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The long-term extensive application of chemical fertilizers wreaks havoc on soil bacterial structure and function. To reduce the damage caused by chemical fertilizers, a six-year experiment was performed to study the effects of replacing 0% (CK), 25% (S25), 50% (S50), 75% (S75), and 100% (S100) of 225 kg ha−1 mineral nitrogen fertilizer with an equivalent amount of nitrogen from maize straw on the soil bacterial community structure, diversity, and function. The results showed that Proteobacteria, Acidobacteria, and Gemmatimonadetes were the dominant soil bacterial phyla after the replacement treatments. Replacing mineral nitrogen fertilizer with an equivalent amount of nitrogen from maize straw significantly reduced the number of Photobacterium and bacterial populations involved in genetic information processing in soil, but significantly increased the number of bacterial populations involved in organismal systems, human diseases, and environmental information processing. Compared with other treatments, the relative abundance of TK10 significantly increased by 33.52–76.36% in S25. The number of subgroup 6, Gram-negative, biofilm-forming, potentially pathogenic, and anaerobic bacteria significantly increased, whereas that of Chloroflexi and Blastocatellia subgroup 4 significantly decreased in S50 and S75 compared with CK. The number of TK10 and Blastocatellia subgroup 4 in S50 and S100, respectively, was significantly lower than that in CK. Bacterial species were significantly more present in S25 than in S75. The diversity of bacterial species in S75 was significantly lower than that in CK. S25 and S100 were more favorable to increasing the number of Gram-positive, aerobic, mobile-element containing, and stress-tolerant bacteria. Rhodobacteraceae, Pyrinomonadaceae, Xanthobacteraceae, Nocardioidaceae, and Vulgatibacteraceae with statistical differences in CK, S25, S50, S75, and S100, respectively, could be used as biomarkers. Chloroflexi, Acidobacteria, and Nitrospirae could be used as the main basis for the bacterial classification of soil samples in the equivalent substitution of nitrogen chemical fertilizer with maize straw. S25 is ideal for increasing soil bacterial species richness and abundance. [ABSTRACT FROM AUTHOR]

Swine manure is considered to be a reservoir for antibiotic resistance genes (ARGs) but little is known about the variations in ARGs during the co-composting of swine manure with Chinese medicinal herbal residues (CMHRs). Thus, this study explored the effects of CMHRs on the variations in ARGs during co-composting with swine manure. The results showed that CMHRs could reduce effectively most of the targeted ARGs (0.18–2.82 logs) and mobile genetic elements (MGEs) (0.47–3.34 logs). The correlations indicated that CMHRs might decrease the spread of ARGs via horizontal gene transfer. Redundancy analysis showed that the bacterial communities had more important effects on the variations in ARGs compared with environmental factors and MGEs. The results of this study demonstrate that CMHRs can decrease the abundances of ARGs and MGEs, as well as reducing the risk of ARGs spreading during the application of compost products to farmland. [ABSTRACT FROM AUTHOR]

Superabsorbent polymers (SAPs) are considered suitable amendments for reducing the selection pressure due to heavy metals and the abundances of antibiotic resistance genes (ARGs) during composting. In this study, three SAP (sodium polyacrylate) levels (0, 5, and 15 mg kg −1 of compost) were applied and their effects on the abundances of ARGs, mobile genetic elements (MGEs), and the bacterial community were investigated. After composting, the abundances of ARGs and MGEs decreased to different extent, where the removal efficiencies for tetW , dfrA7 , ermX , aac(6′)-ib-cr and MGEs exceeded 90%. The high SAP concentration significantly reduced the abundances of ARGs and MGEs, and changed the microbial community. Redundancy analysis indicated that the moisture content mainly explained the changes in ARGs and MGEs. Network analysis determined the potential hosts of ARGs and MGEs, and their co-occurrence. The results suggested that applying 15 mg kg −1 SAP is appropriate for reducing ARGs in compost. [ABSTRACT FROM AUTHOR]

This study explored the effects of different mass ratios of swine manure relative to wheat straw (3:7, 5:5, and 7:3, i.e., control reactors C1, C2, and C3, respectively) on variations in antibiotic resistance genes (ARGs) and the microbial community during anaerobic digestion (AD). The cumulative biogas production volumes were 1711, 3857, and 3226 mL in C1, C2, and C3, respectively. After AD, the total relative abundance of ARGs decreased by 4.23 logs in C3, whereas the reductions were only 1.03 and 1.37 logs in C1 and C2, respectively. Network analysis showed that the genera Solibacillus , Enterococcus , Facklamia , Corynebacterium_1 , and Acinetobacter were potential hosts of ermB , sul1 , and dfrA7 . Redundancy analysis showed that the bacterial communities and environmental factors played important roles in the variation in ARGs. Thus, reductions in ARGs should be considered before reusing animal manure treated by AD. [ABSTRACT FROM AUTHOR]

