Study On Antimicrobial Activity Of Pleuromutilins Combined With Tetracycline For Staphylococcus Aureus

With rapidly increasing antibiotic resistance and decline in new antibiotic drug development,the toughest challenge remains to maintain and preserve the efficacy of currently available antibiotics.Combining antibiotics is a promising strategy for increasing treatment efficacy and for controlling resistance evolution.Combination therapies have distinct advantage over monotherapy in terms of broad antibacterial spectrum,synergistic effect and prevention of emergence of drug resistance.Pleuromutilins were separated from the pleuromutilin-producing fungus Pleurotus mutilus,tiamulin and valnemulin are two established derivatives in veterinary medicine for oral and intramuscular administration.But there has been a resurgence in interest to develop this class for human use.Retapamulin and lefamulin are characterized by potent activity against Gram-positive and fastidious Gram-negative pathogens.This indicates that pleuromutilin drugs are getting more and more attention in the period of serious bacterial resistance.However,the relationship between pleuromutilins and other drugs has not been studied.Therefore,this study used Staphylococcus aureus as a model strain to investigate the relationship between pleuromutilin drugs and other drugs by the high throughput method.Strain-specific transcriptome of the possible mechanisms for the synergistic action of valnemulin and tetracycline was analyzed by using RNA-Seq.In this current study,the relationship between ten antibiotics and antibiotics or nine plant extracts against Staphylococcus aureus were investigated.Compared well agar diffusion method and checkerboard method,identified the cut off of diameter of inhibition zone.The results showed that different concentrations of antibiotics which added to the agar wells had linear relationship with inhibition zone diameter.Through the relationship between the change of inhibition zone diameter and the FICI index after drug combination,the results showed that when the inhibition zone diameter increased 2 mm is defined as synergistic,the diameter zone of inhibition is between 1 mm and 2 mm,it is defined as the additive effect,the diameter of the zone of inhibition changes between 0mm and 1mm,it is defined as an irrelevant effect.The diameter of the zone of inhibition when it is reduced,it is defined as antagonism.The time killing curves were consistent with the results.So the well agar diffusion method can be used to screen the relationship between drugs and other drugs or natural extracts.To screen the relationship between pleuromutilin drugs and 13 other drugs with different mechanisms of action against Staphylococcus aureus by the established method of well agar diffusion.Two standard strains of Staphylococcus aureus?ATCC 29213 and ATCC 43300?and two clinical strains?N9 and N54?were selected.In vitro synergistic effects were observed by standard checkerboard and time-kill assays.The effects of combinations also were verfied in an experimental Galleria mellonella model.The results showed that the pleuromutilin drugs had a synergistic effect with tetracycline?FICI?0.05?and had an antagonism with fluoroquinolones?FICI?4?,which had indifference or additive effects with other drugs.Treatment of G.mellonella larvae infected with lethal doses?10⁶ CFU/mL?of S.aureus resulted in significantly enhanced survival rates when pleuromutilin was given with tetracycline compared with drug treatment alone?p<0.05?.Treatment the infected G.mellonella with pleuromutilin and ciprofloxacin,the survival rate of G.mellonella was between 20%and 60%,and there was no significant difference compared with single drug.Six cfr-positive methicillin-resistance S.aureus?MRSA?strains were used to compare efficacy of valnemulin-doxycycline combination in a mouse thigh model.Synergistic activity between valnemulin and doxycycline was evaluated by checkerboard and time-kill assays.Increased susceptibility to valnemulin was observed with doxycycline combination on the study MRSA strains?MIC decreased 4³2 folds vs valnemulin alone?.The combination had synergistic activity?FICI?0.5?against all cfr-positive MRSA strains followed by confirmation of synergism using the time-kill methodology(?2 log₁₀CFU/ml reduction).The addition of doxycycline significantly improved the efficacy of valnemulin in the mouse thigh model due to cfr-positive MRSA strains.The number of bacteria in the thigh muscle could decreased 0.7 log² log after combination theraph.Additionally,the cytokine IL-6,TNF-?,MCP-1 in the serum of the combination therapy group was reduced than monotherapy except IL-10.Which indicating that the drug not only has the antibacterial effect,but also has anti-inflammatory effects,and at the same time the combination of drugs can play a more significant role in anti-inflammatory.To determine changes in the transcriptome of S.aureus following treatment with valnemulin and tetracycline,both alone and in combination was employed by RNA sequencing.After treatment with combination compared to alone that more than 700differentially regulated genes were identified,including many associated with translation and ribosome structural components.The main metabolic pathway involved were ribosome,biosynthesis of secondary metabolites,metabolic pathways and microbial metabolism in diverse environments.In addition,the combination of the two drugs will down-regulate the ABC superfamily efflux pump genes in S.aureus,which may be the possible mechanism of synergy.In summary,this research established the well agar method can quickly evaluate the relationship between drugs and drugs.By this method,the combination of pleuromutilin and tetracycline has a synergistic effect on Staphylococcus aureus and also has a therapeutic effect on the cfr-MRSA strains.The transcriptomic data have tentatively confirmed the synergistic mechanism of valnemulin and tetracycline associated with the overexpression of ribosomal genes and the downregulation of efflux pump genes on S.aureus.This study provides a theoretical basis for clinical drug combination.