| Escherichia coli(E.coli)is clinically one of the most common pathogens.Its antimicrobial resistance is a significant challenge in clinical medicine.The formation of biofilm is one of the major mechanisms leading to E.coli's antimicrobial resistance,and it is also the main cause of the repeated attacks and difficult treatment of chronic infectious diseases.Traditional Chinese medicine has a broad prospect in the application of anti-bacterial BF infection by their wide source,small side effects,long-lasting effect,and non-resistance.Honokiol is an effective component of traditional Chinese medicine Magnolia.Previous researchs showed that Honokiol has many effects such as anti-oxidation,anti-tumor,anti-bacterial and anti-viral.However,the inhibitory effect of Honokiol on E.coli BF and the mechanism of its action has not been widely studied.The previous studies have shown that the mechanism of inhibitory effect of the formation of bacterial BF is different in sub-minimal inhibitory concentrations(sub-MIC)and minimal inhibitory concentrations(MIC)drug concentrations.This thesis conducts a more in-depth study on the mechanism of inhibition of the formation of E.coli10389 BF by the antibacterial and antibacterial concentrations of Honokiol.The specific research results are as follows:1.The formation of E.coli10389 BF was detected by the congo red plate method and crystal violet semi-quantitative method.The result showed that the formation of E.coli10389 BF increased with the increase of incubation time,and the mature BF was formed after 48 h.2.The MIC and MBC of Honokiol inhibition of E.coli10389 BF formation and mature BF formation were determined by the TTC method.3.The inhibitory effect of Honokiol on the formation of E.coli10389 BF was determined by XTT reduction method.The Honokiol MIC and sub-MIC of inhibitory effect on BF formation were determined.The results showed that when the concentration of Honokiol was greater than 32 ?g/mL,the increase of concentration had no significant effect on the inhibition of E.coli10389 BF formation,so 32 ?g/mL was selected as the inhibitory concentration of the subsequent study of Honokiol,The concentration of Honokiol at the inhibition rate of 50% and 70% on E.coli10389 BF,namely 8 ?g/mL and 16 ?g/mL,were selected as the sub-MIC in the study.4.The effect of Honokiol of sub-MIC and MIC on the formation of E.coli10389 BF at different incubation time was determined by XTT reduction method.The results showed that the inhibition rate of sub-MIC Honokiol on E.coli10389 BF was enhanced with the increase of time within 0-9 h,and there was no significant change in the inhibition rate within 9-24 h.At 9 h,Honokiol of 8 and 16 ?g/mL had the best inhibitory effect on the formation of E.coli10389 BF,and the inhibitory rate were 50.01% and 75.01%,respectively(P<0.01).At 24 h,Honokiol of 32 ?g/mL had the maximum inhibitory effect on the formation of E.coli10389 BF,and the inhibitory rate was 97.43%(P<0.01).5.The effect of Honokiol of sub-MIC and MIC on the growth of E.coli10389 was determined by spectrophotometry.The results showed that the Honokiol of sub-MIC had no significant effect on the growth of E.coli10389 within 0-24 h;Honokiol of MIC had a significant inhibitory effect on the growth of E.coli10389.The inhibitory rate is 51.46% at 24 h(P<0.01).The results showed that the inhibitory effect of MIC Honokiol on E.coli10389 is one of the mechanisms that inhibit the formation of BF.However,the inhibitory effect of sub-MIC Honokiol on the formation of E.coli10389 BF has no relationship with the growth of bacteria.The inhibitory effect involves other regulatory mechanisms.6.The effect of Honokiol of sub-MIC and MIC on the expression levels of E.coli10389 BF related genes(hha,YbaJ and ariR)was detected by qRT-PCR method.The results showed that the Honokiol of sub-MIC did not affect the mRNA expression levels of hha,YbaJ,and ariR.However,Honokiol of MIC significantly affected the mRNA expression levels of hha,YbaJ,and ariR.The mRNA expression levels of hha and ariR were increased by 21.51% and 35.99%,respectively,compared with the control group(P<0.05).The mRNA expression levels of YbaJ were reduced by 62.02%(P<0.01).The results showed that Honokiol of MIC could inhibit the formation of E.coli10389 BF by