城市水体中新型病原细菌生态学特征及其溯源研究
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摘要
城市水体中病原微生物潜在生物污染风险,危害人群健康,已引起广泛关注,但是对城市水体中病原微生物的了解仍十分有限。本论文利用分子生物学技术、生物信息技术、细菌分离鉴定技术以及数学分析方法,对城市水体中的主要病原菌的生态学特征,包括病原微生物群落的生物多样性、病原微生物种群结构、时空分布特点进行了系统的研究;对城市污水受纳水体中主要病原菌,特别是优势种群—致病性弓形杆菌(Arcobacter)进行了溯源分析,评估了现有污水处理工艺对城市污水中主要病原菌去除效果,既为城市水体中病原微生物污染控制提供基础数据,也为源头控制致病性弓形杆菌传播奠定了基础。本论文包括以下主要研究内容和结论:
(1)提取水体中微生物总DNA,利用PCR扩增水体细菌的16SrDNA基因片段,构建了16S rDNA基因文库,对基因文库中的克隆测序后进行序列比对,进而分析水体细菌种群结构。镇江市城市受纳水体—内江中细菌分属9大细菌类群,本研究采用的分子生物学方法较为充分地体现了水体中细菌种群的多样性,存在现行水质生物监测指标以外的多种新型病原菌。其中,变形菌(Proteobacteria)为主要细菌类群,Arcobacter为优势菌群,占总检测样品的76.8%,推测其来源于城市污水。
(2)内江水体中主要病原菌生物活性研究。根据水样中16S rDNA分析结果,采用传统的细菌分离鉴定方法,并通过生理生化指标和细菌16S rDNA序列分析,分离并鉴定了11株分别属于产碱杆菌、气单胞菌、变形菌、不动杆菌等变形菌群的Beta和Gamma亚群的细菌。采用23S rDNA上Arcobacter种属特异性引物,通过PCR扩增技术分析了内江水体中致病性Arcobacter的活性,证明水体中致病性Arcobacter具有潜在的增殖能力。上述结果表明:内江水体中主要病原菌具有生物学活性。
(3)对内江水体微生物逐级构建的16S rDNA基因文库,采用多样性估算指数S_(ACE)和S_(Chaol)进行计算,两者之一达到稳定或两者同时达到稳定时基因库中的克隆数作为全面反映细菌种群多样性的最小库容值。当构建的内江水体16S rDNA基因文库中测序克隆数约为100个时多样性估算达到最大值。
(4)利用本研究建立的水体中细菌群落结构分析方法,对内江水体中细菌种群结构、主要病原微生物的时空分布特征研究发现,不同区域位置水体中细菌类群的总体结构没有明显差异,均以变形菌类群为水中的主要菌群;致病性Arcobacter为水体中的优势菌群,Arcobacter cryaerophilus所占比例最高。在水体不同区域主要的病原细菌的组成与城市污水溢流口的方位和距离有关,推测内江水体中主要病原微生物来源于城市污水。
(5)通过细菌时空分布特征和主要病原菌的生物信息学分析,证明内江水体中微生物种群结构与污水处理厂出水中种群结构相同;内江水体中主要病原菌来源于城市污水,优势细菌菌群Arcobacter主要来自医院污水和居民区生活污水;Trichococcus pasteuri主要来自城市污水中的医院污水;梭菌(Clostridium)则主要来自城市污水中的菜场污水和医院污水;内江水体中其他来自城市污水中的病原菌则与不同来源处的城市污水无明显关系。
(6)以污水处理厂进水水样和污水处理工艺Ⅰ和处理工艺Ⅱ处理后的出水为研究对象,利用分子生物学技术和生物学信息学技术对水体中的细菌种群进行了分析。两种污水处理工艺对细菌群落的组成没有明显改变,但处理工艺Ⅰ对部分主要的病原菌均有明显的消减作用,存在种属差异,并且对优势种群Arcobacter cryaerophilus的控制效果与处理水温有明显关系,推测工艺Ⅰ对Arcobacter cryaerophilus消减作用主要是通过吸附沉淀作用实现的;处理工艺Ⅱ中紫外消毒能有效地消减优势种群Arcobacter cryaerophilus以外的其他病原菌,表明优势种群Arcobacter cryaerophilus对紫外线作用不敏感。
The pathogenic microorganism in the urban water body, which is a latent risk of biological pollution and harmful to public health, has aroused the widespread attention. However, little is known about the pathogenic microorganism in the urban water body. In this thesis, the microorganism population structural feature in the water body was investigated by constructing bacterium's 16S rRNA gene library, and then the massive pathogenic microorganism was discovered. So the pathogenic microorganism's ecological characteristics were studied, including the population structure, space-time distribution characteristic and community's biodiversity of the pathogenic microorganism. Then the origin of the dominant microflora in the water body was explored and the effect had been appraised in removing the pathogenic microorganisms by the existing sewage treatment process. All the research provided the essential data for control the pathogenic microorganism pollution in the urban water body and also indicated the direction for the source control Arcobacter. The research mainly obtained the following conclusion:
