树鼩源碳青霉烯耐药性铜绿假单胞菌的分离鉴定及生物学特性研究

doi:10.16431/j.cnki.1671-7236.2024.04.039

摘要/Abstract

摘要： 【目的】确定养殖过程中死亡树鼩的致病原因并分析病原的生物学特性、致病性和耐药情况,制定防治方法。【方法】对死亡树鼩进行病理剖检,记录肺脏、肝脏、脾脏、肠道等主要脏器的病变情况,通过平板划线法从主要病变脏器中分离单个菌落,观察菌落形态并进行革兰染色镜检,通过生化试验、药敏试验、致病性试验及16S rRNA基因测序和全基因组测序等手段对分离株进行进一步分析。【结果】从死亡滇西树鼩的脾脏、肠道中分离获得1株碳青霉烯耐药性铜绿假单胞菌,分离菌株在LB琼脂培养基中呈现产绿色水溶色素的灰白色菌落,镜检为革兰阴性杆菌;生化试验进一步证实分离菌为非发酵菌;通过序列进化分析可知,分离菌与GenBank收录的土壤来源铜绿假单胞菌F4相似性高达99%。药敏试验结果显示,分离菌对亚胺培南和美罗培南等碳青霉烯类及左氧氟沙星等喹诺酮类抗菌药均具有一定的耐药性。致病性试验结果显示,该分离株对成年昆明小鼠的半数致死量(LD₅₀)为3.382×10⁸ CFU,对死亡小鼠脏器组织进行病理学观察,可见肺脏、肝脏、脾脏、肠道等的炎性损伤和坏死。对各脏器再次进行细菌分离,基于16S rRNA序列分析发现铜绿假单胞菌可在死亡小鼠的脾脏、肠道、血液中存在,具有较高的致病性。全基因组测序结果证实,分离菌具有碳青霉烯耐药性相关基因,并与铜绿假单胞菌高致病参考菌株具有相似的毒力因子基因。【结论】本研究从病死滇西树鼩体内分离出碳青霉烯耐药性铜绿假单胞菌,为树鼩微生物致病研究和驯化养殖提供了新的理论依据。

关键词: 树鼩; 铜绿假单胞菌; 耐药性

Abstract: 【Objective】 This experiment was aimed to determine the cause of death of Tupaia belangeri during the breeding process,analyze the biological characteristics,pathogenicity and drug resistance of the pathogen,and develop prevention and control methods.【Method】 Pathological dissection was performed on the dead Tupaia belangeri and the pathological changes of each major organ were recorded,including lung,liver,spleen and intestine.Single colonies were isolated from the main diseased organs using the streak plate method,and the morphology of the colonies was observed.At the same time,Gram staining was used to observe under the microscope,followed by biochemical and drug resistance tests,and pathogenicity test.Further analysis was conducted on the isolated strains using 16S rRNA gene sequencing and bacteria genome sequencing.【Result】 A carbapenem-resistant Pseudomonas aeruginosa strain was isolated from the spleen and intestine of the deceased Tupaia belangeri chinensis.The isolate presented a gray-white colony producing green water-soluble pigments in LB agar medium,and was Gram-negative under microscope.Biochemical tests further confirmed that it was a non-fermenting bacterium.Sequence evolution analysis showed that it had a high similarity of 99% with the soil source Pseudomonas aeruginosa F4 in GenBank.The drug sensitivity test results showed that the isolated strain had certain resistance to carbapenem antibiotics such as imipenem and meropenem,as well as quinolone antibiotics such as levofloxacin.The pathogenicity test result showed that the median lethal dose (LD₅₀) of the isolated strain to adult Kunming mice was 3.382×10⁸ CFU.The organs and tissues of dead mice were isolated for pathological observation,and inflammatory damage and necrosis of the lungs,liver,spleen,intestine,etc.were observed.Bacterial isolation was conducted again on various organs,and based on 16S rRNA sequence analysis,Pseudomonas aeruginosa could be found in the spleen,intestine and blood of dead mice,with high pathogenicity.The whole genome sequencing results confirmed that the isolate strain had genes related to carbapenem resistance and shared similar virulence factor genes with the highly pathogenic reference strain of Pseudomonas aeruginosa.【Conclusion】 This study isolated one carbapenem-resistant Pseudomonas aeruginosa from the deceased Tupaia belangeri,providing a new theoretical basis for the study of microbial pathogenicity and domestication of Tupaia belangeri.

Key words: Tupaia belangeri; Pseudomonas aeruginosa; drug resistance

中图分类号:

S852.61

穆政融, 程晴晴, 张东洁, 翟珊珊, 李竹欣, 陶俊宇, 冷静. 树鼩源碳青霉烯耐药性铜绿假单胞菌的分离鉴定及生物学特性研究[J]. 中国畜牧兽医, 2024, 51(4): 1717-1728.

MU Zhengrong, CHENG Qingqing, ZHANG Dongjie, ZHAI Shanshan, LI Zhuxin, TAO Junyu, LENG Jing. Isolation,Identification and Biological Characteristics of Carbapenem-resistant Pseudomonas aeruginosa from Tupaia belangeri[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1717-1728.

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