水生动物源嗜芳香环弧菌的鉴定及耐药性分析
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摘要
本研究从患病的半滑舌鳎分离得到5株弧菌,对其进行种属鉴定,并研究其耐药性及分子水平耐药机制。采用生化检测方法和分子生物学方法(基因16S rRNA、HSP60和mreB)进行鉴定,结果显示,5株弧菌均为嗜芳香环弧菌。通过肉汤稀释法检测菌株对抗菌药物的耐药性,通过PCR扩增和测序技术检测菌株中耐药基因和4类整合子并对其携带基因盒进行分析。结果显示,5株嗜芳香环弧菌均对环丙沙星、链霉素、红霉素、氟苯尼考、氨苄西林、磺胺甲恶唑、复方新诺明和利福平耐药,且均携带qnr VC、bla CARB-17、strA、strB和sul耐药基因。此外,菌株JS291和JS295携带Ⅰ类整合子、qacEΔ1和sul1耐药基因;可变区携带基因盒分别为dfr16-aad A2和arr-3-dfr A27。5株嗜芳香环弧菌为多重耐药菌株,且多重耐药表型与耐药基因密切相关。
This paper studied the identification, drug resistance and the molecular resistance mechanism of five Vibrio strains isolated from diseased Cynoglossus semilaevis. The five strains were identified as V. cyclotrophicus by a biochemical test combined with a molecular method(16 S rRNA, HSP60 and mreB genes). The broth dilution method was used to determine the antimicrobial susceptibility of the strains to 14 antimicrobials. The PCR amplification and DNA sequencing were used to detect antimicrobial resistance genes, integrons and the gene cassettes. The results indicated that the five strains were resistant to ciprofloxacin, streptomycin, erythromycin,florfenicol, ampicillin, SMZ, TMP/SMZ and rifampicin, and carried qnr VC, bla CARB-17, strA, strB, sul genes.Class I intergron, qacEΔ1 and sul1 genes were present in JS291 and JS295 strains. The integron-gene cassettes of JS291 were encoding dihydrofolatereductase(dfr16), aminoglycoside-(3″)(9)-adenylyltransferase(aadA2), and the cassettes of JS295 were encoding rifampin ADP-ribosylating transferase(arr-3), dihydrofolate reductase(dfrA27),respectively. These findings suggested that the five strains exhibited multi-drug resistance and the multi-drug resistance phenotype may be closely related to the antimicrobial resistance genes.
This paper studied the identification, drug resistance and the molecular resistance mechanism of five Vibrio strains isolated from diseased Cynoglossus semilaevis. The five strains were identified as V. cyclotrophicus by a biochemical test combined with a molecular method(16 S rRNA, HSP60 and mreB genes). The broth dilution method was used to determine the antimicrobial susceptibility of the strains to 14 antimicrobials. The PCR amplification and DNA sequencing were used to detect antimicrobial resistance genes, integrons and the gene cassettes. The results indicated that the five strains were resistant to ciprofloxacin, streptomycin, erythromycin,florfenicol, ampicillin, SMZ, TMP/SMZ and rifampicin, and carried qnr VC, bla CARB-17, strA, strB, sul genes.Class I intergron, qacEΔ1 and sul1 genes were present in JS291 and JS295 strains. The integron-gene cassettes of JS291 were encoding dihydrofolatereductase(dfr16), aminoglycoside-(3″)(9)-adenylyltransferase(aadA2), and the cassettes of JS295 were encoding rifampin ADP-ribosylating transferase(arr-3), dihydrofolate reductase(dfrA27),respectively. These findings suggested that the five strains exhibited multi-drug resistance and the multi-drug resistance phenotype may be closely related to the antimicrobial resistance genes.
引文
[1]Jiang Y, Yao L, Li F, et al. Characterization of antimicrobial resistance of Vibrio parahaemolyticus from cultured sea cucumbers(Apostichopus japonicas)[J]. Letters in Applied Microbiology, 2014, 59(2):147-154.
[2]Letchumanan V, Yin W F, Lee L H, et al. Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shrimps in Malaysia[J].Frontiers in Microbiology, 2015, 6:33.
[3]Bennett P M. Plasmid encoded antibiotic resistance:acquisition and transfer of antibiotic resistance genes in bacteria[J]. British Journal of Pharmacology, 2008,153(S1):S347-S357.
