Fate of antibiotic resistance genes in sewage treatment plant revealed by metagenomic approach

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have attracted great concerns worldwide. Wastewater treatment plants (WWTPs) are reservoirs of ARGs while wastewater/sludge treatment processes are considered as important means to control these emerging biological pollutants. However, the full profiles of ARGs in WWTPs or the removal efficiency of ARGs by wastewater/sludge treatment process was not well characterized yet. Thus, the major tasks in this study were (1) to reveal the broad-spectrum profiles of ARGs in WWTPs; (2) to investigate the removal efficiencies of ARGs using wastewater/sludge treatment process; (3) to examine the differences and similarities of ARGs in samples from WWTPs and other environments; (4) to develop an efficient platform for examination on the broad-spectrum profiles of environmental ARGs through metagenomic analysis.
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\nActivated sludge is an important segment for harboring ARGs and horizontal gene transfer of ARGs among different microbial communities. Existence of six-lactam resistance genes were found through PCR and qPCR approaches in activated sludge collected from 15 WWTPs in East Asia and North America. Abundances of these resistance genes ranged from 5.34×101 copies/ng DNA to 5.49×104 copies/ng DNA. Broad-spectrum profiles of ARGs in activated sludge from a local WWTP in a period of four years were investigated through metagenomic analysis. A total of 16 ARGs types were detected. During the 4-year period, a descending trend of total ARGs abundance was found but the abundances of _-lactam and quinolone resistance genes increased. Moreover, resistance genes of tetracycline, sulfonamide and vancomycin showed seasonal variation.
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\nMetagenomic analysis was also applied to study the fate of ARGs in wastewater/sludge treatment processes in a full-scale municipal WWTP. Results suggested that 99.82% of ARGs in influent could be removed by wastewater treatment process while only 20.70% of ARGs could be removed in sludge anaerobic digestion. The distribution of ARGs in WWTP was significantly correlated with six genera and five of which included potential pathogens.
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\nEffect of temperature on ARGs removal from activated sludge by anaerobic digestion was examined through metagenomic analysis. No significant change was found for the removal efficiency of total ARGs using bench-scale thermophilic or mesophilic anaerobic digestion reactor. But results suggested that more ARGs subtypes can be effectively removed by anaerobic digestion under mesophilic condition.
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\nARGs profiles in 10 typical environments, including different sections in WWTPs, river water, drinking water, soils, sediments and faeces from livestock and human, were revealed through metagenomic analysis. Generally the total ARGs abundances in different environments increased with the impacts of anthropogenic activities. Co-occurrence of ARGs subtypes in different environments was studied using network analysis, tetM and aminoglycoside resistance genes were recognized as the indicators to estimate the abundances of other co-occurred ARGs subtypes.
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\nThe processes in metagenomic analysis were improved to shorten the analysis time and simplify the classification of ARGs-like sequences. Error and redundant sequences were removed from the widely used reference database of ARGs. A structured database was then constructed to realize the automatic classification of ARGs in metagenomic data. A hybrid alignment process was optimized for rapid ARGs or other specific functional genes annotation as well.

It is becoming clear that the wastewater treatment plants (WWTPs) is a key repository for antibiotic resistance genes (ARGs), and WWTPs also represent an important node for limiting ARGs spreading via altering the magnitude and distribution of ARGs in environment. This review provides an overview of the ARGs degradation performance both in sewage sludge and wastewater in WWTPs. The ARGs reduction behaviors during sludge treatment processes including anaerobic digestion (AD), composting, aerobic digestion, bio-drying and air-drying were reviewed, and parameters affecting ARGs reducing efficiency and the probable mechanisms were concluded. In particular, pretreatment approaches such as microwave, thermal hydrolysis and ozone before sludge AD are found to provide the extra benefits for ARGs removal. Additionally, ARGs fates and reduction mechanisms during sewage biological treatments such as anaerobic, aerobic, anaerobic combined with aerobic process and physical-chemical treatment including coagulation, advanced oxidation and disinfection processes were appraised. Furthermore, problems or doubts of previous study are proposed, and some in-depth mechanism researches are anticipated to elucidate the ARGs decay in sewage and sludge treatment process. This can help further enhance the ARGs removal efficiency in WWTPs.

Distribution of the microbial community and antibiotic resistance genes in farmland surrounding gold tailings: A metagenomics approach.

The prevalence and persistence of antibiotic resistance genes in wastewater treatment plants (WWTPs) is of growing interest, and residual sludge is among the main sources for the release of antibiotic resistance genes (ARGs). Moreover, heavy metals concentrated in dense microbial communities of sludge could potentially favor co-selection of ARGs and metal resistance genes (MRGs). Residual sludge treatment is needed to limit the spread of resistance from WWTPs into the environment. This study aimed to explore the fate of ARGs and MRGs during thermophilic two-phase (acidogenic/methanogenic phase) anaerobic digestion by metagenomic analysis. The occurrence and abundance of mobile genetic elements were also determined based on the SEED database. Among the 27 major ARG subtypes detected in feed sludge, large reductions (> 50%) in 6 ARG subtypes were achieved by acidogenic phase (AP), while 63.0% of the ARG subtypes proliferated in the following methanogenic phase (MP). In contrast, a 2.8-fold increase in total MRG abundance was found in AP, while the total abundance during MP decreased to the same order of magnitude as in feed sludge. The distinct dynamics of ARGs and MRGs during the two-phase anaerobic digestion are noteworthy, and more specific treatments are required to limit their proliferation in the environment.

