猪源奇异变形杆菌的分离鉴定及特征分析

为探究广西地区猪源奇异变形杆菌的毒力和耐药情况，本研究从不同规模养殖场收集病死猪病料98份，通过分离培养、形态学观察、染色镜检、16S rRNA测序对分离菌进行鉴定；采用微量肉汤稀释法进行药敏试验；PCR检测毒力基因、耐药基因和整合子及其可变区；可变区扩增产物克隆至pMD^TM19-T载体进行测序。鉴定结果显示，22株细菌在TSA培养基上弥漫性生长，在SS培养基上形成中心黑色边缘白色的单菌落，镜检为革兰氏阴性短杆菌，变形杆菌属特异性基因（TUF）阳性。16S rRNA测序结果显示，21株分离菌与奇异变形杆菌同源性达99%，1株分离菌与彭氏变形杆菌同源性达99%。药敏结果显示，21株奇异变形杆菌对四环素、多西环素、氨苄西林、磺胺甲噁唑耐药率均为100%，对头孢氨苄、头孢呋辛、头孢噻肟、亚胺培南、卡那霉素耐药率在57.1%以上，所有分离株均为多重耐药菌。PCR结果显示，21株分离菌毒力基因atfA、hpmA、ireA、mrpA、pmfA、rsb、ureC、zapA、ptA检出率均为100%，ucaA检出率为95.2%；ESBLs菌株为bla_TEM型、bla_CTX型或bla_TEM型和bla_CTX型，AmpC菌株均为bla_DHA型，携带ESBLs或AmpC基因的菌株比例为57.1%、14.3%，同时携带ESBLs和AmpC基因的菌株比例为14.3%；qnrA、qnrB、qnrS和aac（6’）-Ⅰb-cr检出率分别为9.5%、0、4.8%和80.1%。21株分离菌Ⅰ类整合子（intⅠ1）阳性率61.9%，检测到9种基因盒阵列（aadA2-linF、estX、dfrA32-ereA-aadA2、drfA5、drfA12-orfF-aadA2、arr3-aac （6’）Ⅰb-cr5、aac （6’）Ⅰb-cr5-bla_OXA-1-catB3-aar3、drfA1-orfC和aadA2）；Ⅱ类整合子（intⅠ2）阳性率76.2%，检测到1种基因盒阵列（drfA1-sat1-aadA1）。本研究结果表明，广西地区猪源奇异变形杆菌毒力高且耐药严重，为后期针对奇异变形杆菌的防治和研究提供数据支持。

Isolation,Identification and Biological Characteristics of Proteus mirabilis of Swine

In order to investigate the virulence and drug resistance of Proteus mirabilis from swine in Guangxi province,98 samples of diseased swine were collected from farms with different scales in this study,and identified the isolated bacteria through isolation and culture,morphological observation,straining microscopy,16S rRNA sequencing.Drug susceptibility testing was performed by broth microdilution method.PCR was used to detect virulence genes,drug resistance genes,integrons and variable regions.The variable region amplification products were cloned into pMD^TM19-T for sequencing.The identification results showed that 22 strains of bacteria grew diffusely on the TSA medium,formed a center black marginal white single colony on the SS medium,and microscopic examination showed Gram-negative short bacillus and Proteus specific gene (TUF) positive.The results of 16S rRNA sequencing showed that 21 isolates had 99% homology with Proteus mirabilis,and one isolate had 99% homology with Proteus penneri.The drug sensitivity results showed that the drug resistance rate of 21 strains of Proteus mirabilis to tetracycline,doxycycline,ampicillin and sulfamethoxazole reached 100%,the drug resistance rate of cefalexin,cefuroxime,cefotaxime,imipenem and kanamycin was above 57.1%,and all the isolated strains were multiple drug resistant bacteria.PCR results showed that the virulence gene detection rates of atfA,hpmA,ireA,mrpA,pmfA,rsbA,ureC,zapA and ptA were all 100% and that of ucaA was 95.2%.The strains of ESBLs were bla_TEM type,bla_CTX type or bla_TEMtype and bla_CTX type,and the AmpC strains were bla_DHAtype.The proportion of strains carrying ESBLs or AmpC genes was 57.1%, 14.3%, and the proportion of strains carrying both ESBLs and AmpC genes was 14.3%.The detection rates of qnrA,qnrB,qnrS and aac(6')-Ⅰb-cr were 9.5%,0,4.8% and 80.1%,respectively.21 strains of isolated bacteria classⅠ integron (intⅠ1) positive rate was 61.9%,nine different gene cassette arrays were detected (aadA2-linF,estX,dfrA32-ereA-aadA2,drfA5,drfA12-orfF-aadA2,arr3-aac(6')-Ⅰb-cr5,aac(6')-Ⅰb-cr5-bla_OXA-1-catB3-aar3,drfA1-orfC and aadA2);classⅡ integron (intⅠ2) positive rate was 76.2%,only one gene cassette array (drfA1-sat1-aadA1) was detected.The results of this study showed that the virulence and drug resistance of swine-origin Proteus mirabilis were high in Guangxi,which provided data support for the prevention and control of Proteus mirabilis.