Frequency Of Conjugative Transfer Research Articles

Chlorite and bromate alter the conjugative transfer of antibiotic resistance genes: Co-regulation of oxidative stress and energy supply

The widespread use of disinfectants during the global response to the 2019 coronavirus pandemic has increased the co-occurrence of disinfection byproducts (DBPs) and antibiotic resistance genes (ARGs). Although DBPs pose major threats to public health globally, there is limited knowledge regarding their biological effects on ARGs. This study aimed to investigate the effects of two inorganic DBPs (chlorite and bromate) on the conjugative transfer of RP4 plasmid among Escherichia coli strains at environmentally relevant concentrations. Interestingly, the frequency of conjugative transfer was initially inhibited when the exposure time to chlorite or bromate was less than 24 h. However, this inhibition transformed into promotion when the exposure time was extended to 36 h. Short exposures to chlorite or bromate were shown to impede the electron transport chain, resulting in an ATP shortage and subsequently inhibiting conjugative transfer. Consequently, this stimulates the overproduction of reactive oxygen species (ROS) and activation of the SOS response. Upon prolonged exposure, the resurgent energy supply promoted conjugative transfer. These findings offer novel and valuable insights into the effects of environmentally relevant concentrations of inorganic DBPs on the conjugative transfer of ARGs, thereby providing a theoretical basis for the management of DBPs.

Environmental concentrations of acetaminophen and its metabolites promote the spread of antibiotic resistance genes through pheromone signaling pathway

The massive use of acetaminophen (APAP), an antipyretic and analgesic drug, especially during the COVID-19 pandemic, has caused the environmental residue and pollution of acetaminophen and its metabolites. However, the environmental ecological and health issues they may cause are still unclear. In this study, we found that environmental concentrations of APAP and its metabolites could promote the conjugative transfer of antibiotic resistance genes mediated by plasmid pCF10 through pheromone-like effect rather than the commonly mechanism found in current studies. Conjugative transfer studies showed that APAP and its metabolites increased the frequency of conjugative transfer of ARGs by up to 3.3 times, reaching 3.2 × 10-2. We explored the underling mechanisms using qPCR, western blot, and SEM images. This study demonstrates that APAP stimulates pheromone secretion, subsequently triggering conjugation and accelerating downstream gene expression. Furthermore, APAP functions as a pheromone, initially facilitating plasmid cleavage and peptidoglycan hydrolysis, followed by the promotion of adhesion protein PrgB expression and T4SS scaffold construction, ultimately enhancing DNA strand transfer via T4CP. This study provides a theoretical basis for understanding the environmental hazards of APAP and the environmental sustainability.

The effect of triclosan on intergeneric horizontal transmission of plasmid-mediated tigecycline resistance gene tet(X4) from Citrobacter freundii isolated from grass carp gut

The transmission of antibiotic resistance genes (ARGs) in pathogenic bacteria affects culture animal health, endangers food safety, and thus gravely threatens public health. However, information about the effect of disinfectants - triclosan (TCS) on ARGs dissemination of bacterial pathogens in aquatic animals is still limited. One Citrobacter freundii (C. freundii) strain harboring tet(X4)-resistant plasmid was isolated from farmed grass carp guts, and subsequently conjugative transfer frequency from C. freundii to Escherichia coli C600 (E. coli C600) was analyzed under different mating time, temperature, and ratio. The effect of different concentrations of TCS (0.02, 0.2, 2, 20, 200 and 2000 μg/L) on the conjugative transfer was detected. The optimum conditions for conjugative transfer were at 37 °C for 8h with mating ratio of 2:1 or 1:1 (C. freundii: E. coli C600). The conjugative transfer frequency was significantly promoted under TCS treatment and reached the maximum value under 2.00 μg/L TCS with 18.39 times that of the control group. Reactive oxygen species (ROS), superoxide dismutase (SOD) and catalase (CAT) activities, cell membrane permeability of C. freundii and E. coli C600 were obviously increased under TCS stress. Scanning electron microscope showed that the cell membrane surface of the conjugative strains was wrinkled and pitted, even broken at 2.00 μg/L TCS, while lysed or even ruptured at 200.00 μg/L TCS. In addition, TCS up-regulated expression levels of oxidative stress genes (katE, hemF, bcp, hemA, katG, ahpF, and ahpC) and cell membrane-related genes (fimC, bamE and ompA) of donor and recipient bacteria. Gene Ontology (GO) enrichment demonstrated significant changes in categories relevant to pilus, porin activity, transmembrane transporter activity, transferase activity, hydrolase activity, material transport and metabolism. Taken together, a tet(X4)-resistant plasmid could horizontal transmission among different pathogens, while TCS can promote the propagation of the resistant plasmid.

