鸡源致病性奇异变形杆菌的分离鉴定与遗传进化分析
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摘要
为了解鸡源奇异变形杆菌的耐药情况、致病力和遗传特征,本试验从凉山地区分离得到2株细菌(YLF1、WS),通过培养特征、镜检特征和16SrRNA序列测定对分离菌进行鉴定及致病性试验;采用(K-B)法检测分离菌对14种常用抗生素(包括β-内酰胺类、头孢烯类、氨基糖苷类、四环素类、氟喹诺酮类、大环内酯类)的敏感性;并与GenBank中公布的21种不同来源的变形杆菌16SrRNA基因序列进行遗传进化分析。结果表明,YLF1、WS菌株均具有迁徙性和β-溶血生长特征,镜检为革兰氏阴性长短不一的杆菌或球菌,经16SrRNA基因鉴定与GenBank中奇异变形杆菌同源性均在99%以上,YLF1、WS菌株确定为奇异变形杆菌,均表现为多重耐药,耐药率分别为78.6%(11/14)和71.4%(10/14);雏鸡致死率均为80%;YLF1、WS菌株与日本人体分离株、中国新疆黄牛分离株和中国广东土壤分离株同源性最高,达99.9%;与来自中国河南、中国新疆及印度的鸡源奇异变形杆菌同源性较高(98.9%~99.3%),遗传关系密切。表明凉山地区鸡群中存在奇异变形杆菌感染,分离株有较高的致病力和较强的耐药性,也提示分离菌可能来源于环境、感染鸡或其他动物,同时存在人畜共患的潜在危险。
In order to understand the drug resistance,virulence and genetic characteristics of chicken source Proteus mirabilis,2 strains of bacteria(YLF1 and WS strains)were prepared by isolation from the Liangshan area,and they were identified by culture characteristics,microscopic characteristics and 16 SrRNA sequence determination,and pathogenicity test.Then the sensitivity of the bacteria to 14 commonly used antibiotics(includingβ-lactams,ceftenes,aminoglycosides,tetracyclines,fulro quinolones and macrolides)was determined using the(K-B)method;The phylogenetic analysis of the 16 SrRNA gene sequences of Proteusfrom 21 different sources published in GenBank was carried out.The results showed that the YLF1 and WS strains all had migratory andβ-hemolytic growth characteristics.The microscopic examination showed gram-negative bacilli or cocci varied in length,the homology of them with Proteus mirabilis in GenBank through the16 SrRNA gene identification was above 99%,YLF1 and WS strains were identified as Proteus mirabilis;YLF1 and WS strains showed multiple drug resistance,the drug resistance rates were78.6%(11/14)and 71.4%(10/14),respectively,the mortality rate of chicks was 80%,the YLF1 and WS strains had the highest homology with Japanese isolate from human,Xinjiang isolate from cattle and Guangdong isolate from soil(99.9%),and they were highly homologous to chicken source Proteus mirabilis from Henan,Xinjiang and India(98.9%to 99.3%),and the genetic relationship was close.These results indicated that Proteus mirabilis infection existed in the chicken populations of Liangshan area.The isolates had high virulence and strong resistance,it also suggested that the isolates might be from the environment,infected chickens or other animals,and there was a potential risk of zoonotic diseases.
In order to understand the drug resistance,virulence and genetic characteristics of chicken source Proteus mirabilis,2 strains of bacteria(YLF1 and WS strains)were prepared by isolation from the Liangshan area,and they were identified by culture characteristics,microscopic characteristics and 16 SrRNA sequence determination,and pathogenicity test.Then the sensitivity of the bacteria to 14 commonly used antibiotics(includingβ-lactams,ceftenes,aminoglycosides,tetracyclines,fulro quinolones and macrolides)was determined using the(K-B)method;The phylogenetic analysis of the 16 SrRNA gene sequences of Proteusfrom 21 different sources published in GenBank was carried out.The results showed that the YLF1 and WS strains all had migratory andβ-hemolytic growth characteristics.The microscopic examination showed gram-negative bacilli or cocci varied in length,the homology of them with Proteus mirabilis in GenBank through the16 SrRNA gene identification was above 99%,YLF1 and WS strains were identified as Proteus mirabilis;YLF1 and WS strains showed multiple drug resistance,the drug resistance rates were78.6%(11/14)and 71.4%(10/14),respectively,the mortality rate of chicks was 80%,the YLF1 and WS strains had the highest homology with Japanese isolate from human,Xinjiang isolate from cattle and Guangdong isolate from soil(99.9%),and they were highly homologous to chicken source Proteus mirabilis from Henan,Xinjiang and India(98.9%to 99.3%),and the genetic relationship was close.These results indicated that Proteus mirabilis infection existed in the chicken populations of Liangshan area.The isolates had high virulence and strong resistance,it also suggested that the isolates might be from the environment,infected chickens or other animals,and there was a potential risk of zoonotic diseases.
