大连典型娱乐海水浴场重要病原细菌筛选及其在不同介质中的分布
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摘要
为研究海水浴场环境中病原细菌的种类及其分布,以大连金石滩浴场和傅家庄浴场为研究区域,通过可培养、基因芯片、高通量测序等技术对其表层海水、沉积物、湿砂、干砂中病原细菌进行筛选,并分析指示细菌和常见病原细菌的空间分布特征。结果表明:不论金石滩还是傅家庄浴场,优势菌属皆为弧菌Vibrio、芽孢杆菌Bacillus、假单胞菌Pseudomonas等,可能的病原菌属为弧菌、假单胞菌、克雷伯氏菌Klebsiella等菌属中的某些种类;基因芯片检测结果显示,两浴场的主要病原细菌为溶藻弧菌V.alginolyticus、副溶血弧菌V.parahaemolyticus、哈维氏弧菌V.harveyi、鳗弧菌V.anguillarum、粪肠球菌Enterococcus faecalis、气单胞菌属Aeromonas、志贺氏菌属Shigella和大肠杆菌Escherichia coli等,会对游客身体健康造成巨大的潜在威胁;空间分布特征分析显示,粪便污染指示细菌粪大肠菌群和肠球菌主要分布在海水和干砂中,其中,排污口处细菌浓度最高,潜在病原细菌金黄色葡萄球菌Staphylococcus aureus主要分布在海水和干砂中,可培养弧菌主要分布在海水中。研究表明,排污口是大连金石滩和付家庄海水浴场病原细菌主要的污染来源,海滩是其重要的非点源污染来源,建议海滩管理者在泳季高峰期定期监测海滩与排污口周围病原细菌的污染情况,本研究结果可为海水浴场环境管理和保护提供重要参考依据。
The pathogenic bacteria were screened in surface seawater, sediment, wet sand and dry sand at Dalian Jinshitan Bathing Beach and Fujiazhuang Bathing Beach by culturable, gene chip and high-throughput sequencing technologies, and the spatial distribution characteristics of indicator bacteria and common pathogenic bacteria were analyzed to investigate the species and distribution of pathogenic bacteria in the beach. High-throughput sequencing results showed that the dominant genus of both the Jinshitan and Fujiazhuang bathing beaches were Vibrio, Bacillus, Pseudomonas and possible pathogens of which were some species in Vibrio, Pseudomonas, and Klebsiella. The gene chip detection revealed that the main pathogenic bacteria in these two beaches were V.alginolyticus, V.parahaemolyticus, V.harveyi, V.anguillarum, Enterococcus faecalis, Aeromonas, Shigella and Escherichia coli, which posed a huge potential threat to the health of entertainers. Analysis of spatial distribution indicated that fecal indicator bacteria, fecal coliforms and Enterococcus were mainly distributed in seawater and dry sand, among all sites, with the maximal concentration of pathogenic bacteria in the sewage outlet site. The potential pathogenic bacterium Staphylococcus aureus was primarily found in seawater and dry sand, and the cultarable Vibrio was mainly observed in seawater. Since the sewage outlet is the main source of pathogenic bacteria in Dalian Jinshitan and Fujiazhuang beaches as an important non-point source pollution, it is recommended that pathogenic bacteria around beaches and sewage outlets should be regularly monitored during the peak of swimming season.
