Elimination Of Antibiotic Resistance Genes Research Articles

Efficient Removal of Antibiotic Resistance Genes through 4f‐2p‐3d Gradient Orbital Coupling Mediated Fenton‐Like Redox Processes

AbstractPeroxymonosulfate (PMS) mediated radical and nonradical active substances can synergistically achieve the efficient elimination of antibiotic resistance genes (ARGs). However, enhancing interface electron cycling and optimizing the coupling of the oxygen‐containing intermediates to improve PMS activation kinetics remains a major challenge. Here, Co doped CeVO4 catalyst (Co−CVO) with asymmetric sites was constructed based on Ce 4f−O 2p−Co 3d gradient orbital coupling. The catalyst achieved approximately 2.51×105 copies/mL of extracellular ARGs (eARGs) removal within 15 minutes, exhibited ultrahigh degradation rate (k=1.24 min−1). The effective gradient 4f‐2p‐3d orbital coupling precisely regulates the electron distribution of Ce−O−Co active center microenvironment, while optimizing the electronic structure of Co 3d states (especially the occupancy of eg), promoting the adsorption of oxygen‐containing intermediates. The generated radical and nonradical generated by interfacial electron cycling enhanced by the reduction reaction of PMS at the Ce site and the oxidation reaction at the Co site achieved a significant mineralization rate of ARGs (83.4 %). The efficient removal of ARGs by a continuous flow reactor for 10 hours significantly reduces the ecological risk of ARGs in actual wastewater treatment.

Efficient Removal of Antibiotic Resistance Genes through 4f-2p-3d Gradient Orbital Coupling Mediated Fenton-Like Redox Processes.

Peroxymonosulfate (PMS) mediated radical and nonradical active substances can synergistically achieve the efficient elimination of antibiotic resistance genes (ARGs). However, enhancing interface electron cycling and optimizing the coupling of the oxygen-containing intermediates to improve PMS activation kinetics remains a major challenge. Here, Co doped CeVO4 catalyst (Co-CVO) with asymmetric sites was constructed based on Ce 4f-O 2p-Co 3d gradient orbital coupling. The catalyst achieved approximately 2.51×105 copies/mL of extracellular ARGs (eARGs) removal within 15 minutes, exhibited ultrahigh degradation rate (k = 1.24 min-1). The effective gradient 4f-2p-3d orbital coupling precisely regulates the electron distribution of Ce-O-Co active center microenvironment, while optimizing the electronic structure of Co 3d states (especially the occupancy of eg), promoting the adsorption of oxygen-containing intermediates. The generated radical and nonradical generated by interfacial electron cycling enhanced by the reduction reaction of PMS at the Ce site and the oxidation reaction at the Co site achieved a significant mineralization rate of ARGs (83.4%). The efficient removal of ARGs by a continuous flow reactor for 10 hours significantly reduces the ecological risk of ARGs in actual wastewater treatment.

Oxygen vacancy engineered molecular imprinted TiO2 for preferential florfenicol remediation by electro-reductive approach: Enhanced dehalogenation performance and elimination of antibiotic resistance genes

To achieve the precision removal of trace florfenicol (FLO) with presence of various high-concentration interferents in water matrices, an oxygen vacancy engineered molecular imprinted TiO2 (MI-TiO2−x) cathode material was customized rationally. Benefiting from the increased recognition and the accelerated interfacial charge transfer contributed by molecular imprinted sites and oxygen vacancy, respectively, MI-TiO2−x deeply dehalogenates 10 mg L−1 FLO via direct electron transfer with the degradation rate constant of 0.021 min−1. Such electrode material outperforms most of the recent electrocatalysts, and show resistance to co-existing interferents (e.g. reduced sulfur species). In an electro-reductive and biological coupling system, MI-TiO2−x removes the antibacterial activity of 2 mg L−1 FLO in swine wastewater and thus eliminates the abundance of FLO resistance genes. This study provides insights into not only rational design of the noble-metal-free electrocatalyst for preferential remediation of low-concentration organic halides, but also the significance of mitigating ecological risk by dehalogenation treatment.

