The very large production and utilization of engineered nanoparticles (NPs) in a variety of fields may directly or indirectly lead to an increase in residual NP concentrations in the environment, which may pose potential effects on various organisms in aquatic or terrestrial ecosystems and attracts more attention on their ecological risks (Bundschuh et al., 2018). For example, due to their unique structural and optical properties (Collins et al., 2012), copper (Cu) NPs are widely added to catalysts and electronics. One of the first commercial NPs, zinc oxide (ZnO) NPs are widely used in textile, cosmetic, pigment, food additive, and medical industries (Piccinno et al., 2012). It has been reported that metal-based NPs (M-NPs) are commonly found as colloids in aquatic environments, and dissolved metal ions or small inorganic complexes produced by M-NPs may be toxic to tested organisms (Liu et al., 2016; Turan et al., 2019). The effect of exposure to single M-NPs, such as Cu or ZnO NPs, has been investigated, and in contrast to studies that suggesting that the dissolved metal species of Cu and ZnO NPs were more toxic than particle forms (Barjhoux et al., 2012; Adam et al., 2014; Hua et al., 2014), it has been reported that the cytotoxic effects were most likely due to the particulate forms of Cu and ZnO NPs (Fernaíndez-Cruz et al., 2013; Song et al., 2014). Notably, the exposure of organisms to multiple contaminants is likely to take place in the natural environment (Wang et al., 2016; Guo et al., 2018a). Nevertheless, it is a phenomenon that Cu and ZnO NPs naturally occurred in the environment; for instance, in freshwater, soil, air, and landfills (Keller and Lazareva, 2014; Pu et al., 2016), but the knowledge of the joint effects of Cu and ZnO NPs on organisms is still lacking.

Antibiotic resistance contamination has become a worldwide challenge since nearly all classes of antibiotics and a wide range of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have been observed in various environmental matrices (Berglund, 2015; Amarasiri et al., 2019; Mills and Lee, 2019), among which surface water is an important reservoir of ARGs and ARB (Stoll et al., 2012; Zhang et al., 2016). In surface water, ARGs and ARB could be easily transmitted via microorganisms between humans and other animal species, and ARGs could also be spread by microbial community succession and mobile gene elements (MGEs); i.e., integrons, plasmids, and transposons (MacLean and San Millan, 2019).

There has been emerging evidence that single M-NPs such as Al₂O₃, TiO₂, Fe₂O₃, CuO, and ZnO could promote antibiotic resistance spread via horizontal gene transfer (HGT) in pure culture (Qiu et al., 2012; Qiu et al., 2015; Wang et al., 2018; Zhang et al., 2019), suggesting the potential risk of ARG dissemination in the presence of M-NPs. Moreover, the metal ions released from M-NPs (e.g., Ag, CuO, ZnO) likely contribute to the promotion of conjugation frequencies, and the conjugation of ARGs facilitated by M-NPs has occurred through mechanisms including intracellular reactive oxygen species production, the SOS response, and cell membrane permeability (Wang et al., 2018; Zhang et al., 2019; Lu et al., 2020). Additionally, the natural environment is a complex heterogeneous matrix comprised of heterogenous chemical and bacterial compositions: determining the effects of M-NPs on the propagation of ARGs in a natural environment is a topic of importance. Recent studies, although limited, had reported that microbial communities and ARG profiles could be altered in various environmental matrices (e.g., wastewater, estuarine water, sludge) when exposed to specific M-NPs (e.g., Au, Ag, CuO, ZnO) (Ma et al., 2016; Metch et al., 2018; Chen et al., 2019; Zhang et al., 2020). Moreover, it is likely that the mixture of various pollutants may have a greater impact on the test organism than that of individual chemicals (Huang et al., 2018; Malandrakis et al., 2020).

Estuary and nearby coastal areas have great ecological and economic significance and often receive considerable pollutant inputs (e.g., ARGs, ARB, NPs, heavy metals, organic pollutants) from urban runoff, river runoff, and sewage outfalls (Czekalski et al., 2014; Guo et al., 2018b; Tou et al., 2021). ARG pollution in estuaries worldwide has been investigated, among which the Yangtze River Estuary is the most frequently examined in China, due to its significant economic and ecological value (Lin et al., 2015; Guo et al., 2018b). However, it is currently unclear how the presence of multiple M-NPs co-exposure influences the microbial communities and ARGs in the estuarine environment. It is crucial to better understand the ARG dissemination risk in estuarine waters under the stress of M-NP co-exposure.

