| BackgroundAt present,the infection of Acinetobacter baumannii（AB）in hospital is not optimistic,especially the multi-drug resistance Acinetobacter baumannii（MDR-AB）,which has become one of the important factors threatening the life of patients in the ICU.In the study of multiple drug resistance mechanisms of AB,more attention has been paid to the role of it’s biofilms.Nowadays,the clinical treatment of MDR-AB,especially carbapenem-resistant Acinetobacter baumannii（CR-AB）,is relatively single.Polymyxin B has become the first-line treatment for CR-Ab.In the study,we investigated the inhibition of polymyxin B combined with tigacycline or azithromycin on ATCC BAA-1605 and the inhibitory activity on its biofilm,aiming to provide a new combination drug regimen for clinical treatment of CR-AB.Objective1.To observe the growth curve,measure the biofilm formation curve of ATCC BAA-1605 and establish it’s biofilm model.2.To measure the MIC of Polymyxin B,Tecycline and Azithromycin against standard strain ATCC BAA-1605.3.To investigate the antibacterial effect of polymyxin B combined with tigecycline or azithromycin on ATCC BAA-1605.4.To investigate the inhibitory effect of polymyxin B,tigecycline and azithromycin on biofilm formation of ATCC BAA-1605 and the destruction of it’s mature biofilm.Methods1.To observe the growth curve and the biofilm formation curve of ATCC BAA-1605and to establish it’s biofilm model:ATCC BAA-1605 was resuscitated and cultured in96-well plate.200μl bacterial liquid was taken out every 2 hours,measured the absorbance at 600nm and then it’s growth curve was obtained.ATCC BAA-1605 was cultured in the polyvinyl chloride（PVC）plate and each group of 3 holes was taken out after 24h.Then observed the biofilm by the crystal violet staining method,measured absorbance value at 595nm.The biofilm growth curve was measured,and the ATCC BAA-1605 biofilm model was established.2.The MIC of polymyxin B,tigecycline and azithromycin against ATCC BAA-1605was determined by microbroth dilution method.And the effect of polymyxin B combined with tigecycline or azithromycin on ATCC BAA-1605 was determined.3.The antibacterial effects of polymyxin B combined with Tigecycline,polymyxin B4.combined with Azithromycin,and Tigecycline combined with Azithromycin were detected by micro checkerboard dilution method.The strain of ATCC BAA-1605 was inoculated on the sterilized 96-well PVC plate and incubated in 37℃constant temperature incubator.Polymyxin B,tigacycline and azithromycin were given separately or in combination for 24 hours at not only 0h but also 24h,and measured by crystal violet staining.The morphology of it’s biofilm and the viable/dead bacteria in biofilm could be surveyed Confocal laser scanning microscope（CLSM）.Results1.ATCC BAA-165,cultured in Luria-Bertani（LB）medium for 0～4h was the slow growth phase of the strain,after 4h,it entered the logarithmic growth phase with a logarithmic growth cycle of 10h,after continuous culture for 14h,the growth of the bacteria tended to be stable,and then the strain entered the stable growth phase;The standard strain of ATCC BAA-1605 was inoculated into sterilized PVC 96-well plates,after 24h,a small amount of purple membrane was observed in the 96-well plate after crystal violet staining,after that,the density of the membrane gradually increased with the extension of culture time,when the culture was about 4d,a high-density biofilm was formed in the 96-well plate.2.MIC of antimicrobial drugs:Polymyxin B 2mg/L,Tigecycline 2mg/L and Azithromycin>256mg/L.3.Fractional inhibitory concentration index（FICI）:Polymyxin B combined with Tigecycline was 1.0625;Polymyxin B combined with Azithromycin was 1.5,these results indicated that the combination of Polymyxin B and Tigecycline or Azithromycin have no effect on ATCC BAA-1605.4.The standard strain of ATCC BAA-1605 was inoculated in PVC 96-well plate,and then treated with different concentrations of polymyxin B,tigacycline and azithromycin for 24h in 0h.The inhibitory rates of polymyxin B,tigecycline and azithromycin against the biological susceptibility of this strain were 75.9%,67.3%and 36.8%at MIC concentration,respectively.When the concentration increased to 2MIC,the inhibition rates were 81.7%,94.9%and 44.4%,respectively.5.The standard strain of ATCC BAA-1605 was inoculated in 96-well PVC plate,after24h incubation,the biofilm of ATCC BAA-1605 was significantly inhibited under crystal purple staining,and the OD595 value was significantly lower than that of the control group（P<0.001）;The biofilm growth of AB was also significantly inhibited by the combination of Tentigecycline（4～2mg/L）and Polymycin B（4～2mg/L）,when the concentration of Azithromycin（128～64mg/L）was fixed,with the concentration of Polymyxin B gradually decreased,the inhibition ability of antibiotics on AB biofilm gradually decreased.6.Observed with CLSM:The ratio of live/dead bacteria in the biofilm was not significantly changed when Azithromycin（256mg/L）was applied alone compared with the control group,however,when polymyxin B（4～2mg/L）or ticacycline（2mg/L）was applied alone,the number of dead bacteria in the biofilm increased significantly.When polymyxin B（4～2mg/L）was combined with tigecycline（2mg/L）or azithromycin（256mg/L）,the number of dead bacteria in the biofilm increased more obviously.Conclusion1.The vitro experiments confirmed that polymyxin B combined with tigecycline or azithromycin had no effect on ATCC BAA-1605.2.At the MIC concentration,polymyxin B and ticacycline not only inhibited the growth of ATCC BAA-1605,but had obvious inhibitory effect on the growth of its biofilm.At the MIC concentration of azithromycin,the inhibition of biofilm was poor.3.At the initial stage of bacterial culture（i.e.0h）,the combination of polymyxin B with tigecycline or azithromycin can effectively inhibit the formation of the biofilm of ATCC BAA-1605.4.At early stage of biofilm formation（i.e.24h）,the combination of polymyxin B with tigecycline or azithromycin could inhibit the further maturation of biofilm and kill the living bacteria in the biofilm. |
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