Metabolite Cross-Feeding Promoting NADH Production and Electron Transfer during Efficient SMX Biodegradation by a Denitrifier and S. oneidensis MR-1 in the Presence of Nitrate.

Antibiotics often coexist with other pollutants (e.g., nitrate) in an aquatic environment, and their simultaneous biological removal has attracted widespread interest. We have found that sulfamethoxazole (SMX) and nitrate can be efficiently removed by the coculture of a model denitrifier (Paracoccus denitrificans, Pd) and Shewanella oneidensis MR-1 (So), and SMX degradation is affected by NADH production and electron transfer. In this paper, the mechanism of a coculture promoting NADH production and electron transfer was investigated by proteomic analysis and intermediate experiments. The results showed that glutamine and lactate produced by Pd were captured by So to synthesize thiamine and heme, and the released thiamine was taken up by Pd as a cofactor of pyruvate and ketoglutarate dehydrogenase, which were related to NADH generation. Additionally, Pd acquired heme, which facilitated electron transfer as heme, was the important composition of complex III and cytochrome c and the iron source of iron sulfur clusters, the key component of complex I in the electron transfer chain. Further investigation revealed that lactate and glutamine generated by Pd prompted So chemotactic moving toward Pd, which helped the two bacteria effectively obtain their required substances. Obviously, metabolite cross-feeding promoted NADH production and electron transfer, resulting in efficient SMX biodegradation by Pd and So in the presence of nitrate. Its feasibility was finally verified by the coculture of an activated sludge denitrifier and So.

High-order time lacunarity feature-aided multiple hypotheses tracking for underwater active small targets in high-clutter harbor environment.

Active tracking of underwater small targets is a great challenge with kinematic information alone. This is because the active sonar often encounters multipath propagation and the induced clutter can even mask target echoes. Recently, high-order time lacunarity (HOT-Lac) has shown its ability in effectively highlighting "blob" targets from high clutter harbor environments. Hence, this paper proposes a HOT-Lac aided track scoring mechanism to solve the ambiguity of data association within the framework of Multiple Hypotheses Tracking (MHT). Specifically, the trajectory consistency of potential targets is captured by a momentum accumulation of the HOT Lac feature, which can inherit the historical information for the whole track. Meanwhile, due to the separability of the distribution of target and clutter in the HOT-Lac feature space, the probabilities of the target hypothesis and null hypothesis are modeled by the online computation of the HOT-Lac feature. Finally, the cumulative likelihood ratio based on HOT-Lac is integrated into MHT to score the potential tracks. Experiments in several real-world harbor scenarios demonstrate that the proposed HOT-Lac feature-aided tracker can suppress false tracks accurately and quickly.

Amino Acid Configuration Affects Volatile Fatty Acid Production during Proteinaceous Waste Valorization: Chemotaxis, Quorum Sensing, and Metabolism.

During proteinaceous waste valorization to produce volatile fatty acids (VFAs), protein needs to be hydrolyzed to amino acids (AAs), but the effects of the configuration of AAs on their biotransformation and VFA production have not been investigated. In this study, more residual d-AAs than their corresponding l-AAs were observed after VFAs were produced from kitchen waste in a pilot-scale bioreactor. For all AAs investigated, the VFA production from d-AAs was lower than that from corresponding l-AAs. The metagenomics and metaproteomics analyses revealed that the l-AA fermentation system exhibited greater bacterial chemotaxis and quorum sensing (QS) than d-AAs, which benefited the establishment of functional microorganisms (such as Clostridium, Sedimentibacter, and Peptoclostridium) and expression of functional proteins (e.g., substrate transportation cofactors, l-AA dehydrogenase, and acidogenic proteins). In addition, d-AAs need to be racemized to l-AAs before being metabolized, and the difference of VFA production between d-AAs and l-AAs decreased with the increase of racemization activity. The findings of the AA configuration affecting bacterial chemotaxis and QS, which altered microorganism communities and functional protein expression, provided a new insight into the reasons for higher l-AA metabolism than d-AAs and more d-AAs left during VFA production from proteinaceous wastes.

Reductive Stress Boosts the Horizontal Transfer of Plasmid-Borne Antibiotic Resistance Genes: The Neglected Side of the Intracellular Redox Spectrum.

