Characteristics And Mechanism Of Resistance Genes Pollution Caused By Oxytetracycline Manure Application In Soil Environment

In order to better understand ARGs pollution characteristics caused by low concentration of long-term antibiotics exposure in the agricultural soil, to provide important theoretical basis and data support of ecological safety assessment, tetracycline resistance genes (TRGs) were focused as main object in this paper. Variation of tetracycline resistance genes abundance caused by three tetracyclines (TCs:OTC, TC, CTC) residues exposed in the agricultural soils was explored used simulation approach in laboratory. The influence of the concentrations of oxytetracycline (OTC) in manure and different fertilization frequency on the tetracycline resistance genes was also studied. Furthermore, by adding quorum sensing inhibitors into soils, the mechanism of the formation of resistance genes pollution in soils based on quorum sensing theory were discussed. The main results obtained in this research were as follows: (1) Three TCs, entered into the soils with the manure Fertilization, showed a continuous decrease, the dissipation rate reached90%at day140, and there was no significant difference of dissipation rate among three different TCs treatments in the soil environment (p>0.05). Antibiotic exposure significantly affect the soil microbial community structure especially in CTC treatment, and there is no significant difference between TC and OTC treatment according to diversity analysis and cluster analysis (p>0.05). Although the tet(A), tet(L), tet(M), tet(Q) abundance were different from each other, but it all showed a tendency of decrease after an initial increase. Its abundance were ranged from10-5to10-3. As exposed to these three TCs, tet(L) and tet(M) abundance both have no significant difference from each other, while tet(A) and tet(Q) not, but always higher than CK (p<0.05)(2) The dissipation rate of OTC in soils was always around50%. Repeated application of different concentrations of manure had caused an accumulation of OTC residues in the soil environment. However, continued exposure to different low-level OTC did not significantly alter microbial community structure, the diversity of the population structure of three concentrations treatments had no statistical difference (p>0.05). The abundance of tet(M) and tet(Q) were significantly positively correlated with residual OTC concentrations (R2=0.965, p<0.01; R2=0.932, p<0.01). While OTC levels<40μg/kg, tet(A) and tet(L) increased slowly as OTC accumulated. At OTC levels>40μg/kg, tet(A) and tet(L) were sharply upregulated.(3) Different manure application frequency significantly influenced the accumulation of OTC residues. The residual concentration was65.05μg/kg,137.78μg/kg and315.50μg/kg in single,5times and10times application treatments, respectively. Higher application frequency caused more OTC residues in soils when added the same concentration. Three application frequency treatments did not change microbial community structure significantly. For tet(A) and tet(L), the higher frequency caused more accumulated abundance. While for tet(M) and tet(Q), it had the highest abundance in treatment with manure application every14days, but accumulated the lowest in treatment with manure application every28days. Manure application frequency significantly influence the TRGs abundance. It is necessary for controlling manure application frequency reasonably.(4)4-NPO and halogenated furanone inhibits the release of AHLs signaling molecules.4-NPO application into soils inhibited C4-HSL, C6-HSL and C8-HSL significantly, while no effect for3-oxo-C6-HSL. halogenated furanone can inhibited all these four signaling molecules. Residual OTC concentrations had no significant difference among treatments before and after inhibitors application when compared with CK2(p>0.05), indicated no effect on OTC dissipation. Two quorum sensing inhibitors caused a decrease trend of intI1abundance, especially applied4-NPO to soil continuously. It shows that quorum sensing can mediate horizontal gene transfer which associated with spread of TRGs. Efflux pump genes (tet(A), tet(L)) abundance appeared significantly attenuated after repeated application of4-NPO, while it had limited effect for controlling ribosomal protein genes (tet(M) and tet(Q)). Controlling for tet(L) and tet(Q) is more pronounced with halogenated furanone application, but it had limited effect of suppressing for the tet(A) and tet(M). It suggested that quorum sensing promote the increase of ARGs abundance, but different quorum sensing inhibitors have different effect for controlling the spread of specific ARGs.