凡纳滨对虾和罗氏沼虾肠道微生物及抗生素抗性基因多样性分析
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摘要
探究凡纳滨对虾和罗氏沼虾肠道微生物及抗生素抗性基因(antibiotic resistance genes, ARGs)种类的差异。通过高通量测序和变性梯度凝胶电泳(denaturing gradient gel electrophoresis, DGGE)技术分析2种虾肠道微生物群落结构差异和微生物多样性,并运用PCR方法检测了2种虾肠道细菌常见38种ARGs的携带情况。结果显示,获得凡纳滨对虾和罗氏沼虾肠道细菌有效序列分别为42 795和40 713条,物种注释单元(operational taxonomic unit, OTU)数目分别为124和82,分类地位明确的细菌种类分别隶属5个门、17个属和5个门、16个属。凡纳滨对虾肠道细菌的优势类群为变形菌门,所占比例为75.45%,优势菌属为副球菌属(25.83%)和不动杆菌属(25.24%);罗氏沼虾肠道细菌的优势类群是厚壁菌门(49.74%),优势菌属为乳球菌属(49.01%)和弧菌属(29.98%)。凡纳滨对虾肠道细菌(2.19)Shannon指数高于罗氏沼虾肠道细菌(1.78),表明前者肠道细菌多样性大于后者。DGGE图谱的分析结果与高通量测序一致,2种虾肠道细菌种类差异很大。PCR结果显示,凡纳滨对虾肠道细菌携带15种ARGs,罗氏沼虾肠道细菌携带14种ARGs。本实验表明凡纳滨对虾肠道细菌的群落种类多样性、OTU丰富度、物种总数和ARGs种类均高于罗氏沼虾肠道细菌,为后续肠道微生物资源的挖掘提供了理论依据。
To compare the differences in gut microbiota and antibiotic resistance genes(ARGs) between Litopenaeus vannamei and Macrobrachium rosenbergii, microbiota community structure and microbiota diversity of two shrimps were analyzed by high-throughput sequencing and denaturing gradient gel electrophoresis(DGGE),and 38 kinds of ARGs in the gut bacteria of two shrimps were detected. The effective bacterial sequences of L. vannamei and M. rosenbergii were 42 795 and 40 713. The results showed that the numbers of operational taxonomic units(OTUs) were 124 and 82, and the bacterial types with clear classification status were 5 phyla, 17 genera and 5 phyla, 16 genera. The dominant group in the gut bacteria of L. vannamei is Proteobacteria, which accounts for75.45%. The dominant genera are Paracoccus(25.83%) and Acinetobacter(25.24%). The dominant group in gut bacteria of M. rosenbergii is Firmicutes(49.74%), and the dominant genera are Lactococcus(49.01%) and Vibrio(29.98%). The Shannon index of the gut bacteria(2.19) of L. vannamei was higher than that of M. rosenbergii(1.78), indicating that the gut bacteria diversity of the former was greater than that of the latter. The analysis results of DGGE fingerprint were consistent with those of high-throughput sequencing, and the bacterial diversity of the two shrimps was significantly different. The PCR results showed that the gut bacteria of L. vannamei carried 15 ARGs and gut bacteria of M. rosenbergii carried 14 ARGs. Our study revealed that the community diversity, OTU richness, total number of species and ARGs in the gut bacteria of L. vannamei were higher than those of M.rosenbergii, which provided a theoretical basis for the subsequent excavation of gut microbial resources.
To compare the differences in gut microbiota and antibiotic resistance genes(ARGs) between Litopenaeus vannamei and Macrobrachium rosenbergii, microbiota community structure and microbiota diversity of two shrimps were analyzed by high-throughput sequencing and denaturing gradient gel electrophoresis(DGGE),and 38 kinds of ARGs in the gut bacteria of two shrimps were detected. The effective bacterial sequences of L. vannamei and M. rosenbergii were 42 795 and 40 713. The results showed that the numbers of operational taxonomic units(OTUs) were 124 and 82, and the bacterial types with clear classification status were 5 phyla, 17 genera and 5 phyla, 16 genera. The dominant group in the gut bacteria of L. vannamei is Proteobacteria, which accounts for75.45%. The dominant genera are Paracoccus(25.83%) and Acinetobacter(25.24%). The dominant group in gut bacteria of M. rosenbergii is Firmicutes(49.74%), and the dominant genera are Lactococcus(49.01%) and Vibrio(29.98%). The Shannon index of the gut bacteria(2.19) of L. vannamei was higher than that of M. rosenbergii(1.78), indicating that the gut bacteria diversity of the former was greater than that of the latter. The analysis results of DGGE fingerprint were consistent with those of high-throughput sequencing, and the bacterial diversity of the two shrimps was significantly different. The PCR results showed that the gut bacteria of L. vannamei carried 15 ARGs and gut bacteria of M. rosenbergii carried 14 ARGs. Our study revealed that the community diversity, OTU richness, total number of species and ARGs in the gut bacteria of L. vannamei were higher than those of M.rosenbergii, which provided a theoretical basis for the subsequent excavation of gut microbial resources.
