中华条颈龟致病性维氏气单胞菌的分离鉴定及药敏试验

doi:10.16431/j.cnki.1671-7236.2019.09.032

摘要/Abstract

摘要： 为明确一起致中华条颈龟发病死亡的病原，本研究从患病的中华条颈龟体内分离出一株优势菌株，通过对分离菌的培养、革兰氏染色观察、生化试验、16S rRNA序列比对分析、动物攻毒试验和药物敏感试验对其进行鉴定。结果显示，分离菌在普通营养琼脂培养基上呈灰白色、不透明、圆形、边缘整齐、表面湿润且光滑、中间凸起的菌落；在SS琼脂培养基上形成浅橘红色、圆形、光滑的菌落；革兰氏染色结果显示，分离菌为革兰氏阴性菌，短杆状，菌体两端钝圆，单个或成对排列，并将该分离菌命名为HNJ002；生化试验结果显示，该菌具有运动性，且葡萄糖、蔗糖、半乳糖、麦芽糖、果糖、精氨酸双水解酶、鸟氨酸脱羧酶、D-葡萄糖产酸、D-葡萄糖产气、明胶水解、氧化酶、吲哚、V-P试验反应为阳性；进化树分析显示，其与GenBank中登录的维氏气单胞菌聚为一簇，且同源性达99%以上；动物攻毒试验显示，该菌对中华条颈龟有较强的致病性；药物敏感试验显示，其对多数抗菌药较为敏感，对罗红霉素、妥布霉素、新霉素中度敏感，仅对少数抗菌药物如氨苄西林、青霉素及克林霉素耐药。研究结果可为防治中华条颈龟维氏气单胞菌感染提供参考依据。

关键词: 中华条颈龟; 维氏气单胞菌; 分离鉴定; 药敏特性

Abstract: In order to identify the pathogen of a serious disease occurred in Mauremys sinensis,a dominant bacteria was isolated from the diseased Mauremys sinensis.It was identified by bacterial isolation and culture,Gram staining microscopy,biochemical test,16S rRNA sequence analysis,animal regression test and drug susceptibility.The isolate was gray-white,opaque,round,neat edge,and had wet and smooth surface with middle protruding colonies in general nutrient agar medium,and orange-red,round,smooth colonies in SS agar medium.Gram staining microscopy showed that the isolate was Gram-negative,short rod-shaped,obtuse round at both ends of the body,single or double-existent.Biochemical tests showed that the isolate was mobilized with positive reactions such as glucose,sucrose,galactose,maltose,fructose,arginine dihydrolase,ornithine decarboxylase,D-glucose acid production,D-glucose gas production,gelatin hydrolysis,oxidase,indole and V-P test.The sequence analysis of 16S rRNA showed that the isolate was clustered with Aeromonas veronii registered in GenBank,and had 99% homology with that reference strains.The animal regression test showed that HNJ002 was highly pathogenic to Mauremys sinensis.Drug sensitivity test showed that the isolate was sensitive to most antibacterial,moderately sensitive to roxithromycin,tobramycin and neomycin,and only resistant to a few antibiotics such as ampicillin,penicillin and clindamycin.This study would provide a scientific basis for the prevention and treatment of Aeromonas veronii in Mauremys sinensis.

Key words: Aeromonas veronii; Mauremys sinensis; isolation and identification; antibiotic sensitivity

中图分类号:

S943

方振华, 曾玉梅, 洪美玲, 丁利. 中华条颈龟致病性维氏气单胞菌的分离鉴定及药敏试验[J]. 中国畜牧兽医, 2019, 46(9): 2760-2768.

FANG Zhenhua, ZENG Yumei, HONG Meiling, DING Li. Identification and Susceptibility Test of Pathogenic Aeromonas veronii Isolated from Mauremys sinensis[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2760-2768.