The pathogenic bacteria were screened in surface seawater, sediment, wet sand and dry sand at Dalian Jinshitan Bathing Beach and Fujiazhuang Bathing Beach by culturable, gene chip and high-throughput sequencing technologies, and the spatial distribution characteristics of indicator bacteria and common pathogenic bacteria were analyzed to investigate the species and distribution of pathogenic bacteria in the beach. High-throughput sequencing results showed that the dominant genus of both the Jinshitan and Fujiazhuang bathing beaches were Vibrio, Bacillus, Pseudomonas and possible pathogens of which were some species in Vibrio, Pseudomonas, and Klebsiella. The gene chip detection revealed that the main pathogenic bacteria in these two beaches were V.alginolyticus, V.parahaemolyticus, V.harveyi, V.anguillarum, Enterococcus faecalis, Aeromonas, Shigella and Escherichia coli, which posed a huge potential threat to the health of entertainers. Analysis of spatial distribution indicated that fecal indicator bacteria, fecal coliforms and Enterococcus were mainly distributed in seawater and dry sand, among all sites, with the maximal concentration of pathogenic bacteria in the sewage outlet site. The potential pathogenic bacterium Staphylococcus aureus was primarily found in seawater and dry sand, and the cultarable Vibrio was mainly observed in seawater. Since the sewage outlet is the main source of pathogenic bacteria in Dalian Jinshitan and Fujiazhuang beaches as an important non-point source pollution, it is recommended that pathogenic bacteria around beaches and sewage outlets should be regularly monitored during the peak of swimming season.
引文
[1] 张振冬,王秀娟,朱琳.海洋环境中病原微生物不同检测方法的比较研究[J].海洋环境科学,2011,30(2):292-295.
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[3] Bonilla T D,Nowosielski K,Esiobu N,et al.Species assemblages of Enterococcus indicate potential sources of fecal bacteria at a South Florida recreational beach[J].Mar Pollut Bull,2006,52(7):807-810.
[4] Hartz A,Cuvelier M,Nowosielski K,et al.Survival potential of Escherichia coli and enterococci in subtropical beach sand:implications for water quality managers[J].J Environ Qual,2008,37(3):898-905.
[5] Yamahara K M,Walters S P,Boehm A B.Growth of enterococci in unaltered,unseeded beach sands subjected to tidal wetting[J].Appl Environ Microbiol,2009,75(6):1517-1524.
[6] Heaney C D,Sams E,Wing S,et al.Contact with beach sand among beachgoers and risk of illness[J].Am J Epidemiol,2009,170(2):164-172.
[7] 李江宇,樊景凤,穆贵强,等.利用PCR-DGGE分析海水浴场细菌多样性[J]. 海洋环境科学,2013,32(4):523-528.
[8] 郭建丽.海水浴场常见致病菌PCR方法和基因芯片检测方法的建立及应用[D].大连:大连海洋大学,2014.
[9] ISO.ISO 7899-2-2000 Water quality-detection and enumeration of intestinal enterococci-Part 2:membrane filtration method[S].Geneva,Switzerland:ISO,2000.
[10] United States Environmental Protection Agency.EPA 440/5-84-002 ambient water quality criteria for bacteria[S].Washington,DC:Office of Water Regulations and Standards Criteria and Standards Division,1986.
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[16] Alm E W,Burke J,Hagan E.Persistence and potential growth of the fecal indicator bacteria,Escherichia coli,in shoreline sand at Lake Huron[J].J Great Lakes Res,2006,32(2):401-405.
[17] Boehm A B,Ashbolt N J,Colford Jr J M,et al.A sea change ahead for recreational water quality criteria[J].J Water Health,2009,7(1):9-20.
[18] Charoenca N,Fujioka R S.Association of staphylococcal skin infections and swimming[J].Water Sci Technol,1995,31(5-6):11-17.
[19] Elmir S M,Wright M E,Abdelzaher A,et al.Quantitative evaluation of bacteria released by bathers in a marine water[J].Water Res,2007,41(1):3-10.
[20] Plano L R W,Garza A C,Shibata T,et al.Shedding of Staphylococcusaureus and methicillin-resistant Staphylococcus aureus from adult and pediatric bathers in marine waters[J].BMC Microbiol,2011,11:5.
[21] Abdelzaher A M,Wright M E,Ortega C,et al.Daily measures of microbes and human health at a non-point source marine beach[J].Journal of Water and Health,2011,9(3):443-457.