引文
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[24] Lou Y, Liu H Q, Zhang Z H, et al. Mismatch between antimicrobial resistance phenotype and genotype of pathogenic Vibrio parahaemolyticus isolated from seafood[J]. Food Control,2016, 59:207-211.
[25] Li H, Tang R, Lou Y, et al. A comprehensive epidemiological research for clinical Vibrio parahaemolyticus in Shanghai[J]. Frontiers in Microbiology, 2017, 8:1043.
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[27] Oxley A P A, Shipton W, Owens L, et al. Bacterial flora from the gut of the wild and cultured banana prawn,Penaeus merguiensis[J]. Journal of Applied Microbio-logy, 2002, 93(2):214-223.
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[29] Zhou Z, He S, Liu Y, et al. Do stocking densities affect the gut microbiota of gibel carp Carassius auratus gibelio cultured in ponds?[J]. Journal of Aquaculture Research&Development, 2011,(S1):003.
[30] Akinbowale O L,Peng H,Barton M D. Antimicrobial resistance in bacteria isolated from aquaculture sources in Australia[J]. Journal of Applied Microbiology,2006,100(5):1103-1113.
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[2]New M B, Singholka S. Freshwater prawn farming:amanual for the culture of Macrobrachium rosenbergii[R].FAO Fisheries Technical Paper, 1982.
[3]杨国梁,陈雪峰,王军毅,等.罗氏沼虾产业在中国持续增长的经济与社会原因分析[J].浙江海洋学院学报(自然科学版),2011,30(5):450-457.Yang G L, Chen X F, Wang J Y, et al. Social and economical factors of sustained growth of Macrobrac hium rosenbergii industry in Mainland China[J]. Journal of Zhejiang Ocean University(Natural Science), 2011,30(5):450-457(in Chinese).
[4] Li X H, Zhou L, Yu Y H, et al. Composition of gut microbiota in the gibel carp(Carassius auratus gibelio)varies with host development[J]. Microbial Ecology,2017, 74(1):239-249.
[5] Wu S G, Gao T H, Zheng Y Z, et al. Microbial diversity of intestinal contents and mucus in yellow catfish(Pelteo bagrus fulvidraco)[J]. Aquaculture, 2010, 303(1-4):1-7.
[6] Ravel J, Blaser M J, Braun J, et al. Human microbiome science:vision for the future, Bethesda, MD, July 24 to26, 2013[J]. Microbiome, 2014, 2(1):16.
[7] Li T T, Long M, Gatesoupe F J, et al. Comparative analysis of the intestinal bacterial communities in different species of carp by pyrosequencing[J]. Microbial Ecology,2015, 69(1):25-36.
[8] Rungrassamee W, Klanchui A, Maibunkaew S, et al.Characterization of intestinal bacteria in wild and domesticated adult black tiger shrimp(Penaeus monodon)[J].PLoS One,2014, 9(3):e91853.
[9] Cardona E, Gueguen Y, Magre K, et al. Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris in a biofloc system[J].BMC Microbiology,2016, 16(1):157.
[10] Wu L Y, Wen C Q, Qin Y J, et al. Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis[J]. BMC Microbiology,2015, 15(1):125.
[11] You J, Wu G, Ren F P, et al. Microbial community dynamics in Baolige oilfield during MEOR treatment,revealed by Illumina MiSeq sequencing[J]. Applied Microbiology&Biotechnology,2016,100(3):1469-1478.
[12] Ampe F, ben Omar N, Moizan C, et al. Polyphasic study of the spatial distribution of microorganisms in mexican pozol, a fermented maize dough, demonstrates the needfor cultivation-independent methods to investigate traditional fermentations[J]. Applied&Environmental Microbiology,1999, 65(12):5464-5473.
