| Presently, the biological and physical variable of microflora in pig manure is not clear yet. In this study, denaturing gradient gel electrophoresis was optimized in the running parameters including level electrophoresis, electrophoresis time and dying time, respectively. The optimized DGGE method was subsequently performed to analyze the diversity of the manure samples from 5 main running scenorios of 5 pig farms distributed along Jialing River. And then, bacterial 16S rDNA clone library of corresponding manures was established. Moreover, antibiotics resistance genes in microbial community DNA extracted from manure samples were amplified by using multiplex PCR detection kit of 4 different antibiotics resistance genes in bacteria isolated from animals. What’s more, it’s also concerned with isolation of antibiotics resistance detection of Staphylococcus and E.coli. from the manure samples.This research will benefit further study on wide microbial diversity, manure management practices, inestimable number of combinations of potential pathogen fates in environment, and environmental factors that influence the presence, persistence, survival, and transport of pathogens.1. Optimization of denaturing gradient gel electrophoresis for bacterial communities in manure from pig farm Ploymerase chain reaction and denaturing gredient gel electrophoresis (PCR-DGGE) technique was used to reveal the bacterial community composition and diversity associated with manure from pig farm. In order to improve the accuracy, sensitivity and repeatability of DGGE based on 16S rDNA V3 region, the running parameters of DGGE were optimized by level electrophoresis, electrophoresis time and dying time, respectively. The results showed that the best range of denaturing gradients was 35-60% in 8% polyacrylamide gels, running time was 12 hours, and the dying time was 30 min using SYBR Green Fluorescent Dye. Furthermore, bacterial communities from 5 excrement samples and 5 slurry samples were also performed by using these optimized reaction conditions. It was found that 10 manure samples exhibited effective separation and rich microbial diversity. This revealed that different manure samples result in the distinct changes of microbial diversity and microbial community structure. PCR-DGGE is proven to be a powerful tool for describing the bacterial flora in manure from pig farms.2. Study on the bacterial abundance of manure sample in pig farms using DGGEDenaturing gradient gel electrophoresis (DGGE) technology was used in studying the diversity of manure samples from 5 main running scenario of 5 pig farm continuously distributed along Jialing River. The detection result showed that the abundance of manure samples was not very rich, while the DGGE bands of manure samples were around 10 respectively. And as for the bacteria diversities and community, there is no positive relationship among the same scenario of difference pig farms, the successive scenario in the same pig farm, and the pig farm distributing along the same river. Furthermore, after recovery and identification the bands from DGGE profiles, the results revealed that above 70% recovery band were Staphylococcus, Escherichia coli and uncultured microorganisms. Therefore, it could be concluded that Staphylococcus, Escherichia coli and uncultured microorganisms were the majority of bacterial species in manure from pig farms.3. Generation and characterization of 16S rDNA library of the manure form pig farmsBacterial 16S rDNA clone library of manure form pig farms was constructed in this research. From cloned PCR products,200 randomly-selected clones containing inserts were sequenced. After removing the vector sequence and trimming the poor-quality sequences, we obtained 106 qualified sequences, with an average length of approximately 1400bp. Tentative annotations were performed by using BLASTX and results were manually validated. According to comparison analysis, Escherichia coli,Staphylococcus and Pseudomonas were the main species. The Homology analysis could be not more than 97% between 50% of 16S rDNA sequences and known DNA sequences in GenBank. It showed that there may be a new bacteria or species. The result indicated that diversiform bacteria maybe exist in manure form pig farms.4. Detection of antibiotics resistance genes in the microbial community DNA extracted from the manure of pig farmsThe antibiotics resistance genes were amplified in microbial community DNA extracted from 25 manure samples from 5 main running scenario of 5 pig farm distributed along Jialing R;ver by using multi-PCR detection kit of Choramphenicols resistance genes in animal original bacteria, the multi-PCR amplification kit of bacteria Aminoglycoside-resistance genes detection, the multi-PCR detection kit of Sulfonamides-resistance genes in bacteria isolated from animals and the multi-PCR detection kit of Tatracycline-resistance genes in bacteria isolated from animals. The result showed that the integrative detection rate of the antibiotics resistance genes from the sewage outlet, biogas pool and oxidation pond is 100%. The antibiotics resistance genes of Choramphenicols and Tatracyclines decreased remarkably after land application, and there was no antibiotics resistance genes detection in underground samples. On the other hand, the fertilization had poor effect on the elimination of the antibiotics resistance genes of Sulfonamides and Aminoglycosides, and the detection rate was above 90% yet in under ground water. It indicated that the resistance genes of often-used antibiotics spread universally, such as Choramphenicols, Tetracycline, Sulfonamides and Aminoglycosides. Furthermore, land application may be a most important scenario of reducing the antibiotics resistance genes.5. Isolation and antibiotics resistance gene detection of Staphylococcus and E.colL in the manure from pig farms This research was concerned with isolation of antibiotics resistance detection of Staphylococcus and E.coli. from 25 manure samples. The result indicated that the isolation rate of E.coli. was 44% in 25 manure samples, the integrative detection rate of drug-resistance genes of Choramphenicols, Tetracycline, Sulfonamides and Aminoglycosides was respectively 82%,100%,100% and 100%. The isolation rate of Staphylococcus was 64% in the manure of pig farms, the integrative detection rate of drug-resistance genes of Choramphenicols, Tetracycline, Sulfonamides and Aminoglycosides was 94%,94%,100% and 100% respectively. The research result indicated that Staphylococcus and E.coli. were commonly found in manure from pig farms, and they might be a important preserver of antibiotics resistance genes in the agricultural environment, and the reasonable application of antibiotics could be obtained special attention in the livestock production process. Moreover, Staphylococcus could be a candidate bacteria indicator similar to E.coli. for manure management of livestock production. |
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