Roles of micro/nanoplastics in the spread of antimicrobial resistance through conjugative gene transfer

Domain: neuroscience
Published: 2025-12-24
Editorial outcome: major_revision

Abstract
The role of micro/nanoplastics (M/NPs) in the dissemination of antimicrobial resistance (AMR) remains insufficiently understood. Here, we examine how polystyrene (PS) M/NPs of varying sizes and concentrations affect AMR gene (ARG) transfer in model systems with gram-negative (Escherichia coli) and gram-positive (Enterococcus faecalis) donors. In these systems, the ARG transfer frequency is higher for intrageneric pairs than for intergeneric pairs. The 20- and 120-nm-sized PS broadly facilitate conjugation, whereas the 1-μm-sized PS selectively promotes ARG transfer to E. coli recipients, in addition to altering the expression of conjugation- and pili-associated genes. Notably, an environmentally relevant (0.1 mg/L) concentration of PS M/NPs facilitates AMR transfer in the tested systems, which correlates with increased reactive oxygen species levels, ATP levels, and cell membrane permeability in both donors and recipients. Collectively, our findings underscore the role of M/NPs in facilitating AMR spread in specific bacterial systems, providing valuable insights for understanding their potential ecological risk in water environments.

Introduction
Plastic pollution is a global environmental issue that has attracted considerable attention worldwide1. Microplastics (MPs), which are particles derived from the degradation of larger plastic materials, are defined as particles with diameters less than 5 mm. MPs can be fragmented into nanoplastics (NPs), which typically have particle sizes less than 1 μm in diameter. Micro/nanoplastics (M/NPs), which are recognised as emerging contaminants of significant international concern, are widespread, long-lasting and nearly irreversible in various ecosystems2,3, including aquatic environments4–6 and the human gut7. Their high longevity8, with half-lives ranging from decades to more than a thousand years, makes them far more durable than most natural particles9. This persistence allows M/NPs to continuously transport and accumulate contaminants, including harmful chemicals and pathogens, across various ecosystems, thus becoming a serious and enduring environmental concern10. Thus, the ecological risks M/NPs pose and the mechanisms through which they mediate these risks are in urgent need of investigation11. Analogously, water systems can act as sources of antibiotics, facilitating the rapid development and dissemination of antimicrobial resistance (AMR)12,13. The transfer of antimicrobial resistance genes (ARGs) can lead to the acquisition of multidrug resistance by pathogens14,15, causing approximately 700,000 deaths and increased healthcare costs every year, thereby increasing the challenge of preventing and treating bacterial infections16. Hence, AMR and ARGs have become significant concerns for human health17. Various nonantibiotic substances, such as disinfectants, nonantibiotic pharmaceuticals and nanoparticles18–21, have been shown to impact the horizontal gene transfer (HGT) of plasmid-borne ARGs22. The aquatic environment serves as a reservoir for both M/NPs and ARGs, acting as a link with other ecosystems and posing potential risks to public health and the environment. Thus, investigating the ways in which ubiquitous M/NPs impact the dissemination and development of AMR is highly important and can help promote water security23.

ARGs and M/NPs can be emitted or circulated into natural and artificial environments via similar sources and pathways24. The micro-scale mechanisms underlying the influence of M/NPs as environmental factors on the transfer of ARGs remain to be explored. Most studies have shown that PS M/NPs promote ARG transfer through transformation25 and intrageneric conjugation26. Liu et al.27 reported that PS can inhibit the transfer of ARGs. However, to date, whether M/NPs can facilitate intergeneric conjugation, one of the primary mechanisms for the emergence of new multidrug-resistant pathogens, has not been reported26. Moreover, additional bacterial strains and plasmids should be tested to assess the generalisability of the observed M/NP-induced promotion of ARG transfer. It has been argued that particle size can affect the dissemination of AMR, despite its crucial role in governing the interaction between plastics and bacteria. However, existing research presents varying results regarding the impact of M/NP particle size on the transfer of ARGs. Additionally, the impact of environmentally relevant concentrations of M/NPs on the dissemination of AMR has been largely overlooked, despite its high ecological significance25. Thus, exploring the effects of varying sizes and environmentally relevant concentrations28 of PS M/NPs on both intragenus27 and intergenus AMR transfer through conjugation is environmentally significant. Determining whether environmentally relevant concentrations and representative sizes of M/NPs affect the HGT of ARGs via conjugation is highly important.

To fill this knowledge gap, we investigated the effects of bacterial evolutionary distance, particle size (20 nm, 120 nm, and 1 μm), and subminimum inhibitory concentration (0.1, 1, 10, 50, and 100 mg/L; Table S1) on the transfer of ARGs through conjugation between bacteria and explored the underlying mechanisms. PS pellets were chosen as representative plastic pellets because they are produced in large quantities and are often environmentally tested25. Among the three particle sizes, 20 nm PS can enter bacteria directly, 120 nm PS can enter cells through endocytosis, and 1 μm PS cannot enter cells29 (Fig. S1). PS was detected via pyrolysis‒gas chromatography time-of-flight mass spectrometry (Py-GC ToF), with a semi-quantifiable result of 0.2 mg/L in river water30. PS concentrations of 0.1 and 1 mg/L are environmentally relevant28,30,31. In this study, we established an intrageneric (donor E. coli K-12 LE392 and recipient E. coli BL21 (DE3)) and an intergeneric (donor E. coli K-12 LE392 and recipient P. putida KT2440) conjugative model of RP4, which encodes resistance to ampicillin, tetracycline, and kanamycin. Using gram-positive Enterococcus faecalis OG1RF carrying the pCF10 plasmid as the donor, plasmid-free E. faecalis OG1RS as the intrageneric recipient, and E. coli DH5α as the intergeneric recipient, we further tested the ability of M/NPs to promote AMR dissemination. Detailed information on the strains and plasmids can be found in Text S1 and Table S2. The underlying mechanisms promoting conjugative frequencies were revealed by phenotypic and genotypic analyses, which included a culture-based conjugation assay and the detection of reactive oxygen species (ROS), cell membrane permeability, ATP production and transcriptome responses. These findings provide evidence that PS M/NPs can promote the propagation of AMR, which is dependent on particle size, the concentration of PS and the evolutionary distance between bacteria. This study highlights the role of M/NPs as active facilitators of HGT, potentially enhancing the spread of ARGs in microbial communities, with profound ecological and human health implications, and emphasises the need for targeted strategies to mitigate M/NP pollution and AMR risk.

Results
The intrageneric conjugation frequency exceeded the intergeneric transfer frequency in the tested model bacterial systems
Plasmid transfer frequencies depend on host genotypes and plasmid‒host interactions32, which favour conjugation among phylogenetically close microorganisms33. This study further verified that conjugative transfer is more likely to occur among microorganisms with a relatively close phylogenetic distance. We found that regardless of changes in the concentration and particle size of PS, microorganisms with closer evolutionary relationships were more likely to undergo conjugation (Fig. 1). The spontaneous intrageneric transfer frequency for RP4 of ARGs was 5.20 × 10−6, whereas the spontaneous intergeneric transfer frequency was 4.71 × 10−8 (Table S3, 4). This observation also held for pCF10 (0.87 × 10-1 and 5.63 × 10−5 for spontaneous intrageneric and intergeneric transfer frequency, respectively; Table S5). Taking 20 nm PS as an example, the intrageneric conjugation transfer frequency was significantly greater than that of intergeneric conjugation at all concentrations used (Fig. 1a, b).

There was no significant difference in ROS production (Fig. 1c, d) or cell membrane permeability (Fig. 1e, f) between donors and recipients for RP4-related systems. However, compared with its intergeneric counterpart, the intrageneric conjugation system resulted in increased ROS production (Fig. 1g, h) and cell membrane permeability (Fig. 1i, j) in pCF10-related systems. Specific gene expression profiles are provided in Tables S6–S43. There was substantial enrichment of genes associated with the cell membrane in the donor, indicating that when strains with similar phylogenetic distances came into contact, the permeability of the donor cell membrane increased. With respect to the recipients, genes related to redox states were more enriched in the intrageneric recipients. These findings suggest that the ROS generated in the intrageneric conjugation system have a more pronounced effect on the recipient. In contrast, intergeneric conjugation exhibited the opposite pattern, with a more pronounced oxidative stress response in the donor and greater changes in the permeability of the recipient cell membrane. We speculate that the increased permeability of the donor cell membrane is more conducive to the occurrence of conjugation reactions. Although we failed to determine the conjugation frequency using P. putida KT2440 with RP4 as the donor, which may have occurred because of the detection baseline, the increased intrageneric reverse transfer ratio indicated that PS M/NPs could also promote the transfer of newly acquired ARGs to other strains, facilitating the spread of AMR (Fig. S2).

Twenty-nanometre PS broadly promoted ARG dissemination in the tested systems, whereas 120 nm PS had intermediate effects, and 1 μm PS significantly enhanced ARG transfer in E. coli recipient systems
Conjugative transfer experiments of RP4 involved exposing bacteria to various concentrations of three sizes (20 nm, 120 nm, and 1 μm) of PS microspheres for 8 or 2 h. Notably, 20 nm PS can directly enter cells29; 120 nm PS requires endocytosis for uptake, whereas 1 μm PS cannot penetrate cells, as shown by the TEM images in Fig. S1. TEM images of the conjugation system upon exposure to 120 nm PS are shown in Fig. S3. After plasmids from the donors and transconjugants were extracted, PCR amplification of the blaTEM-1 and tetA genes located on the plasmids was performed, followed by electrophoresis of both the plasmids and the PCR amplicons to confirm that the RP4 plasmids had been successfully transferred to the transconjugants (Fig. S4). The details of the independent-sample t-test analysis for comparing RP4-relevant conjugative transfer to the control in gram-negative bacteria can be found in Tables S3–S4. To assess the broader applicability of PS-facilitated ARG transfer, we tested an additional model conjugation system. We used the clinically relevant gram-positive strain E. faecalis OG1RF (carrying plasmid pCF10) as the donor, with plasmid-free E. faecalis OG1RS as the intrageneric recipient and E. coli DH5α as the intergeneric recipient. These strains were exposed to polystyrene (PS) particles of three sizes (20 nm, 120 nm, and 1 μm) at four concentrations (0, 0.1, 1, and 10 mg/L). The results indicated increased conjugative transfer frequencies under PS exposure across all tested sizes and concentrations (Fig. S5). To determine whether it is the size or the chemical properties of substances that play a critical role in promoting HGT, we conducted conjugation experiments with exposure to nano-clay montmorillonite, which is a moderate chemical to bacteria, at 0, 0.1, 1, and 10 mg/L. Across all tested concentrations, nano-clay did not produce a statistically significant increase in conjugative transfer frequency compared to the control (Fig. S6, p > 0.05). By contrast, the literature reports significant increases in transfer frequency upon exposure to metal nanoparticles, such as nano-TiO234, nano-Ag/Ag20, as well as Nano-CeO₂35. Correspondingly, these nanoparticles markedly promote ROS production in all donor-recipient systems.

The influence of PS particle size on ARG transfer showed distinct and concentration-dependent patterns across different conjugation systems. At 0.1 mg/L (Fig. 2a), a nonmonotonic trend was observed: the transfer frequency increased with 20 nm PS, decreased with 120 nm PS, and increased again with 1 μm PS in E. coli-based intra- and intergeneric systems, as well as in intergeneric E. faecalis. In contrast, intrageneric E. faecalis conjugation was similarly promoted by 20 and 120 nm PS, but this effect declined with 1 μm particles. At 1 mg/L (Fig. 2b), similar patterns persisted, particularly in systems involving E. coli recipients. Under 10 mg/L (Fig. 2c), the size effect diverged further; e.g., the abundance of the intragenus E. faecalis increased with increasing size, whereas in the intergeneric system, the trend reversed. Notably, in E. coli systems, 120 nm PS began to suppress ARG transfer, whereas 1 μm PS maintained a promoting role, albeit weaker than that of 20 nm. These observations indicate a nonuniform and concentration-sensitive size effect, suggesting that both PS size and exposure level modulate the frequency of horizontal gene transfer in a system-specific manner.

To rule out the possibility that the tested M/NPs affect cell viability, we calculated the total number of recipient cells as well as the cell membrane permeability and found that PS M/NPs did not significantly affect the number of recipients during the conjugation process (independent-sample t-test, p > 0.05; Fig. S7). PS M/NPs have a limited inhibitory effect on the growth of recipient cells, especially at the early log phase, when we collected the cells for conjugation experiments (Fig. S8).

In addition to enhancing ARG transfer frequency, PS particles of different sizes exhibited distinct effects on cellular processes associated with conjugation. Importantly, we further investigated the physiological responses potentially associated with PS-facilitated ARG transfer. Among the tested parameters, ROS generation showed the strongest correlation with transfer frequency, particularly in E. faecalis-based systems, where the trends in the levels of ROS corresponded to increases in ARG transfer frequency (Fig. 3a–c). In E. coli-based systems, both ROS and membrane permeability appeared to contribute, with 1 μm PS particles notably inducing significant changes in membrane permeability (Fig. 3d–f). This finding implies a recipient-specific mechanism, possibly involving physical interactions between large PS particles and bacterial surfaces or membrane destabilization under stress. To further validate the role of ROS, we conducted ROS scavenger assays, which revealed that quenching ROS significantly reduced the PS-induced increase in conjugation frequency (Fig. S9). These findings support a model in which oxidative stress is correlated with enhanced ARG transfer under PS exposure, suggesting that ROS may contribute to the observed phenotype in the tested systems. In support of this interpretation, transcriptomic profiling revealed the upregulation of stress-responsive pathways. Excessive ROS production can drive the development and propagation of multidrug resistance36. PS M/NPs induce ROS production, DNA damage, and oxidative stress37. Notably, the addition of PS to the culture medium increased ROS production (Fig. 3a–c and S10-S11). PS M/NPs increased ROS production in both the intrageneric and the intergeneric conjugative systems, suggesting that they induced an overproduction of ROS, enhanced ROS detoxification, and stimulated the oxidative stress response (SOS response) (Fig. 3g, h). This effect correlated with increased intra- and intergeneric conjugative transfer frequencies in the tested systems. Specifically, in E. faecalis systems, the size-dependent variation in ROS production closely matched the observed changes in conjugative transfer frequency. For example, at 0.1 mg/L PS (Fig. 3a), intrageneric transfer increased, but it then decreased with increasing particle size, peaking at 120 nm, a trend mirrored by ROS levels, which reached 1.27-fold that of the control under the same conditions. These findings suggest that ROS may play a primary role in regulating conjugation in gram-positive bacteria. Conversely, in E. coli systems, the observed transfer frequencies appeared to be shaped by both ROS production and membrane permeability. While 20 nm and 120 nm PS induced significant increases in both indicators, the effect of 1 μm PS was reflected mainly in enhanced membrane permeability rather than elevated ROS. Specifically, compared with the control, 1 μm PS increased permeability by 1.26-fold in intrageneric systems. This may be attributed to the physical size of the particles, which is comparable to that of bacterial cells and could promote membrane disruption or surface interaction-based mechanisms, thereby facilitating ARG transfer via a distinct pathway. This change coincided with changes in transfer frequency, where donor- and recipient-generated ROS collectively promoted the transfer of ARGs. To determine the primary ROS involved, four types of ROS were measured in this study. Among them, singlet oxygen (1O2) and hydroxyl radicals (•OH) had the most substantial effects (Fig. S11)38. Concentrations of 0.1 and 10 mg/L 20 nm PS significantly increased the intrageneric conjugation transfer frequency, with a notable increase in singlet oxygen production at these concentrations. This suggests that singlet oxygen plays a significant role in promoting intrageneric conjugation transfer. PS with a diameter of 120 nm had a less pronounced effect on donor ROS production, with only 20 nm PS significantly increasing hydroxyl radical production in intergeneric recipients. However, as the concentration of PS increased, the concentration of hydroxyl radicals significantly increased, potentially inhibiting conjugation. This excessive hydroxyl radical production might lead to microbial death39, causing a decrease in transfer frequency.

In intrageneric donors, genes involved in type IV conjugative transfer (e.g., traD, traI, and traX) were upregulated under 1 μm PS treatment, except for sfmA, whose expression was downregulated by 120 nm PS (Table S31)40. With respect to intergeneric conjugation, larger PS particles inhibited donor pili production, whereas the expression of csgA, which is related to adhesion41, was suppressed by both 120 nm and 1 μm PS. Conjugative transfer via the RP4 plasmid involves the control of global regulatory genes and conjugation-related genes42. Overall, the expression of genes related to plasmid conjugation increased (Fig. 2d–g). traL, trbH, and trbM were upregulated by 20 nm PS, whereas traL was suppressed by 120 nm PS. When exposed to 0.1 and 10 mg/L PS particles, during intrageneric conjugation, 1 μm PS inhibited htrE, ppdD, and sfmH, which are related to enabling fimbrial usher porin activity (Table S16). Notably, type IV pili genes (hofN, ppdD, sfmF, and yadN) were consistently upregulated across all PS particle sizes43. In the case of intergeneric conjugation donors, the expression of genes associated with oxidative stress (Table S10), such as rclA, trxC, and ytfE, significantly increased under the influence of all the tested PS particle sizes. However, 120 nm PS notably inhibited the expression of yfcG, a gene related to oxidative detoxification, while 1 μm PS significantly downregulated the expression of yafO, a gene associated with the SOS response. This difference could contribute to the weaker promotion of intergeneric conjugation by 1 μm PS compared to its promotion of intrageneric conjugation.

