Soil Antibiotic Resistance Research Articles

Characteristics of antibiotic resistance genes in full-scale anaerobic digesters of food waste and the effects of application of biogas slurry on soil antibiotic resistance genes.

The fate of antibiotic resistance genes (ARGs) in full-scale anaerobic digestion (AD) of food waste (FW) and in the soil applied with biogas slurry has not been fully understood. In this study, 12 targeted ARGs and intI1 in FW, intermediate product, and biogas slurry from three full-scale ADwere analyzed. The results showed that subcritical water pretreatment was aneffective method for ARG attenuation, by which the absolute abundance of total targeted ARGs was removed by 99.69%. The predominant ARGs (ermB, tetM, and tetW) in FW were removed more than 99% after subcritical water pretreatment. The result of field experiments with biogas slurry as fertilizer showed that the absolute abundance of several ARGs (sul2, tetM, blaOXA-1, blaTEM) and intI1 accumulated significantly compared to the control group (CK) during three consecutive growth stages of the rice. The detected abundance of ARGs in paddy field soil increased from 190.50 (CK) to 8.87 × 104 copies/g (wet weight) (soil) during tillering stage, and increased from 4102.65 (CK) to 4.38 × 104 copies/g (wet weight) (soil) during heading time. Biogas slurry improved the soil nutrients (TN, AN, TP, and AP); meanwhile, the concentrations of total salt and Cl- increased. Network analysis indicated that 28 genera were the possible hosts of ARGs; variation partitioning analysis (VPA) indicated that microbial communities (contribution 59.30%) were the main factors that affected the fate of ARGs and intI1.

Fumigation practice combined with organic fertilizer increase antibiotic resistance in watermelon rhizosphere soil

Chemical fumigants and organic fertilizer are commonly used in facility agriculture to control soil-borne diseases and promote soil health. However, there is a lack of evidence for the effect of non-antibiotic fumigants on the distribution of antibiotic resistance genes (ARGs) in plant rhizosphere soils. Here, the response of a wide spectrum of ARGs and mobile genetic elements (MGEs) to dazomet fumigation practice in the rhizosphere soil of watermelon was investigated along its branching, flowering and fruiting growth stages in plastic shelters using high-throughput quantitative PCR approach. Our results indicated that soil fumigation combined with organic fertilizer application significantly increased the relative abundance of ARGs and MGEs in the rhizosphere soil of watermelon plant. The positive correlations between the relative abundance of ARGs and MGEs suggested that soil fumigation might increase the horizontal gene transfer (HGT) potential of ARGs. This result was further confirmed by the enhanced associations between ARG and MGE subtypes in the networks of fumigation treatments. Moreover, bipartite associations between ARGs/MGEs and microbial communities (bacteria and fungi) revealed a higher percentage of linkage between MGEs and microbial taxa in the fumigated soils. Structural equation model analysis further suggested that the increases in antibiotic resistance after fumigation and organic fertilizer application were mainly driven by MGEs and fungal community. Together, our results provide vital evidence that dazomet fumigation process combined with organic fertilizer in plastic shelters has the great potential to promote ARGs' dissemination in the rhizosphere, and raise cautions of the acquired resistance by soil-borne fungal pathogen and the potential spreading of ARGs along soil-plant continuum.

Response of antibiotic resistance to the co-exposure of sulfamethoxazole and copper during swine manure composting

Heavy metals driven co-selection of antibiotic resistance in soil and water bodies has been widely concerned, but the response of antibiotic resistance to co-existence of antibiotics and heavy metals in composting system is still unknown. Commonly used sulfamethoxazole and copper were individually and jointly added into four reactors to explore their effects on antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), heavy metal resistance genes (MRGs) and bacterial community structure. The abundance of total ARGs and MGEs were notably decreased by 68.64%–84.95% and 91.27–97.38%, respectively, after the composting. Individual addition of sulfamethoxazole, individual addition of copper, simultaneously addition of sulfamethoxazole and copper increased the abundance of ARGs and MGEs throughout the composting period. Co-exposure of sulfamethoxazole and copper elevated the total abundance of ARGs by 1.17–1.51 times by the end of the composting compared to individual addition of sulfamethoxazole or copper. Network analysis indicated that the shifts in potential host bacteria determined the ARGs variation. Additionally, MGEs and MRGs had significant effects on ARGs, revealing that horizontal gene transfer and heavy metals induced co-resistance could promote ARGs dissemination.

