DNA-based stable isotope probing deciphered the active denitrifying bacteria and triclosan-degrading bacteria participating in granule-based partial denitrification process under triclosan pressure.

Granule-based partial denitrification (PD) is a technology that can supply stable nitrite for applying anaerobic ammonia oxidation in wastewater treatment, and triclosan (TCS) is a frequently detected antibacterial agent in wastewater treatment plants, therefore it is possible that TCS could enter into wastewater that is treated using PD technology. However, the active microorganisms responsible for PD and TCS removing in granule-based PD system have not been clearly identified and it is currently not clear how TCS affects the PD process. In this study, the impacts of TCS on PD performance, PD microbial community, antibiotic resistance genes (ARGs), active PD bacteria and TCS-degrading bacteria in a granule-based PD system were investigated. 3 mg/L TCS had adverse influence on PD process, but PD system could recover gradually after inhibiting of 10 days. After a period of domestication, PD granular sludge could achieve 10.66% of TCS degradation efficiency and 43.62% of TCS adsorption efficiency. Microbes might increase their resistance to TCS by increasing the secretion of extracellular polymeric substances, and the secretion of protein might play a more pivotal role than the secretion of polysaccharides in resisting TCS. The short-term shock of TCS might cause the propagation of acrA-03, while the long-term operation of TCS could propagate fabK and intI1. DNA stable isotope probing assay indicated that Thauera was active PD bacteria and TCS-degrading bacteria in the granule-based PD system, and it could contribute to nitrite accumulation and TCS degradation, simultaneously.

Synergistic effect of sulfidated nanoscale zerovalent iron in donor and recipient bacterial inactivation and gene conjugative transfer inhibition.

Antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) are widespread in urban wastewater treatment plants (UWTPs). In this research, a horizontal transfer model of recipient (Pseudomonas. HLS-6) and donor (Escherichia coli DH5α carries RP4 plasmid) was constructed to explore the effect of sulfidated nanoscale zerovalent iron (S-nZVI) on the efficiency of plasmid-mediated horizontal transfer. When the S/Fe was 0.1, the inactivation efficiency of 1120 mg/L S-nZVI on the donor and recipient bacteria were 2.36 ± 0.03 log and 3.50 ± 0.17 log after 30 min, respectively (initial ARB concentration ≈ 5 ×107 CFU/mL). Effects of treatment time, S/Fe molar ratio, S-nZVI dosage and initial bacterial concentration were systemically studied. S-nZVI treatment could increase the extracellular alkaline phosphatase and malondialdehyde content of the ARB, cause oxidative stress in the bacteria, destroy the cell structure and damage the intracellular DNA. This study provided evidence and insights into possible underlying mechanisms for reducing conjugative transfer, such as hindering cell membrane repair, inducing the overproduction of reactive oxygen species, inhibiting the SOS response, reducing the expression of ARGs and related transfer genes. S-nZVI could inhibit the gene conjugative transfer while inactivating the ARB. The findings provided an alternative method for controlling antibiotic resistance.

Enhancement of antibiotic resistance dissemination by artificial sweetener acesulfame potassium: Insights from cell membrane, enzyme, energy supply and transcriptomics.

The abuse of antibiotics on animals could induce the development of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB), and acesulfame potassium (ACE) is the widely used artificial sweetener in animal feed. Generally speaking, ACE and ARB often coexist in livestock wastewater, however, the impact of the co-occurrence of ACE and ARB on the transmission of ARGs is still unknown. In this study, the effects of ACE on vertical gene transfer (VGT) and horizontal gene transfer (HGT) were both evaluated. For VGT, ACE may hinder the spread of sul gene in Pseudomonas HLS-6 by blocking ARB growth. As for HGT (from Escherichia coli DH5α to Pseudomonas HLS-6), environmentally relevant ACE concentration could facilitate the conjugative transfer. The underlying mechanisms of HGT were characterized by enhanced cell membrane permeability, reactive oxygen species overproduction, SOS response, energy supply, which were all further verified by the changes in transcription levels of related genes. Interestingly, intracellular Mg2+ in donor strain was found for the first time as an indicator for the conjugation occurrence in ACE treated mating system. This study may provide new insights into the role of ACE on ARGs proliferation and highlight its potential environmental impacts.

The removal of antibiotic resistant bacteria and genes and inhibition of the horizontal gene transfer by contrastive research on sulfidated nanoscale zerovalent iron activating peroxymonosulfate or peroxydisulfate.