[4]Liu M, Wong M H Y, Chen S. Molecular characterisation of a multidrug resistance conjugative plasmid from Vibrio Parahaemolyticus[J]. International Journal of Antimicrobial Agents, 2013,42(6):575-579.
[5]刘彦,窦海鸽.半滑舌鳎生物学特征及其增养殖发展前景[J].中国水产,2005(3):63-64.Liu Y, Dou H G. The biology characteristics and enhancement foreground of Cynoglossus semilaevis[J]. China Fisheries, 2005(3):63-64(in Chinese).
[6]陈君.工厂化循环水养殖半滑舌鳎主要细菌性疾病及其控制[D].上海:上海海洋大学,2012.Chen J. Bacterial diseasesand controlof tongue fish(Cynoglossus semilaevis)in recirculating aquaculture system[D]. Shanghai:Shanghai Ocean University,2012(in Chinese).
[7]徐先栋,谢珍玉,欧阳吉隆,等.褐点石斑鱼脱鳞病病原菌的分离与鉴定[J].海洋科学,2012, 36(2):67-74.Xu X D, Xie Z Y, Ouyang J L, et al. Isolation and identification of bacterial pathogens from Epinephelus fuscoguttatus with scale-peeled syndrome disease[J].Marine Sciences, 2012, 36(2):67-74(in Chinese).
[8]陈晓燕,胡超群,陈偿,等.人工养殖点带石斑鱼弧菌病病原菌的分离及鉴定[J].海洋科学,2003, 27(6):68-72.Chen X Y, Hu C Q, Chen C, et al. Characteristics of pathogenic bacteria of Vibriosis in caged Malabar grouper, Epinephelus malabaricus[J]. Marine Sciences,2003, 27(6):68-72(in Chinese).
[9]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.Dong X Z, Cai M Y. Manual of Determinative common bacteriology[M]. Beijing:Science Press, 2001(in Chinese).
[10] CLSI. M45-A2 Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria:approved guideline-second edition[S]. Wayne, PA:National Committee for Clinical Laboratory Standards, 2010.
[11] Nilsson W B, Paranjype R N, Depaola A, et al. Sequence polymorphism of the 16S rRNA gene of Vibrio vulnificus is a possible indicator of strain virulence[J]. Journal of Clinical Microbiology,2003, 41(1):442-446.
[12]李林桂,周俊芳,李新苍,等.对虾源副溶血弧菌强毒株HNBX-1的分离鉴定与毒性分析[J].海洋渔业,2013, 35(4):479-484.Li L G, Zhou J F, Li X C, et al. Identification and toxicity analysis of a virulent Vibrio parahaemolyticus strain isolated from Litopenaeus vannamei[J]. Marine Fisheries, 2013, 35(4):479-484(in Chinese).
[13] Sawabe T, Kita-Tsukamoto K, Thompson F L. Inferring the evolutionary history of Vibrios by means of multilocus sequence analysis[J]. Journal of Bacteriology, 2007,189(21):7962-7936.
[14]国宪虎.耐药整合子细菌在水环境中的散播调查及其多样性分析[D].济南:山东大学,2011.Guo X H. Investigation and diversity analysis of the antibiotic resistant integron-bearing bacteria in aquatic environment[D]. Ji'nan:Shandong University, 2011(in Chinese).
[15] Sandvang D,Aarestrup F M,Jensen L B. Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DTI-04[J]. FEMS Microbiology Letters,1998, 160(1):37-41.
[16] Hedlund B P, Staley J T. Vibrio cyclotrophicus sp. nov.,a polycyclic aromatic hydrocarbon(PHA)-degrading marine bacterium[J]. International Journal of Systematicand Evolutionary Microbiology, 2001, 51:61-66.
[17] Sawabe T, Koizumi S, Fukui Y, et al. Mutation is the main driving force in the diversification of the Vibrio Splendidus clade[J]. Microbes and Environments, 2009,24(4):281-285.
[18]王轶南,刘艳萍,常亚青.患病与健康虾夷马粪海胆(Strongylocentrotus intermedius)体腔液菌群的PCRDGGE分析比较[J].中国农业科技导报,2011,13(1):111-116.Wang Y N, Liu Y P, Chang Y Q. Comparative analysis of bacterial community composition in coelomic fluid between sick and healthy Stronylocentrotus intermedius by PCR-DGGE[J]. Journal of Agricultural Science and Technology, 2011,13(1):11 1-116(in Chinese).
