Viral Communities Contribute More to the Lysis of Antibiotic-Resistant Bacteria than the Transduction of Antibiotic Resistance Genes in Anaerobic Digestion Revealed by Metagenomics.

Ecological role of the viral community on the fate of antibiotic resistance genes (ARGs) (reduction vs proliferation) remains unclear in anaerobic digestion (AD). Metagenomics revealed a dominance of Siphoviridae and Podoviridae among 13,895 identified viral operational taxonomic units (vOTUs) within AD, and only 21 of the vOTUs carried ARGs, which only accounted for 0.57 ± 0.43% of AD antibiotic resistome. Conversely, ARGs locating on plasmids and integrative and conjugative elements accounted for above 61.0%, indicating a substantial potential for conjugation in driving horizontal gene transfer of ARGs within AD. Virus-host prediction based on CRISPR spacer, tRNA, and homology matches indicated that most viruses (80.2%) could not infect across genera. Among 480 high-quality metagenome assembly genomes, 95 carried ARGs and were considered as putative antibiotic-resistant bacteria (pARB). Furthermore, lytic phages of 66 pARBs were identified and devoid of ARGs, and virus/host abundance ratios with an average value of 71.7 indicated extensive viral activity and lysis. The infectivity of lytic phage was also elucidated through laboratory experiments concerning changes of the phage-to-host ratio, pH, and temperature. Although metagenomic evidence for dissemination of ARGs by phage transduction was found, the higher proportion of lytic phages infecting pARBs suggested that the viral community played a greater role in reducing ARB numbers than spreading ARGs in AD.

Response of prokaryotic, eukaryotic and algal communities to heavy rainfall in a reservoir supplied with reclaimed water.

The global climate change made the heavy rainfall happen more frequently, and the non-point source pollution caused by it would exacerbate the risk to the water ecological environment. In this study, we took a reservoir (Shahe reservoir, Beijing, China) supplied with reclaimed water as an exapmle to investigate how spatiotemporal changes in the quantity and diversity of prokaryotic, eukaryotic, and algal communities respond to heavy rainfall. Results showed that heavy rainfall could directly impact the composition of the prokaryotic community by introducing amounts of runoff closely associated bacterium especially for the human potential pathogens such as Aliarcobacter, Aeromonas and Pseudomonas in the Shahe reservoir area. While the eukaryotic community was rather stable, and the development and changes in algal communities occurred in the last few days after heavy rainfall. The microbial source tracking through FEAST indicated that Nansha river (S) was the major contributor to the development of all the three concerned communities in the reservoir. The co-occurrence analysis showed that the modules with the highest cumulative abundance in each community were all strongly and positively connected with Chl-a, pH, turbidity, COD and TOC, but negatively correlated with NO3-N (p < 0.01). The network analysis showed that the eukaryotes played a key role in the interaction network among the three communities, and were more likely to interact with algae and prokaryotes. It was suggested that the controlling of human potential pathogens associated with prokaryotic community should be emphasized at the beginning of the heavy rainfall, but the prevention of the eutrophication bloom should be another focus after the heavy rainfall. This study provided valuable information concerning the role of heavy rainfall on the water ecological environment from the perspective of microbial community.

Ferric chloride further simplified the horizontal gene transfer network of antibiotic resistance genes in anaerobic digestion.

The role of ferric chloride (FC) on the reduction of antibiotic resistance genes (ARGs) in anaerobic digestion (AD) system was investigated from the perspective of vertical (VGT) and horizontal gene transfer (HGT) network through the high-throughput qPCR (HT-qPCR). Although FC showed limited impacts on methane production in AD of swine manure, the tetracycline and MLSB resistance genes were specifically reduced at the end, where tetQ of antibiotic target protection and ermF of antibiotic target alteration contributed the most to the reduction. Both VGT and HGT network were divided into three modules, and the complexity of HGT network was largely reduced along with AD, where the HGT connection was reduced from 683 (Module III) to 172 (Module I), and FC addition could further reduce the relative abundance of ARG hosts in Module I. The contribution of VGT and HGT to the changes of ARGs in AD was further deciphered, and although the VGT reflected by the changes of microbial community contributed the most to the dynamics of ARGs (68.0 %), the HGT contribution could further be reduced by the FC addition. This study provided a new perspective on the fate of ARGs response to the FC addition in the AD system.

Metagenomics revealed the mobility and hosts of antibiotic resistance genes in typical pesticide wastewater treatment plants.

