Effect And Mechanism Of Action Of AL-1 As A Single Agent And In Combination With Antibiotics Against P.aeruginosa Biofilm Formation

ObjectivesAndrographolide, one of the active components of the plant Andrographis paniculata, has potent antitumor, antibacterial and other biological activities. AL-1 is a derivative of andrographolide. AL-1 inhibits bacterial biofilm formation and virulence factor production of Pseudomonas aeruginosa without much effect on growth. The purpose of the present study was to further evaluate the effect of AL-1 on Pseudomonas aeruginosa biofilm formation. We also investigated the effect of AL-1 when it is combined with other antibiotics and its possible mechanism of action.Methods1. Appearance of Pseudomonas aeruginosa biofilm was seen by electronic scanning microscope.2. Biofilm formation and quantification was determined by crystal violet assay followed by MIC determination.3. Prior to examination by CLSM, biofilms extracellular polysaccharides were fluorescently stained with fluorescein isothiocyanate conjugated concanavalin A (FITC-ConA).4. For mechanisms of action study, we used phenol-sulfuric acid method to analyze the quantity of EPS produced by Pseudomonas aeruginosa.5. For mechanisms of action study, we also performed to evaluate inhibition of bacteria quorum sensing system and virulence factors. Results1. AL-1 and andro had little activity on Pseudomonas aeruginosa growth.2. After treatment by AL-1, the surfaces of Pseudomonas aeruginosa were smooth and no mature bio film are seen. However, when AL-1 was combined with antibiotics, the inhibitory effect of AZM, GEN, CIP, FOF and STR were greatly improved.3. AL-1 inhibited Pseudomonas aeruginosa bio film formation and was more potent than its parent andro. AL-1 also increased the susceptibility of P. aeruginosa biofilms to antibiotics when combined with azithromycin, gentamicin, ciprofloxacin, fosfomycin and streptomycin at the tested concentration.4. AL-1 not only inhibited the EPS, bacterial adhesion and bacterial aggregate but also increased the inhibitory effect of antibiotics on EPS.5. EPS produced by P. aeruginosa was significantly reduced after treatment with AZM, GEN, CIP, FOF and STR in combination with AL-1.6. AL-1 not only inhibited the production of pyocyanin and exoproteinases, but also increased the inhibitory effect of antibiotics on pyocyanin and exoproteinases production.ConclusionsThe novel AL-1 inhibits the bio film formation, QS system and virulence factor production of Pseudomonas aeruginosa without much effect on growth. AL-1 also increases susceptibility of P. aeruginosa biofilms to antibiotics and decreases production of polysaccharides when combined with AZM, GEN, CIP, FOF and STR. AL-1 did not directly interact with the autoinducers in the QS pathway but that it significantly reduces the expression of two regulators, LasI and RhlI. Thus AL-1 is synergiwith conventional antimicrobial agent in the treatment of P. aeruginosa infections.