[19]杨扬.应用噬菌体防治弧菌引发的刺参腐皮综合征[D].大连:大连理工大学,2013.Yang Y. Using bacteriophages to control skin ulceration syndrome of the Sea Cucumber(Apostichopus japonicus)caused by Vibrio spp.[D]. Dalian:Dalian University of Technology, 2013(in Chinese).
[20] Deng H, He C B, Zhou Z C, et al. Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicus[J]. Aquaculture, 2009, 287(1-2):18-27.
[21] Hidalgo R B, Cleenwerck I, Balboa S, et al. Diversity of Vibrios associated with reared clams in Galicia(NW Spain)[J]. Systematic and Applied Microbiology, 2008,31(3):215-222.
[22] Austin B, Austin D, Sutherland R, et al. Pathogenicity of vibrios to rainbow trout(Oncorhynchus my kiss, Walbaum)and Artemia nauplii[J]. Environmental Microbiology, 2005, 7(9):1488-1495(in Chinese).
[23]杨求华,葛辉,方旅平,等.池塘养殖刺参病原菌塔式弧菌的分离与鉴定[J].南方水产科学,2014, 10(4):45-51.Yang Q H, Ge H, Fang L P, et al. Identification of Vibrio tubiashii isolated from diseased pond-cultured sea cucumbers(Apostichopus japonicus)[J]. South China Fisheries Science,2014, 10(4):45-51(in Chinese).
[24]王春艳,阎斌伦.连云港市赣榆县水产养殖环境中哈氏弧菌耐药性研究[J].淮海工学院学报(自然科学版),2013, 22(1):88-91.Wang C Y, Yan B L. Study on the antibiotic resistance of Vibrio harveyi isolates from aquaculture environmentin Ganyu County, Lianyungang[J]. Journal of Huaihai Institute of Technology(Natural Sciences Edition),2013, 22(1):88-91(in Chinese).
[25] Ceccarelli D, Salvia A M, Sami J, et al. New cluster of plasmid-located class 1 integrons in Vibrio cholerae 01and a dfrA15 cassette-containing integron in Vibrio parahaemolyticus isolated in Angola[J]. Antimicrobial Agents and Chemotherapy, 2006, 50(7):2493-2499.
[26]写腊月,胡琳琳,房文红,等.海水养殖源弧菌耐药性调查与分析[J].海洋渔业,2011,33(4):442-446.Xie L Y, Hu L L, Fang W H, et al.Investigation and analysis of drug resistance of Vibrios from mariculture source[J]. Marine Fisheries, 2011, 33(4):442-446(in Chinese).
[27] Shah S Q A, Cabello F C, L'Abee-Lund T M, et al.Antimicrobial resistance and antimicrobial resistance genes in marine bacteria from salmon aquaculture and non-aquaculture sites[J]. Environmental Microbiology,2014, 16(5):1310-1320.
[28] Chiou J,Li R C,Chen S. CARB-17 family ofβ-lactamases mediates intrinsic resistance to Penicillins in Vibrio parahaemolyticus[J]. Antimicrobial Agents and Chemotherapy,2015, 59(6):3593-3595.
[29] Liu M,Wong M H Y,Chen S. Mechanisms of fluoroquin olone resistance in Vibrio parahaemolyticus[J]. International Journal of Antimicrobial Agents, 2013, 42(2):187-188.
[30] Mazel D. Integrons:agents of bacterial evolution[J].Nature Reviews Microbiology, 2006, 4(8):608-620.
[31] Lukkana M, Wongtavatchai J, Chuanchuen R. Class 1integrons in Aeromonas hydrophila isolates from farmed Nile tilapia(Oreochromis nilotica)[J]. The Journal of Veterinary Medical Science, 2012, 74(4):435-440.
[32]李绍戊,王荻,刘红柏,等.鱼源嗜水气单胞菌多重耐药菌株整合子的分子特征[J].中国水产科学,2013,20(5):1015-1022.Li S W, Wang D, Liu H B, et al. Molecular characteriz ation of integron-gene cassettes in multi-drug resistant Aeromonas hydrophila from fish[J]. Journal of Fishery Sciences of China, 2013, 20(5):1015-1022(in Chinese).
[2]Letchumanan V, Yin W F, Lee L H, et al. Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shrimps in Malaysia[J].Frontiers in Microbiology, 2015, 6:33.
