Intracellular Antibiotic Resistance Genes Research Articles

Deciphering the reduction of antibiotic resistance genes (ARGs) during medium-chain fatty acids production from waste activated sludge: Driven by inhibition of ARGs transmission and shift of microbial community

Medium-chain fatty acids (MCFAs) production from waste activated sludge (WAS) by chain extension (CE) is a promising technology. However, the effects and mechanisms of CE process on the fate of antibiotic resistance genes (ARGs) remain unclear. In this study, the results showed that the removal efficiency of ARGs was 81.15 % in CE process, suggesting its efficacy in reducing environmental risks. Further, the observed decrease in mobile genetic elements (MGEs) indicated that CE process restricted the horizontal gene transfer (HGT). Complementing this, the increase in soluble organic matters and extracellular 16 S rDNA confirmed that MCFAs production caused bacterial damage. Decreased intracellular ARGs and increased extracellular ARGs further revealed that MCFAs production impaired ARGs hosts, thereby limiting the vertical gene transfer (VGT) of ARGs. Shift of microbial community combined with co-occurrence network analysis demonstrated that functional bacteria without host potential for ARGs were enriched, but potential ARGs and MGEs hosts decreased, showing the role of functional bacterial phylogeny and selection pressure of MCFAs in reducing ARGs. Finally, partial least squares path model was used to systematic verify the mechanism of ARGs removal in CE process, which was attributed to the inhibition of ARGs transmission (HGT and VGT) and shift of microbial community.

Removal of intracellular antibiotic resistance genes by activating peroxymonosulfate with Co3O4@CNT catalytic membrane

The removal of antibiotic resistance genes (ARGs) is crucial to prevent the proliferation of antibiotic resistance. In this study, we successfully removed intracellular ARGs using the PMS/Co@CM system, which involved activating peroxymonosulfate (PMS) with a catalytic membrane composed of Co3O4 and carbon nanotubes. In the PMS/Co@CM system, 1O2 was the primary reactive oxygen species (ROS) responsible for intracellular ARGs removal. However, the presence of sulfamethoxazole (SMX) in the water samples significantly reduced the efficiency of removing intracellular ARGs in the PMS/Co@CM system although SMX was degraded by •SO4−. This can be attributed to the selective pressure of SMX, which elevated intracellular Superoxide Dismutase (SOD) levels while decreasing Propidium Iodide (PI) levels in cells. Consequently, this led to heightened antioxidant defense and reduced membrane permeability, thereby enhancing cell resistance to 1O2 generated by the PMS/Co@CM system. Encouragingly, a typical UV disinfection process facilitated the removal of residual intracellular ARGs in the effluent of the PMS/Co@CM system, attributed to decreased SOD levels and increased intracellular 1O2 by UV irradiation. These findings contribute to the theoretical understanding of effective ARGs removal and aid in the development of more efficient removal technologies.

Removal of Intracellular and Extracellular Antibiotic Resistance Genes and Virulence Factor Genes Using Electricity-Intensified Constructed Wetlands