In the current study, Cu- and ZnO- NPs were selected as representative M-NPs to investigate the changes in microbial community and ARG abundance influenced by the coexistence of those two NPs under environmentally relevant concentrations in the Yangtze River Estuary. Those two M-NPs were chosen on the basis that (1) Cu and Zn are heavy metals of great environmental concern that show significant correlations with the concentrations of ARGs in various environment matrices (e.g., water, sediment, and biofilm) (Ji et al., 2012; Guo et al., 2018a; Chen et al., 2020), and (2) Cu and ZnO are the most commonly used nanomaterials in the coatings, chemical sensors, catalysis, and cosmetics industries (Collins et al., 2012; Piccinno et al., 2012). In particular, ZnO NPs have been widely detected in the water and sediment of the Yangtze River Estuary (Chen et al., 2020; Tou et al., 2021). To this end, the associations among the concentrations of the released metal ions, MGEs, and ARGs as well as the microbial community were investigated. The objectives of this study were (1) to evaluate the effect of the coexistence of Cu and ZnO NPs on variations in the microbial community and ARG profiles in estuarine waters and (2) to explore the underlying mechanisms of the selection of ARGs during Cu and ZnO NPs co-exposure.

Previous studies posited that the M-NPs were involved in the shaping of ARG profiles (Chen et al., 2019; Su et al., 2019; Shi et al., 2019), but effect of M-NPs co-exposure on ARG profiles in natural environment is still lacking. In this study, effect of Cu and ZnO NPs co-exposure on ARG profiles and microbial community in the Yangtze River Estuary was investigated. Considering our previous study, the 22 ARGs corresponding to five antibiotic classes were commonly found in the waters collected from the Yangtze River Estuary (Guo et al., 2018b). Therefore, those 22 ARGs were analyzed, and the results showed that only six ARGs (sul1, sul2, tetA, tetW, ermB, and aac(6’)-Ib) were abundant in the samples; consequently, they were selected for our further study.

In the present study, the presence of Cu NPs reduced the ARGs. Previous study also reported that Cu NPs can inhibit ARG (sul1 and aadA1) dissemination in leachate (Su et al., 2019). In addition, our results suggested that the existence of ZnO NPs (at a concentration of 0.5 mg/L) caused a decrease in the ARG abundance. In previous publications, ZnO NPs with tested concentrations in a wide magnitude ranging from 0.2 mg/L (an environmentally relevant concentration) to 500 mg/L (a relatively high concentration) were observed to have an increased ARG abundance in natural waters, sludge, and landfill leachate (Chen et al., 2019; Huang et al., 2019; Shi et al., 2019), and ZnO NPs could facilitate dissemination of ARGs via enhancing the conjugative frequencies of plasmid RP4 (Wang et al., 2018). This difference may have been caused by the physicochemical differences among these environmental matrices that led to a diverse microbial community which is the major host and transmitter of ARGs and the different fates of NPs (aggregation, bioavailability, or toxicity), which would influence their effects toward microbes. Additionally, the density of donors and recipients directly influenced the formation of new transconjugants (Sorensen et al., 2005). The bacteria density in the present study was lower (by at least two orders of magnitude) than those in the previous studies, which may be resulted in the lower HGT frequencies of ARGs during the NP exposure. Nevertheless, a similar observation was found by Su et al. (2019), which implied that the ZnO NP (5, 50, and 100 mg/L) exposure prompted the rate of ARG attenuation and reduced the abundance of leachate microbiota. Considering these facts, there were multiple possible mechanisms involved in the ZnO NPs affecting ARG dynamics in various environments. Therefore, more studies are required to better understand the ARG dissemination risk in various environments under the stress of M-NP exposure.

Notably, the mixture of Cu and ZnO NPs was likely to induce the enrichment of ARGs at the exact concentration of 0.5 mg/L of NP exposure. Also, it has been reported that M-NPs, such as CuO NPs, ZnO NPs, nano-Al₂O₃, and nano-TiO₂ exposures can enhance the conjugative transfer of ARGs, which is mainly contributed to the oxidative stresses caused by NPs or the released ions, with cell membrane permeability increasing, SOS response activation, and genes involved in conjugative transfer up-regulation (Qiu et al., 2012; Qiu et al., 2015; Wang et al., 2018). The biomass (16S rRNA gene copies) in the Cu and ZnO NP co-exposure group was significantly lower than single NP exposure ( Figure S3 ), which could be attributed to the enhanced toxicity in the present study. Therefore, the enhanced toxicity of the Cu and ZnO NP co-exposure may have induced more oxidative stress on the microbes and stimulated the ARG transfer in the microbial community, which resulted in the ARG enrichment under the co-exposure of Cu and ZnO NPs. Furthermore, the natural aquatic environment is complex, involving multiple concentration ratios of Cu and ZnO NPs and various environmental matrices and bacterial compositions. Therefore, to determine the combined effects of multiple NPs on ARGs and the underlying mechanism in the natural environment, more studies concerning controlled single and multiple environmental factors are required.

