Shifts Of Microbial Community And Antimicrobial Resistance Under Antibiotic Selection Pressure

The widespread use of antibiotics inevitably leads to antibiotic residues in the environment.Aminoglycoside antibiotics(AGs)and ?-lactam antibiotics(BLAs)are widely used classes of antibiotics.However,the effects of these two classes of antibiotics on bacterial resistance,microbial community and antibiotic resistance genes(ARGs)have not been clarified.Therefore,kanamycin(KAN,belonging to AGs antibiotics)and ampicillin(AMP,belonging to BLAs antibiotics)were selected in this study to establish a sequencing batch reactor to study the effects of these two antibiotics on bacterial resistance,microbial community and ARGs in activated sludge under different concentration gradients.The main research contents are as follows: After continuous exposure to KAN and AMP antibiotics in sequencing batch reactor,the changes of bacterial resistance,microbial community and occurrence characteristics of ARGs in activated sludge under antibiotic selection pressure were revealed by combining traditional culture-method,16 S r RNA gene based on high-throughput sequencing and metagenomic analysis.We further analyzed whether the risk of resistance caused by selection pressure would persist when antibiotics were stopped.This provides reference for evaluating the risk of antibiotics to ecological environment and theoretical support for rational use of antibiotics.The main results are as follows:(1)Three different concentration gradients of KAN(0.5,5,50 mg/L)and AMP(1.5,15,150 mg/L)were added to the laboratory reactor,and the effects of KAN and AMP on bacterial antibiotic resistance were analyzed culture-based method.The results showed that the enrichment effect of 5 mg/L KAN on TET resistant bacteria was the highest(20.1 times),and that of 150 mg/L AMP on SMZ resistant bacteria was the highest(4.7 times).However,when antibiotics were removed for 16 days,the enrichment effect of TET-resistant bacteria was the largest in AMP 15 mg/L group(7.2times).Compared with the other four antibiotic resistant bacteria,KAN 50 mg/L group had the highest enrichment effect on KAN resistant bacteria(up to 2.0 times).Enrichment of SMZ-resistant bacteria was observed in the three AMP treatment groups after antibiotics were removed,especially in the AMP 150 mg/L group(2.6 times).The results showed that different types and concentrations of antibiotics in activated sludge system could produce different enrichment effects of antibiotic-resistant bacteria.(2)16S r RNA high-throughput sequencing combined with bioinformatics was used to analyze the influence of antibiotic selection pressure on microbial community.The results showed that the selection of antibiotics with high concentration of KAN and AMP could significantly reduce the microbial community diversity of activated sludge,especially when AMP was 150 mg/L(45.2%).Proteobacteria and Bacteroidetes accounted for more than 50% of the relative abundance of all activated sludge samples.Compared with the control,the abundance of proteobacteria decreased by 0.2 times and increased by 1.3 times under the treatment of 50 mg/L KAN for 16 days,while the abundance of proteobacteria increased by 0.3 times under the treatment of 150 mg/L AMP.At the genus level,the relative abundance of Trachelomonas increased by 8.5times when treated with 50 mg/L KAN.However,the relative abundance of Pseudomonas only increased 1.8 times under AMP 150 mg/L.Even after 16 days of antibiotics withdrawal,the relative abundance of Trachelomonas in the 50 mg/L KAN group increased by 5.0-fold.However,the relative abundance of Pseudomonas in AMP150 mg/L group increased by 4.8 times.The results showed that aminoglycoside and sulfonamides ARGs were significantly enriched at high concentrations of KAN(50mg/L)and AMP(150 mg/L),and the enrichment effect could be maintained when KAN was removed.(3)Metagenomics combined with bioinformatics analysis showed that long-term(16 days)high concentration exposure of KAN and AMP resulted in the highest enrichment effect of ARGs in activated sludge system.Compared with the control,aminoglycoside-ARGs was significantly enriched(5.6 times)under 50 mg/L KAN treatment.The enrichment ratio of sulfonamides-ARGs reached 1.4 times under AMP150 mg/L.In addition,the addition of KAN and AMP antibiotics increased the diversity and abundance of ARGs in activated sludge.After the removal of antibiotics,aminoglycoside-ARGs and sul1 in the 50 mg/L KAN treatment group were enriched by 13.8 times and 4.0 times,respectively.However,aminoglycosides-ARGs and sul1 were not enriched when AMP 150 mg/L was removed.The results showed that aminoglycoside-and sulfonamides-ARGs were significantly enriched at high concentrations of KAN(50 mg/L)and AMP(150 mg/L),and continued to be enriched when KAN was removed.