水产环境中一株多抗生素抗性菌的分离与耐药鉴定
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摘要
目的:从水产养殖污水中分离潜在抗生素抗性菌,研究其耐药特性。方法:富集和纯化目标菌种,观察菌种形态,测定16S rDNA序列和比较同源性,鉴定菌株的生长特性、抗生素抗性和抗性极限。结果:在水产养殖场鱼塘底部的成熟活性淤泥与水混合液中,筛选分离出一株双抗生素抗性菌(头孢霉素+青霉素抗性),其适宜生长环境偏酸性。16S rDNA序列分析和革兰氏染色分析显示该菌株是变形菌门苍白杆菌属的革兰氏阴性菌,无芽孢。同时该菌株对四环素、头孢霉素、青霉素、卡那霉素、氯霉素均存在抗性。结论:本论文研究结果表明,水产养殖场因使用抗生素,在其底泥中更易沉积多抗生素抗性菌。
Objective: Antibiotic-resistant bacteria would be identified from an aquaculture environment in Guangzhou City.Method: First, the aimed bacteria were enriched and purified using a series of multi-antibiotic medium, and the colonial morphologieswere also compared in the harvested bacteria colonies. Second, the 16S rDNA sequence of a represented bacterium strain was identified by sequencing and homology analysis. Third, the growth effect factors, multiple-antibiotic resistances and resistance-limits of the isolated bacterium strain was further evaluated in the present study.Results: A bacterium strain with the high resistances to both Cephamycin and penicillin was successfully harvestedfrom the mixture of mature activated sludge and water from a typical aquaculture farm, and its optimal growth condition was acidic medium. By 16S rDNA sequencing, it was confirmed to be genus Ochrobactrum. Meanwhile, it was further identified as Gram-negative bacteria and also observedwithout forming spores. Most interestingly, it was showed to be resistant to multiple antibiotics, including tetracycline, tetracycline, Cephamycin, penicillin, kanamycin and chloramphenicol. Conclusion: These results suggests that the antibiotics used frequently easily result in the deposition of the multiple antibiotic resistant bacteria in the sediment of the typical aquaculture farm.
Objective: Antibiotic-resistant bacteria would be identified from an aquaculture environment in Guangzhou City.Method: First, the aimed bacteria were enriched and purified using a series of multi-antibiotic medium, and the colonial morphologieswere also compared in the harvested bacteria colonies. Second, the 16S rDNA sequence of a represented bacterium strain was identified by sequencing and homology analysis. Third, the growth effect factors, multiple-antibiotic resistances and resistance-limits of the isolated bacterium strain was further evaluated in the present study.Results: A bacterium strain with the high resistances to both Cephamycin and penicillin was successfully harvestedfrom the mixture of mature activated sludge and water from a typical aquaculture farm, and its optimal growth condition was acidic medium. By 16S rDNA sequencing, it was confirmed to be genus Ochrobactrum. Meanwhile, it was further identified as Gram-negative bacteria and also observedwithout forming spores. Most interestingly, it was showed to be resistant to multiple antibiotics, including tetracycline, tetracycline, Cephamycin, penicillin, kanamycin and chloramphenicol. Conclusion: These results suggests that the antibiotics used frequently easily result in the deposition of the multiple antibiotic resistant bacteria in the sediment of the typical aquaculture farm.
引文
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[11]冯广达,陈美标,羊宋贞,等.用于PCR扩增的细菌DNA提取方法比较[J/OL].华南农业大学学报,2013,34(03):439-442.
[12]Weisburg W G,Barns S M,Pelletier D A,et al.16S ribosoma-l DNA amplification for phylogenetic study[J].J Bacteriol,1991,173(2):697-703.
[13]Cherif S,Mnif S,Hadrich F,et al.A newly high alkaline lipa-se:an ideal choice for application in detergent formulations[J].Li-pids Health Dis,2011,10:221.
[14]Nishizawa M,Shimizu M,Ohkawa H,et al.Stereoselectivepr-oduction of(+)-trans-chrysanthemic acid by a microbial esterase:cloning,nucleotide sequence,and overexpression of the esterase g-ene of Arthrobacterglobiformis in Escherichia coli[J].Appl Envir-on Microbiol,1995,61(9):3208-3215.
[15]李工厂,屈平华,陈东科,等.30株苍白杆菌的分离鉴定和药物敏感性分析[J].临床检验杂志,2014(12):948-951.
[2]Chen Hui,Liu Shan,Xu Xiangrong,et al.Antibiotics in typical marine aquaculture farms surrounding Hailing Island,South China:Occurrence,bioaccumulationand human dietary exposure[J].Marine Pollution Bulletin,2015,90(1-2):181-187.
[3]Zhao Jianliang,Liu Yousheng,Liu Wangrong,et al.Tissue-specific bioaccumulation of human and veterinary antibiotics in bile,plasma,liver and muscle tissues of wild fish from a highly urbanized region[J].Environmental Pollution,2015,198:15-24.
[4]俞慎,王敏,洪有为.环境介质中的抗生素及其微生物生态效应[J].生态学报,2011,31(15):4437-4446.
[5]Rico A,Oliveira R,Mcdonough S,et al.Use,fateand ecological risks of antibiotics applied in tilapia cagefarming in Thailand[J].Environmental Pollution,2014,191:8-16.
[6]Sassman S A,Lee L S.Sorption of three tetracyclinesbyseveral soils:assessing the role of p H and cation exchange[J].Environmental Science&Technology,2005,39(19):7452-7459.
[7]张冬梅,阿颖利,宋静.青霉素的抗菌作用机制及合理使用分析[J].求医问药:学术版,2012,10(8):149-149.
[8]Spratt B G.Distinct Penicillin Binding Proteins Involved in the Division,Elongation,and Shape of Escherichia coli K12[J].Proc-eedings of the National Academy of Sciences of the United States of America,1975,72(8):2999-3003.
[9]吴向文.头孢菌素类药物的作用特点不良反应及合理应用[J].基层医学论坛,2009,13(17):560-562.
[10]薛雨,陈宇瑛.头孢菌素类抗生素的最新研究进展[J].中国抗生素杂志,2011,36(2):86-92.
[11]冯广达,陈美标,羊宋贞,等.用于PCR扩增的细菌DNA提取方法比较[J/OL].华南农业大学学报,2013,34(03):439-442.
[12]Weisburg W G,Barns S M,Pelletier D A,et al.16S ribosoma-l DNA amplification for phylogenetic study[J].J Bacteriol,1991,173(2):697-703.
[13]Cherif S,Mnif S,Hadrich F,et al.A newly high alkaline lipa-se:an ideal choice for application in detergent formulations[J].Li-pids Health Dis,2011,10:221.
[14]Nishizawa M,Shimizu M,Ohkawa H,et al.Stereoselectivepr-oduction of(+)-trans-chrysanthemic acid by a microbial esterase:cloning,nucleotide sequence,and overexpression of the esterase g-ene of Arthrobacterglobiformis in Escherichia coli[J].Appl Envir-on Microbiol,1995,61(9):3208-3215.
[15]李工厂,屈平华,陈东科,等.30株苍白杆菌的分离鉴定和药物敏感性分析[J].临床检验杂志,2014(12):948-951.