Constructed wetland (CW) is considered a promising technology for the removal of emerging contaminants. However, its removal performance for antibiotic resistance genes (ARGs) is not efficient and influence of virulence factor genes (VFGs) have not been elucidated. Here, removal of intracellular and extracellular ARGs as well as VFGs by electricity-intensified CWs was comprehensively evaluated. The two electrolysis-intensified CWs can improve the removal of intracellular ARGs and MGEs to 0.96- and 0.85-logs, respectively. But cell-free extracellular ARGs (CF-eARGs) were significantly enriched with 1.8-logs in the electrolysis-intensified CW. Interestingly, adding Fe-C microelectrolysis to the electrolysis-intensified CW is conducive to the reduction of CF-eARGs. However, the detected number and relative abundances of intracellular and extracellular VFGs were increased in all of the three CWs. The biofilms attached onto the substrates and rhizosphere are also hotspots of both intracellular and particle-associated extracellular ARGs and VFGs. Structural equation models and correlation analysis indicated that ARGs and VFGs were significantly cooccurred, suggesting that VFGs may affect the dynamics of ARGs. The phenotypes of VFGs, such as biofilm, may act as protective matrix for ARGs, hindering the removal of resistance genes. Our results provide novel insights into the ecological remediation technologies to enhance the removal of ARGs. Environmental ImplicationConstructed wetland (CW) has been considered a promising technology for the removal of emerging contaminants. But its removal performance for antibiotic resistance genes (ARGs) is controversial. Here, we proposed electrolysis-intensified CW to enhance the removal of intracellular and extracellular ARGs. This strategy significantly promotes the removal of resistance genes. In addition, we evaluated the correlations between virulence factor genes (VFGs) and ARGs, suggesting that VFGs may affected the removal of ARGs using biofilm- or substrate-based technologies. These results lead significant guidance for the enhancement removal of ARGs using CWs, and highlight the need to take VFGs into account.

Enhanced sensitivity of extracellular antibiotic resistance genes (ARGs) to environmental concentrations of antibiotic

As emerging contaminants, antibiotics are frequently present in various environments, particularly rivers, albeit often at sublethal concentrations (ng/L∼μg/L). Assessing the risk associated with these low levels, which are far below the lethal threshold for most organisms, remains challenging. In this study, using microcosms containing planktonic bacteria and biofilm, we examined how antibiotic resistance genes (ARGs) in different physical states, including intracellular ARGs (iARGs) and extracellular ARGs (eARGs) responded to these low-level antibiotics. Our findings reveal a positive correlation between sub-lethal antibiotic exposure (ranging from 0.1 to 10 μg/L) and increased prevalence (measured as ARG copies/16s rDNA) of both iARGs and eARGs in planktonic bacteria. Notably, eARGs demonstrated greater sensitivity to antibiotic exposure compared to iARGs, with a lower threshold (0.1 μg/L for eARGs versus 1 μg/L for iARGs) for abundance increase. Moreover, ARGs in biofilms demonstrates higher sensitivity to antibiotic exposure compared to planktonic bacteria. To elucidate the underlying mechanisms, we established an integrated population dynamics-pharmacokinetics-pharmacodynamics (PD-PP) model. This model indicates that the enhanced sensitivity of eARGs is primarily driven by an increased potential for plasmid release from cells under low antibiotic concentrations. Furthermore, the accumulation of antibiotic in biofilms induces a greater sensitivity of ARG compared to the planktonic bacteria. This study provides a fresh perspective on the development of antibiotic resistance and offers an innovative approach for assessing the risk of sublethal antibiotic in the environment.

Assessing intracellular and extracellular distribution of antibiotic resistance genes in the commercial organic fertilizers

Compost-based organic fertilizers often contain high levels of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Previous studies focused on quantification of total ARGs and MGEs. For a more accurate risk assessment of the dissemination risk of antibiotic resistance, it is necessary to quantify the intracellular and extracellular distribution of ARGs and MGEs. In the present study, extracellular ARGs and MGEs (eARGs and eMGEs) and intracellular ARGs and MGEs (iARGs and iMGEs) were separately analyzed in 51 commercial composts derived from different raw materials by quantitative polymerase chain reaction (qPCR) and metagenomic sequencing. Results showed that eARGs and eMGEs accounted for 11–56% and 4–45% of the total absolute abundance of ARGs and MGEs, respectively. Comparable diversity, host composition and association with MGEs were observed between eARGs and iARGs. Contents of high-risk ARGs were similar between eARGs and iARGs, with high-risk ARGs in the two forms accounting for 6.7% and 8.2% of the total abundances, respectively. Twenty-four percent of the overall ARGs were present in plasmids, while 56.7% of potentially mobile ARGs were found to be associated with plasmids. Variation partitioning analysis, null model and neutral community model indicated that the compositions of both eARGs and iARGs were largely driven by deterministic mechanisms. These results provide important insights into the cellular distribution of ARGs in manure composts that should be paid with specific attention in risk assessment and management.

