一株脓毒血症患者血液分离的罕见螺原体鉴定和生物学特性分析

Identification and characterization of one Spiroplasma species isolated from blood culture of a rare caseof sepsis

目的:对一株自脓毒血症患者血液中分离的罕见螺原体DGKH1进行菌种鉴定和生物学特性分析。方法:对DGKH1进行常规细菌培养、染色与形态学观察、生理生化鉴定、16S rRNA基因测序和系统发育分析,以及基因组测序和基因组相关指数分析,从而确定其分类学地位;参考人型副支原体和解脲脲原体进行药敏试验。结果:分离株DGKH1可以在哥伦比亚血平板、巧克力平板和嗜血巧克力平板上微弱生长,在含有四环素(4 μg/ml)、多西环素(1 μg/ml)、米诺环素(1 μg/ml)、交沙霉素(2 μg/ml)、罗红霉素(1 μg/ml)、克拉霉素(1 μg/ml)和特利霉素(1 μg/ml)的支原体液体培养基中不生长。在商品化的支原体固体培养基上,DGKH1可形成"油煎蛋样菌落",与人型副支原体相类似。采用常规革兰染色,DGKH1不着色;以磷酸盐缓冲液为基质进行透射电镜观察,其菌体呈螺旋形。16S rRNA基因测序显示,分离株与中华绒螯蟹螺原体CCTCC M 207170 T相似性最高(99.85%),但尿素分解试验阳性,与已知螺原体属菌种存在差异。全基因组系统发育分析显示,该菌株与中华绒螯蟹螺原体CCTCC M 207170 T聚类为同一个小的分支,但两者之间的全基因组平均核苷酸一致性(ANI)和数字DNA-DNA杂交(dDDH)值分别为94.14%和56.00%,均低于原核生物的种鉴定阈值。 结论:分离株DGKH1隶属于螺原体属,是一种与中华绒螯蟹螺原体亲缘关系密切的潜在新种,其部分微生物学特性与支原体相类似,但自然宿主来源和流行病学资料还有待于更深入研究。

Objective To identify and characterize one Spiroplasma strain(designated as DGKH1)isolated from the blood of a patient with sepsis.Methods The traditional bacterial culture,staining,morphological observation,physiological and biochemical identification,16S rRNA gene sequencing,phylogenetic analysis,genome sequencing,and the genome-related index analysis were performed to accurately determine the taxonomic status of the strain DGKH1.Antibiotic susceptibility testing was performed using a specific kit for culturing and testing Ureaplasma urealyticum/Metamycoplasma hominis.Results The strain DGKHI could weakly grow on Columbia blood agar,chocolate agar,and Haemophilus chocolate 2 agar.However,it did not grow in liquid culture medium containing tetracycline(4μg/ml),doxycycline(1μg/ml),minocycline(1μg/ml),josamycin(2μg/ml),,roxithromycin(1μg/ml),clarithromycin(1μg/ml),or telithromycin(1μg/ml).DGKH1 resembling Metamycoplasma hominis formed"fried egg-like colonies"on Mycoplasma solid culture medium.DGKHI could not be stained by Gram staining.When observed under transmission electron microscopy(TEM)using phosphate buffer as the matrix,the bacteria were spiral-shaped.Results of 16S rRNA gene sequence alignment showed that DCKH1 was highly similar(99.85%)to Spiroplasma eriocheiris CCTCC M 207170T.However,the urea decomposition test was positive,which was different from all of the known Spiroplasma species.The phylogenetic analysis based on whole genome showed that DGKH1 was clustered in a small branch along with Spiroplasma eriocheiris CCTCC M 207170T.However,the average nucleotide identity(ANI)and digital DNA-DNA hybridization(dDDH)values between the two strains were 94.14%and 56.00%,respectively,both below the threshold for prokaryotic species identification.Conclusions DGKH1 represented a potential new species of genus Spiroplasma,closely related to Spiroplasma eriocheiris.Some microbiological characteristics of DGKH1 were similar to Mycoplasmas.However,the natural host and epidemiological data of DGKH need to be further studied.