(1)The 16S rDNA gene bank was constructed by extracting the total bacterial DNA in the water body and amplifying 16S rRNA gene fragment using PCR. The sequence alignment was carried on after cloning and sequencing, and then bacterium population structure was analyzed. The bacterium in Neijiang water body belongs to 9 bacterium class groups. The 16S rDNA gene bank constructed by molecular biology method fully manifests the bacterium population's multiplicity of the water body. The Neijiang water body had many kinds of pathogenic microorganisms outside the water quality biological monitor index, in which Proteobacteria was the main bacterium class group and Arcobacter was the superiority bacteria colony. Arcobacter was presumably from the urban sewage.
(2)According to the result by the molecular and biological method, the main pathogenic bacteria in Neijiang water body were isolated and identified by traditional bacteria isolation and identification method, specific and selective medium method and morphological and biochemical method. The isolated strains were identified as Alcaligenes faecalis, Providencia stuartii, Aeromonas hydrophila、Aeromonas salmonicida , Acinetobacter junii、Acinetobacter rhizosphaera, and so on. The activity of pathogenic Arcobacter spp. was detected through PCR amplification technique using species-specific primers, which amplify the most variable areas of 23S rRNA gene. The result proved that pathogenic Arcobacter spp. in the water had the potential proliferative ability.
(3) The diversity estimators S_(ACE) and S_(Chaol) were carried on computation for the progressive construction of 16S rDNA gene bank of the microorganisms in Neijiang water body. The best library size for analyzing bacterial diversity could been achieved when one of both index reached stable or both simultaneously. The research result showed that the species richness estimators reached the maximum when the number of clones sequenced in bacterial 16S rDNA library of Neijiang water body was about 100.
(4)The characteristics of bacterial space-time distribution of main pathogenic microorganisms from Neijiang water body were studied by population structure analysis method constructed in the study. The result showed that that the bacterium gross structure didn't have the obvious difference in the region Proteobacteria was the main class group and pathogenic Arcobacter was the the dominant bacteria colony in which Arcobacter cryaerophilus held the high proportion. The type and proportion of the main pathogenic bacteria at different position were related with the orientation and the distance between the urban sewage overflow site and other monitoring sites. The result indicated that the main pathogenic microorganisms probably originated from the urban sewage.
(5) Through the analysis of bacterial space-time distribution characteristics and bioinformatics of the main pathogenic bacteria, it proved that the characteristic of the microorganism population structure in Neijiang water body was the same as that of in the effluent from the wastewater treatment plant. The dominant genus Arcobacter in Neijiang water body came from the hospital sewage and the resident sanitary sewage. Trichococcus pasteuri mainly came from the hospital sewage. Clostridium spp.mainly came from the food market sewage and the hospital sewage. Other pathogenic microorganisms in the Neijiang water body was not obviously related with the sewage from different places.