Treatment with exogenous additives during composting can help to alleviate the accumulation of antibiotic resistance genes (ARGs) caused by the direct application of pig manure to farmland. In addition, nano-cellulose has an excellent capacity for adsorbing pollutants. Thus, the effects of adding 300, 600, and 900 mg/kg nano-cellulose to compost on the bacterial communities, mobile genetic elements (MGEs), and ARGs were determined in this study. After composting, treatment with nano-cellulose significantly reduced the relative abundance of ARGs, which was lowest in the compost product with 600 mg/kg added nano-cellulose. Nano-cellulose inhibited the rebound in ARGs from the cooling period to the maturity period, and weakened the selective pressure of heavy metals on microorganisms by passivating bio-Cu. The results also showed that MGEs explained most of the changes in the abundances of ARGs, and MGEs had direct effects on ARGs. The addition of 600 mg/kg nano-cellulose reduced the abundances of bacterial genera associated with ermQ , tetG , and other genes, and the number of links (16) between ARGs and MGEs was lowest in the treatment with 600 mg/kg added nano-cellulose. Therefore, adding 600 mg/kg nano-cellulose reduced the abundances of ARGs by affecting host bacteria and MGEs. The results obtained in this study demonstrate the positive effect of nano-cellulose on ARG pollution in poultry manure, where adding 600 mg/kg nano-cellulose was most effective at reducing the abundances of ARGs. [Display omitted] • Adding 600 mg/kg nano-cellulose effectively removed ARGs during composting. • 600 mg/kg nano-cellulose can reduce the selective pressure of Cu on microorganisms. • 600 mg/kg nano-cellulose can weaken the correlation between MGEs and ARGs. • 600 mg/kg nano-cellulose reduced the abundance of potential host bacteria of ARGs. [ABSTRACT FROM AUTHOR]

• MAR improved the efficiency of reducing the relative abundances of ARGs and MGEs. • MGEs were mainly responsible for the changes in the abundances of ARGs. • ARGs and MGEs have the same potential host bacteria. • MAR added at 5% can reduce the risk of ARGs in swine manure composting. Swine manure is considered a reservoir for antibiotic resistance genes (ARGs), which may enter the soil and then the food chain to endanger human health. This study investigated the effects of adding 0%, 5%, and 15% (w/w) macroporous adsorption resin (MAR) on ARGs and the bacterial community during composting. The results showed that the addition of MAR reduced the abundances of ARGs (14.14–99.44%) and mobile genetic elements (MGEs) (47.83–99.48%) after swine manure composting. Significant positive correlations were detected between ARGs and MGEs, and thus the variations in MGEs may have led to the changes in ARGs. Redundancy analysis showed that MGEs had stronger effects on ARGs than environmental factors and the bacterial community. Network analysis suggested that ARGs and MGEs co-existed in common host bacteria. In conclusion, the results showed that adding 5% MAR can reduce the risk of ARG transmission. [ABSTRACT FROM AUTHOR]