In order to assess whether the main cause of ARG variation was the dissolved metals or the NPs, the concentrations of Cu²⁺ and Zn²⁺ released from Cu and ZnO NPs were determined in estuarine waters, and the effects of these two metal ions on ARGs abundances were investigated. The results showed that dissolved Cu²⁺ caused most ARG attenuations followed by Zn²⁺ and Cu²⁺ and Zn²⁺ co-exposure ( Figures 1B, C ). Previous study also observed that under the exposure of the same doses (5, 50, and 100 mg/L) of Cu or ZnO NPs, the corresponding dissolved Cu²⁺ reduced the ARG abundances to a greater extent than Zn²⁺ did (Su et al., 2019). Taken together, due to the different trends between the groups with the exposure of metal NPs and their corresponding metal ions, the dissolved metal ions from M-NPs may not be the dominant factor for ARG profiles in this study.

It has been reported that genes involved in heavy metal and antibiotic resistance are likely to be located on the same MGEs, which could promote the HGT of ARGs between microbial communities (Zhang et al., 2018a; Ding et al., 2019). Correspondingly, changes in MRGs and MGEs may indicate a variation in the ARGs. Therefore, we investigated three MRGs corresponding to Cu and Zn (Guo et al., 2018b; Adekanmbi and Adeleke, 2020) and two MGEs which were generally occurred in the estuarine environment (Chen et al., 2019). The co-selection of ARGs and MRGs may have occurred and MGEs may participate in the dissemination of these ARGs in the estuarine water in the presence of Cu and ZnO NPs individually or together in the present study. Similarly, the co-selection of heavy metal and ARGs has been commonly reported in various environments such as soil (Zhao et al., 2019), water (Zhang et al., 2018a) and gut microbiota (Ding et al., 2019).

Changes in the composition of the microbial community are closely related to the variations in the abundance of ARGs, MRGs, and MGEs (Guo et al., 2018b; Chen et al., 2019; Shi et al., 2019). The variations in the microbial community under the co-exposure of Cu and ZnO NPs were mainly attributed to the metal ion dissolution and that the Zn²⁺ contributed by the ZnO NPs had more significant effects than Cu²⁺ did. Proteobacteria, one of the dominant bacterial phyla, showed a decreasing trend in most treatment ( Table S6 ). Likewise, previous studies have reported that abundance of Proteobacteria decreased under stress from the M-NPs (e.g., ZnO and Fe⁰ NP) in OCO reactor and landfill leachate (Liu et al., 2018; Shi et al., 2019). The trend of Actinobacteria changes was similar to ARGs changes, such as sul1, aac(6’)-Ib, and ermB, which implies that Actinobacteria may have been responsible for the increasing trend of ARG in the samples. These findings were also consistent with those of our previous study, which indicated that Actinobacteria are important hosts for carrying and disseminating ARGs in the Yangtze River Estuary (Chen et al., 2019). As for the response of top ten abundant genera in each treatment, most genera were found to belong to the Proteobacteria and Actinobacteria phyla ( Table S7 ). These two bacterial phyla were considered to be important hosts of ARGs and participate in the spread of ARGs (Zhang et al., 2018b). For example, the Nocardioides genus, which belongs to the Actinobacteria phylum, has been identified as an important host of ARGs (e.g., sul1, tetA, tetW, ermB) and MGEs (intI1) in our previous studies (Guo et al., 2018b; Chen et al., 2019). Nevskia belonging to the Proteobacteria phylum was resistant to trimethoprim (Low et al., 2016). In this study, the abundance of Nocardioides, Nevskia, and Panacagrimonas (belonging to Proteobacteria phylum) showed an increasing trend in the samples of Group B, which suggests that these genera may be involved in the enrichment of ARGs in samples in Group B.

