| Objective:Screening traditional Chinese medicine monomers(TCMMs)with quorum sensing(QS)inhibitory activity;To study the carrying status of regulatory genes and signal molecules in the QS system of Acinetobacter baumannii,and evaluate the correlation between the QS system and drug resistance of A.baumannii;To investigate the effects of quorum-sensing inhibitor(QSI)of TCMMs on the motility,adhesion ability,biofilm formation ability and the expression of virulence factor-related genes(aba I,aba R,cus E,bap,pga A)of extensively drug-resistant A.baumannii(XDRAB).Based on comparing the synergistic antibacterial effects of QSIs with imipenem,meropenem,azithromycin,polymyxin B and tigecycline on XDRAB,this study also discusses the synergistic antibacterial effects of QSIs combined with a single antibacterial agent against XDRAB.Methods:Using the qualitative and quantitative detection method of biosensor bacteria(Chromobacterium violaceum 026),the TCMMs with QS inhibitory activity were screened from 34 TCMMs.1.The minimum inhibitory concentration(MIC)of TCMMs and antibacterial drugs was determined by the broth microdilution method.2.PCR amplification and agarose gel electrophoresis were used to detect the carriage of QS regulatory genes aba I and aba R in 60 strains of A.baumannii.3.The signal molecule production of 60 strains of A.baumannii was detected by parallel streaking between biosensor bacteria(Agrobacterium tumefaciens KYC55)and A.baumannii.4.The growth of 9 TCMM with QS inhibitory activity within 24 hours of XDRAB at 1 MIC,1/2 MIC,1/4 MIC and 1/8 MIC was determined by turbidimetry,and the concentration that did not inhibit the growth of bacteria was selected as the working concentration for subsequent experiments.5.Colony plate counting was used to determine the effects of 9 TCMMs on the adhesion ability of XDRAB at working concentrations.6.By measuring the migration diameter of colonies on motility plates,the effects of 9 TCMMs on the motility of XDRAB at working concentrations were studied.7.Crystal violet staining was used to assess the biofilm formation ability of A.baumannii,and to study the effects of 9 TCMMs on the biofilm formation ability of XDRAB at working concentrations.8.Quantitative reverse transcription-polymerase chain reaction(q RT-PCR)was used to determine the effect of 9 TCMMs on the expression of XDRAB virulence factor-related genes(aba I,aba R,csu E,bap,pga A)at working concentrations.9.Micro checkerboard dilution method and partial inhibitory concentration index(FICI)were used to evaluate the synergistic antibacterial effect of 9 TCMMs combined with imipenem,meropenem,tigecycline,polymyxin B,and azithromycin on XDRAB,respectively;The Modified micro checkerboard dilution method and FICI were used to evaluate the synergistic antibacterial effect of pairwise combinations of TCMMs and triple combinations of five antibacterial drugs on XDRAB,respectively.Results:1.Among the 34 TCMMs,21 TCMMs formed specific inhibition zones on CV026 plates and showed different degrees of QS inhibition,which belonged to QS activity inhibitors;9 TCMMs(coumarin,4-terpineol,vanillin,caffeic acid,cinnamic acid,myricetin,matrine,hordenine,kaempferol)had significant inhibitory effects on the production of CV026 violacein at subinhibitory concentrations,and the inhibitory effect was better than that of QSI furanone-C30.2.60 strains of A.baumannii clinically isolated were resistant to 16 antimicrobial agents to varying degrees,with a resistance rate of more than 80.0% to β-amides,including 81.7% and 83.3% to carbapenem imipenem and meropenem,85.0% to aminoglycosides and tetracyclines,more than 75.0% to quinolones,and 0 to polymyxin and tigecycline.According to the definition of resistant bacteria proposed by CLSI 2022 and related consensus,53 resistant bacteria accounted for 88.3% of the total strains,including 39 extensively drug-resistant strains,accounting for 65.0% of the total strains.3.Among 60 strains of A.baumannii,54 strains were detected with QS regulatory genes aba I and aba R,accounting for 90% of the total strains;1 strain was detected with only gene aba I,but aba R was not detected;The remaining 5 strains were not detected with genes aba I and aba R;Among 60 strains of A.baumannii,a total of 56 strains could detect signal molecules,accounting for 93.3% of the total strains,and only 4 strains failed to detect signal molecules.4.9 TCMMs inhibited the motility,adhesion,and biofilm formation ability of XDRAB to varying degrees at 1/8 MIC(myricetin 32 μg/m L,cinnamic acid 256 μg/m L,caffeic acid 256 μg/m L,vanillin 256 μg/m L,hordenine 128 μg/m L,kaempferol 64μg/m L,matrine 256 μg/m L,coumarin 32 μg/m L,4-terpineol 64 μg/m L)and 1/16 MIC,respectively,and showed concentration-dependent tolerance;In addition,the 9 TCMMs significantly inhibited the expression of XDRAB virulence factor-related genes(aba I,aba R,csu E,bap,pga A)at working concentrations,except coumarin,which upregulated the biofilm-related gene bap.5.9 TCMMs combined with five antimicrobial agents(imipenem,meropenem,azithromycin,polymyxin B,tigecycline)showed different degrees of synergy or additive effects on XDRAB,including 100% synergistic antibacterial effects when caffeic acid,vanillin,and myricetin were combined with imipenem,83.0%,92.0%,and92.0% when combined with meropenem,100.0%,92.0%,and 83.0% when combined with tigecycline,and 100.0%,92.0%,and 83.0% when combined with azithromycin and polymyxin B,they mainly showed additive or unrelated effects;Hordenine combined with imipenem and meropenem had 100% synergistic effects,and 58% when combined with polymyxin B,and tigecycline combined with azithromycin.In addition,cinnamic acid,matrine,kaempferol,coumarin,and 4-terpineol showed mainly additive effects in combination with five antimicrobial agents.6.Hordenine combined with coumarin has a synergistic antibacterial effect on XDRAB,and further triple therapy with imipenem,meropenem,tigecycline,azithromycin,and polymyxin B showed 100% synergistic antibacterial effect,of which the combined effect with imipenem is particularly significant,and the MIC value of hordenine decreased from 1,024 μg/m L to 4 μg/m L ～ 8 μg/m L after combination,the MIC value of coumarin decreased from 256 μg/m L to 8 μg/m L ～ 16 μg/m L,and the MIC value of imipenem decreased from 16 μg/m L ～ 128 μg/m L to 0.5 μg/m L ～ 4μg/m L.Conclusion:1.Among 34 TCMMs,coumarin,4-terpineol,vanillin,caffeic acid,cinnamic acid,myricetin,matrine,hordenine and kaempferol had significant QS inhibitory activity.2.The detection rate of extensively drug-resistant strains in 60 strains of A.baumannii was more than 65%;the carriage of QS regulatory genes and the production of signaling molecules in A.baumannii were related to drug resistance.3.9 TCMMs interfere with the QS system and inhibit the motility,adhesion and biofilm formation of XDRAB by down-regulating QS regulatory genes and virulence factor-related genes,thereby reducing bacterial virulence and bacterial pathogenicity.4.The combination of 9 TCMMs with imipenem,meropenem,tigecycline,polymyxin B and azithromycin showed different degrees of synergy or additive effect on XDRAB,respectively;The combination of hordenine combined with coumarin showed a synergistic antibacterial effect on XDRAB,and further combined with 5antimicrobial agents showed 100% synergistic antibacterial effect when combined with the three drugs. |
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