Antibiotic resistant bacteria (ARB) and the antibiotic resistance genes (ARGs) dissemination via plasmid-mediated conjugation have attracted considerable attentions. In this research, sulfidated nanoscale zerovalent iron (S-nZVI)/peroxymonosulfate (PMS) and S-nZVI/peroxydisulfate (PDS) process were investigated to inactivate ARB (Escherichia coli DH5α with RP4 plasmid, Pseudomonas. HLS-6 contains sul1 and intI1 on genome DNA sequence). S-nZVI/PMS system showed higher efficiency than S-nZVI/PDS on ARB inactivation. Thus, the optimal condition 28 mg/L S-nZVI coupled with 153.7 mg/L (0.5 mM) PMS was applied to remove both intracellular ARGs (iARGs) and ARB. The oxidative damage of ARB cell was systemically studied by cell viability, intracellular Mg2+ levels, the changes of extracellular and internal structure, integrity of cell walls and membranes and enzymatic activities. S-nZVI/PMS effectively inactivated ARB (~7.32 log) within 15 min. These effects were greatly higher than those achieved individually. Moreover, removal efficiencies of iARGs sul1, intI1 and tetA were 1.52, 1.79 and 1.56 log, respectively. These results revealed that S-nZVI and PMS have a synergistic effect against ARB and iARGs. The regrowth assays illustrated that the ARB were effectively inactivated. By verifying the inhibitory impacts of S-nZVI/PMS treatment on conjugation transfer, this work highlights a promising alternative technique for inhibiting the horizontal gene transfer.

Chloroxylenol at environmental concentrations can promote conjugative transfer of antibiotic resistance genes by multiple mechanisms.

The intergeneric conjugative transfer of antibiotic resistance genes (ARGs) is recognized as an important way to the dissemination of antibiotic resistance. However, it is unknown whether the extensive use of chloroxylenol (para-chloro-meta-xylenol, PCMX) in many pharmaceutical personal care products will lead to the spread of ARGs. In this study, the abilities and mechanisms of PCMX to accelerate the intergeneric conjugative transfer were investigated. Results showed that exposure of bacteria to environmental concentrations of PCMX (0.20-1.00 mg/L) can significantly stimulate the increase of conjugative transfer by 8.45-9.51 fold. The phenotypic experiments and genome-wide RNA sequencing revealed that 0.02-5.00 mg/L PCMX exposure could increase the content of alkaline phosphatase and malondialdehyde, which are characteristic products of cell wall and membrane damage. In addition, PCMX could lead to excessive production of reactive oxygen species (ROS) by 1.26-2.00 times, the superoxide dismutase and catalase produced by bacteria in response to oxidative stress were not enough to neutralize the damage of ROS, thus promoting the conjugative transfer. Gene Ontology enrichment analysis indicated that cell membrane permeability, pili, some chemical compounds transport and energy metabolism affected conjugative transfer. This study deepened the understanding of PCMX in promoting propagation of ARGs, and provided new perspectives for use and treatment of personal care products.

Metagenomics combined with DNA-based stable isotope probing provide comprehensive insights of active triclosan-degrading bacteria in wastewater treatment.

The biotransformation of triclosan (TCS) during wastewater treatment occurred frequently, while little researches are known the identity of microorganisms involved in the biodegradation process. In this work, DNA-based stable isotope probing (DNA-SIP) was occupied to investigate the TCS assimilation microbes originated from a full-scale cyclic activated sludge system in Beijing. Results of TCS removal pathway showed that the TCS removal in nitrification process was mainly contributed by the metabolism of heterotrophic bacteria, accounting for about 18.54%. DNA-SIP assay indicated that Sphingobium dominated the degradation of TCS. Oligotyping analysis further indicated that oligotype GCTAAT and ATGTTA of Sphingobium played important roles in degrading TCS. Furthermore, the Kyoto Encyclopedia of Genes and Genomes functional abundance statistics based on PICRUSt2 showed that glutathione transferase was the most prevalent enzyme involved in TCS metabolism, and TCS might be removed through microbial carbon metabolism. Metagenomics made clear that Sphingobium might play irrelevant role on the propagation of antibiotics resistance genes (ARGs), even though, it could degrade TCS. Thauera and Dechloromonas were identified as the key hosts of most ARGs. This study revealed the potential metabolic pathway and microbial ecology of TCS biodegradation in nitrification process of wastewater treatment system.

Microplastics affect the ammonia oxidation performance of aerobic granular sludge and enrich the intracellular and extracellular antibiotic resistance genes.

