Transfer Of Antibiotic Resistance Genes Research Articles

A scientific research training programme for teaching biomedical students to identify the horizontal transfer of antibiotic resistance genes.

Worldwide prevalence of multi-antibiotic resistant bacteria is rapidly increasing, and the education of undergraduates and graduates about antibiotic resistance and its associated horizontal gene transfer is critical in the general effort to confront the spread of antibiotic resistance. In this study, a deeper understanding of antibiotic resistance and horizontal gene transfer was achieved by biomedical undergraduate students through a scientific research programme. The enthusiasm of students to participate in the training programme was very high, and results revealed that each student could identify the antibiotic resistance integrative and conjugative element from the Stenotrophomonas maltophilia MER1 genome. Each student could also draw the phylogenetic relationship of the antibiotic resistance integrative and conjugative element. In addition, students proved the horizontal transfer of antibiotic resistance genes from S. maltophilia MER1 to Escherichia coli strain 25DN through conjugation and PCR assays. Each group of students was able to obtain the expected results, indicating that the outcome of the scientific research programme was highly reproducible. This programme improved the theoretical knowledge about antibiotic resistance and horizontal gene transfer and the research skills of biomedical sciences students. Through this programme, students learned that antibiotic resistance genes can be horizontally transferred among different bacteria, laying a solid foundation for students to value the importance of the appropriate use of antibiotics in their future work and life.

Viral community and antibiotic resistance genes carried by virus in soil microbial fuel cells

Soil microbial fuel cells (MFCs) can control the horizontal transfer of antibiotic resistance genes (ARGs) by reducing the abundance of mobile genetic elements. However, little is known about the effect of soil MFCs on the horizontal transfer pathway of ARGs transduced by viruses. In this study, the average abundance of ARGs in soil MFCs was 11 % lower than that in the open-circuit control. Lower virus abundance in soil MFCs suggested less detriment of microbial communities. The structure of the viral community was respectively shifted by the introduction of electrodes and the stimulation of biocurrent, especially for the top three viral genera Oslovirus, Tequatrovirus and Incheonvrus in soil. The ARGs aac(6)-I, cat chloramphenicol acetyltransferase, qnrA and vanY were found as the highest health risk (Rank I), and their total abundance showed the lowest in MFCs, with a decrease of 91–99 % compared to the controls. As the main carrier of ARGs, the abundance of Caudoviricetes showed a significant positive correlation with ARGs. Viral integrase was identified respectively coexisting with arnA and vanR (Rank III) in the same contig, which might aggravate their horizontal transfer. Proteobacteria was the main host of viruses carrying ARGs, which exhibited the lowest abundance in the soil MFC. The genus Pseudomonas was the host of viruses carrying ARGs, whose amount reduced by soil MFCs. This study provides an insight into the bioelectrochemical control of ARGs horizontal transfer.

Quantitative microbial risk assessment of antibiotic-resistant Salmonella enterica contaminating hydroponic leafy vegetables

Hydroponics plays an important role in addressing food security concerns, particularly in countries aiming to increase food self-sufficiency. However, it is vulnerable to microbial contamination, and biofilms formed in hydroponic facilities may promote horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). Eventually, the bacteria are internalized into the edible parts of the vegetable through the roots, which can lead to human exposure to antibiotic-resistant pathogenic bacteria. Microbial risk assessment can play a pivotal role in microbial risk management; however, it has not been conducted for hydroponic systems. In this study, a quantitative microbial risk assessment of hydroponic vegetables was performed using literature values regarding the concentration of Salmonella spp. in hydroponics, efficiency of HGT, probability and rate of internalization, vegetable consumption patterns, and dose-response relationships. Furthermore, a sensitivity analysis was performed to identify the factors that had a significant impact on the infection probability per single exposure event for all Salmonella spp. by calculating Spearman's correlation coefficients. The estimated annual probability of infection per person by all Salmonella spp. was 2.04×10−1, while the estimated probability of infection from Salmonella spp. that acquired ARGs was 2.54×10−6. Our sensitivity analysis showed the correlation between the occurrence of internalization and hydroponic contamination levels, highlighting the need for increased awareness and regulatory action.

