林麝源酪黄肠球菌的分离鉴定及全基因组序列分析

Isolation, identification, and whole genome sequencing of Enterococcus casseliflavus from forest musk deer

摘要:

【背景】 酪黄肠球菌(Enterococcus casseliflavus)为肠球菌属(Enterococcus)中的一种机会性致病菌，其广泛存在于自然界中，但是目前林麝源酪黄肠球菌的相关研究甚少。【目的】 对从病死林麝肝脏中分离出的一株酪黄肠球菌进行分离鉴定和全基因组测序，以期为林麝相关细菌性疾病的预防和治疗奠定基础。【方法】 将病原菌分离并纯化后，对其进行生化试验、药敏试验、16S rRNA基因分析和细菌载量试验，并根据全基因组测序结果进行耐药基因、毒力基因注释及遗传进化分析。【结果】 分离菌株经16S rRNA基因分析和平均核苷酸一致性(average nucleotide identity, ANI)分类后判断其为酪黄肠球菌，生化试验结果与酪黄肠球菌的一般特性相符合，将分离株命名为Dec0527。菌株Dec0527对头孢氨苄、氨曲南、丁胺卡那、妥布霉素等药物耐药，对多西环素、四环素、氯霉素、复方新诺明和部分β-内酰胺类、氨基糖苷类、喹诺酮类药物敏感。细菌载量结果表明菌株Dec0527对肝脏的侵袭力明显高于心脏、肺脏、脾脏和肾脏。该菌株的基因组长度为3 345 060 bp，G+C含量为42.55%。菌株Dec0527携带ebpC、tufA、groEL、cpsA、cap8E、psaA等多种毒力因子，同时还携带对万古霉素、环丙沙星等多种药物耐药的基因，菌株Dec0527对氨基糖苷类、大环内酯类和β-内酰胺类药物存在耐药基因与耐药表型不完全相符的现象。此外，菌株Dec0527基因组还包含2个环状质粒和一个比较完整的噬菌体区域。【结论】 从林麝肝脏分离出一株酪黄肠球菌并对其完成了全基因组测序及分析，为林麝相关疾病的防治提供了参考依据。

Abstract:

[Background] Enterococcus casseliflavus is an opportunistic pathogenic bacterium with wide distribution in the nature, while there are few studies about this bacterium in forest musk deer. [Objective] We isolated, identified, and sequenced a strain of E.casseliflavus from the liver of a death forest musk deer, aiming to lay a foundation for the prevention and treatment of related diseases in forest musk deer. [Methods] Biochemical tests, drug susceptibility tests, 16S rRNA gene analysis, and bacterial load tests were conducted for the isolate. The results of whole genome sequencing were used to annotate gene functions and analyze genetic evolution. [Results] The isolated strain was identified based on 16S rRNA gene analysis and average nucleotide identity as E.casseliflavus, which was consistent with the results of biochemical tests. The strain was named Dec0527. The strain was resistant to cephalexin, aztreonam, amikacin, and tobramycin, and it was sensitive to doxycycline, tetracycline, chloramphenicol, sulfamethoxazole trimethoprim, some β-lactams, aminoglycosides, and quinolones. The bacterial load test results showed that the invasiveness of strain Dec0527 to the liver was significantly higher than that to the heart, lung, spleen, and kidney. The genome length of this strain was 3 345 060 bp, with the G+C content of 42.55%. Strain Dec0527 carried a variety of virulence genes such as ebpC, tufA, groEL, cpsA, cap8E, and psaA, as well as the genes for resistance to vancomycin and ciprofloxacin. The resistance genes of strain Dec0527 against aminoglycosides, macrolides, and β-lactams were not completely consistent with the drug resistance. In addition, the genome of the strain Dec0527 had 2 circular plasmids and a relatively complete phage region. [Conclusion] We isolated a strain of E. casseliflavus from the liver of a dead forest musk deer and sequenced and analyzed the whole genome of this strain, providing a reference for the prevention and control of diseases in forest musk deer.