| Antibiotic resistance has become a major global public health issue in recent years. Antibiotic resistance genes(ARGs) are increasingly regarded as emerging environmental contaminants. ARGs have been found to be able to spread among bacteria via vertical transfer and horizontal transfer, and distribute from human and animal sources to receiving environments. Growing scientific evidence show that antibiotics used in livestock industry may increase the development and abundance of ARGs, which will be transferred into the receiving environments through waste disposal processes such as manure application on land. However, the diversity of ARGs in livestock operations and their dissemination mechanisms to the environment still remain to be explored. This study investigated the occurrence, distribution and dissemination mechanism of ARGs in 11 broiler feedlots, 3 swine feedlots, aiming to assess their impacts on the receiving environments due to discharge of wastewater into rivers and application of wastes on land.We examined the occurrence, abundance and variation of ARGs conferring resistance to chloramphenicols, sulfonamides and tetracyclines in the environments of two representative types of broiler feedlots(free range and indoor) by quantitative PCR, and assessed their dissemination mechanisms. The results showed the prevalence of various types of ARGs in the environmental samples of the broiler feedlots including manure/litter, soil, sediment, and water samples, with the first report of five chloramphenicol resistance genes(cmlA, floR, fexA, cfr, and fexB) in broiler feedlots. Overall, chloramphenicol resistance genes and sulfonamides sul genes were more abundant than tetracyclines tet genes. The ARG abundances in the samples from indoor boiler feedlots were generally different to the free range feedlots, suggesting the importance of feeding operations in ARG dissemination. Pearson correlation analysis showed significant correlations between ARGs and mobile genetic element genes(int1 and int2), and between the different classes of ARGs themselves, revealing the roles of horizontal gene transfer and coselection for ARG dissemination in the environment. Further regression analysis revealed that fexA, sul1 and tetW could be reliable indicator genes to surrogate anthropogenic sources of ARGs in boiler feedlots(correlations of fexA, sul1 and tetW to all ARGs: R = 0.95, 0.96 and 0.86, p < 0.01). Meanwhile, significant correlations were also identified between indicator ARGs and their corresponding antibiotics. In addition, some ARGs were significantly correlated with typical metals(e.g., Cu, Zn, and As with fexA, fexB, cfr, sul1, tetW, tetO, tetS: R = 0.52-0.71) and some environmental parameters(e.g., TOC, TN, TP, NH3-N with fexA, fexB, cfr, sul1, tetW, tetO, tetQ, tetS: R = 0.53-0.87)(p < 0.01). Further redundancy analysis demonstrated that the distribution and transportation of ARGs from the boiler feedlots to the receiving environments were correlated with environmental variables. The findings highlight the contribution of some chemicals such as antibiotics and metals to the development of ARGs in broiler feedlots environments; and the observed ARG dissemination mechanism in the broiler feedlots facilitates the development of effective mitigation measures.Swine feedlots are widely considered as a potential hotspot for promoting the dissemination of ARGs in the environment. ARGs could enter the environment via discharge of animal wastes, thus resulting in contamination of soil, water, and food. We investigated the dissemination and diversification of 22 ARGs conferring resistance to sulfonamides, tetracyclines, chloramphenicols, and macrolides as well as the occurrence of 18 corresponding antibiotics from three swine feedlots to the receiving water, soil environments and vegetables. Most ARGs and antibiotics survived the on-farm waste treatment processes in the three swine farms. Elevated diversity of ARGs was observed in the receiving environments including river water and vegetable field soils when compared with respective controls. The variation of ARGs along the vertical soil profiles of vegetable fields indicated enrichment and migration of ARGs. Detection of various ARGs and antibiotic residues in vegetables fertilized by swine wastes could be of great concern to the general public. This research demonstrated the contribution of swine wastes to the occurrence and development of antibiotic resistance determinants in the receiving environments and potential risks to food safety and human health.To explore the different influence of swine and broiler feedlots to the receiving environment, comparative analysis on prevalence of 26 ARGs and the 2 integron genes was perfromed. The environmental impact of swine and broiler feedlots was showed primarily by total ARG input in receiving soils. The total ARG load in swine manure was significant higher than that in chicken manure(p < 0.01). ARG FODs of manures/soils