The dissemination of plasmid-borne antibiotic resistance genes (ARGs) among bacteria is becoming a global challenge to the "One Health" concept. During conjugation, the donor/recipient usually encounter diverse stresses induced by the surrounding environment. Previous studies mainly focused on the effects of oxidative stress on plasmid conjugation, but ignored the potential contribution of reductive stress (RS), the other side of the intracellular redox spectrum. Herein, we demonstrated for the first time that RS induced by dithiothreitol could significantly boost the horizontal transfer of plasmid RP4 from Escherichia coli K12 to different recipients (E. coli HB101, Salmonella Typhimurium, and Pseudomonas putida KT2440). Phenotypic and genotypic tests confirmed that RS upregulated genes encoding the transfer apparatus of plasmid RP4, which was attributed to the promoted consumption of intracellular glutamine in the donor rather than the widely reported SOS response. Moreover, RS was verified to benefit ATP supply by activating glycolysis (e.g., GAPDH) and the respiratory chain (e.g., appBC), triggering the deficiency of intracellular free Mg2+ by promoting its binding, and reducing membrane permeability by stimulating cardiolipin biosynthesis, all of which were beneficial to the functioning of transfer apparatus. Overall, our findings uncovered the neglected risks of RS in ARG spreading and updated the regulatory mechanism of plasmid conjugation.

Widespread of Potential Pathogen-Derived Extracellular Vesicles Carrying Antibiotic Resistance Genes in Indoor Dust.

Extracellular vesicles (EVs) are newly recognized as important vectors for carrying and spreading antibiotic resistance genes (ARGs). However, the ARGs harbored by EVs in ambient environments and the transfer potential are still unclear. In this study, the prevalence of ARGs and mobile genetic elements (MGEs) in EVs and their microbial origins were studied in indoor dust from restaurants, kindergarten, dormitories, and vehicles. The amount of EVs ranged from 3.40 × 107 to 1.09 × 1011 particles/g dust. The length of EV-associated DNA fragments was between 21 bp and 9.7 kb. Metagenomic sequencing showed that a total of 241 antibiotic ARG subtypes encoding resistance to 16 common classes were detected in the EVs from all four fields. Multidrug, quinolone, and macrolide resistance genes were the dominant types. 15 ARG subtypes were exclusively carried and even enriched in EVs compared to the indoor microbiome. Moreover, several ARGs showed co-occurrence with MGEs. The EVs showed distinct taxonomic composition with their original dust microbiota. 30.23% of EV-associated DNA was predicted to originate from potential pathogens. Our results indicated the widespread of EVs carrying ARGs and virulence genes in daily life indoor dust, provided new insights into the status of extracellular DNA, and raised risk concerns on their gene transfer potential.

Nitric Oxide: A Neglected Driver for the Conjugative Transfer of Antibiotic Resistance Genes among Wastewater Microbiota.

The dissemination of plasmid-borne antibiotic resistance genes (ARGs) in wastewater is becoming an urgent concern. Previous studies mainly focused on the effects of coexisting contaminants on plasmid conjugation, but ignored the potential contribution of some byproducts inevitably released from wastewater treatment processes. Herein, we demonstrate for the first time that nitric oxide (NO), an intermediate of the wastewater nitrogen cycle, can significantly boost the conjugative transfer of plasmid RP4 from Escherichia coli K12 to different recipients (E. coli HB101, Salmonella typhimurium, and wastewater microbiota). Phenotypic and genotypic tests confirmed that NO-induced promotion was not attributed to the SOS response, a well-recognized driver for horizontal gene transfer. Instead, NO exposure increased the outer membrane permeability of both the donor and recipient by inhibiting the expression of key genes involved in lipopolysaccharide biosynthesis (such as waaJ), thereby lowering the membrane barrier for conjugation. On the other hand, NO exposure not only resulted in the accumulation of intracellular tryptophan but also triggered the deficiency of intracellular methionine, both of which were validated to play key roles in regulating the global regulatory genes (korA, korB, and trbA) of plasmid RP4, activating its encoding transfer apparatus (represented by trfAp and trbBp). Overall, our findings highlighted the risks of NO in spreading ARGs among wastewater microbiota and updated the regulation mechanism of plasmid conjugation.

Stepwise artificial evolution of an Sw-5b immune receptor extends its resistance spectrum against resistance-breaking isolates of Tomato spotted wilt virus.

