Decay Of Tetracycline Resistance Genes And Shift Of Bacterial Community Structure In Receiving River Contaminated By LivestockWastewater

The uncontrolled use and misuse of antibiotics have received growing concerns and can contribute emergence and concentration of various antibiotic resistant bacteria (ARB) in the animal gut. The ARB has been considered as the major source of antibiotic resistance genes (ARGs) in the environment. Moreover, the discharge of ARB along with animal feces can accelerate the development and spread of antibiotic resistance genes. Nowadays, tetracyclines are one type of antibiotics commonly used in Chinese swine farms to treat disease and promote growth, and the large-scale use of tetracyclines contributes to the occurrence and spread of tetracycline resistance genes (TRGs) in the environment, posing a great threat to human health and environment.In this study, water samples were collected from a small pig farm in Taihu Lake Basin which located in Wujin District, Changzhou City of Jiangsu Province, and the basic water quality parameters were analyzed according to the standard methods. Polymerase chain reaction (PCR), quantitative real-time PCR (qPCR) and 454 pyrosequencing were then simultaneously employed to detect the TRGs diversity, quantify the relative abundance of TRGs, figure out the relationship between TRGs and fecal contamination, and explored the change of bacterial community in livestock wastewater and the receiving river water. The detailed results of this study are listed as follows:(1) The receiving river water was severely contaminated by the untreated swine wastewater. Generally, the water quality cannot meet the lowest requirements of Environmental Quality Standards for Surface Water in China. The detection of host-specific genetic marker revealed that the rivers were only contaminated by swine feces. Although the results of qPCR demonstrated that fecal pollution gradually reduced along the river flow, the pig-specific Bacteroidales genetic marker was still detected in the farthest sampling site located in the Taige River.(2) The bacterial diversity indices showed a generally decreasing trend along the river flow, demonstrating that self-purification and hydro logical dilution may reduce the bacterial diversity. Generally, microbial community structure of water samples shifted along with seasons change. The proportion of Proteobacteria and Bacteroidetes were slightly different in all the three months, but they all predominated in all the samples. In addition, Pseudomonas, Arcobacter, Fibrobacter, Treponema and Oscillibacter were detectable in all the wastewater samples, suggesting that those kinds of genera were indicative bacteria in the livestock wastewater.(3) Twelve types of TRGs, including tet(B), tet(C), tet(D), tet(E), tet(G), tet(L), tet(M), tet(O), tet(Q), tet(W), tet(S) and tet(X), were present in the livestock wastewater and receiving river water samples of all the three months. The relative abundance of the genes encoding four ribosomal protection proteins (RPPs) (tet(O), tet(Q), tet(M) and te/(W)) decreased with downstream flow. The relative abundance of these genes ranged from 10-3 to 10-2 copies/16S rRNA copies at the last three sampling locations, which are still higher than previous reports on the natural water environment.(4) In general, the relative abundance of tet(O), tet(Q), tet(M) and tet(W) showed good positive linear correlations with the pig-specific Bacteroidales genetic marker in all samples of different seasons, so the results demonstrated that the abundance of host-specific microbial genetic markers can indicate the distribution and abundance of ARGs in the complex environmental system. What’s more, the source of TRGs in receiving river water was also identified by using this method. The conclusion is of great significance in ARGs source tracking and rapid analysis.In conclusion, this study firstly identified the source of fecal pollution in the wastewater and receiving river water. The bacterial community structure of the wastewater and receiving river water was then comprehensively analyzed by 454 pyrosequencing, and the diversity and abundance of TRGs were also quantified by using PCR and qPCR Finally, the relationship between TRGs and fecal pollution was also preliminarily explored. In the future, Illumina high-throughput sequencing technology will be applied to comprehensively characterize the diversity and abundance of ARGs. The correlation among ARGs, fecal pollution, tetracycline concentrations, mobile genetic elements and bacterial community structure also will be statistically analyzed.