In Vitro And In Vivo Assessment Of The Antibacterial Activity Of Colistin Alone And In Combination With Other Antibiotics Against Acinetobacter Baumannii And Escherichia Coli

Objetive:According to the resistance of gram-negative bacteria and limited therapeutic options,to investigate the resistance and ability of biofilm formation of clinically isolated Acinetobacter baumannii,to systematically evaluate the activity of colistin as monotherapy and in combination with other.antibiotics against Acinetobacter baumannii in vitro,and to preliminarily establish a murine model of ascending urinary tract infection(UTI)to assess the antibacterial activity of intravenous colistimethate sodium(CMS)in vivo.Methods:(1)The MICs of colistin,sulbactam,meropenem,imipenem and doripenem were determined using the broth microdilution method.Monte Carlo simulation was used to calculate the probability of target attainment(PTA)of CMS at a specific dose against a specific MIC value by crystal ball software.(2)The presence of the carbapenemase gene blaOXA-23 was tested by PCR.Chequerboard assay and time-kill assay were used to assess the synergistic effects of seven combinations of five antimicrobials(colistin,sulbactam,meropenem,imipenem and doripenem).Ability of biofilm formation of A.baumannii and the inhibitory activity of drug combinations on biofilms were evaluated by the semi-quantitative crystal violet staining.The erythrocyte lysis assay was used to determine the cytotoxicity of the optimal antimicrobial combination.(3)The susceptibility of colistin against urinary tract pathogenic Escherichia coli(UPEC)was determined using the broth microdilution method.The bladders were injected with UPEC suspension to induce an ascending UTI model.The bacterial counts and histopathological examination results were conducted to evaluate the efficacy of intravenous CMS against UTI.Results:(1)Of 106 A.baumannii isolates,104(98.1%)were susceptible to colistin.The rate of resistance of A.baumannii to sulbactam,meropenem,imipenem and donipenem reached 81.2%,79.3%,77.4%and 79.3%,respectively.The results of Monte Carlo simulation showed that when the MIC of colistin was<0.25 ?g/ml,even the minimum dose(90 mg CBA/day)can achieve the ideal PTA value;when the MIC of colistin was?1 ?g/ml,even the maximum dose(300 mg CBA/day)cannot achieve satisfactory effect.(2)The carbapenemase resistance gene blaoxA-23 was present in all 25 A.baumannii isolates tested.The combination of colistin and donipenem in the chequerboard assay showed the highest rate of synergism(60%).No antagonism and cytotoxicity were observed.In the time-kill assay,colistin monotherapy showed rapid bacterial killing against susceptible strain followed by bacterial regrowth.All colistin-based combinations were able to inhibit or slow bacterial re-growth.In the biofilm inhibition assay.84%of Acinetobacter baumannii isolates were able to produce biofilm and the antibiotic combinations based on colistin showed the greatest inhibitory effect on the biofilm.(3)In the in vivo activity study,intravenous CMS reduced not only the bacterial load of urine,bladders and kidneys but also degree of inflammation and maintained structural integrity of infected bladders and kidneys.Conclusion:The high rate of susceptibility in vitro and significant antibacterial activity in vivo suggested that intravenous CMS will be an effective and available therapeutic strategy for UTI due to multidrug-resistant gram-negative bacteria.In-depth in vitro data supports the combination of colistin and donipenem for the treatment of serious infections induced by multidrug-resistant gram-negative bacteria,especially when the MIC of colistin is?1 ?g/ml.