| Antibiotic resistance has become the hot issue in the world.In coastal zone,the developed economy and intensive human activities have led to large discharge of pollutants to coastal areas,resulting in potential ecological and healthy risks.Antibiotics have been widely used to treat human and animal diseases,and promote the growth of animals.However,the abuse of antibiotics has led the pollution and pressures to environment,resulting in the accumulation and spread of antibiotic resistance genes（ARGs）,antibiotic resistant bacteria（ARB）and multi-antibiotic resistant bacteria（MARB）,and posing potential threat to ecological environment and human health.To the best of our knowledge,little systematic research on the ARG pollution in coastal areas in China has been reported.Therefore,the distribution characteristics,the typical interface processes,and the prevention and control mechanisms of ARGs in coastal areas in China have been investigated in this research.The research content in this paper could be divided into three parts.（1）The distribution and source apportionment of ARGs in coastal waterA large-scale sampling was performed in the Bohai Sea,the Yellow Sea and the corresponding coastal zone（Yalu River Estuary to Yangtze River Estuary）.The quantification result of real-time PCR（qPCR） showed that the spatial distribution of ARGs demonstrated the trend of?coastal water>the Bohai Sea>the Yellow Sea,inshore>offshore and inner bay>bay mouth.The abundances of target ARGs in coastal water were in the range of 1.23×104-3.94×105 copies/mL,which were 1-4 orders of magnitude higher than those in the Bohai Sea and the Yellow Sea,and they fluctuated greatly in coastal zone and the Yellow Sea.The abundances of sulfonamide resistance genes were the highest in the sea,and the abundances of quinolone resistance genes were the highest in coastal zone.The abundances of potential MARB were higher in the Yellow Sea by co-occurrence network analysis.The distribution of ARGs in coastal zone（Yalu River Estuary to Qinzhou Bay）has been further investigated by metagenomics and qPCR.The abundances of ARGs demonstrated significant spatio-temporal variation.The quantification result by qPCR showed that the averages of ARG abundances were 8.79×104 and 1.39×105 copies/mL in summer and winter,which were much lower than those in the tail water from mariculture systems.Quinolone resistance genes hold the highest abundances,and the ARG abundances were comparable with those in other countries and regions by literature research.The research on ARG pollutions in coastal zone in China could provide useful information for further studies.In a typical semi-closed bay（Sishili Bay）influenced by intensive human activities,the ARG abundances differed significantly in different functional areas,and the abundances of ARGs were up to 6.1×106 and 6.2×107 copies/m L in coastal water influenced by the discharge of wastewater treatment plants and inflow rivers.There were strong and significant correlations between environmental factors（total nitrogen and nitrite,etc.）and ARGs.The connections between bacteria and ARGs identified in the areas influenced by sewage discharge were more complex than those in other areas.The potential hosts of ARGs were prevalent in the areas influenced by sewage discharge.Mariculture systems were the important sources of pollutions in coastal areas.The distribution of ARGs in conventional mariculture system without recirculation unit（ZZ）and recirculating mariculture systems（DF and IRMC）was investigated by metagenomics.A total of 247 ARG subtypes were identified in the samples collected from IRMC system according to the SARG v2.0 database.The quantification of ARG abundances by normalization to 16S rRNA gene reads number,cell number and total metagenome sequences showed that the ARG abundances in IRMC system were 0.04-0.14 copies/16S rRNA gene copies,0.04-0.19 copies/cell and 15.5-16.7 ppm,respectively.ARGs encoding resistance to tetracyclines,sulfonamides and multiple antibiotics,etc.,were ubiquitous and abundant in mariculture systems.ARGs were more abundant in aquacultural ponds and wastewater treatment units,and Vibrio,etc.,were the potential pathogenic MARB.Industrial mariculture systems were important reservoirs of ARGs and ARB in coastal areas.