Pesticide showed a crucial selective pressure of antibiotic resistance genes (ARGs) in the environmental dimension, especially in the pesticide wastewater treatment process, where the information on the mobility and hosts of ARGs was very important but limited. This study tried to clarify the mobile antibiotic resistome and ARG hosts in three typical pesticide wastewater treatment plants (PWWTPs) through metagenomics. Results showed that ARGs associated with antibiotic efflux and multi-drug resistance generally dominated in the PWWTPs, and the relative abundance of ARGs was generally higher in the water phase than that in sludge phase. The mobile antibiotic resistome accounted for 43.6% ± 16.2% and 44.8% ± 18.0% of the total relative abundance of ARGs in the water phase and sludge phase, respectively. The tnpA, IS91 and intI1 were the dominant mobile genetic elements (MGEs) closely associated with ARGs. MCR-5 and MCR-9 were first identified in the PWWTPs and located together with the tnpA, tnpA2 and int2. The potential human pathogens belonging to Citrobacter, Pseudomonas, Enterobacter, Acinetobacter, and Kluyvern were the major ARG hosts in the PWWTPs. Statistical analysis indicated that microbial community contributed the most to the occurrence of antibiotic resistome, and the reduction of the major ARG hosts was crucial from the perspective of ARGs control.

Coupled mechanism of enhanced and inhibitory effects of nanoscale zero-valent iron on methane production and antibiotic resistance genes in anaerobic digestion of swine manure.

In this study, the turning point for nanoscale zero-valent iron's (NZVI) promotion and inhibition effects of methane production coupled with the reduction of antibiotic resistance genes (ARGs) was investigated. Adding 150 mmol/L NZVI increased methane production by maximum of 23.8 %, which was due to the chemical reaction producing H2 and enhancement of direct interspecies electron transfer (DIET) by NZVI. NZVI350 dramatically repressed methane generation by 48.0 %, which might be associated with the large quantity of reactive oxygen species (ROS) and excessive H2 inhibiting the functioning of microorganisms. The fate of ARGs was significantly related to daily methane production, indicating that the more methane production finally generated, the less the abundance of ARGs at last left. The reduction of ARGs was enhanced by maximum of 61.0 %, which was attributed to the inhibition of vertical gene transfer (VGT) and horizontal gene transfer (HGT) caused by steric hindrance associated with NZVI corrosion.

Zero valent iron improved methane production and specifically reduced aminoglycoside and tetracycline resistance genes in anaerobic digestion.

It is unadvisable to discuss the antibiotic resistance genes (ARGs) reduction in anaerobic digestion (AD) system neglecting its main purpose-methane production. The methane production improvement coupling with antibiotic resistance genes (ARGs) reduction in anaerobic digestion (AD) by zero valent iron (ZVI) were simultaneously investigated. Whether the role of ZVI on the ARGs fate was random or specific was clarified through the high-throughput qPCR (HT-qPCR). Results indicated that ZVI improved methane production and ARGs reduction by 23.9% and 25.0%, respectively. The improved methane production was associated with chemical reaction and variances of microbial community caused by ZVI, where DIET between Petrimonas, Clostridium and Syntrophomonas, Methanosarcina was established along with ACAS being enriched. ZVI specifically, not randomly, facilitated the reduction of aminoglycoside resistance genes of antibiotic inactivation and tetracycline resistance genes of ribosomal protection proteins. The specifical reduction could be attributed to enzyme activity inhibition and intracellular ionic disturbance caused by higher amounts of ZVI, although most of ARGs fate could be well explained by microbial community which contributed the most to ARGs dynamics as a whole. ZVI-based AD was a promising way for the improvement of methane production coupling ARGs reduction.

Enhancement of methane production and antibiotic resistance genes reduction by ferrous chloride during anaerobic digestion of swine manure.

In this study, effects of ferrous chloride (FeCl2) addition on methane production and antibiotic resistance genes (ARGs) reduction were investigated during anaerobic digestion (AD) of swine manure. FeCl2 could both improve the accumulative methane production and reduce the abundance of total ARGs, i.e., the maximum increase of CH4 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 H2 utilization and direct interspecies electron transfer (DIET), where DIET was further enhanced by the reaction of the FeCl2 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, FeCl2-based AD is a feasible and attractive way to improve methane production and ARG reduction.

Effects of ferric oxide on the microbial community and functioning during anaerobic digestion of swine manure.

