长薄鳅恶臭假单胞菌的分离鉴定及其感染的病理损伤
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摘要
恶臭假单胞菌(Pseudomonas putida)是广泛分布于土壤、水体中的一种条件致病菌,可感染两栖类、鱼类和甲壳类等多种水生动物。2017年8月,四川省西昌市某养殖场的长薄鳅出现一种以体表溃烂、鳍条出血为主要特征的传染病。为探究其病因,本研究进行了病原菌分离、人工感染、分离菌表型特征测定、16S r RNA与gyr B基因序列分析。从病鱼肝、脾、肾中分离到优势菌株(BMH170820),人工感染试验证实了其病原性。根据其菌落形态、生理生化特性,结合16S r RNA与gyr B基因序列分析鉴定其为恶臭假单胞菌。组织病理学观察发现,其感染长薄鳅对鳃、肾、肝、心、脾与消化道等多处组织器官造成损伤,表现出明显的变性、坏死和炎症反应,致全身多器官功能障碍而死亡。药物敏感试验显示,该菌对新霉素、氧氟沙星以及强力霉素等敏感,而对阿莫西林、头孢氨苄与氟苯尼考等耐药。
Pseudomonas putida is a conditional pathogen that widely distributed in water and soil,which can infect aquatic animals such as amphibians,fish and crustaceans. During August in 2017,a serious infection that characterized by ulceration of body surface and bleeding of fins occurred in a loach farm located in Xichang,Sichuan Province of China. To determine the etiology of this outbreak,pathogen isolation,experimental infection,physical and chemical characteristics of isolated bacteria and sequence analysis of 16 S rRNA and gyrB gene were carried out. Results showed that a gram-negative,rod-shaped bacillus( designated as BMH170820) was isolated from the diseased fish and its pathogenicity were confirmed by experimental infection test for its pathological changes were consistent with that of natural infection. Based on the colony morphology,physiological and biochemical characteristics,combined with 16 S rRNA and gyrB gene sequence analysis,Pseudomonas putida was confirmed as the causative factor of this outbreak. Histopathological observation revealed that Pseudomonas putida infection in Leptobotia elongate were followed as obvious degeneration,necrosis and inflammation in gill,kidney,liver,heart,spleen and digestive tract,the extensive and serious multiple organ dysfunction might lead to the death of Leptobotia elongate. Drug sensitivity tested results showed that this isolated strain of Pseudomonas putida was sensitive to neomycin,ofloxacin,doxycycline,etc,but resistant to amoxicillin,cefalexin and florfenicol.
Pseudomonas putida is a conditional pathogen that widely distributed in water and soil,which can infect aquatic animals such as amphibians,fish and crustaceans. During August in 2017,a serious infection that characterized by ulceration of body surface and bleeding of fins occurred in a loach farm located in Xichang,Sichuan Province of China. To determine the etiology of this outbreak,pathogen isolation,experimental infection,physical and chemical characteristics of isolated bacteria and sequence analysis of 16 S rRNA and gyrB gene were carried out. Results showed that a gram-negative,rod-shaped bacillus( designated as BMH170820) was isolated from the diseased fish and its pathogenicity were confirmed by experimental infection test for its pathological changes were consistent with that of natural infection. Based on the colony morphology,physiological and biochemical characteristics,combined with 16 S rRNA and gyrB gene sequence analysis,Pseudomonas putida was confirmed as the causative factor of this outbreak. Histopathological observation revealed that Pseudomonas putida infection in Leptobotia elongate were followed as obvious degeneration,necrosis and inflammation in gill,kidney,liver,heart,spleen and digestive tract,the extensive and serious multiple organ dysfunction might lead to the death of Leptobotia elongate. Drug sensitivity tested results showed that this isolated strain of Pseudomonas putida was sensitive to neomycin,ofloxacin,doxycycline,etc,but resistant to amoxicillin,cefalexin and florfenicol.