Production of medium chain fatty acids from antibiotic fermentation residuals pretreated by ionizing radiation: Elimination of antibiotic resistance genes

The propagation of antibiotic resistance genes (ARGs) restricts the application of antibiotic fermentation residues (AFRs). This study investigated medium chain fatty acids (MCFA) production from AFRs, focusing on the effect of ionizing radiation pretreatment on the fates of ARGs. The results indicated that ionizing radiation pretreatment not only stimulated the MCFA production, but also inhibited the proliferation of ARGs. Radiation at 10–50 kGy decreased ARGs abundances by 0.6–21.1% at the end of fermentation process. Mobile genetic elements (MGEs) exhibited higher resistance to ionizing radiation, radiation over 30 kGy was required to suppress the proliferation of MGEs. Radiation at 50 kGy achieved an adequate inhibition to MGEs, and the degradation efficiency was 17.8–74.5% for different kinds of MGEs. This work suggested that ionizing radiation pretreatment could be a good option to ensure the safer application of AFRs by eliminating the ARGs and preventing the horizontal gene transfer of ARGs.

Degradation of Antibiotic Resistance Genes by VADER with CRISPR-Cas Immunity.

The evolution and dissemination of antibiotic resistance genes (ARGs) are prompting severe health and environmental issues. While environmental processes, e.g., biological wastewater treatment, are key barriers to prevent the spread of ARGs, they are often sources of ARGs at the same time, requiring upgraded biotechnology. Here, we present VADER, a synthetic biology system for the degradation of ARGs based on CRISPR-Cas immunity, an archaeal and bacterial immune system for eliminating invading foreign DNAs, to be implemented for wastewater treatment processes. Navigated by programmable guide RNAs, VADER targets and degrades ARGs depending on their DNA sequences, and by employing an artificial conjugation machinery, IncP, it can be delivered via conjugation. The system was evaluated by degrading plasmid-borne ARGs in Escherichia coli and further demonstrated via the elimination of ARGs on the environmentally relevant RP4 plasmid in Pseudomonas aeruginosa. Next, a prototype conjugation reactor at a 10-mL scale was devised, and 100% of the target ARG was eliminated in the transconjugants receiving VADER, giving a proof of principle for the implementation of VADER in bioprocesses. By generating a nexus of synthetic biology and environmental biotechnology, we believe that our work is not only an enterprise for tackling ARG problems but also a potential solution for managing undesired genetic materials in general in the future. IMPORTANCE Antibiotic resistance has been causing severe health problems and has led to millions of deaths in recent years. Environmental processes, especially those of the wastewater treatment sector, are an important barrier to the spread of antibiotic resistance from the pharmaceutical industry, hospitals, or civil sewage. However, they have been identified as a nonnegligible source of antibiotic resistance at the same time, as antibiotic resistance with its main cause, antibiotic resistance genes (ARGs), may accumulate in biological treatment units. Here, we transplanted the CRISPR-Cas system, an immune system via programmable DNA cleavage, to tackle the antibiotic resistance problem raised in wastewater treatment processes, and we propose a new sector specialized in ARG removal with a conjugation reactor to implement the CRISPR-Cas system. Our study provides a new angle for resolving public health issues via the implementation of synthetic biology in environmental contexts at the process level.

