| Occurrence and accumulation of antibiotic resistance genes(ARGs) are significantly promoted by animal production activities. Antimicrobials used in animal feedlots not only select lots of ARGs in animal intestine, but also pose selective pressure on indigenous microorganisms when they are introduced to the environment via animal manure. What is important, ARGs in high background levels contaminate the soils and downstreams adjacent to animal feedlots via manure, and are also transferred to farm lands and vegetable bases via manure fertilizer, potentially threatening public health. Under this background, this study investigated the diversity and arrangement of ARGs in broiler intestine, and also investigated the occurrence and accumulation of ARGs in manure-polluted soil and water bodies; at last we measured ARG pollution in the Liuxi River in Guangzhou city. The main content and results in this study are as follows: 1 Arrangement and distribution of ARGs in animal intestineThis study investigated the diversity, arrangement and distribution of ARGs in broiler intestine, and investigated the variations of ARGs after broilers were treated by chlortetracycline under normal administration. The 54 broilers were randomly and averagely divided to three groups, including the control group, the low dose group, and the treatment dose group. Each group had three replicates, and each replicate had six broilers. Broiler manure was collected at different time points before and after chlortetracycline treatment. We extracted metagenome of broiler manure. We analyzed the abundance and diversity of ARGs, located the hosts carrying ARGs, and determined the arrangement of ARGs in integron, plasmid and phage. The results showed that ARGs were very abundant(3.1 ARGs/16 S r RNA gene at average) in the intestines of broilers that were not treated by antimicrobials. ARGs in broiler intestine exposed to the selective pressure of chlortetracycline according to a normal drug administration, did not have dramatically significant increase, particular in tetracycline resistance genes. The results indicated that reasonable treatment of antimicrobials in broilers farming may not significantly increase ARGs in the intestines. The main ARGs were multidrug resistance genes(53.1%) followed by tetracycline resistance genes(9.3%) in broiler intestine. It was noted that the disinfectant acriflavine resistance genes(7.6%) were heavily polluted. The hosts carrying most abundance of ARGs were Escherichia(83.2%), followed by Klebsiella(6.0%) and Shigella(5.0%). We observed that class I integron carried streptothricin acetyltransferase(Sat1), aminoglycoside adenyltransferase(Aad A1 and Aad A2), chloramphenicol(Cml A), quarternary ammonium compound(Qac) and sulfonamide resistance genes in the experiment. We also observed that phage carried fosmidomycin(Fsm) resistance genes and the disinfectant acriflavine resistance genes(Acr A and Acr B). This study is the first report to analyze the arrangement and distribution of ARGs in broiler intestine. The results guided reasonable administration in the clinical, and provided strong evidences of spread mechanism of ARGs in broiler intestine, such as ARG spread by phage. 2 Effect of manure application on ARGs and bacterial community in soilsThis study investigated the occurrence and accumulation of tetracycline, sulfonamide and plasmid-mediated quinolone resistance(PMQR) genes in soils after application of swine manure containing veterinary drugs that are commonly used. And we also analyzed the variations of bacterial community composition. The soils treated with manure and antimicrobials were set up as the treatment groups, including tetracycline, sulfonamide and fluoroquinolone treatment groups, respectively. The soils only treated with manure was the manure group, and the soils without the treatment of manure and antimicrobials served as the control group. The abundance of ARGs in groups on day 0, 30 and 60 was analyzed by using real time quantitative PCR(q PCR). The bacterial community composition in groups on day 30 was analyzed by using metagenomic sequencing based on 16 s r RNA gene. The results showed that most ARGs(tetracycline resistance genes: tet M, tet O, tet W, tet S, tet Q and tet X; sulfonamide resistance genes: sul1, sul2 and sul3; and PMQR genes: oqx A, oqx B, aac(6’)-Ib-cr and qnr S) significantly increased after manure application(P<0.05). The dissipation rates of tetracycline resistance genes(tet M, tet O, tet W, tet Q and tet X), sulfonamide resistance genes(sul1, sul2 and sul3) and PMQR genes(oqx A, oqx B, aac(6’)-Ib-cr and qnr S) in the manure groups were-38% to-97%,-78% to-83%, and-76% to-94% per month, and these ARGs in the antimicrobials treated groups were 48% to-93%,-50% to-66%, and-58% to-84% per month, respectively. The manure containing antimicrobials significantly selected for special bacterial communities. For example, Steroidobacter, Bradyrhizobium, Nitrospira, Gp4, Gp6, Clostridium and Stenotrophomonas were significantly increased after sulfonamides treatment. The results indicated that the occurrence and accumulation of ARGs in soils were promoted by manure application. Antimicrobials in the manure slowed down the dissipation of corresponding ARGs introduced by manure in the manured soil. And the manure containing different classes of antimicrobials selected for special bacteria in soils. 