Inoculation of thermophilic bacteria from giant panda feces into cattle manure reduces gas emissions and decreases resistance gene prevalence in short-term composting. - Related Documents




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806601.0000Inoculation of thermophilic bacteria from giant panda feces into cattle manure reduces gas emissions and decreases resistance gene prevalence in short-term composting. Here, thermophilic bacteria (TB) with cellulose degradation functions were screened from composting panda feces and applied to cattle manure composting. TB (Aeribacillus pallidus G5 and Parageobacillus toebii G12) inoculation led to remarkable improvement of the compost temperature, prolonging of the thermophilic stage and shortening of the composting process, resulting in increased manure harmlessness (GI ≥ 70%), compost humification, and greenhouse gas emission reduction (14.19%-22.57%), compared with the control compost, within 15 days of composting. In particular, G5 inoculation reduced NH(3) emissions by 41.97% relative to control composts over 15 days. G5 was capable of rapidly colonizing in the composts, and its inoculation immediately enriched the genera of Firmicutes, and simultaneously decreased the genera of Proteobacteria, contributing to the elimination of harmful microorganisms. Notably, this strain lacked antibiotic resistance genes, and the absolute abundances of resistance genes and mobile genetic genes (MGEs) decreased the most (by 80.84%). Metagenomic analysis revealed that enzymes capable of producing CO(2), N(2)O, and NH(3) were generally inhibited, while CO(2) fixation and N(2)O and NH(3) reduction enzymes were enriched in the G5 compost, since metagenome-assembled genomes of Proteobacteria harbored more key genes and enzymes in complete pathways for producing N(2)O, NH(3), and CO(2). Moreover, Proteobacteria, such as Pseudomonas and Halopseudomonas, were the main host of resistance genes and MGEs. Overall, the gas emission could be reduced, and more efficient control of resistance genes could be achieved by inhibited the abundance of Proteobacteria during composting. This study provides a safe and effective microbial agent (A. pallidus) for manure treatment.202539642832
801010.9995Co-occurrence pattern of ARGs and N-functional genes in the aerobic composting system with initial elevated temperature. Animal manure is known to harbor antibiotic resistance genes (ARGs). Aerobic composting is a prevalent cost-effective and sustainable method to treat animal waste. However, the effect of initially elevated temperature on antibiotic resistome during the composting process is unclear. In this study composting was subjected to initial external heating (EHC) for a period of 5 days compared to conventional composting (CC). After composting ARGs abundance was significantly reduced by 2.43 log in EHC and 1.95 log in CC. Mobile genetic elements (MGEs) also exhibited a reduction of 1.95 log in EHC and 1.49 log in CC. However, during the cooling phase, the genes resisting macrolide lincosamide and streptogramin B (MLSB) rebounded by 0.04 log in CC. The potential human pathogenic bacteria Pseudomonas (41.5-61.5%) and Actinobacteria (98.4-98.8%) were significantly reduced in both treatments and the bulk of targeted antibiotics were eliminated by 80.74% in EHC and 68.98% in CC. ARGs and N-functional genes (NFGs), mainly denitrification genes, were carried by the same microbial species, such as Corynebacterium sp. and Bacillus sp., of the dominant phylum. Redundancy analysis (RDA) revealed that CC microbial communities played a key role in the enrichment of ARGs while in EHC the variation of ARGs was attributed to the composting temperature. The number of high-risk ARGs was also lower in EHC (4) compared with CC (6) on day 30. These results provide insight into the effects of an initially enhanced temperature on ARGs removal and the relationship between ARGs and NFGs during the composting process.202337229868
807220.9995Response of microbial communities based on full-scale classification and antibiotic resistance genes to azithromycin and copper combined pollution in activated sludge nitrification laboratory mesocosms at low temperature. This study aimed to investigate the short-term response of abundant-rare genera and antibiotic resistance genes (ARGs) to azithromycin (AZM, 0.05-40 mg/L) and copper (1 mg/L) combined pollution in activated sludge nitrification system at low temperature. Nitrification was as expected inhibited in stress- and post-effects periods under AZM concentration higher than 5 mg/L. Abundant and rare taxa presented dissimilar responses based on full-scale classification. Conditionally rare or abundant taxa (CRAT) were keystone taxa. Relative abundance of ammonia-oxidizing archaea increased, and three aerobic denitrifying bacteria (Brevundimonas, Comamonas and Trichococcus) were enriched (from 9.83% to 68.91% in total). Ammonia nitrogen assimilating into Org-N and denitrification may be nitrogen pathways based on predict analysis. 29 ARGs were found with more co-occurrence patterns and high concentration of AZM (greater than 5 mg/L) caused their proliferation. Importantly, expect for some abundant taxa, rare taxa, potential pathogens and nitrogen-removal functional genera were the main potential hosts of ARGs.202134523571
