The Relationship Between Resistance Of Ab And Formation Of BF And The Effect Of Antibiotics On BF

Objective:To investigate the relationship between the drug resistance of Acinetobacter baumanii(Ab)and the forming ability of bacterial biofilm(BF),and the influence of antibiotics on the BF of multiple drug resistant Acinetobacter baumannii(MDR-Ab).Methods:The strains of Ab were isolated from intensive care units(ICU)of Tianjin First Central Hospital(TFCH)in this study,The experimental design includes two parts:Part ?: admeasurement of Ab-BF forming ability.This part was divided into two groups based on the resistance of bacterial: one is MDR-Ab(n=20),the other is sensitive drug Acinetobacter baumanni(S-Ab)(n=20).BF was established in 96-well and 24-well plates by modified plate method.After 8h,12 h,24h,48 h,72h of incubation,the optical dencity(OD)was separately tested by crystal violet staining(CVS)at 570 nm.In addition,the space structure of plankton bacteria and BF were observed by scanning electron microscope(SEM).Part ?: the effect of antibiotics on BF of MDR-Ab.The strains with biofilm formation capacities were selected from the experiment of Part I.Firstly,the minimal inhibitory concentration(MIC)and minimal biofilm inhibitory concentration(MBIC)of CL,TGC,IPM were measured separately by broth microdilution method.Then the relationship of MIC and MBIC was analysed.Secondly,the BF were intervened by antibiotics at the concentration of MBIC.This test was divided into four groups:group A was the negative control,neither BF nor antibiotic;group B was the positive control,it was given BF but no antibiotic;group C was given BF and a single antibiotic,including Azithromycin(AZM),CL,TGC and IPM;group D was given combined antibiotics based on AZM,that is,AZM+CL,AZM+TGC and AZM+IPM.The OD values of the four group were measured by CVS,besides,the inhibition rate of antibiotic and combined antibiotics on BF was calculated.Thereby,screening the antibiotics with stronger biofilm inhibition ability and guiding the clinical medication.Results:Results:Part ?: the OD values of MDR-Ab and S-Ab were increased with time at 570 nm.In MDR-Ab group,the OD value began to increased at 8h,and the difference was significantly(P<0.05)at 12 h.In S-Ab group,the OD value began to increased at 24 h,and the difference was statistically significant(P<0.05)at 48 h.In addition,the OD values of MDR-Ab were at a higher level at each time point compared with S-Ab,and the difference was significantly(P<0.05)at 12 h.Besides,at 72 h,the positive rates of MDR-Ab and S-Ab BF were 100% and 80% respectively,the difference was statistically significant(P<0.05).The results of SEM show there was no significant difference of the plankton structure between MDR-Ab and S-Ab.As for BF,we can find a large number of filamentous substances around it,they are combine with each other and then becoming a massive and spatial structure.Comparisons between the MDR-Ab and S-Ab find that the BF of MDR-Ab is more compact,producing more filamentous substances,and the degree of bacterial deformation is greater.Part ?: The results of broth dilution method show that MBIC of MDR-Ab was significantly higher than MIC for each antibiotic.Among which the MBIC of CL ranged from 8 to 64?g/ml,and the MIC ranged from 0.5 to 8?g/ml,the MBIC is increased by 8-16 times compared with MIC.The MBIC and MIC of TGC were4-32?g/ml and 0.5-4 ?g/ml respectively,increased by 8 times.The MBIC and MIC of IPM were 512-1024 ?g/ml and 32-256?g/ml respectively,increased by 4-16 times.Intervening the BF of MDR-Ab with a single antibiotic at concentration of MBIC,the results showed that TGC has the highest inhibition rate(34.9%),the second is CL(20.1%).AZM and IPM had no inhibitory effect on BF.Combined with AZM alone,AZM+CL has the highest inhibition rate(65.9%),the second is AZM+TGC(52.7%),and the third is AZM+IPM(39.6%),the differences were statistically significant(P<0.05).Conclusion:1.This experiment proved that the MDR-Ab has a stronger forming ability,forms at an earlier time,and it's structure is more compact and complex.Meanwhile,the resistance to antibiotics of BF is increases than planktonic state.It indicated that there is a positive relationship between the drug resistance of Ab and the forming ability of BF.2.This test showed that the use of TGC and CL alone have varying degreesinhibition ability on BF,Among which TGC has the strongest effect.Further more,the inhibition rates of AZM-based combined use of CL,TGC and IPM are better than single use of corresponding antibiotic,among which AZM combined with CL has the strongest inhibitory effect.