The Effect Of Antibacterial And Anti-biofilm Activity Of IDR-1018 Combined With Meropenem Or Sulbactam

Objective:(1)To observe the growth curve and biofilm formation curve of Acinetobacter baumannii(AB)standard strain ATCC BAA-1605 and establish AB biofilm model.(2)The minimum inhibitory concentration(MIC)of the antibacterials meropenem,cefoperazone sodium,cefoperazone/sulbactam sodium,sulbactam and the antibacterial peptide IDR-1018 against ATCC BAA-1605 were tested.(3)To clarify the antibacterial effect of IDR-1018 with meropenem or sulbactam.(4)To investigate the inhibitory effect of IDR-1018 and meropenem and sulbactam on the biofilm formation of AB and the destructive effect on the formed biofilm.Methods:(1)AB growth curve and biofilm formation curve,the biofilm model was established: AB was cultured in 96-well plate,200μl was taken every 2h,the absorbance value was measured at 600 nm to get AB growth curve;AB was cultured in a polyvinyl chloride(PVC)96-well plate,one group(3 wells in each group)was taken out every 24 h and the biofilm was observed by the crystal violet staining method.AB biofilm model was set up and the curve of biofilm formation was drawn.(2)The minimum inhibitory concentration(MIC)of meropenem,cefoperazone sodium,cefoperazone/sulbactam sodium,sulbactam and antibacterial peptide IDR-1018 on ATCC BAA-1605 were detected by microbroth dilution method.(3)The micrdilution checkerboard techniques was used to determine the antibacterial effects of IDR-1018 with meropenem or with sulbatan.(4)PVC material was use as a carrier,and LB was use as a liquid medium.The bacteria were inoculated on the carrier,and then incubated in 37℃ without shaking.Meropenem,sulbactam and IDR-1018 were individually or jointly used to treat the bacteria from the time point of 0h and 24 h respectively for 24 h.The morphology of biofilm and the number of viable/dead bacteria in biofilm were observed by crystal violet staining and Confocal laser scanning microscope.Results:(1)AB was inoculated into LB liquid medium,0-4h was the growth retardation phase,4 hours later entering the logarithmic growth phase,and the bacteria entered the stable growth phase after 14 hours;AB is cultured in PVC 96-well plate to24 h,low-density purple film formation could be seen,forming dense and stable biofilm in about 4 days.(2)MIC: Meropenem64μg/ml,Cefoperazone sodium>256μg/ml,Cefoperazone/sulbactam sodium>256μg/ml,sulbactam32μg/ml and IDR-1018 32μg/ml.(3)Fractional inhibitory concentration index(FICI): IDR-1018 combined with meropenem was 1.25,the combination of the two for AB drug susceptibility results are irrelevant;IDR-1018 combined with sulbactam was 1.5,the combination of the two for AB drug susceptibility results are also irrelevant.(4)Meropenem,sulbactam and IDR-1018 were added into the medium when bacteria were inoculated,and 24 hours later,we can observed that the phytoplankton were inhibited,no obvious inhibitory effect on the biofilm,while the concentration of meropenem was 64μg/ml,and sulbactam was 32 μg/ml;32μg/ml of IDR-1018 could inhibit phytoplankton,while inhibiting 47.8%of the biofilm formation.The formation of biofilm could be completely inhibited after the combination of IDR-1018(16μg/ml)with meropenem(16μg/ml)or with sulbactam(8μg/ml).(5)Meropenem,sulbactam and IDR-1018 were added into the medium when bacteria were inoculated for 24 h,and 24 hours later,no obvious biofilm inhibition was observed when the three antibacterial agents were used alone.After the combination,the inhibitory effect on the biofilm of AB gradually decreased with the decrease of IDR-1018 concentration.With confocal laser scanning microscopy,compared with the control group,the ratio of viable/dead bacteria in the biofilm in meropenem(64μg/ml)alone group and sulbactam(32μg/ml)alone group,did not change significantly.When meropenem or sulbactam combined with IDR-1018,in both IDR-1018(64μg/ml or 32μg/ml)plus meropenem(64μg/ml)group and IDR-1018(64μg/ml or 32μg/ml)plus sulbactam(32μg/ml)group,the bacteria in the biofilm were mainly dead bacteria,and the number of bacteria decreased significantly compared with the control group.Conclusion:(1)Using PVC as carrier,biofilm models were successfully builded.(2)In vitro experiments IDR-1018 combined with meropenem or sulbactam,the combined drug susceptibility results for AB were both irrelevant.(3)Biofilm is an important reason for resistance of bacteria.When the concentrations of meropenem and sulbactam were of MIC value,they could only inhibit the growth of phytoplankton,while the effect of biofilm was poor.(4)IDR-1018 combined with meropenem or sulbactam could effectively inhibit biofilm formation.(5)In the early stage of biofilm(24h),IDR-1018 combined with meropenem or sulbactam could inhibit the further maturation of biofilms.