Effect Of AcrR On Persister Formation In Aeromonas Veronii

Persister is a small group of dormant bacteria formed by bacteria in response to high concentrations of antibiotics.The formation of persister is associated with chronic infections,revertive infections,and biofilm infections that are difficult to eradicate clinically.The effect of multidrug efflux pump on the formation of persister has been widely reported,but the effect of its upstream regulatory factors on the formation of persister has been rarely mentioned.Acr R(acriflavine resistance regulator)is a ubiquitous bacterial protein with a highly conserved sequence.It is recognized as a typical negative regulator of multidrug efflux pump Acr AB.Aeromonas veronii is a pathogenic bacteria that infects economically farmed fish,and has the characteristics of multiple drug resisitance,which is harmful to the aquaculture industry.Therefore,in this study,Aeromonas veronii strain with deletion of acrR gene was taken as the main research object to explore the function of acrR in the formation of persister.On the one hand,the regulatory relationship between acrR and acr AB gene of efflux pump was explored.The acrR gene knockout strain(ΔacrR)and the acrR gene replacement strain(ΔacrR-p BBR-acrR)of Aeromonas veronii were firstly constructed.By measuring the growth curves,we found that the absence of Acr R in M9 liquid medium didn’t affect the growth of Aeromonas veronii.Through the RT-q PCR experiment,the expression levels of acr AB gene in WT,ΔacrR and ΔacrR-p BBR-acrR strains were compared.The results showed that the expression level of acr AB gene in the ΔacrR strain was significantly higher than that in the WT strain in the logarithmic phase,but no obvious difference was observed between the two strains in the stationary phase.Further research found that Acr R has a negative regulatory effect on the acr AB,meaning that acrR could down-regulate the expression of acr AB gene.On the other hand,the effect of acrR on persistence level was clarified.Firstly,the efflux pump activity of WT,ΔacrR,ΔacrR-p BBR-acrR,Δacr A and Δacr B strains were measured.In the logarithmic phase,the efflux activity of ΔacrR strain was significantly higher than that of WT strain,while in the stationary phase the efflux activities between two strains had no significant difference.Regardless of the logarithmic or stationary phase,the efflux activities of Δacr A and Δacr B strains were significantly lower than those of other three strains.Secondly,the susceptibility curves of strains under antibiotic treatment were measured,it was found that in the logarithmic phase the ΔacrR strain had a higher persister cell formation rate than the WT strain under the treatments of chloramphenicol,tetracycline and cefotaxime.While in the stationary phase,there was no significant difference between WT and ΔacrR strains.Whether in the logarithmic or stationary phase,the Δacr A and Δacr B strains showed lower persister cell formation rate than the WT andΔacrR strains.Finally,the susceptibility curves of WT and ΔacrR strains pretreated by the efflux pump inhibitor CCCP were measured.Compared with the strains that were not pretreated by CCCP,the formation of persister cells was significantly reduced in the strains pretreated by CCCP.Above results indicate that acrR affects the efflux activity of bacteria by inhibiting the expression of acr AB gene in the logarithmic phase,thereby reducing the formation of persister cells.At the same time,acrR has no regulatory effect on acr AB gene in the stationary phase,so there is no significant difference in the persister cell formation rate of WT and ΔacrR strains.This study proposes the molecular mechanism of persister formation affected by acrR,extends the function of acrR in pathogenic bacteria,and provides potential drug targets and new prevention strategies for the treatment of aquatic diseases caused by Aeromonas veronii,drug resistance and bacterial persistence.