Study On Mechanism Of Tigecycline-resistance In Acinetobacter Baumannii& Antibacterial Effect Of TCM Monomers Against Tigecycline-resistant Acinetobacter Baumannii

Objective To investigate tigecycline resistance in clinically isolated Acinetobacter baumannii strains.To explore the distribution of eight RND efflux pump genes and their relevance to tigecycline resistance in A.baumannii.To examine the antibacterial activity of five Traditional Chinese Medicine（TCM）monomers on tigecycline-resistant A.baumannii.To analyze the effect and mechanism of sensitization enhancement of tigecycline combined with TCM monomers.To provide experimental basis for effectively preventing and controlling the spread and infection of resistant A.baumannii in hospitals,and also clues to potential new treatments.Material and Methods A.baumannii isolates were identified by PCR amplification and sequence analysis of 16S-RNA and rpo B genes.Minimum inhibitory concentrations（MIC）of tigecycline for 120 A.baumannii clinical isolates was examined by broth microdilution method.A.baumannii isolates were accordingly divided into tigecycline-resistant（TRAB）and sensitive（TSAB）A.baumannii.1.MICs of 18 antimicrobial agents for TRAB were measured by agar dilution methods.2.Molecular epidemiology of TRAB was performed by multilocus sequence typing（MLST）approach.3.Eight efflux pump genes and their regulatory genes（ade ABC-ade RS、ade FGH-ade L、ade IJK-ade N、ACICU-02904、ACICU-00143、ACICU-03412、ACICU-03066、ACICU-03646）were amplified by PCR.4.Expression level of efflux pump genes（ade B、ade J、ade G、ACICU₀0143、ACICU₀3066、ACICU₀3646）was investigated by quantitative real-time PCR.5.Antibacterial activity of five TCM monomers against TRAB was determined by broth microdilution method.6.MIC value of plumbagin combined with tigecycline was determined by chessboardmethod and fractional inhibitory concentration index（FICI）value was subsequently obtained by calculation.7.Five efflux pump inhibitors（EPIs）,including CCCP,PAβN,NMP,reserpine and verapamil,were used for evaluation of bacterial efflux inhibition effect against TRAB.The efflux inhibition effect of EPIs was further compared with that of plumbagin against TRAB.Tigecycline resistance for 120 A.baumannii clinical isolates was examined by broth microdilution method.A.baumannii isolates including tigecycline-resistant A.baumannii（TRAB）and tigecycline-sensitive A.baumannii（TSAB）.Results1.The MIC distribution range of 120 A.baumannii clinical isolates to tigecycline was<0.0625 8 μg/ml.Among the 120 isolates,we identified eight TRAB and the other 112 TSAB isolates,with a resistance rate of 6.7%.2.TRAB showed high resistance to 18 antimicrobial agents,especially higher resistance to aminoglycosides and tetracyclines.3.Among eight TRAB isolates,five were assigned to ST-195 type,the rest were ST-837,ST-92 and ST-908 type based on MLST approach.All eight TRAB isolates belonged to clonal complex 92（CC92）.4.Six RND efflux pump genes and regulatory genes（ade ABC-ade RS,ade FGHade L,ade IJK-ade N,ACICU₀0143,ACICU₀3066 and ACICU₀3646）were detected in all eight TRAB isolates.For TSAB isolates,three canonical efflux pump operons（ade ABC-ade RS,ade FGH-ade L and ade IJK-ade N）were detected at a rate of 38.4%,and three newly identified genes（ACICU₀0143,ACICU₀3066 and ACICU₀3646）of 50.9%.5.Mean expression levels of ade B,ade J,ACICU₀3066 and ACICU₀3646 in TSA B isolates were observed to be 23.5-,3.75-,1.7-and 1.44-fold higher than in TRA B,respectively.Only the relative expression of ade B increased significantly in TR AB isolates（p=0.014）.6.Plumbagin displayed the best antibacterial activity in five TCM monomers,with theMIC value range of 16³2 μg/ml.7.Plumbagin could reverse the resistance effect of TRAB as demonstrated by MIC values of plumbagin and tigecycline alone or combination,which showed 75%additive effect and 25% irrelevant effect.8.Positive rates for EPIs CCCP,PAβN,verapamil and plumbagin were 50%,37.5%,12.5% and 50% in TRAB isolates,respectively.Upon addition of NMP and reserpine,the MIC value of tigecycline in TRAB showed no change.Conclusion1.Six RND efflux pump genes（ade ABC,ade FGH,ade IJK,ACICU₀0143,ACICU₀3066 and ACICU₀3646）were detected in all eight TRAB isolates.2.Ade B expression level has a tight correlation with bacterial MIC against tigecycline in A.baumannii,indicating that increased activity of efflux pump gene ade B may be a pivotal mechanism for tigecycline resistance in this pathogen.3.Plumbagin has a certain antibacterial activity against TRAB in vitro;and plumbagin-tigecycline combination can reduce the resistance of TRAB with additive effect in majority.We also suggested efflux pump inhibition play a role in the sensitization enhancement effect of plumbagin.