Cu and Zn exert a greater influence on antibiotic resistance and its transfer than doxycycline in agricultural soils

Livestock manure is a main source of heavy metals, antibiotics and antibiotic resistance genes (ARGs) in agricultural soils. The co-existence of heavy metals and ARGs needs to be systematically studied, since manure application is greatly encouraged. In this study, we examined soils for alterations in antibiotic resistance where doxycycline, Cu, and Zn were added equivalent to those found in typical pig manure applications. The results indicated that high levels of Cu inhibited soil respiration and urease for the first 10 days. Metagenomic analysis demonstrated that Cu and Zn additions caused profound alterations in bacterial community, metal resistance genes (MRGs) and mobile genetic elements. Among the differential ARGs, efflux pump genes took a significantly high ratio compared with control for the first 5 days, emphasizing their important roles in the profile of antibiotic resistance. Moreover, the number of differential MRGs was < 30 for doxycycline treatment, but 66–87 for Cu and Zn treatments. The number of differential integrative and conjugative elements was 3 for doxycycline treatment, and 6–13 for Cu and Zn treatments. Overall, high Cu and Zn levels caused a greater influence than did doxycycline on bacterial communities and transfer of antibiotic resistance in soil.

Rainfall facilitates the transmission and proliferation of antibiotic resistance genes from ambient air to soil

Antibiotic resistance is common in bacterial communities and appears to be correlated with human activities. However, the source of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in remote regions remains unclear. In this study, we examined the abundance of ARGs in fine particulate matter (PM2.5) as a carrier throughout the rainfall process (4 mm rain/h) to observe the effects of rainfall on the transmission of ARGs. The results suggested that rainwater served as a reservoir that facilitated the spread of ARGs and that wind and particulate matter (PM) concentrations might be meteorological parameters that influence the distribution of ARGs in rainwater. In addition, soil microcosm experiments were performed to investigate the influence of rainfall on antibiotic resistance in soils with different environmental backgrounds. Rainwater facilitated the proliferation of ARGs and mobile genetic elements (MGEs) from ambient air to soil, and this influence was more obvious in heavy metal-contaminated soil. This is the first study to investigate the routes by which rainfall acts as a mobile reservoir to facilitate the transmission and proliferation of ARGs, and the results indicate the potential source of ARGs in remote regions where humans rarely interfere.

Field aging alters biochar's effect on antibiotic resistome in manured soil

Current understanding of biochar's effect on antibiotic resistance genes (ARGs) in soil is limited, and whether the effect could change after long-term field aging remains largely unknown. In this study, we employed high-throughput quantitative PCR to assess the effect of biochar amendment on soil resistome as affected by three years of field aging. Application of fresh biochar significantly elevated the number and abundance of ARGs in the manured soil, but did not show such effect under pakchoi cultivation. The presence of aged biochar caused a marked reduction of ARGs only in the planted manured soil. Results of principal coordinate analysis and structural equation modeling indicate that biochar's effect on soil ARG profile was changed by field aging through altering soil microbial composition. These results highlight the necessity of considering aging effect of biochar during its on-farm application to mitigate soil antibiotic resistance.

Soil phage and their mediation on the horizontal transfer of antibiotic resistance genes: A review

The spread of antibiotic resistance in soil is a global threat to public health and food safety, challenging the prevention and treatment of human infectious disease. The horizontal transfer of ARGs mediated by bacteriophages (phages) is an important pathway for the spread of antibiotic resistance genes (ARGs). However, the knowledge on the contribution of phages to ARGs transmission in soil is elusive. Here, we reviewed the distribution characteristics of phages in soil and its driving factors. We summarized the main methods for purification and enrichment of soil phage, reviewed recent achievements in the mechanism of phage-mediated horizontal transfer of ARGs in soil and proposed some outstanding questions. This review would contribute to understanding the important ecological role of phages in driving the horizontal transfer of ARGs, and provide a basis for developing management strategies to mitigate ARGs pollution.