Metagenomics and selective culture reveal the major transfer and prevalence of antibiotic resistance genes in Tibetan Plateau animals

The Tibetan Plateau, despite being the highest and least populated plateau on the Earth, harbors antibiotic-resistant bacteria (ARBs) as well as antibiotic resistance genes (ARGs) in its pristine environment and animals. There is, however, limited knowledge regarding antibiotic resistance in Tibetan Plateau animals. Here, fecal samples of grazing yaks and intestinal content samples of wild rats were collected at altitudes of 2920m and 4360m and subjected to selective culture, PCR, and metagenomic sequencing. The results revealed a higher antibiotic resistance rate in low-altitude regions than in high-altitude regions. The simultaneous presence of bacteria exhibiting different antibiotic resistance phenotypes across multiple samples suggests the potential emergence of multidrug-resistant strains within the animal gut microbiota. Pseudomonas spp. and Escherichia coli were the predominant ARBs. In contrast to low-altitude regions with frequent human activities, ARG dissemination in the Tibetan Plateau did not show a significant correlation with mobile genetic elements. ARGs in high-altitude regions relied more on vertical gene transmission. This study expands our knowledge of antibiotic resistance in grazing and wild animals inhabiting the Tibetan Plateau, thereby highlighting the potential threat posed by antibiotic resistance in this region.

Strategy to develop and validate digital droplet PCR methods for global antimicrobial resistance wastewater surveillance.

According to World Health Organization (WHO), antimicrobial resistance (AMR) is currently one of the world's top 10 health threats, causing infections to become difficult or impossible to treat, increasing the risk of disease spread, severe illness, disability, and death. Accurate surveillance is a key component in the fight against AMR. Wastewater is progressively becoming a new player in AMR surveillance, with the promise of a cost-effective real-time tracking of global AMR profiles in specific regions. One of the most useful analytical methods for wastewater surveillance is currently based on real-time PCR (qPCR) and digital droplet PCR (ddPCR) technologies. As stated in the EU Wastewater Treatment Directive proposal, methodological standardization, including a workflow for method development and validation, will play a crucial role in global monitoring of AMR in wastewater. However, according to our knowledge, there are currently no qPCR and ddPCR methods for AMR surveillance available that have been validated according to international standard performance criteria. Therefore, this study proposes a workflow for the development and validation of PCR-based methods for a harmonized and global AMR surveillance, including the construction of specific sequence databases and microbial collections for an efficient method development and method specificity evaluation. Following this strategy, we have developed and validated four duplex ddPCR methods responding to international standard performance criteria, focusing on seven AMR genes (ARG's), including extended spectrum beta-lactam (blaCTX-M), carbapenem (blaKPC-2/3), tetracycline (tet(M)), erythromycin (erm(B)), vancomycin (vanA), sulfonamide (sul2), and aminoglycoside (aac(3)-IV), as well as one indicator of antibiotic (multi-) resistance and horizontal gene transfer, named the class I integron (intl1). The performance of these ddPCR methods was successfully assessed for their specificity, as no false-positive and false-negative results were observed. These ddPCR methods were also considered to be highly sensitive as showing a limit of detection below 25 copies of the targets. In addition, their applicability was confirmed using 14 wastewater samples collected from two Belgian water resource recovery facilities. The proposed study represents therefore a step forward to reinforce method harmonization in the context of the global AMR surveillance in wastewater. PRACTITIONER POINTS: In the context of wastewater surveillance, no PCR-based methods for global AMR monitoring are currently validated according to international standards. Consequently, we propose a workflow to develop and validate PCR-based methods for a harmonized and global AMR surveillance. This workflow resulted here in four duplex ddPCR methods targeting seven ARGs and one general indicator for mobilizable resistance genes. The applicability of these validated ddPCR methods was confirmed on 14 wastewater samples from two Belgian water resource recovery facilities.