Inactivation efficiency of P. Aeruginosa and ARGs removal in UV/NH2Cl process: Comparisons with UV and NH2Cl

UV/NH2Cl process is an emerging combined disinfection method. This study comprehensively analyzed the inactivation of P. aeruginosa in UV/NH2Cl process, which is much better than that of UV alone or NH2Cl alone. The better inactivation performance was resulted from the intracellular reactive oxygen species (ROS) and reactive species produced in UV/NH2Cl process, including HO•, and reactive chlorine species (RCS). Except for the log reduction, inactivation of P. aeruginosa was displayed through the increase of membrane permeability, effluence and degradation of DNA and proteins, and destruction of cellular surface morphology. Also, the dark repair potential after UV alone and UV/NH2Cl processes were assessed and UV/NH2Cl process showed better performance. We also explored the effects of different factors on P. aeruginosa inactivation. The results showed that there was a negative correlation between P. aeruginosa inactivation and pH, and NH2Cl-UV got better inactivation effect than UV/NH2Cl. Besides, comparing the elimination of antibiotic resistance genes (ARGs) in UV, NH2Cl and UV/NH2Cl processes, it was found that UV/NH2Cl process was highly effective in simultaneous removal of P. aeruginosa and ARGs. Overall, our present study supplies much useful information for the potential application of UV/NH2Cl process.

Metagenomic Analysis Reveals Changes in Bacterial Communities and Antibiotic Resistance Genes in an Eye Specialty Hospital and a General Hospital Before and After Wastewater Treatment.

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in hospital wastewater poses a great threat to public health, and wastewater treatment plants (WWTPs) play an important role in reducing the levels of ARB and ARGs. In this study, high-throughput metagenomic sequencing was used to analyze the bacterial community composition and ARGs in two hospitals exposed to different antibiotic use conditions (an eye specialty hospital and a general hospital) before and after wastewater treatment. The results showed that there were various potential pathogenic bacteria in the hospital wastewater, and the abundance and diversity of the influent ARGs in the general hospital were higher than those in the eye hospital. The influent of the eye hospital was mainly composed of Thauera and Pseudomonas, and sul1 (sulfonamide) was the most abundant ARG. The influent of the general hospital contained mainly Aeromonas and Acinetobacter, and tet39 (tetracycline) was the most abundant ARG. Furthermore, co-occurrence network analysis showed that the main bacteria carrying ARGs in hospital wastewater varied with hospital type; the same bacteria in wastewater from different hospitals could carry different ARGs, and the same ARG could also be carried by different bacteria. The changes in the bacterial community and ARG abundance in the effluent from the two hospitals showed that the activated sludge treatment and the direct chlorination disinfection can effectively remove some bacteria and ARGs in wastewater but have limitations. The species diversity increased significantly after the activated sludge treatment, while the direct chlorination disinfection did not increase the diversity. The activated sludge treatment has a better effect on the elimination of ARGs than the direct chlorination disinfection. In summary, we investigated the differences in bacterial communities and ARGs in wastewater from two hospitals exposed to different antibiotic usage conditions, evaluated the effects of different wastewater treatment methods on the bacterial communities and ARGs in hospital wastewater, and recommended appropriate methods for certain clinical environments.

Simultaneous elimination of antibiotics resistance genes and dissolved organic matter in treatment wetlands: Characteristics and associated relationship

Constructed wetlands (CWs) are widely used for treating swine wastewater containing antibiotic resistance genes (ARGs). However, the relation between ARGs removal and dissolved organic matter (DOM) characteristics in CWs remains unknown. This study aimed to assess the removal of ARGs and the dynamitic transformation of DOM from anaerobically-digested swine wastewater in biochar-based CWs with and without forced aeration. Incorporating biochar and aeration into CWs could promote the ARGs (tetA, tetM, tetO, tetW) elimination (0.53–2.14 log) under various influent strengths. Compared with non-aerated CWs, aerated CWs improved the target ARGs removal, while biochar-based CWs without aeration increased the proliferation of ARGs (tetA, tetO, tetW). More accumulation of ARGs was found especially in non-aerated CWs with biochar. Fluorescence excitation-emission spectrophotometry with parallel factor analysis revealed that DOM, mainly including humic-like, protein-like and tryptophan-like components, was significantly reduced with a higher removal efficiency (72.2%) in biochar-based CWs with aeration. Moreover, DOM removal showed an obvious correlation with the elimination of ARGs, except tetW, which might not be influenced by the humification processes. The evolution of microbial communities in different CWs indicated that Actinobacteria, Proteobacteria and Bacteroidetes might play an important role in ARGs and DOM removal. This study could provide new insights on the mechanism of ARGs and DOM elimination by biochar-based CWs with aeration for treating swine wastewater.