The overuse of antibiotics in livestock farms is general, leading to a wide distribution of antibiotic resistance genes (ARGs) in aquatic environment adjacent to livestock farms. However, researches of the distribution and types of ARGs in aquatic environment of China are still in the initial stage. In this study, wastewater and surface water samples were collected from 12 livestock farms (four pig farms, four cattle farms, and four chicken farms) in Jiangsu Province of China. The prevalence, abundance, and distribution of 22 ARGs were investigated, which were categorized into six groups, including nine tetracyclin resistance genes, three sulfonamides resistance genes, three quinolone resistance genes, two macrolide resistance genes, three aminoglycoside resistance genes, and two multidrug resistance genes, employing quantitative real-time PCR (qPCR). The results suggested that all of the 22 ARGs were detected in samples. Sul1, sul2, and tetM were the most abundant with the average concentration of 3.84 × 10(1) copies/16S recombinant RNA (rRNA) gene copies, 1.62 × 10(1) copies/16S rRNA gene copies, 2.33 × 10(1) copies/16S rRNA gene copies, respectively. Principle component analysis revealed that the comprehensive pollution of ARGs in northern Jiangsu was more serious. ARGs in wastewater were more abundant when compared to that in surface water. A preliminary study regarding the fate of ARGs after an aerobiotic process showed that tetA, tetC, sul1, sul2, oqxB, and qnrS were significantly increased. And, among the tetracycline resistance genes, the efflux pump genes were enriched while the ribosomal protection protein encoding genes were decreased in the aerobiotic process. The prevalance of ARGs in water environment is of concern; more surveillance is required to determine the pollution level and pattern of antibiotic resistance genes.

Microplastics deteriorate the removal efficiency of antibiotic resistance genes during aerobic sludge digestion

Contrasting dynamics of manure-borne antibiotic resistance genes in different soils

Distribution of antibiotic resistance genes in soil amended using Azolla imbricata and its driving mechanisms.

Sewage sludge in the wastewater treatment plants contains considerable amount of antibiotic resistance genes (ARGs). A few studies have reported that anaerobic digestion (AD) could successfully remove some ARGs from sewage sludge, but information on the fate of ARGs in sludge pretreatment-AD process is still very limited. In this study, three sludge pretreatment methods, including alkaline, thermal hydrolysis and ultrasonic pretreatments, were compared to investigate the distribution and removal of ARGs in the sludge pretreatment-AD process. Results showed that the ARGs removal efficiency of AD itself was approximately 50.77%, and if these three sludge pretreatments were applied, the total ARGs removal efficiency of the whole pretreatment-AD process could be improved up to 52.50%–75.07%. The ultrasonic pretreatment was more efficient than alkaline and thermal hydrolysis pretreatments. Although thermal hydrolysis reduced ARGs obviously, the total ARGs rebounded considerably after inoculation and were only removed slightly in the subsequent AD process. Furthermore, it was found that the total ARGs concentration significantly correlated with the amount of 16S rRNA gene during the pretreatment and AD processes, and the bacteria carrying ARGs could be mainly affiliated with Proteobacteria.

Soil type shapes the antibiotic resistome profiles of long-term manured soil

Fate, risk and sources of antibiotic resistome and its attenuation dynamics in the river water-sediment system: Field and microcosm study

Metagenomic analysis reveals the fate of antibiotic resistance genes in two-stage and one-stage anaerobic digestion of waste activated sludge

Wastewater treatment systems (WWTS) are considered to be the main source of antibiotic resistance genes (ARGs) spreading into the environment. In this study, samples were collected from WWTS influent, biological treatment tank effluent, and recycled water treatment plant (RTP) effluent during summer and winter, followed by metagenomic sequencing. The study investigated the differences in antibiotic resistance gene transfer between two typical wastewater treatment plants (WWTPs) processes and the impact of recycled water irrigation on ARG dissemination in soil. The WWTS (HD and MD) adopting two combined processes of "Adsorption-Biodegradation Process(AB )+ Anaerobic-Anoxic-Oxic Process(AAO)" and "AAO + Membrane Bioreactor(MBR)" as the research objects for the first time.The primary ARGs types identified were multidrug resistance, tetracycline, macrolide, and aminoglycoside resistance genes. The top three ARGs subtypes by relative abundance in the influent, biological treatment tank effluent, and total effluent were msrE, mphE, and ANT(6)-Ia, respectively. Seasonal variations did not significantly influence the distribution of ARGs in the two WWTSs. The AAO and AB processes increase the relative abundance and diversity of ARGs, while ARGs relative abundance decreases after RTP treatment but may proliferate new ARGs subtypes. Additionally, the efficiency of reducing the relative abundance of ARGs in summer is higher than that in winter. The two WWTSs were able to efficiently remove msrE and mphE. The abundance and diversity of ARGs and microorganisms were maximum in soil samples from the RTP. The microbial genera significantly related to ARGs may become its potential host, such as Rhodanobacter had significant correlations with ropB2, carA, and oleB. These results provide new insights into the control of ARGs contamination and focus on the risks associated with irrigated wastewater.

Antibiotic resistance genes might serve as new indicators for wastewater contamination of coastal waters: Spatial distribution and source apportionment of antibiotic resistance genes in a coastal bay

Size-dependent effects of microplastics on antibiotic resistance genes fate in wastewater treatment systems: The role of changed surface property and microbial assemblages in a continuous exposure mode