Potential for synergism of sequential UV-NaClO disinfection on ARGs removal in municipal sewage: Effectiveness, kinetics, and mechanisms

Conventional disinfection technologies have limitations in effectively addressing the challenges posed by the emerging pollutant of antibiotic resistance genes (ARGs). To reduce the risk of antibiotic resistance in wastewater treatment plant (WWTP) effluent, this study investigated the effectiveness of ultraviolet (UV) irradiation followed by Sodium hypochlorite (NaClO) (sequential UV-NaClO) disinfection in removing six classes and 18 subtypes of ARGs in the secondary effluent of a municipal WWTP. The study showed that UV disinfection could reduce tetracycline ARGs (TCs-ARGs) subtype tetQ by 0.98 log, while it was ineffective in removing sulfonamide ARGs (SAs-ARGs). NaClO disinfection was very effective in removing ARGs. Increasing the disinfectant dosage is more effective than extending the disinfection time. The highest removal was observed for the SAs-ARGs subtype sul1 with 4.15 log and the β-lactams-ARGs(β-LACs-ARGs) subtype blaCTX-M1 with 3.87 log. Sequential UV-NaClO disinfection has the potential for synergistic effects in disinfection. Sequential UV-NaClO disinfection with a UV dosage of 15 mJ/cm2 and a NaClO dosage of 10 mg/L can effectively remove 1.46 log of the total 18 subtypes of ARGs, which is 0.73 log higher than the removal achieved by standalone UV irradiation at a fluence of 100 mJ/cm2, and equivalent to the results obtained with NaClO disinfection alone at a dose of 30 mg/L. Additionally, sequential UV-NaClO disinfection can reduce the repair efficiency of ARGs. The proposed modified Hom model can be used to analyze the kinetics of sequential UV-NaClO disinfection synergy. Analysis of microbial community structure and co-occurrence of ARGs showed synchronized and consistent removal of ARGs and their potential bacterial hosts. Sequential UV-NaClO disinfection could achieve 99 % removal of more than half of the genera. Overall, our results indicate that sequential disinfection is a promising disinfection technology for disinfecting municipal wastewater and reducing the spread and transfer of ARGs.

Tire wear particles enhance horizontal gene transfer of antibiotic resistance genes in aquatic ecosystems

Microplastics (MPs) have introduced new surfaces for biofilm development and gene exchange among bacteria. We investigated Tire Wear Particles (TWPs) for their involvement in horizontal gene transfer (HGT), particularly in relation to associated metals in the matrices of TWPs. We employed red-fluorescently tagged E. coli strain as a donor with green-fluorescently tagged, broad-host-range plasmid pKJK5, resistant to trimethoprim. As a recipient, we utilized Pseudomonas sp. and a natural lake microbial community. HGT activity on TWPs was determined and compared with that on polystyrene (PS) (with and without metals), and chitosan, which was used as a natural surface. Exposure to TWPs significantly enhanced HGT frequency of antibiotic resistance gene (ARG) from donor to recipient compared to PS and chitosan, and metals of TWPs further promoted HGT. HGT frequency on TWPs with Pseudomonas sp. was found to be 10−3 at 30 °C. in the lake community, it was similarly high already at 25 °C suggesting a higher permissiveness of the natural microbial community towards ARG at lower temperatures. This study sheds light on the potential impact of TWPs in promoting HGT, forming the basis for health risk assessments of TWPs and more generally of MP pollution in various aquatic ecosystems.

How nanoscale plastics facilitate the evolution of antibiotic resistance?

