The Intervention Effect Of Fosfomycin On Biofilm Of Acinetobacter Baumannii In Vitro

Part I Establishment and analysis the model of Acinetobacter baumannii biofilmObject To establish the model of acinetobacter baumannii biofilm in vitro, observe and quantitatively analyze the dynamic course of the biofilm formation.Method Biofilm model of biofilm-producing strain acinetobacter baumannii from clinical isolates(2814) was formed on glass plate. After cultivation for 6h,12h,24h,48h,72h,96h, using crystal violet staining method to quantity biofilm. The structure of the biofilm was detected by scanning electron microscope(SEM).Result The model of acinetobacter baumannii biofilm in vitro could established on glass plate. The data of semi-quantitative biofilm at the different time intervals (12h,24h,48h,72h,96h) were 0.145±0.031, 0.503±0.110,0.910±0.043,1.518±0.064,and 0.894±0.011.There werw significan difference between any two group. After cultivation 24h, under SEM there are a large number of bacter adherence, micro-colonies have been formed by bacteria aggregation in partial, a small amount of BF begian to format, when at the 72h, the structure of biofilm become more complex than 24h, and we can see a there-dimensional structure of the mature BF.Conclusions The model of acinetobacter baumannii biofilm in vitro could established on glass coverslip. The formation of Acinetobacter baumannii BF is a dynamic course. The early biofilm was formed at the 24th hour,and the mature bioflim was formed at the 72th hour. The semi-quantitative biofilm and SEM are the ideal methods to observe and quantitatively analyze the biofilm model in vitro.Part II The restraining effect of fosfomycin on biofilm formation of Acinetobacter baumanniiObject To observe the inhibitory effect of fosfomycin on acinetobacter baumannii adhering to the surface of carries, and to restrain biofilm formation.Method Minimal inhibitory concentrations(MIC) were determined by minimal broth dilution method. The in vitro model of acinetobacter baumannii biofilm was established.This experiment divided into control group,FOS group and EM group. FOS(1/2MIC) and EM(1/4MIC) were added at the beginning of making biofilm models.24 hours later, the biofilm on the surface of carriers was examined under microscope and the viable bacterial counts within biofilm were determined. After 3 days, using SEM to observe the form of biofilm, semi-quantity biofilm by crystal violet staining, serial dilution method for detecting viable bacteria counts in the biofilm.Result The MIC of FOS to acinetobacter baumannii is 128μg/ml, EM is 256μg/ml. After 24 hours, the micro-colonies can be seen in the control group under microscope. While both in the FOS group and EM group there are only a small amount of acinetobacter baumannii.The viable bacteria counts showed that the FOS group and EM group are less than control group(P<0.01) and the FOS group in less than EM group(P<0.05). After cultivating 3 days, there are a large number of mature biofilm in the control group, but a few of thin biofilm can be seen both in the FOS group and EM group. The semi-quantitative biofilm by crystal violet staining in FOS group is less than the control group(P<0.01) and EM group(P<0.05). The viable bacteria counts showed that the FOS group is less than control group(P<0.01) and the EM group(P<0.05).Conclusions FOS can inhibit acinetobacter baumannii adhering to the surface of carriers, then restrain it from forming biofilm. The influence of the FOS is stronger than EM.Part III The destruct effect of fosfomycin on biofilm formation of Acinetobacter baumannii and the synergy effect with levofloxacinObject The study is to observe the antibacterial activity of fosfomycin combined with levofloxacin against multiple drug-resistant acinetobacter baumannii in vitro.To observe the destruct effect of fosfomycin on mature biofilm, and its synergy effect with levofloxacin.Method (1)The in vitro antibacterial activities of the drugs were examined by determination of the minimum inhibitory concentrationJoint drug susceptibility testing according to the design board.Records two-drug combination the minimum inhibitory concentration, MIC a drug combination and MIC b drug combination, and then to calculate FIC index. (2)The levofloxacin’s minimal inhibitory concentrations were determined by minimal broth dilution method. (3)The in vitro model of acinetobacter baumannii biofilm was established. This experiment was divided into control group; FOS group; EM group; LFX group; FOS+ LFX group; EM+LFX group. After cultivating 3 days, adding the above-mentioned drug combinations respectively. When the surface of carriers were processed with drug for 24 hours, observing biofilm under SEM. At the drug process 0 hour,4 hour,8 hour,24 hour, the viable bacteria counted by the serial dilution method and the semi-quantitative biofilm by crystal violet stain.Result (1)FICI values are≤0.5 by 57.5%;FICI values are 0.5-1.0 by 40.6%;FICI values are 1.0-2.0 by 1.9%;FICI values are>2 by 0.(2)The MIC of LFX to acinetobacter baumannii is 16μg/ml. (3)The destructive effect of drugs on biofilm under SEM:After the mature biofilm was processed with drugs 24 hours, mucus-like substance both in the FOS group and EM group were less than the control group significantly. And the FOS group is less than EM group.The LFX group compare to control group the changes are small.The FOS+LFX group and EM+LFX group were less than the control group. At all the points, the semi-quantitative biofilm by crystal violet staining in LFX group has small changes compare to control group(P>0.05).At the 24 hour point,the semi-quantitative biofilm by crystal violet staining in FOS group and EM group are less than the control group(P<0.01),also the FOS group is less than EM group.At the 8 hour and the 24 hour points, the FOS+LFX group and the EM+LFX group are less than the control group(P<0.01),also the FOS+LFX group is less than EM+LFX group. The results of the viable bacteria counts:At all the points, there are no significant difference among of control group,FOS group,EM group(P>0.05).At all the points, the FOS+LFX group and the EM+LFX group are less than the control group(P<0.05);and the FOS+LFX group is less than EM+LFX group.Conclusions (1)There is synergistic or additive effect upon the combined uses of fosfomycin and levofloxacin.(2)Fosfomycin can destroy the mature biofilm of acinetobacter baumannii in vitro, and enhance the bactericidal effect of levofloxacin on acinetobacter baumannii within the biofilm.