腹泻标本中肺炎克雷伯菌的分离、鉴定及16S rDNA系统发育分析

[Isolation, identification and 16S rDNA phylogenetic analysis of Klebsiella pneumonia from diarrhea specimens].

作者信息

Guo Xiao-lin, Wang Duo-chun, Zhang Yan-min, Wang Xiao-mei, Zhang Yi, Zuo Ying, Zhang Dong-mei, Kan Biao, Wei Lai, Gao Yan

机构信息

Department of Infectious Disease, People's Hospital, Peking University, Beijing 100044, China.

出版信息

Zhonghua Liu Xing Bing Xue Za Zhi. 2008 Dec;29(12):1225-9.

PMID:19173969

Abstract

OBJECTIVE

To understand the biochemical characteristics and 16S rDNA genetic sequence evolution of strains isolated from diarrhea specimens so as to provide basis for classification and identification of Klebsiella pneumoniae.

METHODS

Specimens were cultured using MacConkey and SS medium. All isolates were identified as K. pneumoniae by automated biochemical tests. DNA was extracted, 1500 bp fragments of the 16S rDNA gene were by amplified PCR and sequenced with K. pneumoniae 16S rDNA primer, after being cut. Fragments of 1000 bp overlapping sequences were analyzed by Blast to confirm the identity of the isolates. A phylogenetic tree was constructed by PHYLIP process to analyze the 16S rDNA sequence of the isolated strain with other relative bacteria species in the GenBank databases.

RESULTS

Among 113 specimens of infectious diarrhea, 25 K. pneumoniae strains were identified by biochemical tests, of which 21 subsp. pneumoniae and 4 subsp. ozaenae, no subsp. of rhinoscleroma were isolated. Strains of subsp. pneumoniae were found having nature of resistance. All isolates were resistant to penicillin G and susceptible to polymyxin with some strains were resistant to Nitrofurantoin, Cephalothin, Kanamycin, Tobramycin. After searching in GenBank of 16S rDNA, strains biochemical identified as subsp. ozaenae shared high similarity with Salmonella strains and other intestinal bacteria. 16S rDNA phylogenetic analysis could be used to confirm subsp. pneumoniae, but could not separate other subspecies of K. pneumoniae completely.

CONCLUSION

16S rDNA phylogenetic analysis useful in identifying and classifying K. pneumoniae.

摘要

目的

了解腹泻标本中分离菌株的生化特性及16S rDNA基因序列进化情况，为肺炎克雷伯菌的分类鉴定提供依据。

方法

标本用麦康凯和SS培养基培养。通过自动化生化试验将所有分离株鉴定为肺炎克雷伯菌。提取DNA，用肺炎克雷伯菌16S rDNA引物经PCR扩增16S rDNA基因的1500 bp片段并测序，酶切后对1000 bp重叠序列片段进行Blast分析以确认分离株的身份。用PHYLIP软件构建系统发育树，分析分离菌株与GenBank数据库中其他相关细菌物种的16S rDNA序列。

结果

在113份感染性腹泻标本中，经生化试验鉴定出25株肺炎克雷伯菌，其中肺炎亚种21株、臭鼻亚种4株，未分离到鼻硬结亚种。肺炎亚种菌株具有耐药性。所有分离株对青霉素G耐药，对多粘菌素敏感，部分菌株对呋喃妥因、头孢噻吩、卡那霉素、妥布霉素耐药。在GenBank中检索16S rDNA后发现，生化鉴定为臭鼻亚种的菌株与沙门菌属菌株及其他肠道细菌有高度相似性。16S rDNA系统发育分析可用于确认肺炎亚种，但不能完全区分肺炎克雷伯菌的其他亚种。