Seasonal change is a major driver of soil resistomes at a watershed scale

Soils harbor the most diverse naturally evolved antibiotic resistomes on Earth that threaten human health, ecosystem processes, and food security. Yet the importance of spatial and temporal variability in shaping the distribution of soil resistomes is not well explored. Here, a total of 319 topsoil samples were collected at a watershed scale during four seasons (spring to winter) and high-throughput quantitative PCR (HT-qPCR) was used to characterize the profiles of soil antibiotic resistance genes (ARGs). A significant and negative correlation was observed between soil ARG profiles and seasonal dissimilarity, which along with seasonally dependent distance-decay relationships highlight the importance of seasonal variability in shaping soil antibiotic resistomes. Significant, though weak, distance-decay relationships were identified in spring, summer and winter, for ARG similarities with geographic distances. There were also strong interactions between specific soil ARGs and Actinobacteria, Firmicutes and Proteobacteria. Moreover, we found that the relative abundance of soil Actinobacteria, Firmicutes and Proteobacteria correlated significantly with annual mean temperature and annual mean precipitation at a watershed scale. A random forest model showed that seasonal change rather than spatial variation was the most important predictor of the composition of soil ARGs. Together, these results constitute an advance in our understanding of the relative importance of spatial and temporal variability in shaping soil ARG profiles, which will provide novel insights allowing us to forecast their distribution under a changing environment.

Dissipation of antibiotic resistance genes in manure-amended agricultural soil

Soil antibiotic resistance due to animal manure application is of great concern in recent years. Little is known about the fate of antibiotic resistance genes (ARGs) in agricultural soils associated with long-term manure application. Here we used soil microcosms to investigate the dissipation of ARGs and the change of bacterial community in agricultural soil originated from a vegetable field which had received 24 years' swine manure application. Soil microcosms were conducted at different soil moistures and with or without biochar over a testing period of two years in lab. Results showed that continuous manure application induced an accumulation of ARGs in soil, wherein the dissipation of ARGs differed from those in non-manure amended soil. ARGs persisted in soils at least two years, although their abundance declined gradually. Meanwhile, soil moisture and biochar had significant impact on the fate of ARGs. ARGs dissipated faster in soil with higher moisture. Biochar amendment contributed to the maintenance of bacterial diversity. Within the two years of simulation experiment, biochar enhanced soil ARG retention as they dissipated slowly in the soil amended with biochar. Succession of microbial community may have sustained the transfer and resilience of ARGs. This study provides insight into the dissipation of antibiotic resistance genes in manure-applied agricultural soil.

Antibiotic Resistance in Agricultural Soil and Crops Associated to the Application of Cow Manure-Derived Amendments From Conventional and Organic Livestock Farms.

The application of organic amendments to agricultural soil can enhance crop yield, while improving the physicochemical and biological properties of the recipient soils. However, the use of manure-derived amendments as fertilizers entails environmental risks, such as the contamination of soil and crops with antibiotic residues, antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). In order to delve into these risks, we applied dairy cow manure-derived amendments (slurry, fresh manure, aged manure), obtained from a conventional and an organic farm, to soil. Subsequently, lettuce and wheat plants were grown in the amended soils. After harvest, the abundance of 95 ARGs and MGE-genes from the amended soils and plants were determined by high-throughput qPCR. The structure of soil prokaryotic communities was determined by 16S rRNA amplicon sequencing and qPCR. The absolute abundance of ARGs and MGE-genes differed between treatments (amended vs. unamended), origins of amendment (conventional vs. organic), and types of amendment (slurry vs. fresh manure vs. aged manure). Regarding ARG-absolute abundances in the amendments themselves, higher values were usually found in slurry vs. fresh or aged manure. These abundances were generally higher in soil than in plant samples, and higher in wheat grain than in lettuce plants. Lettuce plants fertilized with conventional amendments showed higher absolute abundances of tetracycline resistance genes, compared to those amended with organic amendments. No single treatment could be identified as the best or worst treatment regarding the risk of antibiotic resistance in soil and plant samples. Within the same treatment, the resistome risk differed between the amendment, the amended soil and, finally, the crop. In other words, according to our data, the resistome risk in manure-amended crops cannot be directly inferred from the analysis of the amendments themselves. We concluded that, depending on the specific question under study, the analysis of the resistome risk should specifically focus on the amendment, the amended soil or the crop.

Cross-comparison of methods for quantifying antibiotic resistance in agricultural soils amended with dairy manure and compost