According to the RDA ( Figure 3 ) and partial RDA analysis, the microbial community was found to be the key contributor to the variance in ARG profiles, and changes in the abundance of sul1, ermB, and aac(6’)-Ib are likely to be related to the microbes classified into Actinobacteria and Acidobacteria. Xu et al. (2020) reported that most chosen ARG numbers (e.g., sul1, sul2, tetA, ermB) were correlated to Acidobacteria in a drinking water system. The dissemination of ARGs through HGT and co-selection commonly exists in the environment (Bengtsson-Palme et al., 2018). In this study, sul2, tetA, and tetW are significantly correlated with intI1, zntA, and zntB, which indicates that the shifts of these three ARGs may be related to intI1, zntA, and zntB, which were involved in the HGT of ARGs and co-selected by metals. Accordingly, whether Cu NPs or ZnO NPs were exposed alone or together, the variance in the ARG profiles was mainly affected by the abundance of potential host bacteria and the HGT of ARG among microbial communities in estuarine water (Chen et al., 2019; Shi et al., 2019). In terms of the potential host bacteria for ARGs ( Figure 4 ), most bacterial genera belong to Proteobacteria were identified for sul1, which indicates that the changes in the abundance of Proteobacteria may be responsible for sul1. The result was consistent with the conclusion of a previous study, which reported that Proteobacteria was significantly correlated to SAs-ARGs in a large aquaculture pond (Shen et al., 2020). As other hosts for sul1, the abundance of Panacagrimonas and Mycobacterium in the samples in Group B showed an increasing trend, which were likely corresponded to the increasing of sul1 in Group B. The results indicate that Panacagrimonas and Mycobacterium in the estuary water may be involved in the regulation of the sul1 variation. In addition, Mycobacterium was found to be significantly related to a variety of ARGs (sul1, sul2, ermB) in a pig farm (He et al., 2019). Moreover, Haliangium, one kind of moderately halotolerant bacterium able to survive extreme conditions, was found to be related to sul1, resulting in a high risk of ARG dissemination. It is well known that Escherichia coli is the most common enteric pathogen in our daily life, and antibiotic resistance in E. coli is of particular concern (Adekanmbi and Adeleke, 2020). One previous study has reported that multi-resistant E. coli is widespread in tropical estuaries and India, and more than 95% of the isolated strains are resistant to more than three antibiotics (Chandran et al., 2008). In this study, it was found that E. coli was significantly related to sul1. Therefore, the presence of such pathogenic bacteria in natural waters will enhance the direct or indirect risks of human infection. The abundance of Arenimonas in the samples in Group A showed a decreasing trend over time, whereas the abundance of Arenimonas showed a trend of first decline and then arise, in the samples in Group B, which implies that Arenimonas may be related to the variations in sul2 during the NP exposure. Cyanobacteria was demonstrated to act as a significant reservoir for diverse ARGs (e.g., tetA, tetW) dissemination in aquatic environments (Wang et al., 2020). Consistently, Chlorella sp. CC-Bw-9 belonging to Cyanobacteria was identified as a potential host for tetW.

It is unclear the concentrations of Cu and ZnO NPs that are present in the estuarine environment, however, the production of NPs is exponentially increasing, making possible higher inputs into the aquatic environments in the future. In this study, the effects of Cu and ZnO NPs exposure individually or combined under environmentally relevant concentrations on the changes in ARG abundance and microbial composition in estuarine waters were investigated. It was found that under the exact doses (0.5 mg/L) of NP exposure, Cu NPs or ZnO NPs exposed alone mainly resulted in the attenuation of target ARGs in the estuarine water, while a mixture of Cu NPs and ZnO NPs was likely to induce the enrichment of ARGs. Further analysis indicated that changes in the microbial community were the dominant factors driving ARG propagation, followed by dissolved metal ions, MRGs, and MGEs. Network analysis showed that most potential hosts belonged to the Proteobacteria and Actinobacteria phyla, such as Mycobacterium and E. coli., which are likely to be pathogenic to humans and animals. Therefore, in contrast to individual Cu- and ZnO-NPs, combined Cu and ZnO NPs seemed to induce the increase of ARGs, which would increase the risk of ARG transmission into different environmental niches including normal human intestinal flora.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI Sequence Read Archive, SRP409221.

Y-RC: Methodology, validation, formal analysis, investigation, data curation, writing - original draft, writing - review & editing, visualization. R-RS: Investigation. X-LS: Investigation. X-PG: Formal analysis, investigation, data curation, writing - review & editing, supervision, project administration, funding acquisition. YY: Writing - review & editing, supervision. All authors contributed to the article and approved the submitted version.