Pyrite from acid mine drainage promotes the removal of antibiotic resistance genes and mobile genetic elements in karst watershed with abundant calcium carbonate

More information is needed to fully comprehend how acid mine drainage (AMD) affects the phototransformation of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in karst water and sewage-irrigated farmland soil with abundant carbonate rocks (CaCO3) due to increasing pollution of AMD formed from pyrite (FeS2). The results showed FeS2 accelerated the inactivation of ARB with an inactivation of 8.7 log. Notably, extracellular and intracellular ARGs and mobile genetic elements (MGEs) also experienced rapid degradation. Additionally, the pH of the solution buffered by CaCO3 significantly influenced the photo-inactivation of ARB. The Fe2+ in neutral solution was present in Fe(II) coordination with strong reducing potential and played a crucial role in generating •OH (7.0 μM), which caused severe damage to ARB, ARGs, and MGEs. The •OH induced by photo-Fenton of FeS2 posed pressure to ARB, promoting oxidative stress response and increasing generation of reactive oxygen species (ROS), ultimately damaging cell membranes, proteins and DNA. Moreover, FeS2 contributed to a decrease in MIC of ARB from 24 mg/L to 4 mg/L. These findings highlight the importance of AMD in influencing karst water and sewage-irrigated farmland soil ecosystems. They are also critical in advancing the utilization of FeS2 to inactivate pathogenic bacteria.

Impact of uranium on antibiotic resistance in activated sludge

The emergence of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in the environment is well established as a human health crisis. The impact of radioactive heavy metals on ecosystems and ultimately on human health has become a global issue, especially for the regions suffering various nuclear activities or accidents. However, whether the radionuclides can affect the fate of antibiotic resistance in bacteria remains poorly understood. Here, the dynamics of ARB, three forms of ARGs—intracellular ARGs (iARGs), adsorbed extracellular ARGs (aeARGs), and free extracellular ARGs (feARGs)—and microbial communities were investigated following exposure to uranium (U), a representative radioactive heavy metal. The results showed that 90-d of U exposure at environmentally relevant concentrations of 0.05 mg/L or 5 mg/L significantly increased the ARB concentration in activated sludge (p < 0.05). Furthermore, 90-d of U exposure slightly elevated the absolute abundance of aeARGs (except tetO) and sulfonamide iARGs, but decreased tetracycline iARGs. Regarding feARGs, the abundance of tetC, tetO, and sul1 decreased after 90-d of U stress, whereas sul2 showed the opposite trend. Partial least-squares path model analysis revealed that the abundance of aeARGs and iARGs under U stress was predominantly driven by increased cell membrane permeability/intI1 abundance and cell membrane permeability/reactive oxygen species concentration, respectively. Conversely, the changes in feARGs abundance depended on the composition of the microbial community and the expression of efflux pumps. Our findings shed light on the variations of ARGs and ARB in activated sludge under U exposure, providing a more comprehensive understanding of antibiotic resistance risks aggravated by radioactive heavy metal-containing wastewater.

Nanowire electroporation-induced cell pores on antibiotic-resistant bacteria to promote chlorine permeation for eliminating intracellular antibiotic resistance genes