When exposed to 20 nm PS at 1 mg/L, the intrageneric ARG transfer frequency increased 2.86-fold compared with that of the controls (p = 0.0004). In the intragenus gram-positive model system (E. faecalis), 20 nm PS exposure at 1 mg/L was associated with a 2.86-fold increase in intrageneric ARG transfer frequency compared with that of the controls (p = 0.0004). In contrast, 1 μm PS promoted even greater transmission from E. faecalis OG1RF to E. coli DH5α. At 1 mg/L, 1 μm PS increased the transfer frequency to 2.97 times that of the control group. Mechanistically, the suppression of prgX expression correlated with PS-enhanced conjugation (Tables S42–S43). prgX is a key transcriptional repressor in the pheromone-responsive system of E. faecalis, and its downregulation alleviates the repression of downstream genes essential for conjugation, thereby facilitating the transfer of ARGs44. With respect to the transmission of AMR between Enterococcus species, PS notably promoted this process by increasing the expression of genes related to conjugation. Specifically, in the E. faecalis intraspecific system, 20 nm and 120 nm PS exposure correlated with the upregulation of type IV secretion system-related genes, such as mobC, pcfB, pcfJ, prgB, prgM, and prgO45 and increased ARG transfer frequency. In contrast, for interspecies transfer, PS exposure correlated with reduced expression of prgX, potentially alleviating its inhibitory role in conjugation. This effect coincided with increased ARG transfer frequency (Fig. 2a–c).

PS M/NPs also promoted ROS production in pCF10-related conjugation systems. PS (20 nm and 120 nm) significantly promoted ROS production in both the intrageneric and the intergeneric conjugation systems (Fig. 2a–b). The 1 µm PS particles significantly increased ROS production in the intergeneric conjugation system but had little effect on ROS production in the Enterococcus intrageneric conjugation system. Specifically, 1 mg/L of 20 nm and 0.1 mg/L of 120 nm PS increased ROS production in the intraspecies conjugation system by 1.6-fold (p = 0.0194) and 1.28-fold (p = 0.0459) that of the control group, respectively (Fig. 3c). In the intergeneric conjugation system, all concentrations of 1 µm PS particles significantly promoted ROS production, with the greatest increase reaching 1.34-fold (p = 0.0146) of the original value (Fig. 3d). With respect to the effect of PS on individual bacteria, PS significantly affected ROS production in the three types of bacteria, but the promotion factor was relatively low for the donor (Figure. S12a). Compared with the control, the 20 nm PS particles significantly promoted ROS production in the intraspecies recipient E. faecalis OG1RS (Fig. S12b), with a maximum increase of 2.31-fold (p = 0.0367). On the other hand, 1 µm PS particles increased ROS production in the interspecies recipient E. coli DH5α by 1.40- to 1.50-fold (p = 0.0015–0.0301) the original level (Fig. S12). Specifically, 20 nm PS significantly increased ROS production (Fig. 3a-c), cell membrane permeability (Fig. 3d-f and S13-15), and ATP levels (Fig. S16) across conjugative systems, suggesting a comprehensive increase in cellular conditions that favour gene transfer. In contrast, 120 nm PS induced moderate increases in membrane permeability and ATP production (Fig. S18), with no marked difference between the intra- and intergeneric systems, indicating a more uniform but less potent biological response. Notably, 1 μm PS specifically enhanced membrane permeability (Fig. 3d–f) in systems involving E. coli, particularly when E. coli served as the recipient, which may represent a distinct mechanism by which larger particles promote ARG dissemination. This size-specific effect warrants further mechanistic investigation to determine whether physical interactions or particle uptake pathways differ depending on the recipient cell type and particle size.

Environmentally relevant concentrations of PS M/NPs increased the ARG transfer frequency in the tested systems
The impact of PS M/NPs on the conjugative transfer of ARGs correlated with fluctuations in concentrations, all of which remained below the minimum inhibitory concentration (Table S1). Despite varying fold changes in transfer frequency across different treatments, we observed a nonlinear concentration-dependent effect of PS particles on the conjugative transfer of ARGs (Fig. 4a, b). In E. coli-donor systems, the transfer frequency initially increased with increasing PS concentration, peaked at moderate concentrations, and then decreased at higher concentrations. For instance, compared with the control, 100 mg/L 20 nm and 120 nm PS significantly suppressed ARG transfer, indicating a threshold beyond which PS may exert inhibitory effects. In contrast, when E. faecalis served as the donor, 0.1 mg/L PS of all the tested sizes significantly enhanced ARG transfer, but the promoting effect diminished or plateaued with increasing concentrations. These findings suggest that low concentrations of PS facilitate ARG dissemination, whereas high concentrations may interfere with this process, possibly because of particle aggregation, toxicity, or saturation of cellular responses. Moreover, the concentration sensitivity appeared to differ between gram-negative and gram-positive donor systems, implying that concentration effects are both nonlinear and species specific. The most substantial increase in conjugative transfer was achieved at environmentally relevant concentrations (0.1 and 1 mg/L) of PS. For instance, 0.1 mg/L PS at 20 nm, 120 nm and 1 μm significantly increased the intrageneric transfer frequency by 6.52- (p = 0.0002), 3.36- (p = 0.0006), and 10.81-fold (p = 0.0005), respectively (Fig. 4a). Conversely, 100 mg/L PS at 20 nm and 120 nm reduced the transfer frequency by 0.43-fold (p = 0.0217) and 0.79-fold (p = 0.0470), respectively.

Bacterial cell membranes serve as a barrier for horizontal material transfer, with their permeability affecting the movement of substances46. We found that the addition of thiourea had no significant effect on cell membrane permeability (Fig. S13), which suggests that M/NPs influence membrane permeability primarily through ROS-related mechanisms and that the role of thiourea is to specifically scavenge ROS activity without introducing any independent effect on cell membranes. Environmentally relevant concentrations of PS (0.1 and 1 mg/L) significantly promoted an increase in cell membrane permeability. (Fig. 4e, f and Fig. S14, S15). Treatment with 1 mg/L 20 nm PS M/NPs increased donor cell membrane permeability by up to 1.29-fold (p = 0.0013) (Fig. S14). This effect extended to recipients, with up to 1.35- and 1.32-fold increases (p = 0.0002–0.0075) (Fig. 4e, f). At 0.1 mg/L, the three sizes of PS increased the donor permeability by 1.38-fold (p = 0.0419), 1.30-fold (p = 0.0490), and 1.33-fold (p = 0.0381), in ascending order of particle size. However, 10 mg/L PS resulted in minor increases of 1.17-fold (20 nm), 1.13-fold (120 nm), and 1.05-fold (1 μm). The impact of higher concentrations—50 mg/L and 100 mg/L—of PS of different sizes on the permeability of the donor varied between the intrageneric and intergenic models. For intrageneric recipients (Fig. 4e), the effects of 0.1 mg/L PS of all sizes were similar. For the intergeneric recipients (Figs. 4f), 1 mg/L 20 nm PS had the greatest effect, with a 1.61-fold increase in permeability. These results suggest that the cell membrane permeability of recipient cells plays a crucial role in the conjugation process. However, 0.1 mg/L 120 nm PS downregulated the expression of aaeX and sanA (Fig. 4g). The expression levels of the phoE gene and the yghE gene differed minimally, suggesting that 10 mg/L 120 nm PS may prevent the intergeneric transfer of RP4 plasmids (Fig. 4g). In the presence of 0.1 mg/L of 1 μm PS, the expression of aaeX, pdeG (related to periplasmic enzyme activity), and the efflux pump-related gene emrY increased significantly, potentially promoting intrageneric conjugation. In intergeneric conjugation donors (Fig. 4h), 0.1 mg/L 20 nm PS had a more pronounced effect on cell membrane permeability, where genes such as bhsA, gltF, pdeG, and yjbF were significantly upregulated under the influence of all three particle sizes (Tables S14–S21).

PS could increase ATP generation in both gram-positive and gram-negative bacteria (Fig. S16 and S17). Compared with the control, 0.1–50 mg/L 1 μm PS increased the production of ATP in the donor by 2.24-fold (p = 0.0046), 3.67-fold (p = 0.0007), 2.74-fold (p = 0.0029), and 1.38-fold (p = 0.0373) (Fig. S14). The highest levels of ATP production in intrageneric recipients were observed at a concentration of 0.1 mg/L for all three particle sizes of PS, where ATP production increased to 1.33-fold, 1.50-fold, and 1.34-fold, respectively, compared with that in the control group (Fig. S16b). With respect to the intergeneric recipients (Fig. S16c), 0.1 mg/L 20 nm PS promoted the highest ATP production; however, with increasing concentrations of PS, ATP production decreased. These results confirmed the influence of PS on ATP production and highlighted that donor ATP production has a greater effect on donor transfer frequency than on that of recipients (Fig. S16). With respect to genes related to ATP generation and utilisation, most genes were upregulated under 0.1 and 10 mg/L PS within the same genus (Tables S22–29). M/NPs upregulated the expression of the citC gene, which encodes citrate lyase synthetase, in E. coli, thereby affecting the tricarboxylic acid cycle and promoting ATP production in bacterial cells (Tables S22 and S24).

In summary, environmentally relevant concentrations of PS M/NPs promoted the conjugation frequency through enhancing ROS production, promoting cell membrane permeability and facilitating ATP generation. Intragenus conjugation systems of RP4 were characterised by substantial changes in recipient cell membrane permeability (Figs. S14, S15), accompanied by oxidative state alterations and increased ATP generation (Fig. S16, S17) in donors, which correlated with increased ARG transfer frequencies. In contrast, intergenus conjugation involved oxidative stress-induced changes and ATP production (Fig. S16–S18) both in donors and recipients, whereas recipients exhibited more favourable cell membrane (Fig. S14) conditions for plasmid uptake. In pCF10-related conjugation, PS M/NPs exhibited a greater promoting effect on recipients.

Methods
Bacterial strains, growth conditions, and nano-clay montmorillonite
Detailed information on the strains and plasmids can be found in Text S1 and Table S2. For RP4-related conjugation, the donor strains used in this study were E. coli K-12 LE392—carrying RP4 plasmids containing resistance genes for ampicillin (Amp), kanamycin (Kan), and tetracycline (Tet)—and E. faecalis OG1RF carrying pCF10 (TetR). The recipient strains E. coli BL21 (DE3) (ChlR), E. faecalis OG1RS (StrR), P. putida KT2440 (ChlR), and E. coli DH5α (EryR) were selected for intrageneric and intergeneric conjugation experiments. E. coli K-12 LE392 and P. putida KT2440 were sourced from the Institute of Urban Environment, Chinese Academy of Sciences. E. faecalis OG1RF and E. faecalis OG1RS were kindly provided by the Tianjin Institute of Environmental and Operational Medicine66. E. coli BL21 (DE3) and E. coli DH5α were obtained from Beijing BIOBW Company. The culture conditions for the donor and recipient bacteria are shown in Text S1. PS particles of three different sizes (120 nm, 1 μm, and 100 μm) were obtained from Huge Biotechnology (Shanghai, China). Nano-clay montmorillonite (50 nm) was purchased from Sigma (Germany).

Conjugation experiments in the presence of PS M/NPs or nano-clay montmorillonite
This study established two mating models to explore whether PS M/NPs could enhance ARG transfer. In the environmentally relevant model from E. coli K-12 LE391 with RP4 to E. coli BL21 (DE3) or P. putida KT2440, donors and recipients at 108 cfu/mL were mixed 1:1 in PBS, for a total volume of 20 mL. Various concentrations of PS at different sizes (20 nm, 120 nm, and 1 μm) were added to the mating system, including environmentally relevant and sub-MIC concentrations, i.e., 0, 0.1, 1, 10, 50, and 100 mg/L28. For nano-clay montmorillonite, we did the conjugation experiments under 4 different concentration (0, 0.1, 1 and 10 mg/L). After 8 h of incubation at 25 °C without shaking, 100 μL of the mixture was spread onto LB selection plates containing antibiotics to enumerate transconjugants and recipients. The transfer frequency was calculated by dividing the number of transconjugants by the total number of recipients. To investigate the role of ROS, an ROS scavenger that can remove ROS from the system, thiourea67, was added at a final concentration of 100 μM in some experiments. Detailed information is described in Text S2.

For conjugation from E. faecalis OG1RF with pCF10 to E. faecalis OG1RS or E. coli DH5α, the mating systems were mixed 1:1 using brain heart infusion (BHI) medium and mated for only 2 h to avoid vertical gene transfer. Twenty microlitres of the mixture was spread onto BHI selection plates containing antibiotics to enumerate transconjugants and recipients, with detailed information described in Text S2.

Reverse conjugation experiments under exposure to PS M/NPs
Transconjugant bacteria obtained from the conjugation experiment (E. coli BL21 (DE3) and P. putida KT2440 with RP4 plasmid) were used as the donors, and E. coli NK5449, with resistance to Rif, was used as the recipient. The conjugation mating systems were set up as described above and included different sizes of PS M/NPs at environmentally relevant concentrations. The mating systems were then mixed by vortexing and incubated at 25 °C for 8 h without shaking. Afterwards, the mating systems were mixed and used to inoculate selection plates containing antibiotics, which were subsequently incubated as described above. Finally, the colonies of the transconjugants and recipients were counted, and the transfer frequencies were determined as described above.

Transmission electron microscopy (TEM)
TEM (Tecnai G2 Spirit, USA) at 120 kV was used to observe the effect of PS M/NPs on bacterial cells. TEM samples were collected from conjugation systems supplemented with 1 mg/L 120 nm PS or E. coli K-12 LE392 after 2 h of mating with either 1 mg/L 20 nm, 120 nm or 1 μm PS. Details are provided in Text S3.

Measurement of reactive oxygen species (ROS)
The bacterial strains were subsequently washed twice with PBS and resuspended in PBS to 106 cfu/mL. To detect total ROS, the bacterial strains were incubated with 2’,7’-dichlorofluorescein diacetate (DCFDA; final concentration, 20 μM; Abcam®) for 30 min at 37 °C in the dark. Afterwards, 100 μL of bacteria stained with DCFDA were treated with different concentrations of PS M/NPs. For the controls, 1.5% H2O2 was used as the positive control, and ethanol was used as the negative control. After complete mixing, the mixture was incubated at 25 °C in the dark for 2 h, after which the absorbance was measured at 488 nm using a microplate reader (Spark 10M, Tecan). All the detections were conducted in triplicate (Text S4).

Detection of cell membrane permeability
For cell membrane permeability detection, 100 μL of the strain was exposed to different concentrations of PS M/NPs and incubated at 25 °C for 2 h. The strains were then stained with 1 μL of propidium iodide (PI; 2 mM; Life Technologies) and incubated in the dark for 15 min before being measured at 561 nm. To rule out the possibility that thiourea may affect cell membrane permeability and thus affect conjugation frequency, the cell membrane permeability of RP4-relevant bacterial strains was detected after the addition of thiourea and PS M/NPs as described above. All the detections were conducted in triplicate (Text S5).

Detection of ATP generation
To measure ATP content (Text S5), cells were collected by centrifugation in a centrifuge tube, and the supernatant was discarded. The cells were gently resuspended and then lysed. Chemiluminescence intensity was measured using a microplate reader to determine the changes in ATP production under the influence of different particle sizes and concentrations of PS M/NPs. All the detections were conducted in triplicate (Text S6).

RNA extraction, genome-wide RNA sequencing, and transcriptomic analysis
RNA extraction was carried out after the conjugation systems were treated with three different sizes of PS (0, 0.1, and 10 mg/L) for 2 h. Each system was replicated in biological triplicates. After being subjected to quality control, the pellets were sent to MAGIGENE Co. (Shenzhen, China) for RNA extraction, construction of strand-specific cDNA libraries, and Illumina sequencing of paired-end genomes. An Illumina HiSeq PE150 was used. An NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina was used for library construction. Sequence reads consisting of at least 85% bases were then progressively trimmed at the 3’-ends until a quality value ≥ 20 was maintained. Downstream analyses were performed using the generated clean reads that were no shorter than 75 bp. The clean reads of each sample were aligned to the E. coli reference genome (NC_000913), P. putida reference genome (NC_002947), and IncPα plasmid reference genome (L27758) using Bowtie2. RSeQC was used to calculate the strand-specific coverage for each gene and to analyse the differential expression in triplicate bacterial cell cultures. The statistical analyses and visualisation were conducted using edgeR and the CummeRbund package in R. Gene expression was calculated as fragments per kilobase of a gene per million mapped reads (FPKM, a normalised value generated from the frequency of detection and the length of a given gene). The difference in gene expression between the control group (without the addition of PS M/NPs) and the PS-treated groups is represented as the log2-fold change (LFC), which is defined as the base-2 logarithm of the ratio of expression levels in the treatment group relative to the control group. The significance of the transcriptomic data was determined by a false discovery rate (FDR)-adjusted p value of less than 0.05. All samples were prepared in triplicate.

Plasmid verification
To verify that the plasmids had entered the cells, the plasmids of the donors and transconjugants were extracted using the Invitrogen PureLink Quick Plasmid Miniprep Kit (Life Technologies, USA). The blaTEM-1 and tetA genes of the RP4 plasmid were amplified by PCR, and the resulting amplicons as well as the RP4 plasmid were checked using 1% agarose gel electrophoresis. The PCR primers and conditions are described in Text S7.

