肺炎克雷伯氏菌强毒株的分离鉴定及16-23SrRNAITS序列分析

为确诊疑似仔猪肺炎克雷伯氏菌(K.pneumonia)感染,并研究其病原的致病性、耐药性、16-23SrRNA ITS系统进化特征,本研究从云南因肺炎、腹泻而大量死亡的仔猪中分离到1株革兰氏阴性短粗杆菌,命名为KP14013,对其进行生化鉴定、16SrRNA鉴定,研究其对小白鼠和仔猪的致病性,并对其16-23SrRNA ITS基因进行测序和遗传进化分析。结果显示,KP14013分离株生化特征与肺炎克雷伯氏菌相符,其16SrRNA与GenBank中23株肺炎克雷伯氏菌代表株之间的同源性均为99%,将KP14013鉴定为肺炎克雷伯氏菌。KP14013对小白鼠半数致死量(LD50)为3×101.8 CFU,腹腔注射3×108 CFU可使仔猪100%致死。16-23SrRNA ITS系统进化关系结果表明,KP14013与GenBank中收录的15株肺炎克雷伯氏菌形成进化树的一个分支,属于同一个亚群,它们之间的核苷酸同源性为98.4%~99.2%。本研究证实了肺炎克雷伯氏菌是该起仔猪腹泻大量死亡的病原;KP14013分离株为毒力极强菌株,具有多重耐药性,其16-23SrRNA ITS与GenBank中收录的肺炎克雷伯氏菌代表株之间核苷酸存在差异,可用于肺炎克雷伯氏菌菌株间的鉴别。

Isolation,Identification and 16-23S rRNA ITS Sequence Analysis of A Strain of Highly Virulent Klebsiella pneumoniae

To confirm a suspected Klebsiella pneumonia (K.pneumoniae) infection,and study the pathogenicity,drug sensitivity and homologies of the 16-23S rRNA ITS genes of the pathogen,one Gram-negative,rod-shaped bacteria named as KP14013 was isolated from livers and gut of the piglets with diarrhea,high-mobility and high-mortality.The isolate was preliminarily identified as K.pneumoniae by biochemical test and further identified as K.pneumoniae by 16S rRNA sequence analysis which shared 99% homology with 23 representative isolates from GenBank.Lethality tests indicated that the 50% lethal doses (LD₅₀) in mice were 3×10^1.8 colony-forming units (CFU) for the isolate and that 100% mortality in piglets by intraperitoneal inoculation with 3×10⁸ CFU.The 16-23S rRNA ITS sequence analysis indicated that KP14013 belonged to one subgroup with 15 represent isolates from GenBank and shared homologies of 98.4% to 99.2%.This study confirmed the K.pneumoniae infection with diarrhea and high mobility and high motality in piglets.One highly virulent K.pneumoniae strain was isolated from liver and gut of piglets.The KP14013 isolate was high virulent to mice and piglets, and showed multiple-drug resistance among common antibiotics.The 16-23S rRNA ITS of KP14013 proved to be a feasible and rapid way for identification of different K.pneumoniae species for its moderate conservatism and variability.