The plastic can enhance the proliferation of antibiotic resistance genes (ARGs), however, the effect of nanoplastics (NPLs) on bacterial antibiotic resistance has not been clearly explained. Herein, we explored the effects and mechanisms of NPLs of different sizes (200 and 600 nm) on the evolution of antibiotic resistance in Serratia marcescens. The results indicated that the evolution of bacterial antibiotic resistance could be promoted under NPLs exposure, which the median of relative abundance of ARGs was 1.11–1.46 times compared to the treatment without NPLs. Transcriptomic analysis showed that the larger size of NPLs mainly increased the permeability of bacterial cell membranes to efflux antibiotics, thus potentiating antibiotic resistance. While, the smaller NPLs is more than that, its enhanced the expression of antibiotic resistance by modulating bacterial metabolic processes. The genome SNP analysis found that the NPLs could cause the genetic mutation occurrence to alter the membrane transport and metabolism processes, and it increased at a size of 200 nm more than at 600 nm NPLs. Importantly, we demonstrated that the horizontal transfer of ARGs was augmented due to the NPLs could dock to bacterial surface proteins and pull their movement to contact with other bacteria (binding energy of membrane proteins: −8.54 kcal/mol), especially the smaller size. It suggests that NPLs will also contribute to the proliferation of ARGs in the environment. This study provides data for understanding the risk of bacterial resistance.

A TaqMan real-time PCR assay for detection of qacEΔ1 gene in Gram-negative bacteria

The transfer of biocide and antibiotic resistance genes by mobile genetic elements is the most common mechanism for rapidly acquiring and spreading resistance among bacteria. The qacEΔ1 gene confers the resistance to quaternary ammonium compounds (QACs). It has also been considered a genetic marker for the presence of class 1 integrons associated with multidrug-resistant (MDR) phenotypes in Gram-negative bacteria. In this study, a TaqMan real-time PCR assay was developed to detect the qacEΔ1 gene in Gram-negative bacteria. The assay has a detection limit of 80 copies of the qacEΔ1 gene per reaction. No false-positive or false-negative results have been observed. Simultaneous amplification and detection of the 16S rRNA gene is performed as an endogenous internal amplification control (IAC). The TaqMan real-time PCR assay developed is a rapid, sensitive, and specific method that could be used to monitor resistance to QACs, the spread of class 1 integrons, and the prediction of associated MDR phenotypes in Gram-negative bacteria.

Antibiotic resistance genes are transferred from manure-contaminated water bodies to the gut microbiota of animals through the food chain

Fecal-contaminated water may enter the food chain and become an important route for the transmission of antibiotic resistance genes (ARGs) to the human microbiome. However, little is known about the spread of ARGs from fecal contamination in water bodies along the aquatic food chain. In this study, laboratory-raised Daphnia magna and Aristichthys nobilis were used to investigate the effects of the addition of manure on target ARGs in water and their intestinal contents to determine the potential transmission route of ARGs in the aquatic food chain system. The abundance of target ARGs in water as well as D. magna and A. nobilis intestinal contents significantly increased when fecal contamination was present. ARGs bioaccumulated along the food chain, with four ARGs (tetM-01, tetX, qnrS, and sul2) detected regularly. Mn and Cr were key environmental factors that promoted the transfer of ARGs along the food chain. Fecal addition significantly changed the structure of microbial communities in water, D. magna gut, and A. nobilis gut. The ARG spectrum was significantly correlated with the composition and structure of the bacterial community. Proteobacteria, Bacteroidetes, and Firmicutes were identified as the main host bacteria and were likely to act as carriers of ARGs to promote the spread of antibiotic resistance in the food chain. The composition and structure of bacterial communities, along with mobile genetic elements, were two key drivers of ARG transfer. These findings provide new insights into the distribution and spread of ARGs along the freshwater food chain.

Aged Microplastics and Antibiotic Resistance Genes: A Review of Aging Effects on Their Interactions.