(6) The microorganisms in the influent of wastewater treatment plant and the effluents from treatment process I and II were investigated by molecular and biological methods and bioinformatics methods. The analysis results showed that there were no significant difference of the community structure of total bacterial population between the influents and the effluents, but partial main microorganisms were removed by treatment process I, and the effect of the treatment process to control the dominant genera Arcobacter cryaerophilus was related to the water temperature. It is guessed that partial bacteria are removed by an adsorption precipitation process in the treatment method. And in treatment process II some other bacterial species decreased except the dominant genus Arcobacter cryaerophilus by ultraviolet irradiation, which meaned Arcobacter cryaerophilus was not sensitive with ultraviolet irradiation.
(1)提取水体中微生物总DNA,利用PCR扩增水体细菌的16SrDNA基因片段,构建了16S rDNA基因文库,对基因文库中的克隆测序后进行序列比对,进而分析水体细菌种群结构。镇江市城市受纳水体—内江中细菌分属9大细菌类群,本研究采用的分子生物学方法较为充分地体现了水体中细菌种群的多样性,存在现行水质生物监测指标以外的多种新型病原菌。其中,变形菌(Proteobacteria)为主要细菌类群,Arcobacter为优势菌群,占总检测样品的76.8%,推测其来源于城市污水。
(2)内江水体中主要病原菌生物活性研究。根据水样中16S rDNA分析结果,采用传统的细菌分离鉴定方法,并通过生理生化指标和细菌16S rDNA序列分析,分离并鉴定了11株分别属于产碱杆菌、气单胞菌、变形菌、不动杆菌等变形菌群的Beta和Gamma亚群的细菌。采用23S rDNA上Arcobacter种属特异性引物,通过PCR扩增技术分析了内江水体中致病性Arcobacter的活性,证明水体中致病性Arcobacter具有潜在的增殖能力。上述结果表明:内江水体中主要病原菌具有生物学活性。
(3)对内江水体微生物逐级构建的16S rDNA基因文库,采用多样性估算指数S_(ACE)和S_(Chaol)进行计算,两者之一达到稳定或两者同时达到稳定时基因库中的克隆数作为全面反映细菌种群多样性的最小库容值。当构建的内江水体16S rDNA基因文库中测序克隆数约为100个时多样性估算达到最大值。
(4)利用本研究建立的水体中细菌群落结构分析方法,对内江水体中细菌种群结构、主要病原微生物的时空分布特征研究发现,不同区域位置水体中细菌类群的总体结构没有明显差异,均以变形菌类群为水中的主要菌群;致病性Arcobacter为水体中的优势菌群,Arcobacter cryaerophilus所占比例最高。在水体不同区域主要的病原细菌的组成与城市污水溢流口的方位和距离有关,推测内江水体中主要病原微生物来源于城市污水。
(5)通过细菌时空分布特征和主要病原菌的生物信息学分析,证明内江水体中微生物种群结构与污水处理厂出水中种群结构相同;内江水体中主要病原菌来源于城市污水,优势细菌菌群Arcobacter主要来自医院污水和居民区生活污水;Trichococcus pasteuri主要来自城市污水中的医院污水;梭菌(Clostridium)则主要来自城市污水中的菜场污水和医院污水;内江水体中其他来自城市污水中的病原菌则与不同来源处的城市污水无明显关系。
(6)以污水处理厂进水水样和污水处理工艺Ⅰ和处理工艺Ⅱ处理后的出水为研究对象,利用分子生物学技术和生物学信息学技术对水体中的细菌种群进行了分析。两种污水处理工艺对细菌群落的组成没有明显改变,但处理工艺Ⅰ对部分主要的病原菌均有明显的消减作用,存在种属差异,并且对优势种群Arcobacter cryaerophilus的控制效果与处理水温有明显关系,推测工艺Ⅰ对Arcobacter cryaerophilus消减作用主要是通过吸附沉淀作用实现的;处理工艺Ⅱ中紫外消毒能有效地消减优势种群Arcobacter cryaerophilus以外的其他病原菌,表明优势种群Arcobacter cryaerophilus对紫外线作用不敏感。
The pathogenic microorganism in the urban water body, which is a latent risk of biological pollution and harmful to public health, has aroused the widespread attention. However, little is known about the pathogenic microorganism in the urban water body. In this thesis, the microorganism population structural feature in the water body was investigated by constructing bacterium's 16S rRNA gene library, and then the massive pathogenic microorganism was discovered. So the pathogenic microorganism's ecological characteristics were studied, including the population structure, space-time distribution characteristic and community's biodiversity of the pathogenic microorganism. Then the origin of the dominant microflora in the water body was explored and the effect had been appraised in removing the pathogenic microorganisms by the existing sewage treatment process. All the research provided the essential data for control the pathogenic microorganism pollution in the urban water body and also indicated the direction for the source control Arcobacter. The research mainly obtained the following conclusion:
(1)The 16S rDNA gene bank was constructed by extracting the total bacterial DNA in the water body and amplifying 16S rRNA gene fragment using PCR. The sequence alignment was carried on after cloning and sequencing, and then bacterium population structure was analyzed. The bacterium in Neijiang water body belongs to 9 bacterium class groups. The 16S rDNA gene bank constructed by molecular biology method fully manifests the bacterium population's multiplicity of the water body. The Neijiang water body had many kinds of pathogenic microorganisms outside the water quality biological monitor index, in which Proteobacteria was the main bacterium class group and Arcobacter was the superiority bacteria colony. Arcobacter was presumably from the urban sewage.
