Mechanism Of Tobramycin Potentiation By CCCP Or Ethanol Against Stationary-phase Escherichia Coli

The widespread dissemination of antibiotic-resistant microorganisms poses a significant threat to humanity’s ability to combat bacterial infections in the 21st century.With the development of new antibiotics currently at a standstill,it is crucial to enhance the bactericidal efficacy of existing antibiotics to slow the rate of bacterial resistance and reduce the pressure on the evolution of antibiotic resistance.This study aims to increase the sensitivity of stationary-phase Escherichia coli to antibiotics and improve the lifespan of existing antibiotics.The first part of the study focused on carbonyl cyanide m-chlorophenyl hydrazone(CCCP)to investigate its application conditions and molecular mechanism in enhancing antibiotic bactericidal activity.CCCP can reduce the proton motive force(PMF)and ATP levels of exponential-phase E.coli,which inhibits the bactericidal activity of antibiotics.However,this study found that CCCP can promote the bactericidal activity of tobramycin against stationary-phase E.coli and only synergized aminoglycoside antibiotics,but notβ-lactam or quinolones antibiotics.Among the 16 bacterial strains currently validated,this method can effectively kill 4 types of bacteria,including Gram-negative bacteria E.coli,Shigella flexneri and Vibrio alginolyticus,and Gram-positive bacteria Staphylococcus aureus.In addition,among the 18 strains of E.coli,5 strains of S.aureus and 17 strains of MRSA isolated clinically,10 strains,1 strain and 14 strains could be effectively killed by combined treatment,respectively.The mechanism of CCCP enhancing tobramycin sterilization has been further revealed.First of all,through the bacteriostatic zone experiment and tobramycin ELISA kit,it is found that CCCP potentiates tobramycin against stationary-phase E.coli not by increasing antibiotic uptake,but can cause the leakage of the intracellular protein.Obvious protein leakage is detected stationary-phase E.coli treated with CCCP,and leakage is more serious when CCCP co-treated with tobramycin.Combined with the results of PI staining,it demonstrates that combined treatment can cause serious damage to the bacterial cell membrane.In addition,since CCCP has been detected to reduce the PMF and ATP levels of the stationary-phase E.coli,eight reagents that could lower bacterial PMF or ATP levels are evaluated,and only FCCP and triclosan can effectively enhance the bactericidal activity of tobramycin.Considering that PMF across the cytoplasmic membrane of bacteria is composed of two components,i.e.,the transmembrane proton gradient(Δp H)and the transmembrane electric potential(ΔΨ),Nigericin which depletesΔp H and valinomycin which dissipatesΔΨ,both can enhance the bactericidal action of tobramycin against stationary-phase E.coli,indicating that the two components of PMF play an important role in CCCP-enhanced killing.Finally,ROS probes have been detected that CCCP induces the production of large amounts of superoxide and hydroxyl radicals in stationary-phase E.coli,At the same time,antioxidants such as NAC,vitamin Cand glutathione suppress the bactericidal efficacy of CCCP in combination with tobramycin,and NAC significantly inhibited the content of hydroxyl radicals but did not affect the superoxide.These results indicate that the accumulation of hydroxyl radicals may be an important factor of tobramycin potentiation by CCCP.The second part of this study is to explore the molecular mechanism of tobramycin potentiation by ethanol and evaluate its potential application value.Ethanol has been shown to enhance the killing of stationary-phase tolerant bacteria by aminoglycoside antibiotics in a concentration-dependent manner.Currently,this method has been validated to be effective against eight types of bacteria,including Gram-negative bacteria such as E.coli,Pseudomonas aeruginosa,Salmonella typhimurium,S.flexneri,V.alginolyticus,as well as Gram-positive bacteria S.aureus,Streptococcus epidermidis,and Micrococcus luteus.In addition,ethanol combined with tobramycin or gentamicin exhibits varying degrees of killing effect against 11 of the 13 clinical isolates of E.coli.Further studies showed that ethanol can reduce the level of NAD~+,increase the level of ATP,promote the uptake of tobramycin,and significantly weaken the bactericidal synergism of ethanol dehydrogenase-deficient strainΔadh P,the single knockout strain of alcohol dehydrogenase.These results suggest that stationary-phase E.coli may increase ATP levels through the catabolism of ethanol,thereby promoting tobramycin’s entry into the bacteria,and eventually resulting in a killing effect against E.coli of up to 4 orders of magnitude.In conclusion,this study found that CCCP and ethanol potentiates aminoglycosides against stationary-phase E.coli and the synergistic bactericidal mechanism has been preliminarily discussed.The findings are of great significance to the enhancement and detoxification of aminoglycoside antibiotics and provide new ideas for the development of antibiotic adjuvants which is significant for enhancing the efficacy and reducing the toxicity of aminoglycoside antibiotics,and provides a new strategy for the development of antibiotic adjuvants.