Agricultural soils are often amended with livestock manure, making them a key reservoir of antibiotic resistance genes (ARGs). Given that soils are among the most microbially-diverse environments on the planet; effective characterization and quantification of the effects of manure-derived amendments on soil resistomes is a major challenge. This study examined the effects of dairy manure-derived amendments on agricultural soils via two strategies: quantification of anthropogenic ARG markers via qPCR and shotgun metagenomic resistome profiling; and these strategies were compared to a previously published antibiotic resistant fecal coliform dataset. Soil samples were collected throughout a 120 day complete block field experiment to compare the effects of amendment type on antibiotic resistance. Results of all three measurements were consistent with the hypothesis that the application of composted manure reduced antibiotic resistance in soil relative to the application of raw manure, although some differences were noted in comparing the patterns of the three measurements with time. Raw dairy manure-amended soils yielded high sul1 and tet(W) relative abundances on Day 0 (following amendment application), but significantly decreased to background levels by Day 67 (harvest) and Day 120 (study completion). Shotgun metagenomics similarly detected a decrease in the relative abundances of sulfonamide and tetracycline-associated ARGs over time in the raw manure- and compost-amended soils; however, these levels were significantly lower than those estimated by qPCR. Interestingly, although patterns of sulfonamide and tetracycline resistance among culturable fecal coliforms echoed those observed via qPCR and metagenomics; erythromycin resistant coliforms were directly recovered by culture in amended soils, but corresponding ARGs were not detected by qPCR or metagenomics. This study supports both composting and time restrictions as means of reducing the potential for antibiotic resistance in manure to spread via soil application. Results suggest some differences in finer conclusions drawn depending on which antibiotic resistance monitoring target is selected.

Antibiotic resistance levels in soils from urban and rural land uses in Great Britain.

Although soil is one of the largest microbial diversity reservoirs, the processes that define its microbial community dynamics are not fully understood. Improving our understanding of the levels of antibiotic resistance in soils with different land uses in Great Britain is not only important for the protection of animal health (including humans), but also for gaining an insight into gene transfer levels in microbial communities. This study looked at the levels of antibiotic-resistant bacteria (ARB) able to survive inhibitory concentrations of chloramphenicol, erythromycin and vancomycin, as well as subinhibitory (10 µg ml−1) erythromycin concentrations. Soils from nine different sites across Great Britain with three distinct land uses (agricultural, urban and semi-natural) were sampled and the percentage of ARB was calculated for each site. Statistical analyses confirmed a significant difference in the level of ARB found in agricultural land compared to urban or semi-natural sites. The results also showed that resistance levels to vancomycin and chloramphenicol in the agricultural and urban sites sampled were significantly higher than those for erythromycin, whilst in semi-natural sites all three antibiotics show similar resistance levels. Finally, although the levels of resistance to a subinhibitory (10 µg ml−1) erythromycin concentration were significantly higher across land use types when compared to the levels of resistance to an inhibitory (20 µg ml−1) concentration, these were much less marked in soil from agricultural land compared to that from urban or semi-natural land use soil.

Long-term biogas slurry application increased antibiotics accumulation and antibiotic resistance genes (ARGs) spread in agricultural soils with different properties

Animal manures are commonly applied to soil which possibly promote the spread of antibiotic resistance from soil to human beings via food chains. Biogas slurry is an end product of anaerobic digestion of animal manures, which has been widely applied as fertilizers in the agricultural soil. However, effect of long-term biogas slurry application on the soil antibiotic resistance and the associated mechanism still remains unclear. The present study characterized antibiotics, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and bacterial community, in different agricultural soils unamended (BS-) and amended (BS+) with biogas slurry (8–18 years) in five field experiments. Our results indicated that long-term application of biogas slurry largely increased the concentrations of tetracyclines in soils, and greatly increased the abundances of ARGs, transposase gene (Tn916/1545) and ARGs-associated bacteria. Long-term application of biogas slurry led to tetracyclines accumulation and ARGs enrichment in agricultural soil, and the selection pressure from tetracyclines and the increase of Tn916/1545 abundace become potential contributors for the increase of soil antibiotic resistance via promoting the enrichment of ARG-associated bacteria. The results of the present study should be taken into consideration to develop policy and practice for mitigating the enrichment and spread of antibiotic resistance during the recycling of biogas slurry into agricultural soil.

Coexistence between antibiotic resistance genes and metal resistance genes in manure-fertilized soils

Antibiotics and metals introduced during manure application are believed to be the major drivers of the bloom and spread of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) in agricultural environments. However, the coexistence of multidrug resistance (MDR) and metal resistance and the effects of manure application on the coexistence are less studied. Here, we profiled the coexistence patterns of 9919 MDR genes (MDRGs) and 25,312 MRGs in paddy fields with manure or inorganic fertilization. The abundances of both MDRGs and MRGs were higher under manure fertilization than inorganic fertilization. Network analysis determined that coexistence between MDRGs and MRGs was much more frequent in manure-fertilized soils than in inorganic-fertilized soils. Manure application drove this coexistence by not only directly introducing resistance genes but also increasing co-selection through antibiotics and metals, and stimulating their potential common hosts, including species of Actinobacterium WWH12, Bacillus, Geobacter, Solirubrobacter, Acidobacteriales, Bacillales, Chloroflexi, Methyloligellaceae and Xanthobacteraceae. Soil antibiotic resistance was significantly predicted by the coexistence of major multidrug efflux genes (mfs, mex, abc, mate and smr) and MRGs. The coexistence between MDRGs and MRGs explained 18% of the increase in soil antibiotic resistance. Such coexistence is nonnegligible and permits the promotion of soil antibiotic resistance, thus posing a potential threat to both agroecosystems and humans.

