林麝源蜡样芽孢杆菌SCBCM001株的分离鉴定及全基因组序列分析

Isolation, identification, and whole genome sequencing of Bacillus cereus SCBCM001 from forest musk deer (Moschus berezovskii)

摘要:

【背景】细菌性疾病是林麝规模化养殖的重要制约因素，蜡样芽孢杆菌曾在林麝化脓灶中检出，但是目前对林麝源蜡样芽孢杆菌的研究报道很少。【目的】对分离自病死林麝肝脏中的一株疑似蜡样芽孢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，对病原菌进行生化试验、药敏试验和小鼠致病性试验；并通过第3代单分子测序技术进行全基因组测序，根据测序结果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】该病原菌经平均核苷酸相似度分类和系统发育树分析属于蜡样芽孢杆菌群，生化结果符合蜡样芽孢杆菌的一般特征，将分离菌株命名为SCBCM001。该菌株对小鼠的半数致死量为8.3×10⁷ CFU，对大多数β-内酰胺类、四环素和磺胺异噁唑耐药，对氨基糖苷类、头孢氨苄、头孢哌酮和亚胺培南等药物敏感；全基因组测序结果表明该菌株的染色体大小为5 292 570 bp，GC含量为35.37%，多位点序列分型显示该菌株属于ST427序列类型。在菌株SCBCM001基因组内发现hblA、hblC、hblD、nheA、nheB、clo和cytK等多种毒力因子，同时菌株携带对β-内酰胺类、万古霉素和四环素等多种药物的耐药基因，与耐药表型不完全相符。菌株SCBCM001基因组还包含6个质粒，其中一个质粒上存在一个较完整的噬菌体区域。【结论】该研究分离了林麝源蜡样芽孢杆菌并对其进行了全基因组测序及分析，为林麝细菌性疾病防治和治疗提供了参考依据。

关键词:林麝;蜡样芽孢杆菌;致病性;全基因组序列分析

Abstract:

[Background] Bacterial diseases are major factors limiting the large-scale cultivation of forest musk deer. Bacillus cereus has been detected in abscesses of forest musk deer. However, there are few studies about the B. cereus from forest musk deer. [Objective] We isolated, identified, and sequenced the whole genome of a suspected B. cereus strain from the liver of a dead forest musk deer, aiming to provide a foundation for the prevention and treatment of diseases cause by B. cereus in forest musk deer. [Methods] After strain purification, we conducted biochemical tests, drug sensitivity tests, and mouse pathogenicity tests on the pathogen. We then employed the third-generation sequencing to reveal its whole genome and phylogenetic relationship. Furthermore, gene function annotation and genetic evolution analysis were conducted. [Results] The average nucleotide identity (ANI) and phylogenetic tree suggested that the pathogen belonged to the B. cereus group, and the biochemical test results of the strain conformed to the general characteristics of B. cereus. The isolate was thus named SCBCM001. The strain showed the median lethal dose of 8.3×10⁷ CFU for mice. It was resistant to most β-lactams, tetracycline, and sulfamethoxazole and sensitive to cephalexin, cefoperazone, imipenem, and aminoglycosides. The chromosome size of the strain was 5 292 570 bp, with the GC content of 35.37%. Multilocus sequence typing showed that SCBCM001 was a strain of ST427. The genome of SCBCM001 carried a variety of virulence genes such as hblA, hblC,hblD, nheA, nheB, clo, and cytK and the genes conferring the resistance to antibiotics such as β-lactams, vancomycin, and tetracycline, which were not completely consistent with the drug-resistant phenotype. In addition, the genome of strain SCBCM001 contained 6 plasmids, one of which had a relatively complete phage region. [Conclusion] The whole genome of a B. cereus strain isolated from forest musk deer was sequenced and analyzed, which provided a basis for the prevention and treatment of bacterial diseases in forest musk deer.

Key words:forest musk deer;Bacillus cereus;pathogenicity;whole genome sequencing