| In recent years,with the irrational use of antibiotics worldwide,leading to veterinary clinical major pathogenic bacteria to common drugs has produced serious resistance,while the rate of new drug development in recent years has been declining year by year,causing a serious threat to the livestock breeding industry.In response to this dilemma,it is urgent to screen the commonly used drug resistance inhibitor targets that are now in the resistance window and develop new antimicrobial potentiators to delay the life of existing drugs.As one of the major pathogens of bovine respiratory diseases,Pasteurella mutocida(Pm)has developed serious resistance to commonly used veterinary drugs.Previous studies by our group have shown that the expression of different drug-resistant phenotypes of bovine podococcal A Pm SOS pathway genes(rec O,ruv A,etc.)mediates the retention of Pm and can be used as potential targets for drug resistance inhibitors.To this end,the present study was conducted to screen potential drug-derived molecules with rec A,a key initiator gene of SOS response,as a target to enhance the therapeutic effects of fluoroquinolones and prolong their lifespan.The main study includes the following three parts:Firstly,the rec A gene of bovine capsule type A Pm was used as the target gene,and its homologous modeling was carried out by bioinformatics method.Inhibitors targeting Rec A protein were screened from small molecule compound library according to different precision scoring principles of molecular docking.The results showed that small molecule compound B4964 scored low(glide gscore=-7.916),which speculated that it might target Rec A protein;The inhibitory effect of B4964 on rec A gene was confirmed by q PCR at m RNA level.The results showed that B4964 at sub-inhibitory concentration could significantly inhibit the overexpression of Pm rec A gene induced by ciprofloxacin(P < 0.001);The combined antibacterial index of B4964 and ciprofloxacin was determined by chessboard method.The results showed that B4964 and ciprofloxacin had additive effect(FIC ≤ 1),which proved that B4964 could enhance the therapeutic effect of ciprofloxacin.Secondly,in order to further evaluate the pharmacodynamics of small molecule compound B4964 in vitro,The effects of subinhibitory concentration B4964 on the formation time,mutation selection window,mutation frequency,biofilm formation ability,antibiotic tolerance and bacterial retention of bovine capsule type A Pm induced by ciprofloxacin were detected by routine pharmacological experiments.The results showed that B4964 could inhibit the expression of rec A gene,prolong the formation time of Pm resistance to ciprofloxacin,reduce the mutation selection window of ciprofloxacin and the mutation frequency of bovine capsule type A Pm with different drug resistance phenotypes,inhibit the biofilm formation ability of bovine capsule type A Pm,and inhibit the formation of bovine capsule type A Pm retention bacteria.At the same time,based on the fluorescence activity reporter vector of SOS reaction promoter and the expression vector of Rec A protein,it was found that B4964 combined with ciprofloxacin could significantly inhibit the initiation of SOS reaction induced by ciprofloxacin and inhibit the expression of Rec A.Finally,the safety of B4964 combined with ciprofloxacin was preliminarily detected by cytotoxicity test and hemolytic activity test.The results showed that the combined use did not have toxic effect on RAW264.7 cells and 293 cells,and did not have hemolytic activity on rabbit red blood cells;The infection model and treatment model of P3-pneumonia in mice were constructed,It was found that the combined medication could enhance the survival rate of mice,quickly kill pathogenic bacteria in target organs and tissues,inhibit the formation of persistent bacteria in vivo induced by ciprofloxacin treatment,inhibit the formation of mutant strains,reduce the mutation frequency of pathogenic bacteria in vivo,and enhance the therapeutic effect of ciprofloxacin in vivo.In a word,this study used bovine capsule type A Pm as a model strain to construct a screening platform for SOS response resistance inhibitors,and screened B4964,a potential drug source molecule that can inhibit SOS response,and proved that it can inhibit the production of bovine capsule type A Pm retention bacteria,which laid a foundation for the research and development of fluoroquinolone resistance inhibitors. |
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