Study On The Mechanism Of Antibiotic Co-metabolic Degradation And Antibiotic Resistance Genes Action By Antibiotic-resistant Denitrifying Bacteria

With the increase of antibiotic production and usage,antibiotic resistance genes influence on human health and ecological environment became the research hot spot,in order to explore the antibiotic resistance type of denitrifying bacteria,based on the research on the impact of different concentrations of antibiotics on antibiotics-resistant bacteria activity,on the basis of further carry out the research of antibiotics-resistant bacteria to antibiotics to remove.The culture sludge with specific carbon source was taken as the control group,and the culture sludge with long-term exposure to antibiotics was taken as the experimental group.The concentration of antibiotics(ofloxacin,norfloxacin)was increased by gradient.The influence of antibiotics on the denitrifying sludge and the removal of antibiotics in this process were observed under different experimental conditions.To develop two groups of denitrification of the sludge as the research object,based on 16S rDNA sequencing,macro genome sequencing and high-throughput fluorescence quantitative PCR techniques such as different training period and the succession of microbial community in sludge,sludge in different microbial diversity analysis,gene expression,the difference of the key functional genes and antibiotic resistance genes species belong to spend.At the same time,starting from the actual sewage treatment plant,the diversity and abundance of microorganisms in the activated sludge of sewage treatment plant with different carbon sources,and the number and abundance of antibiotic resistance genes in different sludge were investigated.The aim is to provide theoretical support for optimizing the removal of antibiotics and antibiotic resistance genes by antibiotics-resistant bacteria in wastewater treatment plants,and to provide theoretical reference for the denitrification process of wastewater treatment plants containing antibiotic pollutants.The wastewater treatment plant can efficiently and stably complete biological nitrogen removal while reducing the risk of new pollutants such as antibiotics and antibiotic resistance genes to human and ecological environment in the effluent.Based on the above studies,the results of the study on the co-metabolic degradation of antibiotics by antibiotics-resistant denitrifying bacteria and the mechanism of action of antibiotic resistance genes are as follows:(1)with methanol,acetic acid sodium as electron donor,cultivation of methanol and potassium nitrate under the condition of non-resistant denitrifying sludge(Me-non OFL),methanol,potassium nitrate and trace ofloxacin(OFL)under the condition of resistance type denitrifying sludge(Me-trace OFL),acetic acid sodium and potassium nitrate under the condition of non-resistant denitrifying sludge(Na Ac-non OFL),sodium acetate,potassium nitrate,and traces of ofloxacin(OFL)under the condition of resistance type denitrifying sludge(Na Ac-trace OFL).The results of antibiotic inhibition test showed that carbon source had an influence on the tolerance of denitrifying bacteria.For antibiotics-resistant bacteria,sodium acetate as carbon source(Na Ac-trace OFL)had a better tolerance,and the nitrate removal rate could reach 100%under the condition of low concentration OFL.For non-antibiotics-resistant bacteria,Me-non OFL with methanol as carbon source had better tolerance,and the nitrate removal rate was higher than 90%at both low and high concentrations of OFL.Whether methanol or sodium acetate was used as specific carbon source,denitrifying bacteria without OFL stress could also acquire a certain tolerance to antibiotics(as compared with inoculation sludge),and denitrifying bacteria with trace amount of OFL stress could acquire a tolerance to high concentration of OFL.The effect of the same antibiotic(NOR)on the denitrification process of Me-non OFL sludge and Me-trace OFL sludge was the same as that of OFL.(2)The results of antibiotic removal test showed that the removal of OFL in activated sludge was mainly by adsorption,and the antibiotics-resistant bacteria domesticated under low OFL condition could still remove the high-concentration OFL to a certain extent.Compared with non-antibiotics-resistant bacteria,antibiotics-resistant bacteria had certain advantages in the treatment of antibiotic pollutants.The two kinds of denitrifying sludge also showed a certain ability to remove the same antibiotic(NOR),indicating that the sludge cultured under single antibiotic condition can also remove the same antibiotic to a certain extent.(3)Based on 16S rDNA amplicon sequencing technology,the microbial composition of each culture sludge in the experimental stage was obtained.The results showed that Proteobacteria,Firmicutes and Bacteroidota appeared in all the sludge samples,and the total number of bacteria in the three phyla and accounted for more than 91%of the total number of microorganisms in the sludge samples,indicating that they were the core denitrification microflora.The dominant bacteria in Me-non OFL sludge include Enterococcus,Methylovellis,Aquimonas,Escherichia-Shigella,Denitratisoma,Flavobacterium and Lentimicrobium.The dominant bacteria in Me-trace OFL sludge include Mangroviflexus,Aquamicrobium,SM1A02,Hyphomicrobium,Paludibacter,Hydrogenophaga,Bacteroides,while the dominant bacteria in Na Ac-non OFL sludge include Thauera,Ruminococcus,Faecalibacterium,Azonexus and Akkermansia.The dominant bacteria genus in Na Ac-trace OFL sludge are Lentimicrobium,Desulfomicrobium,Ignavibacterium,Truepera and Erysipelothrix.