801230.9994Sensitive response mechanism of ARGs and MGEs to initial designed temperature during swine manure and food waste co-composting. The rapid aerobic composting process has been used to reduce organic wastes, but the associated risks of antibiotic resistance genes (ARGs) need to evaluate in an efficient way. The primary objective of this work was to explore the underlying mechanism of initial adjustment in composting temperature on the variation of ARGs, mobile genetic elements (MGEs), and microbial composition during co-composting. The co-composting was initially externally heated (T2) for 5 days. The results showed that ARGs abundance in conventional composting (T1) was reduced by 49.36%, while multidrug was enriched by 86.16% after a period of 30 days. While in T2 ARGs were removed by 79.46% particularly the fraction of sulfonamide, multidrug, and vancomycin resistance genes were >90% without rebounding of any ARGs. Whereas, MGEs were reduced by 68.12% and 93.62% in T1 and T2, while the half-lives of ARGs and MGEs were lower in T2 compared to T1 (86.3%,86.7%). T2 also affected the metabolism function by regulating carbohydrate metabolism (9.62-10.39%) and amino acid metabolism (9.92-10.93%). Apart from this, the potential human pathogenic bacteria Pseudomonas was reduced by 90.6% in T2 and only 32.9% in T1 respectively. Network analysis showed that Ureibacillus, Weissella, Corynebacterium, Escherichia-Shigella, Acinetobacter were the main host of multiple genes. Structural equation models exhibited that bacterial communities were mainly responsible for the enrichment of ARGs in T1, whereas, it was directly affected by MGEs in T2. Similarly, ARGs variation was directly related to composting temperature. With this simple strategy, ARGs associated risk can be significantly reduced in composting.202336208781
759340.9994Microbial community functional structure in an aerobic biofilm reactor: Impact of streptomycin and recovery. Antibiotics can affect microbial community structure and promote antibiotic resistance. However, the course of microbial community recovery in wastewater treatment systems after antibiotic disturbance remains unclear. Herein, multiple molecular biology tools, including 16S amplicon sequencing, GeoChip 5.0, quantitative polymerase chain reaction (qPCR), and metagenomic sequencing, were used to investigate the year-long (352 d) recovery of the microbial community functional structure in an aerobic biofilm reactor. Nitrification was completely inhibited under 50 mg/L of streptomycin spiking (STM_50) due to the significant reduction of ammonia-oxidizing bacteria, but recovered to original pre-disturbance levels after streptomycin removal, indicating the high resilience of ammonia-oxidizing bacteria. Bacterial community richness and diversity decreased significantly under STM_50 (p < 0.05), but recovered to levels similar to those observed before disturbance after 352 d. In contrast, bacterial composition did not recover to the original structure. The carbon degradation and nitrogen cycling functional community significantly changed after recovery compared to that observed pre-disturbance (p < 0.05), thus indicating functional redundancy. Additionally, levels of aminoglycoside and total antibiotic resistance genes under STM_50 (relative abundance, 0.33 and 0.80, respectively) and after one year of recovery (0.12 and 0.29, respectively) were higher than the levels detected pre-disturbance (0.04 and 0.24, respectively). This study provides an overall depiction of the recovery of the microbial community functional structure after antibiotic exposure. Our findings give notice that recovery caused by antibiotic disturbance in the water environment should be taken more seriously, and that engineering control strategies should be implemented to prevent the antibiotic pollution of wastewater.202032417519
806050.9994Responses of bacterial communities and antibiotic resistance genes to nano-cellulose addition during pig manure composting. 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.202134649327