[13] Ercolini D. PCR-DGGE fingerprinting:novel strategies for detection of microbes in food[J]. Journal of Microbiological Methods, 2004, 56(3):297-314.
[14] Vignesh R, Karthikeyan B S, Periyasamy N, et al.Antibiotics in aquaculture:an overview[J]. South Asian Journal of Experimental Biology, 2011:161-189.
[15] Pruden A, Pei R T, Storteboom et al. Antibiotic resistance genes as emerging contaminants:studies in northern colorado[J]. Environmental Science&Technology, 2006, 40(23):7445-7450.
[16]罗义,周启星.抗生素抗性基因(ARGs)——一种新型环境污染物[J].环境科学学报,2008, 28(8):1499-1505.Luo Y, Zhou Q X. Antibiotic resistance genes(ARGs)as emerging pollutants[J]. Acta Scientiae Circumstantiae,2008,28(8):1499-1505(in Chinese).
[17] Allen H K, Donato J, Wang H H, et al. Call of the wild:antibiotic resistance genes in natural environments[J].Nature Reviews Microbiology, 2010, 8(4):251-259.
[18]巴永兵,娄阳,赵飞,等.水产品批发市场中抗生素抗性基因的污染分析[J].食品与生物技术学报,2017,36(8):800-806.Ba Y B, Lou Y, Zhao F, et al. Research of the pollution of antibiotic resistance genes in aquatic products wholesale market[J]. Journal of Food Science&Biotechnology, 2017, 36(8):800-806(in Chinese).
[19]李欢,张昭寰,汤荣,等.食品与临床分离的致病性副溶血性弧菌耐药性比较[J].中国人兽共患病学报,2016, 32(11):1006-1012.Li H, Zhang Z H, Tang R, et al. Comparison of antimicrobial resistance of pathogenic Vibrio parahaemolyticus in seafood and clinical samples[J]. Chinese Journal of Zoonoses, 2016, 32(11):1006-1012(in Chinese).
[20] Thomas C M, Nielsen K M. Mechanisms of, and barriers to, horizontal gene transfer between bacteria[J]. Nature Reviews Microbiology,2005, 3(9):711-721.
[21] Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene database project:improved data processing and web-based tools[J]. Nucleic Acids Research,2013, 41:D590-D596.
[22] Jami E, Israel A, Kotser A, et al. Exploring the bovinerumen bacterial community from birth to adulthood[J].ISME Journal,2013, 7(6):1069-1079.
[23] Muyzer G, De Waal E C, Uitterlinden A G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[J]. Applied and Environmental Microbiology,1993, 59(3):695-700.
[24] Lou Y, Liu H Q, Zhang Z H, et al. Mismatch between antimicrobial resistance phenotype and genotype of pathogenic Vibrio parahaemolyticus isolated from seafood[J]. Food Control,2016, 59:207-211.
[25] Li H, Tang R, Lou Y, et al. A comprehensive epidemiological research for clinical Vibrio parahaemolyticus in Shanghai[J]. Frontiers in Microbiology, 2017, 8:1043.
[26] Vaughan E E, Schut F, Heilig H G H J, et al. A molecular view of the intestinal ecosystem[J]. Current Issues in Intestinal Microbiology,2000, 1(1):1-12.
[27] Oxley A P A, Shipton W, Owens L, et al. Bacterial flora from the gut of the wild and cultured banana prawn,Penaeus merguiensis[J]. Journal of Applied Microbio-logy, 2002, 93(2):214-223.
[28] Li T T, Long M, Ji C, et al. Alterations of the gut microbiome of largemouth bronze gudgeon(Coreius guichenoti)suffering from furunculosis[J]. Scientific Reports,2016, 6:30606.
[29] Zhou Z, He S, Liu Y, et al. Do stocking densities affect the gut microbiota of gibel carp Carassius auratus gibelio cultured in ponds?[J]. Journal of Aquaculture Research&Development, 2011,(S1):003.
[30] Akinbowale O L,Peng H,Barton M D. Antimicrobial resistance in bacteria isolated from aquaculture sources in Australia[J]. Journal of Applied Microbiology,2006,100(5):1103-1113.
[31] Cabello F C. Heavy use of prophylactic antibiotics in aquaculture:a growing problem for human and animal health and for the environment[J]. Environmental Microbiology, 2006, 8(7):1137-1144.
[32] Frost L S, Leplae R, Summers A O, et al. Mobile genetic elements:the agents of open source evolution[J]. Nature Reviews Microbioloegy, 2005, 3(9):722-732.