Background: Microplastic aging affects the dynamics of antibiotic resistance genes (ARGs) on microplastics, yet no review presents the effects of microplastic aging on the associated ARGs. Objectives: This review, therefore, aims to discuss the effects of different types of microplastic aging, as well as the other pollutants on or around microplastics and the chemicals leached from microplastics, on the associated ARGs. Results: It highlights that microplastic photoaging generally results in higher sorption of antibiotics and ARGs due to increased microplastic surface area and functional group changes. Photoaging produces reactive oxygen species, facilitating ARG transfer by increasing bacterial cell membrane permeability. Reactive oxygen species can interact with biofilms, suggesting combined effects of microplastic aging on ARGs. The effects of mechanical aging were deduced from studies showing larger microplastics anchoring more ARGs due to rough surfaces. Smaller microplastics from aging penetrate deeper and smaller places and transport ARGs to these places. High temperatures are likely to reduce biofilm mass and ARGs, but the variation of ARGs on microplastics subjected to thermal aging remains unknown due to limited studies. Biotic aging results in biofilm formation on microplastics, and biofilms, often with unique microbial structures, invariably enrich ARGs. Higher oxidative stress promotes ARG transfer in the biofilms due to higher cell membrane permeability. Other environmental pollutants, particularly heavy metals, antibacterial, chlorination by-products, and other functional genes, could increase microplastic-associated ARGs, as do microplastic additives like phthalates and bisphenols. Conclusions: This review provides insights into the environmental fate of co-existing microplastics and ARGs under the influences of aging. Further studies could examine the effects of mechanical and thermal MP aging on their interactions with ARGs.

Specific aspects of bacteriophage regulation: international practices and future developments

INTRODUCTION. The increasing prevalence of multidrug-resistant strains of pathogens determines the need for fundamentally new antibacterial agents, including bacteriophage preparations. The consistent implementation of phage therapy is hindered by the lack of generally accepted standardised regulatory documents governing the legal and methodological aspects of the production and preclinical and clinical studies of bacteriophage preparations.AIM. This study aimed to analyse the international experience with the production and lifecycle management of bacteriophage preparations, as well as the main regulatory requirements for the control of their quality, safety, and efficacy.DISCUSSION. It is difficult to develop virulent bacteriophage preparations in accordance with the existing requirements for other medicinal products because of the biological characteristics of bacteriophages, the wide variety of bacteriophage strains, and the potential for rapid changes both in the bacteriophage population and in the pathogen population. Therefore, it is reasonable to develop streamlined marketing authorisation routes for phage therapies and methods for the assessment of their safety and efficacy. As part of these efforts, it is necessary to assess the adverse events specific to this group of medicinal products, such as the risks of lysogeny, resistance to bacteriophages, and antibiotic resistance gene transfer between bacterial strains. The pharmaceutical development of bacteriophage preparations can be based on several approaches. Many countries worldwide, including the United States, are implementing the concept of Quality by Design, considering approaches based on the Biological Master File, and conducting Expanded Access programmes. The Active Substance Master File procedure allows the submission of a separate document package covering only part of the registration dossier for regulatory approval. Expanded Access programmes provide individual patients with access to innovative medicinal products without approved treatment protocols. In the Russian Federation, the commercial production of bacteriophage medicinal products is conducted in accordance with the quality standards specified in the State Pharmacopoeia of the Russian Federation.CONCLUSIONS. There are fundamental differences in the approaches to phage therapy and its regulation around the world and in the Russian Federation. It is reasonable to supplement the current national guidelines for the safety and efficacy evaluation of bacteriophage preparations, in particular, to specify the requirements for conducting preclinical studies.

Microplastic biofilms promote the horizontal transfer of antibiotic resistance genes in estuarine environments