(2)According to the result by the molecular and biological method, the main pathogenic bacteria in Neijiang water body were isolated and identified by traditional bacteria isolation and identification method, specific and selective medium method and morphological and biochemical method. The isolated strains were identified as Alcaligenes faecalis, Providencia stuartii, Aeromonas hydrophila、Aeromonas salmonicida , Acinetobacter junii、Acinetobacter rhizosphaera, and so on. The activity of pathogenic Arcobacter spp. was detected through PCR amplification technique using species-specific primers, which amplify the most variable areas of 23S rRNA gene. The result proved that pathogenic Arcobacter spp. in the water had the potential proliferative ability.
(3) The diversity estimators S_(ACE) and S_(Chaol) were carried on computation for the progressive construction of 16S rDNA gene bank of the microorganisms in Neijiang water body. The best library size for analyzing bacterial diversity could been achieved when one of both index reached stable or both simultaneously. The research result showed that the species richness estimators reached the maximum when the number of clones sequenced in bacterial 16S rDNA library of Neijiang water body was about 100.
(4)The characteristics of bacterial space-time distribution of main pathogenic microorganisms from Neijiang water body were studied by population structure analysis method constructed in the study. The result showed that that the bacterium gross structure didn't have the obvious difference in the region Proteobacteria was the main class group and pathogenic Arcobacter was the the dominant bacteria colony in which Arcobacter cryaerophilus held the high proportion. The type and proportion of the main pathogenic bacteria at different position were related with the orientation and the distance between the urban sewage overflow site and other monitoring sites. The result indicated that the main pathogenic microorganisms probably originated from the urban sewage.
(5) Through the analysis of bacterial space-time distribution characteristics and bioinformatics of the main pathogenic bacteria, it proved that the characteristic of the microorganism population structure in Neijiang water body was the same as that of in the effluent from the wastewater treatment plant. The dominant genus Arcobacter in Neijiang water body came from the hospital sewage and the resident sanitary sewage. Trichococcus pasteuri mainly came from the hospital sewage. Clostridium spp.mainly came from the food market sewage and the hospital sewage. Other pathogenic microorganisms in the Neijiang water body was not obviously related with the sewage from different places.
(6) The microorganisms in the influent of wastewater treatment plant and the effluents from treatment process I and II were investigated by molecular and biological methods and bioinformatics methods. The analysis results showed that there were no significant difference of the community structure of total bacterial population between the influents and the effluents, but partial main microorganisms were removed by treatment process I, and the effect of the treatment process to control the dominant genera Arcobacter cryaerophilus was related to the water temperature. It is guessed that partial bacteria are removed by an adsorption precipitation process in the treatment method. And in treatment process II some other bacterial species decreased except the dominant genus Arcobacter cryaerophilus by ultraviolet irradiation, which meaned Arcobacter cryaerophilus was not sensitive with ultraviolet irradiation.
引文
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