腹泻标本中肺炎克雷伯菌的分离鉴定和16S rDNA系统进化分析 ; Isolation, identification and 16S rDNA phylogenetic analysis of Klebsiella pneumonia from diarrhea specimens

目的 探讨腹泻标本中肺炎克雷伯菌属亚种的分类和鉴定.方法 腹泻标本使用麦康凯和SS培养基划线培养,挑取可疑菌落分纯后使用细菌生化鉴定仪鉴定到亚种;提取菌株的全基因组DNA,以克雷伯菌16S rDNA引物PCR扩增菌株的1500 bp 16S rDNA序列,扩增产物进行测序,测序结果 经截取后,使用Blastn程序在GenBank库中搜索16S rDNA序列的相似性匹配;用PHYLIP程序建立分离菌株及GenBank库中16S rDNA序列的进化发生树并进行分析.结果 113份腹泻标本中,经生化鉴定有25株(分离率22.2%)肺炎克雷伯菌株,其中,肺炎亚种21株(分离率18.6%)、鼻炎亚种4株,未分离到鼻硬结哑种.这些标本中未分离到常见腹泻病原菌.生化鉴定的肺炎亚种菌株均对青霉素G耐药,而对多黏菌素敏感,对呋喃坦叮、头孢噻吩、卡那霉素和妥布霉素有部分耐药.菌株的16S rDNA序列在GenBank库中搜索发现,生化鉴定为鼻炎哑种的4株菌株与沙门菌等肠道致病菌株有最大相似性匹配;16S rDNA序列系统进化树将生化鉴定的肺炎亚种聚为一群,但不能将肺炎克雷伯菌属的3个亚种完全区分开来.结论 16S rDNA序列进化分析在肺炎克雷伯菌的鉴定和分类中有一定意义. ; 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.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.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 ...