藏羊源产气荚膜梭菌的分离鉴定与药物敏感性分析

doi:10.16431/j.cnki.1671-7236.2025.11.041

摘要/Abstract

摘要： 【目的】对采集自西藏拉萨市曲水县疑似产气荚膜梭菌感染致死病例进行病原检测并进行体外药物敏感性分析。【方法】无菌采集病死藏羊肺脏、肝脏、心脏、脾脏、肾脏、血液、胃内容物、肠内容物等12份病料样品，通过厌氧培养进行病原菌的分离培养和纯化，对分离纯化后的菌株进行革兰染色镜检、生化鉴定、16S rRNA基因PCR鉴定，并进行基因序列测定和遗传进化分析，通过PCR检测分离株的毒素基因携带情况并进行毒素分型，进一步开展分离株的体外药物敏感性研究。【结果】试验从12份藏羊病料样品中分离获得10株疑似菌，分离株在RMC培养基中生长旺盛，在TSC培养基中呈中间黑、边缘白的菌落，在5%脱纤维绵羊血平板上呈现双溶血环，革兰染色为阳性杆菌，初步判定为产气荚模梭菌，命名为QS-1～QS-10。生化鉴定结果显示，分离株乳糖、蔗糖、D-核糖、鼠李糖、葡萄糖、麦芽糖、木糖、甘露醇、山梨醇、明胶、尿素、牛乳发酵试验呈阳性，松三糖、棉子糖、阿拉伯糖、M-R、V-P、甘油、水杨苷、硫化氢、过氧化氢、尿素、吲哚试验结果呈阴性。分离株16S rRNA基因序列与产气荚膜梭菌参考株的相似性为98.30%～99.90%，与产气荚膜梭菌参考株处于同一分支，而与大肠杆菌、沙门菌等处于不同分支。毒素基因检测结果显示，8株分离株仅携带cpa基因，为A型产气荚膜梭菌；2株分离株携带cpa和etx基因，为D型产气荚膜梭菌。分离株均对诺氟沙星、氧氟沙星、环丙沙星、氨苄西林、羧苄西林、哌拉西林表现敏感或中介，对其余15种药物均存在不同程度的耐药性，其中9株存在多重耐药性，5重以上耐药的菌株占80.00%，分离株QS-3耐药数量多达13种。【结论】本研究从猝死藏羊病料中分离出10株产气荚膜梭菌，其中2株为D型、8株为A型，分离株耐药情况严重。研究结果为当地藏羊产气荚膜梭菌病的治疗和预防提供了数据指导。

关键词: 藏羊; 产气荚膜梭菌; 分离鉴定; 耐药性

Abstract: 【Objective】 Pathogen detection was conducted on the samples collected from the suspected death of Clostridium perfringens infection in Qushui county,Lhasa city,Tibet,and in vitro drug sensitivity analysis was performed. 【Method】 Sterilely collected 12 samples of pathological specimens,including lungs,livers,hearts,spleens,kidneys,blood,stomach contents,and intestinal contents from dead sheep.The isolation,cultivation and purification of pathogenic bacteria were carried out through anaerobic culture.The isolated and purified strains were subjected to Gram staining microscopy,biochemical identification,16S rRNA gene PCR identification,and genetic sequence determination and genetic evolution analysis.The toxin genes of the isolates was detected by PCR and toxin typing was carried out,and the drug sensitivity of the isolates in vitro was studied. 【Result】 Ten suspected strains were isolated from 12 Tibetan sheep samples.The isolates grew vigorously in RMC medium,and showed white colonies with black edges in the middle on TSC medium.They showed double hemolytic rings on 5% defibrinated sheep blood plates.Gram staining was positive for bacteria.They were preliminarily determined to be Clostridium perfringens,named QS-1 to QS-10.The biochemical identification results showed that the isolates were positive for lactose,sucrose,D-ribose,rhamnose,glucose,maltose,xylose,mannitol,sorbitol,gelatin,urea,and milk fermentation test,while the results of the tests for sophorose,carobose,arabinose,M-R,V-P,glycerol,salicin,hydrogen sulfide,peroxide,urea,and indole were negative.The similarity of the 16S rRNA gene sequence of the isolates to the reference strain of Clostridium perfringens was 98.30% to 99.90%,and they were in the same branch as the reference strain of Clostridium perfringens,while they were in different branches from Escherichia coli,Salmonella,etc.The toxin gene test results showed that 8 isolates only carried cpa gene and were of the A type of Clostridium perfringens，2 isolates carried both cpa and etx genes and were of the D type of Clostridium perfringens.The isolates were sensitive or intermediate to norfloxacin,ofloxacin,ciprofloxacin,ampicillin,carbenicillin and piperacillin,and had different degrees of drug resistance to the other 15 drugs. Among them,9 isolates exhibited multi-drug resistance,and 80.00% of the isolates had resistance to more than 5 drugs.The isolates QS-3 exhibited resistance to 13 drugs. 【Conclusion】 In this study,10 strains of Clostridium perfringens were isolated from the sudden death of Tibetan sheep,including 2 strains of type D and 8 strains of type A.The drug resistance of the isolates was serious.This results provided data guidance for the treatment and prevention of Clostridium perfringens disease of Tibetan sheep in local area.

Key words: Tibetan sheep; Clostridium perfringens; isolation and identification; drug resistance

中图分类号:

S852.61⁺6.3

王冬经, 曾江勇, 苏中华, 巴桑次仁, 尼玛潘多, 王登云, 马弘财. 藏羊源产气荚膜梭菌的分离鉴定与药物敏感性分析[J]. 中国畜牧兽医, 2025, 52(11): 5465-5475.

WANG Dongjing, ZENG Jiangyong, SU Zhonghua, BASANG Ciren, NIMA Panduo, WANG Dengyun, MA Hongcai. Isolation,Identification and Drug Sensitivity Analysis of Clostridium perfringens from Tibetan Sheep[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5465-5475.

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