一株产酸克雷伯氏菌的分离鉴定及其药敏检测

Isolation and Identification of a Nitrogen-fixing Bacteria Klebsiella oxytocaand Screening of Sensitive Antimicrobials

采集崇左市江门林场10年桉树林地土壤并从中分离自生固氮菌,通过常规细菌鉴定、16SrRNA基因可变区（V1～V3区）序列测序及Biolog自动微生物鉴定系统确定其种属,并利用药敏纸片琼脂扩散法（K-B法）测定其耐药性,结果表明：从所采土壤中分离得到1株自生固氮菌KO108,提交到Genbank获得登陆号为KM460926.1,与GenBank中产酸克雷伯氏菌基因序列相似性达到99%以上;Biolog自动微生物鉴定系统相似值分别为0.509和0.610,大于0.5,因此鉴定该菌株为革兰氏阴性产酸克雷伯氏菌Klebsiella oxytoca,属于肠杆菌科Enterbateriaceae克雷伯氏菌属Klebsiella;药敏实验结果显示该菌对呋喃妥因、链霉素、头孢曲松、卡那霉素、氧氟沙星、强力霉素、环丙沙星、氯霉素敏感;对复方新诺明中度敏感;对多粘菌素、青霉素G、四环素、红霉素耐药。本实验所获得菌株可作为基因改造的工程菌,为林业生产中的固氮微生物的改造和利用提供了实验依据。

The aim of this study was to isolate bacterial strains of azotobacter from the soil of Eucalyptus plantations with 10-year-old stands in Dongmen town,Fusui county,Chongzuo city,Guangxi Zhuang Autonomous Region,China,and to identify its genus by conventional methods,sequencing the Variable region（V1～V3）of gene of 16 SrRNA and Biolog Automatic Microbial Identification System.The Variable region sequences of 16 SrRNA gene was cloned by PCR method,KO108 is submitted to NCBI database with the accession number of KM460926.1,and the similarity values of 16 SrRNA sequences between KO108 and Klebsiella oxytoca was above 99%.The similarity is greater than 0.5,which were0.509 and 0.610 by using Biolog automatic microbial identification system respectively.The strain KO108 was indentified as Gram-negative acid-producing Klebsiella oxytoca belonging to the family of Enterbateriaceae genus Klebsiella.In addition,drug resistance of them was also detected by the disc agar diffusion test. The results showed that it was sensitive to nitrofurantoin,streptomycin,ceftriaxone,kanamycin,ofloxacin,doxycycline,ciprofloxacin,chloromycetin;and moderately sensitive to cotrimoxazole but resistant to polymyxin,penicillin G,tetracycline and erythromyc.The strain could be used as an object for genetic engineering, which laid the foundation for further study and transformation of nitrogen fixation microorganisms in forestry.