Elevated CO2 concentration modifies the effects of organic fertilizer substitution on rice yield and soil ARGs

Antibiotic resistance and rising CO2 levels are considered among the most significant challenges we will face in terms of global development over the following decades. However, the impact of elevated CO2 on soil antibiotic resistance has rarely been investigated. We used a free-air CO2 enrichment system to investigate the potential risks posed by applying mineral and organic fertilizers to paddy soil at current CO2 concentration (370 ppm) and future elevated CO2 (eCO2, 570 ppm predicted for 2100). Organic fertilizer substitution (substituting the mineral fertilizer by 50% N) alone increased the plant uptake and soil residue of sulfamethazine, and enriched sulfonamide resistance genes (sul1, sul2), tetracycline resistance genes (tetG, tetM) and class 1 integron (intl1). But it decreased the rice grain yield (by 7.6%). Comparatively, eCO2 decreased the sul2, tetG and intl1 gene abundances by organic fertilizer substitution, and meanwhile increased grain yield (by 8.4%). Proteobacteria and Nitrospirae were potential hosts of antibiotic resistance genes (ARGs). Horizontal gene transfer via intl1 may play an important role in ARGs spread under eCO2. Results indicated that future elevated CO2 concentration could modify the effects of organic fertilizer substitution on rice yield and soil ARGs, with unknown implications for future medicine and human health.

Impact of electrokinetic remediation of heavy metal contamination on antibiotic resistance in soil

Electrokinetic remediation is an effective technology for soil contaminated with heavy metals. However, little is known about the fate of antibiotic resistance in the process under heavy metal stress, since antibiotic resistance genes (ARGs) are widely distributed and can be co-selected with heavy metals. This study focused on antibiotic resistant bacteria and ARGs over different remediation periods (1, 2, and 5 days), voltages (0.4 and 0.8 V cm−1), and initial concentrations (250–1,000 mg kg−1 for Cu, and 1,000–3,000 mg kg−1 for Zn). The application of polarity-reversal maintained a suitable pH, eliminating possible negative effects on soil quality. In addition to a decrease in total metals, the speciation was modified as residual forms decreased while reactive forms increased. Compared with anti-oxytetracycline bacteria, anti-sulfamethoxazole bacteria were more resistant to the electric field, which might be ascribed to greater constraints on their resistance enzymes. The presence of heavy metals accelerated the spread of ARGs, with a 2.67-fold increase for tetG, and a 3.86-fold increase for sul1. Among the ARGs studied, tetM and tetW, as well as sul genes were more easily removed than tetC and tetG genes. Finally, a significant correlation was found between ARGs and Cu, consistent with the relatively stronger toxicity of Cu and its high potential to induce the SOS response. This study advances the understanding of how electrokinetics influences antibiotic resistance in soil with heavy metals, which has important implications for the simultaneous control of these pollutants in soil.

Grazing does not increase soil antibiotic resistome in two types of grasslands in Inner Mongolia, China

Long-term livestock grazing often comes with inputs of manure and urine of animals to grassland soils and can influence the microbial communities. However, the effects of grazing on the diversity and abundance of soil antibiotic resistance genes (ARGs) are still obscure. Here we profiled soil resistome using high-throughput quantitative PCR analysis from desert and typical grasslands (Siziwang and Xilingol steppe, respectively) in Inner Mongolia, each with four grazing intensities (no grazing, light grazing, moderate grazing, and heavy grazing). Totally, 54 and 39 ARGs were detected in the Siziwang steppe and the Xilingol steppe, respectively. The relative abundance of ARGs significantly decreased in the Siziwang steppe or remained unchanged in the Xilingol steppe along the gradient of increasing grazing intensity. Mobile genetic elements (MGEs) were significantly and positively correlated with ARGs in the Siziwang steppe but not in the Xilingol steppe. Structural equation model revealed that grazing indirectly affected ARGs through its impact on MGEs, bacterial diversity, and bacterial community composition. These findings suggest that livestock grazing can impact soil resistome via regulating microbiome in grassland soils. However, grazing intensity has not resulted in a significant impact on the soil resistomes in these desert and typical grasslands.