Regulation of quorum sensing for the manipulation of conjugative transfer of antibiotic resistance genes in wastewater treatment system

The emergence and transmission of antibiotic resistance genes (ARGs) through plasmid-mediated conjugation has become a significant worldwide public health threat. Biofilms are widely recognized as the primary reservoirs for ARGs, providing favorable conditions for horizontal gene transfer. Quorum sensing (QS) plays a critical role in bacterial biofilm formation, which further influences the spread of bacterial resistance. In this study, we examined the effects of vanillin, a QS inhibitor (QSI), at subinhibitory concentrations (sub-MICs) ranging from 0 – 0.1 g/L, on the transfer of ARGs between Escherichia coli and Pseudomonas aeruginosa. Our findings indicated that vanillin at sub-MICs inhibited the conjugative transfer frequency of the RP4 plasmid. This inhibition was supported by the downregulation of plasmid transfer genes. The suppression of conjugation can mainly be attributed to the inhibition of biofilm formation, the synthesis of extracellular polymeric substances (EPS), and the secretion of virulence factors, all of which are regulated by the bacterial QS system. On the other hand, the levels of ROS and cell membrane permeability were not primary explanations for this phenomenon. Furthermore, vanillin also reduced the conjugative transfer frequency of ARGs in wastewater effluent, providing a potential approach to alleviate bacterial resistance in water environments. These findings underscore the regulatory role of QSI in controlling ARGs transfer and have significant implications for manipulating the dissemination of bacterial resistance in the environment.

Carbonaceous particulate matter promotes the horizontal transfer of antibiotic resistance genes.

There is growing concern about the transfer of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in airborne particulate matter. In this study, we investigated the effects of various types of carbonaceous particulate matter (CPM) on the transfer of ARGs in vitro. The results showed that CPM promoted the transfer of ARGs, which was related to the concentration and particle size. Compared with the control group, the transfer frequency was 95.5, 74.7, 65.4, 14.7, and 3.8 times higher in G (graphene), CB (carbon black), NGP (nanographite powder), GP1.6 (graphite powder 1.6 micron), and GP45 (graphite powder 45 micron) groups, respectively. Moreover, the transfer frequency gradually increased with the increase in CPM concentration, while there was a negative relationship between the CPM particle size and conjugative transfer frequency. In addition, the results showed that CPM could promote the transfer of ARGs by increasing ROS, as well as activating the SOS response and expression of conjugative transfer-related genes (trbBp, trfAp, korA, kroB, and trbA). These findings are indicative of the potential risk of CPM for the transfer of ARGs in the environment, enriching our understanding of environmental pollution and further raising awareness of environmental protection.

Size- and surface charge-dependent hormetic effects of microplastics on bacterial resistance and their interactive effects with quinolone antibiotic

