18 ST types,presenting high diversity.Among them,six ST types were the new types discovered in this study.The dead embryo isolates of P.aeruginosa were mainly ST-260,and the environmental sample isolates were mainly ST-2100 and ST-3202.Genetic evolution analysis showed that P.aeruginosa strains in environment were closely related to those in dead embryos.【Conclusion】There are different degrees of P.aeruginosa infection or contamination in dead embryos and surrounding environment of three pheasant farms,and the isolates have strong drug resistance.Therefore,it is recommended that we should not only strengthen the breeding management and raise the awareness of bio-safety in the process of breeding,but also use antibacterial drugs reasonably according to the results of the drug sensitivity test.