Statistical analysis
All experiments were conducted independently at least in biological triplicate. All phenotypic data are expressed as the mean ± SD and were analysed with GraphPad Prism 10 and SPSS 25.0 (SPSS, Chicago, USA). The results were analysed by analysis of variance and two-sided independent-sample t-test methods with Benjamini–Hochberg correction. A corrected p value less than 0.05 was considered to indicate statistical significance.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_2": "This study investigates an important and timely environmental health issue – the potential role of micro/nanoplastics (M/NPs) in facilitating the dissemination of antimicrobial resistance (AMR). The findings suggesting polystyrene (PS) M/NPs, particularly at environmentally relevant concentrations, can promote intra- and intergeneric AMR gene transfer via induced physiological stress are intriguing and potentially significant. However, two major concerns regarding the scope of the bacterial models used for intergeneric transfer and the mechanistic evidence provided limit the strength and generalizability of some conclusions.", "reviewer_4": "The revision of the manuscript is commendably thorough, with the authors having addressed numerous points raised by previous reviewers in a serious and considered manner. However, I feel that the discussion section remains comparatively weak, and would benefit from greater attention and refinement. Specifically, the discussion covers many critical aspects concerning the dissemination of AMR via micro- and nanoplastics (M/NPs), especially focusing on polystyrene (PS) particles and their effects across evolutionary distances. The overall experimental narrative and linkages to environmental and public health concerns are timely and relevant. However, to meet the standards expected by Nature Communications, improvements are needed in structure, logic, conceptual clarity, and English idiom. Below are the main issues and corresponding suggestions.\n\nThe discussion is, at times, excessively dense and repetitive; key mesages are diluted amid tangential information. The structure could be improved by more clearly grouping themes—first describing major results, then their mechanistic implications, and finally the broader significance. Morever, to me several sentences are convoluted, imprecise, or awkwardly constructed. For a high-impact journal, prose should be concise, neutral, logical and idiomatic.\n\nThe discussion is largely descriptive, lacking sufficient critical comparison with prior literature or critical appraisal of the study's own limitations and possible confounders. For instance, mechanistic links (e.g., between particle size, ROS, membrane permeability, ATP production, and conjugation) are asserted but not rigorously interrogated or contextualised against the field’s understanding.", "reviewer_5": "Dear Authors,\nthank you for having exhaustively replied to the questions. The study has been further improved. Still, there are a few issues to fix, in my opinion, mainly related to the statistical significance of the results. My recommendation is major revision. Please note that the comments refer to the last merged version of the manuscript (file named: 611238_0_merged_1748258182.pdf).\n\n1. General comment:\nConsider making uniform as much as possible the tenses of the manuscript. Too many variations from past to present tense and vice versa.\nWhen using “ARGs” in an adjective form (e.g., “ARGs transfer”), consider using it in the singular (e.g., “ARG dissemination”).\nPlease check the whole manuscript.\nSince you have defined ARGs as “antimicrobial resistance genes”, please change, throughout the manuscript, AMR genes to ARGs.\n\n2. Main comment:\nTables S6-47: All the tables referring to changes in gene expression reported, under the table, an explanation of statistical symbols. However, no stars are present next to the fold change values. Given this, I do not know if the results shown in the manuscript are significant or not. If not, they should not be mentioned. Some examples in the following comments. Please check everywhere this type of result is presented.\n\n3. Line 32: consider changing “on the dissemination of antimicrobial resistance (AMR) dissemination remains” to “on the dissemination of antimicrobial resistance (AMR) remains”.\n\n4. Line 61: consider changing “water systems serve as critical hubs for antibiotics” to “water systems could act as a source of antibiotics”.\n\n5. Line 62: “AMR” has never been abbreviated in the main text (Introduction). Please correct.\n\n6. Line 131 and Figure 1: you wrote “The intrageneric conjugation system displayed more ROS production (Fig. 1c-d, g-h) and cell membrane permeability (Fig. 1e-f, i-j) than its intergeneric counterpart”. However, no significance symbols are displayed for Fig. 1c-j. Does it mean that there is no significant difference between systems or that the statistical analysis was not performed? In the first case, you cannot affirm that ROS production and cell membrane permeability were higher (“more”) in the intrageneric conjugation system. In the second case, please carry out the statistical test.\nMoreover, I think that Fig. 1 g-h is referred to Enterococcus-based system (as for the panels i and j of Fig. 1) and not to E. coli-based system (otherwise, they showed the same information as panels c and d).\n\n7. Line 163: consider changing “is proved by the TEM images shown in Fig. S1” to “is shown by the TEM images in Fig. S1”.\n\n8. Lines 163-167: the main verb is missing, in my opinion. Consider changing “PCR amplicons to confirm that the RP4” to “PCR amplicons, were done to confirm that the RP4” or “PCR amplicons, were performed to confirm that the RP4”.\n\n9. Line 173: the unit of measurement of the concentration is missing. Please add.\n\n10. Line 174: please specify the three sizes of PS.\n\n11. Line 217: please remove the point between the bracket and the comma.\n\n12. Fig. S9: please add the explanation of the statistical symbols, as for the other figures.\n\n13. Lines 253-268: you reported in Fig. 2 and 3 captions that only significantly up- or down-regulated genes were shown. However, in the main text, genes (e.g., sfmA) not present in the figures are reported. Genes that did not significantly differ should not be mentioned and/or discussed. Which criteria have been used to select the genes displayed in Fig. 2 and 3?\nMoreover, for Fig. 2 and 3, the references present in the text are wrong. For example, line 258: “(Fig. 2f-h)” but panel h does not exist for Fig.2; line 264: should be “Fig. 3h” and not “Fig. 3f”. Please check carefully here and throughout the manuscript.\n\n14. Lines 270-284: given that no “significance stars” are reported next to the fold change values of tables S42-43, I do not know if the results shown in this section (Lines 270-284) are significant or not. If not, they should not be mentioned. Please check here and where this type of result is presented.\n\n15. Lines 276-277: consider using italics for the gene name (with the p in lowercase) and for E. faecalis.\n\n16. Lines 287-295: as for comment #13, the figure references present in the text seem to be wrong and should be referred to the right panels. Please check carefully.\n\n17. Lines 329-330: consider changing “fluctuation concentrations” to “fluctuation in concentrations” or “varying concentrations”.\n\n18. Lines 362-374: as for comment #14, I cannot understand if the results are significant or not when looking at what is reported in the supplementary tables (no starred values). Moreover, also considering that all the genes in the tables were significantly up- or down-regulated, there are discrepancies between the genes cited in the text and the (significant) genes shown in panels g and h of Fig. 4. Which criteria have been used to select the genes displayed in Fig. 4?\n\n19. Lines 386-389: the same as the comments #14 and 18 about the significance of the results.\n\n20. Line 421: consider changing “species” to “genera”.\n\n21. Line 446: consider changing “ROS” to “ROS production”.\n\n22. Lines 464-466: consider changing “However, PS M/NPs at higher concentrations (10, 50, and 100 mg/L) may have a negative effect on AMR transmission, which may potentially hinder cell-to-cell contact or block conjugative pili, thereby reducing conjugation efficiency” in “However, PS M/NPs at higher concentrations (10, 50, and 100 mg/L) may potentially hinder cell-to-cell contact or block conjugative pili, thereby reducing conjugation efficiency and affecting negatively AMR transmission”.\n\n23. Line 486: consider changing “negligible and, may” to “negligible, it may”.\n\n24. Line 503: consider changing “of PS” to “for PS”.\n\n25. Line 537: consider changing “LE392 (carrying” to “LE392, carrying”.\n\n26. Line 634: consider changing “The specific blaTEM-1 and tetA” to “The blaTEM-1 and tetA genes”.\n\n27. Line 635: consider changing “RP4 analyzed” to “the plasmid RP4 checked”."}, "author_response": "Response to Reviewers’ comments:\nReviewer #2 (Remarks to the Author):\nThis study investigates an important and timely environmental health issue – the potential role of micro/nanoplastics (M/NPs) in facilitating the dissemination of antimicrobial resistance (AMR). The findings suggesting polystyrene (PS) M/NPs, particularly at environmentally relevant concentrations, can promote intra- and intergeneric AMR gene transfer via induced physiological stress are intriguing and potentially significant. However, two major concerns regarding the scope of the bacterial models used for intergeneric transfer and the mechanistic evidence provided limit the strength and generalizability of some conclusions.\nResponse: We agree and sincerely appreciate the reviewer’s valuable comments, which have significantly strengthened the rigor and clarity of our work. We have fully addressed the two major concerns through additional contextualization, textual revisions, and tempered conclusions. All changes are highlighted in the revised manuscript.\n1. Limitation of Bacterial Models for Intergeneric AMR Transfer: The claim that PS M/NPs promote intergeneric AMR gene transfer is based on experiments involving only two donor strains (one Gram-negative and one Gram-positive) and recipients from different genera. This is a significant limitation. Intergeneric AMR transfer is an extremely complex process governed by numerous factors, including plasmid compatibility, restriction-modification systems, pilus compatibility, cell surface receptors, and fundamental physiological/genetic barriers between diverse genera. Results obtained using only one or two specific donor-recipient pairs cannot adequately represent this complex phenomenon across the vast diversity of environmental bacteria. Consequently, the finding that 1-µm PS primarily facilitates transfer to E. coli, and the associated gene regulation observations, may be highly specific to the particular strains and plasmids used. Extrapolating this to a general size-dependent mechanism for intergeneric transfer or stating that environmentally relevant concentrations \"significantly promote... intergeneric AMR transfer\" overstates the evidence.\nResponse: We thank the reviewer for highlighting this important limitation. In our tested systems, we found that the intrageneric conjugation frequency was much higher than intergeneric one. But we agree that intergeneric conjugation is a multifactorial and might be highly strain-specific process. Therefore, we have toned down our conclusions to the tested strains and acknowledged this limitation in the revised manuscript. Specifically, we have now:\n(1) Explicitly limited all conclusions to the tested systems (e.g., E. coli and E. faecalis donors) throughout the manuscript. As per this comment, the following statements have been changed in the revised manuscript. (Page 2, Line 33-44; Page 4, Line 119-120; Page 7, Line 180-182; Page 7, Line 185-187.)\n(2) Added dedicated parts in the Discussion acknowledging the strains and plasmid specificity of our findings and calling for future studies with broader bacterial diversity. (Page 20, Line 501-503; Page 20, Line 505-517)\n(3) Revised all potential overstated claims. For example, we added ‘potentially’ to some conclusions (Page 8, Line 209-210; Page 20-21, Line 527-531).\nThese changes aim to provide a more accurate interpretation of our data while remaining scientifically rigorous and transparent about the study’s scope.\nPage 2, Line 33-44: Here, we examine how polystyrene (PS) M/NPs of varying sizes and concentrations affect AMR gene (ARG) transfer in model systems with gram-negative (Escherichia coli) and gram-positive (Enterococcus faecalis) donors. In these systems, the ARG transfer frequency was higher for intrageneric pairs than for intergeneric pairs. The 20- and 120-nm-sized PS broadly facilitated conjugation, whereas the 1-μm-sized PS selectively promoted ARG transfer to E. coli recipients, in addition to altering the expression of conjugation- and pili-associated genes. Notably, an environmentally relevant (0.1 mg/L) concentration of PS M/NPs facilitated AMR transfer in the tested systems, which correlated with increased reactive oxygen species levels, ATP levels, and cell membrane permeability in both donors and recipients. Collectively, our findings underscore the role of M/NPs in facilitating AMR spread in specific bacterial systems, providing valuable insights for understanding their potential ecological risk in water environments.\nPage 4, Line 119-120: The intrageneric conjugation frequency exceeded the intergeneric transfer frequency in the tested model bacterial systems\nPage 7, Line 180-182: To assess the broader applicability of PS-facilitated ARG transfer, we tested an additional model conjugation system.\nPage 7, Line 185-187: The results indicated increased conjugative transfer frequencies under PS exposure across all tested sizes and concentrations.\nPage 8, Line 209-210: Importantly, we further investigated the physiological responses potentially associated with PS-facilitated ARG transfer.\nPage 20, Line 501-503: We found that intrageneric conjugation transfer frequencies far exceeded those of their intergeneric counterparts in the tested conjugation systems.\nPage 20, Line 505-517: Moreover, our results reveal that PS particles can enhance conjugative gene transfer not only within species but also across species, including between gram-positive and gram-negative bacteria. These findings contrast with those of single-cell studies by Liu et al.27, who reported inhibitory effects of PS on the intrageneric conjugation of E. coli, highlighting the context-dependent and potentially species-specific nature of interactions between M/NPs and microbes. By demonstrating the PS-induced increase in cross-genus gene transfer, our study advances the current understanding of HGT dynamics in plastic-polluted environments. Despite these novel insights, we should acknowledge that the intergeneric conjugation results were derived from a limited number of donor–recipient pairs and only 2 plasmids (RP4 and pCF10), which may not fully capture the diversity of conjugation mechanisms present in environmental microbiomes. Plasmid compatibility, host restriction systems, and recipient recognition vary substantially across taxa; thus, broader validation with diverse bacterial hosts and mobile genetic elements is needed.\nPage 20-21, Line 527-531: At the molecular level, PS exposure upregulated genes in the tested systems involved in the mating pair formation (Mpf) system (traL, trbH, trbM)56, 60, 61; in efflux pumps51, 62; and, in gram-positive E. faecalis donors, the phosphotransferase system (PTS) and teichoic acid biosynthesis (Table S44–47), potentially contributing to species-specific conjugation responses.\nResponse: We appreciate the reviewer’s insightful comments. In our study, we observed a correlation between enhanced AMR gene transfer frequency and increased levels of ROS, cell membrane permeability, and ATP generation. We fully agree that correlation does not imply causation. As suggested, in our revised manuscript:\n(1) We have clarified throughout the Results and Discussion section that the increase of ROS, ATP production, and membrane permeability are correlated with increased transfer frequencies, but are not all causally proven. (Page 8, Line 209-213; Page 8, Line 228-229; Page 20, Line 519-524:)\n(2) For ROS, we conducted ROS scavenger experiments (Fig. S7), which demonstrated a reduction in transfer frequency upon ROS quenching, providing partial causal evidence. This is now emphasized more clearly in the revised text. (Page 7, Line 214-215)\n(3) For ATP levels and membrane permeability, we now use more cautious and tempered language such as ‘correlated’ or ‘correlative’ and have removed phrasing that implied a direct mechanistic role. (Page 15-16, Line 414-417)\n(4) In the Discussion section, we also added the statement acknowledging that further studies are needed to confirm the sufficiency and necessity of these physiological changes for observed conjugation promotion. (Page 21, Line 534-538)\nAdditionally, we recognize that some stressors might induce similar physiological responses but not necessarily increase conjugative transfer frequency. However, previous research has shown that certain non-antibiotic chemicals, such as disinfectants, non-antibiotic pharmaceuticals, metals1, 2, 3, 4 can indeed promote the transfer of ARGs through mechanisms related to ROS generation, ATP modulation, and altered membrane permeability (Page 3, Line 67-69). We reference this in the revised manuscript to underscore the plausibility of our findings while acknowledging the need for further mechanistic investigation.\nThese revisions aim to ensure that our mechanistic interpretations remain evidence-based and appropriately restrained.\nPage 3, Line 67-69: Various nonantibiotic substances, such as disinfectants, nonantibiotic pharmaceuticals and nanoparticles18, 19, 20, 21, have been shown to impact the horizontal gene transfer (HGT) of plasmid-borne ARGs22.\nPage 8, Line 209-213: Importantly, we further investigated the physiological responses potentially associated with PS-facilitated ARG transfer. Among the tested parameters, ROS generation showed the strongest correlation with transfer frequency, particularly in E. faecalis-based systems, where the trends in the levels of ROS corresponded to increases in ARG transfer frequency (Fig. 3a–c).\nPage 8, Line 219-221: These findings support a model in which oxidative stress is correlated with enhanced ARG transfer under PS exposure, suggesting that ROS may contribute to the observed phenotype in the tested systems.\nPage 8, Line 228-229: This effect correlated with increased intra- and intergeneric conjugative transfer frequencies in the tested systems.\nPage 15-16, Line 414-417: Intragenus conjugation systems of RP4 were characterized by substantial changes in recipient cell membrane permeability (Figs. S12–S13), accompanied by oxidative state alterations and increased ATP generation (Figs. S14–S15) in donors, which correlated with increased ARG transfer frequencies.