野猪源奇异变形杆菌的分离鉴定及致病性分析

Isolation,Identifi cation and Pathogenicity of Proteus mirabilis Isolates from Wild Boars

为探究贵州省野猪携带病原菌情况,本试验对无菌采集自全省6个地区15头野猪肝脏样本的分离菌株进行培养观察、革兰染色、生化鉴定、16S rRNA基因序列分析、药敏试验和小鼠致病性试验及病理组织切片,同时根据已报道的基因序列合成奇异变形杆菌的mrpA、rsbA、ureC、zapA、rsmA、hpmA、FIiL、ucaA、pmfA、atfA 10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。结果显示:从15份野猪肝脏样本中分离出6株病原菌,分离菌的菌落形态、生化特征和16S rRNA基因序列均与奇异变形杆菌相符,其中优势菌落染色可见单个排列的革兰氏阴性球杆菌;生化试验均可分解葡萄糖产酸产气,产生硫化氢;尿素和赖氨酸阳性;苯丙氨酸和蛋白胨水阴性;经PCR检测,6株分离菌株的16S rRNA扩增片段大小均为1453 bp,与奇异变形杆菌菌株同源性均在98%以上;药敏试验结果显示:分离菌株对多种抗生素呈现不同程度耐药,但对诺氟沙星、氧氟沙星、丁胺卡那、氟苯尼考敏感;10种毒力基因在该致病菌中均被检测到,同时该菌对小鼠有致病性。结果表明,6株分离菌株均为具有较强致病性的奇异变形杆菌,这为贵州野猪流行病学调查提供依据。

This experiment was carried out for isolation and identifi cation of Proteus mirabilis from 15 wild boar liver samples from 6 regions of Guizhou province.Total 6 P.mirabilis isolates were obtained through culture observation,gram staining,biochemical reaction,16S rRNA gene sequence analysis and drug susceptibility.The colony morphology,biochemical characteristics and 16S rRNA gene sequence of these isolated were consistent with P.mirabilis.The dominant colony staining showed a single array of Gram-negative cocci.Biochemical testing of these isolates revealed decomposition of glucose to produce acid and gas,production of hydrogen sulfi de,positive reaction of urea and lysine,and negative reaction for peptone water.The 16S rRNA fragments of these 6 isolates were 1453 bp in length and had more than 98%homology with P.mirabilis in the published accounts.In addition,10 virulence gene primers of P.mirabilis(mrpA,rsbA,ureC,zapA,rsmA,hpmA,FIiL,ucaA,pmfA,atfA)were designed according to the reported sequences and used in PCR amplifi cation.All these virulence genes existed in these 6 isolates,suggesting they were highly pathogenic.Drug susceptibility testing showed that these 6 isolates were resistant to a variety of antibiotics but sensitive to norfl oxacin,ofl oxacin,amikacin,fl orfenicol.Additional experiment results also showed that these isolates were pathogenic to mice.These results from the present study provided a basis for the epidemiological investigation of P.mirabilis infection in wild boars in Guizhou.