一株肉牛源致病肺炎克雷伯菌的分离鉴定

Isolation and Identification of a Pathogenic Klebsiella pneumoniae Strain from Beef Cattle

【目的】确定导致某牛场牛呼吸系统疾病并引起急性死亡的病原。【方法】采集病牛鼻液样品并从中分离出1株优势菌株,进而对分离菌株进行形态鉴定、生化鉴定、16S rRNA鉴定、小鼠接种试验以及耐药性分析。【结果】分离菌镜检为革兰氏阴性球杆菌;除精氨酸双水解试验呈阳性外,其他生化特性均与肺炎克雷伯菌相符合;其16S rRNA序列与GenBank中肺炎克雷伯菌株之间的同源性均达99%。因此,可将分离菌确定为肺炎克雷伯菌,并将其命名为7Y-1。在小鼠接种试验中,用1.01×108 CFU/mL菌液腹腔注射8只SPF级昆明鼠(0.2 mL/只),16~36 h内小鼠全部(8/8)死亡。剖检见肺、肝、脾和肾等多个组织器官充血肿大,肠道积气积液、部分肠段充血、肿胀;组织学观察可见肺、肝、脾淤血、出血、肾小球肾炎变化;药敏试验结果显示:分离菌株对人医常用碳青霉烯类的亚胺培兰、美罗培兰、氨基糖苷类的阿米卡星以及磺胺类的磺胺异噁唑等抗生素耐药,对兽医临床常用的头孢曲松、头孢西丁、头孢唑林、卡那霉素、庆大霉素和氟苯尼考等抗生素高度敏感。【结论】本试验从病牛分离到对小鼠强致死性的肺炎克雷伯菌株具有特殊耐药性,表明该菌可能为引起该肉牛呼吸道感染发病,甚至引起死亡的主要病菌或继发性感染菌,应引起高度重视。

[Purpose]To determine the pathogen causing respiratory diseases and acute death of cattle in a cattle farm.[Method]Samples of sick cows nasal fluid were collected and a dominant strain was isolated.The isolated strain was further identified by morphological identification,biochemical identification,16S rRNA identification,mice inoculation test and drug resistance analysis.[Result]The isolated bacteria were gram negative coccobacteria under microscopic examination.Except that arginine double hydrolysis test showed positive,other biochemical characteristics were consistent with Klebsiella pneumoniae.The homology between 16S rRNA sequence and K.pneumoniae strain in GenBank is 99%.Therefore,the isolated bacteria can be identified as K.pneumoniae and named 7Y-1.In the mice inoculation test,eight SPF-KM mice(0.2 mL/mouse)were intraperitoneally injected with 1.01×108 CFU/mL bacterial solution,the mice all(8/8)died within 16-36 h.Section examination of lung,liver,spleen,kidney and other tissues and organs congestion swelling,intestinal gas and fluid,part of intestinal congestion,swelling;histological observation showed lung,liver congestion,spleen congestion hemorrhage and glomerulonephritis changes;the drug sensitivity test results showed that the isolated strains were resistant to carbapenems,imipenem,meropenem,aminoglycoside amikacin and sulfamethoxazole,and were highly sensitive to antibiotics commonly used in veterinary clinic,such as ceftriaxone,cefoxitin,cefazolin,kanamycin,gentamicin and florfenicol.[Conclusion]K.pneumoniae was isolated from the infected cattle and was highly lethal to mice,and had special drug resistance,indicating that the bacteria might be the main bacteria or secondary infection bacteria that caused the respiratory infection of the beef cattle,or even death,which should be paid high attention to.