(4)With the increase of culture time,the species richness and diversity in the community also changed accordingly.The relative abundance of dominant phyla in microbial communities of sludge samples tended to be stable gradually.Proteobacteria mainly played a role in the sludge of the methanol group in the later stage,while Bacteroidetes was the main phylum playing a role in the sludge of the ethanol group.At the genus level,Hyphomicrobium,Norank?F?A4b,Unclassified?F?Burkholderiaceae,Rhodobacter,and Mehtylobaclis existed in the sludge culture stages of methanol group and had high abundance,while Thauera,Aquimonas,and Flavobacterium existed in the sludge culture stages of sodium acetate group and had high abundance.The abundance of the top 15 species among different samples were significantly different at the genus level(P<0.05),Methylo Vendlis,Azonexus,Hydrogenophaga,Lentimicrobium,Aquimonas,Ignavibacterium,Desulfomicrobium have not been found in the original inoculated sludge samples.Methylo Vendlis and Hydrogenophaga are the dominant strains after methanol domestication,Thauera and Ignavibacterium are the dominant strains after sodium acetate domestication,Desulfomicrobium is relatively low in the non-drug-resistant bacteria and high in the antibiotics-resistant bacteria.(5)Based on metagenomics technology and KEGG database analysis of microbial metabolic function,it can be seen that the dominant function of microbial flora in the culture sludge is metabolism,followed by cell process,genetic information processing and human diseases.Compared with inoculation mud,the abundance values of each functional module of drug-resistant and non-antibiotics-resistant bacteria were significantly increased after culture and domestication for a period of time,and there was little difference between antibiotics-resistant and non-antibiotics-resistant bacteria in each functional module.By annotating the protein function of microbial population in the Cazy database,it can be seen that the microbes in the Me-trace OFL system in the methanol group had stronger carbohydrate decomposition ability,and the microbes in the Na AC-non OFL system in the sodium acetate group had stronger carbohydrate decomposition ability.Based on high-throughput quantitative PCR,134 species of antibiotic resistance genes(ARGs)were detected in Me-non OFL sludge,Me-trace OFL sludge,Na Ac-non OFL sludge,and Na Ac-trace OFL sludge,146,151 and 158,respectively.The corresponding absolute abundance levels of ARGs were 6.52×10⁹copies/g,1.78×10⁹copies/g,3.05×10¹⁰copies/g and1.34×10¹¹copies/g,respectively.(6)The dominant bacteria phyla in sewage sludge samples mainly include Proteobacteria,Actinobacteriota,Bacteroidota,Firmicutes and Chloroflexi.The dominant species in the sludge samples from AY sewage plant are Sulfuritalea,Hgcl?clade and Trichococcus.The dominant species in the sludge samples from SMX sewage plant are Lamia,Conexibacter,Ahniella,Gaiella,Unidentified?Nitrospiraceaeand Candidatus?Microthrix.Candidatus?Competibacter,Hydrogenophagaand Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium were the dominant species in the sludge samples from JD wastewater treatment plant.The dominant species in the sludge samples from DY3 sewage treatment plant are Ottowia,Dialister,Novosphingobium,Faecalibacterium,Lactobacillus,Bifidobacterium and Terrimonas.The dominant species in the sludge samples from QSY sewage treatment plant are Prosthecobacter,Methanosaeta,Terrisporobacter,Thauera and Ferruginibacter.The dominant species in the sludge samples from the XT sewage treatment plant were mainly Helicobacter,966-1,Phaeodactylibacter,Bacteroides,Escherichia-Shigella and Terrisporobacter.(7)The absolute abundance of ARGs in the culture sludge was 1.78×10⁹copies/g?1.34×10¹¹copies/g,and the absolute abundance of ARGs in the sewage sludge sample was1.12×10⁸copies/g?2.21×10¹⁰copies/g.The absolute abundance of ARGs in AY and SMX sewage sludge samples with sodium acetate as carbon source ranged from 7.02×10⁷copies/g to 3.99×10⁹copies/g,and the absolute abundance of ARGs in sludge with sodium acetate as carbon source ranged from 3.05×10¹⁰copies/g to 1.34×10¹¹copies/g.Dosing of the same kind of carbon source,sludge cultivation of sludge in the laboratory environment ARGs absolute abundance than sewage factory,but the sewage plant,the species number of the ARGs is greater than the cultivation of sludge,may be a sewage plant environment is more complicated,although will promote the diversity of the ARGs,but the ARGs absolute abundance compared with the cultivation of the laboratory conditions sludge,laboratory single cultivation environment more can promote the sludge in the absolute abundance of ARGs.