参考文献

[1] 史海涛,洪美玲,傅丽容,等.龟类的养殖与保护[J]生物学通报,2009,44(1):18-21. SHI H T,HONG M L,FU L R,et al.The conservation and captive breeding of turtles[J].Bulletin of Biology,2009,44(1):18-21.(in Chinese)
[2] 洪美玲,付丽容,王锐萍,等.龟鳖动物疾病的研究进展[J].动物学杂志,2003,38(6):115-119. HONG M L,FU L R,WANG R P,et al.The advances in the research of the turtle's disease[J].Chinese Journal of Zoology,2003,38(6):115-119.(in Chinese)
[3] HERNANDEZDIVERS S J,HENSEL P,GLADDEN J,et al.Camus,investigation of shell disease in map turtles (Graptemys spp.)[J].Journal of Wildlife Diseases,2009,45(3):637-652.
[4] HICKMANBRENNER F W,MACDONALD K L,STEIGERWALT A G,et al.Aeromonas veronii,a new ornithine decarboxylase-positive species that may cause diarrhea[J].Journal of Clinical Microbiology,1987,25(5):900-906.
[5] 宋振辉,乔霞,张鹦俊,等.维氏气单胞菌的分离鉴定及系统进化分析[J].中国兽医杂志,2011,47(1):23-25. SONG Z H,QIAO X,ZHANG Y J,et al.Isolation,identification and phylogenetic analysis of Aeromonas veronii[J].Chinese Journal of Veterinary Medicine,2011,47(1):23-25.(in Chinese)
[6] 田浪,温贵兰,张升波,等.斑点叉尾鮰源致病性维氏气单胞菌的分离与生物学特性鉴定[J].中国畜牧兽医,2018,45(11):3203-3210. TIAN L,WEN G L,ZHANG S B,et al.Isolation and biological characterization of the pathogenic Aeromonas veronii from Ictalurus punctatus[J].China Animal Husbandry & Veterinary Medicine,2018,45(11):3203-3210.(in Chinese)
[7] 刘亚,杨锐,陈叶雨,等.达氏鲟维氏气单胞菌的分离鉴定及病理组织学观察[J].南方农业学报,2018,49(6):1235-1241. LIU Y,YANG R,CHEN Y Y,et al.Isolation,identification of Aeromonas veronii in Acipenser dabryanus and its histological observations[J].Journal of Southern Agriculture,2018,49(6):1235-1241.(in Chinese)
[8] 高金伟,梁利国,王亚冰,等.鳜源致病性维氏气单胞菌的鉴定及药敏试验[J].微生物学通报,2016,43(12):2686-2692. GAO J W,LIANG L G,WANG Y B,et al.Identification and susceptibility test of pathogenic Aeromonas veronii isolated from Siniperca chuatsi[J].Microbiology China,2016,43(12):2686-2692.(in Chinese)
[9] 韦阳道,杨军,文衍红,等.罗非鱼竖鳞病病原维氏气单胞菌的分离鉴定与致病性研究[J].中国畜牧兽医,2016,43(11):3047-3052. WEI Y D,YANG J,WEN Y H,et al.Isolation,identification and pathogenicity of Aeromonas veronii from lepidorthosis pathogenic bacteria of Tilapia[J].China Animal Husbandry & Veterinary Medicine,2016,43(11):3047-3052.(in Chinese)
[10] 杨泽晓,周亚,任冉阳,等.白缘(鱼央)维氏气单胞菌的分离鉴定与药敏试验[J].中国畜牧兽医,2012,39(2):92-96. YANG Z X,ZHOU Y,REN R Y,et al.Isolation,identification and drug susceptibility test of Aeromonas veronii from Liobagrus marginatus Günther[J].China Animal Husbandry & Veterinary Medicine,2012,39(2):92-96.(in Chinese)
[11] 张小明,崔文耀,丁少青,等.一株凡纳滨对虾源维氏气单胞菌的分离鉴定及药敏特性[J].微生物学通报,2017,44(12):2795-2804. ZHANG X M,CUI W Y,DING S Q,et al.Isolation,identification and antibiotic sensitivity of Aeromonas veronii from Litopenaeus vannamei[J].Microbiology China,2017,44(12):2795-2804.(in Chinese)
[12] 房海,陈翠珍,张晓君,等.中华绒螯蟹病原维氏气单胞菌的检验[J].中国人兽共患病学报,2008,24(1):45-49. FANG H,CHEN C Z,ZHANG X J,et al.Examination of the pathogenic Aeromonas veronii isolated from crab Eriocheir sinensis[J].Chinese Journal of Zoonoses,2008,24(1):45-49.(in Chinese)
[13] 徐先栋,李燕华,王海华,等.台湾泥鳅幼鱼红点病病原的分离鉴定及药敏分析[J].水产科学,2018,37(4):522-526. XU X D,LI Y H,WANG H H,et al.Isolation,identification and drug sensitivity of a pathogen causing punctate hemorrhag in farmed taiwan loach fingerlings[J].Fisheries Science,2018,37(4):522-526.(in Chinese)
[14] ZHU Z X,JIANG X H,DENG S Z,et al.Isolation,identification and in vitro antimicrobial susceptibility of pathogenic Aeromonas veronii from soft-shelled turtles[J].Agricultural Science,2016,17(4):804-809.