The facilitation of microplastics (MPs) on bacterial resistance has attracted wide concern, due to the widespread presence of MPs in environmental media and their ubiquitous contact with bacteria strains. Furthermore, MPs possibly co-exist with antibiotics to trigger combined stress on bacterial survival. Therefore, it is significant to reveal the dose-responses of MPs and MP-antibiotic mixtures on bacterial endogenous and exogenous resistance. In this study, 0.1 and 5 μm polystyrenes with no surface functionalization (PS-NF, no charge), surface functionalized with amino groups (PS-NH2, positive charge) and carboxyl groups (PS-COOH, negative charge) were selected as the test MPs, and norfloxacin (NOR) was set as the representative of antibiotics. It was found that six types of PS all inhibited the growth of Escherichia coli (E. coli) but induced hormetic dose-responses on the mutation frequency (MF) and conjugative transfer frequency (CTF) of RP4 plasmid in E. coli. Moreover, these hormetic effects exhibited size- and surface charge-dependent features, where 0.1 μm PS-NH2 (100 mg/L) triggered the maximum stimulatory rates on MF (363.63 %) and CTF (74.80 %). The hormetic phenomena of MF and CTF were also observed in the treatments of PS-NOR mixtures, which varied with the particle size and surface charge of PS. In addition, the interactive effects between PS and NOR indicated that the co-existence of PS and NOR might trigger greater resistance risk than the single pollutants. Mechanistic exploration demonstrated that the increase of cellular reactive oxygen species and the variation of cell membrane permeability participated in the hormetic effects of PS and PS-NOR mixtures on bacterial resistance. This study provides new insights into the individual effects of MPs and the combined effects of MP-antibiotic mixtures on bacterial resistance, which will promote the development of environmental risk assessment of MPs from the perspective of bacterial resistance.

Frequency of Conjugal Transfer of Genes Between Non-pathogenic and Pathogenic Agrobacteria

Frequency of Conjugal Transfer of Genes Between Non-pathogenic and Pathogenic Agrobacteria

Simulated Gastric Acid Promotes the Horizontal Transfer of Multidrug Resistance Genes across Bacteria in the Gastrointestinal Tract at Elevated pH Levels.

The assessment of factors that can promote the transmission of antibiotic resistance genes (ARGs) across bacteria in the gastrointestinal tract is in great demand to understand the occurrence of infections related to antibiotic-resistant bacteria (ARB) in humans. However, whether acid-resistant enteric bacteria can promote ARG transmission in gastric fluid under high-pH conditions remains unknown. This study assessed the effects of simulated gastric fluid (SGF) at different pH levels on the RP4 plasmid-mediated conjugative transfer of ARGs. Moreover, transcriptomic analysis, measurement of reactive oxygen species (ROS) levels, assessment of cell membrane permeability, and real-time quantitative assessment of the expression of key genes were performed to identify the underlying mechanisms. The frequency of conjugative transfer was the highest in SGF at pH 4.5. Antidepressant consumption and certain dietary factors further negatively impacted this situation, with 5.66-fold and 4.26-fold increases in the conjugative transfer frequency being noted upon the addition of sertraline and 10% glucose, respectively, compared with that in the control group without any additives. The induction of ROS generation, the activation of cellular antioxidant systems, increases in cell membrane permeability, and the promotion of adhesive pilus formation were factors potentially contributing to the increased transfer frequency. These findings indicate that conjugative transfer could be enhanced under certain circumstances in SGF at elevated pH levels, thereby facilitating ARG transmission in the gastrointestinal tract. IMPORTANCE The low pH of gastric acid kills unwanted microorganisms, in turn affecting their inhabitation in the intestine. Hence, studies on the factors that influence antibiotic resistance gene (ARG) propagation in the gastrointestinal tract and on the underlying mechanisms are limited. In this study, we constructed a conjugative transfer model in the presence of simulated gastric fluid (SGF) and found that SGF could promote the dissemination of ARGs under high-pH conditions. Furthermore, antidepressant consumption and certain dietary factors could negatively impact this situation. Transcriptomic analysis and a reactive oxygen species assay revealed the overproduction of reactive oxygen species as a potential mechanism by which SGF could promote conjugative transfer. This finding can help provide a comprehensive understanding of the bloom of antibiotic-resistant bacteria in the body and create awareness regarding the risk of ARG transmission due to certain diseases or an improper diet and the subsequent decrease in gastric acid levels.