Iron-based materials have been suggested as environmentally-friendly additives that can enhance methane production during anaerobic digestion (AD). In this study, the effects of ferric oxide (Fe2O3) addition on methane production were investigated during swine manure AD. In addition, the effects of Fe2O3 addition on the AD ternary pH buffer system and microbial community were evaluated. Fe2O3 could improve the accumulative methane production by maximum 11.06% when adding 75 mmol of Fe2O3. Higher methane production could be attributed to the enhancement of direct interspecies electron transfer (DIET) and the formation of Fe-S precipitates, but not the addition of Fe2O3 as a nutrient. Furthermore, Fe2O3 addition enhanced methanogenesis rather than acetogenesis, as evinced by analysis of functional genes. Nevertheless, high-throughput sequence analysis of microbial community composition revealed the lack of a significant influence by Fe2O3 addition, and Fe2O3 addition did not significantly affect the ternary pH buffer system.

Effects of magnetite on anaerobic digestion of swine manure: Attention to methane production and fate of antibiotic resistance genes.

Effects of magnetite on methane production and fate of antibiotic resistance genes (ARGs) during anaerobic digestion (AD) of swine manure were investigated. Results showed that methane production was increased by maximum 16.1%, and magnetite could enhance the acetoclastic methanogenesis not hydrogenotrophic methanogenesis reflected by the functional gene quantification and microbial community analysis. The propionate degradation rate was improved, and it was syntrophic oxidized into H+/e-/CO2 for direct interspecies electron transfer (DIET) and acetate, where DIET was further enhanced by magnetite and the acetate was transformed into methane through syntrophic acetate oxidation (SAO) pathway. Magnetite mainly influenced the ARGs at the interim period of AD, where ARGs especially ermF were significantly enriched. Magnetite did not influence the total ARGs abundance at the end, although the tetM was enriched and mefA was reduced finally. Statistical analysis indicated that magnetite influenced the ARGs fate mainly through the changes of microbial community.

Fate of antibiotic resistance genes during anaerobic digestion of sewage sludge: Role of solids retention times in different configurations.

In this study, three anaerobic digestion experiments were established to investigate the effects of solids retention times (SRT) on the fate of antibiotic resistance genes (ARGs) including anaerobic digestion of sewage sludge (CK), one-stage anaerobic digestion of microwave pretreatment sludge (MW) and two-stage anaerobic digestion of microwave pretreatment sludge (Acid stage and CH4 stage). The response of ARGs to the SRT varied significantly from ARG types and reactor configurations. Shorter SRT could avail the ARGs reduction for CK and two-stage digestion, while MW need longer SRT for the ARGs reduction. Concerning the variance of microbial community caused by reactor configurations, the role of SRT was limited. The partial redundancy analysis and structural equation models analysis indicated that the role of SRT on the ARGs fate could be attributed the most to the co-selection from heavy metals.

Response and mechanisms of the performance and fate of antibiotic resistance genes to nano-magnetite during anaerobic digestion of swine manure.

Swine manure is an important reservoir of environmental antibiotic resistance genes (ARGs), and anaerobic digestion (AD) is a commonly used method for swine manure treatment. In this study, the optimized dosage of nano-magnetite to enhance methane production was figured out, the changes of the fate of ARGs response to nano-magnetite were investigated, and the microbial mechanisms were deciphered through the microbial community analysis and key functional genes quantification. Results showed that nano-magnetite could improve the methane production by maximum 6.0%, the maximum daily methane production could be increased by 47.8%, and the AD time could be shortened by above 20.0% at the addition of 75 mmol. The improved performance could be associated with the enhancement of direct interspecies electron transfer (DIET) and the inhibition release due to the formation of Fe-S precipitation not the nutrition elements role of nano-magnetite, and nano-magnetite did not significantly influence the dynamics of microbial community. Nano-magnetite could enhance the methanogenesis instead of the acetogenesis reflected by the functional genes analysis, and the limited effects of nano-magnetite on the fate of ARGs could be associated with its limited influence on the microbial community which determined the fate of ARGs during AD of swine manure.

Which animal type contributes the most to the emission of antibiotic resistance genes in large-scale swine farms in China?

Modern swine farms generally contain several animal types and rely extensively on the feed additives, including antibiotics and heavy metals, to augment animal growth. Nonetheless, as an important reservoir of antibiotic resistance genes (ARGs) in the environment, the ARGs emission of each animal type from swine farms has not been characterized. The goal of this study is to determine which animal type contributes the most to the ARGs emission into the environment in typical swine farms of China. Results showed that chlortetracycline (CTC), Cu and Zn were the typical feed additives, and the concentrations of antibiotics and heavy metals in the feed and swine manure were generally higher in nursery pigs (NP) than other animal types, while the gene copies of ARGs from gestation sows (GS) were the most abundant. GS released the most of antibiotics, ARGs and mobile genetic elements (MGEs) per head per day compared to other animal types. A typical swine farms with the feedstock of 10,000 pigs could release about 4.0±1.3×1017 gene copies of ARGs per day, and concerning the breeding ratio and manure production coefficient, growing and fattening pigs (GFP) released the most of ARGs and antibiotics, whereas gestation sows (GS) released the most of MGEs. The different distribution of ARGs in different animal types was mainly determined by the discrepancy of microbial community composition reflected by mantel test and partial redundancy analysis (pRDA). The dominant phylum in swine manure were Firmicutes and Bacteroidetes, but Proteobacteria, Bacteroidetes and Spirochaetae played the dominant role in shaping the ARGs profiles. Antibiotics and heavy metals could have generated and maintained the ARGs profiles, whereas the proliferation and spread of ARGs could be mainly attributed to microbial community in swine manure.