Plants use intracellular nucleotide-binding leucine-rich repeat immune receptors (NLRs) to recognize pathogen-encoded effectors and initiate immune responses. Tomato spotted wilt virus (TSWV), which has been found to infect >1000 plant species, is among the most destructive plant viruses worldwide. The Sw-5b is the most effective and widely used resistance gene in tomato breeding to control TSWV. However, broad application of tomato cultivars carrying Sw-5b has resulted in an emergence of resistance-breaking (RB) TSWV. Therefore, new effective genes are urgently needed to prevent further RB TSWV outbreaks. In this study, we conducted artificial evolution to select Sw-5b mutants that could extend the resistance spectrum against TSWV RB isolates. Unlike regular NLRs, Sw-5b detects viral elicitor NSm using both the N-terminal Solanaceae-specific domain (SD) and the C-terminal LRR domain in a two-step recognition process. Our attempts to select gain-of-function mutants by random mutagenesis involving either the SD or the LRR of Sw-5b failed; therefore, we adopted a stepwise strategy, first introducing a NSmRB -responsive mutation at the R927 residue in the LRR, followed by random mutagenesis involving the Sw-5b SD domain. Using this strategy, we obtained Sw-5bL33P/K319E/R927A and Sw-5bL33P/K319E/R927Q mutants, which are effective against TSWV RB carrying the NSmC118Y or NSmT120N mutation, and against other American-type tospoviruses. Thus, we were able to extend the resistance spectrum of Sw-5b; the selected Sw-5b mutants will provide new gene resources to control RB TSWV.

Integrated Metagenomic and Metaproteomic Analyses Unravel Ammonia Toxicity to Active Methanogens and Syntrophs, Enzyme Synthesis, and Key Enzymes in Anaerobic Digestion.

During anaerobic digestion, the active microbiome synthesizes enzymes by transcription and translation, and then enzymes catalyze multistep bioconversions of substrates before methane being produced. However, little information is available on how ammonia affects truly active microbes containing the expressed enzymes, enzyme synthesis, and key enzymes. In this study, an integrated metagenomic and metaproteomic investigation showed that ammonia suppressed not only the obligate acetotrophic methanogens but also the syntrophic propionate and butyrate oxidation taxa and their assistant bacteria (genus Desulfovibrio), which declined the biotransformations of propionate and butyrate → acetate → methane. Although the total population of the hydrolyzing and acidifying bacteria was not affected by ammonia, the bacteria with ammonia resistance increased. Our study also revealed that ammonia restrained the enzyme synthesis process by inhibiting the RNA polymerase (subunits A' and D) during transcription and the ribosome (large (L3, L12, L13, L22, and L25) and small (S3, S3Ae, and S7) ribosomal subunits) and aminoacyl-tRNA synthesis (aspartate-tRNA synthetase) in translation. Further investigation suggested that methylmalonyl-CoA mutase, acetyl-CoA C-acetyltransferase, and CH3-CoM reductase, which regulate propionate and butyrate oxidation and acetoclastic methanation, were significantly downregulated by ammonia. This study provides intrinsic insights into the fundamental mechanisms of how ammonia inhibits anaerobic digestion.

Fast compressive beamforming with a modified fast iterative shrinkage-thresholding algorithm.

Compressive beamforming has been successfully applied to direction-of-arrival estimation with sensor arrays. The results demonstrated that this technique achieves superior performance when compared with traditional high-resolution beamforming methods. The existing compressive beamforming methods use classical iterative optimization algorithms in their compressive sensing theories. However, the computational complexity of the existing compressive beamforming methods tend to be excessively high, which has limited the use of compressive beamforming in applications with limited computing resources. To address this issue, this paper proposes a fast compressive beamforming method which combines the shift-invariance of the array beam patterns with a fast iterative shrinkage-thresholding algorithm. The evaluation shows that the proposed fast compressive beamforming method successfully reduces the number of floating-point operations by 3 orders of magnitude when compared with the existing methods. In addition, both the simulations and experiments demonstrate that the resolution limit for discerning closely spaced sources of the introduced fast method is comparable to those of the existing compressive beamforming methods, which use classical iterative optimization algorithms.

The E3 ubiquitin ligase Itch regulates expression of transcription factor Foxp3 and airway inflammation by enhancing the function of transcription factor TIEG1.