807160.9994Impact of ciprofloxacin and copper combined pollution on activated sludge: Abundant-rare taxa and antibiotic resistance genes. This study aimed to explore the impacts of ciprofloxacin (CIP, 0.05-40 mg/L) and copper (3 mg/L) combined pollution on nitrification, microbial community and antibiotic resistance genes (ARGs) in activated sludge system during stress- and post-effect periods. Higher CIP concentration inhibited nitrification and an average of 50% total nitrogen removal occurred under 40 mg/L of CIP pressure. The stress- and post-effects on bacterial diversity and structure were obviously distinct. Abundant genera were more sensitive to combined pollution than rare genera based on full-scale classification and conditionally rare or abundant taxa were keystone taxa in their interactions. Ammonia oxidation genes were inhibited under high CIP level, but some aerobic denitrifying bacteria (Thauera, Comamonas and Azoarcus) and key genes increased. 96 ARG subtypes were detected with complex positive relationships and their potential hosts (abundant-rare-functional genera) changed in two periods. This study highlights the different stress- and post-effects of combined pollution on activated sludge.202235217161
809070.9994Swine Manure Composting With Compound Microbial Inoculants: Removal of Antibiotic Resistance Genes and Their Associations With Microbial Community. In this study, compound microbial inoculants, including three Bacillus strains and one Yeast strain, were inoculated into swine manure composting to explore the effects on antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), microbial community structure, and pathogenic bacteria. The results indicated that the abundances of the detected ARGs ranged from 3.6 × 10(3) to 1.13 × 10(8) copies/g. The ARGs with the highest abundance was sul2, and the lowest was blaCTX. Composting removes most of the ARGs and MGEs by 22.8-99.7%. These ARGs were significantly reduced during the thermophilic phase of compost. The removal rate of ARGs at the different layers of compost pile was different as follows: middle layer > upper layer > lower layer. But some ARGs proliferated significantly in the maturation phase of compost, especially the sulfonamide resistance genes. Compound microbial inoculants increased the temperature of compost, accelerated water loss, nitrogen fixation, and increased the removal rate of β-lactamase resistance genes, the transposon gene tn916 and part of tetracycline resistance genes by 3.7-23.8% in compost. Compound microbial inoculants changed the community structure and increased the Bacillus abundance in the thermophilic phase of compost. And it was helpful for removing pathogens during composting. The addition of compound microbial inoculants causes the decrease of Firmicutes and the increase of Bacteroidetes, which may be related to the removal and proliferation of ARGs.202033250880
808480.9994Metagenomic insights into effects of carbon/nitrogen ratio on microbial community and antibiotic resistance in moving bed biofilm reactor. This study investigated the effects of carbon/nitrogen (C/N) ratio on microbial community in moving bed biofilm reactor (MBBR) using metagenomic analysis, and the dynamic changes of relevant antibiotic resistance genes (ARGs) were also analyzed. The results showed that under low C/N ratio, MBBR exhibited average removal rates of 98.41 % for ammonia nitrogen and 75.79 % for total nitrogen. Metagenomic analysis showed low C/N ratio altered the structure of biofilm and water microbiota, resulting in the detachment of bacteria such as Actinobacteria from biofilm into water. Furthermore, sulfamethazine (SMZ)-resistant bacteria and related ARGs were released into water under low C/N ratio, which lead to the increase of SMZ resistance rate to 90%. Moreover, most dominant genera are potential hosts for both nitrogen cycle related genes and ARGs. Specifically, Nitrosomonas that carried gene sul2 might be released from biofilm into water. These findings implied the risks of antibiotic resistance dissemination in MBBR under low C/N ratio.202438901747
804390.9994Effect of tetracycline on bio-electrochemically assisted anaerobic methanogenic systems: Process performance, microbial community structure, and functional genes. Bio-electrochemically assisted anaerobic methanogenic systems (An-BES) are highly effective in wastewater treatment for methane production and degradation of toxic compounds. However, information on the treatment of antibiotic-bearing wastewater in An-BES is still very limited. This study therefore investigated the effect of tetracycline (TC) on the performance, microbial community, as well as functional and antibiotic resistance genes of An-BES. TC at 1 and 5 mg/L inhibited methane production by less than 4.8% compared to the TC-free control. At 10 mg/L TC, application of 0.5 and 1.0 V decreased methane production by 14 and 9.6%, respectively. Under the effect of 1-10 mg/L TC, application of 1.0 V resulted in a decrease of current from 