[3]Bennett P M. Plasmid encoded antibiotic resistance:acquisition and transfer of antibiotic resistance genes in bacteria[J]. British Journal of Pharmacology, 2008,153(S1):S347-S357.
[4]Liu M, Wong M H Y, Chen S. Molecular characterisation of a multidrug resistance conjugative plasmid from Vibrio Parahaemolyticus[J]. International Journal of Antimicrobial Agents, 2013,42(6):575-579.
[5]刘彦,窦海鸽.半滑舌鳎生物学特征及其增养殖发展前景[J].中国水产,2005(3):63-64.Liu Y, Dou H G. The biology characteristics and enhancement foreground of Cynoglossus semilaevis[J]. China Fisheries, 2005(3):63-64(in Chinese).
[6]陈君.工厂化循环水养殖半滑舌鳎主要细菌性疾病及其控制[D].上海:上海海洋大学,2012.Chen J. Bacterial diseasesand controlof tongue fish(Cynoglossus semilaevis)in recirculating aquaculture system[D]. Shanghai:Shanghai Ocean University,2012(in Chinese).
[7]徐先栋,谢珍玉,欧阳吉隆,等.褐点石斑鱼脱鳞病病原菌的分离与鉴定[J].海洋科学,2012, 36(2):67-74.Xu X D, Xie Z Y, Ouyang J L, et al. Isolation and identification of bacterial pathogens from Epinephelus fuscoguttatus with scale-peeled syndrome disease[J].Marine Sciences, 2012, 36(2):67-74(in Chinese).
[8]陈晓燕,胡超群,陈偿,等.人工养殖点带石斑鱼弧菌病病原菌的分离及鉴定[J].海洋科学,2003, 27(6):68-72.Chen X Y, Hu C Q, Chen C, et al. Characteristics of pathogenic bacteria of Vibriosis in caged Malabar grouper, Epinephelus malabaricus[J]. Marine Sciences,2003, 27(6):68-72(in Chinese).
[9]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.Dong X Z, Cai M Y. Manual of Determinative common bacteriology[M]. Beijing:Science Press, 2001(in Chinese).
[10] CLSI. M45-A2 Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria:approved guideline-second edition[S]. Wayne, PA:National Committee for Clinical Laboratory Standards, 2010.
[11] Nilsson W B, Paranjype R N, Depaola A, et al. Sequence polymorphism of the 16S rRNA gene of Vibrio vulnificus is a possible indicator of strain virulence[J]. Journal of Clinical Microbiology,2003, 41(1):442-446.
[12]李林桂,周俊芳,李新苍,等.对虾源副溶血弧菌强毒株HNBX-1的分离鉴定与毒性分析[J].海洋渔业,2013, 35(4):479-484.Li L G, Zhou J F, Li X C, et al. Identification and toxicity analysis of a virulent Vibrio parahaemolyticus strain isolated from Litopenaeus vannamei[J]. Marine Fisheries, 2013, 35(4):479-484(in Chinese).
[13] Sawabe T, Kita-Tsukamoto K, Thompson F L. Inferring the evolutionary history of Vibrios by means of multilocus sequence analysis[J]. Journal of Bacteriology, 2007,189(21):7962-7936.
[14]国宪虎.耐药整合子细菌在水环境中的散播调查及其多样性分析[D].济南:山东大学,2011.Guo X H. Investigation and diversity analysis of the antibiotic resistant integron-bearing bacteria in aquatic environment[D]. Ji'nan:Shandong University, 2011(in Chinese).
[15] Sandvang D,Aarestrup F M,Jensen L B. Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DTI-04[J]. FEMS Microbiology Letters,1998, 160(1):37-41.
[16] Hedlund B P, Staley J T. Vibrio cyclotrophicus sp. nov.,a polycyclic aromatic hydrocarbon(PHA)-degrading marine bacterium[J]. International Journal of Systematicand Evolutionary Microbiology, 2001, 51:61-66.
[17] Sawabe T, Koizumi S, Fukui Y, et al. Mutation is the main driving force in the diversification of the Vibrio Splendidus clade[J]. Microbes and Environments, 2009,24(4):281-285.
[18]王轶南,刘艳萍,常亚青.患病与健康虾夷马粪海胆(Strongylocentrotus intermedius)体腔液菌群的PCRDGGE分析比较[J].中国农业科技导报,2011,13(1):111-116.Wang Y N, Liu Y P, Chang Y Q. Comparative analysis of bacterial community composition in coelomic fluid between sick and healthy Stronylocentrotus intermedius by PCR-DGGE[J]. Journal of Agricultural Science and Technology, 2011,13(1):11 1-116(in Chinese).