Conventional chlorination (Cl2) is inefficient to eliminate intracellular antibiotic resistance genes (i-ARGs) of antibiotic-resistant bacteria (ARB) due to the chlorine permeation barrier and oxidative competition of cell wall/membrane. Herein, we proposed an efficient strategy by constructing synergistic nanowire-induced electroporation (DC-EP) and Cl2 (DC-EP/Cl2) with H2-reduced TiO2 nanowire (TiOxNW) electrodes, attempting to promote i-ARG removal via DC-EP-induced cell damage for chlorine permeation and i-ARG leakage. DC-EP/Cl2 (∼3.7 log and ∼ 60.5 J/L/log) exhibited excellent synergistic effects for ARB inactivation (Escherichia coli) and i-ARG removal (tet(W), aac(3)-II, and blaTEM–1), which achieved much larger i-ARG removal and lower energy consumption than the individual 1.5 V-DC-EP (∼0.05 log) and 10 mg/L-Cl2 (∼0.5 log and ∼ 262.0 J/L/log). Analyses of i-ARG/e-ARG abundances, bacterial cell integrity, and cell morphology indicated that DC-EP-induced cell pores provided channels of chlorine permeation, which was accompanied with oxidative enlargement of cell pores for i-ARG leakage and subsequent degradation. DC-EP/Cl2 exhibited 15–90 times lower frequency of horizontal gene transfer than the individual Cl2 disinfection. The TiOxNW-based DC-EP/Cl2 exhibited excellent stability of i-ARG removal (undetectable ARG abundance and ∼ 20.3 J/L/log) for treating 103 CFU/mL ARB-contaminated tap water within 7-day continuous operation, suggesting its great application potential to alleviate ARG dissemination risk for drinking water.

Tracking the extracellular and intracellular antibiotic resistance genes across whole year in wastewater of intensive dairy farm

Monitoring the annual variation of antibiotic resistance genes (ARGs) in livestock wastewater is important for determining the high-risk period of transfer and spread of animal-derived antibiotic resistance into the environment. However, the knowledge regarding the variation patterns of ARGs, especially intracellular ARGs (iARGs) and extracellular ARGs (eARGs), over time in livestock wastewater is still unclear. Herein, we conducted a year-round study to trace the profiles of ARGs at a Chinese-intensive dairy farm, focusing on the shifts observed in different months. The results showed significant differences in the composition and variation between iARGs and eARGs. Tetracycline, sulfonamide, and macrolide resistance genes were the major types of iARGs, while cfr was the major type of eARG. The environmental adaptations of the host bacteria determine whether ARGs appear as intracellular or extracellular forms. The total abundance of ARGs was higher from April to September, which can be attributed to the favorable climatic conditions for bacterial colonization and increased antibiotic administration during this period. Integron was found to be highly correlated with most iARGs, potentially playing a role in the presence of these genes within cells and their similar transmission patterns in wastewater. The intracellular and extracellular bacterial communities were significantly different, primarily because of variations in bacterial adaptability to the high salt and anaerobic environment. The intracellular co-occurrence network indicated that some dominant genera in wastewater, such as Turicibacter, Clostridium IV, Cloacibacillus, Subdivision5_genera_incertae_sedis, Saccharibacteria_genera_incertae_sedis and Halomonas, were potential hosts for many ARGs. To the best of our knowledge, this study demonstrates, for the first time, the annual variation of ARGs at critical points in the reuse of dairy farm wastewater. It also offers valuable insights into the prevention and control of ARGs derived from animals.

Fates of extracellular and intracellular antibiotic resistance genes in activated sludge and plastisphere under sulfadiazine pressure

Microplastics, antibiotics, and antibiotic resistance genes (ARGs) represent prominent emerging contaminants that can potentially hinder the efficacy of biological wastewater treatment and pose health risks. Plastisphere as a distinct ecological niche for microorganisms, acts as a repository for ARGs and potential pathogenic bacteria. Nonetheless, the spread pattern of extracellular ARGs (eARGs) and intracellular ARGs (iARGs) in plastisphere under antibiotic exposure was not yet known. This study aimed to investigate disparities in extracellular polymeric substances (EPS) production, extracellular and intracellular microbial community structures, as well as the transmission of eARGs and iARGs between activated sludge and plastisphere in an anaerobic/anoxic/oxic system under sulfadiazine (SDZ) exposure. SDZ was found to enhance EPS production in activated sludge and plastisphere. Interestingly, as SDZ removal efficiency increased, EPS content decreased in activated sludge and plastisphere collected from oxic zone, and continued to increase in plastisphere samples collected from anaerobic and anoxic zones. There were significant differences in microbial community structure between activated sludge and plastisphere, and the DNA fragments of potential pathogenic bacteria were detected in extracellular samples. SDZ exhibited a promoting effect on the propagation of eARGs, which were more abundant in the plastisphere than in activated sludge, thus heightening the risk of ARGs dissemination. Extracellular mobile genetic elements played a pivotal role in driving the spread of eARGs, while the microbial community induced the changes of iARGs. Potential pathogenic bacteria emerged as potential hosts for ARGs and mobile genetic elements within activated sludge and plastisphere, leading to more serious environmental threats.