\nPage 20, Line 519-524: Mechanistically, PS M/NPs facilitate ARG transfer through size-, concentration-, and strain-specific pathways involving oxidative stress, membrane perturbation, and energy metabolism40, 58. Our data revealed that 20 nm PS induced the greatest increase in the production of ROS, particularly •OH59, and increased membrane permeability and ATP levels, whereas 1 μm PS increased E. coli membrane permeability without increasing the corresponding level of ROS, suggesting a ROS-independent route distinct from that reported for metal nanoparticles20.\nPage 21, Line 534-538: While the associations between physiological stress and enhanced transfer were consistent, the partial restoration of conjugation frequency by ROS scavengers supports a partial causal role for oxidative stress, while the direct contributions of ATP production and membrane destabilization remain correlative and should be further clarified through genetic, chemical, or imaging-based approaches.\nReviewer #4 (Remarks to the Author):\nThe revision of the manuscript is commendably thorough, with the authors having addressed numerous points raised by previous reviewers in a serious and considered manner. However, I feel that the discussion section remains comparatively weak, and would benefit from greater attention and refinement. Specifically, the discussion covers many critical aspects concerning the dissemination of AMR via micro- and nanoplastics (M/NPs), especially focusing on polystyrene (PS) particles and their effects across evolutionary distances. The overall experimental narrative and linkages to environmental and public health concerns are timely and relevant. However, to meet the standards expected by Nature Communications, improvements are needed in structure, logic, conceptual clarity, and English idiom. Below are the main issues and corresponding suggestions.\nResponse: We sincerely appreciate the reviewer’s overall positive assessment of our manuscript, especially the valuable comments on how to improve the Discussion section. In response, we have thoroughly improved the structure and logical flow, strengthened the conceptual clarity, and refined the language for clarity and idiomatic correctness of the Discussion section as suggested (EFE3-FD93-E4E6-35B8-A5CP).\nSpecifically, as suggested we reorganized the structure of discussion into more structured thematic sub-paragraphs, expanded comparisons with prior literature, made the key message (concise and neural) stand out of tangential information, acknowledged the limitations and possible confounders of our study.\nMeanwhile, we made every effort to delete the dense and repetitive phrasing, improve the language expression to be concise, neural, logical and idiomatic throughout the revised manuscript to better align with the standards of Nature Communications. We believe these changes have significantly enhanced the coherence and scientific depth of the Discussion section.\nThe discussion is, at times, excessively dense and repetitive; key messages are diluted amid tangential information. The structure could be improved by more clearly grouping themes—first describing major results, then their mechanistic implications, and finally the broader significance. Moreover, to me several sentences are convoluted, imprecise, or awkwardly constructed. For a high-impact journal, prose should be concise, neutral, logical and idiomatic.\nThe discussion is largely descriptive, lacking sufficient critical comparison with prior literature or critical appraisal of the study's own limitations and possible confounders. For instance, mechanistic links (e.g., between particle size, ROS, membrane permeability, ATP production, and conjugation) are asserted but not rigorously interrogated or contextualised against the field’s understanding.\nResponse: We thank the reviewer for this insightful comment on how to improve the discussion part. In response, we have restructured the Discussion section to improve thematic clarity, key messages presentation, logical flow, and conciseness.\nThe revised section now presents a more coherent and logical structure, beginning with a summary of the key findings: PS M/NPs, including environmentally relevant concentrations and varying sizes, can significantly promote the conjugative transfer of ARGs both within and across bacterial genera in tested model systems (Page 18, Line 440-446). Following this, we discussed that 20 nm PS broadly promoted ARG dissemination in tested systems, while 120 nm showed intermediate effects, and 1 μm significantly enhanced transfer in E. coli recipient systems (Page 18-19, Line 448-469). Then, for a specific particle size, the impact of PS N/MPs on AMR is dose dependent (Page 19, Line 474-492). And we clarify that PS M/NPs could facilitate AMR dissemination both in tested intrageneric and intergeneric conjugation systems (Page 20, Line 499-501; Page 20, Line 505-517). We then delve into the underlying mechanisms (Page 20, Line 519-524) and conclude with a discussion of the implications and significance of our study (Page 21, Line 550-558). In addition, we have removed any redundant statements, refined sentences that were previously imprecise or awkward, and improved the overall clarity and fluency of the text to enhance its readability and idiomatic expression.\nFurthermore, we have comprehensively expanded the critical discussion and comparison with existing literature, moving beyond mere description to a rigorous interrogation of our mechanistic findings. We now explicitly acknowledge and differentiate the strength of evidence for each proposed mechanism. Our data implicate that oxidative stress acts as partial causal role. And we now clearly frame the roles of ATP elevation and membrane permeability as correlative, which necessitates future validation through genetic or chemical approaches. We believe this interpretation would address the concern between physiological responses and mechanistic links raised both by you and reviewer 2.\nFor example, we have contextualized these findings within the broader field. Regarding size-effect (Page 19, Line 469-472): Our data reveal a clear size-dependent phenomenon where 20 nm PS elicited the strongest facilitation of conjugation. We now contextualize this finding by citing other studies that also found nanoparticles (NPs) to be of heightened concern. Our work aligns with work by Zhao et. al. 5 who showed NPs efficiently recruit pathogenic ARG hosts in constructed wetlands, and Xu et. al.6 who demonstrated that NPs alter membrane transport systems to increase resistance. Furthermore, the markedly greater impact of NPs over microplastics (MPs) is consistent with the comprehensive soil microbiome study7, which found nano-sized plastics elevated ARG abundance and diversified bacterial hosts more substantially than their micro-sized counterparts. Regarding ROS-dependent pathways: We compare our results with Duan et al. 8 and Chen et al.9 on photo-aged nanoplastics, noting a consensus that ROS burst is a key driver for HGT (Page 20, Line 505-517). However, we also highlight a distinction: their study attributed ROS generation to the nanozyme-like activity of aged particles, whereas our study points to physiological stress responses in bacteria induced by pristine PS. Following the reviewer’s suggestion, by integrating these comparative analyses, we have shifted the discussion from asserting mechanistic links to critically evaluating them against the field's understanding, thereby providing a more balanced and scholarly perspective on how PS particles facilitate ARG transfer.\nWe also added parts addressing the limitations of our study to the corresponding paragraph. For example, we acknowledged all experiments were conducted under simplified laboratory conditions, and thus the effects of aged or weathered M/NPs, multispecies biofilms, and natural aquatic matrices remain to be clarified through further laboratory and in situ validation (Page 19, Line 469-472). Moreover, our findings on intergeneric conjugation were based on a limited set of donor–recipient pairs and two plasmids (RP4 and pCF10), which may not fully reflect the diversity of conjugation mechanisms in complex environmental microbiomes. Given that plasmid compatibility, host restriction systems, and recipient recognition can vary substantially across taxa, broader validation with diverse bacterial hosts and mobile genetic elements will be important (Page 20, Line 505-517). Finally, while our results consistently point to a role of physiological stress in enhancing transfer, the partial rescue of conjugation frequencies by ROS scavengers suggests only a partial causal role for oxidative stress, whereas the contributions of ATP production and membrane destabilization remain correlative and warrant further mechanistic dissection through genetic, chemical, or imaging-based approaches (Page 20-21, Line 527-538). These changes aim to enhance the scientific depth, neutrality, and linguistic quality to meet the high standards of Nature Communications.\nPage 18, Line 440-446: In this study, we demonstrated that PS M/NPs, at environmentally relevant concentrations and varying sizes, can significantly promote the conjugative transfer of ARGs both within and across bacterial genera in the tested model systems. This enhancement was associated with cellular stress responses in recipient bacteria, including elevated levels of ROS, increased levels of intracellular ATP, and increased membrane permeability. These findings reveal a previously underrecognized pathway through which plastic pollution may contribute to the spread of AMR in the environment.\nPage 18-19, Line 448-469: In our study, 20 nm PS broadly promoted ARG dissemination in the tested systems, whereas 120 nm PS had intermediate effects, and 1 μm PS significantly enhanced the transfer in E. coli recipient systems. Given the widespread contamination of M/NPs and ARGs in aquatic and gut environments45 and their coexistence with complex microbial communities, understanding M/NP-mediated AMR dissemination is critical. Current research suggests that M/NPs promote both transformation25 and intragenus conjugation27, 46. Among the various modes of HGT, conjugation is the most prevalent natural means through which ARGs can disseminate22; this allows ARGs to be exchanged within or across genera, thereby creating new ecological niches for microorganisms47. Most studies have focused on plastics with particle sizes exceeding 75 nm25, 48. Importantly, environmental shear forces continuously fragment plastics into smaller, higher-risk nanoparticles49, which is consistent with findings in which PS with particle sizes smaller than 100 nm enhanced exogenous ARG uptake25. Our study reveals a strong size-dependent hierarchy in PS-mediated conjugative transfer. We found that 20 nm PS, which is capable of entering bacterial cells through membrane pores, facilitated the spread of ARGs both within and across genera among the tested gram-negative and gram-positive bacterial strains, thereby increasing the likelihood of the emergence of multidrug-resistant microorganisms. These findings align with those of studies showing that NPs can recruit pathogenic ARG hosts in wetlands50, alter membrane transport to potentiate resistance51 and increase soil ARG abundance to a greater extent than microplastics are capable of52. PS (120 nm) had intermediate effects, mirroring size-dependent risks in constructed wetlands50. Unlike transformation, where large plastics (> 1 μm) have minimal effects25, our study demonstrated that 1 μm PS, which is similar in size to microorganisms, remains consequential for the dissemination of AMR to E. coli.\nPage 19, Line 469-472: However, all the experiments were performed under simplified laboratory conditions. The effects of aged or weathered M/NPs, multispecies biofilms, and natural aquatic matrices remain unknown and warrant further laboratory and in situ validation53.\nPage 19, Line 474-492: For a specific particle size, the impact of PS N/MPs on AMR is dose dependent. Previous studies have typically employed experimental concentrations starting at 5 mg/L27, which are significantly higher than environmental concentrations5. Here, we demonstrated that environmentally relevant concentrations (0.1–1 mg/L) were sufficient to induce significant effects. Indeed, exposure to as low as 0.1 mg/L of 20 nm PS nanoparticles increased the transfer frequency by up to 8.9-fold54. Field evidence also supports this trend, with the abundance of microplastics (14.9 items/g faeces) positively correlated with that of ARGs (6.24 × 10 8 copies/g) and laboratory exposure, increasing conjugation by 1.4–1.7-fold55. Notably, the relationship is nonmonotonic: small NPs can first stimulate but then suppress transfer at higher doses45, and extreme concentrations of microplastics (2000 mg/L) reduce the conjugation frequency by an order of magnitude27. In systems with E. coli carrying RP4, PS M/NPs at higher concentrations (10, 50, and 100 mg/L) may hinder cell-to-cell contact or block conjugative pili and toxicity56, thereby reducing the conjugation efficiency and negatively affecting AMR transmission. For the E. faecalis system, we specifically examined environmentally relevant concentrations but did not evaluate higher concentration exposures. The effects of environmentally relevant concentrations of PS on promoting the dissemination of AMR are not negligible and may have adverse implications for water safety, which underscores the ecological plausibility of our findings and suggests that sublethal exposure to M/NPs may drive nonmonotonic stress responses, with important implications for environmental AMR proliferation57.\nPage 20, Line 499-501: While most previous studies have focused on intrageneric conjugation events, particularly within model organisms such as E. coli25, 46, 48, the role of M/NPs in facilitating intergeneric ARG transfer has remained largely unexamined.\nPage 20, Line 505-517: Moreover, our results reveal that PS particles can enhance conjugative gene transfer not only within species but also across species, including between gram-positive and gram-negative bacteria. These findings contrast with those of single-cell studies by Liu et al.27, who reported inhibitory effects of PS on the intrageneric conjugation of E. coli, highlighting the context-dependent and potentially species-specific nature of interactions between M/NPs and microbes. By demonstrating the PS-induced increase in cross-genus gene transfer, our study advances the current understanding of HGT dynamics in plastic-polluted environments. Despite these novel insights, we should acknowledge that the intergeneric conjugation results were derived from a limited number of donor–recipient pairs and only 2 plasmids (RP4 and pCF10), which may not fully capture the diversity of conjugation mechanisms present in environmental microbiomes. Plasmid compatibility, host restriction systems, and recipient recognition vary substantially across taxa; thus, broader validation with diverse bacterial hosts and mobile genetic elements is needed.\nPage 20, Line 519-524: Mechanistically, PS M/NPs facilitate ARG transfer through size-, concentration-, and strain-specific pathways involving oxidative stress, membrane perturbation, and energy metabolism 40, 58. Our data revealed that 20 nm PS induced the greatest increase in the production of ROS, particularly •OH59, and increased membrane permeability and ATP levels, whereas 1 μm PS increased E. coli membrane permeability without increasing the corresponding level of ROS, suggesting a ROS-independent route distinct from that reported for metal nanoparticles20.\nPage 20-21, Line 527-538: At the molecular level, PS exposure upregulated genes in the tested systems involved in the mating pair formation (Mpf) system (traL, trbH, trbM) 56, 60, 61; in efflux pumps51, 62; and, in gram-positive E. faecalis donors, the phosphotransferase system (PTS) and teichoic acid biosynthesis (Table S44–47), potentially contributing to species-specific conjugation responses. These stress- and metabolism-linked pathways may collectively promote plasmid mobilization, natural transformation25, and both intra- and intergenus conjugation, underscoring the multifactorial nature of PS-mediated AMR propagation. While the associations between physiological stress and enhanced transfer were consistent, the partial restoration of conjugation frequency by ROS scavengers supports a partial causal role for oxidative stress, while the direct contributions of ATP production and membrane destabilization remain correlative and should be further clarified through genetic, chemical, or imaging-based approaches.\nPage 21, Line 550-558: Future work should investigate whether other M/NP types and aged M/NPs exhibit similar or stronger ARG transfer potential, confirm the direct causal mechanisms driving enhanced transfer, incorporate integrative multiomics to elucidate the underlying molecular pathways, and assess species-specific responses in complex habitats such as soil, freshwater, marine ecosystems, wastewater, and gastrointestinal environments. Given the persistence and ubiquity of plastics, even low-level pollution may accelerate microbial evolution by promoting the horizontal dissemination of ARGs. These findings underscore the urgent need for interdisciplinary research to quantify public health risks and to inform mitigation strategies in the context of a One Health framework aimed at curbing the environmental spread of AMR.\nReviewer #5 (Remarks to the Author):\nReviewer #3 (Comments for the Author)\nDear Authors,\nThank you for having exhaustively replied to the questions. The study has been further improved. Still, there are a few issues to fix, in my opinion, mainly related to the statistical significance of the results. My recommendation is major revision. Please note that the comments refer to the last merged version of the manuscript (file named: 611238_0_merged_1748258182.pdf).\nResponse: We sincerely appreciate your positive evaluation of our work and your constructive comments, which have helped us further improve the manuscript much. We acknowledge your concern regarding the statistical significance of the results and have carefully re-analyzed the data to address this issue. Specifically, we have revised the figures/tables to clearly present significant levels. We believe these revisions have strengthened the robustness and clarity of our findings. Detailed responses to each comment are provided below, with the corresponding changes marked in the revised manuscript.\n1. General comment:\nConsider making uniform as much as possible the tenses of the manuscript. Too many variations from past to present tense and vice versa.\nWhen using “ARGs” in an adjective form (e.g., “ARGs transfer”), consider using it in the singular (e.g., “ARG dissemination”). Please check the whole manuscript.