Microplastics exacerbate co-occurrence and horizontal transfer of antibiotic resistance genes

Microplastic pollution is a rising environmental issue worldwide. Microplastics can provide a niche for the microbiome, especially for antibiotic-resistant bacteria, which could increase the transmission of antibiotic resistance genes (ARGs). However, the interactions between microplastics and ARGs are still indistinct in environmental settings. Microplastics were found to be significantly correlated with ARGs (p < 0.001), based on the analysis of samples taken from a chicken farm and its surrounding farmlands. Analysis of chicken feces revealed the highest abundance of microplastics (14.9 items/g) and ARGs (6.24 ×108 copies/g), suggesting that chicken farms could be the hotspot for the co-spread of microplastics and ARGs. Conjugative transfer experiments were performed to investigate the effects of microplastic exposure for different concentrations and sizes on the horizontal gene transfer (HGT) of ARGs between bacteria. Results showed that the microplastics significantly enhanced the bacterial conjugative transfer frequency by 1.4–1.7 folds indicating that microplastics could aggravate ARG dissemination in the environment. Potential mechanisms related to the up-regulation of rpoS, ompA, ompC, ompF, trbBp, traF, trfAp, traJ, and down-regulation of korA, korB, and trbA were induced by microplastics. These findings highlighted the co-occurrence of microplastics and ARGs in the agricultural environment and the exacerbation of ARGs' prevalence via rising the HGT derived from microplastics.

Ozonation enables to suppress horizontal transfer of antibiotic resistance genes in microbial communities during swine manure composting

Although aerobic composting process has been proposed as an efficient approach to treat livestock manure, the evolution patterns of extracellular antibiotic resistance genes (eARGs) and intracellular antibiotic resistance genes (iARGs) and their horizontal transfer during composting are unclear. Here, the variations of iARGs and eARGs during swine manure composting were systematically analyzed. We found that the abundance of ARGs rebounded from thermophilic phase to mature phase. This was due to the release of persistent eARGs and the increase of certain iARGs during cell proliferation. The conjugative transfer frequencies of ARGs in composting communities were (1.68 ± 0.2) × 10-1 Transconjugant/Donor + Recipient (TC/D + R) in composting products, which was comparable to the frequency before the composting. Ozone was then optimized to suppress the rebound and spread of ARGs. We found that the low ozone dosage of 0.4 g/kg FW in the cooling phase could not only significantly remove iARGs (2.02-logs less than the control, 8 of 12 targeted iARGs were not detected) by eliminating the potential hosts, but also decrease the possibilities of both conjugation and transformation among the composting products by eliminating both HGT (conjugation and transformation) efficiencies of microbes and microbes themselves. Collectively, this study proposes the optimal ozonation strategy to mitigate the dissemination of ARGs in the composting products.

Fungicide exposure accelerated horizontal transfer of antibiotic resistance genes via plasmid-mediated conjugation

Co-pollution of soil with pesticide residues and antibiotic resistance genes (ARGs) is increasing due to the substantial usage of pesticides and organic fertilizers in greenhouse-based agricultural production. Non-antibiotic stresses, including those from agricultural fungicides, are potential co-selectors for the horizontal transfer of ARGs, but the underlying mechanism remains unclear. Intragenus and intergenus conjugative transfer systems of the antibiotic resistant plasmid RP4 were established to examine conjugative transfer frequency under stress from four widely used fungicides: triadimefon, chlorothalonil, azoxystrobin, and carbendazim. The mechanisms were elucidated at the cellular and molecular levels using transmission electron microscopy, flow cytometry, RT-qPCR, and RNA-seq techniques. The conjugative transfer frequency of plasmid RP4 between Escherichia coli strains increased with the rising exposure concentrations of chlorothalonil, azoxystrobin, and carbendazim, but was suppressed between E. coli and Pseudomonas putida by a high fungicide concentration (10 µg/mL). Triadimefon did not significantly affect conjugative transfer frequency. Exploration of the underlying mechanisms revealed that: (i) chlorothalonil exposure mainly promoted generation of intracellular reactive oxygen species, stimulated the SOS response, and increased cell membrane permeability, while (ii) azoxystrobin and carbendazim primarily enhanced expression of conjugation-related genes on the plasmid. These findings reveal the fungicide-triggered mechanisms associated with plasmid conjugation and highlight the potential role of non-bactericidal pesticides on the dissemination of ARGs.

