In Vitro Antibacterial Activity And Early Bactericidal Activity Of Rifabutin Against Rifampicin-resistant M. Tuberculosis

Background and Objectives: With the multi-drug resistant tuberculosis(MDR-TB) and extensively drug-resistant tuberculosis(XDR-TB) epidemic gradually severe, the difficulty to control tuberculosis is also increasing. Rifabutin belonging to rifamycins for Mycobacterium tuberculosis and M. avium complex(MAC) had strong bactericidal activity. In this study, we want to determine antimycobacterial activities of rifabutin(RFB) and the relationship between drug-resistance and rpo B mutations for rifampicin-resistant(RIF-R)/TIFArifabutin-susceptible(RFB-S) Mycobacterium tuberculosis clinical isolates so as to offer the reference for the clinical application of rifabutin.Methods: Measured by conventional drug susceptibility test to RIF-R/RFB-S Mycobacterium tuberculosis strains randomly selected 180 clinical isolates in Beijing Chest Hospital Affiliated Capital Medical University from 2011 to 2013, 162 strains were obtained by recovery and culture. The MICs of rifampicin and rifabutin were determined for clinical RIF-R/RFB-S clinical isolates of Mycobacterium tuberculosis(n=162) by microplate-based Alamar blue assay(MABA) method.Additionally, all stains were tested mutations in rpo B gene by DNA sequencing of the PCR amplified target DNA.Results: Of 162 RIF-R/RFB-S clinical isolates tested by tranditional drug susceptibility testing method, 78 isolates were resistant to RFB(MIC>0.5μg/m L) by microplate-based Alamar blue assay method, which were inconsistent with tranditional drug susceptibility testing results, these strains have shown a high degree of rifampicin-resistance(MIC-RIF: ≥32μg / m L); 84 isolates were susceptible to RFB(MIC≤0.5μg/m L),71 isolates rifampicin-MIC range 2-16μg / m L, 13 isolates rifampicin-MIC≥32μg / m L. In addition, 150 isolates(92.60%,150/162) occurred rpo B mutations, in which there were 10 types of single mutations, including codon 531, 526, 516, 533 and so on. Mutations at codons 531(TCG/TTG), 526(CAC/CGC, TGC, GAC), 522(TCG/CAG), 516(GAC/GTC) and 513 in the rpo B gene correlated with resistance to both RIF and RFB. While mutations at codons 511(CTG/CCG), 516(GAC/TAC, GGC), 522(TCG/TTG), 526(CAC/AAC, CTC, GTC, ACC, TAC), 531(TCG/CAG), 533 and 574 possibly influenced the susceptibility to RIF but not to RFB. There were 15 types of double mutations(30/150), the most common is 511/516(16,53.3%). 12 isolates have no mutation in rpo B gene.Conclusions: Of RIF-R/RFB-S clinical isolates tested by tranditional drug susceptibility testing method, still 48% isolates were resistant to RFB by using microplate-based Alamar blue assay method. Some specific gene mutations have abetter reference significance to the clinical judgment of rifabutin sensitivity.Objective: This study was aimed to evaluate the early bactericidal activity of rifabutin(RFB) in patients with rifampin(RIF)-resistant and rifabutin-susceptible pulmonary tuberculosis evidenced by positive sputum smears using consecutive sputum CFU monitoring over 10 days.Methods: Patients with RIF-resistant and RFB-susceptible Pulmonary Tuberculosis Evidenced by Positive Sputum Smears were assigned to receive rifabutin(300 mg daily) for 10 days. 16 hours overnight sputum for quantitative culture was collected on days 0, 3, 6, 10. Counts of colony forming units(CFU) of Mycobacterium tuberculosis in collections were incubated on plates of 7H11 medium. Early bactericidal activity was estimated by measuring the decline in bacilli, defines as the decrease in log10cfu/ml sputum/day during 10 days of treatment.Results: 7 patients with smear-positive rifampin-resistant and rifabutin-susceptible pulmonary tuberculosis were involved in the study according to the inclusion criteria, 6 patients completed the study, 4 patients have early bactericidal activity,Rifabutindemonstrated high bactericidal activity from baseline to day 3 and bactericidal activity from baseline to day 10, 2 patients don’t have early bactericidal activity.Conclusion: Of 6 patients with RIF-resistant and RFB-susceptible Pulmonary Tuberculosis, 4 patients have early bactericidal activity(0-3). Rifabutin demonstrated high bactericidal activity from baseline to day 3 and was well tolerated, and no serious adverse events occurred related to drug.