Abstract This study evaluated how tylosin (TYL), ciprofloxacin (CIP), and sulfadimidine (SM 2) affected biogas and CH 4 production during anaerobic digestion (AD) via their effects on the key genes related to methane production and the methanogenic community. The results showed that TYL, CIP, and SM 2 reduced the production of methane during AD by 7.5%, 21.9%, and 16.0%, respectively. After AD for five days, CIP strongly inhibited the mcrA gene, where its abundance was 49% less than that in the control. TYL and SM 2 decreased the abundances of Spirochaeta and Fibrobacteres during AD. High-throughput sequencing identified 10 methanogen genera, where Methanocorpusculum , Methanobrevibacter , and Methanosarcina accounted for 99.1% of the total archaeal reads. TYL and SM 2 increased the efficiency of the acetoclastic methanogen pathway (Methanosarcina) by 29.04% and 52.79%, respectively. Redundancy analysis showed that Spirochaeta, Fibrobacteres, and Methanosarcina had positive correlations with CH 4 and mcrA. We found that 30 mg kg−1 CIP had a strong inhibitory effect on methane production by influencing the abundances of Methanobrevibacter and Methanosarcina during AD. Graphical abstract Image 1 Highlights • 30 mg kg−1 ciprofloxacin significantly decreased CH 4. • CIP affected the acetic, propionic, and butyric acid utilization pathways. • Methanosarcina was the main methanogen in the later stage of AD. • Spirochaetes and Fibrobacteres had positive correlations with CH 4 and mcrA. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Solid-state anaerobic digestion (SAD) removed over 7/10 ARGs from digestion product. • The variations in the bacterial community were smaller in SAD than LAD. • Firmicutes comprised 62.5% of the potential hosts for ARGs and MGEs. • SAD decreased the environmental risk of ARGs in the manure digestion product. Abstract Livestock manure is a hotspot for antibiotic resistance genes (ARGs). Solid-state anaerobic digestion (SAD) is a high efficiency livestock manure treatment, but the changes in ARGs and microbial communities during SAD are unknown. Therefore, thermophilic SAD (55 °C), mesophilic SAD (35 °C), and liquid anaerobic digestion (35 °C) were conducted to investigate the impacts of SAD on ARGs, mobile genetic elements, and microbial communities. Compared with liquid anaerobic digestion, SAD significantly reduced at least 7/10 ARGs and all of the mobile genetic elements considered, where the total abundance of ARG was 23.7% higher in the thermophilic SAD product than the mesophilic SAD product. Firmicutes and Proteobacteria were the main potential hosts for ARGs, and their decreased abundances were mainly responsible for the reductions in ARGs during SAD. This study demonstrated that SAD is a promising method for decreasing the ARGs risk in livestock manure. [ABSTRACT FROM AUTHOR]

Highlights • Coal gasification slag can enhance the removal of ARGs and MGEs from swine manure. • Decreases in ARGs may be due to reductions in abundances of potential host bacteria. • Bacterial succession responsible for variations in the ARG profiles. • Coal gasification slag added at 10 g/L can reduce the risk of ARGs in swine manure. Abstract Coal gasification slag (GS) is an industrial solid waste with a highly developed pore structure, which can be used in anaerobic digestion (AD) to remove antibiotic resistance genes (ARGs) due to its structure, thereby utilizing this waste resource. This study evaluated the effects of three GS levels (0, 5, and 10 g/L) on the abundances of ARGs, mobile genetic elements, and the bacterial community. With GS added at 10 g/L, the removal rates for ARGs (dfrA7 , sul2 , tetW , ermF , and ermQ) were 24.81–90.48% after AD, and the removal rate for ISCR1 was 95.4%. In addition, 10 g/L GS was more effective at reducing the abundances of potential human pathogens. The variations in ARGs may have been affected by the succession of the microbial community. The results of this study demonstrate that supplementation with 10 g/L GS is more useful for reducing ARGs during AD. [ABSTRACT FROM AUTHOR]

Highlights • Coal gasification slag (CGS) is one of the waste products. • CGS effectively reduced most ARGs and MGEs in swine manure compost. • Fourteen genera were identified as possible hosts of various ARGs. • Adding 10% CGS is a cost-effective option for reducing the abundance of ARGs. Abstract This study investigated the effects of the addition of coal gasification slag (CGS) at three levels (0%, 5%, and 10% w/w) on antibiotic resistance genes (ARGs) and the bacterial community during composting. The addition of CGS effectively facilitated the removal of ARGs and mobile genetic elements (MGEs), where it significantly reduced the relative abundances of 5/11 ARGs and three MGEs in the swine manure composting product. In addition, the enrichment of ARGs and intI1 was lower under the addition of 10% CGS compared with 0% CGS. The bacterial community was distributed according to the composting time under different treatments. Redundancy analysis showed that bacterial community succession and MGE-mediated horizontal gene transfer played important roles in the variations in ARGs. Network analysis indicated the co-occurrence of ARGs and MGEs with specific microorganisms. Thus, 10% CGS may be a suitable additive for reducing the risks of ARGs in compost products. [ABSTRACT FROM AUTHOR]

