Zhou Juan, Xu Mingchao, Guo Wentao, Yang Jing, Pu Ji, Lai Xin-He, Jin Dong, Lu Shan, Zhang Sihui, Huang Yuyuan, Zhu Wentao, Huang Ying, Zheng Han, Xu Jianguo
State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, PR China.
Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University, Nanjing 211166, Jiangsu Province, PR China.
Int J Syst Evol Microbiol. 2021 May;71(5). doi: 10.1099/ijsem.0.004803.
Four Gram-stain-positive, non-motile and asporous bacilli (strains ZJ-599, ZJ-621, MC1420 and MC1482), isolated from animal tissue and environmental samples collected on the Qinghai-Tibet Plateau, PR China, were taxonomically characterized. Based on the results of 16S rRNA gene sequence analyses, the closest relatives of strains ZJ-599 and ZJ-621 were LMM-1653 (97.5 %), M408/89/1 (96.5 %) and OJ8 (96.3 %), whereas strains MC1420 and MC1482 were closest to CCUG 58655 (98.9 %), DSM 20632 (98.4 %) and DSM 44291 (97.9 %). The results of gene sequence similarity analysis indicated that M408/89/1 and CCUG 58655 were closest to strains ZJ-599/ZJ-621 (83.5 %) and MC1420/MC1482 (91.8 %), respectively. The two novel type strains shared a similarity of 95.2 % in 16S rRNA and 81.3 % in gene sequences. The TAP-PCR DNA fingerprint and MALDI-TOF MS spectrum patterns clearly differentiated the novel isolates within and between each pair of strains. Strain ZJ-599 had 21.9-22.4 % digital DNA-DNA hybridization (dDDH) scores with LMM-1653, M408/89/1 and OJ8, and 72.3-72.9 % of average nucleotide identity (ANI) with them. Similarly, strain MC1420 had 22.9-23.7 % dDDH values with CCUG 58655, DSM 20632 and DSM 44291, and 80.4-81.3 % ANI scores with them. Strain ZJ-599 had a 23.1 % dDDH value and 70.5 % ANI score with strain MC1420, both below the corresponding thresholds for species delineation. Strains ZJ-599 and MC1420 both contain mycolic acids and have MK-8(H) and MK-9(H) as the predominant respiratory quinones, -diaminopimelic acid as the diagnostic diamino acid, and C 9 as the main fatty acid. C 8 and C 8 were predominant in strain ZJ-599 in contrast to C 7 being predominant in strain MC1420. The main polar lipids in strain ZJ-599 were diphosphatidylglycerol, phosphatidylinositol and one unidentified glycolipid, while strain MC1420 had diphosphatidylglycerol, phosphatidylglycerol and one unidentified lipid as the major components. Since the two pairs of novel strains (ZJ-599/ZJ-621, MC1420/MC1482) distinctly differ from each other and from their nearest relatives, two novel species of the genus are proposed, namely (type strain ZJ-599=GDMCC 1.1779=JCM 34341) and (type strain MC1420=GDMCC 1.1783=JCM 34340), respectively.
从中国青藏高原采集的动物组织和环境样本中分离出4株革兰氏阳性、无运动性且无芽孢的杆菌（菌株ZJ-599、ZJ-621、MC1420和MC1482），并对其进行了分类学特征分析。基于16S rRNA基因序列分析结果，菌株ZJ-599和ZJ-621的最接近亲缘菌株分别是LMM-1653（相似度97.5%）、M408/89/1（相似度96.5%）和OJ8（相似度96.3%），而菌株MC1420和MC1482最接近CCUG 58655（相似度98.9%）、DSM 20632（相似度98.4%）和DSM 44291（相似度97.9%）。基因序列相似性分析结果表明，M408/89/1和CCUG 58655分别与菌株ZJ-599/ZJ-621（相似度83.5%）和MC1420/MC1482（相似度91.8%）最为接近。这两株新型菌株在16S rRNA基因序列上的相似度为95.2%，在其他基因序列上的相似度为81.3%。TAP-PCR DNA指纹图谱和MALDI-TOF MS质谱图谱能够清晰地区分每对菌株内部和之间的新型分离株。菌株ZJ-599与LMM-1653、M408/89/1和OJ8的数字DNA-DNA杂交（dDDH）得分在21.9 - 22.4%之间，平均核苷酸同一性（ANI）得分在72.3 - 72.9%之间。同样，菌株MC1420与CCUG 58655、DSM 20632和DSM 44291的dDDH值在22.9 - 23.7%之间，ANI得分在80.4 - 81.3%之间。菌株ZJ-599与菌株MC1420的dDDH值为23.1%，ANI得分为70.5%，均低于物种划分的相应阈值。菌株ZJ-599和MC1420均含有分枝菌酸，以MK-8(H)和MK-9(H)作为主要呼吸醌，以L-二氨基庚二酸作为诊断性二氨基酸，以C9作为主要脂肪酸。与菌株MC1420中以C7为主不同，菌株ZJ-599中C8和C8'为主。菌株ZJ-599中的主要极性脂为双磷脂酰甘油、磷脂酰肌醇和一种未鉴定的糖脂，而菌株MC1420以双磷脂酰甘油、磷脂酰甘油和一种未鉴定的脂类为主要成分。由于这两对新型菌株（ZJ-599/ZJ-621、MC1420/MC1482）彼此之间以及与它们最接近的亲缘菌株明显不同，因此分别提出该属的两个新物种，即（模式菌株ZJ-599 = GDMCC 1.1779 = JCM 34341）和（模式菌株MC1420 = GDMCC 1.1783 = JCM 34340）。
