Selection And Characterization Of Disinfectant-tolerant Mutants

Disinfectants are widely used in medical,agricultural,and food fields and they are essential to reduce the postoperative infection rate and control infectious microorganisms from spreading.However,the large-scale use of disinfectants inevitably induces the rapid development of bacteria tolerance/resistance to disinfectants,and the thrust behind the emergence of drug tolerance/resistance is largely attributed to the unnecessary,abusive overuse of disinfectants.Although there have been a few reports on the tolerance/resistance of disinfectants in recent years,with the emergence of multi-drug antibiotic resistant bacteria,extremely drug-resistant bacteria,and even pan-drug resistant bacteria,the potential harm of loss of disinfectant efficacy can’t be ignored.Due to the vague definition of disinfectant-resistant and tolerant bacteria,few researches have focused on relevant mechanism of disinfectant tolerance.Therefore,it is of great significance to find the tolerance gene of disinfectant and to uncover the molecular mechanism underlying bacterial tolerance to disinfectants,which will help guide the rational use of disinfectants,prevent tolerant bacteria from arising,and mitigate the spreading of antimicrobial resistant pathogens..In this study,a classic phenolic disinfectant(phenol)was used to kill Esterichiae coli and enrich mutants tolerant to phenol-mediated killing.After multiple rounds of enrichment and single colony culture purification,a mutant named Phen-T was obtained.We confirmed that Phen-T is a tolerant rather than a resistant mutant by observing no change MIC but significant protection from phenol-mediated killing.Comparative whole genome re-sequencing identified two single-base mutations in Phen-T.The killing experiments using strains having the single-base mutations identified and deletional mutation in each gene identified the key gene responsible for Phen-T tolerance to phenol.Suprisingly,we found that both the Phen-T and key gene deletion mutants conferred tolerance phenotype to diverse types of disinfectants.In addition to E.coli BW25113,several other Gram-negative and Gram-positive bacteria lacking the key gene exhibited tolerance to phenol.Thus,the key gene may involve in a pathway that participate in a mechanism of killing shared by diverse types of disinfectants and many bacterial specieas.Such a shared mechanism of killing might be closely related to the changes of bacterial energy metabolism by antibiotic exposure that produces reactive oxygen species(ROS)as a by-product,because as seen with lethal antibiotic exposure,treatment with phenol also induced over-accumulation of ROS.The phenol-induced ROS over-accumulation was less in the key gene deletion mutant than in wild-type strain.Since ROS is produced as a by-product during energy metabolism that synthezies ATP,we next measured the changes of intracellular ATP levels in wild-type and the key gene deletion mutant,respectively during phenol treatment.The results showed that phenol stimulated the increase of intracellular ATP levels in E.coli,but such an increase was less in the key gene deletion mutant than in the wild-type cells,which is consistent with the suppressed elevation of intracellular ROS accumulation by the key gene deletion mutant after phenol treatment.Since crosstolerance was observed between disinfectants and antimicrobials and since antimicrobial tolerance preceds and promote resistance,we next used sodium hypochlorite,ethanol,chlorhexidine,and phenol to explore the effect of disinfectant treatment on bacterial resistance to antibiotics.The results showed that wild-type strain pre-treated with each disinfectant significantly increased resistance frequency to ciprofloxacin,while such increase was much smaller with the key gene deletion mutant than with wildtype,which indicates that exposure of bacteria to disinfectants increases the propensity of bacterial resistance to antibiotics.The deletion of the key gene affects the bacterial carbon flux and energy metabolism.We found that mutations of several genes upstream and downstream of the key gene involved in phenol tolerance also conferred different degrees of tolerance to phenol.We next used the key gene mutant as a starting strain and selected mutants that confer even higher levels of phenol tolerance.One such super-tolerance mutant,B7-3,was otained and sequenced.Two potential super-tolerance target sites were found.The B7-3 mutant also exhibited super-tolerance to other disinfectants.Mechanism of supertolerance needs further investigation.The discovery and characterization of these disinfectant tolerance genes will guide elucidation of detailed molecular mechanisms underlying disinfectant-mediated killing and help prevent widespread tolerance to disinfectants from arising.