林麝源支气管败血波氏杆菌FMDBb1株的分离鉴定及全基因组序列分析

Isolation,identification and whole-genome analysis of Bordetella bronchiseptica FMDBb1 from forest musk deer(Moschus berezovskii)

【背景】呼吸系统疾病是圈养麝常见疾病之一,其中部分由支气管败血波氏杆菌引起,但目前关于林麝源支气管败血波氏杆菌的研究甚少。【目的】对分离自患病林麝鼻黏液的一株疑似支气管败血波氏杆菌进行分离鉴定和全基因组序列分析,为林麝相关疾病的防治奠定基础。【方法】将病原菌纯化培养后,通过菌落形态和生化试验初步鉴定病原菌类型,然后进行药敏试验和小鼠致病性试验以观察其耐药和毒力表型,最后对其进行全基因组测序,通过平均核苷酸一致性(Average NucleotideIdentity,ANI)比对在全基因组水平进一步评估物种间的亲缘关系,并进行基因功能注释和遗传进化分析。【结果】该病原菌经ANI分类属于经典波氏杆菌亚种,结合菌落形态和生化结果综合鉴定为支气管败血波氏杆菌,菌株命名为FMDBb1,药敏表型显示其对林可霉素、利福平及大多数β-内酰胺类抗生素耐药,对小鼠的半数致死量为8.55×106 CFU。全基因组测序显示该菌基因组大小为5133936 bp,基因功能注释显示其拥有强代谢能力,基因组内检测到65个经典波氏杆菌毒力因子,以及1个粉霉素和1个利福平的靶向耐药基因,同时发现19个插入序列和1个噬菌体区域的存在。序列类型为ST33,克隆复合体类型为CC6,管家基因联合建树分析显示与分离于韩国短毛猫的KVNON-570株相似性最高。【结论】研究分离了林麝源支气管败血波氏杆菌并对其进行了全基因组测序及分析,为该病原菌感染的防治提供了宝贵的信息。

[Background]Respiratory diseases are common in captive musk deer,some of which are caused by Bordetella bronchiseptica(Bb).However,the research on Bb from forest musk deer(FMD) is insufficient.[Objective]To identify a strain of Bb isolated from the nasal mucus of FMD,analyze its whole-genome sequence and lay a foundation for the prevention and control of related diseases in FMD.[Methods]The pathogen was purified by culture method and preliminarily identified based on colony morphology and biochemical properties.Then,drug sensitivity test and mouse pathogenicity test were carried out to analyze the drug resistance and virulence phenotype.Finally,the whole genome was sequenced.The genetic relationship among species was evaluated based on average nucleotide identity(ANI) at the whole genome level.Meanwhile,gene function annotation and phylogenetic analysis were conducted.[Results]The ANI comparison demonstrated that the pathogen belonged to the classical Bordetella subspecies.According to the colony morphology and biochemical properties,the pathogen was identified as Bb and named FMDBb1.The strain was resistant to lincomycin,rifampicin and most β-lactam antibiotics,with LD50=8.55×106 CFU.The whole genome of the strain was 5 133 936 bp,and gene function annotation showed that it had strong metabolic capacity.In the genome,65 classical Bordetella virulence factors and the resistance genes targeting pulvomycin and rifampicin were identified.Meanwhile,19 insertion sequences and 1 phage region were found as well.The strain showed the sequence type 33 and the clone complex 6.The phylogenetic tree constructed based on the housekeeping genes showed that FMDBb1 had the highest homology with KVNON-570 isolated from Korean shorthair cat.[Conclusion]The whole genome of Bb isolated from FMD was sequenced and analyzed,which provided valuable information for the prevention and control of the pathogen infection.