引文
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[16]沈晓静,胡秀彩,兰云,等.锦鲤荧光假单胞菌的分离鉴定及药敏试验[J].水产科学,2014,33(7):443-446.SHEN X J,HU X C,LAN Y,et al.Drug susceptibility,isolation and identification of pathogenic bacterium Pseudomonas fluorescens from Koi Carp(Cyprinus carpio)[J].Fisheries Science,2014,33(7):443-446.(in Chinese with English abstract)
[17]安伟,肖雨,高晓华,等.鳜源致病性荧光假单胞菌的分离与鉴定[J].动物学杂志,2014,49(5):760-765.AN W,XIAO Y,GAO X H,et al.Isolation and identification of pathogenic Pseudomonas fluorescens from Siniperca chuatsi[J].Chinese Journal of Zoology,2014,49(5):760-765.(in Chinese with English abstract)
[18]郭政,赵庆中,张先锋.长江江豚感染铜绿假单胞菌肺炎的诊治[J].兽类学报,2007,27(1):86-91.GUO Z,ZHAO Q Z,ZHANG X F.Diagnosis and treatment of pneumonia caused by Pseudomonas aeruginosa in a Yangtze finless porpoise(Neophocaena phocaenoides asiaeorientalis)[J].Acta Theriologica Sinica,2007,27(1):86-91.(in Chinese with English abstract)
[19]任燕,陈献稿,刘鹏威,等.大黄鱼假单胞菌病病原的分离鉴定及药物敏感试验[J].广东农业科学,2012,39(18):151-154.REN Y,CHEN X G,LIU P W,et al.Isolation,identification and the sensitivity to antimicrobial agents of Pseudomonassis pathogen from Great yellow croaker(Pseudosciaena crocea)[J].Guangdong Agricultural Sciences,2012,39(18):151-154.(in Chinese with English abstract)
[20]LADHANI S,BHUTTA Z A.Neonatal Pseudomonas putida infection presenting as staphylococcal scalded skin syndrome[J].European Journal of Clinical Microbiology and Infectious Diseases,1998,17(9):642-644.
[21]覃章纯,汪崇阳,童杨雯.恶臭假单胞菌致小儿败血症一例[J].中华儿科杂志,2000,38(4):238.QIN Z C,WANG C Y,TONG Y W.A case of aeruginosa caused by pseudomonas putida in children[J].Chinese Journal of Pediatrics,2000,38(4):238.(in Chinese)
[22]孔繁才,崔一峰.早餐奶污染恶臭假单胞菌引起食物中毒一例报告[J].中华流行病学杂志,2009,30(1):49.KONG F C,CUI Y F.Food poisoning caused by Pseudomonas putida in breakfast milk:a case report[J].Chinese Journal of Epidemiology,2009,30(1):49.(in Chinese)
[23]李庆山,邢瑞云,张芳萍,等.首次发现恶臭假单胞菌引起的食物中毒[J].中国公共卫生,2000,16(1):50-51.LI Q S,XING R Y,ZHANG F P,et al.The discovery of food-poisoning gaused by Pseudomonas putida for the first time[J].China Public Health,2000,16(1):50-51.(in Chinese with English abstract)
[24]IWALOKUN B A,AKINSINDE K A,LANLENHIN O,et al.Bacteriocinogenicity and production of pyocins from Pseudomonas species isolated in Lagos,Nigeria[J].African Journal of Biotechnology,2006,5(11):1072-1077.
[25]LOMBARDI G,LUZZARO F,DOCQUIER J D,et al.Nosocomial infections caused by multidrug-resistant isolates of Pseudomonas putida producing VIM-1 Metallo-β-Lactamase[J].Journal of Clinical Microbiology,2002,40(11):4051-4055.
