| Pseudomonas aeruginosa（P.a）,is a common zoonotic opportunistic pathogen.It is resistant to most common antibiotics,and the infection is easy to repeat and difficult to remove.The main reason is that it is easy to form Biofilm.Therefore,it is often used as a model for biofilm research.The growth cycle of P.a biofilm mainly includes adhesion,formation,maturity and dispersion.At present,a large number of studies have been reported on strategy of the P.a biofilm control,which has foundation for the prevention and control of P.a biofilm-related infection,but these studies mainly focus on inhibiting the formation of P.a biofilm.There are still many gaps in the study of the removal of mature biofilms.Traditional antibiotics are difficult to penetrate into the highly structured biofilm to kill bacteria in the biofilm,especially the persister bacteria.In fact,mature biofilms are often faced in clinical practice,and accelerating the research on the removal of mature biofilms has become an urgent problem to be solved.In our previous study,it was found that the modified antimicrobial peptide CRAMP could obviously clear the biomass of P.a wild-type strain PAO1.In this study,label-free quantitative proteomics technology was used to analyze the differential protein expression of the biofilm of PAO1 after treated with CRAMP modified peptide.A series of phenotypic studies were carried out according to the results of proteomics,and the mechanism of CRAMP modified peptide in clearing P.a biofilm was preliminarily clarified.In addition,the combination of CRAMP modified peptide with traditional antibiotics was used to find the best combination of anti-biofilm drugs,and the biofilm after drug intervention was observed by confocal laser scanning microscope.Part Ⅰ Proteomic analysis of PAO1 biofilm treated by CRAMPObjective: To analyze the key differential proteins in PAO1 biofilm by using Labelfree proteomics techniqueMethods: biofilm samples of PAO1 and CRAMP modified peptides were prepared in cell culture flask in large quantities,and the protein peptides of biofilm and biofilm cells were quantified by Label-free quantitative proteomics technique.After data quality control analysis,quantitative peptides were compared with Uniport protein database.The obtained protein quantitative results were analyzed by GO annotation and functional enrichment analysis,KEGG annotation and functional enrichment analysis,differential protein volcanic map analysis,differential protein cluster analysis,etc.Results: After analyzing the missing peptide site,amino acid length and relative molecular weight of protein,the relationship between PSMs and retention time and mass to charge ratio,the quality of Label-free protein quantitative data was proved to be reliable.The correlation analysis between the experimental group and the control group showed that there was no significant difference within the group but significant difference between the groups.A total of 3307 proteins were identified,including 785 differential proteins,including 349 up-regulated proteins and 436 down-regulated proteins.By combing the differential proteins associated with the biofilm,it was found that the clearance of the mature PAO1 biofilm by CRAMP modified peptide was related to rhamnolipid synthesis,c-di-GMP metabolism and flagellar assembly.Conclusion: Through proteomic analysis,it is speculated that CRAMP modified peptide may induce the dispersion of PAO1 biofilm cells through c-di-GMP signaling pathway and rhamnolipid synthesis pathway regulated by QS system.Part Ⅱ Preliminary explore on the dispersion mechanism of PAO1 biofilm induced by modified peptides CRAMPObjective: To elucidate the mechanism of CRAMP modified peptide inducing the dispersion of PAO1 mature biofilm.Methods: The content of rhamnolipid in PAO1 biofilm after treated with CRAMP modified peptide was determined by orcinol-sulfuric acid method.The whereabouts of biofilm cells were investigated by counting biofilm cells and the upper suspension cells.The motility of PAO1 biofilm dispersed cells after the treated with CRAMP modified peptide was detected by semi-solid medium culture.Then,the rhl A and rhl B genes of P.a were knocked out by CRISPR/Cas9 system,and the effect of CRAMP modified peptide on biomass of PAO1（35）rhl AB strain was observed.Results: CRAMP modified peptide could significantly increase rhamnolipid content in PAO1 biofilm.The CRAMP modified peptide could disperse biofilm bacteria into the upper suspension at all concentrations,but it had a significant inhibitory effect on dispersed bacteria at 62.5 μg/m L.Meanwhile,the motility of bacteria in CRAMP modified peptide group was significantly enhanced.The（35）rhl AB strain of PAO1 was successfully constructed by CRISPR/Cas9 system.There was no significant difference between（35）rhl AB strain and PAO1 strain in biofilm formation ability and biofilm clearance rate of CRAMP modified peptide.Conclusion: CRAMP modified peptide can significantly increase rhamnolipid content and motility of PAO1 biofilm bacteria,both of which contribute to the dispersal of biofilm bacteria.However,the rhamnolipid content was not directly affected by rhl A and rhl B genes.Part Ⅲ Effects of modified peptides CRAMP combined with antibiotics on PAO1 biofilmsObjective: To eradicate the mature biofilm of PAO1 by combining antibiotics.Methods: The minimum inhibitory concentration（MIC）of CRAMP modified peptide and 14 antibiotics were measured by microbroth dilution method,and the minimum bactericidal concentration（MBC）and the minimum biofilm eradication concentration（MBEC）of CRAMP modified peptide and 14 antibiotics were measured by plate counting method.Then the MBEC values of CRAMP modified peptide combined each antibiotic were measured respectively.The time killing curve（TKC）of vancomycin,roxithromycin and azithromycin was plotted by plate counting method.Synergistic and antagonism was determined by time killing curve,and the synergistic antagonistic effect was determined by Biliss formula.The PAO1 Biofilm was stained with FilmtracerTMLIVE/DEADTM Biofilm Viability Kit,and the PAO1 Biofilm after treated by drug combination was observed under confocal laser scanning microscope.Biofilm Q software was used to analyze the Biofilm volume,substrate area,dead bacteria（red）,living bacteria（green）fluorescence intensity and other parameters.Results: The MBEC values of CRAMP modified peptides and 14 antibiotics were greater than MIC and MBC values.Except for 2 carbapenems and 4 β-lactam antibiotics,the MBEC values of other experimental antibiotics decreased after combined treatment with CRAMP modified peptide,and vancomycin,roxithromycin and azithromycin showed the most obvious decrease.After the PAO1 biofilm treated by CRAMP combined with vancomycin,the confocal laser scanning microscope observation showed that the number,volume and fluorescence intensity of the biofilm decreased significantly.Only the biofilm area in the combination group was significantly lower than that in the control group.The red fluorescence intensity and area ratio of red fluorescence intensity in the combination group were significantly higher than those in the CRAMP modified peptide group.The green fluorescence intensity to volume ratio in CRAMP group was significantly higher than that in control group and vancomycin group.Conclusion: CRAMP modified peptide can reduce the minimum biofilm eradication concentration of antibiotics.CRAMP modified peptide,roxithromycin and azithromycin have additive effect on PAO1 biofilm.CRAMP modified peptide and vancomycin had synergistic effect on PAO1 biofilm,and the synergistic effect reached the maximum after1 h of treatment.CRAMP modified peptide can significantly enhance the killing effect of vancomycin against PAO1 biofilm bacteria. |