Elimination of antibiotic resistance genes from excess activated sludge added for effective treatment of fruit and vegetable waste in a novel vermireactor

Elimination of antibiotic resistance genes (ARGs) from excess activated sludge (EAS) mixed for effective treatment of different fruit and vegetable waste (FVW) by using a novel vermireactor consisted of substrate and bed compartments was investigated. ARGs (tet G, tet M and sul 1) and mobile genetic element gene (intl 1) were targeted and, through quantitative analysis of their abundances in both the compartments and the fresh cast of earthworms, significant reductions in substrate compartments were confirmed for the treatments for FVW added with EAS and EAS alone even if the reduction extents differed among the types of FVW. Apparent reductions were not found in the bed compartment where the final products accumulated. For the fresh cast, the relative abundances of ARGs and intl 1 against to the total bacterial 16S rDNA decreased markedly. The present study provided an insight for proper controlling of ARGs during vermicomposting of FVW and EAS.

Elimination of antibiotic resistance genes in waste activated sludge by persulfate treatment during the process of sludge dewatering

Two sludge conditioning modes (nanoscale zero valent iron modified by Ginkgo biloba L. leaf (G-nZVI)/sodium persulfate (PS) conditioning with different ratios (1:0, 1:0.1, 1:1 and 1:10) and G-nZVI/PS conditioning with continuous addition) in reducing the specific resistance of filtration (SRF) and removing antibiotic resistant genes (ARGs) were investigated. After 3 min, the SRF values decreased in following order: 2.45 × 108 m/kg (1:10) > 5.95 × 106 m/kg (1:0.1) > 3.72 × 106 m/kg (1:0) > 4.92 × 105 m/kg (1:1). In the continuous addition (1:1), the SRF value decreased from 1.04 × 108 m/kg to 6.47 × 106 m/kg at 9 min. Removal efficiency of ARGs was 2–6 orders of magnitude and no regeneration of ARGs was observed in sludge and water phase. When treated samples were incubated for 36 h, dominant microflora was negatively correlated with ARGs. This study revealed persulfate treatment could achieve dewatering and remove ARGs, simultaneously.

Clinical class 1 integron-integrase gene – A promising indicator to monitor the abundance and elimination of antibiotic resistance genes in an urban wastewater treatment plant

In this study, 295 antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) from the influent, activated sludge (AS), and membrane bioreactor (MBR) permeate were primarily examined in the wastewater treatment plant (WWTP) biweekly over 13 months. The absolute concentrations of ARGs and MGEs respectively ranged from 1.27 × 1010 to 1.94 × 1011 and 8.00 × 109 to 1.24 × 1011 copies/L in the influent, of which were reduced by 2 to 3 orders of magnitude in the permeate. No significant seasonal variation of ARGs and MGEs was found in the WWTP, except that the absolute abundance of ARGs and MGEs in the AS was peaked during spring. The antibiotics affected neither ARGs nor MGEs significantly, suggesting their concentrations may be not high enough to pose a selective pressure. In contrast, the bacterial community had direct effect on the MGEs variation, meanwhile the MGEs influenced the ARG abundance directly. Class 1 integron-integrase gene (intI1), clinical intI1, and Tn21 associated more frequently with ARGs in the AS over long-term, suggesting the potential of them involved in horizontal gene transfer. Both intI1 and clinical intI1 had significantly positive associations with the overall abundance of ARGs, as well as significantly negative relationships with the overall removal rates of ARGs in the MBR. However, the abundances between intI1 and clinical intI1 were significantly different. Meanwhile, clinical intI1 remained rather consistent proportion with the ARG abundance in the AS and permeate, was stronger correlated with human pathogens, and was associated with greater number of ARGs over time. Moreover, clinical intI1 was significantly associated with the removal efficiency of ARGs from all classes. Taken together, clinical intI1 can be adopted as an indicator for the abundance and removal efficiency of ARGs in the WWTP.

