The Effects Of Ethylenediaminotetraacetic Acid On Mucoid Pseudomonas Aeruginosa Biofilm

PartⅠ: Effects of ethylenediaminotetraacetic acid combining with three kinds of antibacterial agents on mucoid Pseudomonas aeruginosa biofilm in vitroObjective To investigate the effects of metal-chelator ethylenediamine tetraacetic acid (EDTA) combination with three kinds of antibacterial agents on mucoid Pseudomonas aeruginosa (PA) biofilm(BF).Methods Plate culture method was used to establish mature bioflim. Mini broth dilution method to measure the minimal inhibitory concentration (MIC) of antibacterial agents and EDTA. The biofilms were treated by EDTA, ciprofloxacin, gentamicin, ampicillin alone or symphysisly for 24 hours, the number of the bacteria colony in biofilm was measured by agar plate method. The EPS in biofilm was stained with FITC-ConA, observed by fluorescence microscope;stained with SYTO9/PI, monitored by Confocal Laser Scanning Microscope (CLSM), quantification of the effect was calculated by Image Structor Analyzer(ISA) software . Results 0.5mg/ml EDTA can reduce the MIC of antibacterial agents against planktonic PA, the MIC of ciprofloxacin and ampicillin were reduced by 30 times. 5MIC EDTA can exerst the best effect against PA biofilm , the number of colonies in biofilm was reduced from 107CFU/ml to 104CFU/ml (p<0.05). 0.1MIC and 5MIC EDTA can enhance the bactericidal activity of ciprofloxacin. EDTA of 5MIC combined with three antibacterial agents make the number of colonies in biofilm reduce from 107 CFU/ml to 103 CFU/ml (P<0.05). Fluorescence microscope and CLSM show: EDTA can reduce the content of extracellular polymeric substances (EPS) and the thickness of the biofilm, raise the ratio of dead bacteria. The result of ISA show that after the administration of EDTA, BF thickness, Average Diffusion Distance (ADD), and Textual Entropy (TE) decreased (P<0.05), while areal porosity (AP) increased(P<0.05).Conclusions EDTA could enhance the activity of antibacterial agents against planktonic PA, and destroy the structure of BF. The combination of EDTA with antibacterial agents has significant activity to remove mucoid PA biofilm.PartⅡ: Effects of Magnesium ions on the mucoid pseudomonas aeruginosa biofilmObjective To investigate the influence of different Mg2+ concentration on biofilm formation by mucoid Pseudomonas aeruginosa.Methods Multifunction fluorometer was used to measure fluorescence of PA at the bottom of 96-well plate. The EPS of PA was stained with FITC-ConA and detected by fluorescence microscope. The biofilm was stained with SYTO9/PI, monitored by Confocal Laser Scanning Microscope(CLSM),quantification of the effect was calculated by Image Structor Analyzer(ISA) sofeware.Results Two days group: after intervention with 1mM mg2+, the fluorescence intensity was increased from 1845.67±45.39 to 2254.78±42.45 ,t=?9.96 ,P<0.05 ; 0.1mM mg2+ could also increase the fluorescence intensity; Other time groups have the approximate tendency as two days group; the EPS in biofilm was significantly increased after intervention with mg2+, which was visualized by fluorescence microscope, based on the FITC-ConA stained . The live bacteria in the biofilm increase, the colony close-up after administration of Mg2+, when observed by CLSM, based on the SYTO9/PI stained. The quantitative data from ISA software showed: after administration of 1 mM Mg2+, for 6 day biofilm ,the depth increase from (25.80±1.16)um to (34.87±1.59)um , t=?13.85 ,P<0.05 , Areal porosity (AP) decrease from 0.96±0.05 to 0.90±0.04,t=2.48, P<0.05 , Average Diffusion Distance (ADD) increase from 1.54±0.15 to 1.92±0.16,t=5.23 ,P<0.05 , Textual Entropy (TE) increase from 3.64±0.57 to 4.70±1.09,t=?2.6 ,P<0.05. The 3 day biofilm have the same tendency. Conclusions Magnesium can increase the initial attachment of PA and alter the subsequent biofilm formation and structure.PartⅢ: Effects of ethylenediaminotetraacetic acid combining with ciprofloxacin on Pseudomonas aeruginosa biofilm in vivoObjective to investigate the effect of EDTA combined with ciprofloxacin on Pseudomonas aeruginose biofilm in vivo.Methods Pseudomonas aeruginosa was inhaled into the lung of guinea pigs and colonized , formed biofilm. After 7 days, the model was treated with ciprofloxacin, EDTA alone , or a combination of them for 7 days. The number of colony in the lungs is measured by agar plate. The pathological change of the lung is observed by hematoxylin and eosin(HE) staining and scanning electron microscope.Results EDTA combined with ciprofloxacin make the number of bacteria colony in the lung reduced from 105CFU/g to 10CFU/g, (t=24.67, P<0.05) , the lung lesion was less-sever histophathologically.Conclusions The combination of EDTA with ciprofloxacin has significant activity to remove mucoid PA biofilm in vivo.