The Effects Of Andrographolide Against Single And Mixed Biofilms And The Synergistic Activity In Combination With Gentamicin

PART I In vitro studiesobjective To investigate the effects of andrographolide (Andro)against Pseudomonas aeruginosa (PA) and Staphylococcus aureus (S.aureus)biofilms and the synergistic activity in combination with Gentamicin(GEN).Methods Turbidimetric method was used to detect the effect of Andro(100μg/ml) on PA and S.aureus growth curves; PA01,Staphylococcusaureus ATCC25923and two clinical strains: CY001and CY002were usedfor single and mixed biofilm formation in vitro, dividing into control group,Andro intervention group, GEN intervention group and Andro combinedwith GEN intervention group, microplate reader was used to detect theOD600nm values and indirectly observe the biofilm formation ability ofeach group; scanning electron microscopy（SME）was used to directlyobserve morphology of biofilms treated with different interventions.Results Under the action of100μg/ml andrographolide, the growth curve of each strain compared with the control group had no difference(P>0.05); No matter according to which ratio of mixed culture, in themixed biofilm of PA and S.aureus, the number of PA was always more thanS.aureus;Except CY002could not form biofilm, there were no significantdifference between groups (P>0.05), the single and mixed biofilms of PAand S.aureus in each intervention groups were less than the control groups(P<0.005), the inhibition effect of Andro was stronger than GEN (P<0.05),and the inhibition effect of the combination drug was obviously strongerthan they were used respectively (P<0.05);Morphology observation underSEM prompted that each intervention condition has inhibitory effect to thesingle and mixed biofilm, the inhibitory effect of the combination durg wasespecially striking.Conclusion PA is predominate in the mixed biofilm of PA andS.aureus. Andro has no significant effect on the growth of PA and S.aureus,but it has excellent inhibitory effect on the single and mixed biofilms of PAand S.aureus, and can significantly enhance the inhibitory effect of GEN. PART II In vivo studiesObjective Establishing chronic PA and S.aureus biofilm woundsinfection model in BALB/c mice, to study the role of Andro on biofilm ofchronic wounds and its effect on wounds healing.Methods PA01,Staphylococcus aureus ATCC25923and two clinicalstrains: CY001and CY002were used for single and mixed biofilmformation in vivo, established chronic PA and S.aureus biofilm III°burnwounds infection model in BALB/c mice, dividing into blank controlgroup,control group, Andro intervention group, GEN intervention groupand Andro combined with GEN intervention group, histopathologicalexamination was performed on the wound tissue; IL-1β,IL-6,TNF-α waschosen as markers for inflammation and measured by standardized ELISAmethod;scanning electron microscopy（SME）was used to directly observemorphology of biofilms treated with different interventions in the wounds.Results Skin tissue pathology analysis showed that，compared withthe control group and gentamicin intervention group,the dermal vacuolesand inflammatory cells of the Andro intervention group and the combinedintervention group were significantly decreased, and the newborngranulation tissue and the capillaries formed were increased with a largenumber;after been burned and infected, the expression of inflammatorycytokines IL-1β, IL-6and TNF-α in skin wound tissue of mice were increased significantly(blank control group compared with control group, P＜0.001）. gentamicin could reduce the expression of inflammatory factorsIL-6and TNF-α weakly, and Andro could significantly reduce the level ofIL-1β, IL-6and TNF-α in wound tissue (P＜0.005),and the reduce effectof the combination drug on the level of IL-6and TNF-α was obviouslystronger than they were used respectively (P<0.05)； Morphologyobservation under SEM prompted that each intervention condition hasinhibitory effect to mixed biofilm, the inhibitory effect of the combinationdurg was especially striking.Conclusion Andro can inhibit biofilm formation in the wound, andreduce the the expression level of IL-1β,IL-6,TNF-α in wound tissue,reduce local inflammatory response, promote the formation of granulationtissue, accelerate wound healing.