The Screening And Characterization Of ?-lactam Tolerant Escherichia Coli Mutant

The advent of penicillin in 1928 opened the door for antimicrobial therapy,which has saved countless lives from bacterial infections.However,due to the lack of awareness of prudent use of antibiotics,the widespread overuse or even misuse of antibiotics in the general population,agriculture,animal husbandry,and hospitals has increased the probability of selection of drug-resistant bacteria,leading to serious abuse of antibiotics.The abuse of antibiotics has led to the emergence of many resistant bacteria,even multidrug resistant bacteria or extremely drug-resistant super bacteria.Bacterial resistance to antibiotics is the main cause of failure of antibiotic treatment.At present,the research progress on the mechanism of drug-resistant bacteria is relatively advanced.However,studies have shown that drug resistance is the result of the bacteria becoming tolerance to exposure to antibiotics before they cause resistance,so it is necessary to explore the mechanism of antibiotic tolerance.Therefore,we chose meropenem,a commonly used member of ?-lactam antibiotic,to enrich for tolerant bacteria from a pooled keio library of more than 4,000 E.coli BW25113 non-essential single-gene knockout strains.After several rounds of successive enrichment,we obtained a mutant having the same MIC(minimum inhibitory concentration)as the BW25113 wildtype strain,but under the same drug concentration pressure,exhibiting a significantly higher survival rate than the wild type.It was subsequently confirmed that the tolerant phenotype of this strain,and the gene deleted in the resistant strain was identified to be a gene involved in purine metabolism that encodes the FAD(flavin adenine dinucleotide)binding subunit.Subsequent bactericidal experiments confirmed that,in the absence of the purine metabolism,there was a significant tolerance phenotype compared to the BW25113 wildtype strain when cells were treated with meropenem.Based on the tolerance phenotype caused by the deletion of the purine metabolism gene,we conducted a series of experiments to explore the tolerance mechanism.The experimental results showed that the deletion of the purine metabolism gene also conferred significant tolerance to other antibiotics and biocides,in addition to tolerance to meropenem.In order to tie the tolerance phenotype to the deletion of the functional purine metabolism gene,we complemented the mutant strain with a plasmid-born wild-type gene.Complementation was successfully achieved.Measurement of intracellular ROS and ATP levels during antimicrobial exposure showed that both were reduced in the mutant than in the wildtype.Since previous literature has implicated the purine metabolism gene in the purine salvage pathway,we next searched all relevant genes related to purine and pyrimidines metabolism on relevant websites,and selected over 80 single-gene knockout strains from E.coli BW25113 single-gene knockout strain library(Keio mutant library)for tolerant phenotype.Several single-gene knockout strains were identified having obvious tolerance phenotype to ciprofloxacin,which provides us more clues for studying the mechanism of antimicrobial tolerance.