The Gram-negative Pseudomonas aeruginosa bacterial pathogen is reputed for its resistance to multiple antibiotics, and this property is strongly associated with the development of biofilms. Bacterial biofilms form by aggregation of microorganisms on a solid surface and secretion of an extracellular polysaccharide substances that acts as a physical protection barrier for the encased bacteria. In addition, the P aeruginosa quorum-sensing system contributes to antibiotic resistance by regulating the expression of several virulence factors, including exotoxin A, elastase, pyoverdin and rhamnolipid. The organosulfur compound allicin, derived from garlic, has been shown to inhibit both surface-adherence of bacteria and production of virulence factors. In this study, the effects of allicin on P aeruginosa biofilm formation and the production of quorum-sensing controlled virulence factors were investigated. The results demonstrated that allicin could inhibit early bacterial adhesion, reduce EPS secretion, and down-regulate virulence factors' production. Collectively, these findings suggest the potential of allicin as a therapeutic agent for controlling P aeruginosa biofilm.
Objective To investigate the influence of different Mg2+ concentration on biofilm formation by mucoid Pseudomonas aeruginosa.Method 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.Result Two days group: after intervention with 1 mmol/L Mg2+,the fluorescence intensity was increased from 1845.67±45.39 to 2254.78±42.45,t=-9.96,P0.05;0.1 mmol/L Mg2+ could also increase the fluorescence intensity;Other time groups showed 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 stain.The live bacteria in the biofilm increase and colony close-up after administration of Mg2+,when observed by CLSM,based on the SYTO9/PI stain.The quantitative data from ISA software showed that after administration of 1 mmol/L Mg2+,the depth of 6 day biofilm increase from(25.80±1.16)μm to(34.87±1.59)μm(t=-13.85,P0.05).Areal porosity decrease from 0.96±0.05 to 0.90±0.04(t= 2.48,P0.05),average diffusion distance increase from 1.54±0.15 to 1.92±0.16(t=5.23,P0.05),and textual entropy increase from 3.64±0.57 to 4.70±1.09(t=-2.6,P0.05).The 3 day biofilm showed the same tendency.Conclusion Magnesium can increase the initial attachment of PA and alter the subsequent biofilm formation and structure.
Objective To investigate the effects of EDTA on the structure of mucoid Pseudomonas aeruginosa(PA) biofilm.Method Plate culture method was used to establish muture bioflim,tube dilusion method to measure the MICs of EDTA and ciprofloxacin.The biofilm was treated by EDTA,ciprofloxacin alone or combined,and the number of the bacteria in biofilm was measured by agar plate.The biofilm was stained by FITC-ConA,observed by fluorescence microscope.The treated biofilm was stained by SYTO9/PI,monitored by Confocal Laser Scanning Microscope(CLSM),quantification of the effect was calculated by Image Structure Analyzer(ISA) sofeware.Result 5 MIC EDTA could exert the best effect upon PA biofilm and reduce the number of colonies from 107 CFU/ml to 104 CFU/ml.0.1 MIC and 5 MIC EDTA could enhance the bactericidal ability of ciprofloxacin.EDTA could break the ESP of biofilm,change the structure of biofilm and raise the ratio of dead bacteria.It decreased the depth of biofilm from(22.59±4.13)μm to(8.97±2.45)μm(t=8.515,P0.05),Average Diffusion Distance(ADD) from 3.08±0.96 to 1.59±0.24(t=4.510,P0.05),Textual Entropy(TE) from 6.25±0.79 to 3.02±0.67(t=9.375,P0.05),and increased areal porosity(AP) from 0.89±0.07 to 0.97±0.02(t=-2.653,P0.05).Conclusion EDTA can destroy the structure of BF and enhance the bactericidal ability of antibiotics.
Objective 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 PA01 in 96-well plate.The adhesion ratio of PA01 was calculated to determine the effects of allitride on PA01 biofilm adhesion,and qualitative study adherence of GFP-PA01 was performed by fluorescence microscope.The fluorescein isothiocyanate(FITC)conjugated concanavalin A(FITC-conA)was used to stain the EPS,in order for the qualitation of EPS by fluorescence microscope;the phenol-sulfuric acid method was used to quantitate the EPS in each group.Results Six-hour 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,P0.05.Low dose of allitride could also affect the adhesion ratio,but not so obvious as high dose.Other time groups except the 9-hour group had an approximate tendency as the 6-hour group,which may be related with unstability of allitride.Compared with normal saline control group,the colony distribution of GFP-PA01 became rarefactive after intervention,especially with high doses.The EPS in biofilm was significantly decreased after intervention with allitride,which was visualized by fluorescence microscope,based on the FITC-conA staining.Quantification of EPS showed 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,P0.05).Conclusions Allitride could decrease the adhesion and EPS producing ability of PA01 significantly.
Objective To investigate the effect of N-(3-oxododecanoyl)-L-homoserine lactone(3-O-C12-HSL,signaling molecule of Pseudomonas aeruginosa biofilm) on the expression of toll like receptors(TLR) 2/4 mRNA and the apoptosis of monocyte.Method Human monocytes,isolated from the peripheral blood of healthy donors,were cultured with 3-O-C12-HSL in different concentration of 0,1,10,50 and 100 μmol/L for 12 hours;the proliferation capacity of cells was observed by MTT.The expression of TLR2/4 mRNA was observed by reverse transcription RT-PCR.Result After 12 h treatment with 3-O-C12-HSL,TLR2/4 mRNA expression was reduced in a dose-dependent manner;the inhibition of 3-O-C12-HSL was most significant at the concentration of 100 μmol/L(0.3494±0.0979,0.3351±0.1121).The proliferation of these monocyte cells was inhibited in a dose-dependent maner.Conclusion 3-O-C12-HSL can induce the monocyte cells apoplosis,decrease the expression of TLRs,and may contribute to immune evasion of the Pseudomonas aeruginosa.
Objective To investigate the effect of quorum-sensing(QS) system on Pseudomonas aeruginosa biofilm formation in vitro.Method In vitro biofilm models of P.aeruginosa wild-type PA01 and lasI rhlI mutants were established on glass slice,then the biofilm development of the two strains tagged with SYT09/PI were detected using confocal laser scanning microscopy on day 3.Quantitative parameters describing biofilm spatial structure were acquired after the image information was calculated by Image Structure Analyzer(ISA) software.Result After being cultured for 3 days,the biofilm was mature in the PAO1,whereas it was poorly formed in △lasI △rhlI group.The quantitative data from ISA software showed that the thickness of △lasI △rhlI biofilm was(6.62±0.31)μm,and that of PAO1 was(21.64±0.57)μm(t=0.205,P0.05),meanwhile the areal porosity(AP) was 0.902±0.006 and 0.970±0.003,respectively;The average diffusion distance(ADD) and textural entropy(TE) were 1.571±0.019 and 6.586±0.110 in △lasI △rhlI biofilm,whereas 1.467±0.015 and 5.213±0.111 in PAO1 biofilm.The AP of △lasI △rhlI biofilm was less than that of PAO1(t=0.335,P0.05),while the ADD and TE of △lasI △rhlI biofilm were larger than those of PAO1(t=0.289,t=0.300,P0.05).Conclusion △lasI △rhlI obviously affects the formation of biofilm of P.aeruginosa.QS system plays an important role in the formation of P.aeruginosa biofilm.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)
