| 马来穿山甲源嗜水气单胞菌亚种的分离鉴定及生物学特性分析 |
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| 引用本文： | 杜雪晴,古河祥,萨家祺,吴亚江,侯方晖,肖嘉杰,翟俊琼,陈武,邹洁建.马来穿山甲源嗜水气单胞菌亚种的分离鉴定及生物学特性分析[J].中国动物检疫,2021,38(4):124-131. |
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| 作者姓名： | 杜雪晴 古河祥 萨家祺 吴亚江 侯方晖 肖嘉杰 翟俊琼 陈武 邹洁建 |
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| 作者单位： | 广州动物园;广东省野生动物救护中心 |
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| 基金项目： | 国家林业与草原局濒危野生动物调查监管与行业规范项目（20190921） |
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| 摘 要： | 为鉴定马来穿山甲(Manis javanica)腿部致病菌并研究其致病性、耐药性和耐药基因,从救护的1只马来穿山甲腿部溃烂处采集样品,无菌分离培养致病菌,进行形态学观察、革兰氏染色及16S rRNA和gyrB基因测序分析,并开展细菌药敏试验和小鼠致病性试验,随后通过PCR扩增6种毒力基因(rtxA、earA、hlyA、alt、act、epa)及19种耐药基因(5种β-内酰胺酶相关基因、6种氨基糖苷类修饰酶基因、6种氯霉素耐药基因、2种磺胺耐药基因)。结果显示:分离的1株优势菌为革兰氏阴性短杆菌,经16S rRNA和gyrB基因测序以及序列进化树分析鉴定为嗜水气单胞菌亚种(Aeromonas dhakensis),其gyrB基因序列与马来西亚分离株高度同源,推测此致病菌可能由非法走私的穿山甲从马来西亚携带至我国。药敏试验显示,其对头孢曲松、头孢克肟、庆大霉素和氧氟沙星等药物敏感,对青霉素、阿莫西林、头孢氨苄、头孢克洛、氯霉素、磺胺甲恶唑、甲氧苄啶和林可霉素耐药。4种耐药基因tem、aac(3)-II、flor、sul2为阳性,与抗生素耐药表型相符。4种毒力基因earA、hlyA、alt、act为阳性,小鼠半数致死量为3.75×10^(6 )CFU/mL。根据药敏结果使用敏感抗菌药物治疗穿山甲,其腿部患处有好转。因此,引起穿山甲腿部溃烂的病原嗜水气单胞菌亚种(Aeromonas dhakensis)可能为国外传入菌株,具有较强的致病性和多重耐药性。本研究为嗜水气单胞菌的流行病学调查及其感染的防治提供了依据。
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| 关 键 词： | 嗜水气单胞菌亚种 马来穿山甲 耐药性 耐药基因 毒力基因 |
Isolation and Identification of Aeromonas dhakensis Derived from Manis javanica and Analysis on Its Biological Characteristics |
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| Du Xueqing,Gu Hexiang,Sa Jiaqi,Wu Yajiang,Hou Fanghui,Xiao Jiajie,Zhai Junqiong,Chen Wu,Zou Jiejian.Isolation and Identification of Aeromonas dhakensis Derived from Manis javanica and Analysis on Its Biological Characteristics[J].China Journal Of Animal Quarantine,2021,38(4):124-131. |
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| Authors: | Du Xueqing Gu Hexiang Sa Jiaqi Wu Yajiang Hou Fanghui Xiao Jiajie Zhai Junqiong Chen Wu Zou Jiejian |
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| Institution: | (Guangzhou Zoo,Guangzhou Wildlife Research Center,Guangzhou,Guangdong 510070,China;Guangdong Wildlife Rescue Center,Guangzhou,Guangdong 510520,China) |
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| Abstract: | In order to identify the pathogenic bacteria in hindlimb muscles of Manis javanica and study its pathogenicity,drug resistance and resistant genes,samples were collected from the leg ulcers of one Manis javanica to isolate and culture the bacteria,followed by morphological observation,gram staining,16S rRNA and gyrB gene sequencing,bacterial drug sensitivity test and mouse pathogenicity test as well as amplification of 6 virulence genes(including rtxA,earA,hlyA,alt,act and epa)and 19 resistant genes(including 5β-lactamases genes,6 aminoglycoside-modifying enzymes genes,6 chloramphenicol resistant genes and 2 sulfanilamide resistant genes).The results showed that one isolated dominant strain was gram-negative brevibacterium,which was identified as Aeromonas dhakensis through 16S rRNA and gyrB gene sequencing and phylogenetic tree analysis.gyrB gene sequence was highly homologous with that of Malaysian strain,so it was speculated that the pathogen might be introduced toChina by illegally smuggled pangolins from Malaysia. By drug susceptibility test,it was found that the isolate washighly susceptible to ceftriaxone,cefixime,gentamicin and enrofloxacin,and resistant to penicillin,amoxicillin,cefalexin,cefaclor,chloramphenicol,trimethoprim and lincomycin. 4 resistant genes including tem,aac(3)-II,florand sul2 were positive,which were consistent with antibiotic resistance phenotype. 4 virulence genes including earA,hlyA,alt and act were positive,50% lethal dose(LD50)in mice was 3.75×106 CFU/mL. The Manis javanica wasadministered with sensitive antimicrobial drugs according to the results of drug sensitivity,and its legs were improved.In conclusion,the isolated Aeromonas dhakensis with strong pathogenicity and multi-drug resistance was possiblyintroduced from other countries. A basis was provided by the study for epidemiological investigation on Seromonashydrophila and control of its infection. |
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| Keywords: | Aeromonas dhakensis Manis javanica drug resistance resistant gene virulence gene |
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