Studies On Characterization Of Pollution Of Antibiotic Resistance Genes In The Typical Estuary

ObjectivesAntibiotics are widely applied in clinical treatment,aquaculture and husbandry,aiming to effectively control and eliminate the threats of infectious bacterial diseases to humans and animlas.Unfortunately,bacteria have evolved with high resistance to antibiotics over a long term of antibiotic use.The World Health Organization(WHO)has announced that antibiotic resistance is one of the most serious environmental health issues in the 21st century,and appealed to the whole human beings to fight against the spread of antibiotic resistance worldwide.Aquatic environments play the key roles in the dissemination of antibiotic resistance genes(ARGs).The estuary attracts more attentions due to the fact that huge amounts of ARGs are released from typical pollution sources(e.g.,clinic wastewater,sewage treatment plants,livestock farms,etc.)with antibiotics and their resistant bacteria,as rapid economic development and urbanization have happened in the nearby area.As a result,the estuaries are considered as an important ’sink’ of ARGs,as well as a nonnegligible source for pathogenic bacteria to acquire ARGs.The objectives of this thesis were to recognize the status and characterization of ARG pollution in the Yangtze River Estuary(YRE)and the Pearl River Estuary(PRE)using various the state-of-art methods and techniques,and further to explore the relationships among ARGs and antibiotics,as well as MGEs.The accomplishments achieved in this thesis could be essential to accurate estimation of environmental health risks caused by ARGs in the estuaries.MethodsVarious methods and techniques related to environmental analytical chemistry,bio-informatics and molecular biology were used in this thesis.An couple system that was comprised of solid phase extraction,high performance liquid chromatography and electron spray ionization tandem mass spectrometry was employed to determine the concentrations of antibiotics in the sediments.Tranditional and real-time quantitative PCR were used to analyze the levels and distribution of tetracycline and sulfonamide resistance genes(tet and sul genes),integron genes(int1)and bacterial 16S rRNA genes in the sediments.Comprehensive characteristics of ARGs,microbial community structures and composition and diversity of ARGs related to plasmids in the sediments collected from deep ocean and estuary were analyzed using second generation DNA sequencing technique-based bioinformatic methods.ResultsOur results demonstrated that the levels of 16S rRNA and int1 genes were 1.34×109 and 3.7×106 copies/g sediment,which were much lower than those in other estuaries and river in China.The total level of sul genes in the YRE sediments was much higher than that of tet genes.Spatial distribution of ARGs was characterized with that high levels of ARGs were found in the area nearby Shanghai City and Hangzhou Bay,and similar spatial pattern was observed forint1 gene.It suggests that int1 gene could play an important role in the dissemination of ARGs in the environments.Further correlation analysis showed that a significant relationship was only found between int1 and sul1 genes.However,analysis of antibiotics exhibited that the occurrence and concentrations of tetracyclines and sulphonamides were both low in the sediment samples from the YRE.Second,another study was conducted to distinguish the differences in characterization of bacterial communities,ARGs and plasmid genome between the PRE and the West Pacific(WP)sediments using a high throughput DNA sequencing technique-based metagenomic method.The results showed that bacteria in the sediments were assigned to 16 phyla,and proteobacteria in the WP sediment accounted for about 95%of bacterial population,which was significantly higher than that in the PRE sediments.The majority of ARGs in the WP sediments were multidrug resistance genes(as high as 77.8%),whereas ARGs in the PRE sediments were mainly related to anthropogenic antibiotics such as sulfonamides,macrolides,aminoglycosides,β-lactams,etc.Such a high diversity of ARGs in the polluted PRE environment was consistent with the reisistance of human pathogens to multiple antibiotics.Furthermore,only 0.3%of ARGs in the sediments of the WP were carried in the plasmids,and ARGs resided in the plasmids in the PRE sediments took up more than 40%of the total ARGs.ConclusionAntibiotic resistance can exist in natural water environment that were less impacted by human activities,and is not originated from wide use of antibiotics.ARGs in the nature environment were characterized with high abundance of multidrug genes and the low possibility of being transferred between bacteria.In highly-impacted estuary,ARG pollution is closely assocated with wide use of antibiotics in the nearby regions,which is mainly released from typical pollution sources such as medical wastewater rather than in situ production.Mobile genetic elements(MGEs)such as integrons and plasmids play irreplaceable roles in the dissemination of ARGs.Due to the ubiquitity of sulfonamide resistance genes,sulfonamides have been abandoned from clinical treatment.