Microplastics (MPs) and antibiotic resistance genes (ARGs), as emerging pollutants, are frequently detected in wastewater treatment plants, and their threats to the environment have received extensive attentions. However, the effects of MPs on the nitrification of aerobic granular sludge (AGS) and the spread patterns of intracellular and extracellular ARGs (iARGs and eARGs) in AGS were still unknown. In this study, the responses of AGS to the exposure of 1, 10 and 100 mg/L of typical MPs (polyvinyl chloride (PVC), polyamide (PA), polystyrene (PS) and polyethylene (PE)) and tetracycline were focused on in 3 L nitrifying sequencing batch reactors. 10 mg/L MPs decreased the nitrification function, but nitrification could recover. Furthermore, MPs inhibited ammonia-oxidizing bacteria and enriched nitrite-oxidizing bacteria, leading partial nitrification to losing stability. PVC, PA and PS stimulated the secretion of extracellular polymeric substances and reactive oxygen species. PE had less negative effect on AGS than PVC, PA and PS. The abundances of iARGs and eARGs (tetW, tetE and intI1) increased significantly and the intracellular and extracellular microbial communities obviously shifted in AGS system under MPs stress. Potential pathogenic bacteria might be the common hosts of iARGs and eARGs in AGS system and were enriched in AGS and MPs biofilms.

Achieving stable and long-term partial nitrification of domestic wastewater by side-stream sludge treatment using a novel nitrite oxidation inhibitor chloroxylenol.

Using inhibitors to selectively suppress the activity of nitrite-oxidizing bacteria (NOB) was an emerging way to rapidly achieve partial nitrification (PN). This study explored the feasibility of inactivating NOB by a novel inhibitor chloroxylenol (PCMX) in real domestic wastewater. Different frequencies (periodic strategy and concentrative time strategy) of PCMX side-stream sludge treatment were used to achieve and maintain PN during 250 days. PN was realized by PCMX treatment once a day about 20 days, due to the inhibition of Nitrospira. PN was completely destroyed after 212 days by periodic strategy, caused by the increase of Candidatus Nitrotoga. PN maintained without PCMX in following 201 days by concentrative time strategy. The risks of PCMX were assessed and almost no PCMX was detected in the effluent of mainstream sequencing batch reactors. These results meant PN realized by PCMX side-stream sludge treatment was feasible and concentrative time strategy was a better operating strategy.

Enhanced removal of antibiotic resistance genes by nanoscale iron-cobalt particles modified with Ginkgo biloba L. leaf: Combining Illumina MiSeq sequencing and oligotyping analysis.

The addition of second metal (Co) to nanoscale iron particles (NIPs) is an attractive strategy to improve catalytic capacity. However, the nanoparticles tend to form chain-like aggregates. In this study, bacterial 16S rRNA gene, antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) from secondary effluent were significantly removed by Ginkgo biloba L. modified nanoscale iron-cobalt particles (GNICPs). When the Co loading, initial pH value, DO and dosage were 10%, 7.33, 8.94 mg/L and 1.12 g/L, some ARGs and MGEs could be reduced below the detection limit at the 2nd or 3rd cycle. Illumina MiSeq sequencing demonstrated that negative correlations were found between ARGs and reaction time/cycles. The predicted microbial functions by FAPROTAX database indicated GNICPs were effective in eliminating human_pathogens_all. Furthermore, oligotyping revealed all ARGs and MGEs were positively correlated with oligotype 10, which indicated GNICPs removed oligotype 10 easily.

Long-term responses of antibiotic resistance genes under high concentration of enrofloxacin, sulfadiazine and triclosan in aerobic granular sludge system.

It is worth to reveal the long-term responses of antibiotic resistance genes (ARGs) in aerobic granular sludge (AGS) system exposed to high level enrofloxacin (ENR), sulfadiazine (SDZ) and triclosan (TCS). In present study, ppm level ENR, SDZ and TCS were added into three AGS reactors, respectively. ARGs in ENR and SDZ systems showed trends of increasing first and then decreasing, which were contrary to that in TCS system. 80%, 56% and 40% ARGs in ENR, SDZ and TCS systems, respectively, were enriched after loading, but several ARGs still kept high enrichment values after the withdrawn of loadings. The dominant bacteria in ENR (Flavobacterium), SDZ (Candidatus_Competibacter and Defluviicoccus) and TCS (Defluviicoccus) systems might contribute to the reductions of ARGs. IntI1 altered the overall ARGs profiles through horizontal gene transfer. The interactions of bacterial communities and environmental factors might be responsible for the different ARGs patterns in ENR, SDZ and TCS systems.