Different fates of extracellular and intracellular antibiotic resistance genes in flocs, granular and biofilm nitrification systems under the stress of acetaminophen

The spread of antibiotic resistance genes (ARGs), including intracellular ARGs (i-ARGs) and extracellular ARGs (e-ARGs), has become a global problem that cannot be ignored. This study clarified the fates of e-ARGs and i-ARGs in floc sludge reactor (FS), granular sludge reactor (GS) and biofilm reactor (BF) under the stress of acetaminophen (APAP). The results showed that the risk of ARGs transmission, especially for e-ARGs, in FS and BF could increase with the increasing times of APAP treatment, except for that in GS. The fates of i-ARGs in three different systems were similar, which were mainly clustered as the efflux pumps mechanism. The secretion and disintegration of extracellular polymeric substances mainly affected the fates of e-ARGs. In the three systems, the complexity of network relationships between ARGs and microbial communities was FS, GS and BF. Partial least-squares path model analysis indicated that bacterial community directly contributed to the variations of e-ARGs and i-ARGs under APAP treatment in the three systems, playing a leading role. And i-ARGs and protein secondary structure showed direct effects on e-ARGs. This study indicated that e-ARGs in complex systems were more susceptible to be influenced, which should be paid more attention to prevent further propagation of ARGs.

Removal of typical sulfonamide resistance genes by ultraviolet/hydrogen peroxide advanced oxidation process in mariculture wastewater

Antibiotics are extensively used in the mariculture sector, leading to the emergence and dissemination of antibiotic resistance genes (ARGs) and posing a serious threat to public health. Therefore, it is urgent to remove and control of ARGs in the mariculture environment. In this study, in order to assess the efficiency and mechanism of ARGs removal, we employed the ultraviolet/hydrogen peroxide (UV/H2O2) advanced oxidation process (AOP) to treat mariculture wastewater containing prevalent sulfonamide resistance genes, namely sul1, sul2, and intl1. The results demonstrated that UV/H2O2 AOP effectively eliminated ARGs, achieving a significant reduction of 2.51–2.71 log for 16S rRNA, intl1, sul1, and sul2 genes after 10 min of treatment with a UV dose of 570 mJ·cm−2, 0.01 mol·L−1 H2O2, and pH 3. The removal of ARGs was influenced by the concentration of the oxidizing agent. Specifically, as the H2O2 concentration increased, the removal of ARGs initially increased and then decreased. Moreover, UV/H2O2 AOP exhibited different removal efficiencies for intracellular ARGs (iARGs) and extracellular ARGs (eARGs). The absolute abundance of iARGs gradually decreased, the eARGs were less impacted by the UV/H2O2 AOP treatment. Furthermore, the microbial community exhibited distinct sensitivities to UV/H2O2 AOP and UV treatment. Proteobacteria and Campylobacterota consistently appeared as dominant phyla, with Sulfurovum being the dominant genus. These findings shed light on the control of ARGs in mariculture wastewater and confirmed the promising prospects of UV/H2O2 AOP as an effective approach.