引文
[1]陆承平.最新动物病毒分类简介[J].中国病毒学,2005,20(6):682-688.LU C P.An introduction to the animal viruses in newreport[J].Virologica Sinica,2005,20(6):682-688.(in Chinese)
[2]胡传伟,贾赟,简中友,等.水貂奇异变形杆菌的分离与鉴定[J].经济动物学报,2006,10(2):77-79.HU C W,JIA Y,JIAN Z Y,et al.Isolation and identification of Proteus mirabilis from mink[J].Journal of Economic Animal,2006,10(2):77-79.(in Chinese)
[3]史同瑞,李丹,刘宇,等.貂奇异变形杆菌的分离及其生物学鉴定[J].中国预防兽医学报,2013,10:817-820.SHI T R,LI D,LIU Y,et al.Isolation and biological identification of Proteus mirabilis from mink[J].Chinese Journal of Preventive Veterinary Medicine,2013,10:817-820.(in Chinese)
[4]王承东,李德生,汤纯香,等.大熊猫生殖道感染奇异变形杆菌一例[J].四川动物,2007,26(1):167.WANG C D,LI D S,TANG C X,et al.A case genital tract infection with Proteus mirabilis of panda[J].Sichuan Journal of Zoology,2007,26(1):167.(in Chinese)
[5]BISGAARD M,DAM A.Salpingitis in poultryⅡ.Prevalence,bacteriology,and possible pathogenesis in egg-laying chickens[J].Nord Veterinary Medecine,1981,33(2):81-89.
[6]江益民,龙塔,侯玉泽,等.鸡奇异变形杆菌病病原的分离与鉴定[J].河南农业科学,1990,7:31-33.JIANG Y M,LONG T,HOU Y Z,et al.Isolation and identification of pathogen of Proteus mirabilis disease in chicken[J].Journal of Henan Agricultural Sciences,1990,7:31-33.(in Chinese)
[7]许海峰.雏鸡奇异变形杆菌病的诊断[J].中国畜禽传染病,1997,5:45-47.XU H F.Diagnosis of Proteus mirabilis disease in chicks[J].Chinese Infectious Disease of Livestock and Poultry,1997,5:45-47.(in Chinese)
[8]谌南辉,邓舜洲,郭松林,等.珍禽源奇异变形杆菌分离株微生物学特征研究[J].中国预防兽医学报,1999,4:241-245.CHEN N H,DENG S Z,GUO S L,et al.Microbiological characteristics of Proteus mirabilis in rare birds[J].Chinese Journal of Preventive Veterinary Medicine,1999,4:241-245.(in Chinese)
[9]段二珍,夏平安,张凤华,等.狐狸奇异变形杆菌的分离与鉴定[J].中国兽医科学,2008,38(12):1050-1054.DUAN E Z,XIA P A,ZHANG F H,et al.Isolation and identification of Proteus mirabilis in fox[J].Chinese Veterinary Science,2008,38(12):1050-1054.(in Chinese)
[10]高正琴,王春玲,邢进,等.鼠奇异变形杆菌的鉴别诊断[J].实验动物科学,2005,22(4):46-49.GAO Z Q,WANG C L,XING J,et al.Identification of Proteus mirabilis in laboratory mice[J].Laboratory Animal Science,2005,22(4):46-49.(in Chinese)
[11]赵振鹏,杨振,林伟东,等.猪源奇异变形杆菌的分离鉴定及集群运动分析[J].中国畜牧兽医,2014,41(10):219-224.ZHAO Z P,YANG Z,LIN W D,et al.Isolation,identification and cluster motion analysis of Proteus mirabilis in pigs[J].China Animal Husbandry&Veterinary Medicine,2014,41(10):219-224.(in Chinese)
[12]陈曦,王远微,王永,等.山羊腹泻粪便中奇异变形杆菌的分离鉴定及系统发育分析[J].中国畜牧兽医,2014,41(8):215-218.CHEN X,WANG Y W,WANG Y,et al.Isolation,identification and phylogenetic analysis of Proteus mirabilis from goat diarrhea feces[J].China Animal Husbandry&Veterinary Medicine,2014,41(8):215-218.(in Chinese)
[13]赵远,杨凤梅,陈丽雄,等.人工哺育期腹泻婴猴奇异变形杆菌的分离与鉴定[J].中国比较医学杂志,2012,22(4):19-22.ZHAO Y,YANG F M,CHEN L X,et al.Isolation and identification of Proteus mirabilis from infantile monkeys during artificial feeding[J].Chinese Journal of Comparative Medicine,2012,22(4):19-22.(in Chinese)
