| Pseudomonas aeruginosa(P.a)is the main pathogen of chronic lung infections,and it is also a common pathogen that causes chronic recurrent infection of the body with low immunity.The death rate of pneumonia caused by infection is as high as 30%,and the mortality caused by sepsis even reached 80%-90%.Resistant to many common antibiotics,the infection is prone to recurring and difficult to clear,the main reason is that it is very easy to form biofilm(BF).Alginate is the main component of P.a biofilm,which is a key factor preventing the immune system reaction and the removal of biofilm in antibiotic therapy,which will lead to higher drug resistance.The bacteria forming biofilm can exhibit up to 1000 times more than planktonic bacteria Antibiotic resistance.In this project,the wild-type standard strain PAO1 of Pseudomonas aeruginosa was used as a test strain.By establishing an in vitro BF model,the effect of ratderived antimicrobial peptide(CRAMP)on PAO1 mature biofilm was explored.In addition,CRAMP was investigated on PAO1 mature organisms.The influence of the content of polysaccharides from hymenopsia and the changes in m RNA levels of genes related to Alginate formation in PAO1 mature biofilms.Part 1 CRAMP inhibition of Pseudomonas aeruginosa mature biofilmObjective: To observe the effect of CRAMP on the mature biofilm of PAO1.Methods: A 96-well cell culture plate was used to construct the PAO1 mature biofilm model in vitro,and different concentrations of antimicrobial peptide CRAMP intervention group,different concentrations of positive peptide(LL-37)control group,and different concentrations of antibacterial drugs(Ciprofloxacin lactate,Enrofloxacin,Gentamicin sulfate)control group,and the blank control group without intervention,the intervention time was 1h.The crystal violet staining method was used to detect the amount of biofilm,and the ideal concentration and conditions were initially screened.Then,a 6-well cell culture plate was used to construct the PAO1 mature biofilm model in vitro,and the combination of crystal violet staining and biofilm viable count was used to quantitatively detect the biofilm to further verify the ideal concentration of the intervention effect.The laser scanning confocal microscope(CLSM)was used to observe the morphological changes of PAO1 biofilm under the intervention of the optimal CRAMP concentration.Statistical analysis was conducted based on the results of three or more independent tests.Results: 96-well cell culture plate: the test wells in the CRAMP group(CRAMP)were compared with the blank control wells in this test group at 4MIC(P<0.01,biofilm reduction rate of 43.75%),2MIC(P<0.01,biofilm Volume reduction rate is 40.63%),1/8MIC(P<0.05,biofilm volume reduction rate is 25%)under the conditions of three concentrations can effectively reduce biofilm volume;LL-37 group and the test group blank Control wells showed no significant reduction in PAO1 biofilm volume at all concentrations(P>0.05).The Gentamicin sulfate group compared with the blank control well of this test group at 4MIC(P<0.01,the bacteriostatic rate is 30.30%),2MIC(P<0.05,bacteriostatic rate is 36.36%)can effectively reduce the amount of biofilm at two concentrations;Enrofloxacin group compared with the blank control wells of this test group(P>0.05),test concentration there was no significant difference.The preliminary results show that the effect of the drug used on the test on the biofilm volume is generally dosetolerant,and the effect of 4 times the MIC concentration is the best.However,considering that the high concentration of the peptide drug may cause cytotoxicity,and subsequent tests will be intervened at this concentration.6-well cell culture plate: the results of biofilm testing showed that the CRAMP(4MIC)group significantly reduced the biofilm volume under the conditions compared with the blank control wells of this test group(P<0.001,the reduction rate of biofilm volume was 76.74%);LL-37(4MIC)group compared with the blank control wells of this test group could significantly reduce the amount of biofilm(P <0.01,the reduction rate of biofilm amount was 51.16%);But the comparison between the two polypeptide groups,CRAMP group of biological the membrane volume was also significantly smaller than that of LL-37(P<0.05),indicating that CRAMP had a better effect on the inhibition of biofilm volume than LL-37 at 4 MIC concentration.In addition,there was no significant difference between the three antibacterial drugs(Ciprofloxacin lactate,Enrofloxacin,Gentamicin sulfate)group and the blank control well group of this test group(P>0.05).Biofilm count results show that the CRAMP group compared with the blank control wells of this test group can significantly reduce the number of viable bacteria in PAO1 biofilm under 4MIC conditions(P <0.001,a reduction of 1.16 log10CFU/ml,that is,more than 90% of the biofilm bacteria were suppressed),and the number of viable biofilm bacteria in the LL-37 group was not significantly changed compared with the blank control wells of this test group(P>0.05).Comprehensive analysis shows that the antimicrobial peptide CRAMP(4MIC)has better intervention effect on PAO1 mature biofilm than LL-37 and three common