3 Effect of manure pollution on ARGs and bacterial community in river waterThis study investigated the occurrence and accumulation of tetracycline, sulfonamide and PMQR genes in river water on day 14 after the treatment of swine manure containing common antimicrobials. And we also investigated the effect of the selective pressure of antimicrobials on bacterial community composition in the sediment and water. The water bodies treated with manure and antimicrobials were set up as the treatment groups, including tetracycline, sulfonamide and fluoroquinolone treatment groups, respectively. The water bodies only treated with manure was the manure group, and the water bodies without the treatment of manure and antimicrobials served as the control group. Of sediment samples, the relative abundances of tetracycline resistance genes(tet M, tet O, tet W, tet Q, tet X, and tet S), sulfonamide resistance genes(sul1, sul2 and sul3) and PMQR genes(oqx A, oqx B, aac(6’)-Ib and qnr S) in the treatment groups were 1.8~4.5, 4.5~7.2 and 1.8~4.2 fold of those in the control group, respectively. Compared with the control group, antimicrobials groups significantly selected for various special bacterial communities. For example, tetracyclines significantly selected for Salmonella, Escherichia/Shigella, Clostridium and Stenotrophomonas in the sediment. The results indicated that manure containing antimicrobials significantly selected for most ARGs in the sediment and water bodies, and also selected for special bacterial communities including the taxa associated with opportunistic pathogens in the water bodies. 4 Analysis of ARGs and bacterial community in the Liuxi RiverWe investigated the abundance of ARGs and bacterial community composition in the Liuxi River with heavy manure pollution in a field study. We collected sediment and surface water samples from upstream and downstream in the Liuxi River. Relative abundance of most detected ARGs(tet M, tet O, tet W, tet S, tet Q, tet X, sul1, sul2, sul3, oqx A, oqx B, aac(6’)-Ib and qnr S) was in the range of 10-5 to 10-2, and the absolute abundance of these ARGs were in the range of 102 to 108 copies/g(or m L). Some taxa associated with opportunistic pathogens were also observed, such as Acinetobacter, Clostridium and Arcobacter observed in the sediment and water, with the abundance up to 29%, 0.68% and 0.58%. The results indicated that the Liuxi River was heavily polluted by ARGs. These ARGs and taxa associated with opportunistic pathogens observed in the river posed the potential to threaten the health of residents in the river bank. 5 ConclusionsAnimal intestines are reservoirs of ARGs. High abundance of ARGs was observed in the intestines of broilers that were not treated by antimicrobials. No dramatically significant increase of ARGs was found in the intestine of broilers that were treated by chlortetracycline under normal administration. The main ARGs were multidrug resistance genes, and the hosts carrying most abundance of ARGs were Escherichia in broiler intestine. Integrons, plasmids and phages carrying ARGs observed in this study is benefit to the horizontal spread of ARGs.Animal manure is an important medium for the spread of ARGs to the environment(farm lands and rivers). Tetracycline resistance genes(tet M, tet O, tet W, tet S, tet Q and tet X), sulfonamide resistance genes(sul1, sul2 and sul3) and PMQR genes(oqx A, oqx B, aac(6’)-Ib-cr and qnr S) significantly increased after manure application. But the durations of significant increase of ARGs introduced by manure to soil are different. Antimicrobials including tetracyclines, sulfonamides and fluoroquinolines in manure slowed down the dissipation of corresponding ARGs introduced by manure in the manured soils, and posed selective pressure on ARGs in sediment and water samples in water environments. Meanwhile, manure containing antimicrobials significantly select for special bacteria including the bacteria associated with opportunistic pathogens in soils and water environments. |
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