affecting the related genes of BF formation.However,the inhibitory effect of sub-MIC Honokiol on E.coli10389 BF has no relationship with the expression of related genes which regulate the formation of BF.The inhibitory effect involves other regulatory mechanisms.7.The effect of sub-MIC and MIC Honokiol on the mRNA expression of related genes in the Luxs/AI-2 system and indole system of E.coli10389 qRT-PCR method were detected by qRT-PCR method.The results showed that both sub-MIC and MIC Honokiol could inhibit the expression of luxs mRNA in Luxs/AI-2 system,but they had little effect on the expression of tnaA mRNA in pfs and indole system in Luxs/AI-2 system.The Honokiol expressions of 8,16 and 32 ?g/mL in luxs mRNA decreased by 26.12%,49.72%,and 49.77%,respectively,compared with the control group(P<0.05).The results showed that both sub-MIC and MIC Honokiol could inhibit the formation of E.coli10389 BF by regulating the expression of luxs mRNA in Luxs/AI-2 system.8.The effect of Honokiol on the transcription of E.coli10389 luxs gene at 0-24 h was detected by qRT-PCR method,and the relationship between the inhibitory effect of sub-MIC and MIC Honokiol on the formation of E.coli10389 BF and luxs was discussed.The results showed that the inhibitory effect of sub-MIC Honokiol on luxs gene transcription increased with the increase of time within 9 h,while the inhibitory effect did not change significantly between 9 and 24 h.Honokiol at 8 ?g/mL and 16 ?g/mL reduced luxs gene transcription by 37.33% and 51.10% respectively(P<0.05)compared with the control group at 9 h.This result was consistent with the effect of sub-MIC Honokiol on the E.coli10389 BF within 0-24 h,which indicated that the inhibition of the formation of E.coli10389 BF by sub-MIC Honokiol was achieved by inhibiting the transcription of luxs gene.However,MIC Honokiol showed no changes in luxs gene transcription within 0-24 h,which was reduced by about 50% compared with the control group(P<0.05).This result was different from the inhibitory effect of MIC Honokiol on the E.coli10389 BF,which was continuously enhanced within 0-24 h.This result indicates that the regulation of luxs gene transcription was not the major mechanism for MIC Honokiol to inhibit the E.coli10389 BF.9.The effect of sub-MIC Honokiol on the secretion of E.coli10389 AI-2 within 0-24 h was detected by bioluminescence method.The results showed that the secretion of sub-MIC Honokiol inhibiting AI-2 increased with time within 0-9 h.However,there was no significant effect on the secretion of AI-2 within 9-24 h.Compared with the control group,the inhibition rates of 8 and 16 ?g/mL Honokiol on AI-2 were 18.64% and 29.32% at 9 h,respectively(P<0.01).This result is consistent with the effect of sub-MIC Honokiol on the formation of E.coli10389 BF within 0-24 h,which indicate that the sub-MIC Honokiol inhibits the formation of E.coli10389 BF by affecting the secretion of AI-2.10.The effect of sub-MIC Honokiol on the mRNA expression level of downstream genes(mqsR,qseBC,flhDC,flic,mcbR and csrA)which regulated by AI-2 signaling molecules was detected by qRT-PCR method.The mechanism of the inhibition the formation of BF was explored.The results showed that the Honokiol of sub-MIC could significantly inhibit the mRNA expression of mqsR,mcbR,csrA,flhDC and flic,but had little inhibitory effect on qseBC.Honokiol of 16 ?g/mL reduce the mRNA expression of mqsR,mcbR,csr A,flhD,flhC and flic by 65.21%,55.01%,73.16%,62.01%,60.30% and 59.71%,respectively,compared with the control group(P<0.01).The results showed that the mechanism of sub-MIC Honokiol inhibiting the formation of E.coli10389 BF was mainly achieved by inhibiting the mRNA expression of mqsR,mcbR and csrA,flhDC and flic.Based on the above results,the mechanism of MIC and sub-MIC Honokiol the inhibition of the formation of E.coli10389 BF was different.MIC Honokiol inhibits the formation of E.coli 10389 BF mainly by inhibiting the growth of bacteria and the mRNA expression of related genes.Sub-MIC Honokiol inhibits the formation of E.coli10389 BF by inhibiting the mRNA expression of Luxs/AI-2 system,which reduce the secretion of AI-2 and the mRNA expression of its downstream genes mqsR,mcbR,csrA,flhDC and flic. |
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