Cardiobacterium valvarum临床分离株的生物学特性研究及16S rRNA鉴定

目的 采用不同的方-法描述Cardiobacterium valvarum(C. valvarum)临床分离株的生物学特性,利用16S rRNA基因测序技术进行分子鉴定。 方-法 转种阳性血培养标本到血琼脂平板上进行细菌培育,革兰染色涂片镜检,用VITEK 2 Compact全自动微生物鉴定分析仪对临床分离株进行细菌鉴定,基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)对分离株的蛋白质进行高通量测定,E-test法对分离株作药敏试验。提取分离株的DNA,采用16S rRNA基因测序技术对PCR的产物进行测序,在NCBI的BLAST网站上与GenBank数据库上的序列做相似性比较,用MEGA7.0.26软件构建该分离株的系统进化树。结果 经细菌培养发现小而圆、光滑、不透明,灰色的菌落;经革兰染色后镜下见到小、两端圆形、革兰阴性的多形性杆菌;VITEK 2 Compact上机、MALDI-TOF-MS技术均未得到该分离株的鉴定结-果;16S rRNA基因测序技术测得该菌株基因全序列约为1 450 bp,与C. valvarum F0432的16S rRNA同源性为99.59%,鉴定为C. valvarum。结论该菌的形态、生化反应均无代表性,采用16S rRNA基因测序技术可以对C. valvarum进行鉴定,对该疾病的诊断具有重要意义。

Biological characteristics and 16S rRNA identification of clinical isolates of Cardiobacterium valvarum

In this study, multiple methods were used to describe the biological properties of Cardiobacterium valvarum clinical isolates, and molecular identification was performed through 16S rRNA gene sequencing. Positive blood culture specimens were transferred to blood agar plates for bacterial cultivation and gram staining smear microscopy. A VITEK 2 Compact analyzer was used for automatic bacterial identification in clinical isolates, MALDI-TOF-MS was used for high-throughput determination of the protein of isolates, and E-tests were used to assess the drug sensitivity of isolated bacteria. The DNA of the isolated strain was extracted, and the PCR products were subjected to 16S rRNA gene sequencing. The sequences were compared with those in the GenBank database through NCBI's BLAST website, and a phylogenetic tree of the isolated strain was constructed in MEGA7.0.26 software. After culture, small, round, smooth, opaque and gray colonies were observed. Gram-negative pleomorphic bacilli with small ends were observed under a microscope. VITEK 2 Compact and MALDI-TOF-MS analyses did not identify the bacteria. Through 16S rRNA gene sequencing, the entire gene sequence of this strain was found to comprise approximately 1 450 bp, and the sequence identity with 16S rRNA of C. valvarum F0432 was 99.59%, thus resulting in the identification C. valvarum. We concluded that the morphological and biochemical reactions of this strain were not representative, and 16S rRNA gene sequencing technology can be used to identify C. valvarum—a capability essential for C. valvarum diagnosis.