Occurrence And Abundance Of Antibiotic Resistance Genes In Sediments Of The Yangtze River Estuary

ARGs（Antibiotic resistance genes,ARGs）are widespread and abundant in the environment,especially in the sediment.At the same time,the sediment composition is complex and the particle size is diverse,which may have different effects on the occurrence of ARGs in different forms.As an important transitional area connecting land and ocean,estuarine area has always been the focus of environmental geography.The interaction of saltwater and fresh water,the strong tidal action,the complex environmental conditions,the changeable coupling process,the complex evolution mechanism and the intense impact of human activities in this area.In this complex ecosystem,the distribution,content and environmental factors of ARGs under long-term time scale and different particle sizes need to be studied.On the basis of the previous research group’s study on the occurrence and distribution of ARGs in the coastal sediments of the Yangtze estuary,this paper further selected two typical sediment samples（LHK and WSK）,and determined and quantified the types and abundance of ARGs in the sediments of different years（2007-2019）by using the common PCR（polymerase chain reaction）and quantitative PCR（qPCR）techniques.UPLC-MS-MS and ICP-MS techniques were used to determine the contents of antibiotic and weak acid extracted metal in sediments.Social and economic data were collected and correlation analysis was conducted with ARGs,antibiotics,weak acid extracted metal and physical and chemical indicators to analyze the relationship between ARGs and socio-economic and environmental factors.Stokes sedimentation siphon method was used to separate sediment particle sizes,and the distribution characteristics and contribution rates of ARGs in different particle sizes were investigated under the conditions of wet sample,dry sample,ultrasonic and oscillation.The intracellular DNA（iDNA）and extracellular DNA（eDNA）were isolated byNa₂HPO₄-SDS-PVPP.iDNA and eDNA were purified and extracted by CTAB method and 3S Kit method,and the distribution characteristics and contribution rates of ARGs with iDNA and eDNA in different particle sizes under corresponding conditions were investigated.Based on the above research,the main findings and understandings of this paper are as follows:（1）ARGs（sul1,sul2,tetA,tetM,tetW,qnrS,aac（6’）-Ib）and its related genes（intI1gene mobile element and metal resistance genes such as zntA and copA）are prevalent in the coastal sediments of the Yangtze estuary in different years,and their annual variation patterns are different.Among them,sul1,sul2,tetM,tetW,qnrS,aac（6’）-Ib and zntA showed an increasing trend year by year.tetA shows a relatively stable trend;intI1 and copA showed no significant change in different years.The spatial variation of ARGs is characterized by that the sampling point（WSK）of urban sewage discharge and urban inland river input is higher than that of urban inland river input（LHK）.（2）Most of the socio-economic indicators showed an increasing trend year by year,while the interannual variation of antibiotic content and metal in the extraction state of weak acid was not obvious.In the physical and chemical properties of sediments,TOC showed an increasing trend year by year,while the interannual changes of pH,salinity and particle size（clay,silt and sand）were basically insignificant.The correlation analysis between ARGs and socio-economic data and environmental factors shows that most socio-economic indicators are positively or significantly positively correlated with their corresponding antibiotic content,metal content of weak acid extraction state and TOC content.Most socio-economic indicators are significantly correlated with several ARGs.There was no significant correlation between the total concentration of antibiotics and the corresponding ARGs,but some specific antibiotics had significant correlation with the corresponding ARGs.The concentration of metal in the weak acid extraction state was significantly correlated with most ARGs.It indicates that socio-economic development may increase the contents of antibiotics,organics,metals and ARGs in sediments,and antibiotics,organics and metals may participate in regulating the occurrence and distribution of ARGs in sediments.At the same time,TOC,pH and clay may also be the main factors affecting the interannual abundance of ARGs in sediments.（3）The particle size of the sediment was separated by wet sample,dry sample,ultrasonic sample and oscillation method respectively.Under the four methods,the distribution characteristics of 16S rRNA,sul1 and tetA were shown as（<1μm）>（1-20μm）>total sediment>（>63μm）>（20-63μm）.According to the mass balance,the contribution ratio of 16S rRNA,sul1 and tetA in the amount of each particle size to the total sediment volume was（1-20μm）>>（1μm）>（20-63μm）>（>63μm）,and 1-20μm dominated.The recovery results show that the wet sample is better than the dry sample,and there is no significant difference between the ultrasonic and the oscillatory sample..（4）The intracellular DNA（iDNA）and extracellular DNA（eDNA）of sediments were separated by Na₂HPO₄-SDS-PVPP binding method,and the DNA of sediments with different particle sizes were extracted and purified by ultrasound,vibration,CTAB method and 3S Kit method respectively.Under the four methods,the distribution characteristics of 16S rRNA,sul1 and tetA in iDNA were taken as（1-20μm）>total sediments>（<1μm）>（20-63μm）>（>63μm）.The distribution characteristics of 16S rRNA,sul1 and tetA in eDNA were（<1μm）>（1-20μm）>total sediment>（>63μm）>（20-63μm）.CTAB method is recommended for iDNA extraction and 3S method is recommended for eDNA extraction.According to the mass balance,the contribution rate of eDNA to the total ARGs was higher than that of iDNA in both the total amount and the different particle sizes,indicating that the eDNA of sediments was the repository of ARGs and had a strong transformation and propagation capacity.