[14]王瑞君,熊筱娟.棘胸蛙烂皮病奇异变形杆菌的分离、鉴定及对药物敏感性研究[J].淡水渔业,2012,42(4):31-34.WANG R J,XIONG X J.Isolation,identification and drug sensitivity of Proteus mirabilis from bark rot of frog[J].Freshwater Fisheries,2012,42(4):31-34.(in Chinese)
[15]张庆华,熊清明,肖琳琳,等.大黄鱼溃烂症的一种致病菌——奇异变形杆菌ZXS02菌株[J].水产学报,2005,29(6):824-830.ZHANG Q H,XIONG Q M,XIAO L L,et al.Proteus mirabilis ZXS02——A pathogen of large yellow croaker ulceration[J].Journal of Fisheries of China,2005,29(6):824-830.(in Chinese)
[16]潘玉善,苑丽,刘建华,等.鸡大肠杆菌和奇异变形杆菌的分离、鉴定及药敏分析[J].江西农业学报,2009,21(12):156-159.PAN Y S,YUAN L,LIU J H,et al.Isolation,identification and drug sensitivity analysis of Escherichia coli and Proteus mirabilis in chicken[J].Acta Agriculturae Jiangxi,2009,21(12):156-159.(in Chinese)
[17]焦振泉,刘秀梅,孟昭赫.16SrRNA序列同源性分析与细菌系统分类鉴定[J].国外医学(卫生学分册),1998,25(1):12-16.JIAO Z Q,LIU X M,MENG Z H.Homology analysis of 16SrRNA sequence and identification of bacterial system[J].Foreign Medical Sciences(Section of Hygiene),1998,25(1):12-16.(in Chinese)
[18]彭涛,温博海,蹇锐,等.分支杆菌临床分离株的16SrRNA基因序列分析[J].中国人兽共患病杂志,2004,20(9):772-776.PENG T,WEN B H,JIAN R,et al.Sequence analysis of 16SrRNA gene in mycobacterium clinical isolates[J].Chinese Journal of Zoonoses,2004,20(9):772-776.(in Chinese)
[19]朱明华,朱瑞良,马荣德,等.鸡奇异变形杆菌的分离鉴定和16SrRNA基因序列测定与系统进化分析[J].中国兽医学报,2011,31(6):804-808.ZHU M H,ZHU R L,MA R D,et al.Isolation and identification of Proteus mirabilis from chicken and sequencing and phylogenetic analysis of 16SrRNA gene[J].Chinese Journal of Veterinary Science,2011,31(6):804-808.(in Chinese)
[20]CLARRIDGE J E.Impact of 16SrRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases[J].Clinical Microbiology Reviews,2004,17(4):840-862.
[21]DRANCOURT M,BOLLET C,CARLIOZ A,et al.16Sribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates[J].Journal of Clinical Microbiology,2000,38(10):3623-3630.
[22]JANDA J M,ABBOTT S L.Bacterial identification for publication:When is enough[J].Journal of Clinical Microbiology,2002,40(6):1887-1891.
[23]禹文海,赵远,黄芬,等.PCR方法检测猕猴奇异变形杆菌[J].中国人兽共患病学报,2013,29(3):278-281.YU W H,ZHAO Y,HUANG F,et al.PCR detection of Proteus mirabilis in rhesus monkeys[J].Chinese Journal of Zoonoses,2013,29(3):278-281.(in Chinese)
[24]曾超锋,余冬令.一起奇异变形杆菌食物中毒的实验室诊断及分析[J].国际医药卫生导报,2008,14(21):89-90.ZENG C F,YU D L.Laboratory diagnosis and analysis of a Proteus mirabilis food poisoning[J].International Medicine and Health Guidance News,2008,14(21):89-90.(in Chinese)
[25]李少彤,孙凤琪,曾德荣,等.一起奇异变形杆菌引起食物中毒的病原学分析[J].中国卫生检验杂志,2002,12(5):620.LI S T,SUN F Q,ZENG D R,et al.Etiological analysis of a food poisoning by Proteus mirabilis[J].Chinese Journal of Health Laboratory Technology,2002,12(5):620.(in Chinese)
[26]COHEN-NAHUM K,SAIDEL-ODES L,RIESENBERG K,et al.Urinary tract infections caused by multi-drug resistant Proteus mirabilis:Risk factors and clinical outcomes[J].Infection,2010,38(1):41-46.
