Enrichment,Screening And Mechanism Characterization Of Antimicrobial Pan-tolerant Mutants

Antibiotics can protect people from infectious diseases.However,because of their misuse,bacterial resistance to these agents is getting more and more serious,gradually eroding efficacy of existing antibacterial drugs,which limits the choice of treatment options.Studies have shown that bacteria can go from susceptible to resistant directly to antibiotics but may also go through tolerance as an intermediate stage that greatly facilitate subsequent development of resistance.Mechanisms of antimicrobial resistance which have been thoroughly studied are relatively well documented,but very limited mechanism of action is known about tolerance to antimicrobials.In-depth study of bacterial tolerance mechanism may help discover new drug targets,prevent tolerance from developing or stop bacteria at the tolerance stage,and reduce the subsequent generation of resistant bacteria.In order to explore the mechanism of pan-tolerance to many types of lethal antimicrobials,we sequentially killed E.coli B W25113 with one representative drug of three major bactericidal antimicrobials to enrich and select mutants exhibiting drastically reduced killing with little change in minimal inhibitory concentration(MIC)to each drug used.After 2 rounds of the above sequential enrichment,a pan-tolerant mutant with strong tolerant phenotype was screened out,and the key target of mutation responsible for tolerance was mapped in bacterial carbohydrate transport system by whole genome resequencing and comparative analysis.This system transports carbohydrate into the cells for energy metabolism.We found that deletional or active site single-base mutations in this transport system conferred pan-tolerance by blocking transportation of several key carbohydrates,reducing energy metabolism,and thereby drastically reducing antimicrobial killing without eliciting changes in minimal inhibitory concentration to antimicrobials or affecting growth rate in the absence of antimicrobials.Molecular details of antibiotic pan-tolerance mediated by this sugar transportation system was further investigated by measuring intracellular ROS and ATP concentrations,by adding exogenous chemicals that inhibit enzymatic activity of this system or ROS accumulation,by constructing rpoS and ROS-related gene deletion strains for tolerance assay,and by comparative transcriptomics analysis(RNA-seq)of the wild-type and the mutant obtained with/without antimicrobial treatment.Results showed that the tolerance phenotype is mainly achieved by regulation of adenylate cyclase activity and subsequent changes in carbon metabolism flux.Carbon flux was shifted from the tricarboxylic acid cycle(TCA cycle)to glycolysis and pentose phosphate pathway(PPP),thereby reducing ATP synthesis and ROS levels under antimicrobial stress.Decreased ROS production/accumulation reduced the toxicity to bacterial cells,allowing bacteria more time to repair antimicrobial-mediated damage to better survive antimicrobial treatment.Since the carbohydrate transportation system plays a key role in mediating antimicrobial tolerance,and since it does not exist in plants and animals,enzymes involved in this system may be used as bacterial-specific drug targets for designing new drugs that mitigate tolerance and suppress resistance from arising.