小熊猫源铜绿假单胞菌耐药性和耐药基因的研究

Study on Resistance and Resistance Gene of Pseudomonas aeruginosa from Red Panda

为了鉴定小熊猫(Ailurus fulgens)肺源致病菌并研究其耐药性和耐药基因,从死亡小熊猫肺部组织中无菌分离培养致病菌,进行革兰氏染色及16S rDNA测序鉴定。采用纸片扩散法(K-B法)进行细菌药敏试验和小鼠致病性试验,应用PCR法检测分离株的28种β-内酰胺酶相关基因、6种氨基糖苷类修饰酶基因(AMEs)、外膜蛋白D2基因(oprD2)、磺胺耐药基因(qacE△1-sul1)、3种整合子基因(intⅠ1、2、3)和主动外排泵调节基因(mexR)等共40种耐药基因。肺组织样品中分离培养的1株菌鉴定为铜绿假单胞菌,革兰氏染色阴性,药敏试验对氯霉素敏感,对β-内酰胺类、头孢类、氨基糖甙类、大环内酯类、硝基咪唑类、喹诺酮类和利福霉素等均具有耐药性,小鼠表现与小熊猫相似的症状而死亡。5种耐药基因TEM、aac(3)-Ⅱ、ant(3″)-Ⅰ、qacE△1-sul1、intⅠ1为阳性,其他35种为阴性。该小熊猫肺源铜绿假单胞菌具有较强的致病性和多药耐药性,其耐药机制与5种耐药基因有关。

We identified the pulmonary pathogens of red panda(Ailurus fulgens)and investigated their drug resistance and resistant-related genes.Bacterial pathogens were isolated and cultured from the lungs of dead red pandas for gram staining and 16S rDNA sequencing.The K-B method and pathogenicity tests in mice were used to test the drug sensitivity of bacteria.Forty drug resistant-related genes including 28β-lactamase related genes,6 aminoglycoside-modifying enzyme genes(AMEs),protein oprD2 gene,sulfanilamide resistance gene(qacE△1-sul1),3 integron genes(intⅠ1,2,3)and one active efflux pump regulatory gene(mexR)were tested by PCR,and verified by DNA sequencing.One strain isolated and cultured from lung tissue samples was identified as Pseudomonas aeruginosa.This strain tested gram-negative,sensitive to chloramphenicol,and resistant toβ-lactams,cefazoles,aminoglycosides,macrolides,nitroimidazoles,quinolones and rifamycin.Mice showed symptoms similar to the red panda and died.Five resistant-related genes TEM,aac(3)-Ⅱ,ant(3″)-Ⅰ,qacE△1-sul1 and intⅠ1 tested positive,the remaining 35 genes tested negative.The strain of P.aeruginosa isolated from red panda lungs showed strong pathogenicity and multi-drug resistance.Its multi-resistant mechanism was mainly related to five resistant-related genes.