[15] 何成伟,钟勇,段保屹,等.黄喉拟水龟致病菌——维氏气单胞菌的鉴定及药敏试验[J].中国兽医杂志,2016,52(1):106-108. HE C W,ZHONG Y,DUAN B Y,et al.Identification and drug susceptibility test of Aeromonas veronii,a pathogen of Mauremys mutica[J].Chinese Journal of Veterinary Medicine,2016,52(1):106-108.(in Chinese)
[16] NAWAZM,SUNG K,KHAN S A,et al.Biochemical and molecular characterization of tetracycline-resistant Aeromonas veronii isolates from catfish[J].Applied Environmental Microbiology,2006,72(10):6461-6466.
[17] 董靖,丁浩,胥宁,等.杂交鲶维氏气单胞菌鉴定及联合药敏试验研究[J].中国预防兽医学报,2017,39(11):897-901. DONG J,DING H,XU N,et al.Identification and synergistic susceptibility test of pathogenic Aeromonas veronii isolated from hybrid catfish[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(11):897-901.(in Chinese)
[18] JOSEPH S W,CARNAHAN A M,BRAYTON P R,et al.Aeromonas jandaei and Aeromonas veronii dual infection of a human wound following aquatic exposure[J].Journal of Clinical Microbiology,1991,29(3):565-569.
[19] SALAM S,GHALY M,BARAKA D,et al.Histopathological changes in diseased catfish (Clarias gariepinus) and treated by ciprofloxacin and clove extract[J].Journal of Veterinary Medicine Allied Science,2017,13(2):321-329.
[20] BALDISSERA M D,SOUZA C F,VERDI C M,et al.Aeromonas caviae inhibits hepatic enzymes of the phosphotransfer network in experimentally infected silver catfish:Impairment on bioenergetics[J].Journal of Fish Diseases,2018,41(3):469-474.
[21] LEE S W,WENDY W.Antibiotic and heavy metal resistance of Aeromonas hydrophila and Edwardsiella tarda isolated from red hybrid tilapia (Oreochromis spp.) coinfected with motile aeromonas septicemia and edwardsiellosis[J].Veterinary World,2017,10(7):803-807.
[22] DONG H T,TECHATANAKITARNAN C,JINDAKITTIKUL P,et al.Aeromonas jandaei and Aeromonas veronii caused disease and mortality in Nile tilapia,Oreochromis niloticus (L.)[J].Journal of Fish Diseases,2017,40(10):1395-1403.
[23] 曹颖颖,杜泓明,白安斌,等.家养观赏地图鱼肺炎克雷伯氏菌、维氏气单胞菌与黏质沙雷氏菌的分离鉴定及耐药性分析[J].中国畜牧兽医,2015,42(1):215-223. CAO Y Y,DU H M,BAI A B,et al.Isolation,identification and drug resistance of Klebsiella pneumoniae,Aeromonas veronii and Serratia marcescens isolated from domesticated ornamental Astronotus ocellatus[J].China Animal Husbandry & Veterinary Medicine,2015,42(1):215-223.(in Chinese)
[24] 彭博文,杨移斌,艾晓辉,等.克氏原螯虾源维氏气单胞菌分离鉴定及药敏特性研究[J].海洋湖沼通报,2018,4:108-114. PENG B W,YANG Y B,AI X H,et al.Isolation and identification of Aeromonas veronii from Procamaeus clarkii and its antibiotic sensitivity[J].Transaction of Oceanology and Limnology,2018,4:108-114.(in Chinese)
[25] VILA J,MARCO F,SOLER L,et al.Figueras,in vitro antimicrobial susceptibility of clinical isolates of Aeromonas caviae,Aeromonas hydrophila and Aeromonas veronii biotype sobria[J].Journal of Antimicrobial Chemotherapy,2002,49(4):701-702.
[26] 马志宏,李铁梁,姜娜,等.中草药对致病性维氏气单胞菌体外抑菌活性及最优组方研究[J].中国畜牧兽医,2011,38(6):155-159. MA Z H,LI T L,JIANG N,et al.Study on the bacteriostatic activity and the best combination of Chinese herbs against pathogenic Aeromonas veronii in vitro[J].China Animal Husbandry & Veterinary Medicine,2011,38(6):155-159.(in Chinese)
[27] 杨永立,王利,吴满林.库拉索芦荟提取物对维氏气单胞菌抑菌作用的研究[J].中国畜牧兽医,2013,40(9):147-150. YANG Y L,WANG L,WU M L.Study on the bacteriostasis of aloe vera extracts against Aeromonas veronii[J].China Animal Husbandry & Veterinary Medicine,2013,40(9):147-150.(in Chinese)
[28] 李铁梁,罗琳,马志宏,等.2种粒径组方中草药对西伯利亚鲟维氏气单胞菌病预防效果研究[J].中国畜牧兽医,2014,41(10):252-257. LI T L,LUO L,MA Z H,et al.Effect of Chinese herbal medicines with two particle size on Aeromonas veronii disease prevention of siberian sturgeon (Acipenser baerii)[J].China Animal Husbandry & Veterinary Medicine,2014,41(10):252-257.(in Chinese)