Transforming growth factor-beta (TGF-beta) signaling in naive T cells induces expression of the transcription factor Foxp3, a 'master' regulator of regulatory T cells (T(reg) cells). However, the molecular mechanisms leading to Foxp3 induction remain unclear. Here we show that Itch-/- T cells were resistant to TGF-beta treatment and had less Foxp3 expression. The E3 ubiquitin ligase Itch associated with and promoted conjugation of ubiquitin to the transcription factor TIEG1. Itch cooperated with TIEG1 to induce Foxp3 expression, which was reversed by TIEG1 deficiency. Functionally, 'TGF-beta-converted' T(reg) cells generated from TIEG1-deficient mice were unable to suppress airway inflammation in vivo. These results suggest TIEG and Itch contribute to a ubiquitin-dependent nonproteolytic pathway that regulates inducible Foxp3 expression and the control of allergic responses.

Unusual subcutaneous dedifferentiated liposarcoma exhibiting coexistence of meningothelial-like whorls and inflammatory myofibroblastic tumor-like structures.

To explore the clinicopathological features of a rare dedifferentiated liposarcoma (DDLPS) with meningothelial-like whorls, we retrospectively analyzed 46 reported cases and 1 case that we encountered. Fluorescence in situ hybridization (FISH) analysis of the MDM2 amplification status of our case was also performed. Our case involved a 73-year-old male patient who had a mass in the upper part of his left arm for 10 years and was treated by surgical ablation of the tumor because of the mass' recent rapid enlargement. Microscopically, the tumor tissues showed coexistence of well-differentiated and dedifferentiated components, the latter of which included meningothelial-like whorls and inflammatory myofibroblastic tumor-like structures. The dedifferentiated components diffusely expressed vimentin, MDM2, CDK4, p16, and smooth muscle actin. They were also focally positive for desmin but negative for S-100, CD117, CD34, ALK, EMA, SOX-10, p53, and ß-catenin. FISH detection showed MDM2 amplification. In conclusion, subcutaneous DDLPS with meningothelial-like whorls and inflammatory myofibroblastic tumor-like features is rare. This case broadens the histopathological lineage of DDLPS, and confirms DDLPS with myogenic differentiation. The use of the combination of MDM2, CDK4, p16, and FISH to detect MDM2 amplification is a reliable basis for the diagnosis of DDLPS with meningothelial-like whorls.

Fast online high-order time lacunarity for characterizing active sonar echographs of harbor environment.

High-order time lacunarity (HOT-Lac) is an effective feature for characterizing active sonar echographs of harbor environments. However, it involves high computational complexity of loop summations. Motivated by the idea of integral image, this Letter extends an echo-intensity integral sequence, a representation of filtering with time domain recursion, permitting fast and online updates of HOT-Lac in a constant number of operations. Evaluated by a set of real-world harbor data, the proposed method is capable of computing HOT-Lac extremely rapidly while maintaining equivalent area under curve performances to its off-line counterpart, demonstrating its potential for real-time surveillance of the harbor.

Detecting moving targets in active sonar echograph of harbor environment using high-order time lacunarity.

This paper presents a feature for detecting potential targets from high level littoral clutters in active sonar echographs. Based on lacunarity, which describes the image texture statistics, an extension to the time domain is made in order to measure the dynamic behavior of target echoes and background clutters. Moreover, as high-order moments have been shown to well characterize the non-Rayleigh tails of littoral clutter, high-order computation is incorporated in the proposed high-order time lacunarity (HOT-Lac). The potential of HOT-Lac is demonstrated using a series of active sonar echographs with diverse cooperative targets detected in real-world harbor environments in the South China Sea. Specifically, it is shown how HOT-Lac can effectively distinguish different moving small targets from high-level background clutter, and how this ability can be exploited to highlight an invasion target in harbor security.

K33-linked polyubiquitination of T cell receptor-zeta regulates proteolysis-independent T cell signaling.

Tagging the cell surface receptor with ubiquitin is believed to provide a signal for the endocytic pathway. E3 ubiquitin ligases such as Cbl-b and Itch have been implicated in T cell activation and tolerance induction. However, the underlying mechanisms remain unclear. We describe that in mice deficient in the E3 ubiquitin ligases Cbl-b and Itch, T cell activation was augmented, accompanied by spontaneous autoimmunity. The double-mutant T cells exhibited increased phosphorylation of the T cell receptor-zeta (TCR-zeta) chain, whereas the endocytosis and stability of the TCR complex were not affected. TCR-zeta was polyubiquitinated via a K33-linkage, which affected its phosphorylation and association with the zeta chain-associated protein kinase Zap-70. The juxtamembrane K54 residue in TCR-zeta was identified to be a primary ubiquitin conjugation site, whose mutation increased its phosphorylation and association of TCR-zeta and Zap-70. Thus, the present study reveals unconventional K33-linked polyubiquitination in nonproteolytic regulation of cell-surface-receptor-mediated signal transduction.