[26]谢朝云,熊芸,孙静,等.91株恶臭假单胞菌流行病学特征及耐药性[J].中国感染控制杂志,2017,16(12):1185-1188.XIE Z Y,XIONG Y,SUN J,et al.Epidemiological characteristics and antimicrobial resistance of 91 strains of Pseudomonas putida[J].Chinese Journal of Infection Control,2017,16(12):1185-1188.(in Chinese with English abstract)
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[2]樊海平.恶臭假单胞菌引起的欧洲鳗鲡烂鳃病[J].水产学报,2001,25(2):147-150.FAN H P.Study on the gill-rot disease of Anguilla anguilla infected by Pseudomonas putida[J].Journal of Fisheries of China,2001,25(2):147-150.(in Chinese with English abstract)
[3]余为一,李槿年,祖国掌.一株中华绒螯蟹病原菌的研究初报[J].安徽农业大学学报,1999,26(2):174-177.YU W Y,LI J N,ZU G Z.A preliminary report on a pathogenic bacterium from Eriocheir sinensi[J].Journal of Anhui Agricultural University,1999,26(2):174-177.(in Chinese with English abstract)
[4]闫茂仓,单乐州,马爱敏,等.条石鲷烂尾白浊症病原菌的分离鉴定及系统发育分析[J].上海海洋大学学报,2010,19(1):50-55.YAN M C,SHAN L Z,MA A M,et al.Identification and phylogenetic analysis of a pathogenic bacterial strain isolated from Oplegnathus fasciatus with tail rot and whitish symptom[J].Journal of Shanghai Ocean University,2010,19(1):50-55.(in Chinese with English abstract)
[5]王高学,黄增荣,袁明.三疣梭子蟹牛奶病病原的分离鉴定[J].西北农林科技大学学报(自然科学版),2007,35(6):29-33.WANG G X,HUANG Z R,YUAN M.Isolation and identification of the pathogen of milk disease cultured in swimmingcrab Portunus tritubercularus(portunidae)[J].Journal of Northwest A&F University(Natural Science Edition),2007,35(6):29-33.(in Chinese with English abstract)
[6]陶保华,石和荣,黄俊文,等.假单胞菌引起罗氏沼虾黄鳃、黑鳃病的研究[J].中山大学学报(自然科学版),2000,39(1):255-259.TAO B H,SHI H R,HUANG J W,et al.Studied on yellow and black gills of Macrobrachium rosenbergii caused by Pseudoonas[J].Acta Scientiarum Naturalium Universitatis Sunyatseni,2000,39(1):255-259.(in Chinese with English abstract)
[7]毛芝娟,王美珍,陈吉刚,等.黑鲷肠炎病原恶臭假单胞菌的分离和鉴定[J].渔业科学进展,2010,31(3):23-28.MAO Z J,WANG M Z,CHEN J G,et al.Isolation and characterization of Pseudomonas putida,a causative agent of gastroenteritis in black sea bream,Sparus macrocephalus[J].Progress in Fishery Sciences,2010,31(3):23-28.(in Chinese with English abstract)
[8]杨圆圆,杨移斌,曹海鹏,等.杂交鲟源恶臭假单胞菌的分离鉴定及药敏特性研究[J].浙江农业学报,2017,29(12):1978-1985.YANG Y Y,YANG Y B,CAO H P,et al.Isolation,identification and antibiotic sensitivity of Pseudomonas putida from hybrid sturgeon(Huso dauricus♀×Acipenser schrenckii♂)[J].Acta Agriculturae Zhejiangensis,2017,29(12):1978-1985.(in Chinese with English abstract)
[9]段亚佼,黄小丽,詹瑰然,等.杂交鲇恶臭假单胞菌的分离鉴定及其病理损伤研究[J].水生生物学报,2017,41(2):371-378.DUAN Y J,HUANG X L,ZHAN G R,et al.Isolation,identification and pathological lesions of Pseudomonas putida from hybrid catfish[J].Acta Hydrobiologica Sinica,2017,41(2):371-378.(in Chinese with English abstract)
[10]许斌福,程海华,池洪树,等.大黄鱼内脏白点病的病原分析与鉴定[J].福建农业学报,2015,30(7):631-635.XU B F,CHENG H H,CHI H S,et al.Identification of pathogen responsible for white-spots disease in Larimichthys crocea[J].Fujian Journal of Agricultural Sciences,2015,30(7):631-635.(in Chinese with English abstract)
[11]ALTINOK I,KAYIS S,CAPKIN E.Pseudomonas putida infection in Rainbow trout[J].Aquaculture,2006,261(3):850-855.
[12]刘军,胡兵,李惠,等.长江上游四种特有鱼类肌肉营养组成与评价[J].水生生物学报,2007,31(5):763-766.LIU J,HU B,LI H,et al.Analysis of the nutritional composition of muscle in four endemic species[J].Acta Hydrobiologica Sinica,2007,31(5):763-766.(in Chinese with English abstract)
[13]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:166-167.
[14]LI R,DEBELLA H J,CARMICHAEL W W.Isolates identifiable as Arthrospira maxima and Arthrospira fusiformis(Oscillatoriales,Cyanobacteria)appear identical on the basis of a morphological study in culture and 16S r RNA gene sequences[J].Phycologia,2001,40(4):367-371.
[15]YAMAMOTO S,HARAYAMA S.PCR amplification and direct sequencing of gyr B genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains[J].Applied and Environmental Microbiology,1995,61(3):1104-1109.
