In the regions where bedaquiline (BDQ) is introduced into the regimen, analysis of MIC and screening for preexisting resistance mutations could be crucial. The high prevalence of isolates with high BDQ MICs without prior exposure to BDQ was worrisome.

In order to shorten the course of treatment and its effectiveness, it is essential to gain an in-depth insight into the drug resistance mechanisms of Mycobacterium tuberculosis (M. tuberculosis). In this study, we evaluated the contribution of 26 drug efflux pumps plus target gene mutations to the drug resistance levels in multi-drug resistant (MDR)/pre-extensively drug-resistant (pre-XDR)/extensively drug-resistant (XDR) and mono-drug resistant clinical isolates of M. tuberculosis. The panels of 25 M. tuberculosis clinical strains were characterized for drug resistance-associated mutations with whole-genome sequencing and antibiotic profiles in the presence and absence of efflux inhibitor verapamil (VP). Different MICs were observed for the same target gene mutations. Out of the 16 MDR/pre-XDR/XDR isolates, 6 (37.5%) and 3 (18.8%) isolates demonstrated a significant decrease in rifampicin (RIF) MIC and isoniazid (INH) MIC due to the VP exposure (64 μg/mL), respectively. Susceptibility to RIF was fully restored in two isolates after VP exposure. Moreover, the efflux pump genes of Rv2938, Rv2936, Rv1145, Rv1146, Rv933, Rv1250, Rv876, Rv2333, Rv2459, Rv849, and Rv1819 were overexpressed in the presence of anti-TB drugs, showing the contribution of these efflux pumps to the overall resistance phenotype. Our results clearly showed that efflux systems, besides spontaneous mutations, play a role in the development of INH/RIF resistance. In addition, although VP was effective in reducing the expression of some efflux pumps, it was not very successful at the phenotypic level.

Mixed strain infections of Mycobacterium tuberculosis make diagnosis, treatment, and control of tuberculosis (TB) more difficult. This study was aimed to evaluate the relationship between mixed infections, antibiotic resistance patterns and treatment of TB patients. In this study, among 2850 suspected TB clinical samples, a total of ninety-six clinical samples from 66 TB confirmed patients were subjected to the 24-locus variable-number tandem repeat method to evaluate the prevalence of mixed infections. For all studied strains, 288 colonies (three individual clones for each sample) were isolated from different colonies and separately analyzed by the Drug Susceptibility Test (DST). For all patients, follow up was done after 6 months of treatment. Based on direct 24 loci MIRU-VNTR, in the 66 TB patients, 53% (35/66) showed mixed infection. In the mixed samples, 45.71% (16/35) showed different antibiotic resistant patterns. Among the mixed infection patients, eight (22.9%; 8/35) showed treatment failure after six- month therapy. Six of these non-treated patients (75%; 6/8) had different antibiotic resistant patterns. We conclude that mixed infections, have a negative impact on treatment of TB patients especially when co-infecting M. tuberculosis strains display heteroresistance.

The emergence and spread of multidrug resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains is a critical global health problem. Between 2014 and 2018, 606 MTBC strains were isolated from 13,892 suspected pulmonary tuberculosis (TB) patients in Tehran, Iran, including 16 (2.6%) MDR-TB cases. A combination of phenotypic and genotypic methods (whole-genome sequencing) was employed for the identification of additional drug resistances and strain-to-strain genetic distances as a marker for recent transmission events. MDR and extensively drug-resistant (XDR) TB cases were almost exclusively infected by lineage 2/Beijing strains (14/16, P < 0.001). We further showed that recent transmission and/or recent introduction of lineage 2/Beijing strains contribute to high XDR-TB rates among all MDR-TB cases and should be considered an emerging threat for TB control in Tehran. In addition, the extensive pre-existing drug resistance profiles of MDR/XDR strains will further challenge TB diagnostics in the region.