Manure fertilization increase antibiotic resistance in soils from typical greenhouse vegetable production bases, China

A large quantity of manure is applied in greenhouse vegetable production (GVP) soils, while manure fertilization often leads to the proliferation of antibiotic resistance genes (ARGs) in soils. However, comprehensive study on the effects of different types of manure on ARGs in GVP soils remains unknown, and the baseline level of ARGs in GVP soil is poorly quantified. This study conducted a comprehensive survey of ARGs in GVP soils using high-throughput quantitative PCR. We found elevated ARG diversity and absolute abundance in fertilized soil, whereas no significant difference in soil ARGs amended with different types of manure. Redundancy analysis indicated that the change of bacterial community compositions and environmental factors contributed partially to the shift in ARG profiles. Bipartite network analysis indicated that one ARG was detected in non-manured soils, while 50 ARGs and 4 mobile gene elements were exclusively detected in fertilized soils, suggesting introduction of ARGs from manure into soils largely explained the increased ARG diversity in fertilized soil. By comparison of ARG absolute abundance between manured and non-manured soil, we estimated the typical level of ARG absolute abundance in non-manured soil, which provided the first rough baseline level of ARGs to assess ARG contamination in GVP soils.

Does reduced usage of antibiotics in livestock production mitigate the spread of antibiotic resistance in soil, earthworm guts, and the phyllosphere?

The overuse of antibiotics in animal husbandry is widespread and believed to significantly contribute to the selection of antibiotic resistance genes (ARGs) in animals. Thus, there is a global drive to reduce antibiotic use in the agricultural sector. However, it has not been established whether a reduction in the use of antibiotics in livestock production would be effective in reducing the spread of ARGs. A microcosm approach was used to determine how the addition of manure with either reduced antibiotic levels or with typical antibiotic levels could affect the spread of antibiotic resistance genes between soil, earthworms and the phyllosphere. When compared to the control soil, earthworm and phyllosphere samples had the greater increase in ARG abundance in conventional manure treatments (P < 0.05). Reduced antibiotic manure also enriched the abundance of ARGs in the phyllosphere and soil but not earthworm guts when compared to the control (P < 0.05). In both soil and earthworm guts, the enrichment of ARGs was lower in reduced antibiotic manure than in conventional manure. This study has identified bacterial transfer through the soil-earthworm-phyllosphere system as a potential means to spread ARGs between habitats after fertilization with livestock derived manures.

Temporal effects of repeated application of biogas slurry on soil antibiotic resistance genes and their potential bacterial hosts

Biogas slurry, a liquid end product of animal manure fermentation, is widely used as fertilizer in crop fields. Land application may introduce antibiotics and related resistance genes from livestock production into agricultural soil. Nevertheless, changes in antimicrobial resistance in soil where biogas slurry has been repeatedly applied are not fully understood. In the present study, 13 veterinary antibiotics were analyzed in soils that were repeatedly sprayed with biogas slurry, and simultaneously, temporal changes in antibiotic resistance genes (ARGs) and bacterial community composition were investigated using a real-time quantitative PCR assay and MiSeq sequencing. Long-term repeated application of biogas slurry did not result in excessive accumulation of antibiotic residuals in the soil but increased the abundance of ARGs and facilitated ARG transfer among potential hosts. Although the quantitative PCR assay showed a decreasing trend for the relative abundance of ARGs over time, a relevance network analysis revealed highly complex bacteria-ARG co-occurrence after long-term application, which implied that repeated application might intensify horizontal gene transfer (HGT) of ARGs among different bacterial hosts in soil. The increased relative abundance of the intl1 gene supported the shift in ARG-bacteria co-occurrence. Furthermore, ordination analysis showed that the distributions of antibiotic resistance bacteria (ARB) and ARGs were closely related to application duration than to the influence of antibiotic residuals in the biogas slurry-treated soil environment. Additionally, natural level of ARG abundance in untreated soils indirectly suggested the presence/absence of antibiotics was not a key determinant causing the spread of antimicrobial resistance. This study provides improved insight into the effects of long-term repeated application of biogas slurry on the shift in ARG abundances and bacteria-ARG co-occurrence in soils, highlighting the need to focus on the influence of changed soil environment on the ARG transfer.