[22] Halliday E,Griffith J F,Gast R J.Use of an exogenous plasmid standard and quantitative PCR to monitor spatial and temporal distribution of Enterococcus spp.in beach sands[J].Limnol Oceanogr:Methods,2010,8(4):146-154.
[23] Scholthof K B G.The disease triangle:pathogens,the environment and society[J].Nat Rev Microbiol,2007,5(2):152-156.
[24] Bonilla T D,Nowosielski K,Cuvelier M,et al.Prevalence and distribution of fecal indicator organisms in South Florida beach sand and preliminary assessment of health effects associated with beach sand exposure[J].Mar Pollut Bull,2007,54(9):1472-1482.
[25] Cui Henglin,Yang Kun,Pagaling E,et al.Spatial and temporal variation in enterococcal abundance and its relationship to the microbial community in Hawaii Beach Sand and water[J].Appl Environ Microbiol,2013,79(12):3601-3609.
[26] USEPA.Recreational water quality criteria[R].Washington,DC:USEPA,2012.
[27] Heaney C D,Sams E,Dufour A P,et al.Fecal indicators in sand,sandcontact,and risk of enteric illness among beachgoers[J].Epidemiology,2012,23(1):95-106.
[28] Ishii S,Hansen D L,Hicks R E,et al.Beach sand and sediments are temporal sinks and sources of Escherichia coli in Lake Superior[J].Environ Sci Technol,2007,41(7):2203-2209.
[29] Clark A,Turner T,Dorothy K P,et al.Health hazards due to pollution of waters along the coast of Visakhapatnam,east coast of India[J].Ecotoxicol Environ Saf,2003,56(3):390-397.
[30] Esiobu N,Green M,Echeverry A,et al.High numbers of Staphylococcus aureus at three bathing beaches in South Florida[J].Int J Environ Health Res,2013,23(1):46-57.
[31] Abreu R,Figueira C,Rom?o D,et al.Sediment characteristics and microbiological contamination of beach sand—A case-study in the archipelago of Madeira[J].Sci Total Environ,2016,573:627-638.
[32] Fan Jingfeng,Ming Hongxia,Li Lili,et al.Evaluating spatial-temporal variations and correlation between fecal indicator bacteria (FIB) in marine bathing beaches[J].J Water Health,2015,13(4):1029-1038.
[33] Feng Zhixuan,Reniers A,Haus B K,et al.A predictive model for microbial counts on beaches where intertidal sand is the primary source[J].Mar Pollut Bull,2015,94(1-2):37-47.
[34] Oshiro R,Fujioka R.Sand,soil,and pigeon droppings:sources of indicator bacteria in the waters of Hanauma Bay,Oahu,Hawaii[J].Water Sci Technol,1995,31(5-6):251-254.
[35] Yan Tao,Goto D K,Feng Fan.Concentration Dynamics of Fecal Indicators in Hawaii's Coastal and Inland Sand,Soil,and Water during Rainfall Events[M].Alexandria,VA,USA.Publishing,2011.
[36] Baty III A M,Eastburn C C,Techkarnjanaruk S,et al.Spatial and temporal variations in chitinolytic gene expression and bacterial biomass production during chitin degradation[J].Appl Environ Microbiol,2000,66(8):3574-3585.
[37] Obiri-Danso K,Jones K.Intertidal sediments as reservoirs for hippurate negative campylobacters,salmonellae and faecal indicators in three EU recognised bathing waters in North West England[J].Water Res,2000,34(2):519-527.
[38] Ghinsberg R C,Bar Dov L,Rogol M,et al.Monitoring of selected bacteria and fungi in sand and sea water along the Tel Aviv coast[J].Microbios,1994,77(310):29-40.
[39] 黄宝华,陈庆森,庞广昌.志贺氏菌研究及其快速检测技术发展现状[J].食品科学,2004,25(11):333-336.
[40] Ferguson D M,Moore D F,GetrichM A,et al.Enumeration and speciation of enterococci found in marine and intertidal sediments and coastal water in southern California[J].J Appl Microbiol,2005,99(3):598-608.
