Impact Of Nanoparticles On The Antibiotic Resistance Genes(ARGs) In Estuarine Water:ARG Variations And Their Association With The Microbial Community

Due to the unique physicochemical properties,nanoparticles?NPs?could interact with other pollutants and affect their fate in the environment.Recent years,antibiotic resistance genes?ARGs?,as a new pollutant,has become a great public health concern worldwide.In addition,previous publications have found that nanoparticles could facilitate the frequency of horizontal gene transfer?HGT?of plasmid-borne ARGs between different bacteria strain,thereby increasing the dissemination risk of ARGs.However,the current knowledge on how NPs impact ARG profiles in natural aquatic environments is largely unknown.In particular,estuarine and coastal environment are important transition areas of ocean,rivers and continent,with important ecological functions and the geographical values,which have been a hot area for environmental geoscience research.It was reported that estuarine and coastal environment were natural reservoir for various contaminants such as NPs and ARGs.Therefore,the occurrence,distribution and abundance of ARGs,and the impact of NPs on ARGs remain to be studied.In the present study,the Yangtze Estuary was taken as the representative study area.Firstly,we conducted a supplementary analysis to investigate the occurance and distribution of ARGs and the related environmental factors in the Yangtze Estuary.Then,the impact of typical NPs?ZnO NPs?exposure to microbial community and ARG profiles in the waters?offshore and the nearby sea?of the Yangtze Estuary was analyzed,and the potential mechanism was elucidated by exploring the relationship between ARGs,mobile genetic elements?MGEs?,metal resistance genes?MRGs?,dissolved metal ions and microbial communities.Furthermore,the response of microbial communities and ARGs to the combined exposure of Cu NPs and ZnO NPs in the estuarine waters was investigated.Given the above studies,we drawn the following conclusions:?1?ARGs were ubiquitous in the study area.The frequency and abundance of most ARGs showed different spatial variation patterns in the water and sediment phases.The relative abundances of total ARGs were relatively higher in the turbidity maximum zone?TMZ?than in the other samples in the water phase but showed an increasing trend from inlet to outlet in the sediment phase.This may imply that suspended particulate matter?SPM?is a reservoir for ARGs and microbes associated with ARGs in waters.A MGE?intI1?was positively correlated with ARGs in both waters and sediments,which implied that intI1 may be related to the spatial variations of ARGs in the Yangtze Estuary.The spatial variations of estuarine ARGs were positively correlated with various environmental factors.For water samples,concentrations of sulfonamide antibiotics,dissolved metals and metals in SPM,and other sample properties,including temperature,SPM,NO₃^-and chlorophyll a were significantly correlated with ARGs.Metal concentrations,especially bioavailable metals,as well as other environmental factors,including temperature,salinity,pH,sand,silt and clay,could regulate the occurrence and distribution of ARGs in the sediment phase.?2?In the offshore of the Yangtze Estuary,the abundances of total ARGs increased after exposure to ZnO NPs,particularly at low doses?0.2 and 1 mg/L?.Relatively low and environmentally relevant concentrations of ZnO NPs likely induced the selection of sul1,tetA,ermB and qnrS,whereas a high concentration?10 mg/L?of ZnO NPs was more selective for sul2 and tetW.After normalization of the abundances of ARGs exposed to NPs to those of the corresponding blanks,the ARGs in the DH samples with relatively high salinity and relatively low NP doses showed the highest normalized values by the end of the exposure period,which indicated the high dissemination potential of ARGs in relatively brackish water.A significant positive correlation was found between ARGs and MGEs?Tn916/1545 and int I1?,which indicated the high potential of ARG dissemination via HGT in estuarine waters after exposure to ZnO NPs.The response of the microbial community to ZnO exposure was dose-and time-dependent at both the phylum and genus levels.Although the absolute abundance of microbes showed a decreasing trend after exposure to ZnO NPs,the absolute abundance values normalized to the corresponding blanks generally showed a trend consisting of an initial inhibition followed by a rebound.RDA analysis showed that the microbial community contributed more to the variation in ARGs in estuarine waters after exposure to ZnO NPs than MGEs and dissolved Zn²⁺.A network analysis evaluated the possible host bacteria for the detected ARGs,and the results revealed that some of these bacteria were associated with multiple ARGs and might pose a high risk for the dissemination of ARGs in estuarine environments.?3?After exposure to environmentally relevant concentration of ZnO NPs,ARGs?sul1,sul2,and tetA?were enriched,paticularly in the S13 sample which located in the outlet of the Yangtze Estuary.This indicates that during the exposure of environmentally relevant concentrations of ZnO NPs,the ARGs in the outlet water samples were more ready to accumulate and spread ARGs than those in the inlet and TMZ regions.However,in comparison,ARGs in the offshore water samples are more likely to enrich and spread ARGs than the estuary water samples.RDA analysis found that the microbial community contributed most to the variation in ARGs profiles.The abundance of bacteria in the S4 and S9 samples were generally inhibited after exposure to ZnO NPs,and the relative abundance decreased,but the abundance of most bacteria in the S13 samples showed an increasing trend,which may be related to the increase of ARGs abundance in the S13 sample.Network analysis identified a variety of potential host bacteria for ARGs.Paraperlucidibaca is a multi-resistance bacterium and a potential host for two ARGs?sul1 and tetA?.Notably,Paraperlucidibaca is widely distributed from inlet to outlet of the Yangtze Estuary.In addition,ARBs,Sphingomonas sp.and Marinobacter sp.,Which were resistance to sulfadiazine and tetracycline were obtained from the raw water samples by traditional plate culture separation methods.These two bacteria were also identified as the host of three ARGs?sul1,tetA and tetW?in the network analysis.Therefore,all the analysis had proved that these multi-resistance bacteria are highly related to the dissemination of ARGs in the surface water in the Yangtze Estuary.?4?Environmentally relevant concentration of Cu NPs exposure facilitated the ARGs attenuation in estuarine waters.In contrast to the Cu²⁺control group,we found that the released Cu²⁺from NPs contributed most to the ARGs attenuation.In addition,correlation analysis showed that MGEs were significantly correlated with the abundance of ARGs,suggesting that exposure to Cu NPs might decrease the spread of ARGs via HGT.While,when both of Cu NPs and ZnO NPs were added,the abundance of ARGs and two zinc-resistance genes?zntA and zntB?increased,and ARGs were significantly related to zntA and zntB.This maybe imply that the co-exist of Cu NPs and ZnO NPs could induce co-selection of heavy metals to enhance the enrichment and propagation of ARGs in the estuarine waters.In addition,the microbial community changed significantly at both the phylum and genus level.RDA analysis showed that in addition to dissolved Cu²⁺,microbial communities,MRGs,MGEs,and dissolved Zn²⁺were all related to the changes in ARGs abundance.The results of network analysis indicate that some pathogenic bacteria?such as:Mycobacterium,Escherichia coli?were the potential hosts for sul1,and these pathogenic bacteria may increase the risk of ARGs dissemination in the estuarine water environment and threaten the potential health of humans.The paper clarified the occurrence of ARGs in the acquatic environment of the Yangtze Estuary,revealed the potential environmental factors that affect the dissemination of ARGs,and explored the impact of typical NPs single or combine exposure to the microbial community and ARGs in estuary waters.This study provided basis and reference for understanding the effect of NPs on the distribution and dissemination of ARGs in the natural environment.