| In 2006,antibiotic resistance genes(ARGs)were first proposed as emerging pollutants.It has been widely concerned because of their replicability,propagation and persistent presence in the environment,and potential risk to human health.According to previous studies,ARGs were widely distributed in different environmental media such as rivers,sediments,air,medical waste water,sewage,farmland,Arctic frozen soil,and they had been detected in the feces of human and animals.The ARGs pollution has become more and more serious and can not be underestimated.As a transition zone from land to sea,the mangrove ecosystem has special ecological functions.However,human activities seriously polluted the mangrove ecosystem.Composition and spatial distribution of mangrove species have been destroyed at varying degrees.To our knowledge,no studies have been repored on the ARGs in mangroves.In the present paper,we collected sediment samples from the mangrove area of Dongzhai Harbor.The main research subjects were typical ARGs,including chloramphenicol resistance genes(catal,cata2,cml_el,cml_e3),sulfonamide resistance genes(sull,su12,dfrA1),and tetracycline resistance genes(tetA,tetC,tetG,tetM)and quinolone resistance genes(qnrS,gepA,aac(6')-Ib).The abundance of ARGs in mangrove ecosystem was studied via the real-time quantitative PCR(RT-qPCR).Through the study,the basic pollution status and changes of ARGs in the sediment environment could be obtained,and their distribution characteristics and source analysis were explored.High-throughput sequencing methods were used to perform 16S rRNA sequencing analysis on sediment samples in this area to investigate the distribution of microorganisms in the mangrove area.Furthermore,the correlation between ARGs and microbial diversity could be obtained,and at the same time,the special relationship between ARGs and certain specific bacteria groups could be sought.Through the determination of physicochemical properties of sediment samples,the distribution of environmental factors in the region was grasped,and the correlation between ARGs and environmental factors was thoroughly explored.The main research results of this paper are as follows:(1)The domestic wastewater was the mainly antibiotic pollution source in sampling sites S1and S2 close to the living area.Sampling sites from S6 to S10 gradually extends into the sea with less residual antibiotics.Compared with other sampling sites,the pollution from S3 to S5 was relatively serious,especially S4.There are many fishponds nearby the S4 which might be the main reason with a high pollution level.Therefore,the mangrove area close to S4 was selected for further study.(2)Quantitative analysis of typical ARGs found that target resistance genes were detected in all sediments and the abundance of ARGs was relatively high compared with others' studies,with a range of 1.0×101 copies·g-1?1.0×108 copies-g-1.Typical ARGs in this region had a special distribution pattern.The basic manifestation of each sample was that the copy number of ARGs was highest in the surface layer and lowest in the lower layer,which showed a good vertical distribution.The abundance of ARGs was the highest at D1 sampling sites and decreased gradually with the sampling sites away from the D1.The abundance of ARGs in the rainy season was higher than dry season.This phenomenon might be related to the large-scale aquaculture in the area,the special ecological functions of mangroves,the climate of Hainan,and various environmental factors.The proportions of the total ARGs in the four typical antibiotics were different.The results showed that the chloramphenicol resistance genes,tetracycline resistance genes,sulfa resistance genes,and quinolone resistance genes accounted for 40.05%,38.52%,21.43%and 0.003%of the total resistance genes,respectively.There were significouldt differences in the composition ratios of the various resistance genes.In different sampling sites,tetA,cml_e3,and sul2 genes predominated,followed by sull,tetC,and catal genes,and the remaining proportion was extremely low.It indicated that the active efflux mechanism and gene mutations or loss of enzyme genes might be dominant in ARGs in the mangrove area of Dongzhai Harbor.There was an intrinsic correlation between ARGs,and there was a very significouldt positive correlation between ARGs of the same type of antibiotics.There was a significouldt positive correlation and positive correlation between ARGs of different types of antibiotics.(3)16S rRNA sequencing analysis showed that the amount of sequencing data was reasonable and the sequencing results were reliable,covering most species information.The species information annotation results showed that 64 phylum,130 class,221 order,339 family,and 505 genus were identified in the samples.Among them,Proteobacteria and Chloroflexi were the main phylum,followed by Planctomycetes in the phylum level.At the class level,Deltaproteobacteria,Gammaproteobacteria and Dehalococcoidia were the predominant microflora in sediment samples.Other classifications could also be subdivided in orders.Alpha diversity analysis showed high species richness in the surface and middle layers.The results of beta diversity analysis showed that different environmental samples from the mangrove presented three clusters,and mangrove marginal sediments were closer to mudflat sediments.There were closely correlation between ARGs and microbial diversity.ARGs are related to specific bacteria.The aac(6')-Ib,tetM,and catal genes escaped the entire network,forming small branches each.For example,the tetM gene had only a positive correlation with Atribacteria.The relationship about the sull gene in the network was extremely complex(eg,the sull gene had a strong positive correlation with Cyanobacteria,Saccharibacteria,and negative correlation with Thaumarchaeota,Aerophobetes,and Chloroflexi,etc.).Network relationship of other genes was also more complicated(tetG gene,dfrA1 gene,etc.).A target gene could be matched a specific phylum or multiple phylum.The relationship between ARGs and bacteria could be either positive or negative,and even stronger or weaker.The ARGs are related to some specific bacteria and can be further explored basis on this study.(4)The results of environmental factor analyses are as follows.Different environmental factors had their own distribution rules in the Dongzhaigang mangrove area.The correlation analysis between environmental factors and ARGs showed that nitrite was negatively correlated with most of the target ARGs ?for example,there was a highly significouldt negative correlation with the cml_el gene(p =-0.654,r = 0.0032)and the tetA gene(p =-0.629,r = 0.0052)}.Nitrate was positively correlated with the target ARGs{for example,there was a highly significouldt positive correlation between the dfrAl gene(p=0.597,r = 0.0088)and the tetA gene(p = 0.613,r = 0.068)}.However,ammonium and total phosphorus were in partial positively or negatively correlated with the target resistance genes {There was a significouldt negative correlation between ammonium and aac(6')-Ib genes(p =-0.565,r = 0.0145),and there was a significouldt positive correlation between total phosphorus and sul2 genes(p = 0.475,r = 0.0466)}.There were basically negative correlations between pH,salinity,moisture content and the ARGs. |
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