Elimination of antibiotic resistance genes and human pathogenic bacteria by earthworms during vermicomposting of dewatered sludge by metagenomic analysis

This study used a metagenomic approach to investigate the effects of earthworms on ARGs and HPB during the vermicomposting of dewatered sludge. Results showed that 139 types of ARGs were found in sludge vermicompost, affiliated to 30 classes. Compared with the control, the total abundance of ARGs in sludge vermicompost decreased by 41.5%. Moreover, the types and sequences of plasmids and integrons were also decreased by vermicomposting. Proteobacteria and Actinobacteria were the most dominant hosts of ARGs in sludge vermicompost. In addition, earthworms reduced the total HPB abundance and modified their diversity, thus leading to higher abundance of Enterobacteriaceae in sludge vermicompost. However, the sludge vermicompost was still ARG and HPB enriched, indicating a remaining environmental risk for agricultural purpose. The observed change of microbial community and the reduction of mobile genetic elements caused by earthworm activity are the main reasons for the alleviation of ARG pollution during vermicomposting.

Elimination of antibiotic resistance genes and control of horizontal transfer risk by UV-based treatment of drinking water: A mini review.

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have been recognized as one of the biggest public health issues of the 21st century. Both ARB and ARGs have been determined in water after treatment with conventional disinfectants. Ultraviolet (UV) technology has been seen growth in application to disinfect the water. However, UV method alone is not adequate to degrade ARGs in water. Researchers are investigating the combination of UV with other oxidants (chlorine, hydrogen peroxide (H2O2), peroxymonosulfate (PMS), and photocatalysts) to harness the high reactivity of produced reactive species (Cl·, ClO·, Cl2·-, ·OH, and SO4·-) in such processes with constituents of cell (e.g., deoxyribonucleic acid (DNA) and its components) in order to increase the degradation efficiency of ARGs. This paper briefly reviews the current status of different UV-based treatments (UV/chlorination, UV/H2O2, UV/PMS, and UV-photocatalysis) to degrade ARGs and to control horizontal gene transfer (HGT) in water. The review also provides discussion on the mechanism of degradation of ARGs and application of q-PCR and gel electrophoresis to obtain insights of the fate of ARGs during UV-based treatment processes.

Impacts of advanced treatment processes on elimination of antibiotic resistance genes in a municipal wastewater treatment plant

Antibiotic resistance genes (ARGs) pose a serious threat to public health. Wastewater treatment plants (WWTPs) are essential for controlling the release of ARGs into the environment. This study investigated ARG distribution at every step in the treatment process of a municipal WWTP located in Harbin for six consecutive months. Changes in ARG distribution involved in two advanced secondary effluent treatment processes, ozonation and granular activated carbon (GAC) adsorption, were analyzed. Biological treatment resulted in the highest ARG removal (0.76–1.94 log reduction), followed by ultraviolet (UV) disinfection (less than 0.5-log reduction). Primary treatment could not significantly remove ARGs. ARG removal efficiency increased with an increase in the ozone dose below 40 mg/L. However, amorphous GAC (AGAC) adsorption with a hydraulic retention time (HRT) of 1 h showed better removal of ARGs, total organic carbon (TOC), total nitrogen (TN), and total phosphorus (TP) than ozonation at a 60 mg/L dose. UV treatment could efficiently reduce the relative ARG abundance, despite presenting the lowest efficiency for the reduction of absolute ARG abundance compared with GAC and ozone treatments. The combination of ozone and AGAC can significantly improve the removal of ARGs, TOC, TN and TP. These results indicate that a treatment including biological processing, ozonation, and AGAC adsorption is a promising strategy for removing ARGs and refractory organic substances from sewage.