Long-term effects of engineered nanoparticles on enzyme activity and functional bacteria in wastewater treatment plants.

The pervasive use of engineered nanoparticles (NPs) in a wide range of fields raises concerns about their potential environmental impacts. Previous studies confirmed that some NPs had already entered wastewater treatment plants (WWTPs). Wastewater nutrient removal depends on the metabolisms of activated sludge bacteria and their related key enzymes. Therefore, this study compared the possible influences of Al2O3, SiO2, TiO2, and ZnO NPs on the key enzymes activities and microbial community structures involved in wastewater treatment facilities. It was found that long-term exposure to these NPs significantly affected the microbial communities and changed the relative abundances of key functional bacteria, such as ammonia-oxidizing bacteria. Also, the gene expressions and catalytic activities of essential enzymes, such as ammonia monooxygenase, nitrite oxidoreductase, nitrate reductase, and nitrite reductase, were decreased, which finally resulted in a lower efficiency of biological nitrogen removal.

Effects of titanium dioxide and zinc oxide nanoparticles on methane production from anaerobic co-digestion of primary and excess sludge.

Anaerobic co-digestion of primary and excess sludge is regarded as an efficient way to reuse sludge organic matter to produce methane. In this study, short-term and long-term exposure experiments were conducted to investigate the possible effects of titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) on methane production from anaerobic co-digestion of primary and excess sludge. The data showed that TiO2 NPs had no measurable impact on methane production, even at a high concentration (150 mg/g total suspended solids (TSS)). However, short-term (8 days) exposure to 30 or 150 mg/g-TSS of ZnO NPs significantly decreased methane production. More importantly, these negative effects of ZnO NPs on anaerobic sludge co-digestion were not alleviated by increasing the adaptation time to 105 days. Further studies indicated that the presence of ZnO NPs substantially decreased the abundance of methanogenic archaea, which reduced methane production. Meanwhile, the activities of some key enzymes involved in methane production, such as protease, acetate kinase, and coenzyme F420, were remarkably inhibited by the presence of ZnO NPs, which was also an important reason for the decreased methane production. These results provide a better understanding of the potential risks of TiO2 and ZnO NPs to methane production from anaerobic sludge co-digestion.

N2O recovery from wastewater and flue gas via microbial denitrification: Processes and mechanisms.

Nitrous oxide (N2O) is increasingly regarded as a significant greenhouse gas implicated in global warming and the depletion of the ozone layer, yet it is also recognized as a valuable resource. This paper comprehensively reviews innovative microbial denitrification techniques for recovering N2O from nitrogenous wastewater and flue gas. Critical analysis is carried out on cutting-edge processes such as the coupled aerobic-anoxic nitrous decomposition operation (CANDO) process, semi-artificial photosynthesis, and the selective utilization of microbial strains, as well as flue gas absorption coupled with heterotrophic/autotrophic denitrification. These processes are highlighted for their potential to facilitate denitrification and enhance the recovery rate of N2O. The review integrates feasible methods for process control and optimization, and presents the underlying mechanisms for N2O recovery through denitrification, primarily achieved by suppressing nitrous oxide reductase (Nos) activity and intensifying competition for electron donors. The paper concludes by recognizing the shortcomings in existing technologies and proposing future research directions, with an emphasis on prioritizing the collection and utilization of N2O while considering environmental sustainability and economic feasibility. Through this review, we aim to inspire interest in the recovery and utilization of N2O, as well as the development and application of related technologies.

EV-COMM: A database of interspecies and intercellular interactions mediated by extracellular vesicles.

