石河子地区某规模化奶牛场肺炎克雷伯菌的分离鉴定及耐药性分析

doi:10.16431/j.cnki.1671-7236.2021.09.039

摘要/Abstract

摘要： 为了阐明引起石河子地区某规模化奶牛场犊牛呼吸道症状的主要细菌性病原体及其生物学特性，本研究采集2~6月龄犊牛鼻拭子与肛拭子各39份，通过细菌分离培养、形态学观察、生化分析、PCR扩增16S rRNA基因和溶血酵素（khe）基因、药敏试验和小鼠致病性试验等方法对分离菌株的生物学特性进行分析。结果显示，分离菌株中有3株在MIAC平板上形成紫红色带有沉淀环的菌落（鼻拭子1株，肛拭子2株），且镜检为革兰氏阴性短杆菌，疑为肺炎克雷伯菌。全自动微生物分析系统显示，分离株与肺炎克雷伯菌相似性均为96%；PCR扩增16S rRNA基因测序结果显示，分离株与GenBank数据库中肺炎克雷伯菌核苷酸相似性在96.5%~99.8%之间，肺炎克雷伯菌特异性基因khe阳性且相似性达99%以上；药敏结果显示，分离菌株对青霉素类、头孢菌素类、一代和二代氨基糖苷类、四环素类、一代大环内酯类、磺胺类、多烯类、林可酰胺类抗菌药呈现出不同程度的耐药；对三代氨基糖苷类、二代大环内酯类、氯霉素类、多肽类、喹诺酮类抗菌药敏感，且呈现不同程度的多重耐药性；致病性试验结果表明，分离菌株均可不同程度导致小鼠死亡且以鼻拭子分离株致病性较强。本研究成功分离鉴定石河子地区规模化奶牛场引起犊牛呼吸道症状的肺炎克雷伯菌3株，并阐明分离菌的部分生物学特性，为新疆地区牛源肺炎克雷伯菌病的检测、诊断及临床治疗提供技术支撑。

关键词: 肺炎克雷伯菌; 分离; 鉴定; 致病性; 耐药性

Abstract: In order to clarify the main bacterial pathogens causing respiratory symptoms of calves in a large-scale dairy farm in Shihezi area and their biological characteristics, 39 nasal swabs and 39 anal swabs of 2-6 months old calves were collected. The biological characteristics of the isolated strains were analyzed by bacterial isolation and culture, morphological observation, biochemical analysis, PCR amplification of 16S rRNA and khe genes, drug sensitivity test and mouse pathogenicity test. The results showed that 3 of the isolated strains formed purplish red colonies with sedimentation rings on the MIAC plate (1 nasal swab and 2 anal swabs), and were Gram-negative Brevibacterium and suspected to be Klebsiella pneumoniae by microscopic examination. The automatic microbiological analysis system showed that the similarity between isolates and Klebsiella pneumoniae was 96%. PCR amplified 16S rRNA sequencing results showed that the nucleotide homology between the isolates and Klebsiella pneumoniae in the GenBank database was between 96.5% and 99.8%, and the specific gene khe of Klebsiella pneumoniae was positive and the similarity was over 99%. The results of drug sensitivity showed that the isolated strains were resistant to penicillins, cephalosporins, first-generation and second-generation aminoglycosides, tetracyclines, first-generation macrolides, sulfonamides, polyenes and lincamides. They were sensitive to the third generation of aminoglycosides, the second generation of macrolides, chloramphenicols, polypeptides and quinolones, and showed different degrees of multi-drug resistance. The pathogenicity test showed that the isolated strains could lead to the death of mice in varying degrees, and the nasal swab isolates were highly pathogenic. This study successfully isolated and identified three strains of Klebsiella pneumoniae causing respiratory symptoms of calves in large-scale dairy farms in Shihezi area, clarified some biological characteristics of the isolated bacteria, and provided technical support for detection, diagnosis and clinical treatment of bovine Klebsiella pneumoniae in Xinjiang.

Key words: Klebsiella pneumoniae; isolation; identification; pathogenicity; drug resistance

中图分类号:

S852.61

王哲红, 吴桐忠, 赵玉宾, 张星星, 韩猛立, 钟发刚, 胡建军, 黄新. 石河子地区某规模化奶牛场肺炎克雷伯菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2021, 48(9): 3464-3472.

WANG Zhehong, WU Tongzhong, ZHAO Yubin, ZHANG Xingxing, HAN Mengli, ZHONG Fagang, HU Jianjun, HUANG Xin. Isolation,Identification and Drug Resistance Analysis of Klebsiella pneumoniae in a Large-scale Dairy Farm in Shihezi Area[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3464-3472.

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