应用不同微生物源追踪方法追踪水库中粪便污染来源

Tracking sources of fecal pollution from reservoir by using two different microbial source tracking methods

目的:用两种不同的微生物源追踪(Microbial source tracking,MST)方法对江苏省盱眙县桂五水库粪便污染来源进行追踪。方法:于春、夏、秋、冬4季分别采集水库周边已知来源粪便标本,分离大肠埃希菌并作为指示菌,分别建立已知源指示菌细菌基因组重复序列PCR(repetitive Extragenic Palindromic-Polymerase chain reaction,rep-PCR)特征指纹库和已知源指示菌抗生素敏感性(Antibiotic Resistance Analysis,ARA)数据库,并分别用统计分析及试验设计软件(JMP7.0软件)和基因聚类分析软件(Bionumerics 4.0软件)计算平均正确归类率(Average Rate of Correct Classification,ARCC),以检验各自的宿主来源区分效果。同期采集水样,膜过滤法分离并确认指示菌,分别进行rep-PCR扩增和ARA试验,并分别与已知源指示菌rep-PCR特征指纹库和ARA数据库进行统计学比对,判断桂五水库中粪便污染来源。结果:将已知源指示菌rep-PCR特征指纹库和ARA数据库分别分为2、3、4、5和9类时,平均正确归类率分别为:89.19%、77.58%、76.69%、75.25%、70.92%和88.59%、84.98%、80.81%、79.58%、76.71%。两个已知源指示菌数据库均可较好地区分指示菌的不同来源。rep-PCR微生物源追踪和ARA微生物源追踪分别对534和547株水样指示菌进行了分析,判别分析结果分别显示人、鸡、鸭、鹅、狗、猪、牛、羊和野生动物各占26.49%、14.74%、7.77%、5.78%、3.98%、7.97%、10.96%、8.76%、8.57%和32.72%、8.78%、1.28%、2.01%、9.51%、12.98%、1.83%、28.34%、1.83%。结论:水体标本监测结果显示桂五水库粪便污染来源种类繁多,两种方法追踪结果不尽相同,rep-PCR法提示人、鸡和牛为主要污染源,ARA法结果显示人、羊和猪是最主要的污染贡献者,这可能与两法所选指示菌不完全相同有关,也可能是方法本身的差异导致源追踪结果有差异。

Objective：To track the fecal pollution sources from Guiwu reservoir at Xuyu,Jiangsu Province by using two different microbial source tracking methods（MST）.Methods：Fresh fecal samples from human and animals（poultry,livestock,dogs,and wild animals） were collected throughout villages around Guiwu reservoir in Spring,Summer,Autumn and Winter of a year.Escherichia coli were isolated and identified by API20E as indicator.A known-sources fingerprinting library and a known-source antibiotic resistance library containing all kinds of animal and human feces which had the chances to pollute the Guiwu reservoir were developed respectively.Then,a library self-cross with Bionumerics 4.0 and discriminant analysis with JMP7.0 software was used to calculate average rates of correct classification to evaluate the above library respectively.Water samples were collected once a month,and Escherichia coli were isolated by membrane filtration and identified by API20E as indicator.Then Escherichia coli were subject to rep-PCR amplification and antibiotic resistance test respectively.The fecal pollution sources from Guiwu reservoir were identified statistically by compared the rep-PCR fingerprinting and the profiles of ARA of each Escherichia coli from water samples with those from the above two known-source library respectively.Results：When the known-source fingerprinting library was classified as 2,3,4,5 and 9 groups respectively,the average rates of correct classification were respectively 89.19%,77.58%,76.69%,75.25% and 70.92%.When the known-source ARA library was classified as 2,3,4,5 and 9 groups respectively,the average rates of correct classification were respectively 88.59%,84.98%,80.81%,79.58% and 76.71%.Discriminant analysis showed that the division of different groups was clearly.534 indicators from water samples by using rep-PCR MST were identified its pollution source derived from human（26.49%）,chicken（14.74%）,ducks（7.77%）,goose（5.78%）,dogs（3.98%）,pigs（7.97%）,cattle（10.96%）,goats（8.76%）,and wild animals（8.57%） respectively.547 indicators from water samples by using ARA MST showed its pollution source derived from human（32.72%）,chicken（8.78%）,ducks（1.28%）,goose（2.01%）,dogs（9.51%）,pigs（12.98%）,cattle（1.83%）,goats（28.34%）,and wild animals（1.83%） respectively.Conclusion：Although the results of fecal pollution sources with the two MST methods was different,the fecal pollution sources from Guiwu reservoir was shown to be complex.The feces from human,chicken,and cattle were identified as the main pollution sources with rep-PCR MST,while those from human,goats,and pigs were the main pollution sources with ARA MST.It is possible that the indicators analyzed by the two methods were different partly,or that the difference resulted from the two methods themselves.