As emerging pollutants, microplastics can aggregate microorganisms on their surfaces and form biofilms, enriching antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Consequently, microplastic biofilms have become a focal point of research. Horizontal gene transfer is one of the primary mechanisms by which bacteria acquire antibiotic resistance, with much of the research focusing on suspended bacteria. However, microplastic biofilms, as hotspots for horizontal gene transfer, also merit significant investigation. This study primarily explored and compared the frequency of ARG conjugative transfer between suspended bacteria and microplastic biofilms. The results demonstrated that, compared to suspended bacteria, microplastic biofilms enhanced the frequency of ARG conjugative transfer by 7.2–19.6 times. Among them, biofilms on polyethylene microplastics showed the strongest promotion of conjugation. After the formation of microplastic biofilms, there was a significant increase in bacterial density within the biofilms, which raised the collision frequency of donor and recipient bacteria. Then microplastic biofilms facilitated the gene expression levels of outer membrane proteins, enhanced bacterial gene transfer capabilities, promoted the synthesis of conjugative pili, accelerated the formation of conjugative pairing systems, and elevated the expression levels of genes related to DNA replication and transfer systems, thereby enhancing the conjugative transfer of ARGs within microplastic biofilms. Among different types of microplastic biofilms, polyethylene biofilms exhibited the highest bacterial density, thus showing the highest frequency of ARG conjugation. This study highlights the risks associated with ARG conjugative transfer following the formation of microplastic biofilms and provides insights into the risks of microplastic and antibiotic resistance propagation in estuarine environments.

Insights into the removal of sulfamethazine and sulfonamide-resistant bacteria from wastewater by Fe-Mn spinel oxide modified cow manure biochar activated peroxymonosulfate: A nonradical pathway regulated by enhanced adsorption and 3d orbital electron reconstruction

Effectively regulating nonradical pathways is crucial for adapting to complex water environments in sulfate radical-based advanced oxidation processes (SR-AOPs). This study prepared manganese ferrite-modified cow-manure biochar composites (BC-FM) as peroxymonosulfate activators to remove sulfamethazine (SMT) and sulfonamide-resistant bacteria (SA-ARB) from wastewater. The extensive conjugated graphitic structure in biochar induced Fe 3d orbital electron reconstruction, promoting empty orbitals formation and enhancing peroxymonosulfate inner-sphere complexation. This facilitated 100 % electron-transfer-dominated nonradical regulation. BC-FM exhibited remarkable catalytic performance, with apparent rate constant of 0.170 min−1 for SMT, inactivating ∼8.6 log of SA-ARB, and effectively blocking horizontal transfer of antibiotic resistance genes in both single and complex pollution systems (SMT/SA-ARB). Moreover, ion leaching experiments, cycling tests, real-water experiments, fluidized-bed and fixed-bed experiments demonstrated BC-FM’s potential for prolonged utilization and practical implementation. This study offers new insights into designing high-performance, environment-friendly bimetal catalysts and provides a basis for remediating organic contaminants with SR-AOPs in livestock wastewater.

Acetylshikonin reduces the spread of antibiotic resistance via plasmid conjugation

The plasmid-mediated conjugative transfer of antibiotic resistance genes (ARGs) stands out as the primary driver behind the dissemination of antimicrobial resistance (AMR). Developing effective inhibitors that target conjugative transfer represents an efficient strategy for addressing the issue of AMR. Here, we studied the effect of acetylshikonin (ASK), a botanical derivative, on plasmid conjugation. The conjugative transfer of RP4-7 plasmid inter and intra species was notably reduced by ASK. The conjugation process of IncI2 and IncX4 plasmids harboring the mobile colistin resistance gene (mcr-1), IncX4 and IncX3 plasmids containing the carbapenem resistance gene (blaNDM-5), and IncFI and IncFII plasmids possessing the tetracycline resistance gene [tet(X4)] were also reduced by ASK. Importantly, the conjugative transfer frequency of mcr-1 positive IncI2 plasmid in mouse peritoneal conjugation model and gut conjugation model was reduced by ASK. The mechanism investigation showed that ASK disrupt the functionality of the bacterial cell membrane. Furthermore, the proton motive force (PMF) was dissipated. In addition, ASK blocked the electron transmission in bacteria's electron transport chain (ETC) through disturbing the quinone interaction, resulting in an insufficient energy supply for conjugation. Collectively, ASK is a potential conjugative transfer inhibitor, providing novel strategies to prevent the spread of AMR.

