一株林麝肺源创口博德特氏杆菌全基因组及细胞致死性肿胀毒素分析

Whole genome sequence and cytolethal distending toxin analysis of a Bordetella trematum from the lung of a forest musk deer

【目的】为探究林麝的死亡原因,本研究无菌采集1只死亡林麝肺脏后进行细菌的分离鉴定。【方法】通过细菌分离纯化、生化试验和16S rRNA序列分析,对分离菌株进行鉴定;然后通过药敏试验、全基因组测序与分析以及细胞致死性肿胀毒素(cytolethal distendin toxin subunit B,CDTB)分析,对分离菌株耐药性和致病性进行研究。【结果】在死亡林麝肺脏中分离出1株革兰阴性菌,经鉴定为创口博德特氏杆菌,命名为ZL0001。经过细胞培养观察和CDTB分析表明该菌为胞内寄生菌,含有细胞致死性肿胀毒素,能导致细胞凋亡。药敏结果表明该分离株对氨苄西林、亚胺培南、链霉素和四环素等药物敏感;对氨曲南、林可霉素、克林霉素和呋喃妥因耐药。全基因组测序分析结果表明,该菌株与其他创口博德特氏杆菌的平均核苷酸一致性(average nucleotide identity,ANI)值均>97%,基因组大小为4350644 bp,共发现22个耐药相关基因。此外,该菌株基因组包含63个毒力相关基因,其参与鞭毛蛋白、脂多糖、铁摄取、抗血清蛋白以及细胞致死性肿胀毒素等毒力因子的合成。【结论】本研究首次在动物呼吸道中分离出创口博德特氏杆菌,并证明其为胞内寄生菌,对林麝肺炎的发病机理研究具有重要意义。

[Objective]In order to investigate a forest musk deer’s death,we collected the musk deer’s lung tissue to isolate and identify bacteria.[Methods]We identified the strains by bacterial isolation and purification,biochemical test and 16S rRNA sequence analysis.Then,we designed the whole genome and cytolethal distendin toxin subunit B analysis to learn the drug resistance and pathogenicity of the isolated strain.[Results]First,a gram-negative strain was isolated from the lungs of the musk deer,and identified as Bordetella trematum and named as ZL0001.It was an intracellular parasitic bacterium containing cytolethal distendin toxin subunit B.According to drug sensitivity test,the isolate was sensitive to ampicillin but resistant to amtriaxone.The average nucleotide identity values of the strain compared to other Bordetella trematum were>97%,and the genome size was 4350644 bp.In the whole-genome,22 drug-resistant genes were found.Moreover,the genome contains 63 virulence genes that participated in many processes,such as iron intake.[Conclusion]Bordetella trematum was isolated in the respiratory tract of forest musk,and proved as intracellular bacterium.