The Selection And Dissemination Mechanism Of Antibiotic Resistance For Bacteria Under Environmental Pressure In The Ba River

The urban river is a natural water to accept a variety of waste and recycled water.It also accepts the emerging pollution of antibiotic resistant genes（ARGs）that results from the extensive use of antibiotics by humans.The antibiotic resistance of bacteria will affect the river ecosystem and even threat to human health.It is worth asking how the aquatic environmental pressures caused by pollutants such as antibiotics and heavy metals select antibiotic resistance in the natural environment and how they affect the dissemination of ARGs.In this study,we chose a pristine river and an urban river.The techniques of real-time PCR,16S r RNA gene,metagenomics and Binning methods were used to study the characteristics of ARGs,heavy metal resistance genes（MRGs）and mobile genetic elements（MGEs）,host bacteria and co-resistance of ARGs in water samples,sediments and Hemiculter leucisculus gut samples,respectively.Chemical analysis methods such as ultra-high-performance liquid chromatography coupled with a triple quadrupole mass spectrometry,inductively coupled plasma mass spectrometer,and continuous flow analysis were used to determine the content of antibiotics,heavy metals,and nutrient salts in natural water bodies,respectively.To explore the mechanism of acquired bacterial antibiotic resistance under environmental pressures from antibiotics,heavy metals and other ecological factors.To analyze the potential gene transfer pathway of ARGs from bacteria of water column or sediment to the gut bacteria of fish.The main results are as follows:1.Bacterial community structure and characteristics of ARGs in water and sediments under different selection pressuresIn water samples,sulfonamides,tetracyclines,macrolides,quinolones and chloramphenicol antibiotics all showed an increasing trend from upstream to downstream.Sulfonamides and macrolides were as high as 1839.75 ng/L and 429.20 ng/L,respectively.In sediments,all 6 categories of antibiotics showed a low level in the upstream,and the middle and downstream sediments showed an increasing trend.Among them,tetracyclines appeared at very high levels of 454.02μg/kg in the middle and lower sediments.Based on the analysis of 16S r RNA genes,the results indicated that Proteobacteria and Bacteroidetes were the dominant phyla in all samples,and sediment samples had higher bacterial diversity and richness than water.Canonical correspondence analysis（CCA）indicated that Acinetobacter,Arcobacter and Aeromonas appeared to be linked with As,Cu,trimethoprim,sulfamerazine and oxytetracycline.The advantage ARGs of tet C,bla _TEM,erm F,sul1,cml A and gyr A had higher pollution abundance ranging from 10^-4 to 10⁰ copies/16S r RNA gene copies in all samples.The absolute abundance of ARGs in sediments was 1-3 orders of magnitude higher than in water.The CCA results of antibiotic residues and ARGs found that tet Z and tetracycline antibiotics,bla _TEM and bla _IMP4 andβ-lactam antibiotics,qnr S and norfloxacin had a corresponding positive correlation.The correlations between other resistance genes and antibiotics did not correspond,suggesting that the abundance of ARGs may not be completely determined by antibiotics in the river.2.Dissemination mechanism of ARGs in sediments under different selective pressuresMetagenomic assembly indicated that environmental pressures significantly increased the relative abundance of macrolide-lincosamide-streptogramins,vancomycin,β-lactam and sulfonamide resistance genes.At the lower reach of the Ba River,the higher ratios for contigs carrying more than one ARG suggested that environmental pollution favored the co-occurrence of multiple ARGs.All samples had a higher ratio of contigs carrying ARGs（ARCs）identified as plasmid than chromosomal ARCs,and all types of ARGs were detected.Further,the ratio of ARCs carrying MRGs identified as plasmid sequences was higher than that of chromosomal ARCs carrying MRGs,suggesting the co-transfer ability of ARGs and MRGs.Environmental pressures also increased the relative abundance of advantaged hosts,including Chloroflexi,Firmicutes and Euryarchaeota.By metagenomic binning,a total of 23 high quality genome bins carrying ARGs were reconstructed from sediment samples.At the lower reach of the Ba River,Romboutsia timonensis carrying multiple ARGs and ICEs was successfully recovered,posing a serious threat to human health by affecting the metabolism of gut microbiomes.And Methanothrix soehngenii affiliated to archaea carrying multiple ARGs,MRGs and ICEs were also recovered from the lower Ba River.The partial least squares path modeling（PLS-PMA）revealed that MGEs were the most predominant factors inducing the ARG profiles.Furthermore,environmental factors could indirectly impact the ARG profiles by first influencing the ARGs’hosts.3.Mechanism of ARGs transfer from water to fish gut under different selection pressuresThe metagenomic assembly indicated that the decrease of ARG abundances from pristine to anthropogenic regions was attributed to the reduction in the proportion of multidrug resistance genes in water microbiomes along the environmental gradient.Whereas anthropogenic impact contributed to the enrichment of ARGs in fish gut microbiomes.The increasing trends in the proportion of the ARCs concomitant with plasmids along environmental gradient indicated that plasmids act as efficient mobility vehicles to enhance the spread of ARGs under environmental pressures.The Procrustes analysis and Mantel test showed a significant correlation between the ARG and MRG profiles.Moreover,the higher co-occurrence of ARGs and MRGs on plasmids in water and fish gut samples revealed that environmental pressures accelerated the co-transfer potential of ARGs and MRGs and the enrichment of ARGs.By metagenomic binning,a total of 27 and 23 high quality genome bins carrying ARGs were reconstructed from water and fish gut samples,respectively.Potential pathogens Vibrio parahaemolyticus,Enterobacter kobei,Aeromonas veronii and Microcystis aeruginosa＿C with multiple ARGs,MRGs and ICEs were retrieved from fish gut microbes in the lower reach of the Ba River.PLS-PMA revealed that water ARGs host bacteria were the most predominant factors affecting the fish gut ARG profiles.And environmental factors significantly affected water host bacteria,indicating that environmental pressures could shape fish gut antibiotic resistome mainly via affecting ARGs host bacteria in water microbiomes.In summary,this study found that with the increase of selection pressure caused by environmental pollution,the relative abundance of dominant ARGs in water increased,ARGs were enriched in fish gut,and HGT mediated by plasmids or ICEs promoted the dissemination of ARGs.Environmental pressures could shape fish gut antibiotic resistome mainly via affecting ARGs host bacteria in water microbiomes.The results of the study will contribute to figuring out the dissemination features for the bacteria’s antibiotic resistance under environmental pressures from all kinds of pollutants in the natural ecosystem.The results will guide the prevention and control of ARGs pollution in the water ecosystem.