The removal of antibiotic resistant bacteria and antibiotic resistance genes by sulfidated nanoscale zero-valent iron activating periodate: Efficacy and mechanism

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have drawn much more attention due to their high risk on human health and ecosystem. In this study, the performance of sulfidated nanoscale zero-valent iron (S-nZVI)/periodate (PI) system toward ARB inactivation and ARGs removal was systematically investigated. The S-nZVI/PI system could realize the complete inactivation of 1 × 108 CFU/mL kanamycin, ampicillin, and tetracycline-resistant E. coli HB101 within 40 min, meanwhile, possessed the ability to remove the intracellular ARGs (iARGs) (including aphA, tetA, and tnpA) carried by E. coli HB101. Specifically, the removal of aphA, tetA, and tnpA by S-nZVI/PI system after 40 min reaction was 0.31, 0.47, and 0.39 log10copies/mL, respectively. The reactive species attributed to the E. coli HB101 inactivation were HO• and O2•−, which could cause the destruction of E. coli HB101 morphology and enzyme system (such as superoxide dismutase and catalase), the loss of intracellular substances, and the damage of iARGs. Moreover, the influence of the dosage of PI and S-nZVI, the initial concentration of E. coli HB101, as well as the co-existing substance (such as HCO3−, NO3−, and humic acid (HA)) on the inactivation of E. coli HB101 and its corresponding iARGs removal was also conducted. It was found that the high dosage of PI and S-nZVI and the low concentration of E. coli HB101 could enhance the disinfection performance of S-nZVI/PI system. The presence of HCO3−, NO3−, and HA in S-nZVI/PI system showed inhibiting role on the inactivation of E. coli HB101 and its corresponding iARGs removal. Overall, this study demonstrates the superiority of S-nZVI/PI system toward ARB inactivation and ARGs removal.

Efficient Degradation of Intracellular Antibiotic Resistance Genes by Photosensitized Erythrosine-Produced 1O2.

Intracellular antibiotic resistance genes (iARGs) constitute the important part of wastewater ARGs and need to be efficiently removed. However, due to the dual protection of intracellular DNA by bacterial membranes and the cytoplasm, present disinfection technologies are largely inefficient in iARG degradation. Herein, we for the first time found that erythrosine (ERY, an edible dye) could efficiently degrade iARGs by producing abundant 1O2 under visible light. Seven log antibiotic-resistant bacteria were inactivated within only 1.5 min, and 6 log iARGs were completely degraded within 40 min by photosensitized ERY (5.0 mg/L). A linear relationship was established between ARG degradation rate constants and 1O2 concentrations in the ERY photosensitizing system. Surprisingly, a 3.2-fold faster degradation of iARGs than extracellular ARGs was observed, which was attributed to the unique indirect oxidation of iARGs induced by 1O2. Furthermore, ERY photosensitizing was effective for iARG degradation in real wastewater and other photosensitizers (including Rose Bengal and Phloxine B) of high 1O2 yields could also achieve efficient iARG degradation. The findings increase our knowledge of the iARG degradation preference by 1O2 and provide a new strategy of developing technologies with high 1O2 yield, like ERY photosensitizing, for efficient iARG removal.

The fates of antibiotic resistance genes and their association with cell membrane permeability in response to peroxydisulfate during composting

The aims of this study were to use metagenomics to reveal the fates of antibiotic resistance genes (ARGs) during composting under the regulation of peroxydisulfate and clarify the relationship between ARGs and cell membrane permeability. Results showed that peroxydisulfate increased cell membrane permeability by effectively regulating the expression of outer membrane protein and lipopolysaccharide related genes. Besides, it reduced polysaccharides and proteins in extracellular polymer substances by 36% and 58%, respectively, making it easier for intracellular ARGs (i-ARGs) to reach the extracellular environment, among which the absolute intracellular abundance of mphK, Erm(31), and tet(44) decreased to 1.2, 1.0, and 0.89 fold of the control, respectively. Finally, variation partitioning analysis showed that i-ARGs dominated the removal of ARGs. These results revealed that the removal of i-ARGs by activated peroxydisulfate was the key to the removal of ARGs and increased cell membrane permeability played a key role for peroxydisulfate to remove i-ARGs during composting.