[2]胡传伟,贾赟,简中友,等.水貂奇异变形杆菌的分离与鉴定[J].经济动物学报,2006,10(2):77-79.HU C W,JIA Y,JIAN Z Y,et al.Isolation and identification of Proteus mirabilis from mink[J].Journal of Economic Animal,2006,10(2):77-79.(in Chinese)
[3]史同瑞,李丹,刘宇,等.貂奇异变形杆菌的分离及其生物学鉴定[J].中国预防兽医学报,2013,10:817-820.SHI T R,LI D,LIU Y,et al.Isolation and biological identification of Proteus mirabilis from mink[J].Chinese Journal of Preventive Veterinary Medicine,2013,10:817-820.(in Chinese)
[4]王承东,李德生,汤纯香,等.大熊猫生殖道感染奇异变形杆菌一例[J].四川动物,2007,26(1):167.WANG C D,LI D S,TANG C X,et al.A case genital tract infection with Proteus mirabilis of panda[J].Sichuan Journal of Zoology,2007,26(1):167.(in Chinese)
[5]BISGAARD M,DAM A.Salpingitis in poultryⅡ.Prevalence,bacteriology,and possible pathogenesis in egg-laying chickens[J].Nord Veterinary Medecine,1981,33(2):81-89.
[6]江益民,龙塔,侯玉泽,等.鸡奇异变形杆菌病病原的分离与鉴定[J].河南农业科学,1990,7:31-33.JIANG Y M,LONG T,HOU Y Z,et al.Isolation and identification of pathogen of Proteus mirabilis disease in chicken[J].Journal of Henan Agricultural Sciences,1990,7:31-33.(in Chinese)
[7]许海峰.雏鸡奇异变形杆菌病的诊断[J].中国畜禽传染病,1997,5:45-47.XU H F.Diagnosis of Proteus mirabilis disease in chicks[J].Chinese Infectious Disease of Livestock and Poultry,1997,5:45-47.(in Chinese)
[8]谌南辉,邓舜洲,郭松林,等.珍禽源奇异变形杆菌分离株微生物学特征研究[J].中国预防兽医学报,1999,4:241-245.CHEN N H,DENG S Z,GUO S L,et al.Microbiological characteristics of Proteus mirabilis in rare birds[J].Chinese Journal of Preventive Veterinary Medicine,1999,4:241-245.(in Chinese)
[9]段二珍,夏平安,张凤华,等.狐狸奇异变形杆菌的分离与鉴定[J].中国兽医科学,2008,38(12):1050-1054.DUAN E Z,XIA P A,ZHANG F H,et al.Isolation and identification of Proteus mirabilis in fox[J].Chinese Veterinary Science,2008,38(12):1050-1054.(in Chinese)
[10]高正琴,王春玲,邢进,等.鼠奇异变形杆菌的鉴别诊断[J].实验动物科学,2005,22(4):46-49.GAO Z Q,WANG C L,XING J,et al.Identification of Proteus mirabilis in laboratory mice[J].Laboratory Animal Science,2005,22(4):46-49.(in Chinese)
[11]赵振鹏,杨振,林伟东,等.猪源奇异变形杆菌的分离鉴定及集群运动分析[J].中国畜牧兽医,2014,41(10):219-224.ZHAO Z P,YANG Z,LIN W D,et al.Isolation,identification and cluster motion analysis of Proteus mirabilis in pigs[J].China Animal Husbandry&Veterinary Medicine,2014,41(10):219-224.(in Chinese)
[12]陈曦,王远微,王永,等.山羊腹泻粪便中奇异变形杆菌的分离鉴定及系统发育分析[J].中国畜牧兽医,2014,41(8):215-218.CHEN X,WANG Y W,WANG Y,et al.Isolation,identification and phylogenetic analysis of Proteus mirabilis from goat diarrhea feces[J].China Animal Husbandry&Veterinary Medicine,2014,41(8):215-218.(in Chinese)
[13]赵远,杨凤梅,陈丽雄,等.人工哺育期腹泻婴猴奇异变形杆菌的分离与鉴定[J].中国比较医学杂志,2012,22(4):19-22.ZHAO Y,YANG F M,CHEN L X,et al.Isolation and identification of Proteus mirabilis from infantile monkeys during artificial feeding[J].Chinese Journal of Comparative Medicine,2012,22(4):19-22.(in Chinese)