antibacterial drugs(Ciprofloxacin lactate,Enrofloxacin,Gentamicin sulfate).CLSM: Through the calculation of fluorescence intensity,the CRAMP(4MIC)group could significantly reduce the total fluorescence intensity of bacteria compared with the blank control wells of this test group(the total fluorescence intensity of SYTO and PI reflects the total amount of dead bacteria)(P<0.05,A reduction of 42.41%),the difference between the LL-37(4MIC)group and the blank control well of this test group was not significant(P>0.05);And the total fluorescence intensity of bacteria in CRAMP(4MIC)group was significantly lower than that in LL-37(4MIC)group(P<0.05).It shows that CRAMP has a clear effect on the total dead and live bacteria of PAO1 mature biofilm,and the effect is better than LL-37.Compared with the blank control wells of the experimental group,the ratio of PI/SYTO(dead bacteria fluorescence intensity/live bacteria fluorescence intensity)in the CRAMP(4MIC)group increased significantly(P<0.01,15.36% in the blank group and 32.50% in the CRAMP group),The LL-37(4MIC)group was significantly different from the blank control wells of this test group(P<0.01,ratio was 30.84%).It shows that CRAMP has a clear scavenging effect on the mature biofilm of PAO1,and also has a significant inhibitory effect on live biofilm bacteria,and the effect is better than LL-37.Conclusion: A certain concentration of CRAMP can inhibit PAO1 mature biofilm in a short time,and can effectively remove mature biofilm and kill a large number of biofilm bacteria at 4 MIC,the effect is better than the three common antibacterial drugs and typical Antimicrobial peptide LL-37.Part 2 CRAMP inhibits extracellular polysaccharides in the mature biofilm of Pseudomonas aeruginosaObjective: To observe the effect of CRAMP on extracellular polysaccharide(EPS)in mature PAO1 biofilm.Methods: A 6-well plate was used to construct a PAO1 mature biofilm model in vitro,and a 4MIC concentration antimicrobial peptide CRAMP intervention group,a4 MIC concentration positive peptide(LL-37)control group,and a blank control group without intervention were set up.The phenolsulfuric acid method was used to detect the polysaccharide content in the mature PAO1 biofilm and the Foline-phenol reagent method was used to detect the protein content in the PAO1 mature biofilm.Results: Compared with the blank control wells of the experimental group,the content of extracellular polysaccharides in the CRAMP group was significantly reduced(P<0.05,the content was reduced by 36.64%).The difference between the LL-37 group and the blank control wells of the experimental group was not significant(P>0.05),and the extracellular polysaccharide in CRAMP group was significantly reduced compared with LL-37(P<0.05,the reduction rate was 34.38%).Conclusion: CRAMP(4MIC)can effectively reduce the content of extracellular polysaccharide in PAO1 mature biofilm,and the effect is better than LL-37.Part 3 The effect of CRAMP on Alginates in PAO1 mature biofilmObjective: To observe the effect of CRAMP on the content of Alginates(the main component of mature biofilm extracellular polysaccharides)in PAO1 mature biofilms and to detect changes in m RNA levels of Alginate synthesis-related genes in PAO1 mature biofilms.Methods:(1)Construct PAO1 mature biofilm model in vitro with 6-well plate,and set up 4MIC concentration of antimicrobial peptide CRAMP intervention group,4MIC concentration positive peptide(LL-37)control group,and non-intervention blank control group(2)UV Spectrophotometry was used to detect the polysaccharide content of biofilm algae;(3)RT-PCR was used to detect the changes of m RNA levels of genes related to Alginate synthesis(Alg D,Alg U,Muc A)in PAO1 mature biofilm.Results:(1)The effect of CRAMP on the content of Alginates in the mature biofilm of PAO1: Compared with the blank control wells of the experimental group,the content of Alginates in the CRAMP group decreased significantly(P<0.05,a decrease of 78.41%).Compared with the blank control wells in this test group,the difference was not significant(P>0.05).In addition,the CRAMP group was also significantly lower than that of the LL-37 group(P<0.05,a decrease of 54.09%),it Shows that CRAMP can significantly reduce the production of PAO1 mature biofilm algae polysaccharide,and the effect is better than LL-37;(2)Changes in m RNA levels of genes related to Alginate formation in PAO1 mature biofilm:analysis of the relative expression of each gene shows that there is no significant difference in the effects of CRAMP on m RNA levels of Alginate-related genes in mature biofilm.There was no significant difference in the up/down regulation of alg D,alg U,Muc A gene.Conclusion: CRAMP(4MIC)has an obvious intervention effect on Alginate,an important extracellular polysaccharide of PAO1 mature biofilm,but the m RNA level of algae polysaccharide synthesis-related genes has no significant change.Which initially indicates that CRAMP has an effect on PAO1 mature biofilm.The intervention effect of the Alginate is not exerted through its synthetic pathway,it is speculated that may affect the regulation of Alginate transport or cleavage. |
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