Activity Of Bedaquiline,Delamanid And Clofazimine Against Non-tuberculosis Mycobacteria In Vitro

Objective:To determine the in vitro susceptibility of Non-tuberculosis mycobacteria?NTM?against bedaquiline?Bdq?,delamanid?Dlm?and clofazimine?Cfz?,which are the three most new anti-tuberculosis agents.The mutation within the reported drug resistant genes was also analyzed.Methods:A total of 45 standard strains of NTM spceimes and 205 NTM clinical isolates were recruited.All clinical isolates were identified into species levels by homologue gene comparasions.The drug susceptibility was tested by microplate gradient dilution assay.The DNA sequencing of Mar R and Atp E gene was conducted to identify the relevance with Bedaquiline and Clofazimine resistance,respectively.Results:Among all the recruited NTM isolates,35 were M.intracellulare,40 were M.abscessus,33 were M.fortuitum,22 were M.avium,45 were M.kansassii,10 were M.gordonae,21 belonged to other unusual NTM species.Bdq demonstrated uniform strong antibacterial activity against almost all of the tested SGM species with MICs far below 1?g/ml,except 2 species?2?g/m L,Bdq also exhibited very potent in vitro activity against the recruited RGM reference stains,26 out of 30 RGM species had MICs?1?g/m L,four had MICs>2?g/ml.Dlm exhibited highly variable antimicrobial activities against the 15 tested SGM species.Although,8 species had MIC lower than0.25?g/ml,6 species had MICs greater than 32?g/ml and 2?g/ml for one species.However,Dlm did not present evident inhibitory activity against most of the tested RGM species,24 out of 30 tested RGM isolates had MICs greater than 32?g/ml.Only three RGM species had MICs below 1?g/ml.Cfz demonstrated uniform strong inhibitory activity against almost all of the tested SGM species,with MICs far below 1?g/ml,except one species MIC>8?g/m L.Cfz exhibited diverse activity against the recruited RGM species.eighteen out of the 30 RGM species had MICs lower than 1?g/m L,whereas other three RGM species had MICs of?4?g/m L.The Bdq susceptibility testing revealed that 11.43%?4/35?of M.intracellulare isolates were resistant to Bdq,9.09%?3/33?for M.fortuitum,20%?8/40?for M.abscessus,0%?0/22?for M.avium,2.22%?1/45?for M.kansassii,20%?2/10?for M.gordonae.The Cfz susceptibility testing revealed that among M.intracellulare isolates,48.57%?17/35?were resistant to Cfz,87.88%?29/33?for M.fortuitum,90%?36/40?for M.abscessus,22.73%?5/22?for M.avium,4.44%?2/45?for M.kansassii,30%?3/10?for M.gordonae.The Dlm susceptibility testing revealed that 91.43%?32/35?of M.intracellulare isolates were resistant to Dlm,100%?33/33?for M.fortuitum,92.50%?37/40?for M.abscessus,68.18%?15/22?for M.avium,13.33%?6/45?for M.kansassii,40%?4/10?for M.gordonae.A comparison of MIC₅₀ and MIC₉₀ values acquired Bdq<Cfz<Dlm.Sequencing analysis of 17 strains of M.intracellulare Mar R gene found that 2 strains of nucleotide mutations?Asp92Glu,Ala153Pro?,14 had synonym mutation,1 strains had no mutation.Among the 5 strains of M.avium,1 had synonymous mutation,2 strains had nucleotide mutation?Asp127Asn?,1 strains had no mutation.Analysis of Atp E gene found that there was no mutation of Atp E gene in15 strains of Bdq strains.Conclusion:This method have great clinical value.In our study,Bdq presented excellent inhibitory effect on most NTM clinical strains,and Cfz had good inhibitory effect on most slow growing mycobacteria?SGM?clinical strains.Dlm had better inhibitory effect on some SGM clinical strains,but had a little inhibitory effect on most rapid growing mycobacteria?RGM?clinical strains.According to the MIC distributions,the tentative epidemiological cut-off?ECOFF?values for M.kansassii,M.avium and M.intracellulare were defined at 0.5?g/ml,1?g/ml and 2?g/ml,respectively.The NTM resistance mechanism of Bdq and Cfz resistance of NTM is related to Mar R gene mutation in Rv0678 transcription region.