Effects And Primary Mechanisms Of Allitride Against Biofilms Of Pseudomonas Aeruginosa

PARTⅠEFFECTS OF ALLITRIDE ON ADHESION AND EXOPOLYSACCHARIDE OF PSEUDOMONAS AERUGINOSA BIOFILMSObjective To investigate the effects of allitride on adhesion of Pseudomonas aeruginosa biofilm at early stage and Extracellular Polymeric Substances (EPS).Methods Multifunction fluorometer was used to measure fluorescence of green fluorescent protein (GFP) in pGFPuv transformated PAO1 in 96-well plate. The adhesion ratio of PAO1 was calculated to determine the effects of allitride on PAO1 biofilm adhesion and qualitation adherence of GFP-PAO1 by fluorescence microscope. The fluorescein isothiocyanate (FITC) conjugated concanavalin A (FITC-conA) was used to stain the EPS. In order to qualitation the EPS by fluorescence microscope; the phenol-sulfuric acid method was used to quantitate the EPS in each group. Results Six hours group: after intervention with high dose of allitride, the adhesion ratio was decreased from 0.70±0.03 to 0.50±0.01, t=15.014, P<0.05, low dose of allitride could also affect the adhesion ratio, but not so obvious as high dose. Other time groups except 9 hours have the approximate tendency as 6 hours group, which may be related with unstability of allitride. For normal saline control group, GFP-PAO1 showed colony distribution, while it became rarefaction after intervention, with high dose was more obvious. The EPS in biofilm was significantly decreased after intervention with allitride, which was visualized by fluorescence microscope, based on the FITC-conA stained. Quantification of EPS show that total EPS decreased from (602.66±21.94)μg to (181.19±1.59)μg after high dose of allitride intervention, t=60.589, P<0.05.Conclusions allitride could decrease the adhesion and EPS produce ability of PAO1 significantly. PARTⅡEFFECTS OF ALLITRIDE ON FORMING PROCESS AND QUANTIFICATION OF STRUCTURE OF PSEUDOMONAS AERUGINOSA BIOFILMSObjective To investigate the effect of allitride on biofilms forming process of PAO1 by Confocal Laser Scanning Microscopy (CLSM).To quantify the BF structure with Image Structure Analyer (ISA) software.Methods In vitro models of 1d, 3d, 7d of PAO1 biofilms were established. The effects of normal saline (NS, control groμp), allitride at both concentration of 128μg/ml and 10μg/ml on BF were monitored by CLSM respectively, based on fluorophores from PAO1 with nucleic acid SYTO9/PI stained. Quantification of the effect could be calculated by Image Structure Analyzer (ISA) software.Results (1) NS control group: the thickness was 12.94±0.55μm, 23.98±0.99μm, 28.83±0.67μm at 1d, 3d, 7d respectively, indicating an increasing tendency; the areal porosity (AP) was 0.89±0.01, 0.85±0.02, 0.76±0.128 respectively, showing a decreasing tendency. The Average Diffusion Distance (ADD) was 1.68±0.13, 1.71±0.08, 2.54±0.33 respectively with slightly increase. The Textual Entropy (TE) was 5.62±0.89, 6.15±0.82, 6.78±0.0.93 respectively, giving an increasing tendency. (2) In BF model at 7d after administration of allitride in concentration of 10μg/ml, BF thickness was decreased from (28.83±0.67)μm to (16.50±0.69)μm. AP was increased from (0.76±0.10) to (0.92±0.02). TE was decreased from (6.78±0.93) to (1.76±0.19). All the differences are significant comparing to control group. The effect of allitride at concentration of 10μg/ml has the same tendency as allitride in 128μg/ml group, but not so obviously. (3) After administration of allitride, BF at the 1d and 3d models showed the same tendency as that at the 7d model.Conclusions ISA software can quantify the structure of PA01 biofilms at different stage. Allitride can affect PAO1 biofilm at different stage. The effect of 128μg/ml allitride was more significant than 10μg/ml allitride group. PARTⅢEFFECTS OF ALLITRIDE COMBINED WITH CIPROFLOXACIN ON ANTIBACTERIAL ACTIVITY AND BIOFILM OF PSEUDOMONAS AERUGINOSAObjective To establish a biofilm model of Pseudomonas aeruginosa in vitro and observe effects of allitride combined with ciprofloxacin on antibacterial activity and biofilm of Pseudomonas aeruginosa.Methods The protocol was evaluated by checkerboard synergy tests ,Minimal Inhibitory Concentration (MIC) of allitride combined with ciprofloxacin under kinds of concentration were determined by broth microdilution,the fractional inhibitory concentration(FIC) index was calculated according to MIC results.The combined effects were confirmed by FIC index.The suma of both FICs in each well was used to classify the combination of antimicrobial agents as synergistic effect when FIC indexes were≤0.5; partial synergy FIC >0.5 but < 1; additive FIC = 1.0; indifferent effect when values were >1 and < 4 and antagonistic when values were≥4.0 The assessment of the activity effects of combination of allitride and ciprofloxacin against Pseudomonas aeruginosa biofilms with MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide and fluorescence microscope.Results MICs of allitride and ciprofloxacin significantly decreased after combination, The FIC index was 0.2578125. 1/2, 1/4 allitride could obviously increase effects of 1/2, 1/4 ciprofloxacin on antimicrobial activity against Pseudomonas aeruginosa biofilms.The live bacteria on slide significantly reduced after the combination,but the application of two separate antibiotics was the colony mainly; the same as the saline control group.Conclusions The results suggested that synergistic effect of allicin combined with ciprofloxacin against Pseudomonas aeruginosa biofilms. PARTⅣEFFECTS OF ALLITRIDE ON QUORUM SENSING-CONTROLED VIRULENCE FACTORS PRODUCTION IN PSEUDOMONAS AERUGINOSAObjective Quorum-sensing (QS) system is a key regulator of virulence and biofilm development in Pseudomonas aeruginosa. Allitride was examined in this study for its effects on P. aeruginosa virulence factors and biofilm formation in vitro.Methods Enzeme-linked immunosorbent assay (ELISA) systems for the quantification of exotoxin A, Expression of elastase and pyoverdin were assessed semi-quantitatively using elastin Congo red and fluorescence respectively.Production of rhamnolipid was determined by H2SO4-anthrone analysis method, absorbance at 553nm. We used a confocal laser scanning microscope (CLSM) to study the effects of allitride on mature biofilm in vitro.Results Allitride down regulated virulence factors: exotoxin A, elastase, rhamnolipid and pyoverdin, production of exotoxin A, elastase, rhamnolipid and pyoverdin deceased from (9.630±0.573)pg/μl, (0.467±0.003), and (2.009±0.063)g/L, (9325.833±367.675) to (6.529±0.289)pg/μl, (0.032±0.001), 0.269±0.009)g/L and (7819.167±111.800) respectively For normal sodium control group Pseudomonas aeruginosa formed mature mushroom srtuctures, thickness of BF was 29μm, while ormed flat undifferentiated biofilm, thickness of BF was 18μm, susceptible to treatment with allitride using CLSM.Conclusions Allitride attenuates its virulence significantly and supressed biofilm formation by interventing Quorum-sensing system of P. aeruginosa.