in both swine and broiler were significant higher than those of control soils. Shannon’ diversity index of swine and broiler manure was equal to their receiving soils. However, swine receiving soils had higher diversity index than control soils(p < 0.05), while broiler receiving soils had slightly higher diversity index than control soils, but not significant(p > 0.05), indicating that the influence of livestock feedlots on environment was showed on the ARG diversification. Swine feedlots, by comparison, played more role in facilitating diversification of ARGs. The distribution of five classes of ARGs in manure and their corresponding receiving soils was found to be consistent with the use of antibiotic. ARGs in the water environment of swine and broiler feedlots exhibited different distribution. Total relative abundance in swine water environment was higher than that in broiler water environment. Water generally contained more ARGs than their sediments. Generally, swine feedlots appeared to impact environment more deeply than broiler feedlots as showed by the distribution of ARGs in manure, receiving soil and water environment.16S rRNA gene sequencing was performed to investigate microbiome in 24 samples from the three swine feedlots, including wastewater along the treatment units, well water, fishpond water, receiving soil and vegetables. Bacterial diversity increased after lagoon, anaerobic digester treatment units. Residue of most bacteria were build up on ready harvest vegetables regardless of ordinary cleanness. Swine feedlots affected the microbiome of well water, vegetables soil, fishpond water and sediment. Eighty-nine bacteria at genus were enriched in vegetables soil. Nineteen genus of core bacteria were shared by swine wastewater and vegetables soil. Animal originated Proteobacteria, Firmicutes, Bacteroidetes were introduced into soil by land application of swine wastes, and altered soil microbiome subsequently, posing potential risk to eco-environment and hunman health.High-throughput sequencing-based metagenomic approach was applied to investigate the broad-spectrum profiles and fate of ARGs in 8 samples, including soil vertical profiles at three layers adjacent or far away from swine feedlots and their corresponding irrigation water. Totally, 79 ARGs types belonging to 12 ARGs classes were identified the broad investigation of metagenomic analysis. Total abundance of ARGs in three layer of swine vegetables soil was 5.70(surface horizon soil), 14.7(subsoil), 5.93(substratum) times more than that of reference plot. Total abundance of ARGs in irrigation water of swine vegetables soil was 11.9 times more than that of reference plot. The most difference of ARG abundance in irrigation water was those resistant to tetracycline, followed by aminoglycoside and MLSB. ARGs conferring resistance to aminoglycoside, chloramphenicol, sulfonamide and tetracycline were found to be enriched in surface horizon soil. Multidrug resistance efflux pump, sulfonamide, tetracycline resistance genes were enriched in subsoil. And aminoglycoside, bacitracin, sulfonamide resistance genes exhibited to be more abundant in substratum layer. In general, ARGs conferring resistance to sulfonamide had the most highest fold changes in the whole vertical soil cores, followed by those conferring resistance to tetracycline and multidrug resistance efflux pump. A total of 59 pathogenic bacteria species was obtained from the 8 samples, belonging to 8 phylum, 12 class, 18 order, 27 family and 30 genus at taxonomic composition. The 59 species in three layer of swine vegetables soil was-0.38~64.0(surface horizon soil), 0.58~200(subsoil),-1.00~1.94(substratum) times more than that of reference plot. A total of 451 antibiotic resistance bacteria(ARB) species was obtained from the 8 samples, belonging to 18 phylum, 35 class, 79 order, 150 family and 254 genus at taxonomic composition. The 451 species in three layer of swine vegetables soil was-1.00~255(surface horizon soil),-1.00~848(subsoil),-1.00~113(substratum) times more than that of reference plot. Piper plot showing the relative distribution of 150 ARB at family level and 12 classes of ARGs in soil profiles. In the reference vegetables field, most ARB and ARGs distributed at substratum layer, few were found to equally prevelant through the vertical soil profiles. However, the distribution pattern in the vegetables plot adjacent to swine feedlots exhibited remarkable difference from the reference vegetables field. The three layers of soil profiles were settled by ARB and ARGs. This results indicated that the distribution of ARB and ARGs was altered by swine wastes irrigation. ARGs were introduced into receiving soil by swine manure/wastewater, migrated down into the deep layer and enriched through the vertical profiles. The bacteria community composition structured the prevalence of ARGs and shaped the soil resistomes. |
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