| Antibiotics, in addition to uses for human disease treatment, have been widely used in the rearing of livestock and aquatic products as well as meat production since the beginning of the1950s. About70%of total output of the antibiotics is used in animal husbandry all round the world. Because of the long-term abuse of veterinary medicines in animal husbandry and aquaculture, many drug-resistant strains coding antibiotic resistance genes have been induced in the gut of farmed animals, which are the most important source of antibiotic resistance genes in the environment.According to the conventional manure application rates in agricultural soils, we set up an application rate (20000kg·hm-2PG8),2times the conventional application amount (40000kg·hm-2, PG16) as well as four times the conventional application amount (80000kg·hm-2, PG32) different fertilization levels, and set the corresponding three levels of sterilized pig manure (SPG8, SPG16, SPG32). Additionally, based on the average residue levels of tetracycline in pig manure (0.533mg·kg-1), we also set0.5mg·kg-1(TC0.5),5mg·kg-1(TC5) and50mg·kg-1(TC50) and other three treatments of pollution of antibiotics. Through the outdoor soil column experiment, the number of resistant bacteria and resistance genes in soil were analyzed for a period of six months after livestock and poultry manure carrying the resistance genes were applied in the soil by the use of traditional culture methods based and PCR approach. The results are as follows:(1) In day0of swine manure application, the number of resistant bacteria of PG8, PG16and PG32in the arable layer of soil were6.3x104CFU·g-1,3.6x105CFU·g-1and6.1×105CFU·g-1, which is104,105and105times higher than the control. Pig manure significantly increased the number of resistant bacteria in the arable layer of soil and the number of the resistance bacteria increase with the amount of manure application. The number of resistant bacteria is in a continuous decreasing trend against time; the number of the bacteria in the treatment of PG8and PG16reduced to control levels by day150, while in the PG32treatment the number of resistant bacteria reduced to control levels by day180.(2) In day0of sterilized swine manure application, the resistant bacteria of3different levels of treatments of SPG8, SPG16and SPG32in the arable layer of soil were not detected. After30days of application, we detected a large number of drug-resistant bacteria of the treatments of SPG8, SPG16and SPG32, and the number is as follows:4.6×103CFU·g-1,2.5×104CFU·g-1and1.6×105CFU·g-1which is the highest value in the entire experimental period. The number of resistant bacteria is in a continuous decreasing trend against time; the number of resistant bacteria of the treatments of SPG8and SPG16reached the control level in the day150, while the treatment of SPG32achieved control level in day180. Relatively the same level of fertilization, the number of resistant bacteria between the different detection time of pig manure and sterilized pig manure shows:the number of resistant bacteria in soil applied with sterilized soil is more than halved.(3) Of analyzing the impact of the TC on the number of resistant bacteria in the soil, the results show that low concentrations of TC had a significant role in promoting drug-resistant bacteria of the30-days and60-days soil with sterilized swine manure, while to the resistant bacteria of the60-days soil with pig manure and the bacteria of the0-day and the60-days to120-days soil with pig manure, it was significantly inhibited; the middle level concentration of TC had a significant role in promoting drug-resistant bacteria of the30-days,60-days and90-days soil with sterilized swine manure, while to the resistant bacteria of the0day to60-days soil with pig manure and the bacteria of the0-day to the30-days and the90-days to120-days soil with pig manure, it was significantly inhibited; the high level concentrations of TC had a significant role in promoting drug-resistant bacteria of the30-days and60-days soil with sterilized swine manure, while to the resistant bacteria of the60-days soil with pig manure and the bacteria of the30-days and the60-days soil with pig manure, it was significantly inhibited. In general, from day0to day120of the treatment, all concentrations of TC had a general inhibition on the number of resistant bacteria in the soil fertilized with pig manure. However, all had a general role in promoting resistant bacteria in the soil with sterilized pig manure and did not affect the formation of resistant bacteria in the soil without pig manure.(4) Through4track determinations of the number of resistant bacteria in the soil columns of each treatment (20-40cm) and lower layer (40-60cm) in period of0～150days, the results showed that:In day0, the resistant bacteria of3levels of pig manure (PG) and three levels of sterilization pig manure (SPG) as well as different TC levels in20-40cm and40-60cm soil layer were not detected; after30days or60days, resistant bacteria can be detected in20-40cm and40-60cm soil layer. This phenomenon indicated a decreasing trend of the resistant bacteria; with time goes on, the number of drug-resistant bacteria was reduced to the level of the control in day150.(5) By using of7encoding ribosomal protection protein genes which include let(M). tet(O)、 tet(Q), tet(S),tet(T), tet(W) and tet(B/P)and7efflux genes include tet(A),tet(B), tet(C), tet(D), tet(G), tet(Y) and tet(Z), we did a PCR amplification of each treatment. The results of PCR amplification on the pig manure sample showed that there are3ribosomal protection protein genes which include tet(B/P), tet(M) and tet(W) and4efflux genes include tet(B), tet(D), tet(G) and tet(Z) in the pig manure. According to the results, we did a PCR amplification on the DNA extract of the bacteria cultured by the antibiotic tablet. The results showed that all the5kinds except tet(Z) and tet(D) of resistance genes can be detected in the culturable bacteria of the arable layer of soil with the conventional fertilization of pig manure in the whole fertilization cycle. There was a downward migration of the resistance gene, but the rate of gene migration among the genes is inconsistent. There was still some resistance genes left in the soil at the end of the fertilization cycle. High concentrations of tetracycline pollution in soil did not produce resistance genes in the short term. |
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