This study investigated the effects of adding copper at 3 treatment levels (0 (control: CK), 200 (low: L), and 2000 (high: H) mg·kg−1 treatments) on the bacterial communities during swine manure composting. The abundances of the bacteria were determined by quantitative PCR and their compositions were evaluated by high-throughput sequencing. The results showed that the abundance of bacteria was inhibited by the H treatment during days 7-35, and principal component analysis clearly separated the H treatment from the CK and L treatments. Actinobacteria, Firmicutes, and Proteobacteria were the dominant bacterial taxa, and a high copper concentration decreased the abundances of bacteria that degrade cellulose and lignin (e.g., class Bacilli and genus Truepera), especially in the mesophilic and thermophilic phases. Moreover, network analysis showed that copper might alter the co-occurrence patterns of bacterial communities by changing the properties of the networks and the keystone taxa, and increase the competition by increasing negative associations between bacteria during composting. Temperature, water-soluble carbohydrates, and copper significantly affected the variations in the bacterial community according to redundancy analysis. The copper content mainly contributed to the bacterial community in the thermophilic and cooling phases, where it had positive relationships with potentially pathogenic bacteria (e.g., Corynebacterium_ 1 and Acinetobacter). [ABSTRACT FROM AUTHOR]

Swine manure is an important reservoir for antibiotic resistance genes (ARGs) but anaerobic co-digestion (AcoD) can potentially reduce the abundance of these ARGs. However, few studies have considered the effects of Chinese medicinal herbal residues (CMHRs) on the variations in ARGs and mobile genetic elements (MGEs) during AcoD. Thus, this study explored the fate of ARGs and MGEs during the AcoD of CMHRs and swine manure. The results showed that CMHRs effectively reduced the abundances of the main ARGs (excluding ermF , qnrA , and tetW ) and four MGEs (by 36.7–96.5%) after AcoD. Redundancy analysis showed that changes in the bacterial community mainly affected the fate of ARGs rather than horizontal gene transfer by MGEs. Network analysis indicated that 17 bacterial genera were possible hosts of ARGs. The results of this study suggest that AcoD with CMHRs could be employed to remove some ARGs and MGEs from swine manure. [ABSTRACT FROM AUTHOR]

Inoculation with microorganisms is an effective strategy for improving traditional composting processes. This study explored the effects of inoculation with lignocellulose-degrading microorganisms (LDM) on the degradation of organic matter (OM), methane (CH 4) emissions, and the microbial community (bacteria and methanogens) during composting. The results showed that LDM accelerated the degradation of OM (including the lignocellulose fraction) and increased the CH 4 releases in the later thermophilic and cooling stages during composting. At the ending of composting, LDM increased the CH 4 emissions by 38.6% compared with the control. Moreover, LDM significantly increased the abundances of members of the bacterial and methanogenic community during the later thermophilic period (P < 0.05). In addition, LDM promoted the growth and activity of major bacterial genera (e.g., Ureibacillus) with the ability to degrade macromolecular OM, as well as affecting key methanogens (e.g., Methanocorpusculum) in the composting system. Network analysis and variance partitioning analysis indicated that OM and temperature were the main factors that affected the bacterial and methanogen community structures. Structural equation modeling demonstrated that the higher CH 4 emissions under LDM were related to the growth of methanogens, which was facilitated by the anaerobic environment produced by large amounts of CO 2. Thus, aerobic conditions should be improved during the end of the thermophilic and cooling composting period when inoculating with lignocellulose-degrading microorganisms in order to reduce CH 4 emissions. • Compost inoculated with lignocellulose-degrading microorganisms (LDM). • CH 4 emissions and organic matter (OM) degradation assessed under LDM treatment. • Temperature and OM main contributors to bacterial and methanogenic communities. • The anaerobic environment created by CO 2 under LDM promoted the release of CH 4. • Aerobic conditions should be improved to optimize LDM application in composting. [ABSTRACT FROM AUTHOR]

Livestock manure is a major source of antibiotic resistance genes (ARGs) that enter the environment. This study assessed the effects of inoculation with mature compost (MC) on the fates of ARGs and the bacterial community during swine manure composting. The results showed that MC prolonged the thermophilic period and promoted the decomposition of organic matter, which was due to the rapid growth and reproduction of thermophilic bacteria (Bacillus , Thermobifida , and Thermobacillus). MC significantly reduced the relative abundances of ARGs (1.02 logs) and mobile genetic elements (MGEs) (1.70 logs) after composting, especially sulfanilamide resistance genes. The total ARGs removal rate was 1.11 times higher in MC than the control. Redundancy analysis and structural equation modeling showed that horizontal gene transfer mediated by MGEs (IS CR1 and intI1) was the main direct factor related to the changes in ARGs during composting, whereas the C/N ratio and pH were the two most important indirect factors. Network analysis showed that members of Firmicutes comprising Romboutsia , Clostridisensu_stricto_1 , and Terrisporobacter were the main bacterial hosts of ARGs and MGEs. MC reduced the risk of ARGs transmission by decreasing the abundances of bacterial hosts. Thus, MC is a promising strategy for reducing the proliferation risk of ARGs. [Display omitted] • Mature compost increased the abundance of thermophilic bacteria. • Mature compost is an efficient strategy for reducing ARGs and MGEs. • Mature compost decreased ARGs transmission risk by reducing bacterial host. • MGEs (IS CR1 and intI1) were the main direct factors related to changes in ARGs. [ABSTRACT FROM AUTHOR]

