Inhibitory Ability Of Ambroxol Combined With Levofloxacin On Klebsiella Pneumoniae Biofilm Formation

Objective:1.To study the ability of Klebsiella pneumoniae(KP)biofilms with different resistance mechanisms to provide evidence for clinically antagonizing Klebsiella pneumoniae biofilms.2.To study the inhibitory and clearance effects of ambroxol combined with levofloxacin on the bacterial biofilm of KP with different resistance mechanisms,aiming to provide more clinical strategies to antagonize and treatment of biofilms.Methods:1.The collected KP with different resistance mechanisms identified by the Vitek2 automatic bacterial identification instrument in Yijishan Hospital of Wannan Medical College were divided into sensitive group and extended-spectrum beta-lactamase producing group(ESBLs)and Carbapenem-resistant Klebsiella pneumoniae(CRKP)group.15 strains in each group,and semi-quantitative determination of its biofilm by crystal violet staining method.2.Three strains with similar biofilm yields were selected,and the minimum inhibitory concentration(MIC)of the two drugs against KP was determined by the micro broth dilution method.Then use the micrdilution checkerboard techniques to determine the effect of different concentrations of ambroxol on the MIC of levofloxacin and Calculate the Fractional inhibitory concentration index(FIC)to select the best synergistic concentration.3.The effects of ambroxol and levofloxacin on KP biofilm were observed by crystal violet method combined with Confocal laser scanning microscopy.Results:1.The three groups of bacterial biofilms all reached maturity on the 5th day,and the sensitive group was easier to form and produced more than the ESBLs and CRKP groups(F=3.725,P=0.032).2.The checkerboard dilution method showed that with the increase of the concentration of ambroxol,the geometric mean of the MIC values of the three groups of bacteria levofloxacin decreased significantly.And the Pearson Correlation Coefficient were-0.531(sensitive),-0.876(ESBLs),-0.907(CRKP).3.The MIC value when the two drugs were combined was smaller than the MIC value measured by the single drug(P<0.05).And the calculation results of the FIC index of each bacteria were all ≤0.5,and the combination of the two drugs showed a synergistic effect.4.In the biofilm test,as the concentration of ambroxol increased,the yield of biofilm on the fifth day gradually decreased until the concentration of ambroxol reached 1.875mg/ml,there was basically no biofilm formation.Its inhibition rate reached more than75%(When ambroxol was 0.95mg/ml,the inhibition rate of each group was 75.32% for sensitive,91.09% for ESBLs,and 90.42% for CRKP).In the synergistic concentration,the inhibition rate reached more than 85%(At synergistic concentrations,the inhibition rate of each group was 86.18% for sensitive,85.23% for ESBLs,and 91.56% for CRKP).5.After the biofilm reached maturity,it was found that the biofilm clearance rate gradually increased with the increase of the concentration of ambroxol drug.But its removal was not complete enough,even when the ambroxol concentration reached 3.75mg/ml,its removal rate still did not reach 70%(When ambroxol was 3.75mg/ml,the biofilm clearance rates were 69.95% in the sensitive,62.81% in the ESBLs,and 66.77% in the CRKP).The synergistic concentration group also did not see obvious clearing effect(Clearance rates of each group were sensitive group: 41.65%,ESBLs group:46.14%,and CRKP group: 39.77%).It was no significant difference with 0.49,0.95mg/ml ambroxol group,and MIC levofloxacin group(P>0.05).Conclusion:1.Compared with ESBLs group and CRKP group,the sensitive group is easier to form biofilm and produces more.2.Our study support that ambroxol combined with levofloxacin can inhibit the bacterial biofilm of KP early.3.Its optimal synergistic concentration is 0.49mg/ml(ambroxol)+ 4μg/ml(levofloxacin).