42.3 to 2.8 mA. TC was mainly removed by adsorption; its removal extent increased by 19.5 and 32.9% with application of 0.5 and 1.0 V, respectively. At 1.0 V, current output was not recovered with the addition of granular activated carbon, which completely removed TC by adsorption. Metagenomic analysis showed that propionate oxidizing bacteria and methanogens were more abundant in electrode biofilms than in suspended culture. Antibiotic resistance genes (ARGs) were less abundant in biofilms than in suspended culture, regardless of whether voltage was applied or not. Application of 1.0 V resulted in the enrichment of Geobacter in the anode and Methanobacterium in the cathode. TC inhibited exoelectrogens, propionate oxidizing bacteria, and the methylmalonyl CoA pathway, leading to a decrease of current output, COD consumption, and methane production. These findings deepen our understanding of the inhibitory effect of TC in An-BES towards efficient bioenergy recovery from antibiotic-bearing wastewater, as well as the response of functional microorganisms to TC in such systems.202235533856
8087100.9994Effect of different biochars on antibiotic resistance genes and bacterial community during chicken manure composting. Rice straw biochar (RSB) and mushroom biochar (MB) were added to lab-scale chicken manure composting to evaluate their effects on the behaviors of antibiotic resistance genes (ARGs) and on total and bio-available heavy metals (Cu, Zn and As). The associated bacterial community was characterized by 16SrRNA high-throughput sequencing. The abundance of pathogenic bacteria was also calculated. At the end of the control composting experiment, the average removal rate of ARGs was 0.86 log units and the removal rate of pathogenic bacteria was 57.1%. MB addition resulted in a higher removal rate than that in the control composting experiment. However, RSB addition yielded opposite results, which may be due to the higher abundance of Erysipelotrichaceae, Lactobacillaceae, Family_XI_Incertae_Sedis (belonging to Firmicutes carrying and disseminating ARGs) and pathogenic bacteria carrying ARGs. Furthermore, the correlations between bio-available heavy metals and ARGs were more obvious than those between total heavy metals and ARGs.201626720134
8091110.9994Changes in antibiotic resistance genes and mobile genetic elements during cattle manure composting after inoculation with Bacillus subtilis. This study explored the effects of Bacillus subtilis at four levels (0, 0.5%, 1%, and 2% w/w compost) on the variations in ARGs, mobile genetic elements (MGEs), and the bacterial community during composting. The composting process had a greater impact on ARGs than Bacillus subtilis. The main ARG detected was sul1. The addition of Bacillus subtilis at 0.5% reduced the relative abundances of ARGs, MGEs, and human pathogenic bacteria (by 2-3 logs) in the mature products. Network and redundancy analyses suggested that intI1, Firmicutes, and pH were mainly responsible for the changes in ARGs, thus controlling these factors might help to inhibit the spread of ARGs.201931442833
8080120.9993Fates of antibiotic resistance genes and bacterial/archaeal communities of activated sludge under stress of copper: Gradient increasing/decreasing exposure modes. Effect of copper (Cu) on antibiotic resistance genes (ARGs) and bacterial/archaeal community of activated sludge under gradient increasing (0.5-10 mg/L) or decreasing exposure (10-0.5 mg/L) modes was explored. Here, 29 genes were detected among 48 selected ARGs and mobile gene elements (MGEs). Two exposure modes showed dissimilar effects on ARGs and distribution was more affected by environmental concentrations of Cu, which promoted transmission of ARGs (multiple drug resistance and sulfonamide). Cellular protection was main resistance mechanism, which was less inhibited than efflux pumps. The tnpA-02, as main MGE, interacted closely with ARGs (sul2, floR, etc.). Gradient increasing exposure mode had more effects on bacterial/archaeal structure and composition. Bacteria were main hosts for specific ARGs and tnpA-02, while archaea carried multiple ARGs (cmx(A), adeA, etc.), and bacteria (24.24 %) contributed more to changes of ARGs than archaea (19.29 %). This study clarified the impacts of Cu on the proliferation and transmission of ARGs.202236096328
