1株鸵鸟铜绿假单胞菌的分离鉴定与进化树分析

Isolation,identification and phylogenetic analysis of a Pseudomonas aeruginosa strain

目的对内蒙古自治区通辽市某养殖场幼龄鸵鸟致死的致病菌进行分离鉴定与进化树分析。方法无菌采集幼龄病死鸵鸟的内脏,进行细菌分离培养。将分离菌株进行小鼠致病性试验、生化鉴定及药物敏感性试验,并对其进行16S r RNA基因测序,选取比对结果同源性相近的20条序列进行系统进化树分析;PCR扩增ExoT、ExoU、ExoY、ExoS 4个毒力基因,分析分离菌株毒力基因携带情况。结果经细菌形态学鉴定、16S rRNA基因序列Blast比对、BD PhoenixTM100全自动微生物鉴定和药敏系统及部分毒力基因PCR检测得出,该致病菌为铜绿假单胞菌。3种毒力基因ExoT、ExoY、ExoS在该致病菌中被检测,未携带ExoU基因;该菌对环丙沙星、庆大霉素和莫西沙星等13种药物敏感,对头孢唑啉和氯霉素等7种药物耐药。系统进化树分析表明,分离菌株与铜绿假单胞菌中国湖南株(EU915713.1)和中国四川株(KY962357.1)遗传距离最近,并命名为TLTL1。结论该致病菌经鉴定为铜绿假单胞菌,为一种条件致病菌。本研究对铜绿假单胞菌的鉴定及用药提供了参考。

Objective To isolate,identify and analyze the phylogenesis of pathogenic bacteria killing young ostriches in a farm in Tongliao City,Inner Mongolia Autonomous Region,China.Methods The viscera of young dying ostriches were collected aseptically,from which the bacterial strain was isolated and cultured.The isolated strain was subjected to pathogenicity test in mice,biochemical identification and drug sensitivity test,of which the 16 S rRNA gene was sequenced,and 20 sequences with high homology were used to establish a phylogenetic tree.Four virulence genes,ExoT,ExoU,ExoY and ExoS 4,were amplified by PCR and analyzed for carrying in isolated strain.Results Bacterial morphology identification,16 S r RNA gene sequence Blast alignment,BD Phoenix TM 100 automatic microbial identification and drug susceptibility system as well as PCR detection of partial virulence genes showed that the pathogenic bacterial strain was Pseud omonas aeruginosa.Three virulence genes,ExoT,ExoY,and ExoS,were detected in the pathogenic bacterial strain,while no ExoU gene was detected.The bacterial strain was sensitive to 13 drugs including ciprofloxacin,gentamicin and moxifloxacin,while was resistant to 7 drugs including cefazolin and chloramphenicol.Phylogenetic tree showed that the isolated strain,named as TLTL1,was the closest to the Chinese Hunan strain(EU915713.1)and Chinese Sichuan strain(KY962357.1)of P.aeruginosa.Conclusion The pathogen was identified as P.aeruginosa,which was a conditional pathogen.This study provides a reference for the identification and medication of P.aeruginosa.