（2）The typical interface processes of ARGs in coastal areasThe investigations on the typical interface processes of ARGs between sediment-water phases showed that the distribution coefficients of ARGs in sediments and water were higher than 1.0 L/g,indicating that ARGs were inclined to be accumulated in sediments.In the Bohai Sea,the total abundances of target ARGs reached 4.9×108 copies/g.Sulfonamides resistance genes were the most abundant and ubiquitous with the abundances in the range of 5.7×104-1.8×107 copies/g.The abundances ofβ-lactams resistance genes were relatively lower.The contour map showed that the target ARGs were more abundant in Laizhou Bay,parts of Bohai Bay and the eastern of the Bohai Sea.Co-occurrence network analysis showed that the potential hosts of ARGs were more prevalent and abundant in coastal zone.Microplastics were representative among the detected suspended particles collected from the coastal industrial recirculating mariculture system.The typical interface processes of ARGs between microplastic-water phases showed that the ARG abundances in microplastic samples were in the range of 1.59?×?109-1.83?×?109copies/g,which were 3-4 orders of magnitude higher than those in water.The diversity of bacteria was significantly higher in microplastic samples than that in water.More potential hosts of ARGs and MARB were identified in microplastic samples.It showed that microplastics were important carriers of ARGs and potential hosts of ARGs.The typical interface processes of ARB between microplastic-water phases showed that the counts of cultivable ARB in microplastic samples were 100-5000 times higher than those in water,and the percentages of ARB were also higher in microplastics than in water.High-throughput sequencing demonstrated that the diversity and abundances of cultivable ARB were higher in microplastic samples,and the dominant bacteria were Vibrio,Muricauda and Ruegeria,etc.Among the 160isolates,the detection rates of ARGs,the diversity of int I1-associated gene cassettes and arrays were all higher in microplastic samples.It showed that microplastics were important carriers of ARB and MARB.（3）The prevention and control mechanisms of ARGs in coastal areasA wastewater treatment plant was selected as the object and the conventional anaerobic-anoxic-oxic（AAO）system was served as the contrast.The removal efficiencies of ARGs in the reclaimed water reuse system with integrated membrane process（IMR）were investigated.The total ARG abundance was 4.03?×?104 copies/m L in the effluent of IMR system,which was 2-3 orders of magnitude lower than those in the influent.The daily flux of ARGs decreased to（1.02?±?1.37）×1014 copies/day in the effluent of IMR system,which was 1-3 orders of magnitude lower than those in the effluent of AAO system.Co-occurrence network analysis revealed that the IMR system achieved high performance for the removal of potential MARB.The IMR system was one of the effective technologies for the removal of ARGs and potential MARB.Using the tail water of coastal aquaculture system as the object,a simultaneous partial nitrification,anammox and denitrification system has been constructed under high dissolved oxygen conditions（HO-SNAD）.The HO-SNAD bioreactor could be carried out without the addition of extra carbon.After 60-day operation,the bioreactor reached the stable stage,and the average concentrations of ammonia,nitrate,nitrite and chemical oxygen demand（COD）in the effluent were 0.26,0.75,0.47 and 0.27mg/L,respectively.There were significant differences in the abundances of nitrogen bacteria between different plants.Complex and strong significant correlations were identified between the nitrogen functional genes and bacteria.ARG abundances decreased with 0.46-0.76 and 0.27-1.24 log values in water and biofilm samples collected from the outlet of the bioreactor.With the synergistic effect of bacteria and functional genes,the HO-SNAD bioreactor achieved high performance for the simultaneous removal of nitrogen and ARGs.The experiment of the regulation of antibacterial peptide to the ARGs in aquaculture system showed that the ARGs,encoding resistance to sulfonamides,tetracyclines,macrolides,quinolones,aminoglycosides andβ-lactams were detected with lower abundances in water,faeces and fish samples in the group of antibacterial peptide.The abundances of potential MARB were lower in the group of antibacterial peptide by the co-occurrence network analysis.Antibacterial peptide could regulate the aquaculture environment and weaken the accumulation of ARGs and potential MARB to some extent in aquaculture systems. |
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