Antibiotic resistance genes transfer risk: Contributions from soil erosion and sedimentation activities, agricultural cycles, and soil chemical contamination

The transfer of antibiotic resistance genes (ARGs) pose environmental risks that are influenced by soil activity and pollution. Soil erosion and sedimentation accelerate degradation and migration, thereby affecting soil distribution and contamination. This study quantified the vertical and horizontal transfer capabilities of ARGs and simulated soil environments under various scenarios, such as erosion, agricultural cycles, and chemical pollution. The results showed that slope, runoff, and sediment volume significantly affected soil erosion and ARG transfer risks. The response of environmental factors to the transfer risk of ARGs is as follows: the promotion effect of soil deposition (average: 21.41 %) is significantly greater than the inhibitory effect of soil erosion (average: −11.31 %); the planting period (average: −64.654) is greater than the harvest period (average: −56.225); the response to soil chemical pollution is: the impact of phosphate fertilizer residues, antibiotics, and pesticide pollution is more significant. This study constructed a vertical and horizontal transfer system of ARGs in soil erosion and sedimentation environments and proposed a response analysis method for the impact of factors, such as soil erosion and sedimentation activities, agricultural cycles, and soil chemical pollution, on ARGs transfer capabilities.

The effects of small plastic particles on antibiotic resistance gene transfer revealed by single cell and community level analysis

Small plastic particles with sizes comparable to bacterial cells, widely exist in environment. However, their effects on antibiotic resistance gene (ARG) dissemination remain unclear. Using polystyrene (PS) particles (0.2µm, 2µm, 5µm, 10µm, 15µm, 20µm) as models, conjugative transfer of ARGs between the donor E. coli and different recipients (E. coli or sludge bacterial community) was investigated. Compared to the pure strain, the sludge bacterial community exposed to PS particles showed higher transfer frequencies (1.67 to 14.31 times the blank control). The transfer frequencies first decreased and then increased with particle size, and plastics similar in size to bacteria (e.g., 2µm) appear to be a transitional zone with minimal impact on ARG transmission. Furthermore, using microfluidics, in-situ observation at single cell level found that 2µm plastics can act as barriers between donor and recipient bacteria inhibiting growth, but conjugation events mostly occurred around them. Conversely, nanoplastics (e.g., 0.2µm) and larger microplastics (e.g., 20µm) significantly promote conjugation, mainly due to increased reactive oxygen species production and cell membrane permeability, or facilitating bacterial adhesion and biofilm formation, respectively. This study aids in assessing environmental risks of small plastic particles on ARG dissemination.

The removal of antibiotic resistance bacteria and genes and inhibition of RP4 plasmid-mediated conjugative transfer by citric acid chelated Fe(II) catalyzing peroxymonosulfate systems: Performance and mechanisms

The excessive and inappropriate usage of antibiotics accelerated the appearance and proliferation of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs), posing a considerable threat to human health and ecological systems. The objectives of this study were to construct Fe(II) chelated by citric acid (CA) catalyzing peroxymonosulfate (PMS) systems with different dosages for the elimination of ARB (E. coli DH5α) and ARGs (tetA, tetR, and blaTEM-1), and to reveal the mechanisms on ARGs conjugation transfer. The processes still maintained high ARB/ARGs removal rates in various water matrices (tap water, river water, sewage, and farm wastewater). The results demonstrated Fe(II)-CA/PMS system in high dosages ([Fe(Ⅱ)] = 0.10 mM; [CA] = 0.08 mM; [PMS] = 0.40 mM) significantly enhanced the removal of ARB (8-log) and ARGs (5.11-log tetA, 1.01-log tetR, and 0.62-log blaTEM-1) by promoting the production of reactive oxygen species (ROSs), thereby inhibiting conjugative transfer completely. Interestingly, Fe(II)-CA/PMS treatment at low dosages ([Fe(Ⅱ)] = 0.0025 mM; [CA] = 0.002 mM; [PMS] = 0.02 mM) still inhibited the transfer of ARGs (2.26 × 10−4, 0.65-fold), mainly through downregulation of genes related with ROS synthesis, DNA damage repair (SOS response), ATP synthesis, and transfer-related genes, as well as upregulation of globally regulatory genes. These results presented a hopeful strategy for holding back the dissemination of antibiotic resistance in environments.