Intra- and inter-organismal interactions play a crucial role in the maintenance and function of individuals, as well as communities. Extracellular vesicles (EVs) have been identified as effective mediators for the communication both within and between species. They can carry and transport molecular cargoes to transmit biological messages. Several databases (ExoBCD, ExoCarta, EVpedia, EV-TRACK, Vesiclepedia) complied the cargoes information including DNA, RNA, protein, lipid and metabolite associated with EVs. Databases that refer to the complete records on both donor and recipient information are warranted to facilitate the understanding of the interaction across cells and species. In this study, we developed a database that compiled the records equipped with a structured process of EV-mediated interaction. The sources of donor and recipient were classified by cell type, tissues/organs and species, thus providing an extended knowledge of cell-cell, species-species interaction. The isolation and identification methods were presented for assessing the quality of EVs. Information on functional cargoes was included, where microRNA was linked to a prediction server to broaden its potential effects. Physiological and pathological context was marked to show the environment where EVs functioned. At present, a total of 1481 data records in our database, including 971 cell-cell interactions belonging to more than 40 different tissues/organs, and 510 cross-species records. The database provides a web interface to browse, search, visualize and download the interaction records. Users can search for interactions by selecting the context of interest or specific cells/species types, as well as functional cargoes. To the best of our knowledge, the database is the first comprehensive database focusing on interactions between donor and recipient cells or species mediated by EVs, serving as a convenient tool to explore and validate interactions. The Database, shorten as EV-COMM (EV mediated communication) is freely available at http://sdc.iue.ac.cn/evs/list/ and will be continuously updated.

Efficient nitrate and Cr(VI) removal by denitrifier: The mechanism of S. oneidensis MR-1 promoting electron production, transportation and consumption.

When Cr(VI) and nitrate coexist, the efficiency of both bio-denitrification and Cr(VI) bio-reduction is poor because chromate hinders bacterial normal functions (i.e., electron production, transportation and consumption). Moreover, under anaerobic condition, the method about efficient nitrate and Cr(VI) removal remained unclear. In this paper, the addition of Shewanella oneidensis MR-1 to promote the electron production, transportation and consumption of denitrifier and cause an increase in the removal of nitrate and Cr(VI). The efficiency of nitrate and Cr(VI) removal accomplished by P. denitrificans as a used model denitrifier increased respectively from 51.3% to 96.1% and 34.3% to 99.8% after S. oneidensis MR-1 addition. The mechanism investigations revealed that P. denitrificans provided S. oneidensis MR-1 with lactate, which was utilized to secreted riboflavin and phenazine by S. oneidensis MR-1. The riboflavin served as coenzymes of cellular reductants (i.e., thioredoxin and glutathione) in P. denitrificans, which created favorable intracellular microenvironment conditions for electron generation. Meanwhile, phenazine promoted biofilm formation, which increased the adsorption of Cr(VI) on the cell surface and accelerated the Cr(VI) reduction by membrane bound chromate reductases thereby reducing damage to other enzymes respectively. Overall, this strategy reduced the negative effect of chromate, thus improved the generation, transportation, and consumption of electrons. SYNOPSIS: The presence of S. oneidensis MR-1 facilitated nitrate and Cr(VI) removal by P. denitrificans through decreasing the negative effect of chromate due to the metabolites' secretion.

Disinfectant polyhexamethylene guanidine triggered simultaneous efflux pump antibiotic- and metal-resistance genes propagation during sludge anaerobic digestion.

The environmental transmission of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) exerted devastating threats to global public health, and their interactions with other emerging contaminants (ECs) have raised increasing concern. This study investigated that the abundances of ARGs and MRGs with the predominant type of efflux pump were simultaneously increased (8.4-59.1%) by disinfectant polyhexamethylene guanidine (PHMG) during waste activated sludge (WAS) anaerobic digestion. The aggregation of the same microorganisms (i.e., Hymenobacter and Comamonas) and different host bacteria (i.e., Azoarcus and Thauera) were occurred upon exposure to PHMG, thereby increasing the co-selection and propagation of MRGs and ARGs by vertical gene transfer. Moreover, PHMG enhanced the process of horizontal gene transfer (HGT), facilitating their co-transmission by the same mobile genetic elements (20.2-223.7%). Additionally, PHMG up-regulated the expression of critical genes (i.e., glnB, trpG and gspM) associated with the HGT of ARGs and MRGs (i.e., two-component regulatory system and quorum sensing) and exocytosis system (i.e., bacterial secretion system). Structural equation model analysis further verified that the key driver for the simultaneous enrichment of ARGs and MRGs under PHMG stress was microbial community structure. The study gives new insights into the aggravated environmental risks and mechanisms of ECs in sludge digestion system, providing guidance for subsequent regulation and control of ECs.