广东省猪传染性胸膜肺炎放线杆菌分离鉴定及耐药表型和耐药基因检测与分析

【目的】了解广东省部分规模化猪场引起猪传染性胸膜肺炎(PCP)的病原菌猪传染性胸膜肺炎放线杆菌(APP)的耐药表型和耐药基因携带情况,并分析比较其相关性,为有效防控该病提供理论依据。【方法】将2019-2021年从广东省不同地区规模化猪场采集的疑似猪传染性胸膜肺炎病死猪病料通过病原分离培养、革兰氏染色、生化试验、PCR扩增等方法进行病原菌分离鉴定和测序分析,随后对分离株进行药敏试验并通过PCR检测其耐药基因,确定其耐药表型和耐药基因的携带率,分析比较两者的一致性。【结果】分离菌需在含有血清和NAD的培养板中生长;革兰氏染色结果显示分离菌为红色,可判定为革兰氏阴性细菌,通过生化试验、PCR结果及测序分析确定分离到20株APP,且没有表现出明显的地域特性。所有菌株均呈现多重耐药且约50%菌株呈8重及以上耐药;其中对四环素类、磺胺类、氯霉素类和大环内酯类药物耐药率较高,分别为77.5%、65.0%、55.0%和48.8%;进一步分析发现,分离菌主要对四环素、磺胺异噁唑、氟苯尼考和林可霉素等抗菌药物耐药,而对头孢唑啉(先锋Ⅴ)和阿奇霉素敏感。23种主要耐药基因中共检测到bla_CMY、aph(2″)-Ⅰb、sul1、sul2、sul3、tetA、tetB、tetM、tetO、tetR和floR这11种耐药基因,携带率分别为85%、85%、50%、60%、75%、30%、100%、85%、100%、70%和80%;未检测到喹诺酮类、大环内酯类和林可胺类相关耐药基因。【结论】从广东省猪群中分离到的APP表现出广泛耐药性,且为多重耐药,耐药基因与耐药表型基本一致,表明耐药基因的携带是细菌对抗菌药物产生耐药的主要原因之一,但也可能存在其他未检测的耐药基因,或存在新的耐药机制。

Isolation and Identification of Actinobacillus pleuropneumoniae from Guangdong Province and Detection and Analysis of Drug Resistance Phenotype and Gene

【Objective】 This study was aimed to investigate the drug resistance phenotype and drug resistance gene carrying status of Acinetobacter pleuropneumoniae (APP),a pathogen of porcine contagious pleuropneumia (PCP) in some large-scale pig farms in Guangdong province,and to analyze and compare their correlation,so as to provide theoretical basis for effective prevention and control of PCP.【Method】 The dead pig materials of suspected PCP collected from large-scale pig farms in different regions of Guangdong province from 2019 to 2021 were isolated,identified and sequenced by pathogen isolation and culture,Gram staining,biochemical test,PCR amplification and other methods.Then the drug sensitivity test was carried out on the isolated strains,and their drug resistance genes were detected by PCR to determine their drug resistance phenotype and the carrying rate of drug resistance genes,and the consistency between them was analyzed and compared.【Result】 The isolates needed to be grown in culture plates containing serum and NAD.Gram staining results showed that the isolated bacteria were red,which could be determined as Gram-negative bacteria.Through biochemical test,PCR results and sequencing analysis,20 strains of APP were isolated,and did not show obvious regional characteristics.All strains showed multiple drug resistance,and about 50% of the strains showed 8 or more drug resistance.The drug resistance rates to tetracyclines,sulfonamides,chloramphenicols and macrolides were 77.5%,65.0%,55.0% and 48.8%,respectively.Further analysis showed that the isolated bacteria were mainly resistant to tetracycline,sulfaisoxazole,florfenicol and lincomycin,but sensitive to cefazolin (Pioneer V) and azithromycin.bla_CMY, aph(2″)-Ⅰb,sul1,sul2,sul3,tetA,tetB,tetM,tetO,tetR and floR were detected in 23 main drug resistance genes,and the carrying rates were 85%,85%,50%,60%,75%,30%,100%,85%,100%,70% and 80%,respectively.No quinolones,macrolides and lincomycin related resistance genes were detected.【Conclusion】 APP isolated from pigs in Guangdong province showed extensive drug resistance and multiple drug resistance.The drug resistance gene was basically consistent with the drug resistance phenotype,indicating that the carrying of drug resistance gene was one of the main reasons for bacterial resistance to antibiotics,but there might also be other undetected drug resistance genes or new drug resistance mechanisms.