\nSince you have defined ARGs as “antimicrobial resistance genes”, please change, throughout the manuscript, AMR genes to ARGs.\nResponse: We thank the reviewer for pointing out inconsistencies in tense usage. We have carefully reviewed the entire manuscript and revised the text to ensure tense consistency. Specifically, all descriptions of experiments and results have been standardized to the past tense, while established knowledge and general scientific facts are now consistently presented in the present tense. Regarding the use of “ARGs” in adjectival contexts, we have carefully revised the manuscript and replaced all instances of “ARGs transfer” with more grammatically appropriate and idiomatic expressions such as “ARG transfer” or “ARG dissemination” and conducted a comprehensive search throughout the manuscript and replaced all instances of “AMR genes” with “ARGs” to ensure consistent terminology, as previously defined.\n2. Main comment:\nTables S6-47: All the tables referring to changes in gene expression reported, under the table, an explanation of statistical symbols. However, no stars are present next to the fold change values. Given this, I do not know if the results shown in the manuscript are significant or not. If not, they should not be mentioned. Some examples in the following comments. Please check everywhere this type of result is presented.\nResponse: We thank the reviewer for carefully examining the supplementary tables and for identifying this inconsistency. We have now carefully re-checked all tables from S6 to S47 and added statistical significance symbols (e.g., *, **, ***) next to the fold change values wherever applicable. These were derived from the adjusted p-values of the differential expression analysis. For clarity, only results with p < 0.05 are now highlighted and discussed in the main text.\nIn addition, we revised the relevant sections of the manuscript and figure legends to ensure that only statistically significant results are emphasized. Non-significant findings have been either removed or rephrased to avoid overinterpretation.\n3. Line 32: consider changing “on the dissemination of antimicrobial resistance (AMR) dissemination remains” to “on the dissemination of antimicrobial resistance (AMR) remains”.\nResponse: Changed, as suggested.\n4. Line 61: consider changing “water systems serve as critical hubs for antibiotics” to “water systems could act as a source of antibiotics”.\nResponse: Changed, as suggested.\n5. Line 62: “AMR” has never been abbreviated in the main text (Introduction). Please correct.\nResponse: Changed, as suggested.\n6. Line 131 and Figure 1: you wrote “The intrageneric conjugation system displayed more ROS production (Fig. 1c-d, g-h) and cell membrane permeability (Fig. 1e-f, i-j) than its intergeneric counterpart”. However, no significance symbols are displayed for Fig. 1c-j. Does it mean that there is no significant difference between systems or that the statistical analysis was not performed? In the first case, you cannot affirm that ROS production and cell membrane permeability were higher (“more”) in the intrageneric conjugation system. In the second case, please carry out the statistical test.\nMoreover, I think that Fig. 1 g-h is referred to Enterococcus-based system (as for the panels i and j of Fig. 1) and not to E. coli-based system (otherwise, they showed the same information as panels c and d).\nResponse: Thank you very much for the constructive comments. We apologize for the lack of clarity in our previous description. Statistical analyses were indeed performed for Fig. 1c–j, and the corresponding significance levels have now been added to the revised figure. The results indicate that there was no significant difference in ROS production (Fig. 1c–d) or cell membrane permeability (Fig. 1e–f) between donors and recipients in the RP4-related systems. In contrast, the intrageneric pCF10-related conjugation system displayed significantly higher ROS production (Fig. 1g–h) and cell membrane permeability (Fig. 1i–j) compared with its intergeneric counterpart. Accordingly, we have updated both the figure and the legend to make these findings clearer (Page 5 Line 133-136).\nRegarding the labelling of Fig. 1g–h, we sincerely apologize for the confusion. These panels indeed refer to the E. faecalis-based system (consistent with panels i–j), rather than the E. coli-based system. We have corrected this mislabelling in the revised figure and the corresponding text (Page 5–6, Lines 147–160).\nRegarding the labelling of Fig. 1g–h, sincere sorry for the confusion. Yes, these panels refer to the E. faecalis-based system (consistent with panels i–j), rather than the E. coli-based system. We have corrected this mislabeling in the revised figure and corresponding text (Page 6 Line 151-166).\nPage 5 Line 133-136: There was no significant difference in ROS production (Fig. 1c–d) or cell membrane permeability (Fig. 1e–f) between donors and recipients for RP4-related systems. However, compared with its intergeneric counterpart, the intrageneric conjugation system resulted in increased ROS production (Fig. 1g–h) and cell membrane permeability (Fig. 1i–j) in pCF10-related systems.\nPage 6 Line 151-166:\nFig. 1 Effect of phylogenetic distance between microorganisms on the transfer of ARGs.\na Conjugative transfer frequency within and across genera under 20 nm PS exposure for RP4.\nb Conjugative transfer frequency within and across genera under 20 nm PS exposure for pCF10.\nc ROS production of bacterial strains in the RP4-related conjugation system without PS. d ROS production in the intrageneric and intergeneric conjugation system for RP4 without PS. e Cell membrane permeability of bacterial strains in the RP4-related conjugation system without PS. f Cell membrane permeability in the intrageneric (donor E. coli K-12 LE392 and recipient E. coli BL21(DE3)) and intergeneric (donor E. coli K-12 LE392 and recipient P. putida KT2440) conjugation systems for RP4 without PS. g ROS production of bacterial strains in the pCF10-related conjugation system without PS. h ROS production in the intrageneric and intergeneric conjugation systems for pCF10 without PS. i Cell membrane permeability of bacterial strains in the pCF10-related conjugation system without PS. j Cell membrane permeability in the intrageneric and intergeneric conjugation systems for pCF10 without PS. Statistically significant differences between intra- and intergeneric conjugation were tested with an independent-sample t test with Benjamini–Hochberg correction: *p < 0.05 and **p < 0.01.\n7. Line 163: consider changing “is proved by the TEM images shown in Fig. S1” to “is shown by the TEM images in Fig. S1”.\nResponse: Changed, as suggested.\n8. Lines 163-167: the main verb is missing, in my opinion. Consider changing “PCR amplicons to confirm that the RP4” to “PCR amplicons, were done to confirm that the RP4” or “PCR amplicons, were performed to confirm that the RP4”.\nResponse: Changed, as suggested.\n9. Line 173: the unit of measurement of the concentration is missing. Please add.\nResponse: Added, as suggested.\n10. Line 174: please specify the three sizes of PS.\nResponse: Revised, as suggested.\n11. Line 217: please remove the point between the bracket and the comma.\nResponse: Revised, as suggested.\n12. Fig. S9: please add the explanation of the statistical symbols, as for the other figures.\nResponse: Added, as suggested\n13. Lines 253-268: you reported in Fig. 2 and 3 captions that only significantly up- or down-regulated genes were shown. However, in the main text, genes (e.g., sfmA) not present in the figures are reported. Genes that did not significantly differ should not be mentioned and/or discussed. Which criteria have been used to select the genes displayed in Fig. 2 and 3? Moreover, for Fig. 2 and 3, the references present in the text are wrong. For example, line 258: “(Fig. 2f-h)” but panel h does not exist for Fig.2; line 264: should be “Fig. 3h” and not “Fig. 3f”. Please check carefully here and throughout the manuscript.\nResponse: Sorry for the confusion and appreciate the reviewer’s comments.\n(1) The genes displayed in Fig. 2 are plasmid-borne genes only. The gene sfmA is located on the donor chromosome, not on the plasmid; therefore, it was not included in the main figures but is reported in the Supplementary Information (Table S31).\n(2) For Figs. 2 and 3, only genes that were significantly up- or down-regulated in the donors with |LFC| ≥ 1 are shown. Statistical significance of the transcriptome data was determined by FDR-adjusted p-value of < 0.05.\n(3) We have carefully checked and corrected all figure/panel references in the manuscript. Specifically:\nLine 258: corrected from “Fig. 2f–h” to “Fig. 2d–g.” (Page 11 Line 273-274)\nLine 264: corrected from “Fig. 3f” to “Table S10.” (Page 11 Line 278-281)\nIn addition, we performed a manuscript-wide audit to ensure all figure citations and captions are accurate.\nPage 11 Line 273-274: Overall, the expression of genes related to plasmid conjugation increased (Fig. 2d–g).\nPage 11 Line 278-281: In the case of intergeneric conjugation donors, the expression of genes associated with oxidative stress (Table S10), such as rclA, trxC, and ytfE, significantly increased under the influence of all the tested PS particle sizes.\n14. Lines 270-284: given that no “significance stars” are reported next to the fold change values of tables S42-43, I do not know if the results shown in this section (Lines 270-284) are significant or not. If not, they should not be mentioned. Please check here and where this type of result is presented.\nResponse: In the revised version, we have added significance stars to all supplementary tables, including Tables S42–S43, to clearly indicate which genes are significantly regulated. We also confirm that all genes mentioned in the main text (Lines 270–284) are significantly regulated under at least one treatment condition, and thus all results described are statistically supported.\n15. Lines 276-277: consider using italics for the gene name (with the p in lowercase) and for E. faecalis.\nResponse: Revised, as suggested.\n16. Lines 287-295: as for comment #13, the figure references present in the text seem to be wrong and should be referred to the right panels. Please check carefully.\nResponse: Corrected from “Fig. 3c–d” to “Fig. 2a–b.”\nPage 12 Line 304-306: PS (20 nm and 120 nm) significantly promoted ROS production in both the intrageneric and the intergeneric conjugation systems (Fig. 2a–b).\n17. Lines 329-330: consider changing “fluctuation concentrations” to “fluctuation in concentrations” or “varying concentrations”.\nResponse: Changed, as suggested.\n18. Lines 362-374: as for comment #14, I cannot understand if the results are significant or not when looking at what is reported in the supplementary tables (no starred values). Moreover, also considering that all the genes in the tables were significantly up- or down-regulated, there are discrepancies between the genes cited in the text and the (significant) genes shown in panels g and h of Fig. 4. Which criteria have been used to select the genes displayed in Fig. 4?\nResponse: Thank you for pointing out this important comment and sorry for the confusion.\nOn statistical significance in the supplementary tables: All the genes with star indicators listed in the supplementary tables were significantly up- or down-regulated based on the adjusted p-value (FDR < 0.05) and fold-change thresholds (|LFC| ≥ 1) specified in the Methods. In the revised version, we have added significance indicators (e.g., stars or notes) to make this clearer.\nOn criteria for gene selection in Fig. 4: Panels g and h of Fig. 4 display a subset of significantly regulated genes chosen based on their functional relevance to conjugation-related membrane response pathways in recipients, rather than an exhaustive list of all significant genes. This approach was used to highlight representative genes for the main figures, while the complete list of significant genes remains available in the supplementary tables.\nOn consistency between text and figures: We have now carefully revised the text to ensure that only genes shown in Fig. 4 are discussed in the main body. Genes not displayed in Fig. 4 are now referred to in the Supplementary Information, where the complete dataset is provided.\nWe appreciate the reviewer’s feedback, which helped us improve the clarity of both the figures and the supplementary data presentation.\n19. Lines 386-389: the same as the comments #14 and 18 about the significance of the results.\nResponse: In the revised version, we have added significance stars to all supplementary tables. All the genes with star indicators listed in the supplementary tables were significantly up- or down-regulated based on the adjusted p-value (FDR < 0.05) and fold-change thresholds (|LFC| ≥ 1) specified in the Methods. We also confirm that all genes mentioned in the main text (Lines 386-389) are significantly regulated under at least one treatment condition, and thus all results described are statistically supported.\n20. Line 421: consider changing “species” to “genera”.\nResponse: Changed, as suggested.\n21. Line 446: consider changing “ROS” to “ROS production”.\nResponse: Changed, as suggested.\n22. Lines 464-466: consider changing “However, PS M/NPs at higher concentrations (10, 50, and 100 mg/L) may have a negative effect on AMR transmission, which may potentially hinder cell-to-cell contact or block conjugative pili, thereby reducing conjugation efficiency” in “However, PS M/NPs at higher concentrations (10, 50, and 100 mg/L) may potentially hinder cell-to-cell contact or block conjugative pili, thereby reducing conjugation efficiency and affecting negatively AMR transmission”.\nResponse: Changed as suggested.\n23. Line 486: consider changing “negligible and, may” to “negligible, it may”.\nResponse: Changed, as suggested.\n24. Line 503: consider changing “of PS” to “for PS”.\nResponse: Changed, as suggested.\n25. Line 537: consider changing “LE392 (carrying” to “LE392, carrying”.\nResponse: Changed, as suggested.\n26. Line 634: consider changing “The specific blaTEM-1 and tetA” to “The blaTEM-1 and tetA genes”.\nResponse: Changed, as suggested.\n27. Line 635: consider changing “RP4 analyzed” to “the plasmid RP4 checked”\nResponse: Changed, as suggested."}, {"round": 2, "reviewer_comments": {"reviewer_2": "Thank you for your detailed response to my previous comments. The revisions have improved the manuscript. However, I have one remaining major concern regarding the interpretation of the results.\n\nThe study compellingly demonstrates that polystyrene (PS) M/NPs, particularly at nano-scale (20 nm and 120 nm), can facilitate antimicrobial resistance (AMR) gene transfer. The authors attribute this, in part, to the \"nano-size\" feature of the particles. This raises a critical question about the specificity of the findings: Is the observed effect unique to plastic nanoparticles, or is it a general phenomenon associated with the presence of any nano-sized particles in the environment?\n\nAs the authors note, nanoparticles are abundant in natural and engineered water environments, originating from various sources such as natural clay minerals, metal oxides, and other anthropogenic colloids. If the primary mechanism is related to physical stress induced by the nano-scale itself (e.g., through increased membrane permeability and oxidative stress), then other common environmental nanoparticles could potentially exert a similar effect, making the AMR facilitation risk a much broader environmental issue.\n\nTo address this and solidify the ecological relevance of the findings, I strongly recommend that the authors include an additional control experiment. The study would be significantly strengthened by comparing the effects of PS-NPs with a naturally abundant nanoparticle control, such as nano-clay (e.g., montmorillonite) or a common metal oxide nanoparticle (e.g., TiO2, SiO2) at a similar size range and concentration.", "reviewer_5": "Dear Authors,\nI appreciate your effort to answer my questions. The study has been improved, and I have no further suggestions.\n\nMinor comment:\nLine 149 of the file named 611238_1_merged_1757492039.pdf: I assume the sentence refers to Figure S17 (never mentioned in the text) and not Figure S16."}, "author_response": "Response to Reviewers’ comments:\nReviewer #2 (Remarks to the Author):\nThank you for your detailed response to my previous comments. The revisions have improved the manuscript. However, I have one remaining major concern regarding the interpretation of the results.\n\nThe study compellingly demonstrates that polystyrene (PS) M/NPs, particularly at nano-scale (20 nm and 120 nm), can facilitate antimicrobial resistance (AMR) gene transfer. The authors attribute this, in part, to the \"nano-size\" feature of the particles. This raises a critical question about the specificity of the findings: Is the observed effect unique to plastic nanoparticles, or is it a general phenomenon associated with the presence of any nano-sized particles in the environment?\n\nAs the authors note, nanoparticles are abundant in natural and engineered water environments, originating from various sources such as natural clay minerals, metal oxides, and other anthropogenic colloids. If the primary mechanism is related to physical stress induced by the nano-scale itself (e.g., through increased membrane permeability and oxidative stress), then other common environmental nanoparticles could potentially exert a similar effect, making the AMR facilitation risk a much broader environmental issue.\n\nTo address this and solidify the ecological relevance of the findings, I strongly recommend that the authors include an additional control experiment. The study would be significantly strengthened by comparing the effects of PS-NPs with a naturally abundant nanoparticle control, such as nano-clay (e.g., montmorillonite) or a common metal oxide nanoparticle (e.g., TiO2, SiO2) at a similar size range and concentration.\nResponse: We sincerely thank the reviewer for this thoughtful and valuable comment. As suggested, we performed additional control experiments exploring the effect of nano-clay montmorillonite on the AMR gene transfer. These experiments showed that nano-clay montmorillonite had no significant effect on ARG conjugative transfer (Page 7 line 187-194, Supporting Information: Page 16 line 424-432, Fig. S6; Page 32 line 524-527, Table S3; Page 34 line 533-536, Table S5). In line with our experimental results, several published papers reported that metal nanoparticles1-5 (e.g., TiO ) enhanced conjugative transfer.