[19]杨扬.应用噬菌体防治弧菌引发的刺参腐皮综合征[D].大连:大连理工大学,2013.Yang Y. Using bacteriophages to control skin ulceration syndrome of the Sea Cucumber(Apostichopus japonicus)caused by Vibrio spp.[D]. Dalian:Dalian University of Technology, 2013(in Chinese).
[20] Deng H, He C B, Zhou Z C, et al. Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicus[J]. Aquaculture, 2009, 287(1-2):18-27.
[21] Hidalgo R B, Cleenwerck I, Balboa S, et al. Diversity of Vibrios associated with reared clams in Galicia(NW Spain)[J]. Systematic and Applied Microbiology, 2008,31(3):215-222.
[22] Austin B, Austin D, Sutherland R, et al. Pathogenicity of vibrios to rainbow trout(Oncorhynchus my kiss, Walbaum)and Artemia nauplii[J]. Environmental Microbiology, 2005, 7(9):1488-1495(in Chinese).
[23]杨求华,葛辉,方旅平,等.池塘养殖刺参病原菌塔式弧菌的分离与鉴定[J].南方水产科学,2014, 10(4):45-51.Yang Q H, Ge H, Fang L P, et al. Identification of Vibrio tubiashii isolated from diseased pond-cultured sea cucumbers(Apostichopus japonicus)[J]. South China Fisheries Science,2014, 10(4):45-51(in Chinese).
[24]王春艳,阎斌伦.连云港市赣榆县水产养殖环境中哈氏弧菌耐药性研究[J].淮海工学院学报(自然科学版),2013, 22(1):88-91.Wang C Y, Yan B L. Study on the antibiotic resistance of Vibrio harveyi isolates from aquaculture environmentin Ganyu County, Lianyungang[J]. Journal of Huaihai Institute of Technology(Natural Sciences Edition),2013, 22(1):88-91(in Chinese).
[25] Ceccarelli D, Salvia A M, Sami J, et al. New cluster of plasmid-located class 1 integrons in Vibrio cholerae 01and a dfrA15 cassette-containing integron in Vibrio parahaemolyticus isolated in Angola[J]. Antimicrobial Agents and Chemotherapy, 2006, 50(7):2493-2499.
[26]写腊月,胡琳琳,房文红,等.海水养殖源弧菌耐药性调查与分析[J].海洋渔业,2011,33(4):442-446.Xie L Y, Hu L L, Fang W H, et al.Investigation and analysis of drug resistance of Vibrios from mariculture source[J]. Marine Fisheries, 2011, 33(4):442-446(in Chinese).
[27] Shah S Q A, Cabello F C, L'Abee-Lund T M, et al.Antimicrobial resistance and antimicrobial resistance genes in marine bacteria from salmon aquaculture and non-aquaculture sites[J]. Environmental Microbiology,2014, 16(5):1310-1320.
[28] Chiou J,Li R C,Chen S. CARB-17 family ofβ-lactamases mediates intrinsic resistance to Penicillins in Vibrio parahaemolyticus[J]. Antimicrobial Agents and Chemotherapy,2015, 59(6):3593-3595.
[29] Liu M,Wong M H Y,Chen S. Mechanisms of fluoroquin olone resistance in Vibrio parahaemolyticus[J]. International Journal of Antimicrobial Agents, 2013, 42(2):187-188.
[30] Mazel D. Integrons:agents of bacterial evolution[J].Nature Reviews Microbiology, 2006, 4(8):608-620.
[31] Lukkana M, Wongtavatchai J, Chuanchuen R. Class 1integrons in Aeromonas hydrophila isolates from farmed Nile tilapia(Oreochromis nilotica)[J]. The Journal of Veterinary Medical Science, 2012, 74(4):435-440.
[32]李绍戊,王荻,刘红柏,等.鱼源嗜水气单胞菌多重耐药菌株整合子的分子特征[J].中国水产科学,2013,20(5):1015-1022.Li S W, Wang D, Liu H B, et al. Molecular characteriz ation of integron-gene cassettes in multi-drug resistant Aeromonas hydrophila from fish[J]. Journal of Fishery Sciences of China, 2013, 20(5):1015-1022(in Chinese).
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