Recipient UvrD helicase is involved in single- to double-stranded DNA conversion during conjugative plasmid transfer.

Dissemination of antibiotic resistance, a current societal challenge, is often driven by horizontal gene transfer through bacterial conjugation. During conjugative plasmid transfer, single-stranded (ss) DNA is transferred from the donor to the recipient cell. Subsequently, a complete double-stranded (ds) plasmid molecule is generated and plasmid-encoded genes are expressed, allowing successful establishment of the transconjugant cell. Such dynamics of transmission can be modulated by host- or plasmid-encoded factors, either in the donor or in the recipient cell. We applied transposon insertion sequencing to identify host-encoded factors that affect conjugative transfer frequency in Escherichia coli. Disruption of the recipient uvrD gene decreased the acquisition frequency of conjugative plasmids belonging to different incompatibility groups. Results from various UvrD mutants suggested that dsDNA binding activity and interaction with RNA polymerase are dispensable, but ATPase activity is required for successful plasmid establishment of transconjugant cells. Live-cell microscopic imaging showed that the newly transferred ssDNA within a uvrD- recipient often failed to be converted to dsDNA. Our work suggested that in addition to its role in maintaining genome integrity, UvrD is also key for the establishment of horizontally acquired plasmid DNA that drives genome diversity and evolution.

High-pressure processing effect on conjugal antibiotic resistance genes transfer in vitro and in the food matrix among strains from starter cultures

This study analyzed the effect of high-pressure processing (HPP) on the frequency of conjugal gene transfer of antibiotic resistance genes among strains obtained from starter cultures. Gene transfer ability was analyzed in vitro and in situ in the food matrix. It was found that the transfer of aminoglycoside resistance genes did not occur after high-pressure treatment, either in vitro or in situ. After exposure to HPP, the transfer frequencies of tetracycline, ampicillin and chloramphenicol resistance genes increased significantly compared to the control sample, both in vitro and in situ. The frequency of resistance genes transfer in the food matrix in the pressurized samples did not differ significantly from the in vitro transfer rate. Minimum Inhibitory Concentrations (MICs) for these antibiotics determined for transconjugants were lower or equal to MICs determined for the donors. No significant differences were observed between the MIC values determined for the transconjugants obtained in vitro and in situ. The results suggest that HPP may contribute to the spread of antibiotic resistance. This points to the need to verify starter cultures strains for their antibiotic resistance and pressurization parameters to avoid spreading antibiotic resistance genes.

Triadimefon Promoted Plasmid-mediated Conjugative Transfer of Multiple Antibiotic Resistance Genes within Bacterial Genus

The rapid spread of antibiotic resistance genes (ARGs) has been a worldwide threat to the public health, especially via horizontal gene transfer (HGT). Conjugation is one of the major pathways of HGT. Triadimefon (TF) is a broad-spectrum antifungal compound used to control rust and powdery mildew on crops. Nevertheless, it is not known whether TF could affect the conjugation of ARGs and potential molecular mechanisms. Here, the effects of TF on conjugative transfer of RP4 plasmid within Escherichia coli were systematically investigated. The results demonstrated that TF increased the transconjugant number by 1.41–2.21 folds and the frequency of conjugative transfer by 1.49–2.22 folds at concentrations of 0.1–10 mg/L. There was no obvious change in the number of donor and recipient strains in the mating system of intra-genus with TF. The main mechanisms include increasing intracellular reactive oxygen species and membrane permeability. Our findings highlight the promotion effect of TF on ARG conjugation, providing evidence of the risk of non-antibiotic agrochemical use in ARG dissemination.

Nanoplastics promote the dissemination of antibiotic resistance through conjugative gene transfer: implications from oxidative stress and gene expression

S-PSNPs facilitated conjugative transfer frequency of ARGs between E. coli strains than L-PSNPs, and the mechanisms include SOS response, membrane permeability and altered related gene expression.

