一株野生马来穿山甲源奇异变形杆菌的分离鉴定及生物学特性分析

Isolation,Identification and Biological Characteristics Analysis of One Proteus mirabilis Strain from Wild Manis javanica

为查明野生动物保护协会送检的1只野生马来穿山甲(Manis javanica)的死亡原因,对其进行剖检,从肺脏组织中分离得到1株病原菌。对分离菌株进行革兰氏染色、生化试验、16S r RNA同源性分析、药敏试验、耐药基因检测、毒力基因检测和小鼠致病性试验等研究。结果显示:分离菌株在麦康凯、SS、血琼脂和LB培养基上均能生长;革兰氏染色镜检为阴性短杆菌;生化鉴定和16S r RNA同源性分析鉴定该菌株为奇异变形杆菌(Proteus mirabilis),命名为YN2018株;生长曲线表明该菌株在2 h后进入对数生长期,16 h后进入稳定期;该菌株对头孢他啶、头孢噻肟和大观霉素较敏感,对庆大霉素、阿米卡星、阿莫西林、妥布霉素和链霉素中度敏感,对罗红霉素、多西环素和复方新诺明等14种抗菌药物耐药;该菌株含有aadA1、aadB、sul1、sul2和dfrA1耐药基因,并携带有mrpA、rsbA、ureC、zapA、rsmA、hpmA、fliL、ucaA、pmfA和atf A毒力基因;小鼠接种菌株浓度为8.50×10^(8)、1.70×10^(9)、3.40×10^(9)、6.80×10^(9)、1.36×10^(10)cfu/m L的死亡率分别为0、50%、75%、100%、100%;死亡小鼠肝脏和肺脏组织病理切片显示发生局灶性细胞坏死及炎性细胞浸润。分离得到的奇异变形杆菌具有多重耐药和强致病性,可能是导致野生马来穿山甲死亡的原因。

In order to find out the death cause of a wild Manis javanica sent by the Wildlife Conservation Association,the dead animal was dissected and then a pathogenic bacteria from its lung tissue was isolated successfully.Gram staining,biochemical test,16S rRNA homology analysis,drug susceptibility test,drug resistance gene test,virulence gene test and mice pathogenicity test were carried out on the isolated strain.The results showed that the isolated strain could grow on MacConkey,SS,blood agar and LB media;gram stain was negative for Brevibacterium;biochemical identification and 16S rRNA homology analysis identified the strain as Proteus mirabilis,and named YN2018 strain;the growth curve showed that the strain entered logarithmic growth phase after two hour,and the stable phase after 16 h;the strain was sensitive to ceftazidime,cefotaxime and spectinomycin,moderately sensitive to gentamicin,amikacin,amoxicillin,tobramycin and streptomycin,and it was resistant to 14 kinds of antibacterial drugs such as roxithromycin,doxycycline and compound sulfa⁃methoxazole;the strain contained aadA1,aadB,sul1,sul2 and dfrA1 drug resistance genes,and carried mrpA,rsbA,ureC,zapA,rsmA,hpmA,fliL,ucaA,pmfA and atfA virulence genes;the death rates of mice inoculated with strain con⁃centration of 8.50×10^(8),1.70×10^(9),3.40×10^(9),6.80×10^(9) and 1.36×10^(10) cfu/mL were 0,50%,75%,100%and 100%re⁃spectively;pathological sections of the liver and lungs of the dead mice showed focal cell necrosis and inflammatory cell in⁃filtration.The isolated Proteus mirabilis has multiple drug resistance and strong pathogenicity,which may be the cause of death of wild Manis javanica.