Degradation characteristics of intracellular and extracellular ARGs during aerobic composting of swine manure under enrofloxacin stress

Composting has been widely recognized as an effective method for removing antibiotic resistance genes (ARGs) from livestock manure. This study investigated the variation in intracellular ARGs (iARGs) and extracellular ARGs (eARGs) during swine manure composting under low (31.44 ± 1.11 mg/kg DM) and high (176.76 ± 1.90 mg/kg DM) enrofloxacin concentration stress. After 25 days of composting, the rates of total iARG and eARG degradation reached 67.30% ∼ 96.13% and 54.70% ∼ 80.30%, respectively. The fluoroquinolone and multidrug ARGs that were degraded included intracellular and extracellular aac(6′)-ib-cr, qnrS, qnrD, parC, oqxB, acrA, acrB and tolC, but i-parE and e-parE were not eliminated. The main reason for degradation were the alteration of bacterial communities and mobile genetic elements (MGEs). The total absolute abundances of iARGs were higher than those of eARGs during composting. iARG degradation was driven by the death and succession of potential hosts, primarily including gram-negative bacteria. Composting time advancement played a greater role in facilitating the microbial succession. High temperatures promoted bacterial death, which caused the release of iARGs and generation of eARGs, but continuous high temperature led to more eARG degradation. The dynamics of longer-term eARGs were more strongly affected by extracellular MGEs (eMGEs), and it is more likely that eARGs enter the environment synchronously with eMGEs and persist in the environment at lower abundances via eMGEs. Once eARGs re-enter bacteria, they may become more mobile and dangerous. This study provides insights for facilitating the removal of ARGs in fertilizer utilization.

The dynamics and removal efficiency of antibiotic resistance genes by UV-LED treatment: An integrated research on single- or dual-wavelength irradiation

Antimicrobial resistance has gained increasing attention, because of the awareness of its potential health risks. Strategies for the removal of antibiotic resistance genes (ARGs) are urgently required. In the present study, UV-LEDs at wavelength of 265 and 285 nm were integrated at five conditions, including single 265 nm UV-LED, single 285 nm UV-LED, and combined 265 nm and 285 nm UV-LED at different intensities, to remove tet A, cat 1, and amp C. The ARGs removal efficiency, gene behavior, and possible cellular mechanism were analyzed using real-time quantitative PCR, flow cytometry, and transmission electron microscopy (TEM). The 265 nm UV-LED is more effective than the 285 nm UV-LED and their combinations in terms of ARGs control, in which 1.91, 1.71, and 1.45 log were removed for tet A, cat 1, and amp C, respectively, at a UV dosage of 500 mJ/cm2. The intracellular gene leakage was detected in all five UV-LED experiment scenarios even when the cell membrane damage was insignificant with the highest increase of 0.69 log ARGs. ROS was generated during the irradiation, and the ROS was strongly negative correlated with intracellular ARGs, which could promote the degradation and removal of ARGs. This study provides a new insight of intracellular ARGs removal, because direct irradiation, ROS oxidation, and leakage to the extracellular serve as the three main pathways under high-dosage UV-LED irradiation. Further research should be focused on the mechanism and optimization of UV technology with 265 nm UV-LED for ARG control.

Simultaneous removal of antibiotic-resistant Escherichia coli and its resistance genes by dielectric barrier discharge plasma

As emerging contaminants, antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have been widely detected in various aqueous environments. For antibiotic resistance to be inhibited in the environment, it is essential to control ARB and ARGs. In this study, dielectric barrier discharge (DBD) plasma was used to inactivate antibiotic resistant Escherichia coli (AR E. coli) and remove ARGs simultaneously. Within 15 s of plasma treatment, 108 CFU/mL of AR E. coli were inactivated by 97.9%. The rupture of the bacterial cell membrane and the increase of intracellular ROS are the main reasons for the rapid inactivation of bacteria. Intracellular ARGs (i-qnrB, i-blaCTX-M, i-sul2) and integron gene (i-int1) decreased by 2.01, 1.84, 2.40, and 2.73 log after 15 min of plasma treatment, respectively. In the first 5 min of discharge, extracellular ARGs (e-qnrB, e-blaCTX-M, e-sul2) and integron gene (e-int1) decreased by 1.99, 2.22, 2.66, and 2.80 log, respectively. The results of the ESR and quenching experiments demonstrated that ·OH and 1O2 played important roles in the removal of ARGs. This study shows that DBD plasma is an effective technique to control ARB and ARGs in waters.