Structural effects of sulfonamides on the proliferation dynamics of sulfonamide resistance genes in the sequencing batch reactors and the mechanism

Antibiotic resistance genes (ARGs) can be easily promoted by antibiotics, however, the structural effects of antibiotics on the proliferation of ARGs dynamic and the associated mechanisms remain obscure in, especially, activated sludge sequencing batch reactors. In the present study, the effects of 9 sulfonamides (SAs) with different structures on the proliferation dynamic of sulfonamide resistance genes (Suls) in the activated sludge sequencing batch reactors and the corresponding mechanisms were determined (30 days), and the results showed that the largest proliferation value (∆AR) of Suls dynamic for SAs (sulfachloropyridazine) was approximately 2.9 times than that of the smallest one (sulfadiazine). The proliferation of Suls was significantly related to the structural features (minHBint6, SssNH, SHBd and SpMax2_Bhm) that represent the biological activity of SAs. To interpret the phenomenon, a mechanistic model was developed and the results indicated that the biodegradation of SAs (T1/2) rather than conjugative transfer frequency or mutation frequency tends to be the key process for affecting Suls proliferation. T1/2 was proved to be dependent on the interactions between SAs and receptors (Ebinding), the cleavage mode (bond dissociation energy), and the site of nucleophilic assault. Besides, the metagenomic analysis showed that SAs posed significant effect on antibiotic resistome and Tnp31 played a vital role in the proliferation of Suls. Overall, our findings provide important insight into a theoretical basis for understanding the structural effects of SAs on the proliferation of ARGs in SBR systems.

Dose-dependent joint resistance action of antibacterial mixtures in their hormetic effects on bacterial resistance based on concentration addition model

The judgment of joint resistance action is significant for evaluating the resistance risk of antibacterial mixture. Using bacterial mutation frequency (MF) and conjugative transfer frequency (CTF) to respectively characterize the bacterial endogenous and exogenous resistance, mutation unit and conjugative transfer unit have been proposed to judge the joint resistance action of antibacterial mixture at a certain dose. However, these methods could not evaluate the antibacterial mixture's joint resistance action at a larger concentration-range. In this study, the concentration addition for bacterial resistance (CA-BR) approach was used to judge the joint resistance actions between kanamycin sulfate (KAN) and some other typical antibacterial agents, including sulfonamides (SAs), sulfonamide potentiators (SAPs), and silver antibacterial compounds (SACs). Through comparing the hormetic dose-response curves of the binary mixtures on the MF (or CTF) in Escherichia coli (E. coli) and the corresponding CA-BR curves calculated from the hormetic dose-responses of the single agents, the joint resistance actions between KAN and other agents were judged to exhibit dose-dependent feature: with the increase of mixture concentration, the joint mutation actions between KAN and SAs (or SAPs) were fixed at synergism, and the joint mutation actions between KAN and SACs varied from antagonism to synergism; the joint conjugative transfer actions between KAN and other agents changed from antagonism to synergism. Mechanistic explanation suggested that the heterogeneous pattern of joint resistance action had a close relationship with the interplays among the agents' modes of action, and meanwhile was significantly influenced by their joint survival pressure on E. coli. This study reveals the dose-dependent feature for the joint resistance action of antibacterial mixture and highlights the importance of exposure concentration, which will benefit clarifying the resistance risk of antibacterial mixture in the environment.

Natural organic matters promoted conjugative transfer of antibiotic resistance genes: Underlying mechanisms and model prediction

Dissemination of antibiotic resistance gene (ARG) is a huge challenge around the world. Natural organic matter (NOM) is one of the most commonly components in aquatic systems. Information regarding ARG transfer induced by NOM is still lacking. In this study, experimental exploration and model prediction on RP4 plasmid conjugative transfer between bacteria under NOM exposure was conducted. Compared with no exposure, the conjugative transfer frequency of RP4 plasmid increased 7.1-fold and 3.2-fold under exposure to 10 kDa and 100 kDa NOM exposure, respectively. NOM exposure with a lower molecular weight and higher concentration promoted gene expressions related to reactive oxygen species generation, cell membrane permeability, intercellular contact, quorum sensing, and energy driving force. Concurrently, the expressions of conjugation genes in RP4 plasmid were also upregulated. Moreover, model prediction demonstrated that the maintenance of the acquired plasmid was shortened to 133 h under 10 kDa NOM exposure compared with the control (200 h). Long-term NOM exposure enhanced transfer frequency and transfer rate of ARG. This study firstly theoretically and experimentally revealed the underlying mechanisms for promoting ARG transfer by NOM.