\n\nIn detail, we conducted nano-clay montmorillonite at the same exposure concentrations (0, 0.1, 1, and 10 mg/L) with intrageneric RP4-related (donor E. coli K-12 LE392 and recipient E. coli BL21 (DE3)) and pCF10-related (donor E. faecalis OG1RF and recipient E. faecalis OG1RS) systems. Across all tested concentrations, we observed that nano-clay montmorillonite did not significantly increase conjugative transfer frequency (p > 0.05) (Page 7, lines 187–194). After observing this phenomenon, to contextualize our findings, we further compiled published data (Supporting Information: Table S48) on other metal nanoparticles1-6, including nano-TiO2, nano-Ag/Ag⁺, nano-CuO/Cu²⁺, nano-CeO₂, nano-Al₂O₃, which further confirmed our results: for metal nanoparticles, sub-lethal concentrations could enhance conjugative transfer frequency, because its detrimental effect to bacterial strains. Moreover, substances such as antibiotics, disinfectants, heavy metals, etc. would enhance HGT by increasing ROS production, damaging cell membranes, or otherwise perturbing bacterial physiology in ways that favour gene transfer. Metal nanoparticles (e.g., nano-TiO ) and PS M/NPs fall into this category: both are more likely to promote ROS production, induce oxidative stress, trigger stress responses, and alter conjugation-related processes. In contrast, nano-clay montmorillonite is comparatively inert and non-toxic under the tested conditions and did not enhance transfer. These collective findings provide solid evidence supporting the interpretation that nanoparticle-mediated ARG transfer results from the chemical nature and bacterial physiological responses rather than being solely attributable to the nanoscale size.\n\nIn the revised manuscript, we have added the following text to the results, discussion, and supplementary information to further clarify this point.\n\nPage 7 line 187-194: To determine whether it is the size or the chemical properties of substances that play a critical role in promoting HGT, we conducted conjugation experiments with exposure to nano-clay montmorillonite, which is a moderate chemical to bacteria, at 0, 0.1, 1, and 10 mg/L. Across all tested concentrations, nano-clay did not produce a statistically significant increase in conjugative transfer frequency compared to the control (Fig. S6, p > 0.05). By contrast, the literature reports significant increases in transfer frequency upon exposure to metal nanoparticles, such as nano-TiO 34, nano-Ag/Ag35, as well as Nano-CeO₂36.\n\nPage 18 line 452-459: Additionally, our results highlight that the promotion of HGT by nanoparticles is primarily driven by their chemical properties. Specifically, their ability to induce oxidative stress and disrupt bacterial membranes (Table S48). PS micro/nanoplastics and metal nanoparticles (e.g., nano-TiO 34) showed significant effects, while nano-clay montmorillonite (Fig. S6), a less reactive material, did not. These findings reveal a previously underrecognized pathway through which plastic pollution may contribute to the spread of AMR in the environment, emphasising the importance of considering the chemical properties of materials in evaluating their ecological impact.\n\nSupporting Information: Page 16 line 424-432\nFig. S6 Effects of nano-clay montmorillonite on plasmid-mediated conjugative transfer. a Fold changes in intrageneric conjugative ARG transfer frequency under 50 nm nano-clay montmorillonite at different concentrations in the intrageneric E. coli system. b Fold changes in intrageneric conjugative ARG transfer frequency under 50 nm nano-clay montmorillonite at different concentrations in the intrageneric E. faecalis system. Statistically significant differences between nanoparticle-treated groups and the control group were tested with the independent-sample t tests with the Benjamini–Hochberg correction and are indicated by *p < 0.05 and **p< 0.01.\n\nReviewer #5 (Remarks to the Author):\nDear Authors,\nI appreciate your effort to answer my questions. The study has been improved, and I have no further suggestions.\n\nMinor comment:\nLine 149 of the file named 611238_1_merged_1757492039.pdf: I assume the sentence refers to Figure S17 (never mentioned in the text) and not Figure S16.\nResponse: Revised, as suggested."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors made great efforts to revise the paper. My previous comments have been addressed and the paper can be published.", "reviewer_2": "1. I acknowledge that the author conducted additional experiments using Enterococcus faecalis as a model organism to verify and demonstrate the broader generalizability of nanoparticles (NPs) in promoting antimicrobial resistance (AMR) dissemination across different bacterial species. While these supplementary experiments successfully confirmed the previously established and well-documented impact of NPs on AMR dissemination in bacterial populations, they did not specifically address or advance our understanding of the primary research objective of this study—namely, the critical relationship between NP size and concentration on AMR development and spread.\n\n2. Secondly, upon careful examination of the experimental results, I observed that the impact of NP concentration on antibiotic resistance gene (ARGs) transfer rates demonstrated small variation across the range of tested concentrations when compared to the control conditions. This finding is particularly relevant given that NPs are ubiquitously present in environmental settings at various baseline levels, suggesting that increased environmental NP concentrations would not necessarily correlate with or result in elevated risks of AMR transfer among bacterial populations.\n\n3. Thirdly, while the author conducted comprehensive analyses of several cellular mechanisms—including reactive oxygen species (ROS) production, cell membrane permeability alterations, and adenosine triphosphate (ATP) generation—which are theoretically linked to ARGs transfer processes, this investigation falls short of providing conclusive experimental evidence demonstrating a direct causal relationship between these cellular mechanisms and increased ARGs transfer rates. The absence of such direct mechanistic evidence represents a significant limitation in establishing the precise role of these processes in facilitating enhanced ARGs transfer.", "reviewer_3": "Dear Authors\nThank you for an exhaustive reply and impressive improvement of the study and manuscript.\nThe lack of species diversity in the study was very nicely complemented by performing additional experiments with Gram-positive species which greatly strengthens the study.\nDescription of the aims of the study along with description of prior work has been well improved.\n\n1. It seems that the initial Figure 1b showing conjugative transfer frequency (not fold changes) with ROS scavenger was removed, apparently not to be found in supplementary data either. why?\n\n2. Line 156-158: Having a PCR verification for the transconjugant was a good addition. However, I am concerned that we cannot fully interpret the gel pictures in Fig S3 without product size information and ladder size information. If pure isolates culture is used for extracting transconjugant DNA I would not expect significant band intensity variations between the tested positive transconjugants. Please consider providing the following: Text S7: add information on the size of the PCR product and how DNA is prepared from the strains (i.e. from regrown transconjugant isolates). Fig S3: please indicate reference information for the ladder used on the gel and ladder size information (at least for the main band). Indicate which band(s) corresponds to the target PCR product. Including a negative control (dH2O sample) on the gel would have been useful to indicate possible presence of primer-dimers.\n\nMinor suggestions:\n3. Line 33 Consider changing “full explored” to “fully explored”.\n4. Line 35 Consider changing “Gram-negative and positive donors” to “Gram-negative and Gram-positive donors”.\n5. Line 39: consider changing “mediated by PS particles with various sizes” to “mediated by PS particles sizes”\n6. Line 43: consider changing “managing” with “understanding”\n7. Line 50: please add a space before “Microplastics”\n8. Line 61: consider changing “AMR and ARG…” to “Hence, AMR and ARG…”\n9. Line 73: consider changing \"…of ARGs, however, …\" to \"…of ARGs. However, …\"\n10. Line 75-76: consider deleting “Meanwhile, more bacterial strains and plasmids should be tested to validate the universality.” Or explain “validate the universality” of what?\n11. Line 79: consider replacing “Simultaneously” with “Additionally”.\n12. Line 99-100: consider replacing “…we further validated the generality of M/NPs in promoting AMR dissemination.” with “…we further tested the influence of M/NPs in promoting AMR dissemination.”\n13. Line 105-108: Since the study was not performed with environmental samples, I would suggest replacing “…significantly enhancing the spread of AMR genes in microbial communities...” with “…potentially enhancing the spread of AMR genes in microbial communities...”. This aspect is properly discussed line 439-458.\n14. Line 113: please remove punctuation before “favoring”.\n15. Figure 1: Y-axis units for c-j are not readable and should be improved. Maybe using RLUx103 would enable bigger fonts for the axis units.\n16. Line 377: “originally non-resistant pathogens that were originally non-resistant»: repeating “originally non-resistant” does not seem useful. Please consider deleting one."}, "author_response": "Response: We sincerely thank the reviewer for their positive feedback and valuable comments, which improved the manuscript much.\n\nReviewer #2 (Comments for the Author):\n1. I acknowledge that the author conducted additional experiments using Enterococcus faecalis as a model organism to verify and demonstrate the broader generalizability of nanoparticles (NPs) in promoting antimicrobial resistance (AMR) dissemination across different bacterial species. While these supplementary experiments successfully confirmed the previously established and well-documented impact of NPs on AMR dissemination in bacterial populations, they did not specifically address or advance our understanding of the primary research objective of this study—namely, the critical relationship between NP size and concentration on AMR development and spread.\nResponse: We would like to thank the valuable and constructive comments from the reviewer regarding the critical relationship between NP size and concentration on AMR development and spread. To answer this question, we conducted further analysis of the conjugation data with a specific focus on how NPs size and concentration influence the transfer of ARGs under various donor-recipient combinations.\n(1) Size effect: Regarding the size effect, we agree with the reviewer that the relationship between particle size and conjugation is complex and context dependent. We conducted a detailed analysis under different concentrations, and the results suggest that the size-dependent trends in ARGs transfer frequency are not uniform across systems. For instance, at 0.1 mg/L, conjugation frequencies in E. coli (both intra- and intergeneric) and intergeneric E. faecalis systems increased with 20 nm PS, decreased at 120 nm, and peaked again with 1 μm PS. However, in intra-genus E. faecalis, 1 μm PS showed a weaker promoting effect. At higher concentrations (1 and 10 mg/L), the trends diverged further, showing size-specific and system-dependent shifts, such as suppressed transfer by 120 nm PS in some systems, and selective enhancement by 1 μm PS in others. These findings reinforce that PS particle size does affect ARGs dissemination, but the effect is strongly influenced by concentration and donor–recipient pairing. Specifically,\nAmong different particle sizes, 20 nm PS consistently could exhibit strong enhancement effects across all conjugation systems. This enhancement was particularly significant in the intergeneric system of E. coli K-12 LE392 with RP4.\nIn contrast, 120 nm PS exhibited a weaker promoting effect compared to the other two particle sizes.\nInterestingly, 1 μm PS particles significantly promoted the transfer of ARGs specifically to E. coli recipients.\nThese results suggest that NPs exhibit a distinct but non-uniform size-dependent pattern to the conjugative transfer of ARGs across different donor-recipient systems. To further clarify this valuable point, we have rephrased our expression in the main text of Results part (Page 6, Line 177-189) and have updated the detailed data in Fig. 2 in the revised manuscript.\nOur results showed that there was no uniform trend in ROS production or membrane permeability with increasing particle size. Instead, the effects varied across conjugation systems.\no In E. faecalis systems, the variation in ROS production closely mirrored the changes in ARGs transfer frequency, suggesting ROS as a major driver in this Gram-positive bacterium.\no However, in E. coli-based systems, both ROS and membrane permeability contributed to the observed transfer patterns.\no Notably, 1 μm PS increased membrane permeability more strongly than ROS, particularly in E. coli recipients, implying a recipient-specific mechanism related to particle–cell surface interaction, possibly involving surface interactions or stress-induced membrane destabilization.\nThese nuanced, system-specific responses highlight the complex interplay between particle size, physiological stress, and microbial identity, and we have clarified this point in the revised manuscript\nWe have added text in the revised manuscript to further clarify the underlying mechanisms to the distinct size effect of PS to AMR transfer (Page 11, Line 301-310).\n(2) Concentration effects: Analysis of Fig. 4a-b and S2 reveals a nonlinear trend: transfer frequencies initially increase with rising M/NPs concentrations but then decrease at higher levels. Notably, 100 mg/L of 20 nm or 120 nm PS significantly inhibited ARGs transfer, suggesting a possible cytotoxic or inhibitory threshold at elevated concentrations.\nMoreover, we extended our concentration-dependent experiments using E. faecalis as the donor. At 0.1 mg/L, PS particles of all sizes significantly enhanced conjugation frequency (Fig. S4). However, with increasing concentrations, the changes in transfer frequency were less pronounced than those observed in the E. coli-based system. This suggests that Gram-positive donors may be less sensitive to NPs concentration changes, possibly due to differences in their cell envelope structures and conjugation machinery.\nThe observed sustained promotion of ARGs transfer across multiple concentration levels reflects the potential of M/NPs to continuously influence horizontal gene transfer (HGT) in microbial communities, thereby accelerating the long-term spread and evolution of AMR.\nNonlinear responses to increasing PS concentration:\nAcross all tested sizes, the physiological indicators—ROS production, cell membrane permeability, and ATP generation—generally followed a concentration-dependent increase at lower doses, followed by a decline at higher doses. While maximum values were not observed, our data showed that 1 μm PS significantly promoted ATP generation in all systems (Fig S14).\nIn most conjugation systems, the peak ROS levels occurred at 0.1 or 1 mg/L, supporting the existence of a stimulatory concentration window (Fig. 4c-d).\nHigher concentrations (e.g., 100 mg/L) may induce excessive stress or aggregation, thereby limiting conjugation (Fig. S2 &7).\nFurthermore, we observed that M/NPs exerted distinct effects on Gram-negative and Gram-positive donor systems. Specifically, transcriptomic analysis revealed that exposure to M/NPs led to the upregulation of genes associated with the phosphotransferase system (PTS) and teichoic acid biosynthesis pathways—mechanisms primarily relevant to Gram-positive bacteria (Fig. S4, Table S44-47). The PTS system is known to regulate energy metabolism and stress responses, potentially enhancing ATP production and the expression of conjugation-related proteins such as pili. Meanwhile, increased teichoic acid biosynthesis may alter the cell surface properties, facilitating closer contact between donor and recipient cells. Together, these changes likely promote the conjugative transfer of ARGs in Gram-positive bacteria by improving both the physiological state and physical interactions necessary for HGT.\nPage 6, Line 177-189: The influence of PS particle size on ARGs transfer showed distinct and concentration-dependent patterns across different conjugation systems. At 0.1 mg/L (Fig. 2a), a non-monotonic trend was observed: transfer frequencies increased with 20 nm PS, decreased with 120 nm, and rose again with 1 μm PS in E. coli-based intra- and intergeneric systems as well as intergeneric E. faecalis. In contrast, intra-genus E. faecalis conjugation was similarly promoted by 20 and 120 nm PS, but this effect declined with 1 μm particles. At 1 mg/L (Fig. 2b), similar patterns persisted, particularly in systems involving E. coli recipients. Under 10 mg/L (Fig. 2c), the size effect diverged further, e.g., intra-genus E. faecalis showed decreasing enhancement with increasing size, while in intergeneric systems, the trend reversed. Notably, in E. coli systems, 120 nm PS began to suppress ARGs transfer, whereas 1 μm PS maintained a promoting role, albeit weaker than that of 20 nm. These observations indicate a non-uniform and concentration-sensitive size effect, suggesting that both PS size and exposure level modulate the efficiency of horizontal gene transfer in a system-specific manner.\nPage 7, Line 215-227: Specifically, In E. faecalis systems, the size-dependent variation in ROS production closely matched the observed changes in conjugative transfer frequencies. For example, at 0.1 mg/L PS (Fig 3a)., intrageneric transfer increased then declined with increasing particle size, peaking at 120 nm, a trend mirrored by ROS levels, which reached 1.27-fold of the control under the same conditions. This suggests ROS may play a primary role in regulating conjugation in Gram-positive bacteria. Conversely, in E. coli systems, the observed transfer frequencies appeared to be shaped by both ROS production and membrane permeability. While 20 nm and 120 nm PS induced significant increases in both indicators, the effect of 1 μm PS was mainly reflected in enhanced membrane permeability rather than elevated ROS. Specifically, 1 μm PS increased permeability to 1.26-fold of the control in intrageneric systems. This may be attributed to the physical size of the particles, which is comparable to bacterial cells and could promote membrane disruption or surface interaction-based mechanisms, thereby facilitating ARGs transfer via a distinct pathway.\nPage 9, Line 240:\nFig. 2 Different sizes and concentrations of PS M/NPs affected plasmid-mediated conjugative transfer. a Fold changes in conjugative ARGs transfer frequencies under 0.1 mg/L PS with different particle sizes.\nPage 12, Line 313:\nFig. 3 Different sizes of PS M/NPs affected ROS production and cell membrane permeability. a ROS production under 0.1 mg/L PS with different particle sizes. b ROS production under 1 mg/L PS with different particle sizes. c ROS production under 10 mg/L PS with different particle sizes. d Cell membrane permeability under 0.1 mg/L PS with different particle sizes. e Cell membrane permeability under 1 mg/L PS with different particle sizes. f Cell membrane permeability under 10 mg/L PS with different particle sizes.