Antibacterial Activity Of Fosfomycin Combined With Polymyxin B Against Multidrug-Resistant Pseudomonas Aeruginosa In Vitro And In Vivo Study

Objective:We evaluated the effects of FOM alone and combined with PB on the antibacterial activity of clinically isolated MDR-PA in vitro.The antibacterial effect of FOM alone and combined with PB on MDR-PA was confirmed by in vivo antibacterial experiments in a mouse acute pneumonia model.This can provide the basic laboratory evidence for the rational application of antibacterial drugs by clinicians.Methods:(1)We determined the MIC of FOM,PB,DOR,CRO and LEW against 117 of MDR-PA isolates in vitro.Among them,the measurement of FOM is applied by the agar dilution,and other antibacterial drugs were used by broth microdilution method;the drug-resistance genes for PB in the clinical isolates of 5 of PA was amplified by PCR.For checkerboard assay,the synergistic bactericidal activity of FOM/PB combination therapy was determined,further evaluate whether the two drugs have the synergy,and the synergistic bactericidal activity of FOM combined with PB was observed by time-kill curves.(2)Six strains of MDR-PA with synergistic effect were selected for the combination of drugs and the bacterial biofilms model was constructed to assess the biofilm formation ability of 15 strains,and the antibiofilm efficacy of FOM alone and in combination with PB was detected by the semi-quantitative crystal violet staining.(3)An MDR-PA pneumonia model of granulocyte-deficient mice was established,evaluating the in vivo activity of both monotherapy and combination was determined by observing the histopathological sections and bacterial colony counts of the mice.Results:(1)Most of the 117 MDR-PA strains tested were resistant to DOR,CRO and LEW,and showed multi-drug resistance.Among them,the resistance rate to the third-generation cephalosporin CRO was the highest,followed by LEW.The DOR resistance rate of carbapenem antibacterials was 74.36%,and the sensitivity rate of FOM was 88.03%,PB showed the highest antibacterial activity,the sensitivity rate was 94.02%,only 5 strains showed resistance to PB from both phenotype and genotype.After the combination of FOM and PB in 28 strains of MDR-PA,most of them showed synergistic effects and there was no antagonistic effect was found.Then the time-kill assay showed that FOM/PB showed synergistic and bactericidal activity,and the bactericidal activity at 0.5×MIC and 1×MIC drug concentrations was significantly better than that of the monotherapy group,the regeneration of PB was inhibited after the combination with FOM.(2)All of 6 strains tested can form biofilms,when treated with FOM and PB monotherapy for 24 h in MDR-PA biofilm carrier,the OD value was lower than that of the control group was a significant difference(P<0.05),and the two-drug combination treatment group compared with each single drug group indicated that the significantly different(P<0.01).(3)Finally,the acute lung injury model was successfully established in mice,when compared with the single drug group,the FOM+PB group had a clear structure of alveolar wall and bronchus,and no obvious histopathological changes were observed.Conclusion:This study had confirmed that FOM and PB combinations have the synergistic antibiotic effect on MDR-PA in vitro and in vivo results,and synergistically to inhibit the pathogenic biofilms produced by MDR-PA.These studies would be offering a new strategy to treat MDR infections.