Reductions in abundances of intracellular and extracellular antibiotic resistance genes by SiO2 nanoparticles during composting driven by mobile genetic elements

Applying exogenous additives during the aerobic composting of livestock manure is effective for slowing down the spread of antibiotic resistance genes (ARGs) in the environment. Nanomaterials have received much attention because only low amounts need to be added and they have a high capacity for adsorbing pollutants. Intracellular ARGs (i-ARGs) and extracellular ARGs (e-ARGs) comprise the resistome in livestock manure but the effects of nanomaterials on the fates of these different fractions during composting are still unclear. Thus, we investigated the effects of adding SiO2 nanoparticles (SiO2NPs) at four levels (0 (CK), 0.5 (L), 1 (M), and 2 g/kg (H)) on i-ARGs, e-ARGs, and the bacterial community during composting. The results showed that i-ARGs represented the main fraction of ARGs during aerobic composting of swine manure, and their abundance was lowest under M. Compared with CK, M increased the removal rates of i-ARGs and e-ARGs by 17.9% and 100%, respectively. SiO2NPs enhanced the competition between ARGs hosts and non-hosts. M optimized the bacterial community by reducing the abundances of co-hosts (Clostridium_sensu_stricto_1, Terrisporobacter, and Turicibacter) of i-ARGs and e-ARGs (by 96.0% and 99.3%, respectively) and killing 49.9% of antibiotic-resistant bacteria. Horizontal gene transfer dominated by mobile genetic elements (MGEs) played a key role in the changes in the abundances of ARGs. i-intI1 and e-Tn916/1545 were key MGEs related closely to ARGs, and the maximum decreases of 52.8% and 100%, respectively, occurred under M, which mainly explained the decreased abundances of i-ARGs and e-ARGs. Our findings provide new insights into the distribution and main drivers of i-ARGs and e-ARGs, as well as demonstrating the possibility of adding 1 g/kg SiO2NPs to reduce the propagation of ARGs.

Distribution and dynamics of antibiotic resistance genes in a three-dimensional multifunctional biofilm during greywater treatment

Antibiotic resistance genes (ARGs) have been identified as serious threats to public health. Despite the widespread in various systems, dynamics of ARGs in three-dimensional multifunctional biofilm (3D-MFB) treating greywater are largely undefined. This work tracked the distributions and dynamics of eight target genes (intI1, korB, sul1, sul2, tetM, ermB, blaCTX-M and qnrS) in a 3D-MFB during greywater treatment. Results showed that hydraulic retention times at 9.0 h achieved the highest linear alkylbenzene sulfonate (LAS) and total nitrogen removal rates at 99.4% and 79.6%, respectively. ARGs presented significant liquid-solid distribution feature, but non-significant with biofilm position. Intracellular ARGs (predominant by intI1, korB, sul1 and sul2) at bottom biofilm were 210- to 4.2 × 104- fold higher than that in cell-free liquid. Extracellular polymeric substances (EPS)-attached LAS showed linear relationship with most of ARGs (R2 > 0.90, P < 0.05). Sphingobacteriales, Chlamydiales, Microthrixaceae, SB-1, Cryomorphaceae, Chitinophagaceae, Leadbetterella and Niabella were tightly bound up with target ARGs. Key is that EPS-attached LAS considerably determines the occurrence of ARGs, and microbial taxa play an important role in the dissemination of ARGs in the 3D-MFB.