一株脓液分离的海岸嗜半胱氨酸菌的鉴定及毒力基因分析

Identification and virulence gene analysis of a Cysteiniphilum litorale strain isolated from pus

目的:通过对1株从1名被海虾刺伤的患者脓液分离的致病菌株JM-1进行准确鉴定、药敏试验和毒力基因分析,为海岸嗜半胱氨酸菌( Cysteiniphilum litorale)作为罕见病原体引起临床相关感染的筛检及改善预后提供实验依据。 方法:对分离株进行生化表型鉴定、基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)鉴定、16S rRNA基因测序和系统发育分析,以及基于全基因组序列的平均核苷酸一致性(average nucleotide identity,ANI)、平均氨基酸一致性(average amino acid identity,AAI)分析和系统发育分析,以准确确定其分类学地位;以 E-test法进行药敏试验,并依据CLSI M45-A3土拉弗朗西斯菌解释标准进行结果判读;以VFDB毒力因子数据库对全基因组进行毒力基因注释和分析。 结果:分离株JM-1在哥伦比亚血平板上培养3 d可见灰白色、中等大小、光滑、圆形、凸起的溶血菌落。革兰染色为着色较淡的阴性球杆菌。API NH鉴定结果提示为卡他莫拉菌(生化编码:3010);Vitek2系统NH卡鉴定无鉴定结果(生物编码:0211002121)。EXS3000质谱仪自建库数据库鉴定为海岸嗜半胱氨酸菌。基于16S rRNA基因和全基因组的系统发育分析均表明该菌株与海岸嗜半胱氨酸菌DSM 101832 T聚类为同一个小的分支,且两者之间的全基因组ANI和AAI值分别为95.07%和95.65%。药敏试验显示分离株JM-1产β-内酰胺酶和青霉素酶,对青霉素耐药,对庆大霉素、链霉素、强力霉素、四环素、环丙沙星、左氧氟沙星、氯霉素敏感。从分离株JM-1基因组注释到了与弗朗西斯菌相似的毒力基因:弗朗西斯菌致病岛(FPI)、Ⅳ型菌毛、荚膜和脂多糖等。 结论:海岸嗜半胱氨酸菌是一种具有弗朗西斯菌毒力相关基因罕见病原体,且其药敏特性亦与弗朗西斯菌相类似。本案例确认了一例由海岸嗜半胱氨酸菌引起的临床感染,自建库的MALDI-TOF质谱系统可用于其快速鉴定。

Objective To identify a pathogenic strain JM-1 isolated from the pus of a patient stabbed by a sea shrimp and to analyze its antibiotic susceptibility and virulence genes,aiming to provide reference for screening clinically related infections caused by Cysteiniphilum litorale as a rare pathogen and improving prognosis.Methods Biochemical phenotype identification,matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS),16S rRNA gene sequencing,analysis of average nucleotide identity(ANI)and average amino acid identity(AAI)based on the whole genome and phylogenetic analysis of the 16S rRNA gene and the whole genome were performed to accurately determine the taxonomic status of the strain JM-1.E-test was used to detect antibiotic susceptibility,and the results were interpreted according to the interpretation standards of Francisella tularensis in CLSI M45-A3.The virulence factor database(VFDB)was used for genome-wide annotation and analysis of virulence genes.Results After culturing the strain JM-1 on the Columbia blood plate for 3 d,some grey-white,medium-sized,smooth,round and convex hemolytic colonies were observed.Gram staining result showed lightly colored Gram-negative Coccobacillus.API NH identification results suggested that the isolate JM-1 was Moraxella catarrhalis(biochemical code:3010),while there was no identification result in Vitek2 system NH card(biochemical code:0211002121).The EXS3000 mass spectrometer self-built database identified the isolate JM-1 as Cysteiniphilum litorale.The phylogenetic analysis based on the 16S rRNA gene and the whole genome showed that the isolate JM-1 and Cysteiniphilum litorale DSM 101832T clustered into the same branch,and the ANI and AAI values between the two strains were 95.07%and 95.65%,respectively.The biochemical phenotype identification indicated the isolate JM-1 producingβ-lactamase and penicillinase.Antibiotic susceptibility test results showed the strain was resistant to penicillin and sensitive to gentamicin,streptomycin,doxycycline,tetracycline,ciprofloxacin,levofloxacin,and chloramphenicol.Genome annotation suggested the virulence genes of the isolate JM-1 were similar to those of Francisella,including Francisella pathogenicity island(FPI),typeⅣfimbriae,capsule and lipopolysaccharide.Conclusions Cysteiniphilum litorale was a rare pathogen with virulence genes similar to those of Francisella,and its antibiotic susceptibility was also similar to that of Francisella.This study confirmed a case of clinical infection caused by Cysteiniphilum litorale.The self-built MALDI-TOF MS system could be used for its rapid identification.