鹅源奇异变形杆菌的分离鉴定及毒力基因检测

Isolation, identification of goose Proteus mirabilis and it’s virulence genes detection

摘要:

【背景】奇异变形杆菌(Proteus mirabilis)是一种人兽共患机会致病菌，革兰氏阴性，兼性厌氧，呈多形性。【目的】探明内蒙古自治区通辽市某养殖场雏鹅发生死亡的病因，研究致病菌的分类地位和毒力基因携带情况。【方法】通过病原菌的筛查和感染试验确定发病原因，对其16S rRNA基因测序，建立病原菌系统进化树，结合BD PhoenixTM 100全自动微生物鉴定仪对病原菌进行分析鉴定，综合判定致病菌的种类及其分类地位；根据已报道的基因序列合成奇异变形杆菌的ureC、zapA、mrpA、ucaA、rsbA、pmfA、atfA、atfC 8个毒力基因引物，通过PCR扩增研究病原菌毒力基因携带情况。【结果】致病茵的16S rRNA基因与已报道的奇异变形杆菌(P. mirabilis)相似性在99%以上，通过构建系统进化树和BD PhoenixTM 100全自动微生物鉴定仪鉴定，确定该菌株为P. mirabilis并编号为AYQ-1；8种毒力基因在该致病菌中均被检测到；雏鹅腹腔注射感染可导致雏鹅死亡，半致死浓度(LD50)为1.51×107 CFU/mL；药敏试验结果显示，该菌对链霉素、氨曲南和氧氟沙星等19种药物敏感，对万古霉素、青霉素和四环素等15种药物耐药。【结论】AYQ-1经鉴定为鹅奇异变形杆菌，毒力基因型为ureC(+)、zapA(+)、mrpA(+)、ucaA(+)、rsbA(+)、pmfA(+)、atfA(+)、atfC(+)型。

Abstract:

[Background] Proteus mirabilis is a Gram-negative and pleomorphic facultative anaerobe, and it is an opportunistic pathogen of zoonosis. [Objective] To confirm the pathogeny that causes goslings’ death at some geese farm in Tongliao, Inner Mongolia Autonomous Region, and characterize the virulence genes of the pathogenic bacteria. [Methods] The pathogenic bacteria were confirmed based on the pathogens screening and artificial challenge experiment, and identified according to 16S rRNA genes sequence and biochemical characteristics. Primers of 8 virulence genes (ureC, zapA, mrpA, ucaA, rsbA, pmfA, atfA, atfC) were designed according to the sequences of Proteus mirabilis reported on the GenBank and used to detect the virulence genes of the pathogenic bacteria by PCR amplification. [Results] The pathogenic bacteria isolated from infected gosling was proved to be the pathogeny to Proteus mirabilis. Analysis of 16S rRNA gene sequences of the AYQ-1 gave highest identity (99%) to P. mirabilis and named AYQ-1. This was further supported by phylogenetic tree and biochemical characteristics, AYQ-1 was identified as P. mirabilis. AYQ-1 gave positive PCR results for 8 virulence genes. Intraperitoneal injection with AYQ-1 could lead to goslings’ death, with a value of the median lethal dose (LD50) of 1.51×107 CFU/mL. Antibiotics sensitivity test showed that among 19 antibiotics tested, AYQ-1 was sensitive to 19 antibiotics such as streptomycin, aztreonam and ofloxacin, but it was resistant to 15 antibiotics such as vancomycin, penicillin and achromycin. [Conclusion] The pathogen was identified to be Proteus mirabilis, and it’s virulence genotype are ureC(+), zapA(+), mrpA(+), ucaA(+), rsbA(+), pmfA(+), atfA(+), atfC(+).