\nPage 13, Line 400:\nFig. 4 Different concentrations of PS M/NPs affected ARGs dissemination. a Fold changes in conjugative ARGs transfer frequencies from E. coli K-12 LE392 to E. coli BL21 (DE3) under varying PS concentrations. b Fold changes in conjugative ARGs transfer frequencies from E. coli K-12 LE392 to P. putida KT2440 under varying PS concentrations. c ROS production of intrageneric recipient: E. coli BL21 (DE3). d ROS production of intrageneric recipient: P. putida KT2440. e Cell membrane permeability of intrageneric recipient: E. coli BL21 (DE3). f Cell membrane permeability of intrageneric recipient: P. putida KT2440.\nPage 11, Line 301-310: Specifically, 20 nm PS significantly increased ROS production (Fig. 3a-c), cell membrane permeability (Fig. 3d-f), and ATP levels (Fig. S14) across conjugative systems, suggesting a comprehensive enhancement of cellular conditions favoring gene transfer. In contrast, 120 nm PS induced moderate increases in membrane permeability and ATP production (Fig. S14), with no marked difference between intra- and inter-generic systems, indicating a more uniform but less potent biological response. Notably, 1 μm PS specifically enhanced membrane permeability (Fig. 3d-f) in systems involving E. coli, particularly when E. coli served as the recipient, which may represent a distinct mechanism by which larger particles promote ARGs dissemination. This size-specific effect warrants further mechanistic investigation to determine whether physical interactions or particle uptake pathways differ depending on recipient cell type and particle size.\nPage 13, Line 331-343: Despite varying fold changes in transfer frequencies across different treatments, we observed a nonlinear concentration-dependent effect of PS particles on the conjugative transfer of ARGs (Fig. 2c-d). In E. coli-donor systems, transfer frequency initially increased with PS concentration, peaking at moderate levels, and then declined at higher concentrations. For instance, 100 mg/L of 20 nm and 120 nm PS even significantly suppressed ARGs transfer compared to the control, indicating a threshold beyond which PS may exert inhibitory effects. Notably, when E. faecalis served as the donor, all tested sizes of PS at 0.1 mg/L significantly enhanced ARGs transfer, and although the promoting effect diminished or plateaued with increasing concentrations, the transfer frequencies remained elevated compared to the control. This suggests that the mere presence of M/NPs—regardless of concentration—can pose a persistent risk by promoting ARGs dissemination from Gram-positive donors. Moreover, the concentration sensitivity appears to differ between Gram-negative and Gram-positive donor systems, implying that concentration effects are both nonlinear and species-specific.\nPage 17, Line 466-469: These results suggest a nonlinear concentration-dependent effect of PS, where moderate doses (0.1–1 mg/L) enhance ROS production, membrane permeability, and ATP generation, whereas higher concentrations may trigger excessive cellular stress or particle aggregation, which in turn limits conjugative transfer in systems with Gram-negative donors.\nSupporting Information Page 15, Line 404:\nFig. S4 Fold changes in conjugative ARGs transfer frequencies for pCF10 from E. faecalis OG1RF to a E. faecalis OG1RS and b E. coli DH5α. c Genes related to phosphotransferase system (PTS) in intrageneric donors E. faecalis OG1RF. d Genes related to phosphotransferase system (PTS) in intergeneric donors E. faecalis OG1RF. e Genes related to teichoic acid biosynthesis in intrageneric donors E. faecalis OG1RF. f Genes related to teichoic acid biosynthesis in intergeneric donors E. faecalis OG1RF. Statistically significant differences between PS M/NPs-treated groups and the control group were tested with the independent-sample t test with the Benjamini–Hochberg correction and are indicated by *p < 0.05 and **p< 0.01. All genes shown in the figure were significantly (p < 0.05) up- or downregulated under exposure to tested PS M/NPs.\nSupporting Information Page 18, Line 425:\nFig. S7 Fold changes of conjugative transfer frequency for RP4 under the treatment of a 20 nm PS; b 20 nm PS with ROS scavenger; c 120 nm PS; d 120 nm PS with ROS scavenger; e 1 μm PS; f 1 μm PS with ROS scavenger. Statistically significant differences between treated and control groups were tested with the independent-sample t test with the Benjamini–Hochberg correction: *p < 0.05 and **p < 0.01.\nSupporting Information Page 77, Line 703:\nTable S44. Fold changes of expression of core genes related to teichoic acid biosynthesis in intrageneric donor bacteria E. faecalis OG1RF after treated by 0.1 mg/L, 1 mg/L and 10 mg/L PS M/NPs of 3 particle sizes (20 nm, 120 nm, 1 μm)\nFold change of gene expressiona\n20 nm 120 nm 1 μm\nGene COG annotation\n0.1 1 10 0.1 1 10 0.1 1 10\nmg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L\nOG1RF_10984 transcriptional regulator 0 8.738 8.877 8.244 9.887 0 9.630 0 0\npsr transcriptional regulator 8.759 9.395 9.236 9.772 8.644 11.178 0 8.852 10.199\nphosphatidylglycerol--membrane- - - - - - - - - -\nOG1RF_11521 oligosaccharide -8.548 0.001 -0.151 1.383 -8.548 -8.548 -8.548 -8.548 0.809\nglycerophosphotransferase\nCDP-glycerol:poly(glycerophosphate)\nOG1RF_11711 8.590 9.443 9.358 9.826 0 10.049 10.381 8.796 7.843\nglycerophosphotransferase\nCDP-glycerol:poly(glycerophosphate)\nOG1RF_11713 0 9.576 9.506 10.086 9.794 9.720 10.239 6.169 7.776\nglycerophosphotransferase\n- - - - - - - - - -\nOG1RF_12071 transcriptional regulator -4.043 -1.468 10.863 3.001 3.124 10.863 10.863 10.863 10.863\nD-alanine--D-alanyl carrier protein ligase\ndltC -3.507 2.585 2.251 3.985 -3.507 -3.507 -3.507 -3.507 -3.507\nsubunit 2\ndtlB\nD-alanine transfer protein DltB 4.518 4.373 4.391 4.494 4.543 3.683 1.998 3.673 4.159\na: Comparing with the control group without PS M/NPs treatment\n*: p < 0.05, **: p < 0.01\nSupporting Information Page 78, Line 708:\nTable S45. Fold changes of expression of core genes related to teichoic acid biosynthesis in intergeneric donor bacteria E. faecalis OG1RF after treated by 0.1 mg/L, 1 mg/L and 10 mg/L PS M/NPs of 3 particle sizes (20 nm, 120 nm, 1 μm)\nFold change of gene expressiona\n20 nm 120 nm 1 μm\nGene COG annotation\n0.1 1 10 0.1 1 10 0.1 1 10\nmg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L\nD-alanyl-lipoteichoic acid biosynthesis\ndelta 0.161 0.051 -0.255 -0.122 0.385 0.118 0.228 0.775 -0.357\nprotein DltD\nD-alanine--poly(phosphoribitol) ligase\ndltC 4.328 3.749 0 0 0 0 0 0.999 3.231\nsubunit DltC\nD-alanine--poly(phosphoribitol) ligase\ndltA 0.121 0.114 -0.094 0.580 0.442 0.036 0.404 0.672 0.519\nsubunit DltA\na: Comparing with the control group without PS M/NPs treatment\n*: p < 0.05, **: p < 0.01\nSupporting Information Page 79, Line 713:\nTable S46. Fold changes of expression of core genes related to phosphotransferase system (PTS) in intrageneric donor bacteria E. faecalis OG1RF after treated by 0.1 mg/L, 1 mg/L and 10 mg/L PS M/NPs of 3 particle sizes (20 nm, 120 nm, 1 μm)\nFold change of gene expressiona\n20 nm 120 nm 1 μm\nGene COG annotation\n0.1 1 mg/L 10 mg/L 0.1 1 mg/L 10 mg/L 0.1 1 mg/L 10 mg/L\nmg/L mg/L mg/L\nPTS family oligomeric\nOG1RF_12533 beta-glucoside porter 0 9.013 9.007 8.979 0 0 11.940 0 0\ncomponent IIC\nPTS family porter\nOG1RF_12479 -0.662 4.839 5.255 4.905 6.054 4.879 6.724 -4.750 -4.750\ncomponent II\nPTS family\nOG1RF_12478 fructose/mannitol (fru) 10.790 9.658 10.170 10.056 11.287 9.681 11.020 4.228 9.424\nporter component IIC\nPTS family N-\nacetylgalactosamine\nOG1RF_12312 0 6.914 6.334 6.310 0 0 10.197 0 0\n(nac) porter component\nIID\nPTS family galactitol\nOG1RF_11559 (gat) porter component 0 8.787 8.398 9.160 11.085 0 10.015 11.397 0\nIIC\nPTS family\nOG1RF_10752 lactose/cellobiose (lac) 2.305 1.867 2.121 1.823 1.551 -1.068 3.264 -8.445 2.191\nporter component IIC\nPTS family\nOG1RF_10746 lactose/cellobiose porter -7.773 1.963 1.545 1.923 -7.773 2.032 2.623 2.399 0.515\ncomponent IIC\nsacX PTS family porter, -4.442 5.120 3.918 4.718 -4.442 2.777 4.546 -4.442 3.526\nenzyme I\nPTS family mannose\nmanX2 -0.059 1.435 1.633 1.492 0.270 0.689 -2.634 -0.859 1.360\nporter, IIAB component\nPTS family fructose\nfruA 1.346 1.372 1.251 1.130 1.025 1.954 1.895 0.382 0.003\nporter, IIABC component\nPTS family\nsorB mannose/fructose/sorbose 0.000 5.519 5.501 6.091 10.016 0.000 8.260 0.000 0.000\nporter, IIB component\nPTS family\nOG1RF_12476 fructose/mannitol (fru) 0.000 7.610 8.399 8.348 0.000 0.000 8.158 0.000 0.000\nporter component IIA\nPTS family\nOG1RF_10021 mannose/fructose/sorbose 2.175 1.346 1.088 0.947 1.320 1.947 1.663 1.677 1.309\nporter component IID\nPTS family beta-\nscrA glucosides porter, IIABC 0.860 1.437 1.329 1.933 0.668 1.813 1.795 2.599 2.205\ncomponent\nPTS family mannitol\nmtlA2 8.051 10.151 9.849 10.145 11.580 9.382 7.756 10.905 10.539\nporter, EIICB component\nPTS family\nsrlE glucitol/sorbitol porter, 8.007 8.117 8.196 8.191 0.000 0.000 6.808 9.152 0.000\nIIB component\nPTS family ascorbate\nOG1RF_12477 0.000 8.578 9.378 9.188 0.000 0.000 6.716 0.000 8.935\nporter, IIB component\nPTS family porter,\nbglF 1.088 0.849 1.469 1.695 2.614 1.980 1.118 2.817 2.634\nenzyme I\nPTS family\nmltF2 fructose/mannitol porter 0.000 7.001 7.613 5.833 0.000 0.000 6.138 0.000 0.000\ncomponent IIA\nmltF PTS family 0.000 7.664 7.244 7.814 0.000 9.036 5.632 4.228 0.000\nfructose/mannitol porter\ncomponent IIA\nPTS family ascorbate\nulaA 0.000 7.554 7.477 7.884 0.000 9.567 5.415 0.000 0.000\nporter, IIC component\nPTS family porter\nOG1RF_11236 0.000 7.815 7.960 8.245 0.000 10.232 4.448 9.577 4.446\ncomponent IIC\na: Comparing with the control group without PS M/NPs treatment\n*: p < 0.05, **: p < 0.01\nSupporting Information Page 82, Line 718:\nTable S44. Fold changes of expression of core genes related to phosphotransferase system (PTS) in intergeneric donor bacteria E. faecalis OG1RF after treated by 0.1 mg/L, 1 mg/L and 10 mg/L PS M/NPs of 3 particle sizes (20 nm, 120 nm, 1 μm)\nFold change of gene expressiona\n20 nm 120 nm 1 μm\nGene COG annotation\n0.1 1 mg/L 10 0.1 1 mg/L 10 0.1 1 mg/L 10\nmg/L mg/L mg/L mg/L mg/L mg/L\nfob 1-phosphofructokinase 0.261 0.068 -0.269 2.138 1.963 1.918 2.123 2.171 2.179\nfructose-specific PTS\nOG1RF_RS02415 0.524 -0.097 -0.076 1.170 1.123 0.981 1.017 1.105 1.250\ntransporter subunit EIIC\nPTS system\nmannose/fructose/sorbose\nOG1RF_RS11845 2.349 1.062 3.043 -2.688 2.493 -2.688 3.650 1.177 3.402\nfamily transporter subunit\nIID\nPTS mannose transporter\nOG1RF_RS12645 0.474 1.742 0.912 2.068 1.921 1.499 2.249 -0.004 0.606\nsubunit IIA\nPTS sugar transporter\nOG1RF_RS07765 3.177 2.790 1.761 -2.688 -2.688 1.803 3.074 2.127 0.543\nsubunit IIC\nPTS sugar transporter\nOG1RF_RS12305 0.032 0.940 1.570 -0.698 0.859 2.190 1.156 -1.079 0.083\nsubunit IIC\nPTS system\nmannose/fructose/sorbose\nOG1RF_RS01855 2.038 4.420 -2.688 2.760 -2.688 1.250 2.816 2.127 2.994\nfamily transporter subunit\nIID\nPTS ascorbate transporter\nOG1RF_RS04730 6.149 5.833 6.399 5.044 0.000 5.865 0.000 6.585 6.668\nsubunit IIC\nPTS sugar transporter\nOG1RF_RS07955 0.000 0.000 5.731 0.000 3.295 3.937 0.000 5.789 0.000\nsubunit IIC\nOG1RF_RS12290 PTS fructose transporter 4.725 4.695 0.000 0.000 6.328 4.890 4.229 5.382 6.090\nsubunit IIA\nPTS fructose transporter\nOG1RF_RS06400 0.219 1.283 -1.559 -5.073 -5.073 0.792 -0.285 -5.073 -0.921\nsubunit IIC\nPTS transporter subunit\nOG1RF_RS08195 0.131 -2.163 -2.012 -0.030 -1.280 -1.259 0.027 0.124 -0.203\nEIIC\nPTS N-\nagaW acetylgalactosamine 0.000 4.695 5.012 3.544 4.779 3.937 3.304 0.000 4.152\ntransporter subunit IIC\nPTS glucitol/sorbitol\nOG1RF_RS13065 1.938 2.095 0.878 -3.571 2.181 -0.542 1.619 -3.571 1.139\ntransporter subunit IIC\nfructose PTS transporter\nOG1RF_RS02310 -3.571 0.727 -3.571 2.670 -3.571 1.631 -3.571 -3.571 1.853\nsubunit IIA\nPTS sugar transporter\nOG1RF_RS02965 -0.441 -1.728 0.254 1.648 1.002 0.199 -1.248 -0.663 0.931\nsubunit IIC\nPTS cellobiose\ncelB 0.860 0.829 1.428 1.597 1.039 1.810 0.215 1.076 0.968\ntransporter subunit IIC\nPTS transporter subunit\nOG1RF_RS12915 -0.134 0.818 -2.130 0.120 1.507 1.529 0.694 -0.261 0.764\nEIIC\na: Comparing with the control group without PS M/NPs treatment\n*: p < 0.05, **: p < 0.01\nWe have added text in the Discussion part of the revised manuscript to further clarify this point (Page 18, Line 478-485).\nPage 18, Line 478-485: Interestingly, M/NPs displayed differential effects on conjugative transfer depending on whether the donor strain was Gram-negative or Gram-positive. Transcriptomic analysis indicated that in E. faecalis-based systems, M/NP exposure upregulated genes involved in the phosphotransferase system (PTS) and teichoic acid biosynthesis, both of which are critical to cell wall structure and metabolism in Gram-positive bacteria. These molecular changes may contribute to the distinct conjugation responses observed between donor types, underscoring the species-specific nature of M/NP-mediated AMR propagation.\nIn summary, the size-dependent effect of PS particles on ARGs transfer was non-uniform across donor–recipient systems, highlighting that the impact of particle size is closely linked to microbial identity and conjugation context. In contrast, the concentration effect showed a nonlinear pattern, with low concentrations generally promoting ARGs dissemination, while higher concentrations may induce inhibitory stress—emphasizing the potential of M/NPs to continuously influence AMR spread under environmentally relevant conditions. Taken together, these results demonstrate that PS concentration plays a critical role in modulating cellular responses relevant to ARGs transfer. The effect is nonlinear and context-dependent, influenced by both PS size and bacterial pairing.\nWe believe this clarification and additional analysis would address your concerns regarding the relationship between PS size and concentration on AMR development and spread.\n2. Secondly, upon careful examination of the experimental results, I observed that the impact of NP concentration on antibiotic resistance gene (ARGs) transfer rates demonstrated small variation across the range of tested concentrations when compared to the control conditions. This finding is particularly relevant given that NPs are ubiquitously present in environmental settings at various baseline levels, suggesting that increased environmental NP concentrations would not necessarily correlate with or result in elevated risks of AMR transfer among bacterial populations.\nResponse: We appreciate a lot for the reviewer’s valuable insights and pointing this great point out. Yes, we agree with the reviewer that increased environmental NPs concentrations would not necessarily correlate with or result in elevated risks of AMR transfer. Indeed, our experimental results also show a complex, nonlinear relationship between ARGs transfer frequency and NPs concentration.\nSpecifically,\n(1) In systems with Gram-negative bacteria as donors, the ARGs transfer frequency initially increases compared to the control as NPs concentration rises, but then decreases, showing an overall trend of first increasing and then declining. Nevertheless, even modest increases in concentration can significantly promote the spread of resistance genes, indicating that NPs contribute importantly to the risk of AMR dissemination.\n(2) In systems with Gram-positive bacteria as donors, the ARGs transfer frequency increases compared to the control as NPs concentration rises, showing an overall trend of continuous increase or only slight variation. This suggests that regardless of concentration, the persistent presence of M/NPs in the environment can continuously promote the spread of AMR and contribute to the long-term risk of AMR dissemination.\nWe note that even small increases in HGT frequency can have significant ecological and clinical consequences. M/NPs are highly persistent and ubiquitous in the environment2 due to their unique physical and chemical properties. With a wide size range (from nanometers to millimeters)3 and high longevity (from decades to over a thousand years)3, they can remain in aquatic environments for extended periods and travel long distances4, thus becoming a significant and enduring environmental concern. Even though the increase in ARGs transfer may not be dramatic, the widespread and persistent nature of NPs in diverse environments could still pose a significant, albeit subtle, consistent risk to microbial resistance dynamics over time.\nWe have added text in the revised manuscript to further clarify this point (Page 2, Line 56-59).\nPage 2 Line 56-59: Their high longevity3, with half-lives ranging from decades to over a thousand years, makes them far more durable than most natural particles2. This persistence allows M/NPs to continuously transport and accumulate contaminants, including harmful chemicals and pathogens, across various ecosystems, thus becoming a significant and enduring environmental concern4.\nPage 17 Line 448-453: NPs are distinct from microplastics and engineered nanomaterials due to their unique transport properties, higher surface molecule fraction, bioavailability, which influence their environmental fate, impact on AMR transfer and human health5. While the increase in ARGs transfer rates due to NPs may be modest, their ubiquitous and persistent presence in the environment means that their contribution to the spread of AMR should not be overlooked.\nWe also added a brief note about the environmental relevance of our tested concentrations based on measured NP levels in aquatic environments.\nPage 3 Line 98-100: Using pyrolysis-gas chromatography time-of-flight mass spectrometry (Py-GCToF), the presence of PS was detected with a semi-quantifiable result of 0.2 mg/L in river water6.\n3. Thirdly, while the author conducted comprehensive analyses of several cellular mechanisms—including reactive oxygen species (ROS) production, cell membrane permeability alterations, and adenosine triphosphate (ATP) generation—which are theoretically linked to ARGs transfer processes, this investigation falls short of providing conclusive experimental evidence demonstrating a direct causal relationship between these cellular mechanisms and increased ARGs transfer rates. The absence of such direct mechanistic evidence represents a significant limitation in establishing the precise role of these processes in facilitating enhanced ARGs transfer.\nResponse: Again, this is a good point. We are grateful for the comment regarding the need for more direct mechanistic evidence linking observed cellular changes to enhanced ARGs transfer. In response, we would like to clarify and expand upon the supporting evidence we have already obtained:\nIn most cases, ROS generation is the primary driver of increased ARGs transfer frequency, particularly in E. faecalis systems where ROS trends closely align with conjugation patterns. In E. coli-based systems, both ROS and membrane permeability contribute to the enhancement of ARGs transfer. Notably, 1 μm PS particles predominantly increase membrane permeability together with ROS production, indicating an additional recipient-specific mechanism, possibly linked to physical interactions with the cell surface or membrane destabilization.