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.

Inactivation of sulfonamide antibiotic resistant bacteria and control of intracellular antibiotic resistance transmission risk by sulfide-modified nanoscale zero-valent iron.

The inactivation of a gram-negative sulfonamide antibiotic resistant bacteria (ARB) HLS.6 and removal of intracellular antibiotic resistance gene (ARG, sul1) and class I integrase gene (intI1) by nanoscale zero-valent iron (nZVI) and sulfide-modified nZVI (S-nZVI) with different S/Fe molar ratios were investigated in this study. The S-nZVI with high sulfur content (S/Fe = 0.05, 0.1, 0.2) was superior to nZVI and the treatment effect was best when S/Fe was 0.1. The ARB (2 × 107 CFU/mL) could be completely inactivated by 1.12 g/L of S-nZVI (S/Fe = 0.1) within 15 min, and the removal rates of intracellular sul1 and intI1 reached up to 4.39 log and 4.67 log at 60 min, respectively. Quenching experiments and flow cytometry proved that reactive oxygen species and adsorption were involved in the ARB inactivation and target genes removal. Bacterial death and live staining experiments and transmission electron microscopy showed that the ARB cell structure and intracellular DNA were severely damaged after S-nZVI treatment. This study provided a potential alternative method for controlling the antibiotic resistance in aquatic environment.

Sulfidated nanoscale zero-valent iron is an efficient material for the removal and regrowth inhibition of antibiotic resistance genes.

Antibiotic resistance genes (ARGs) and mobile gene elements (MGEs), the emerging genetic contaminants, are regarded as severe risks to public health for impairing the inactivation efficacy of antibiotics. Secondary effluents from wastewater treatment plants are the hotspots for spreading these menaces. Herein, sulfidated nanoscale zero-valent iron (S-nZVI) was occupied to remove ARGs and MGEs in secondary effluents and weaken the regrowth capacity of their bacterial carriers. The effects of S/Fe molar ratios (S/Fe), initial pH and dosages on 16S rRNA and ARGs removal were also investigated. Characterization, mass balance and scavenging experiments were conducted to explore the mechanisms of the gene removal. Quantitative PCR (qPCR) and high throughput fluorescence qPCR showed more than 3 log unit of 16S rRNA and seven out of 10 ARGs existed in secondary effluent could be removed after S-nZVI treatment. The mechanisms might be that DNA accepted the electron provided by the Fe0 core of S-nZVI after being adsorbed onto S-nZVI surface, causing the decrease of 16S rRNA, ARGs and lost their regrowth capacity, especially for typical MGE (intI1) and further inhibiting the vertical gene transfer (VGT) and intI1-induced horizontal gene transfer (HGT). Fe0 core was oxidized to iron oxides and hydroxides at the same time. High throughput sequencing, network analysis and variation partitioning analysis revealed the complex correlations between bacteria and ARGs in secondary effluent, S/Fe could directly influence ARGs variations, and bacterial genera made the greatest contribution to ARGs variations, followed by MGEs and operational parameters. As a result, S-nZVI could be an available reductive approach to deal with bacteria and ARGs.

Simultaneous adsorption and degradation of triclosan by Ginkgo biloba L. stabilized Fe/Co bimetallic nanoparticles.

Triclosan (TCS), an antimicrobial agent added in many pharmaceutical and personal care products, can cause some environmental problems due to its bioaccumulation, toxicity and potential antibiotic cross-resistance. In this study, Ginkgo biloba L. leaf extract was used as the green stabilizing agent to synthesize Fe/Co bimetallic nanoparticles (G-Fe/Co NPs), which were applied to remove TCS from aqueous solution. G-Fe/Co NPs were characterized by TEM, EDS, SEM, BET, FTIR, XRD and XPS. G. biloba L. leaf extract improved the dispersion and reduced the passivation of NPs. The TCS removal efficiency followed the order of G-Fe/Co NPs > G-Fe NPs > Co NPs > Fe/Co NPs > Fe NPs. G-Fe/Co NPs can be reused at least eight times. The Co leaching under different initial pH values was negligible. The factors affecting the TCS removal were investigated. The results indicated that the removal of TCS followed pseudo-second-order kinetics, and the removal rate constant decreased with increasing the initial pH value and the initial TCS concentration, and decreasing the Co loading of G-Fe/Co NPs and NPs dosage. The mass balance of TCS removal by G-Fe/Co NPs indicated that adsorption was dominant process and TCS degradation was an accumulative process.