朱鹮源弗氏柠檬酸杆菌分离鉴定及基因组特征分析

doi:10.16431/j.cnki.1671-7236.2025.12.044

摘要/Abstract

摘要： 【目的】明确1例野生死亡朱鹮的主要病原菌,并对其进行耐药性、致病性及生物学特性分析，为相关疾病的防治提供参考。【方法】无菌采集湖南崀山自然保护区内死亡朱鹮的肝脏、小肠及胸腔积液样本进行细菌分离培养。通过菌落形态观察、革兰染色及全自动微生物质谱系统进行菌株鉴定；利用定量悬浮法评估不同消毒剂对分离菌的消杀效果；采用微量肉汤稀释法检测分离菌的药物敏感性；通过全基因组测序分析分离菌耐药基因、毒力因子及多位点序列分型(MLST)情况；利用Parsnp软件构建系统发育树，并通过动物试验检测菌株致病力。【结果】试验从死亡朱鹮小肠中分离得到4株细菌。分离菌在血平板上呈乳白色菌落，在LB固体培养基上为半透明、表面有光泽的菌落,革兰染色镜检结果为革兰阴性短杆菌。全自动微生物质谱检测系统鉴定显示，4株菌均为弗氏柠檬酸杆菌，分别命名为ZhLB2、ZhX1、ZhX4和ZhX5。消毒剂消杀试验结果显示，10种受试消毒剂在推荐浓度下对分离菌均具有良好杀菌效果，其中月苄三甲氯铵和浓戊二醛溶液效果最佳。药敏试验结果显示，4株分离菌对复方新诺明、多黏菌素B、头孢西丁及美罗培南均完全耐药(4/4)，对氨苄西林、恩诺沙星和氟苯尼考高度耐药(3/4)；4株分离菌对头孢噻呋和庆大霉素均表现敏感，但仅分离株ZhLB2对四环素表现敏感。全基因组测序分析结果显示，4株分离株共携带bla_CMY、qnrB等16种耐药基因及44种毒力因子；MLST分型显示，分离株ZhX1、ZhX4属于ST580型，ZhLB2、ZhX5为未知分型。系统发育树结果显示，分离株ZhLB2和ZhX5分别与孟加拉国鸡源和斯里兰卡水源弗氏柠檬酸杆菌处于同一分支，ZhX1、ZhX4均与中国水源弗氏柠檬酸杆菌处于同一分支。1×10⁹ CFU/mL菌液感染小鼠，分离株ZhX1、ZhLB2、ZhX4、ZhX5的致死率分别为70%、40%、20%和10%，病理学检查可见特征性肺部出血和小肠炎症。【结论】本研究从朱鹮小肠中分离获得4株弗氏柠檬酸杆菌，其具有多重耐药性，对小鼠具有致病性。本研究结果为珍稀鸟类疾病防控体系的完善提供了重要理论基础，同时为指导临床合理用药提供了参考。

关键词: 朱鹮; 弗氏柠檬酸杆菌; 分离鉴定; 全基因组测序; 致病性

Abstract: 【Objective】 This experiment was conducted to identify the main pathogenic bacteria of a wild dead Nipponia nippon,and analyze its drug resistance,pathogenicity and biological characteristics,so as to provide reference for the prevention and treatment of related diseases. 【Method】 Sterile samples of the liver,small intestine and pleural effusion were collected from the deceased Nipponia nippon in the Langshan Nature Reserve of Hunan province for bacterial isolation and culture.The strains were identified through colony morphology observation,Gram staining,and the automatic microbial mass spectrometry system.The quantitative suspension method was used to evaluate the disinfection effects of different disinfectants on the isolated bacteria.The drug sensitivity of the isolates was detected using the microbroth dilution method.Whole-genome sequencing was used to analyze the drug resistance genes,virulence factors and multilocus sequence typing (MLST) of the isolates.The phylogenetic tree was constructed using the Parsnp software,and its pathogenicity was tested through animal experiments. 【Result】 Four bacterial strains were isolated from the small intestine of the deceased Nipponia nippon during the experiment.The isolated bacteria formed white colonies on the blood agar plate,and were translucent with a shiny surface on the LB solid medium.The results of Gram staining microscopic examination showed that they were Gram-negative short bacilli.The four strains were identified as Citrobacter freundii by automated microbial mass spectrometry system and were named ZhLB2,ZhX1,ZhX4 and ZhX5,respectively.The results of the disinfectant elimination test showed that all 10 tested disinfectants had good bactericidal effects on the isolates at the recommended concentrations,with the best results for benzyltrimethylammonium and concentrated glutaraldehyde.Drug susceptibility test showed that four isolates were completely resistant to compound sulfamethoxazole,polymyxin B,cefoxitin and meropenem (4/4),and highly resistant to ampicillin,enroxacin and florfenicol (3/4).Four isolates were sensitive to cefotaxime and gentamicin,but only the isolate ZhLB2 was sensitive to tetracycline.The results of whole genome sequencing analysis showed that the four isolates carried a total of 16 drug resistance genes,including bla_CMY and qnrB,and 44 virulence factors.MLST typing showed that isolates ZhX1 and ZhX4 belonged to ST580,and ZhLB2 and ZhX5 were of unknown typing.Phylogenetic tree results showed that isolates ZhLB2 and ZhX5 were in the same branch with Citrobacter freundii from Bangladeshi chicken source and Sri Lankan water source,respectively,ZhX1 and ZhX4 were both in the same branch with Citrobacter freundii from Chinese water source.When mice were infected with 1×10⁹ CFU/mL bacterial solution,the mortality rates of the isolates ZhX1,ZhLB2,ZhX4 and ZhX5 were 70%,40%,20% and 10%, respectively.Pathological examination revealed characteristic pulmonary hemorrhage and intestinal inflammation. 【Conclusion】 In this study,four strains of Citrobacter freundii were isolated from the small intestine of Nipponia nippon.The isolates exhibited multi-drug resistance and were pathogenic to mice.The results of this study provided an important theoretical basis for the improvement of the disease prevention and control system for rare birds,and also offered a reference for guiding rational drug use in clinical practice.

Key words: Nipponia nippon; Citrobacter freundii; isolation and identification; whole genome sequencing; pathogenicity

中图分类号:

S852.61

黄培宏, 王可, 刘建勤, 杨嘉豪, 魏佳欣, 李本睿, 何瑜, 陈小军. 朱鹮源弗氏柠檬酸杆菌分离鉴定及基因组特征分析[J]. 中国畜牧兽医, 2025, 52(12): 6020-6030.

HUANG Peihong, WANG Ke, LIU Jianqin, YANG Jiahao, WEI Jiaxin, LI Benrui, HE Yu, CHEN Xiaojun. Isolation, Identification and Genomic Characterization of Citrobacter freundii from Nipponia nippon[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(12): 6020-6030.

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