Effect Of Candida Albicans’ Supernatant On Biofilm Formation And Virulence Factors Of Pseudomonas Aeruginosa Through Las/rhl System

Objective:To investigate the effect of Candida albicans’（C.albicans）supernatant on the biofilm formation and virulence factors of Pseudomonas aeruginosa（P.aeruginosa）（PAO1）and Pseudomonas aeruginosa’defective strain（Δlas Irhl I andΔlas Rrhl R）.Methods:To divide into PAO1 group,PAO1+C.albicans’supernatant group andΔlas Irhl I group,Δlas Irhl I+C.albicans’supernatant group,Δlas Rrhl R group andΔlas Rrhl R+C.albicans’supernatant group.To observe the biofilm formation by crystal violet semi-quantitative,determine the colony counts in the biofilm by serial dilution,and observe the morphology and structure of early biofilm by the SEM for 1^thand 3^thday.To determine the activities of proteolytic enzymes and elastase by azocasein method and elastase Congo red method.Results:（1）The results of crystal violet semi-quantitative determination of biofilm and continuous dilution biofilm colony plate counts were showed that the biofilm formation ability was significantly higher in PAO1+C.albicans’supernatant group than that in PAO1 group（P<0.05）.On 1^thday,SEM was showed the adhesion and aggregation of PAO1 colonies,no obvious biofilm structure in PAO1 group and PAO1+C.albicans’supernatant group,while more bacterial colonies in PAO1+C.albicans’supernatant group.On the 3^thday,more bacterial colonies,extracellular matrix and early biofilm formation were observed on the surface of carriers in PAO1 group and PAO1+PAO1+C.albicans’supernatant group.In PAO1+C.albicans’supernatant group,bacterial colonies adhered more closely,gathered denser,had more extracellular matrix,and biofilm formed more and more stable.（2）The activities of proteolytic enzyme and elastase in the PAO1+C.albicans’supernatant group were significantly higher than those in PAO1 group on the 1^thand 3^thday（P<0.05）.（3）The biofilm forming ability of PAO1 group was significantly higher than that ofΔlas Irhl I group andΔlas Rrhl R group（P<0.05）by semi-quantitative determination of biofilm and continuous dilution biofilm colony plate counts.After the addition of C.albicans’supernatant,there was no significant difference in biofilm formation ability betweenΔlas Irhl I andΔlas Rrhl R（P>0.05）.SEM observation was showed that the bacterial colonies were much tighter in PAO1 than those inΔlas Irhl I andΔlas Rrhl R group,which was adhered and aggregated more tighter with the intervention of C.albicans’supernatant in all groups.While there was no significant difference in the number and adhesion of bacterial colonies inΔlas Irhl I andΔlas Rrhl R groups.On 3^thday,more bacterial colonies,extracellular matrix and early biofilm were observed.Compared with theΔlas Irhl I group andΔlas Rrhl R group,we could see that adhesion and aggregation were more tighter with PAO1 colonies,and extracellular matrix and stable biofilm in the PAO1 group,which was more in PAO1+C.albicans’supernatant group than others.However,there was no significant difference between theΔlas Irhl I group andΔlas Rrhl R group,regardless of the intervention with C.albicans’supernatant.The activities of proteolytic enzyme and elastase in PAO1 group were significantly higher than those inΔlas Irhl I group andΔlas Rrhl R group（P<0.05）,and the activities of proteolytic enzyme and elastase in PAO1+C.albicans supernatant group were significantly higher than those in PAO1 group（P<0.05）.However,there was no significant difference in the activities of protein hydrolase and elastase betweenΔlas Irhl I andΔlas Rrhl R,regardless with or without it.C.albicans’supernatant.Conclusion:（1）The supernatant of C.albicans could promote the biofilm formation of PAO1,but had no significant effect onΔlas Irhl I andΔlas Rrhl R,indicating that C.albicans’supernatant may be related to las/rhl system to promote P.aeruginosa for biofilm formation.（2）The supernatant of C.albicans could increase the activity of proteolytic enzyme and elastase in PAO1,but had no significant effect inΔlas Irhl I andΔlas Rrhl R,indicating that the enhancement of proteolytic enzyme and elastase activity of P.aeruginosa by supernatant of C.albicans may be also related to regulate las/rhl system.