Effects comparison between the secondary nanoplastics released from biodegradable and conventional plastics on the transfer of antibiotic resistance genes between bacteria

Antibiotic resistance genes (ARGs) have caused widespread concern because of their potential harm to environmental safety and human health. As substitutes for conventional plastics, the toxic effects of short-term degradation products of biodegradable plastics (polylactic acid (PLA) and polyhydroxyalkanoates (PHA)) on bacteria and their impact on ARGs transfer were the focus of this study. After 60 days of degradation, more secondary nanoplastics were released from the biodegradable plastics PLA and PHA than that from the conventional plastics polystyrene (PS). All kinds of nanoplastics, no matter released from biodegradable plastics or conventional plastics, had no significant toxicity to bacteria. Nanoplastic particles from biodegradable plastics could significantly increase the transfer efficiency of ARGs. Although the amount of secondary nanoplastics produced by PHA microplastics was much higher than that of PLA, the transfer frequency after exposure to PLA was much higher, which may be due to the agglomeration of PHA nanoplastics caused by plastic instability in solution. After exposure to the 60d PLA nanoplastics, the transfer frequency was the highest, which was approximately 28 times higher than that of control. The biodegradable nanoplastics significantly enhanced the expression of the outer membrane pore protein genes ompA and ompC, which could increase cell membrane permeability. The expression levels of trfAp and trbBp were increased by repressed major global regulatory genes korA, korB, and trbA, which eventually led to an increase in conjugative transfer frequency. This study provides important insights into the evaluation of the environmental and health risks caused by secondary nanoplastics released from biodegradable plastics.

Chlorination of methotrexate in water revisited: Deciphering the kinetics, novel reaction mechanisms, and unexpected microbial risks

Chlorination of a typical anticancer drug with annually ascending use and global prevalence (methotrexate, MTX) in water has been studied. In addition to the analysis of kinetics in different water/wastewater matrices, high-resolution product identification and in-depth secondary risk evaluation, which were eagerly urged in the literature, were performed. It was found that the oxidation of MTX by free available chlorine (FAC) followed first-order kinetics with respect to FAC and first-order kinetics with respect to MTX. The pH-dependent rate constants (kapp) ranged from 170.00 M−1 s−1 (pH 5.0) to 2.68 M−1 s−1 (pH 9.0). The moiety-specific kinetic analysis suggested that 6 model substructures of MTX exhibited similar reactivity to the parent compound at pH 7.0. The presence of Br− greatly promoted MTX chlorination at pH 5.0–9.0, which may be ascribed to the formation of bromine with higher reactivity than FAC. Comparatively, coexisting I− or humic acid inhibited the degradation of MTX by FAC. Notably, chlorination effectively abated MTX in different real water matrices. The liquid chromatography-high resolution mass spectrometry analysis of multiple matrix-mediated chlorinated samples indicated the generation of nine transformation products (TPs) of MTX, among which seven were identified during FAC oxidation for the first time. In addition to the reported electrophilic chlorination of MTX (the major and dominant reaction pathway), the initial attacks on the amide and tertiary amine moieties with C-N bond cleavage constitute novel reaction mechanisms. No genotoxicity was observed for MTX or chlorinated solutions thereof, whereas some TPs were estimated to show multi-endpoint aquatic toxicity and higher biodegradation recalcitrance than MTX. The chlorinated mixtures of MTX with or without Br− showed a significant ability to increase the conjugative transfer frequency of plasmid-carried antibiotic resistance genes within bacteria. Overall, this work thoroughly examines the reaction kinetics together with the matrix effects, transformation mechanisms, and secondary environmental risks of MTX chlorination.