\nCausal role of ROS in promoting conjugation:\nIn fact, in our experiment, we designed and performed experiments using ROS scavengers to modulate oxidative stress levels during M/NPs exposure (Fig. S2). Conjugation frequencies were then re-quantified under these conditions. The results showed that reducing ROS levels significantly attenuated the M/NPs-induced enhancement in gene transfer, suggesting a direct and functional role for ROS in mediating this process. Transcriptomic evidence further supported mechanistic pathways. In addition, we have conducted whole-genome RNA sequencing (RNA-seq) to explore the broader metabolic responses of donor and recipient cells to M/NPs exposure. These data provide complementary evidence that the proposed mechanisms—oxidative stress (ROS), ATP synthesis, and membrane permeability alterations—are linked to system-wide transcriptional changes, reinforcing their biological relevance in the context of conjugative transfer.\nSupporting Information Page 18, Line 425:\nFig. S7 Fold changes of conjugative transfer frequency for RP4 under the treatment of a 20 nm PS; b 20 nm PS with ROS scavenger; c 120 nm PS; d 120 nm PS with ROS scavenger; e 1 μm PS; f 1 μm PS with ROS scavenger. Statistically significant differences between treated and control groups were tested with the independent-sample t test with the Benjamini–Hochberg correction: *p < 0.05 and **p < 0.01.\nWe have added text in the revised manuscript to further clarify this point (Page 7, Line 196-209).\nPage 7 Line 196-209: In addition to promoting ARGs transfer, PS particles of different sizes exhibited distinct effects on cellular processes associated with conjugation. Importantly, we further investigated the underlying physiological mechanisms by which PS particles promote the conjugative transfer of ARGs. Among the tested parameters, ROS generation emerged as a primary driver, particularly in E. faecalis-based systems, where trends in ROS production closely mirrored increases in ARGs transfer frequency (Fig 3a-c). In E. coli-based systems, both ROS and membrane permeability appeared to contribute, with 1 μm PS particles notably inducing significant changes in membrane permeability (Fig 3d-f). This implies a recipient-specific mechanism, possibly involving physical interactions between large PS particles and bacterial surfaces or membrane destabilization under stress. To further validate the role of ROS, we conducted ROS scavenger assays, which showed that quenching ROS significantly reduced the PS-induced increase in conjugation frequency (Fig. S7). These findings provide direct functional evidence that oxidative stress is causally linked to the observed enhancement of ARGs transfer. Supporting this, transcriptomic profiling revealed the upregulation of stress-responsive pathways.\n\nReviewer #3 (Comments for the Author):\nDear Authors\nThank you for an exhaustive reply and impressive improvement of the study and manuscript.\nThe lack of species diversity in the study was very nicely complemented by performing additional experiments with Gram-positive species which greatly strengthens the study.\nDescription of the aims of the study along with description of prior work has been well improved.\nResponse: We thank the reviewer for their kind and encouraging comments. We are grateful that the reviewer recognizes the value of the additional Enterococcus faecalis experiments in addressing the concern regarding species diversity, and we appreciate the positive feedback on our revised introduction and clarification of study aims.\n1. It seems that the initial Figure 1b showing conjugative transfer frequency (not fold changes) with ROS scavenger was removed, apparently not to be found in supplementary data either. why?\nResponse: We appreciate the reviewer’s careful attention to this point. The original data showing the conjugative transfer frequency with and without ROS scavenger in E. coli was intended to demonstrate that intrageneric transfer frequency is consistently higher than intergeneric transfer frequency, regardless of ROS scavenging. However, following the reviewer’s initial comments, we conducted additional conjugation experiments using Enterococcus faecalis, which also confirmed that intrageneric transfer frequency is higher than intergeneric transfer frequency.\nTo streamline Figure 1, which already contains multiple panels, we decided to remove the ROS scavenger panel to avoid overcrowding. Nevertheless, the relevant data has been preserved and incorporated into the revised supplementary materials, specifically in Table S3 and Table S4, which include quantitative results from the ROS scavenger experiments.\nWe hope this clarifies our rationale and are happy to reintroduce the figure in the supplement if the reviewer or editor deems it necessary.\nSupporting Information Page 30, Line 508:\nTable S3. Comparison between PS M/NPs concentrations and conjugative frequency within genera (Donor: E. coli K-12 LE392, Recipient: E. coli BL21 (DE3))\n20 nm 120 nm 1 μm\nConcentration（mg/L）\nfrequency Significance (2 tailed), p frequency Significance (2 tailed), p frequency Significance (2 tailed), p\nConjugation within genera\n0 5.28×10-6 5.28×10-6 5.28×10-6\n0.1 3.45×10-5 0.0053 1.78×10-5 0.0072 5.70×10-5 0.0015\n1 2.07×10-5 0.0049 9.81×10-6 0.0103 3.90×10-5 0.0029\n10 2.45×10-5 0.0084 8.55×10-6 0.0176 6.74×10-6 0.0709\n50 7.05×10-6 0.1524 2.96×10-6 0.0271 1.26×10-5 0.0085\n100 2.31×10-6 0.0369 4.19×10-6 0.0740 7.50×10-6 0.1135\nConjugation within genera with ROS scavenger\n0 4.14×10-7 4.14×10-7 4.14×10-7\n0.1 5.29×10-7 0.1393 5.24×10-7 0.1059 4.50×10-7 0.1311\n1 4.71×10-7 0.2657 5.38×10-7 0.0499 1.07×10-6 0.0111\n10 2.82×10-8 0.0013 3.62×10-7 0.1256 1.83×10-7 0.0074\n50 4.16×10-8 0.0015 3.17×10-7 0.1174 3.07×10-7 0.0240\n100 2.35×10-8 0.0014 4.96×10-7 0.1134 1.09×10-7 0.0036\nNote: Comparison with p value (Sig. (2-tailed)) < 0.05 were considered as significant (*), p value < 0.01 were considered as very significant (**).\nSupporting Information Page 31, Line 512:\nTable S4. Comparison between PS M/NPs concentrations and conjugative frequency across genera (Donor: E. coli K-12 LE392, Recipient: P. putida KT2440)\n20 nm 120 nm 1 μm\nConcentration（mg/L） Significance (2 Significance (2 Significance (2\nfrequency frequency frequency\ntailed), p tailed), p tailed), p\nConjugation across genera\n0 4.45×10-8 4.45×10-8 4.45×10-8\n0.1 6.62×10-8 0.0040 4.89×10-8 0.0028 7.28×10-8 0.0015\n1 4.73×10-7 0.0009 7.94×10-8 0.0042 5.70×10-8 0.0062\n10 3.00×10-7 0.0002 1.64×10-8 0.0001 5.38×10-8 0.0511\n50 1.02×10-7 0.0008 1.64×10-8 0.0002 4.75×10-8 0.2895\n100 2.63×10-8 0.0074 1.54×10-8 0.0001 5.57×10-8 0.1503\nConjugation across genera with ROS\nscavenger\n0 2.16×10-9 2.16×10-9 2.16×10-9\n0.1 3.55×10-9 0.0351 2.00×10-9 0.2675 2.23×10-9 0.3209\n1 4.85×10-9 0.0045 1.80×10-9 0.1516 2.03×10-9 0.1154\n10 4.90×10-9 0.0042 1.35×10-9 0.0052 1.68×10-9 0.0115\n50 4.70×10-9 0.0026 8.78×10-9 0.0032 1.65×10-9 0.0480\n100 1.55×10-9 0.0156 1.53×10-10 0.0408 1.83×10-9 0.0415\nNote: Comparison with p value (Sig. (2-tailed)) < 0.05 were considered as significant (*), p value < 0.01 were considered as very significant (**).\n2. Line 156-158: Having a PCR verification for the transconjugant was a good addition. However, I am concerned that we cannot fully interpret the gel pictures in Fig S3 without product size information and ladder size information. If pure isolates culture is used for extracting transconjugant DNA I would not expect significant band intensity variations between the tested positive transconjugants. Please consider providing the following: Text S7: add information on the size of the PCR product and how DNA is prepared from the strains (i.e. from regrown transconjugant isolates). Fig S3: please indicate reference information for the ladder used on the gel and ladder size information (at least for the main band). Indicate which band(s) corresponds to the target PCR product. Including a negative control (dH2O sample) on the gel would have been useful to indicate possible presence of primer-dimers.\nResponse: We thank the reviewer for carefully examining the PCR validation data and for these valuable suggestions to improve the clarity and interpretability of Fig. S3 and Text S7. We redid the PCR and gel electrophoresis according to the reference7. In response:\nText S7 has been revised to include the expected PCR product size (bp) and to describe the method used for DNA extraction. Specifically, we now clarify that we first extracted plasmids from regrown transconjugant isolates from selective plates cultured in fresh selective media. Subsequently, PCR was conducted on the extracted plasmids. DNA ladder used was GeneRuler 1kb Ladder (ranging from 75 to 20,000 bp, Thermo ScientificTM).\nPage 6 line 164-167: After extracting plasmids from the donors and transconjugants, PCR amplification of bla and tetA gene located on the plasmid, followed by TEM-1 electrophoresis of both the plasmids and the PCR amplicons to confirm that the RP4 plasmid had been successfully transferred to the transconjugants (Fig. S3).\nSupplementary Text S7. PCR and gel electrophoresis conditions\nThe donors E. coli K-12 LE392 (RP4), and transconjugants obtained from the transconjugant-selective plates (E. coli BL21 (DE3) and P. putida KT2440) were cultured in LB broth overnight at 37 °C. Plasmids were extracted from the prepared cultures using the Invitrogen PureLink Quick Plasmid Miniprep Kit (Life Technologies, USA). The successful transfer of plasmids from the donor to the recipients was confirmed by agarose gel electrophoresis, indicating the formation of transconjugants. Primers for bla (861 bp) are 5′-TTACCAATGCTTAATCAGTGAGGC-3′ (FW) TEM-1 and 5′-ATGAGTATTCAACATTTCCGTGTCG-3′ (RV). Primers for tetA (1200 bp) are 5’-CGTGTATGAAATCTAACAATGCGCT-3’ (FW) and 5’- CCATTCAGGTCGAGGTGGC-3’ (RV)7.\nPCR assays were performed in a 20-μL reaction volume containing 1 μL of extracted plasmids, 10 μL of Platinum Green Hot Start PCR 2× Master Mix (Invitrogen, Thermo Fisher Scientific), 2 μL of Platinum GC Enhancer, 0.2 μL each of forward and reverse primers, and 6.6 μL of Milli-Q water. PCR amplification was conducted on an Applied Biosystems machine under the following conditions: initial denaturation at 94 °C for 10 minutes, followed by 30 cycles of 94 °C for 1 minute, 60 °C for 1 minute, and 72 °C for 1 minute, with a final extension at 72 °C for 7 minutes. ddH O was used as the 2 negative control (NC). Visualization of all bands was conducted by SYBR safe DNA gel staining and the GeneRuler 1 kb Plus DNA Ladder (Thermo ScientificTM).\nFigure S3 has been updated to:\nSupporting Information Page 14, Line 399:\nFig. S3 Gel electrophoresis of a. RP4 and PCR amplicons for b. bla and c. tetA TEM-1 on plasmid RP4 from donor E. coli K-12 LE392, intrageneric transconjugants E. coli BL21 (DE3) and intergeneric transconjugants P. putida KT2440. NC: negative control ddH O. 2\nMinor suggestions:\n3. Line 33 Consider changing “full explored” to “fully explored”.\nResponse: Revised, as suggested.\n4. Line 35 Consider changing “Gram-negative and positive donors” to “Gram-negative and Gram-positive donors”.\nResponse: Revised, as suggested.\n5. Line 39: consider changing “mediated by PS particles with various sizes” to “mediated by PS particles sizes”\nResponse: Revised, as suggested.\n6. Line 43: consider changing “managing” with “understanding”\nResponse: Revised, as suggested.\n7. Line 50: please add a space before “Microplastics”\nResponse: Revised, as suggested.\n8. Line 61: consider changing “AMR and ARG…” to “Hence, AMR and ARG…”\nResponse: Revised, as suggested.\n9. Line 73: consider changing \"…of ARGs, however, …\" to \"…of ARGs. However, …\"\nResponse: Revised, as suggested.\n10. Line 75-76: consider deleting “Meanwhile, more bacterial strains and plasmids should be tested to validate the universality.” Or explain “validate the universality” of what?\nResponse: We thank the reviewer for pointing out the ambiguity in this sentence. Our intention was to suggest that future studies should test additional bacterial strains and plasmids to assess the generalizability of the observed micro/nanoplastics-mediated enhancement of ARGs transfer. We have now revised the sentence to clarify this point as follows:\nPage 3, Line 80-82: Meanwhile, more bacterial strains and plasmids should be tested to assess the generalizability of the observed M/NPs-induced promotion of ARGs transfer.\n11. Line 79: consider replacing “Simultaneously” with “Additionally”.\nResponse: Revised, as suggested.\n12. Line 99-100: consider replacing “…we further validated the generality of M/NPs in promoting AMR dissemination.” with “…we further tested the influence of M/NPs in promoting AMR dissemination.”\nResponse: Revised, as suggested.\n13. Line 105-108: Since the study was not performed with environmental samples, I would suggest replacing “…significantly enhancing the spread of AMR genes in microbial communities...” with “…potentially enhancing the spread of AMR genes in microbial communities...”. This aspect is properly discussed line 439-458.\nResponse: Revised, as suggested.\n14. Line 113: please remove punctuation before “favoring”.\nResponse: Revised, as suggested.\n15. Figure 1: Y-axis units for c-j are not readable and should be improved. Maybe using RLUx103 would enable bigger fonts for the axis units.\nResponse: We thank the reviewer for this valuable suggestion. To improve readability, we have revised the Y-axis labels in panels c–j by adjusting the font size and adopting the notation “RLU × 10³” where applicable. These changes enhance clarity without altering the data presentation.\nPage 5, Line 143:\na FromE. coliK-12LE392 toE.coli BL21 (DE3)b FromE. faecalis OG1RF toE. faecalis OG1RS\nFromE. coliK-12LE392 toP. putida KT2440 FromE. faecalis OG1RF toE. coli DH5α\n4×10-1\nycn e u q e rf re fsn\na rt e vita\ng u jn o C\n)tn e ip\nice\nr re p (\n1 4 . . 5 3 5\n2\n4 6\n× × ×\n×\n× ×\n1 1 1\n1\n1 1\n0 0 0\n0\n0 0\n- - -\n-\n- -\n5 5 5\n7\n7 7\n** ** **\n**\n** **\n**\n** **\n**\n*\n**\n* *\n**\nycn e u q e rf re fsn\na rt e vita\ng u jn o C\n)tn e ip\nice\nr re p (\n1 2 3\n1\n2\n× × ×\n×\n×\n1 1 1\n1\n1\n0 0 0\n0\n0\n- - -\n-\n-\n1 1 1\n4\n4\n** * **\n*\n** **\n**\n* **\n**\n0 0\n0 0.1 1 10 50 100 0 0.1 1 10\nConcentration (mg/L) Concentration (mg/L)\nc d E E . . c c o o l l i i K K - - 1 1 2 2 L L E E 3 3 9 9 2 2 + + P E . . c p o u li ti B da L 2 K 1 T ( 2 D 4 E 40 3) e f E E . . f f a a e e c c a a l l i i s s O O G G 1 1 R R F F + + E E . . c fa o e li c D al H is 5α OG1RS\nn i ytisn e tn i e cn e cse ro u lF 30 1 × U L R ( n o itcu d o rp S O R ) 2 4 E P. . c p o u li ti B da L 2 K 1 T ( 2 D 4 E 4 3 0 ) n i ytisn e tn i e cn e cse ro u lF 30 1 × U L R ( n o itcu d o rp S O R ) 4 8 lle c n i ytisn e tn i e cn e cse ro u lF 1 × U L R ( ytilib a e m re p e n a rb m 4 8 E P. . c p o u li ti B da L 2 K 1 T ( 2 D 4 E 4 3 0 ) lle c n i y tisn e tn i e cn e cse ro u lF 1 × U L R ( ytilib a e m re p e n a rb m 3 6 9\ne e\ng 0 h 0 i m 0 j m 0\nE. coliK-12 LE392+E.coliBL21 (DE3) E. faecalisOG1RF+E. faecalisOG1RS\nE. coliK-12 LE392+P. putida KT2440 E. faecalisOG1RF+E. coliDH5α\nn i ytisn e tn i e cn e cse ro u lF 30 1 × U L R ( n o itcu d o rp S O R )\n1\n0\n4 8\nE\n. .\n.\nc p\nc\no u\no\nli t\nli\ni B d\nK\na L\n-\n2\nL\nD 4\nE\nE 4\n3\n3 0\n9\n)\n2\nlle c n i y tisn e tn i e cn e cse ro u lF\n30\n1 × U L R ( ytilib a e m re p e n a rb m\ne m\n)1\n0\n4 8\n2\nlle c n i y tisn e tn i e cn e cse ro u lF\n30\n1 × U L R ( ytilib a e m re p e n a rb m\ne m\n)1\n0\n2 5 7\n0\n.\n. .\n.\n.0\n0\n5 0 5 E E\nE\n. .\n.\nf c\nf\na\na\no e\ne\nli c\nc\nD a\na\nl\nl\nH i\ni\ns\ns\nO\nG\nG\n1\nR\nS\nF\nn i ytisn e tn i e cn e cse ro u lF 30 1 × U L R ( n o itcu d o rp S O R ) 1\n1\n0\n6 2\na Conjugative transfer frequency within and across genera under 20 nm PS exposure for RP4. b Conjugative transfer frequency within and across genera under 20 nm PS exposure for pCF10. c ROS production of bacteria strains in RP4-related conjugation system without PS. d ROS production in intrageneric and intergeneric conjugation system for RP4 without PS. e Cell membrane permeability of bacteria strains in RP4-related conjugation system without PS. f Cell membrane permeability in intrageneric and intergeneric conjugation system for RP4 without PS. g ROS production of bacteria strains in pCF10-related conjugation system without PS. h ROS production in intrageneric and intergeneric conjugation system for pCF10 without PS. i Cell membrane permeability of bacteria strains in pCF10-related conjugation system without PS. j Cell membrane permeability in intrageneric and intergeneric conjugation system for pCF10 without PS. Statistically significant differences between intra- and intergeneric conjugation were tested with the independent-sample t test with the Benjamini–Hochberg correction: *p < 0.05 and **p < 0.01.\n16. Line 377: “originally non-resistant pathogens that were originally non-resistant»: repeating “originally non-resistant” does not seem useful. Please consider deleting one.\nResponse: Revised, as suggested.\n\nReference\n1. Andersson DI, Hughes D. Microbiological effects of sublethal levels of antibiotics. Nat. Rev. Microbiol. 12, 465-478 (2014).\n2. Allen D, et al. Microplastics and nanoplastics in the marine-atmosphere environment. Nat. Rev. Earth. Environ. 3, 393-405 (2022).\n3. Koelmans AA, Redondo-Hasselerharm PE, Nor NHM, de Ruijter VN, Mintenig SM, Kooi M. Risk assessment of microplastic particles. Nat. Rev. Mater. 7, 138-152 (2022).\n4. Kumar M, et al. Current research trends on micro- and nano-plastics as an emerging threat to global environment: A review. J. Hazardous Mater. 409, 124967 (2021).\n5. Gigault J, et al. Nanoplastics are neither microplastics nor engineered nanoparticles. Nat. Nanotechnol. 16, 501-507 (2021).\n6. Ivleva NP. Chemical Analysis of Microplastics and Nanoplastics: Challenges, Advanced Methods, and Perspectives. Chem. Rev. 121, 11886-11936 (2021).\n7. Yu Z, Wang Y, Lu J, Bond PL, Guo J. Nonnutritive sweeteners can promote the dissemination of antibiotic resistance through conjugative gene transfer. ISME J. 15, 2117-2130 (2021)."}, {"round": 4, "reviewer_comments": {"reviewer_2": "I appreciate the additional experiment to compare the effect of natural and anthropogenic NPs."}, "author_response": "Response: We thank the reviewer for their positive feedback regarding the additional experiment comparing the effects of natural (nano-clay montmorillonite) and anthropogenic (polystyrene) nanoparticles (NPs) on conjugative gene transfer. We agree that this comparison was crucial, as it significantly enhanced the scientific rigor and ecological plausibility of our conclusions."}]

Decision letter