A New Drug Combination Screening For Pasteurella Multocida

Bovine pasteurellosis is an acute,febrile,highly lethal disease characterized by subcutaneous and organic mucosal hemorrhagic sepsis,pneumonia,and acute gastroenteritis in cattle caused by Pasteurella multocida?Pm?.According to the specificity of the capsular antigen,Pm can be divided into 5 capsular types such as A,B,D,E,and F.Bovine respiratory diseases are mainly caused by capsular type A Pm,while bovine hemorrhagic sepsis is mainly caused by type B Pm.At present,the treatment of bovine-derived pasteurosis mainly depends on antimicrobial drugs,but with the long-term use of antimicrobial drugs,resistance problems inevitably arise.To this end,we design a new drug combination and give a treatment plan for bovine respiratory diseases caused by Pasteurella multocida by a combined treatment method.Among many antimicrobial drugs,the bactericidal effect of ciprofloxacin is mainly to inhibit the expression of type IIA topoisomerase?DNA helicase and topoisomerase IV?,which inhibits the growth of bacteria and DNA synthesis,thereby killing bacterial.The antibacterial mechanism of tetracycline is to specifically bind to the 30S subunit of the bacterial ribosome at the A position,preventing the connection of AA-tRNA at this position,thereby preventing peptide chain extension and bacterial protein synthesis.It is reported that tetracycline is also an autolysing inducer,bacterial autolysis is the self-destructive behavior of bacterial cells.The lysosome releases the enzyme to degrade its own cells.The autolytic ability of tetracycline to Pasteurella bovine is worth exploring;thiolidazine is a clinically used drug to treat anxiety disorders.Studies have shown that thioridazine can effectively inhibit the external pump of bacteria and promote the entry of antimicrobial drugs into the cells of the bacteria.This study examined the design of a new drug combination with ciprofloxacin as the main drug and tetracycline and thioridazine as adjuvants in vitro and in vivo,and the evaluation of its safety in vivo and in vitro,as well as its resistance to bovine-derived homicide The pharmacodynamics and pharmacokinetics of the bacillus is intended to provide a basis for its further clinical applications.First,we determined the composition ratio and in vitro pharmacodynamics of the drug combination,and examined the safety of the drug combination in vitro and in vivo.In order to determine the composition ratio of the drug combination and conduct pharmacodynamic studies in vitro,we used the minimal broth dilution method to test the sensitivity of 10 clinically isolated bovine capsular type A Pm with single,double and triple drug combinations.Determination of bacteriostatic concentration?MIC?and graded bacteriostatic concentration index?FICIs?.The results showed that the single-agent MICs of ciprofloxacin,thioridazine,and tetracycline on the detected bovine capsule A type Pm strains were 8³2?g/mL,256⁵12?g/mL,and 128⁵12?g/mL,respectively.When there is significant synergistic effect against bovine pasteurellosis,FICIs of ciprofloxacin,thioridazine,and tetracycline are 0.012⁰.500,and the concentrations of ciprofloxacin,thioridazine,and tetracycline in the composition 0.25²?g/mL,0.5?g/mL,0.5?g/mL,which is 4¹28 times higher than the antibacterial activity of the drug alone.We choose ciprofloxacin,thioridazine and tetracycline 1:1:1.The optimal proportion is used as a combination drug group for all subsequent research work.Next,in order to evaluate the safety of the drug combination in vitro and in vivo,we also performed in vitro cytotoxicity and in vivo acute toxicity tests.The MTT method was used to evaluate the cytotoxicity of the drug combination.The drug combination used high?27?g/mL?,medium?18?g/mL?,and low?9?g/mL?concentrations for 48 hours.The relative growth rate?RGR?of cells?Vero?were all greater than 85%,indicating that the drug combination was less toxic to normal cell lines.The results of the acute toxicity test showed that the single dose of the drug combination was 9 mg/kg,18 mg/kg,and 27 mg/kg,which could not cause the death of the mice.The half-lethal dose(LD₅₀)was not measured.The maximum concentration?27 mg/kg?and the maximum volume?0.4 mL?were administered to mice at one time.During the whole test,no mouse death was found.In summary,it shows that the drug combination has good drug safety.The above results indicate that the drug combination consisting of ciprofloxacin,thioridazine,and tetracycline in a ratio of 1:1:1 is worthy of further in vivo pharmacodynamic and pharmacokinetic research.Second,in order to further study the in vivo pharmacodynamics of bovine capsular type A Pm in combination with drugs,we established a mouse pneumonia model infected with bovine capsular type A Pm.We have established a mouse pneumonia model to study bovine capsular type A Pm respiratory disease caused by mice because the mice are highly susceptible to bovine capsular type A Pm.The mice died acutely after vaccination with bovine capsular type A Pm.As a feature,it can be evaluated with"health/death"as an index,and it is an ideal experimental animal challenge model.We examined the effects of the drug combination on the clinical symptoms,tissue load,histopathology,and immune factors of bovine capsule A-type Pm-infected mice.The results showed that the optimal concentration of the drug combination?9 mg/kg of Ciprofloxacin,Tetracycline and Thioridazine?was continuously administered orally for pneumonia model mice infected with bovine capsule A type Pm infection.After three days of treatment,the ratio of wet to dry weight of the lung was significantly reduced,and pulmonary edema was effectively relieved,indicating that the drug combination can significantly reduce the clinical symptoms of P.multocida infection;the drug combination can significantly reduce the mortality of mice;compared with the blank group,the drug combination can significantly reduce the lung load of mice,indicating that the drug combination Significant bactericidal effect in vivo;pathological histological results show that the drug combination significantly reduces lung damage caused by Pasteurella bovine and effectively relieves local inflammation;inflammatory factors TNF-?,IL-6,IL-1?and IL-18 content test test proves that the drug combination can effectively alleviate the pulmonary inflammation caused by Pasteurella bovine.The above results indicate that the drug combination has significant in vivo pharmacodynamic effects against bovine capsule A-type Pm.Finally,in order to examine the pharmacokinetics of each component of the drug combination in plasma,we used high performance liquid chromatography?HPLC?to detect the concentrations of each drug in mouse plasma at different time points.The results showed that each drug was rapidly metabolized in the body,reaching a peak within 15 to 45 minutes,and each drug stayed in the body for about 8 hours to reach a minimum value,which was not detected in plasma after 24 hours.The maximum concentration of ciprofloxacin was 1.00?g/mL,and the maximum time required for thioridazine to reach the peak was 45 minutes.In summary,this study systematically analyzed the drug combination consisting of ciprofloxacin,thioridazine,and tetracycline in terms of drug safety evaluation,in vitro and in vivo pharmacodynamics,and pharmacokinetics.The results show that the drug combination It is a new type of anti-Pasteurella drug combination with strong bactericidal ability,good healing effect and complete metabolism.This antibacterial drug composition has good clinical development prospects.