The abuse of heavy metals as feed additives in livestock is widespread and it might aggravate the spread of antibiotic resistance genes (ARGs) in the environment. However, the mechanisms that allow heavy metals to increase the transmission of ARGs in the environment remain unclear. Cu is the heavy metal present at the highest concentration in livestock manure, and thus Cu was selected to investigate the responses of ARGs to heavy metals. The effects of the microbial communities, mobile genetic elements (MGEs), and heavy metal resistance genes (HMRGs) on ARGs were determined in the presence of 75 and 227 mg L−1 Cu in a swine manure anaerobic digestion (AD) system. In the AD products, the presence of residual Cu (75 and 227 mg L−1) increased the total ARGs, HMRGs, and some MGEs, and the higher Cu selected more ARGs than the lower Cu treatment. The results demonstrated that Cu could promote the co-selection of HMRGs, ARGs, and MGEs. The different levels of Cu did not change the bacterial community composition, but they influenced the abundances of bacteria during AD. Network analysis showed that the presence of Cu increased the co-occurrence of specific bacteria containing ARGs, HMRGs, and MGEs. Furthermore, the co-occurrence of MGEs and ARGs increased greatly compared with that of HMRGs and ARGs. Therefore, compared HMRGs, the increased MGEs had the main effect on increasing of ARGs. Unlabelled Image • Anaerobic digestion (AD) process effective at reducing the initial load of ARGs. • Residual Cu (75 and 227 mg L−1) increased the total ARGs in the AD products. • Co-occurrence of MGEs with ARGs increased in Cu treatments. [ABSTRACT FROM AUTHOR]

Green tea residues (GTRs) are byproducts of tea production and processing, and this type of agricultural waste retains nutritious components. This study investigated the co-composting of GTRs with swine manure, as well as the effects of GTRs on antibiotic resistance genes (ARGs) and the bacterial community during co-composting. The temperature and C/N ratio indicate compost was mature after processing. The addition of GTRs effectively promoted the reduction in the abundances of most targeted ARGs (tet and sul genes), mobile genetic element (MGE; intI1), and metal resistance genes (MRGs; pcoA and tcrB). Redundancy analysis (RDA) showed that GTRs can reduce the abundance of MRGs and ARGs by reducing the bioavailability of heavy metals. Network analysis shows that Firmicutes and Actinobacteria were the main hosts of ARGs and ARGs, MGEs, and MRGs shared the same potential host bacteria. Adding GTRs during composting may reduce ARGs transmission through horizontal gene transfer (HGT). GTRs affected the bacterial community, thereby influencing the variations in the ARG profiles and reducing the potential risk associated with the compost product. Image 1 • Co-composting is an effective way to treat green tea residues. • Green tea residues effectively reduce tet and sul genes after co-composting. • Firmicutes and Actinobacteria were the main hosts of ARGs. • Some ARGs, MGEs, and MRGs shared the same potential host bacteria. [ABSTRACT FROM AUTHOR]

• Bacterial agent and bentonite can effectively reduce NH 3 and N 2 O emissions. • Additives enhanced nitrification and denitrification processes in the compost. • Bacterial agents promote ammonia oxidation process to reduce NH 3 emissions. • Bentonite affected denitrification process dominated by nirS and nirK genes. • Proteobacteria most important denitrifying microorganisms. This study investigated the effects of adding a bacterial agent (B) and bentonite (BT) on nitrogen transformation, nitrogen functional genes, and the microbial community dynamics during the aerobic composting of pig manure, as well as their contributions to NH 3 and N 2 O emissions. Treatments B, BT, and BT + B reduced the NH 3 emissions by 31.34%, 18.82%, and 23.67%, respectively, and the N 2 O emissions by 53.16%, 72.56%, and 63.41%. N 2 O and NH 3 emissions were strongly related to the functional genes. Adding bacterial agent promoted the ammonia oxidation process to reduce NH 3 emissions, whereas the influence of bentonite on nitrogen conversion was mostly related to nirS and nirK in denitrification processes. Nitrification and denitrification were dominated by different functional microorganisms in various stages of composting, where Proteobacteria comprised the most important denitrifying microorganisms. Thus, the additives reduced NH 3 and N 2 O emissions by regulating nitrification and denitrification processes, and adding both was highly advantageous. [ABSTRACT FROM AUTHOR]