Mechanisms of horizontal gene transfer and viral contribution to the fate of intracellular and extracellular antibiotic resistance genes in anaerobic digestion supplemented with conductive materials under ammonia stress

The addition of conductive materials (CMs) is an effective strategy for mitigating ammonia inhibition during anaerobic digestion (AD). However, the introduction of CMs can result in increased antibiotic resistance genes (ARGs) pollution, potentially facilitated by enhanced horizontal gene transfer (HGT). The complex dynamics of intracellular and extracellular ARGs (iARGs/eARGs) and the mechanisms underlying their transfer, mediated by CMs, in ammonia-stressed AD systems remain unclear. In this study, we investigated the effects of three commonly used CMs—nano magnetite (Mag), nano zero-valent iron (nZVI), and granular activated carbon (GAC)—on the fate of iARGs and eARGs during the AD of waste activated sludge under ammonia stress. The results revealed an unexpected enrichment of iARGs by 1.5 %–10.9 % and a reduction of eARGs by 14.1 %–25.2 % in CM-supplemented AD. This discrepancy in the dynamics of iARGs and eARGs may be attributed to changes in microbial hosts and the horizontal transfer of ARGs. Notably, CMs activated prophages within antibiotic-resistant bacteria (ARB) and their symbiotic partners involved in vitamin B12 provision, leading to the lysis of ARB and the subsequent release of eARGs for transformation. Additionally, the abundance of potentially mobile ARGs, which co-occurred with mobile genetic elements, increased by 56.6 %–134.5 % with CM addition, highlighting an enhanced potential for the HGT of ARGs. Specifically, Mag appeared to promote both transformation and conjugation processes, while nZVI only promoted conjugation. Moreover, none of the three CMs had any discernible impact on transduction. GAC proved superior to both nano Mag and nZVI in controlling the enrichment of iARGs, reducing eARGs, and limiting HGTs simultaneously. Overall, these findings provide novel insights into the role of viruses and the mechanisms of ARG spread in CM-assisted AD, offering valuable information for developing strategies to mitigate ARG pollution in practical applications.

Absence of synergistic effects between microplastics and copper ions on the spread of antibiotic resistance genes within aquatic bacteria at the community level

Microplastics and copper ions (Cu2+) are favorable in accelerating the propagation of antibiotic resistance genes (ARGs) in the plastisphere, however, their combined effects on the ARG spread within the bacterial community of the natural environment were less understood. The influence of microplastic types and Cu2+ concentrations on the horizontal gene transfer (HGT) of ARGs mediated by RP4 plasmid within natural bacterial communities in aquatic environments was investigated. Both biodegradable polybutylene succinate (PBS) and non-biodegradable polyvinyl chloride (PVC) microplastics significantly enhanced the transfer of ARGs, with PBS showing a significant higher effect compared to PVC. Cu2+ also increased transconjugation rates at environmentally relevant concentrations (5 μg L−1), but higher levels (50 μg L−1) lead to decreased rates due to severe bacterial cell membrane damage. The transconjugation rates in the presence of both microplastics and Cu2+ were lower than the sum of their individual effects, indicating no synergistic effects between them on transconjugation. Proteobacteria dominated the composition of transconjugates for all the treatment. Transmission electron microscope images and reactive oxygen species production in bacterial cells indicated that the increased contact frequency due to extracellular polymeric substances, combined with enhanced membrane permeability induced by microplastics and Cu2+, accounted for the increasing transconjugation rates. The study provides valuable insight into the potential effects of microplastics and heavy metals on the spread of ARGs from donors to bacterial communities in natural environments.