The Ability Of Multiple Drug Resistant Acinetobacter Baumannii To Form Biofilm And The Synergistic Effect Of Ambroxol On Biofilm

Objective: To discuss the ability of multiple drug resistant acinetobacterbaumannii to form biofilm which detected in the lower respiratory tract andthe differences in gene expression (abaI and blaPER-1) between colonization andinfection status.By adding ambroxol, to observe the effect of ambroxolbiofilm formation.Methods:1Strain sources:45bacterial strains containing32infection status and13colonization status were included in this study. They were isolated fromOctober2011to January2012, from patients admitted to the ICU of fourteaching hospitals in shi jia zhuang. Based on the Clinical PulmonaryInfection Score (CPIS) and results of quantitative culture, all subjects weredivided into two groups: the infection group and the colonization group.2All bacterial strains were cultured in Luria-Bertani (LB) broth at37°Cin24-wells polyvinylchloride microtiter plates to form biofilm, after stainingwith crystal violet (1%w/v)，the optical density at570nm was taken as aquantitative measure of biofilm mass. Isolates were classified as biofilm-forming if the optical density at570nm were at least twice of the negativecontrols.3By adding different concentration of ambroxol（2mg/ml、4mg/ml）toobserve whether it can inhibit the formation of biofilms, and through theelectron microscope to observe the morphology of biofilm before and afteradding ambroxol.4PCR amplification of the two biofilm-related genes (abaI and blaPER-1):Synthesis the upstream and downstream primers on the basis of references.abaI: F5’CCGCCATGAATATTATT-3’ R5’-GCCTTACACATCAATCAAGCATGC-3’blaPER: F5’-ATGAATGTCATTATAAAAGC-3’R:5’-AATTTGGGCTTAGGG CAGAA-3’5Amplificated products were put in1%agarose gel to electrophoresis,then save the results of imaging.Results:1Of all the45isolates studied,34(75.6%) formed biofilm in vitro,11(24.4%) did not form biofilm. In infection group,24strains formed biofilm,therate of biofilm is75%,10strains in colonization group formed biofilm,the rateof biofilm is76.9%. Through Chi-square test，p>0.05，there is no statisticaldifferences beween the two groups.2The means optical density at570nm of the three groups respectivelyare0.52±0.28,0.30±0.16and0.19±0.09,by ANOVA method analyze,p=0.000,there were statistical differences of optical density between the three groups.And by S-N-K method there were statistical differences between any twogroups, indicating that ambroxol can inhibit acinetobacter baumannii to formbiofilm，the more the concentration the more the inhibiting effect.3The results of PCR: In infection group,the positive rate of abaI gene is90.6%, while in colonization group is92.3%, through Chi-square test analyze,p=1.000, there were no statistical differences beween the two groups.Ininfection group,the positive rate of blaPER-1gene is37.5%, while in colon-ization group is46.2%;through Chi-square test statistical method analyze, p=0.591, there were no statistical differences between the two groups.In abaIgene positive and negative group, the positive rate of biofilm is80.5%(33/41)and25%(1/4)，through Chi-square test statistical method analyze, p=0.04，there were statistical differences between the two groups. In blaPER-1genepositive group,77.8%(14/18) isolates formed biofilm, in blaPER-1genenegative group,74.1%(20/27) isolates formed biofilm，through Chi-square teststatistical method analyze, p=1.000，there were no statistical differencesbetween the two groups.The means optical density at570nm of blaPER-1genepositive and negative group is0.60±0.35and0.46±0.21, through t test, p =0.025, there were statistical differences between the two groups。Conclusions:1In our study,75.6%of multiple drug resistant acinetobacter baumanniiisolates detected in the lower respiratory tract formed biofilm, but there is nostatistical differences between infection and colonization group.2Ambroxol can inhibit acinetobacter baumannii to form biofilms, there isa dose-dependent effect, the more concentration the greater of the inhibitioneffect.3abaI and blaPER-1gene are related to the formation of biofilm, whilethere is no statistical difference in the expression of abaI and blaPER-1genebetween infection and colonization group.