Occurrence And Abundance Of Antibiotic Resistance Genes In Aquatic Environments Along The Yangtze Estuary

As an emerging pollutant,antibiotics resistance genes?ARGs?have become one of the global hot environmental issues because they could be absorbed by bacteria that affect human health and ecological security.Moreover,the estuarine and coastal area are important transition areas for the intersection of oceans and rivers or lands.Due to their special geographical location and important ecological value,they have always been the focus of environmental geoscience research.In addition,these regions often have complicated hydrological conditions,strong tidal action,diverse environmental medias,and are affected by human activities.Meanwhile,they always receive considerable pollutant inputs including ARGs from land-based sources via river off and sewage outfalls.Therefore,the occurrence,abundance,distribution and transformation of ARGs in this complex ecosystem need to be studied.In the present study,we will focus on the biofilm,sediment and surface water were collected from the Yangtze Estuary and its coastal areas.The occurrence and abundance of ARGs were qualitated and quantitated by PCR?polymerase chain reaction?and qPCR?quantitative-polymerase chain reaction?.The concentrations of antibiotics and metals in biofilm,sediment and water were determined using the UPLC-MS-MS and SP-ICP-MS,respectively.The microbial community structure and composition in sediment were analyzed using the Illumina Miseq high-throughput sequencing platform and the antibiotics resistance bacteria were cultured on the LB agar plated with different antibiotics.The environmental physicochemical parameters were measured using the suitable instruments,such as a protable water quality instrument,a automatic water quality analyzer and so on.The methods including PCA and RDA were used to analysis the seasonal and spatial distribution and the influence environmental factors of ARGs.Finally,the effects of metal and metal nanomaterials on the occurrence,abundance and migration of ARGs were researched.Based on these above researchs,the following conclusions and undertandings are obtained:?1?Antibioticsincludingsulfonamides,tetracyclines,macrolides,fluotoquinolones and chloramphenicol and their corresponding resistance genes?sul1?sul2?sul3;tetA?tetB?tetC?tetE?tetL?tetM?tetO?tetQ?tetW?tetX;ermB;qnrB?qnrS?acc?6'?-Ib and Cm?were ubiquitous in the intertidal aquatic environments along the Yangtze Estuary.The detection frequency and abundance of most ARGs showed the following order:biofilm>sediment>water.Moreover,the target genes including mobile genetic elements?intI1,intI2,Tn916/154 and trb C?and anti-metal resistance genes?zntA,zntB and copA?were also ubiquitous in the intertidal sediments,and the seasonal variations of these genes as well as most ARGs showed:summer>autumn?spring>winter.In addition,the characteristics of spatial variation showed as:ARGs abundance in samples affected by urban sewage discharge or urban inland river input were higher?such as SDK and WSK?,and others were relatively lower?such as CY,DH and XP?,indicating that anthropogenic activities may be the major factor that regulated the occurrence and abundance of ARGs in the esturine environments.?2?In most sampling sites,the ARG abundances were contributed by extracellular DNA?eDNA?in biofilm and sediment samples,while the most of ARGs abundances were contributed by intracellular DNA?iDNA?in biofilms at SDK and SCK.Moreover,the partitioning coefficients of ARGs in eDNA between biofilm and water were higher than those between sediment and water.Therefore,the biofilm could be considered as a more significant sink than sediment,which was as a sink for ARGs,and this theory provides new insights for the study of ARGs in the environments.In addition,ARGs in eDNA were regulated by TOC and non-MBC,and it may be more stable and easy to persist in the environment when eDNA is combined with organic matter,thereby increasing its potential for absorption and utilization by bacteria so that promoting the spread of ARGs.The ARGs in iDNA were regulated by bacterial density and MBC in both biofilm and sediment samples.?3?Among so many environmental factors,TOC,bacterial density,antibiotics and metals were the dominant factors that regulating