Identification Of Swine-specific Microbial Genetic Markers Using A Metagenomic Approach For Microbial Source Tracking

Quality of water environment has important impact on the quality and safety of aquatic products and public health.However,non-point sources pollution including human fecal and animal fecal pollution has become the major concern of environmental management,marine ecology and the development of aquaculture industry.Therefore,it is particularly urgent to establish a microbial source tracking(MST)method that can be used as an efficient indicator of fecal pollution with high sensitivity and strong specificity.In animal guts,genes which are directly involved in bacterium-host interactions may display increased levels of host-associated genetic variations,making them promising candidates for fecal source tracking.The aim of this work was to apply a genome fragment enrichment(GFE)method to enrich swine-specific metagenomic regions that differ from the fecal metagenome of other animal species as well as establish the corresponding Real-Time PCR assays,and eventually promote the development of microbial source tracking studies in unknown fecal sources of water environment.The work indicated that these host-specific assays could be used for sensitive tracking of fecal pollution.The major results are as follows:1.A total of 260 fecal DNA samples from 9 animal species were isolated and used for enrichment of swine-specific metagenomic regions that differ from fecal metagenome of other animal species and directly involved in bacterium-host interactions.Enriched sequences were assigned bacterial class annotations based on the top BLASTx hit(lowest E-value score)in GenBank non-redundant(NR)database.A total of 384 non-redundant swine-specific metagenomic sequences were retrieved and analyzed,of which 82%are similar to genes present in intestinal bacteria belonging to Bacteroidetes(43.2%),Clostridia(19.5%)and Bacilli(8.6%).The putative protein transcript of each sequence was analyzed based on the biochemical function of similar gene sequences using BLASTx with GenBank NR database.Approximately 61.5%of swine-specific metagenomic sequences can be assigned to previously characterized proteins,including proteins involved in information storage and processing(7.6%),cellular processes(12.8%)and metabolism(22%).It is concluded that genes putatively encode surface proteins,membrance-associated or secretory proteins and carbohydrate metabolism proteins of dominant bacterial classes could be regard as targets for swine-specific microbial genetic markers.2.Twenty-six sequences from the swine clone libraries were selected to develop swine fecal-specific PCR assays.The host specificity of PCR assays were tested against fecal DNA form swine and other non-target animals.A total of four PCR assays(1-38,2-95,2-109,3-53)were restricted to swine specificity,while the rests were cross-reacted with at least one non-target domestic animal,suggesting that the four specific primers were good candidates to develop following swine fecal-specific PCR assays.3.Four optimal conditions for each primer and probe pair were conducted to establish the corresponding real-time PCR assays.The overall host specificity values of the 1-38,2-95,2-109,2-53 markers to differentiate between swine and other non-target host groups were 93%,73%,79%and 90%.The overall host sensitivity values for these markers,including swine wastewater and individual swine fecal DNA samples,were 94%,88%,72%and 90%,respectively.The results showed the real-time PCR assays base on 1-38 and 3-53 exhibit better detection effects,and are potential to be used for microbial source tracking.4.The water samples collected from 27 different places of Zhejiang province were tested for the FIB(E.coli and Enterococcus spp.)using culture-based methods and for potential zoonotic bacterial pathogens(Campylobacter spp.,E.coli 0157,and Salmonella spp.)using real-time PCR assays.Based on Bayes' theorem,the probability of detection of the 1-38 and 3-53 markers in water samples that were due to true swine fecal pollution instead of non-target hosts was 92.5%and 91.7%respectively,indicating the validity of the real-time PCR assays based on swine-specific markers(1-38,3-53).Among the 64 water samples tested,70.3%,17.2%and 51.5%samples were PCR positive for E.coli 0157 rfbE,Campylobacter 16S rRNA and SalmonellainvA,respectively.These results suggested that the presence of the swine feces-associated markers along with the presence of potential zoonotic pathogens indicates swine fecal pollution in the reservoir water samples.