8093130.9993Acidic conditions enhance the removal of sulfonamide antibiotics and antibiotic resistance determinants in swine manure. Manure pH may vary depending on its inherent composition or additive contents. However, the effect of pH on the fate of antibiotics and antibiotic resistance determinants in manure remains unclear. This work demonstrated that pH adjustment promoted the removal of different sulfonamide antibiotics (SAs) within swine manure under incubation conditions, which increased from 26-60.8% to 75.0-86.0% by adjusting the initial pH from neutral (7.4) to acidic (5.4-4.8). Acidification was also demonstrated to inhibit the accumulation of antibiotic resistance genes in manure during incubation. Acidified manure contained both lower absolute and relative abundances of sul1 and sul2 than those at a neutral pH like 7.4. Further investigation indicated that acidification promoted the reduction of sul genes in manure by restricting sulfonamide-resistant bacteria (SRB) proliferation and inhibiting IntI1 accumulation. Furthermore, pH adjustment significantly influenced the composition of the manure bacterial community after incubation, which increased Firmicutes and decreased Proteobacteria. Close relationships were observed between pH-induced enrichment of the Firmicutes bacterial phylum, enhanced SAs degradation, and the fates of antibiotic resistance determinants. Overall, lowering the pH of manure promotes the degradation of SAs, decreases sul genes and SRB, and inhibits horizontal sul gene transfer, which could be a simple yet highly-effective manure management option to reduce antibiotic resistance.202032302890
8063140.9993Enhancement of methane production and antibiotic resistance genes reduction by ferrous chloride during anaerobic digestion of swine manure. In this study, effects of ferrous chloride (FeCl(2)) addition on methane production and antibiotic resistance genes (ARGs) reduction were investigated during anaerobic digestion (AD) of swine manure. FeCl(2) could both improve the accumulative methane production and reduce the abundance of total ARGs, i.e., the maximum increase of CH(4) production of 21.5% at FC5, and the maximum ARGs reduction of 33.3% at FC25. The reduction of pathogenic bacteria and metal resistance genes (MRGs) was enhanced. Acetate and propionate utilization were intensified by enhancing H(2) utilization and direct interspecies electron transfer (DIET), where DIET was further enhanced by the reaction of the FeCl(2) and acetic acid. The bacterial community played important role in the evolution of ARGs (68.26%), which were also affected by MRGs, mobile genetic elements (MGEs), and environmental factors. Therefore, FeCl(2)-based AD is a feasible and attractive way to improve methane production and ARG reduction.202031855663
8061150.9993The addition of nano zero-valent iron during compost maturation effectively removes intracellular and extracellular antibiotic resistance genes by reducing the abundance of potential host bacteria. Applying compost to soil may lead to the spread of antibiotic resistance genes (ARGs) in the environment. Therefore, removing ARGs from compost is critical. In this study, for the first time, nano zero-valent iron (nZVI) was added to compost during the maturation stage to remove ARGs. After adding 1 g/kg of nZVI, the abundance of total intracellular and total extracellular ARGs was decreased by 97.62% and 99.60%, and that of total intracellular and total extracellular mobile genetic elements (MGEs) was decreased by 92.39% and 99.31%, respectively. A Mantel test and network analysis indicated that the reduction in potential host bacteria and intI1 after nZVI treatment promoted the removal of intracellular and extracellular ARGs. The addition of nZVI during composting reduced the horizontal transfer of ARGs and improve the total nitrogen and germination index of compost, allowing it to meet the requirements for organic fertilizers.202337352990
8092160.9993Effect of pH on the mitigation of extracellular/intracellular antibiotic resistance genes and antibiotic resistance pathogenic bacteria during anaerobic fermentation of swine manure. Effects of various initial pH values (i.e., 3, 5, 7, 11) during anaerobic fermentation of swine manure on intracellular and extracellular antibiotic resistance genes (iARGs and eARGs) and ARG-carrying potential microbial hosts were investigated. The abundance of almost all iARGs and eARGs decreased by 0.1-1.7 logs at pH 3 and pH 5. The abundance of only three iARGs and eARGs decreased by 0.1-0.9 logs at pH 7 and pH 11. Under acidic initial fermentation conditions (pH 3 and pH 5), the ARG removal effect was more pronounced. Acidic conditions (pH 3 and pH 5) significantly reduced the diversity and abundance of the microbial community, thereby eliminating many potential ARG hosts and antibiotic-resistant pathogenic bacteria (ARPB). Therefore, the study results contribute to the investigation of the effects of swine manure anaerobic fermentation on the removal and risk of contamination of ARGs and ARPB.202336746211