[41] Burton Jr G A,Gunnison D,Lanza G R.Survival of pathogenic bacteria in various freshwater sediments[J].Appl Environ Microbiol,1987,53(4):633-638.
[42] Byappanahalli M N,Nevers M B,Korajkic A,et al.Enterococci in the environment[J].Microbiol Mol Biol Rev,2012,76(4):685-706.
[43] 郭建丽,王玥,李江宇,等.大连重要海水增养殖区粪便污染指示菌和弧菌的时空分布[J].海洋环境科学,2017,36(6):813-819.
[44] 宁喜斌,刘代新,张继伦.副溶血性弧菌的致病性及其快速检测[J].微生物与感染,2008,3(1):53-56.
[45] 刘雪飞.副溶血性弧菌拮抗菌的分离及其抑菌机理研究[D].沈阳:沈阳农业大学,2016.
[46] 杨守明,王民生.嗜水气单胞菌及其对人的致病性[J].疾病控制杂志,2006,10(5):511-514.
[47] Halliday E,Gast R J.Bacteria in beach sands:an emerging challenge in protecting coastal water quality and bather health[J].Environ Sci Technol,2011,45(2):370-379.
[48] Abbott S L,Janda J M,Johnson J A,et al.Vibrio and related organisms[M]//Murray P R,Baron E J,Jorgensen J H,et al.Manual of Clinical Microbiology.9th ed.Washington:American Society for Microbiology,2007:723-733.
[49] Sobrinho P D S C,Destro M T,Franco B D G M,et al.Occurrence and distribution of Vibrio parahaemolyticus in retail oysters in Sao Paulo State,Brazil[J].Food Microbiol,2011,28(1):137-140.
[50] Li Cheng,Wong W H.Model-based analysis of oligonucleotide arrays:expression index computation and outlier detection[J].ProcNatl Acad Sci USA,2001,98(1):31-36.
[2] Esiobu N,Mohammed R,Echeverry A,et al.The application of peptide nucleic acid probes for rapid detection and enumeration of eubacteria,Staphylococcus aureus and Pseudomonas aeruginosa in recreational beaches of S.Florida[J].J Microbiol Meth,2004,57(2):157-162.
[3] Bonilla T D,Nowosielski K,Esiobu N,et al.Species assemblages of Enterococcus indicate potential sources of fecal bacteria at a South Florida recreational beach[J].Mar Pollut Bull,2006,52(7):807-810.
[4] Hartz A,Cuvelier M,Nowosielski K,et al.Survival potential of Escherichia coli and enterococci in subtropical beach sand:implications for water quality managers[J].J Environ Qual,2008,37(3):898-905.
[5] Yamahara K M,Walters S P,Boehm A B.Growth of enterococci in unaltered,unseeded beach sands subjected to tidal wetting[J].Appl Environ Microbiol,2009,75(6):1517-1524.
[6] Heaney C D,Sams E,Wing S,et al.Contact with beach sand among beachgoers and risk of illness[J].Am J Epidemiol,2009,170(2):164-172.
[7] 李江宇,樊景凤,穆贵强,等.利用PCR-DGGE分析海水浴场细菌多样性[J]. 海洋环境科学,2013,32(4):523-528.
[8] 郭建丽.海水浴场常见致病菌PCR方法和基因芯片检测方法的建立及应用[D].大连:大连海洋大学,2014.
[9] ISO.ISO 7899-2-2000 Water quality-detection and enumeration of intestinal enterococci-Part 2:membrane filtration method[S].Geneva,Switzerland:ISO,2000.
[10] United States Environmental Protection Agency.EPA 440/5-84-002 ambient water quality criteria for bacteria[S].Washington,DC:Office of Water Regulations and Standards Criteria and Standards Division,1986.
