A Study On The Role Of Metformin In Restoring The Susceptibility Of Doxycycline-resistant Gram-negative Bacteria

Respiratory,gastrointestinal,and urinary tract infections caused by gram-negative bacilli occur frequently in pet clinics,and to control these diseases,doxycycline is widely used for the treatment of these bacterial diseases because of its broad antibacterial spectrum and low cost.As the use of doxycycline in pet clinics has accumulated,the problem of bacterial resistance has arisen.Currently,restoring the antibacterial activity of doxycycline against resistant bacteria by some compound or antibiotic adjuvant is a promising direction.A recent study has shown that metformin as an anti-diphtheria drug has enhanced antimicrobial activity of doxycycline against multi-drug resistant bacteria(Staphylococcus aureus,Enterococcus faecalis,Escherichia coli and Streptococcus enteritidis),which provides a novel strategy for the clinical treatment of infectious diseases caused by doxycycline-resistant gram-negative bacteria in pets.In this experiment,six representative groups of Gram-negative bacilli,namely Escherichia coli,Klebsiella pneumoniae,Enterobacter cloacae,Pseudomonas aeruginosa,Acinetobacter baumannii,and Acinetobacter chimaera,were isolated from clinical bacterial infection samples in pets,with three strains of each group.Among the clinical isolates,the doxycycline-susceptible bacteria were firstly subjected to in vitro induction of drug resistance,and a doxycycline resistance model was established and preliminary resistance characterization was performed.The biofilm formation ability and efflux pump expression ability of the resistant bacteria were also detected to study the mechanism of resistance development.Then the combination of metformin and doxycycline at doses lower than the MIC multiplier was applied to the acquired resistant and naturally resistant Gram-negative bacilli.By this method,we assessed whether metformin could enhance the antibacterial ability of doxycycline against a variety of Gram-negative resistant bacteria and analyzed the reasons for this.The results obtained from the experiment were as follows.1.among the isolated bacteria,except for Acinetobacter baumannii isolates,which were all sensitive to doxycycline,the rest of the bacteria had resistance status to doxycycline.2.After in vitro induction of resistance in the pet clinically sensitive isolates to doxycycline,the efflux pump characterization test of all resistant strains showed that the MIC of each strain to doxycycline decreased 2-128-fold after the addition of efflux pump inhibition(CCCP).Escherichia coli,Klebsiella pneumoniae and Enterobacter cloacae were all significantly upregulated by doxycycline induction;the naturally resistant Pseudomonas aeruginosa also showed significant upregulation of efflux pump regulatory genes compared to the standard strains;no major efflux pump gene expression was detected in resistant strains of Acinetobacter baumannii and Acinetobacter chimaerae.3.Combined treatment of Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa and Enterobacter cloacae with doxycycline successfully reduced the MIC of the bacteria to doxycycline,while the efflux pump regulatory genes were significantly downregulated and the intracellular doxycycline content of the bacteria was significantly increased(P<0.05).Conclusions: An in vitro model of doxycycline resistance in gram-negative bacilli of pet origin was successfully established.The combination of metformin and doxycycline can reverse the drug resistance of Escherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa and Enterobacter cloacae resistant strains by inhibiting the expression of efflux pump regulatory genes and enhancing the intracellular accumulation of doxycycline in bacteria.