8018170.9993Analysis for microbial denitrification and antibiotic resistance during anaerobic digestion of cattle manure containing antibiotic. This study investigated the effects of tylosin (0, 10, and 100 mg/kg dry weight) on the denitrification genes and microbial community during the anaerobic digestion of cattle manure. N(2) emissions were reduced and N(2)O emissions were increased by 10 mg/kg tylosin. Adding 100 mg/kg tylosin increased the emission of both N(2)O and N(2). The different responses of denitrifying bacteria and genes to tylosin may have been due to the presence of antibiotic resistance genes (ARGs). Network analysis indicated that denitrification genes and ARGs had the same potential host bacteria. intI1 was more important for the horizontal transfer of denitrification genes and ARGs during anaerobic digestion than intI2. The anaerobic digestion of manure containing tylosin may increase nitrogen losses and the associated ecological risk.201931326686
8068180.9993Safety of composts consisting of hydrothermally treated penicillin fermentation residue: Degradation products, antibiotic resistance genes and bacterial diversity. Combining hydrothermal treatment and composting is an effective method to dispose of penicillin fermentation residue (PFR), but the safety and related mechanism are still unclear. In this study, penicillin solution was hydrothermally treated to decipher its degradation mechanism, and then hydrothermally treated PFR (HT-PFR) was mixed with bulking agents at ratios of 2:0 (CK), 2:1.5 (T1), and 2:5 (T2) to determine the absolute abundance of antibiotic resistance genes (ARGs) and the succession of bacterial community. Results showed that penicillin was degraded to several new compounds without the initial lactam structure after hydrothermal treatment. During composting, temperature and pH of the composts increased with the raising of HT-PFR proportion, except the pH at days 2. After 52 days of composting, the absolute copies of ARGs (blaTEM, blaCMY2, and blaSFO) and the relative abundance of bacteria related to pathogens were reduced significantly (P < 0.05). Especially, the total amount of ARGs in the samples of CK and T1 were decreased to equal level (around 5 log(10) copies/g), which indicated that more ARGs were degraded in the latter by the composting process. In the CK samples, Bacteroidetes and Proteobacteria accounted for ~69.8% of the total bacteria, but they were gradually replaced by Firmicutes with increasing proportions of HT-PFR, which can be caused by the high protein content in PFR. Consisting with bacterial community, more gram-positive bacteria were observed in T1 and T2, and most of them are related to manganese oxidation and chitinolysis. As composting proceeded, bacteria having symbiotic or pathogenic relationships with animals and plants were reduced, but those related to ureolysis and cellulolysis were enriched. Above all, hydrothermal treatment is effective in destroying the lactam structure of penicillin, which makes that most ARGs and pathogenic bacteria are eliminated in the subsequent composting.202134492529
7588190.9993Response of viable bacteria to antibiotics in aerobic granular sludge: Resistance mechanisms and behaviors, bacterial communities, and driving factors. The assessment of antimicrobial resistance (AMR) risk by DNA-based techniques mainly relies on total bacterial DNA. In this case, AMR risk recognition is restricted to the genotype level, lacking crucial phenotypic information, such as the distribution of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in dead and viable bacteria. This limitation hinders the recognition of AMR behavior. Herein, based on propidium monoazide (PMA) shielding method, this work firstly quantified the intracellular ARGs/MGEs in viable and dead bacteria, and the impact of viable bacteria composition on the formation of intracellular/extracellular polymeric substance-related /cell-free ARGs (i/e/cARGs) and MGEs (i/e/cMGEs) in aerobic granular sludge (AGS). The shielding efficiency of PMA against dead bacteria was optimized to be as high as 97.5% when the MLSS of AGS was 2.0 g/L. Under antibiotic stimulation, 29.0% ∼ 49.0% of iARGs/iMGEs were carried by viable bacteria, and the remaining proportion were carried by dead bacteria. 18 out of the top 20 dominant genera showed a change in abundance by more than 1% after PMA treatment. 29 viable hosts were identified to associate with 52 iARGs, of which 28 and 15 hosts were also linked to 40 eARGs and 26 cARGs. Also, partial least-squares path model and variance partitioning analysis disclosed that viable bacteria and i/e/cMGEs had a positive effect on i/e/cARGs, with both contributing as much as 64.5% to the total ARGs enrichment. These results better visualized the AMR risk carried by viable bacteria and the categories of viable hosts. This work provides a novel insight into analyzing the actual AMR risk and viable hosts, helping to the reduction and control of AMR in wastewater treatment plants.202337748345