| Pathogenic microorganisms including viruses, rickettsia, mycoplasma, bacteria, and fungi, are commonly present in environment. These pathogens considered as an important cause of infectious diseases mainly existed in water, air and solid waste, such as garbage. Besides, global warming and human activities enhanced the process of lake eutrophication. Furthermore, they might also influence the abundance, virulence and survival of pathogens that are already resident in aquatic ecosystems. Currently, researches about the mechanistic responses of pathogens to increasing nutrients are scarcely refered but maily involved some bacteria (eg. Vibrio cholerae). Legionella spp., one of the familiar pathogens, is the causative agent of Legionnaires' disease and non-pneumonic Pontiac fever. These bacteria cause respiratory disease in humans when a susceptible host inhales aerosolized water containing the bacteria or aspirates contained water. So far, about 50 Legionella species have been defined, nearly half of which have been associated with cases of disease. At present, research about it mainly focused on those in artificial water systems such as cooling towers, sewage treatment plants, water distribution systems, as well as serum. However, few studies were put in the distribution and diversity of Legionella in freshwater ecosystem and in the relationship between Legionella distribution and nutrient concentration.To understand the presence of Legionella spp. in shallow eutrophic lakes, Lake Taihu was selected, which is seriously eutrophic and demonstrated various nutrient states in different parts. The diversity and community structure of Legionella spp. in different regions of Lake Taihu and its relationship with nutrient levels were studied. At first, a rapid detection method nest-PCR-MPN (nested polymerase chain reaction with most probable number) was established to detect Legionella in water samples. The presence of Legionella spp. was screened by this method and the phylogenetic diversity was determined by denaturing gradient gel electrophorese (DGGE) of two different living conditions. Canonical correspondence analysis (CCA) was carried out to infer the relationship between environmental variables and bacterial community structure. The dominant environmental factors which impact the bacterial community in each lake regions were selected, which helped identifying the relationship between distribution Legionella and nutrient gradient. Finally, their community structure was determined by sequencing the excised DGGE bands. The risk of existence of pathogenic Legionella species in different regions of Lake Taihu was speculated. The main results were as followed.1,To directly identify the presence of Legionella in freshwater environments, a nested-PCR-MPN method was set up. Nested-PCR assay applied two pairs of primers to amplify the 16S rRNA gene of Legionella spp.. In the positive amplified samples, the detection limit of this method was evaluated by using decimal dilutions of genomic DNA until the nested-PCR was negative. Clone libraries and phylogenetic affiliation analysis were also used for verifying the specificity of this method. The results showed that our established method was specific to detect Legionella spp. from environmental samples, and the detection limit cloud reach 2.74 fg/μL of Legionella DNA.2,The established nest-PCR-MPN method was applied to investigate the distribution and diversity of Legionella spp. in Lake Taihu. The results showed that the Legionella spp. presented in the whole lake except the free-living bacterial samples of Wuli lake. The detection ratio of Legionella spp. in the northern (eg. Meiliang Bay and Gonghu Lake) and southeast (eg. Southern coastal areas and eastern coastal areas) of the lake was up to 20%. From February to November, the detection rate firstly increased and then decreased. The highest detection rate 75%was detected from the particle-associated bacteria samples at August The lowest detection rate was 12.5%in free-living bacteria samples at November. Overall, the detection rate of the particle-associated bacteria was more than 50%, while that of free-living bacteria was more than 12.5%. Paired-sample t test showed that the detection rate were significant differences between free-living and particle-associated bacterial samples at February and Novembre (p=0.044 and 0.024 respectively), and extremely significant differences in August (p=0.001).3,The genetic diversity of positive Legionella spp. samples were analyzed by DGGE. Legionella spp. band number, population diversity (Shannon-weaver index), dominance (Simpson index) and evenness (Pielou index) of different lake districts were calculated. The results showed that particle-associated bacterial samples in Gonghu bay and western coastal areas were rich in Legionella spp. diversity. DGGE band number increased with the change of seasons, but the Shannon-Wiener index, Simpson index and Pielou index firstly decreased and then increased. The minimum values of three indexes occurred in the February or May, and the maximum occurred in August or November, The free-living bacterial samples in Meiliang Bay, East Taihu Lake and southern coastal areas were rich in diversity. DGGE band number, Shannon-Wiener index and Simpson index increased at first and then decreased. The maximum data of three indexes were in May, and the minimum were in November.4,The typical DGGE bands were excised and sequenced. Totally 68 sequences were obtained, which could be classified into 28 OTUs by 97% similarity. On the basis of bacterial 16S rRNA gene phylogenetic analysis, all sequences belonged to Legionella spp., and most were uncultured Legionella spp. In addition, some sequences were highly similar with the pathogenic Legionella spp., such as Legionella feeleii, Legionella longbeachae,Tatlockia maceachernii and Legionella pneumophila.5,Multivariate statistical methods was applied to analyze the association between Legionella spp. composition and environmental factors. The results showed that the nutritional level of Taihu Lake (TN, TP, turbidity, etc.) and the total biomass of phytoplankton are important factors that influencing the Legionella spp. composition. Therefore, in Taihu Lake, Legionella spp. composition and diversity were certainly correlated with the nutrient gradient. In the northern higher nutrient region (Meiliang Lake, Gong Lake) and south-west region (western coastal areas and southern coastal areas), the detection rate of Legionella spp. and population diversity were higher.It is the first systematic analysis of Legionella spp. composition, spatial and temporal variations of diversity in shallow eutrophic Lake Taihu, and the dominant environmental factors were analyzed. The obtained results promoted to reveal the relationship between lake eutrophication and the distribution and diversity of Legionella spp., which would be a foundation to assess the potential health risks interfered with the pathogenic microorganisms of eutrophic lakes. |
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