In Vitro And In Vivo Antibacterial Effects Of Doxycycline-based Combination Drugs On CRKP

Objective:To study the in vitro antibacterial activity and biofilm formation of doxycycline combined with multiple antibiotics against carbapenem-resistant Klebsiella pneumoniae(CRKP).To study the in vivo therapeutic effect of doxycycline combined with rifampicin on mice infected with Klebsiella pneumoniae.Methods: The clinical isolates of non-repetitive CRKP were collected from Yichong People’s Hospital.1.Broth dilution method was used to determine the minimum inhibitory concentrations(MIC)of multiple antibiotics(doxycycline,rifampicin,fosfomycin,ceftazidime,levofloxacin)against the strains.2.Microdilution checkerboard method was used to determine the minimal inhibitory concentration(MIC)of pairs of antibiotics against the strains.According to the change of MIC of single drug and combination group,the partial inhibitory concentration index(FIC)was calculated to determine the synergistic effect of antibiotic combination.3.Plate colony counting method was used to draw the in vitro timebactericidal curve,and the antibacterial and bactericidal effects of the combination of antibiotics were judged according to the data results combined with the judgment criteria.4.A mouse model of pulmonary Klebsiella pneumoniae infection was established by nasal instillation,and the drug groups were treated by gavage.The changes in lung bacterial concentration in different groups of mice were observed.The serum levels of C-reactive protein(CRP)and interleukin-6(IL-6)were detected by enzyme-linked immunosorbent assay.The inflammatory cells in lung tissue were counted by Image J,and the pathological changes of lung tissue were observed by HE staining.5.Crystal violet staining was used to measure the biofilm formation ability of each strain,and the inhibitory effect of the combination of antibiotics on biofilm formation was observed.The elimination effect of the combination of the two drugs on preformed biofilm was determined by the same method.Results:In vitro experiments showed that all strains were resistant to antibiotics except strain 7,which was sensitive to doxycycline.Checker board test showed that doxycycline combined with rifampicin showed additive or synergistic effects on all strains,and the MIC of doxycycline combined with rifampicin was significantly lower than that of single drug.Doxycycline combined with ceftazidime showed synergistic or additive effects on 6 strains,and the MIC of doxycycline combined with ceftazidime was significantly lower than that of single drug.Doxycycline in combination with fosfomycin or doxycycline in combination with levofloxacin showed synergistic or additive effects on 6 strains.The MIC of doxycycline in combination with fosfomycin or doxycycline in combination with levofloxacin was lower than that of single drug,but the drug concentration was not optimal.The time-killing curve showed that the combination of doxycycline and rifampicin had a synergistic bactericidal or antibacterial effect on the tested bacteria.Doxycycline combined with levofloxacin showed a synergistic inhibitory effect on strain 7.Results of animal experiments: mice in the control group were active and had a normal diet.Compared with the model group and the single drug group,the number of bacteria in the lung tissue,Creactive protein(CRP),IL-6 index and inflammatory cells in the lung tissue of the doxycycline combined with rifampicin group were significantly decreased,and the differences were statistically significant(P<0.05).Compared with the model group,the pathological sections of lung tissue in the doxycycline combined with rifampicin group showed clear alveolar structure,no granulocyte infiltration,a small amount of congestion,and no obvious symptoms of infection.Crystal violet staining showed that doxycycline combined with rifampicin and levofloxacin could inhibit the growth and destroy the bacterial biofilm of the tested strains.Conclusion: In vitro experiments have shown that the combination of doxycycline and rifampicin has synergistic or additive effects against carbapenem-resistant Klebsiella pneumoniae(CRKP),with antibacterial or bactericidal effects.The earlier the drug was used,the more effective the inhibition or destruction of the biofilm was.Animal experiments showed that doxycycline combined with rifampicin had obvious antibacterial effect on mice infected with bacteria in vivo.