[11] 国家环境保护局,国家海洋局.GB 3097-1997海水水质标准[S].北京:环境科学出版社,2004.
[12] 国家海洋局.GB/T 12763.9-2007海洋调查规范第9部分:海洋生态调查指南[S].北京:中国标准出版社,2008.
[13] 国家海洋环境监测中心.GB17378.4-2007海洋监测规范第4部分:海水分析[S].北京:中国标准出版社,2008.
[14] 国家海洋局.HY/T 127-2010滨海旅游度假区环境评价指南[S].北京:中国标准出版社,2010.
[15] Enns A A,Vogel L J,Abdelzaher A M,et al.Spatial and temporal variation in indicator microbe sampling is influential in beach management decisions[J].Water Res,2012,46(7):2237-2246.
[16] Alm E W,Burke J,Hagan E.Persistence and potential growth of the fecal indicator bacteria,Escherichia coli,in shoreline sand at Lake Huron[J].J Great Lakes Res,2006,32(2):401-405.
[17] Boehm A B,Ashbolt N J,Colford Jr J M,et al.A sea change ahead for recreational water quality criteria[J].J Water Health,2009,7(1):9-20.
[18] Charoenca N,Fujioka R S.Association of staphylococcal skin infections and swimming[J].Water Sci Technol,1995,31(5-6):11-17.
[19] Elmir S M,Wright M E,Abdelzaher A,et al.Quantitative evaluation of bacteria released by bathers in a marine water[J].Water Res,2007,41(1):3-10.
[20] Plano L R W,Garza A C,Shibata T,et al.Shedding of Staphylococcusaureus and methicillin-resistant Staphylococcus aureus from adult and pediatric bathers in marine waters[J].BMC Microbiol,2011,11:5.
[21] Abdelzaher A M,Wright M E,Ortega C,et al.Daily measures of microbes and human health at a non-point source marine beach[J].Journal of Water and Health,2011,9(3):443-457.
[22] Halliday E,Griffith J F,Gast R J.Use of an exogenous plasmid standard and quantitative PCR to monitor spatial and temporal distribution of Enterococcus spp.in beach sands[J].Limnol Oceanogr:Methods,2010,8(4):146-154.
[23] Scholthof K B G.The disease triangle:pathogens,the environment and society[J].Nat Rev Microbiol,2007,5(2):152-156.
[24] Bonilla T D,Nowosielski K,Cuvelier M,et al.Prevalence and distribution of fecal indicator organisms in South Florida beach sand and preliminary assessment of health effects associated with beach sand exposure[J].Mar Pollut Bull,2007,54(9):1472-1482.
[25] Cui Henglin,Yang Kun,Pagaling E,et al.Spatial and temporal variation in enterococcal abundance and its relationship to the microbial community in Hawaii Beach Sand and water[J].Appl Environ Microbiol,2013,79(12):3601-3609.
[26] USEPA.Recreational water quality criteria[R].Washington,DC:USEPA,2012.
[27] Heaney C D,Sams E,Dufour A P,et al.Fecal indicators in sand,sandcontact,and risk of enteric illness among beachgoers[J].Epidemiology,2012,23(1):95-106.
[28] Ishii S,Hansen D L,Hicks R E,et al.Beach sand and sediments are temporal sinks and sources of Escherichia coli in Lake Superior[J].Environ Sci Technol,2007,41(7):2203-2209.
[29] Clark A,Turner T,Dorothy K P,et al.Health hazards due to pollution of waters along the coast of Visakhapatnam,east coast of India[J].Ecotoxicol Environ Saf,2003,56(3):390-397.
[30] Esiobu N,Green M,Echeverry A,et al.High numbers of Staphylococcus aureus at three bathing beaches in South Florida[J].Int J Environ Health Res,2013,23(1):46-57.
[31] Abreu R,Figueira C,Rom?o D,et al.Sediment characteristics and microbiological contamination of beach sand—A case-study in the archipelago of Madeira[J].Sci Total Environ,2016,573:627-638.
