多元摩拉菌分离株生物被膜形成能力及其致病和耐药情况研究  

Study on biofilm formation ability,pathogenicity and drug resistance of Moraxella pluranimalium

作　　者：曾君 赵瑞利 孙英峰 于恩远 荣睿璠 李颖[2] 李留安 张东超 王心如 ZENG Jun;ZHAO Ruili;SUN Yingfeng;YU Enyuan;RONG Ruifan;LI Ying;LI Liuan;ZHANG Dongchao;WANG Xinru(College of Animal Science and Veterinary Medicine,Tianjin Agricultural University/Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Breeding,Tianjin 300392,China;Tianjin Animal Disease Prevention and Control Center,Tianjin 300402,China)

机构地区：[1]天津农学院动物科学与动物医学学院/天津市农业动物繁育与健康养殖重点实验室,天津300392 [2]天津市动物疫病预防控制中心,天津300402

出　　处：《黑龙江畜牧兽医》2023年第11期8-14,136,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：天津市应用基础与前沿技术研究计划一般项目(18JCYBJC30100,14JCYBJC30000);天津市高校“中青年骨干创新人才”培养计划项目;天津市“131”创新型人才培养计划项目;天津市奶牛(肉羊)、生猪产业技术体系创新团队建设项目(ITTCRS2021000);天津市高等学校大学生创新创业训练计划项目(202110061017)。

摘　　要：为了研究分离的猪多元摩拉菌生物被膜形成能力及相关特性,了解其致病性和耐药情况,试验采集天津市某猪场因患猪繁殖与呼吸道综合征并继发细菌感染而导致死亡的猪的心脏、肺脏进行细菌分离培养、生化试验、16S rDNA基因序列测定,并对分离菌进行药敏试验、生物被膜及相关基因检测、PK-15细胞黏附试验、致病力研究及毒力基因检测。结果表明:分离菌为革兰氏阴性球菌且多成对排列;氧化酶试验呈阳性,不分解葡萄糖、麦芽糖、蔗糖、蕈糖、木糖,PCR产物大小为868 bp,结合16S rDNA测序结果经BLAST比对鉴定为摩拉菌属成员,与多元摩拉菌(M.pluranimalium)序列的同源性达99.71%,将分离菌命名为M.pluranimalium 1120;M.pluranimalium 1120对氟苯尼考、林可霉素、阿莫西林、恩诺沙星、左氧氟沙星耐药,对头孢噻肟中度敏感,对头孢曲松敏感;在培养至第60小时时生物被膜形成量达到峰值,并检测到生物被膜基因LuxS、LpxM、CRP、nanH、SiaB;对PK-15细胞的平均黏附率为13.3%,黏附能力较弱;对小鼠致病性弱,腹腔注射1×10^(11)cfu/mL菌液0.1 mL的小鼠仅表现饮食欲减退且感染2 d后情况好转,剖检可见肺脏水肿、充血,肝脏、脾脏、肾脏、胃、十二指肠和肠黏膜不同程度淤血、水肿;未检测到毒力基因UspAI。说明M.pluranimalium 1120对多种常见抗生素耐药,生物被膜形成能力强,对PK-15细胞的黏附能力弱,对小鼠致病性低。The purpose of this study was to study the biofilm formation ability and related characteristics of Moraxella pluranimalium,and to understand its pathogenicity and drug resistance.The hearts and lungs of dead pigs suffered from porcine reproductive and respiratory syndrome and secondary bacterial infection were collected from a pig farm in Tianjin.Bacterial isolation and culture,biochemical tests,16S rDNA sequence determination,drug susceptibility test,biofilm and related genes detection,PK-15 cells adhesion test for pathogenicity study and virulence gene detection were conducted.The results showed that the isolates were Gram-negative cocci and most of them were arranged in pairs.The oxidase test was positive and did not decompose glucose,maltose,sucrose,mushroom and xylose.The size of PCR product was 868 bp,and was identified as Moraxella by 16S rDNA BLAST analysis.The sequence homology with Moraxella pluranimalium reached 99.71%.The isolate was named M.pluranimalium 1120.Moraxella pluranimalium 1120 was resistant to penicillin,florfenicol,lincorin,amoxicillin,enrofloxacin,levofloxacin,and moderately sensitive to cefotaxime and sensitive to ceftriaxone.Biofilm formation reached its peak at 60 h of culture,and biofilm genes LuxS,LpxM,CRP,nanH and SiaB were detected.The average adhesion rate to PK-15 cells was 13.3%,indicating that the adhesion ability was weak.The pathogenicity to mice was weak.Mice injected with 0.1 mL of 1×10^(11) cfu/mL bacterial solution intraperitoneally only showed decreased appetite for drinking which was improved after 2 days of infection.Necropsy showed pulmonary edema and congestion,and different degrees of congestion and edema in liver,spleen,kidney,fundus of stomach,duodenum and intestinal mucosa.No virulence gene UspAI was detected.These results indicated that M.pluranimalium 1120 was resistant to many common antimicrobials and had strong biofilm formation ability,which may be related to drug resistance,weak adhesion ability to PK-15 cells and low pathogenicity to mice.

关 键 词：猪 多元摩拉菌 分离 鉴定 耐药性 生物被膜 细胞黏附 致病性 

分 类 号：S852.61[农业科学—基础兽医学]