Rapid startup of thermophilic anaerobic digester to remove tetracycline and sulfonamides resistance genes from sewage sludge

Spread of antibiotic resistance genes (ARGs) originating from sewage sludge is highlighted as an eminent health threat. This study established a thermophilic anaerobic digester using one-step startup strategy to quickly remove tetracycline and sulfonamides resistance genes from sewage sludge. At least 20days were saved in the startup period from mesophilic to thermophilic condition. Based on the results of 16S rDNA amplicons sequencing and predicted metagenomic method, the successful startup largely relied on the fast colonization of core thermophilic microbial population (e.g. Firmicutes, Proteobacteria, Actinobacteria). Microbial metabolic gene pathways for substrate degradation and methane production was also increased by one-step mode. In addition, real-time quantitative PCR approach revealed that most targeted tetracycline and sulfonamides resistance genes ARGs (sulI, tetA, tetO, tetX) were substantially removed during thermophilic digestion (removal efficiency>80%). Network analysis showed that the elimination of ARGs was attributed to the decline of their horizontal (intI1 item) and vertical (potential hosts) transfer-related elements under high-temperature. This research demonstrated that rapid startup thermophilic anaerobic digestion of wastewater solids would be a suitable technology for reducing quantities of various ARGs.

Occurrence and elimination of antibiotic resistance genes in a long-term operation integrated surface flow constructed wetland

Wetland construction is a recommended domestic sewage treatment technique, owing to its simplicity and cost efficiency. Concentrations of 14 antibiotic resistance genes (ARGs) in an integrated surface flow constructed wetland (ICW) steadily operated over 10 years were investigated in the winter and summer. Domestic sewage was observed to be the primary source of ARGs in the ICW, and 77.8% and 59.5% removal rates of total targeted ARGs in the ICW were achieved in the winter and summer, respectively. Concentrations of five ARGs (sul1, tetA, tetC, tetE, and qnrS) in the winter and of six ARGs (sul1, sul3, tetA, tetC, tetE, and qnrS) in the summer were increased throughout the treatment process. Strong correlations were found between ARGs in water and those found in sediments, especially in the summer, indicating that ARGs may be exchanged between water and sediment. Strong positive correlations were also observed between concentrations of intI1 and several ARGs, implying that mobile genetic elements may play a key role in the dissemination of ARGs in an ICW. Our study results suggest aqueous ARGs could be effectively removed via an ICW and that ICWs can also act as reservoirs of specific ARGs.

Anaerobic digestion of nitrogen rich poultry manure: Impact of thermophilic biogas process on metal release and microbial resistances

Poultry manure is a nitrogen rich fertilizer, which is usually recycled and spread on agricultural fields. Due to its high nutrient content, chicken manure is considered to be one of the most valuable animal wastes as organic fertilizer. However, when chicken litter is applied in its native form, concerns are raised as such fertilizers also include high amounts of antibiotic resistant pathogenic Bacteria and heavy metals. We studied the impact of an anaerobic thermophilic digestion process on poultry manure. Particularly, microbial antibiotic resistance profiles, mobile genetic elements promoting the resistance dissemination in the environment as well as the presence of heavy metals were focused in this study.The initiated heat treatment fostered a community shift from pathogenic to less pathogenic bacterial groups. Phenotypic and molecular studies demonstrated a clear reduction of multiple resistant pathogens and self-transmissible plasmids in the heat treated manure.That treatment also induced a higher release of metals and macroelements. Especially, Zn and Cu exceeded toxic thresholds. Although the concentrations of a few metals reached toxic levels after the anaerobic thermophilic treatment, the quality of poultry manure as organic fertilizer may raise significantly due to the elimination of antibiotic resistance genes (ARG) and self-transmissible plasmids.