3种方法对1株临床疑难菌的鉴定及结果比较  

Identification of a clinically difficult bacterial strain by three methods and comparison of results

作　　者：许一丹 谢成彬 苏喆 王频佳 XU Yidan;XIE Chengbin;SU Zhe;WANG Pinjia(Department of Clinical Laboratory,School of Medical Laboratory Science,Chengdu Medical College,Chengdu,Sichuan 610500,China;Department of Laboratory Medicine,Sichuan Provincial Maternitv and Child Health Care Hospital,Chengdu,Sichuan 610045,China)

机构地区：[1]成都医学院检验医学院临床微生物学教研室,四川成都610500 [2]四川省妇幼保健院/成都医学院附属妇女儿童医院检验科,四川成都610045

出　　处：《检验医学与临床》2024年第9期1224-1228,1234,共6页Laboratory Medicine and Clinic

基　　金：四川省大学生创新创业训练计划项目(S202013705085);成都医学院研究生创新基金项目(YCX2022-03-17);成都医学院检验医学院自然科学基金(JYZK202206);四川省妇幼保健院菁英人才科研启动项目(ZC6.16)。

摘　　要：目的通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)、16S rRNA序列分析和全基因组测序(WGS)对从临床标本中分离的1株疑似盐单胞菌进行菌种鉴定,比较3种方法的鉴定结果。方法采用MALDI-TOF MS对待测菌进行蛋白质图谱收集,通过比对图谱特征峰实现菌种鉴定;对菌株进行16S rRNA基因测序,将测序结果与数据库比对;待测菌WGS后进行序列比对分析,具体包括使用ANIm和ANIb 2种方法计算平均核苷酸一致性(ANI)以及与基因组分类数据库(GTDB)进行比对;基于全基因组序列中的16S rRNA序列和看家基因序列构建系统进化树,以及基于COG和KEGG功能对基因组进行聚类分析。结果MALDI-TOF MS对待测菌株的鉴定结果是Halomonas hamiltonii;经16S rRNA序列分析,发现待测菌株与3种盐单胞菌(Halomonas hamiltonii、Halomonas stevensii和Halomonas johnsoniae)的相似度均>99%,且相似度差异非常小,因此无法进行准确的菌种鉴定;基于WGS的基因序列比对、系统发育树、聚类分析均显示待测菌与Halomonas stevensii的亲缘关系更为接近。结论3种方法对分离的1株疑难菌的菌种鉴定结果有差异,MALDI-TOF MS的菌种鉴定操作更加方便、快速,非常适用于临床检验工作;基于WGS的细菌鉴定更适合用于遗传特征相似的菌种之间的鉴别。Objective MALDI-TOF MS,16S rRNA sequence analysis,and whole genome sequencing(WGS)were applied to identify a strain of suspected Halomonas isolated from a clinical specimen and to compare the results of the three methods.Methods MALDI-TOF MS was used to collect protein profiles of the bacteria to be tested,and the identification of microorganisms was achieved by comparing the characteristic peaks of the profiles.The 16S rRNA gene sequencing was performed on the bacteria to be tested,and the sequencing results were compared with database.Based on the genomic sequences obtained from WGS,a comparative analysis was performed,including the calculation of Average Nucleotide Identity(ANI)by ANIm and ANIb,and the comparison with the Genome Taxonomy Database(GTDB).Phylogenetic trees were constructed based on 16S rRNA sequences and housekeeping gene sequences from WGS,as well as cluster analysis based on COG and KEGG functions.Results MALDI-TOF MS resulted in the identification of the strain to be tested as Halomonas hamiltonii.The 16S rRNA gene sequence comparison results showed that the similarity between the bacteria to be tested and the three Halomonas species(Halomonas hamiltonii,Halomonas stevensii and Halomonas johnsoniae)were all above 99%,and the difference in their similarity was so small that accurate species identification could not be performed.The gene sequencing comparison based on WGS,phylogenetic tree and cluster analysis showed that the bacterium was more closely related to Halomonas stevensii.Conclusion The results of the three methods on the strain identification of the isolate are different.MALDI-TOF MS is more convenient and fast for species identification,which is very suitable for clinical testing;WGS-based bacterial identification is particularly useful for distinguishing genetically very similar species.

关 键 词：细菌鉴定 基质辅助激光解吸电离飞行时间质谱 16S rRNA序列 全基因组测序 

分 类 号：R446.5[医药卫生—诊断学]