[32] Fan Jingfeng,Ming Hongxia,Li Lili,et al.Evaluating spatial-temporal variations and correlation between fecal indicator bacteria (FIB) in marine bathing beaches[J].J Water Health,2015,13(4):1029-1038.
[33] Feng Zhixuan,Reniers A,Haus B K,et al.A predictive model for microbial counts on beaches where intertidal sand is the primary source[J].Mar Pollut Bull,2015,94(1-2):37-47.
[34] Oshiro R,Fujioka R.Sand,soil,and pigeon droppings:sources of indicator bacteria in the waters of Hanauma Bay,Oahu,Hawaii[J].Water Sci Technol,1995,31(5-6):251-254.
[35] Yan Tao,Goto D K,Feng Fan.Concentration Dynamics of Fecal Indicators in Hawaii's Coastal and Inland Sand,Soil,and Water during Rainfall Events[M].Alexandria,VA,USA.Publishing,2011.
[36] Baty III A M,Eastburn C C,Techkarnjanaruk S,et al.Spatial and temporal variations in chitinolytic gene expression and bacterial biomass production during chitin degradation[J].Appl Environ Microbiol,2000,66(8):3574-3585.
[37] Obiri-Danso K,Jones K.Intertidal sediments as reservoirs for hippurate negative campylobacters,salmonellae and faecal indicators in three EU recognised bathing waters in North West England[J].Water Res,2000,34(2):519-527.
[38] Ghinsberg R C,Bar Dov L,Rogol M,et al.Monitoring of selected bacteria and fungi in sand and sea water along the Tel Aviv coast[J].Microbios,1994,77(310):29-40.
[39] 黄宝华,陈庆森,庞广昌.志贺氏菌研究及其快速检测技术发展现状[J].食品科学,2004,25(11):333-336.
[40] Ferguson D M,Moore D F,GetrichM A,et al.Enumeration and speciation of enterococci found in marine and intertidal sediments and coastal water in southern California[J].J Appl Microbiol,2005,99(3):598-608.
[41] Burton Jr G A,Gunnison D,Lanza G R.Survival of pathogenic bacteria in various freshwater sediments[J].Appl Environ Microbiol,1987,53(4):633-638.
[42] Byappanahalli M N,Nevers M B,Korajkic A,et al.Enterococci in the environment[J].Microbiol Mol Biol Rev,2012,76(4):685-706.
[43] 郭建丽,王玥,李江宇,等.大连重要海水增养殖区粪便污染指示菌和弧菌的时空分布[J].海洋环境科学,2017,36(6):813-819.
[44] 宁喜斌,刘代新,张继伦.副溶血性弧菌的致病性及其快速检测[J].微生物与感染,2008,3(1):53-56.
[45] 刘雪飞.副溶血性弧菌拮抗菌的分离及其抑菌机理研究[D].沈阳:沈阳农业大学,2016.
[46] 杨守明,王民生.嗜水气单胞菌及其对人的致病性[J].疾病控制杂志,2006,10(5):511-514.
[47] Halliday E,Gast R J.Bacteria in beach sands:an emerging challenge in protecting coastal water quality and bather health[J].Environ Sci Technol,2011,45(2):370-379.
[48] Abbott S L,Janda J M,Johnson J A,et al.Vibrio and related organisms[M]//Murray P R,Baron E J,Jorgensen J H,et al.Manual of Clinical Microbiology.9th ed.Washington:American Society for Microbiology,2007:723-733.
[49] Sobrinho P D S C,Destro M T,Franco B D G M,et al.Occurrence and distribution of Vibrio parahaemolyticus in retail oysters in Sao Paulo State,Brazil[J].Food Microbiol,2011,28(1):137-140.
[50] Li Cheng,Wong W H.Model-based analysis of oligonucleotide arrays:expression index computation and outlier detection[J].ProcNatl Acad Sci USA,2001,98(1):31-36.
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