the occurrence and abundance of ARGs in the aquatic environments along the Yangtze Estuary.Among them,the acid-extracted metal?the bioavailable metal?is more easier to be utilized by microorganisms because it is more easily released from the environment.Therefore,it is recommended to use the metal form in future researches about the relation between ARGs and metals.In addition,compared with the total extracted Zn,the bioavailable Zn was more significantly correlated with most ARGs,and there was also a significant correlation between the abundance of anti-Zn gene and ARGs.So,the Zn ions may be one of the important metals that regulate the occurrence of ARGs in the environment along the Yangtze Estuary.Moreover,the positive correlation between int I1 and sul1 in biofilm and sediment implied int I1 may be related to the occurrence and propagation of sulfonamides resistance genes.Additionally,the bacteria the may carry ARGs in the microbial community and the isolated ARBs may be the important biological impact factors for ARGs in the environments.All of those relationships between ARGs and the environmental factors,especially for the impact of the acid extracted metal on ARGs,provides a new perspective for comprehensive investigation of the environmental factors that may regulate the occurrence and abundance of ARGs.?4?Based on the Illumina Miseq high-throughput sequencing platform,there were 60 and 66 phylum in sediments in winter and summer,respectively.In the most sediments taken along the Yangtze Estuary,the dominant phylum were Proteobacteria and Bacteroidetes in winter and summer,and the Flavobacterium,Phormidium,Luteolibacter,Paracoccus and Dechloromonas in winter and the Methanosaeta,Dechloromonas,WCHB1-05,Nitrospira and Dechloromonsa in summer were dominant respectively.Based on the Bray-Curtis dissimilarity method,the microbial community at WSK and SDK were clustered together in both winter and summer,indicating the more similarity bacterial communities and their influence factors in these two sampling sites.Additionally,Salinity,nutrients,grain size and so on were the vital environmental factors to influence the bacterial community structure and composition.?5?The bacteria may carry different ARGs,MEGs and MRGs in sediments taken along the Yangtze Estuary in winter and summer.In particular,MRGs including cop A and zntA were dominant in winter,while ARGs including sul1,sul2,qnrS,tetA,tetO and tetQ and MGEs including int I1and trbC were dominant in summer.In addition,the anti-sulfonamides bacteria such as Proteus sp.,Enterobacter sp.,Pseudomonas sp.,Morganella sp.,Aeromonas sp.,Aeromonas sp.and Escherichia sp.and the anti-tetracyclines bacteria including Bacillus sp.,Empedobacter sp.and Klebsiella sp.were isolated from the sediments.However,some bacteria of those were not detected through the Illumina Miseq high-throughput sequencing.Therefore,we suggested that combine the high-throughput sequencing method and the plate culture method in the research of ARGs and ARBs in the future.?6?ZnO-NPs and Zn²⁺can reduce the abundance of 16S rRNA,sul1 and tetA in different degrees,and the treatment types,concentrations,times and sampling sites are significantly different.Nevertheless,ZnO-NPs have different mechanisms such as release of Zn²⁺and generation of active oxygen radicals.Therefore,the results of the Zn²⁺treatment group in this experiment cannot fully explain that the effect of ZnO-NPs treatment due to the release of Zn²⁺.In addition,the ARGs in the mud treated ZnO-NPs at LCG had significantly reduced,which may be due to the high salinity that causes ZnO-NPs to release a large amount of zinc ions causing bacterial death and releasing DNA,and then degraded by the action of reactive oxygen species.Meanwhile,ZnO-NPs could promote the migration of the exogenous plasmid-mediated chloramphenicol resistance gene into bacteria in the mud,and the specific mechanism needs further study due to this process may be affected by many factors including the varies of sampling site,bacterial communities,plasmids types and concentrations and nanomaterials.Meanwhile,those results provided new insights and data supports for the composite pollution among the new environmental pollutions,such as the effects of nanoparticles on the occurrence and abundance of ARGs.