[14]王瑞君,熊筱娟.棘胸蛙烂皮病奇异变形杆菌的分离、鉴定及对药物敏感性研究[J].淡水渔业,2012,42(4):31-34.WANG R J,XIONG X J.Isolation,identification and drug sensitivity of Proteus mirabilis from bark rot of frog[J].Freshwater Fisheries,2012,42(4):31-34.(in Chinese)
[15]张庆华,熊清明,肖琳琳,等.大黄鱼溃烂症的一种致病菌——奇异变形杆菌ZXS02菌株[J].水产学报,2005,29(6):824-830.ZHANG Q H,XIONG Q M,XIAO L L,et al.Proteus mirabilis ZXS02——A pathogen of large yellow croaker ulceration[J].Journal of Fisheries of China,2005,29(6):824-830.(in Chinese)
[16]潘玉善,苑丽,刘建华,等.鸡大肠杆菌和奇异变形杆菌的分离、鉴定及药敏分析[J].江西农业学报,2009,21(12):156-159.PAN Y S,YUAN L,LIU J H,et al.Isolation,identification and drug sensitivity analysis of Escherichia coli and Proteus mirabilis in chicken[J].Acta Agriculturae Jiangxi,2009,21(12):156-159.(in Chinese)
[17]焦振泉,刘秀梅,孟昭赫.16SrRNA序列同源性分析与细菌系统分类鉴定[J].国外医学(卫生学分册),1998,25(1):12-16.JIAO Z Q,LIU X M,MENG Z H.Homology analysis of 16SrRNA sequence and identification of bacterial system[J].Foreign Medical Sciences(Section of Hygiene),1998,25(1):12-16.(in Chinese)
[18]彭涛,温博海,蹇锐,等.分支杆菌临床分离株的16SrRNA基因序列分析[J].中国人兽共患病杂志,2004,20(9):772-776.PENG T,WEN B H,JIAN R,et al.Sequence analysis of 16SrRNA gene in mycobacterium clinical isolates[J].Chinese Journal of Zoonoses,2004,20(9):772-776.(in Chinese)
[19]朱明华,朱瑞良,马荣德,等.鸡奇异变形杆菌的分离鉴定和16SrRNA基因序列测定与系统进化分析[J].中国兽医学报,2011,31(6):804-808.ZHU M H,ZHU R L,MA R D,et al.Isolation and identification of Proteus mirabilis from chicken and sequencing and phylogenetic analysis of 16SrRNA gene[J].Chinese Journal of Veterinary Science,2011,31(6):804-808.(in Chinese)
[20]CLARRIDGE J E.Impact of 16SrRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases[J].Clinical Microbiology Reviews,2004,17(4):840-862.
[21]DRANCOURT M,BOLLET C,CARLIOZ A,et al.16Sribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates[J].Journal of Clinical Microbiology,2000,38(10):3623-3630.
[22]JANDA J M,ABBOTT S L.Bacterial identification for publication:When is enough[J].Journal of Clinical Microbiology,2002,40(6):1887-1891.
[23]禹文海,赵远,黄芬,等.PCR方法检测猕猴奇异变形杆菌[J].中国人兽共患病学报,2013,29(3):278-281.YU W H,ZHAO Y,HUANG F,et al.PCR detection of Proteus mirabilis in rhesus monkeys[J].Chinese Journal of Zoonoses,2013,29(3):278-281.(in Chinese)
[24]曾超锋,余冬令.一起奇异变形杆菌食物中毒的实验室诊断及分析[J].国际医药卫生导报,2008,14(21):89-90.ZENG C F,YU D L.Laboratory diagnosis and analysis of a Proteus mirabilis food poisoning[J].International Medicine and Health Guidance News,2008,14(21):89-90.(in Chinese)
[25]李少彤,孙凤琪,曾德荣,等.一起奇异变形杆菌引起食物中毒的病原学分析[J].中国卫生检验杂志,2002,12(5):620.LI S T,SUN F Q,ZENG D R,et al.Etiological analysis of a food poisoning by Proteus mirabilis[J].Chinese Journal of Health Laboratory Technology,2002,12(5):620.(in Chinese)
[26]COHEN-NAHUM K,SAIDEL-ODES L,RIESENBERG K,et al.Urinary tract infections caused by multi-drug resistant Proteus mirabilis:Risk factors and clinical outcomes[J].Infection,2010,38(1):41-46.