The Inhibition Of Staphylococcus Aureus And Pseudomonas Aeruginosa Biofilms By Antimicrobial Peptide 17BIPHE2

ObjectiveTo study the inhibition of Staphylococcus aureus(S.aureu,SA)and Pseudomonas aeruginosa(P.Aeruginosa,PA)biofilms by antimicrobial peptide17BIPHE2 used alone or combined with antibiotics.Furtherly,to explore the changes of the biofilm-related genes at the transcriptional level after the antimicrobial peptide 17BIPHE2 treated.This study provides a new idea for developping drugs and treatment infection of S.aureus or P.Aeruginosa biofilm.Methods1.Congo red plate test(S.aureus)and crystal violet staining use to assess the biofilm formation ability of bacteria;2.The minimum inhibitory concentration(MIC)of 17BIPHE2 peptide against S.aureus and P.Aeruginosa was determined using a microdilution technique in96-well microtiter plate;the minimum bactericidal concentration(MBC)was determined by agar plate assay;3.Biofilm early adhesion experiments were used to observe the effects o17BIPHE2 alone and in combination with antibiotics in the biofilm adhesion phase;4.Biofilm formation experiment were used to observe the effects of17BIPHE2 alone and combined with antibiotics in the biofilm formation stage;5.Scanning electron microscopy(SEM)was used to observe the scavenging effect of 17BIPHE2 alone and combined with antibiotics in mature biofilm.6.Laser confocal scanning microscopy(LCSM)was used to observe the bacterial killing effect of 17BIPHE2 in the P.Aeruginosa mature biofilm;7.Real-Time PCR was used to analyze the effect of 17BIPHE2 on the biofilm-associated genes in transcription level.Results1.Congo red plate test method and crystal violet dyeing showed that SA 05 has a strong ability to form biofilm,but SA 07 didn’t have this ability;crystalline violet staining showed that PA 03 had a strong ability to form biofilm,PA 07didn’t have the ability;2.The MIC of 17BIPHE2 peptide at which they exerted potent bactericidal effects against SA05 was test and was found to be 8μmol/L.In 1/2 × MIC concentration can inhibit the growth of planktonic bacteria effectively.The MIC of17BIPHE2 to inhibit PA03 was 48 μmol / L.In the sub-inhibitory concentration also can inhibit the growth of planktonic bacteria effectively.The MIC of17BIPHE2 combined with antibiotics compared with single antibiotics has decreased;3.In the S.aureus adhesion stage,the 17BIPHE2 treatment group inhibition rate has been reach about 40%.The effect when 17BIPHE2 combined with antibiotics than antibiotics alone has improved obviously.In the P.Aeruginosa adhesion phase,the 17BIPHE2 treatment group inhibition rate nearly 60%.In contrast to the results at above,the inhibition rate of the 17BIPHE2 combined with antibiotic also improved.4.In the S.aureus biofilm formation stage,the inhibition rate was 35% when17BIPHE2 treated alone.But in the P.Aeruginosa biofilm formation phase,theinhibition rate has reached about 80%.It is noteworthy that,17BIPHE2 also can inhibit the ability to produce biofilm significantly in the 1/4 × MIC concentration.The ability to inhibit biofilm production is more improved after 17BIPHE2 combined with antibiotics;5.Scanning electron microscopy revealed that antimicrobial peptide17BIPHE2 can promote the mature biofilm of S.aureus disintegration at 1/4 ×MIC concentration.In the 1 × MIC treatment,the biofilm was disintegrated and the adhesion number of bacteria decreased.Antimicrobial peptide combined with vancomycin treatment group promote biofilm dissipation while a large number of bacterial cytoplasm leakage.17BIPHE2 promotes the biofilm ripening effect of P.Aeruginosa similar to the above.The sub-inhibitory concentration can promote biofilm splitting.With the increase of antimicrobial peptide,the adhesion amount of bacteria also decline dramatically.The effect showed a dose-dependent relationship.When the concentration of 17BIPHE2 reached 1 × MIC,the complete morphology of bacteria almost disappeared.The effect to remove biofilm when peptide combined with 7ciprofloxacin is significant particularly.The biofilm dissipated at the same time cytoplasmic leakaged.6.The laser confocal scanning microscopy showed that the amount of P.Aeruginosa adhered decreased gradually and the number of dead bacteria increased when drug concentration increased at 17BIPHE2 treatment group.This result is also dose-dependent.At 1 × MIC concentration,the number of viable cells and dead bacteria all decreased significantly.7.With the increase of 17BIPHE2,Real-Time PCR showed that the expression level of S.aureus and P.Aeruginosa biofilm-related genes were inhibited at the transcriptional level.The inhibitory effect was more obvious in the high concentration group.ConclusionAntimicrobial peptide 17BIPHE2 inhibits adhesion and aggregation of S.aureus and P.Aeruginosa biofilm.After the biofilm riped,17BIPHE2 can promote disintegration.After 17BIPHE2 combined with antibiotics,the effect of anti-biofilm is improved furtherly.17BIPHE2 can down-regulate the transcription of the biofilm-related genes effectively.This study will provide a new idea for the treatment of related infections caused by S.aureus and P.Aeruginosa biofilm.