Sludge bio-drying followed by land application could control the spread of antibiotic resistance genes.

The process of sludge bio-drying has been adopted in response to the increasing amount of residual sewage sludge. It has been demonstrated that sludge bio-drying effectively reduces both antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), whereas ermF, tetX, and sulII become enriched in response to the dynamic development of the microbial community. The present study further demonstrated that the land application of sludge bio-drying products under current application rate did not cause an increase in the abundance of quantified ARGs in the soil but the persistence of ARB should be paid attention. Although land application introduced ermF, tetX, and tetG into the soil, these soon decreased to control levels. Furthermore, the decay rate varied between soil types, with red soil being the most persistent based on kinetics modeling. The fate of ARGs could also be attributed to the dynamics of the microbial community during land application, and the genus Parasegetibacter, which can degrade extracellular DNA, might play a key role in the control of ARGs. In summary, sludge bio-drying following land application could constitute an effective means of controlling the spread of ARGs, and microbial community changes contributed the most to the fate of the ARGs during the entire treatment chain (residual sewage sludge → bio-drying process → land application).

The role of substrate types and substrate microbial community on the fate of antibiotic resistance genes during anaerobic digestion.

Anaerobic digestion (AD) is regarded as a promising technology in energy recovery and the spread mitigation of antibiotic resistance. However, the performance of AD is dependent on various factors, and substrate type is one of the most important. In this study, the fate of antibiotic resistance genes (ARGs) response to the substrate types was investigated, and three typical environmental reservoirs of ARGs (pig manure, chicken manure and sewage sludge) were selected. The role of substrate microbial community on the fate of ARGs was clarified through the comparison between the AD of the substrates with and without a prior autoclave-disinfected step. Results showed that substrate types significantly influenced the fate of ARGs, while the influence from the substrate microbial community was limited. The concentration of antibiotics, the horizontal gene transfer reflected by intI1 and co-selection from heavy metals reflected by metal resistance genes (MRGs) were all reduced effectively. Microbial community varied from substrate types and dominated the ARGs fate concerning the standardized total effects through the mantel test and SEM analysis. The fate of tetX, ermF, tetM and ermB was mainly determined by the physicochemical parameters and the phyla of Firmicutes and Bacteroides. The phyla of Actinobacteria, pcoA and czcA contributed most to the reduction of blaTEM and mcr-1, and the phyla of Proteobacteria, Chloroflexi, Synergistetes, Euryarchaeote, intI1 and merA correlated significantly with the fate of blaCTX-M, ereA, tetG and sulI. This study highlighted the importance of substrate types when considering the fate of ARGs during AD.

Enhancement of surfactant biodegradation with an anaerobic membrane bioreactor by introducing microaeration.

In controlled ecological life support system (CELSS), 100% water recycle efficiency is needed. Both water and nutrients in wastewater needs reclaiming. In an anaerobic membrane bioreactor (AnMBR) designed to hydrolyze organic matters and convert organic nitrogen, accumulation of anionic surfactants and organic compounds was observed in the supernatant. To solve the problem, microaeration which had rarely been reported to enhance surfactants biodegradation in anaerobic process was introduced and it was proved to be an effective approach that resulted in concentrations of surfactants and soluble chemical oxygen demand (SCOD) in the supernatant decreasing from 9000 mg/L and 40000 mg/L to 2000 mg/L and 10000 mg/L, respectively. And the degradations followed the kinetic characteristics of zero order reaction with R2 values of 0.9472 and 0.949. Three-dimensional excitation emission matrix (3D-EEM) analysis revealed that some activities of microbes were activated and enhanced by microaeration, which resulted in the disintegration of aggregates of large molecules, indicated by the size exclusion chromatography (SEC). After the introduction of microaeration, the emerging genera Aquamicrobium, Flaviflexus, Pseudomonas and Thiopseudomonas in the microbial community might be responsible for the effective biodegradation of the surfactants.