[16]沈晓静,胡秀彩,兰云,等.锦鲤荧光假单胞菌的分离鉴定及药敏试验[J].水产科学,2014,33(7):443-446.SHEN X J,HU X C,LAN Y,et al.Drug susceptibility,isolation and identification of pathogenic bacterium Pseudomonas fluorescens from Koi Carp(Cyprinus carpio)[J].Fisheries Science,2014,33(7):443-446.(in Chinese with English abstract)
[17]安伟,肖雨,高晓华,等.鳜源致病性荧光假单胞菌的分离与鉴定[J].动物学杂志,2014,49(5):760-765.AN W,XIAO Y,GAO X H,et al.Isolation and identification of pathogenic Pseudomonas fluorescens from Siniperca chuatsi[J].Chinese Journal of Zoology,2014,49(5):760-765.(in Chinese with English abstract)
[18]郭政,赵庆中,张先锋.长江江豚感染铜绿假单胞菌肺炎的诊治[J].兽类学报,2007,27(1):86-91.GUO Z,ZHAO Q Z,ZHANG X F.Diagnosis and treatment of pneumonia caused by Pseudomonas aeruginosa in a Yangtze finless porpoise(Neophocaena phocaenoides asiaeorientalis)[J].Acta Theriologica Sinica,2007,27(1):86-91.(in Chinese with English abstract)
[19]任燕,陈献稿,刘鹏威,等.大黄鱼假单胞菌病病原的分离鉴定及药物敏感试验[J].广东农业科学,2012,39(18):151-154.REN Y,CHEN X G,LIU P W,et al.Isolation,identification and the sensitivity to antimicrobial agents of Pseudomonassis pathogen from Great yellow croaker(Pseudosciaena crocea)[J].Guangdong Agricultural Sciences,2012,39(18):151-154.(in Chinese with English abstract)
[20]LADHANI S,BHUTTA Z A.Neonatal Pseudomonas putida infection presenting as staphylococcal scalded skin syndrome[J].European Journal of Clinical Microbiology and Infectious Diseases,1998,17(9):642-644.
[21]覃章纯,汪崇阳,童杨雯.恶臭假单胞菌致小儿败血症一例[J].中华儿科杂志,2000,38(4):238.QIN Z C,WANG C Y,TONG Y W.A case of aeruginosa caused by pseudomonas putida in children[J].Chinese Journal of Pediatrics,2000,38(4):238.(in Chinese)
[22]孔繁才,崔一峰.早餐奶污染恶臭假单胞菌引起食物中毒一例报告[J].中华流行病学杂志,2009,30(1):49.KONG F C,CUI Y F.Food poisoning caused by Pseudomonas putida in breakfast milk:a case report[J].Chinese Journal of Epidemiology,2009,30(1):49.(in Chinese)
[23]李庆山,邢瑞云,张芳萍,等.首次发现恶臭假单胞菌引起的食物中毒[J].中国公共卫生,2000,16(1):50-51.LI Q S,XING R Y,ZHANG F P,et al.The discovery of food-poisoning gaused by Pseudomonas putida for the first time[J].China Public Health,2000,16(1):50-51.(in Chinese with English abstract)
[24]IWALOKUN B A,AKINSINDE K A,LANLENHIN O,et al.Bacteriocinogenicity and production of pyocins from Pseudomonas species isolated in Lagos,Nigeria[J].African Journal of Biotechnology,2006,5(11):1072-1077.
[25]LOMBARDI G,LUZZARO F,DOCQUIER J D,et al.Nosocomial infections caused by multidrug-resistant isolates of Pseudomonas putida producing VIM-1 Metallo-β-Lactamase[J].Journal of Clinical Microbiology,2002,40(11):4051-4055.
[26]谢朝云,熊芸,孙静,等.91株恶臭假单胞菌流行病学特征及耐药性[J].中国感染控制杂志,2017,16(12):1185-1188.XIE Z Y,XIONG Y,SUN J,et al.Epidemiological characteristics and antimicrobial resistance of 91 strains of Pseudomonas putida[J].Chinese Journal of Infection Control,2017,16(12):1185-1188.(in Chinese with English abstract)
[27]HONG J S,YOON E J,SONG W,et al.Molecular characterization of Pseudomonas putida group isolates carrying blaVIM-2 disseminated in a university hospital in Korea[J].Microbial Drug Resistance,2018,24(5):627.
[28]HOLMGAARD D B,HANSEN F,HASMAN H,et al.Characterization of a novel bla IMP gene,bla IMP-58,using whole genome sequencing in a Pseudomonas putida isolate detected in Denmark[J].Diagnostic Microbiology&Infectious Disease,2016,87(1):68-70.
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