• nZVI enhanced the methane yield during anaerobic digestion of cattle manure. • Antibiotic resistance genes (ARGs) effectively removed by 160 mg/L nZVI. • Mobile genetic elements were the main drivers of the changes in ARGs. • Addition of 160 mg/L nZVI reduced abundances of Tn916/1545 and Acinetobacter. This study investigated the effects of adding nano-zerovalent iron (nZVI) at three concentrations (0, 80, and 160 mg/L) on the methane yield and the fate of antibiotic resistance genes (ARGs) during the anaerobic digestion (AD) of cattle manure. The addition of nZVI effectively enhanced the methane yield, where it significantly increased by 6.56% with 80 mg/L nZVI and by 6.43% with 160 mg/L nZVI. The reductions in the abundances of ARGs and Tn916/1545 were accelerated by adding 160 mg/L nZVI after AD. Microbial community analysis showed that nZVI mainly increased the abundances of bacteria with roles in hydrolysis and acidogenesis, whereas it reduced the abundance of Acinetobacter. Redundancy analysis indicated that the changes in mobile genetic elements made the greatest contribution to the fate of ARGs. The results suggest that 160 mg/L nZVI is a suitable additive for reducing the risks due to ARGs in AD. [ABSTRACT FROM AUTHOR]

• Tween-80 greatly influenced the bacterial community structure. • Tween-80 enhanced cellulase activity and bacteria that degrade cellulose and lignin. • Tween-80 enhanced the capacities for carbohydrate and amino acid metabolism. • Order Bacillales closely related to the metabolism of carbon sources. Metabolism by microorganisms is the basis of composting. In this study, the dynamic changes in the enzyme activity levels, bacterial community structure, and metabolism functions were investigated during chicken manure composting with an added bio-surfactant (rhamnolipid) or chemical surfactant (Tween-80). The results showed that rhamnolipid and Tween-80 improved the quality of the finished compost in terms of the C/N ratio, water-soluble carbon content, germination index, E4/E6 ratio, and the cellulase activity, especially with Tween-80. Furthermore, the bacterial communities were determined by high-throughput sequencing, and their metabolism functions were evaluated using the PICRUSt and Biolog methods. Tween-80 greatly influenced the bacterial community structure, where it enhanced the abundances of bacteria that degrade cellulose and lignin (e.g., members of the order Bacillales) and the capacities for carbohydrate and amino acid metabolism. Network analysis also showed that the order Bacillales was closely related to the metabolism of characteristic carbon sources, especially carbohydrates. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two-phase inoculation of PGPR effective in reducing abundances of ARGs (0.26 logs). • PGPR reduced the selective pressure imposed by Cu on readily removed ARGs. • Two-phase inoculation reduced abundances of potentially pathogenic ARG hosts. • Two-phase inoculation inhibited HGT of ARGs by reducing abundance of MGEs. • Two-phase inoculation downregulated expression of functional gene related to ARGs. This study investigated the effects on antibiotic resistance genes (ARGs) and the related mechanisms of different plant growth-promoting rhizobacteria (PGPR) inoculation strategies during composting: no inoculation (CK), inoculation in initial phase (T1), inoculation in cooling phase (T2), and inoculation in both initial and cooling phases (T3). After composting, the total relative abundances (RAs) of ARGs decreased by 0.26 and 0.03 logs under T3 and T2, respectively, but increased by 0.05 and 0.22 logs under T1 and CK. The abundances of eight ARGs were lowest under T3, including some high risk ARGs with clinical importance. Bioavailable Cu significantly affected the readily removed ARGs, and PGPR inoculation decreased the bioavailability of Cu. T3 reduced the abundances of potential pathogen hosts, inhibited horizontal gene transfer by reducing the RAs of mobile gene elements (0.48 logs), and downregulated the expression of genes related to ARG propagation, thereby decreasing the ecological risk of ARGs. [ABSTRACT FROM AUTHOR]