Mamoudou Maiga

Efflux Inhibition with Verapamil Potentiates Bedaquiline in Mycobacterium tuberculosis

ABSTRACT Drug efflux is an important resistance mechanism in Mycobacterium tuberculosis. We found that verapamil, an efflux inhibitor, profoundly decreases the MIC of bedaquiline and clofazimine to M. tuberculosis by 8- to 16-fold. This exquisite susceptibility was noted among drug-susceptible and drug-resistant clinical isolates. Thus, efflux inhibition is an important sensitizer of bedaquiline and clofazimine, and efflux may emerge as a resistance mechanism to these drugs.

Failure to Recognize Nontuberculous Mycobacteria Leads to Misdiagnosis of Chronic Pulmonary Tuberculosis

Background Nontuberculous mycobacterial (NTM) infections cause morbidity worldwide. They are difficult to diagnose in resource-limited regions, and most patients receive empiric treatment for tuberculosis (TB). Our objective here is to evaluate the potential impact of NTM diseases among patients treated presumptively for tuberculosis in Mali. Methods We re-evaluated sputum specimens among patients newly diagnosed with TB (naïve) and those previously treated for TB disease (chronic cases). Sputum microscopy, culture and Mycobacterium tuberculosis drug susceptibility testing were performed. Identification of strains was performed using molecular probes or sequencing of secA1 and/or 16S rRNA genes. Results Of 142 patients enrolled, 61 (43%) were clinically classified as chronic cases and 17 (12%) were infected with NTM. Eleven of the 142 (8%) patients had NTM disease alone (8 M. avium, 2 M. simiae and 1 M. palustre). All these 11 were from the chronic TB group, comprising 11/61 (18%) of that group and all were identified as candidates for second line treatment. The remaining 6/17 (35.30%) NTM infected patients had coinfection with M. tuberculosis and all 6 were from the TB treatment naïve group. These 6 were candidates for the standard first line treatment regimen of TB. M. avium was identified in 11 of the 142 (8%) patients, only 3/11 (27.27%) of whom were HIV positive. Conclusions NTM infections should be considered a cause of morbidity in TB endemic environments especially when managing chronic TB cases to limit morbidity and provide appropriate treatment.

Gene Expression of Mycobacterium tuberculosis Putative Transcription Factors whiB1-7 in Redox Environments

The seven WhiB proteins of Mycobacterium tuberculosis (M.tb) are widely believed to be redox-sensing transcription factors due to their binding of iron-sulfur clusters and similarities to DNA binding proteins. Here, we explored the nature of this hypothesized relationship. We exposed M.tb to physiologic conditions such as gradual hypoxia, nitric oxide (NO), cyclic AMP and in vivo conditions, and measured transcription of the whiB genes. We found whiB3 to be induced both by hypoxia and NO, whiB7 to be induced in macrophage-like cells, and whiB4 to be induced in mouse lung. Cyclic AMP induced whiB1,−2, −4, −6 and −7. Our data indicate that the M.tb whiB genes are induced independently by various stimuli which may add versatility to their suggested redox-sensing properties.

Successful Shortening of Tuberculosis Treatment Using Adjuvant Host-Directed Therapy with FDA-Approved Phosphodiesterase Inhibitors in the Mouse Model

Global control of tuberculosis (TB), an infectious disease that claims nearly 2 million lives annually, is hindered by the long duration of chemotherapy required for curative treatment. Lack of adherence to this intense treatment regimen leads to poor patient outcomes, development of new or additional drug resistance, and continued spread of M.tb. within communities. Hence, shortening the duration of TB therapy could increase drug adherence and cure in TB patients. Here, we report that addition of the United Stated Food and Drug Administration-approved phosphodiesterase inhibitors (PDE-Is) cilostazol and sildenafil to the standard TB treatment regimen reduces tissue pathology, leads to faster bacterial clearance and shortens the time to lung sterilization by one month, compared to standard treatment alone, in a murine model of TB. Our data suggest that these PDE-Is could be repurposed for use as adjunctive drugs to shorten TB treatment in humans.

Risk of Tuberculosis Reactivation with Tofacitinib (CP-690550)

Individuals with latent tuberculosis infection (LTBI) live with a risk of reactivation, and several treatments for chronic inflammatory conditions are highly associated with such reactivation. A new Janus kinase inhibitor, tofacitinib (CP-690550), has shown promising results for treatment of inflammatory disorders, thus raising concerns of risk of active tuberculosis. Our goal was to characterize the impact of tofacitinib on LTBI using a mouse model of contained tuberculosis. Our data indicate that tofacitinib reduces host containment of Mycobacterium tuberculosis and promotes bacterial replication in the lungs, suggesting tuberculosis reactivation. Tofacitinib may carry a significant risk for LTBI reactivation in humans.

Adjuvant Host-Directed Therapy with Types 3 and 5 but Not Type 4 Phosphodiesterase Inhibitors Shortens the Duration of Tuberculosis Treatment

BACKGROUND Shortening tuberculosis treatment could significantly improve patient adherence and decrease the development of drug resistance. Phosphodiesterase inhibitors (PDE-Is) have been shown to be beneficial in animal models of tuberculosis. We assessed the impact of PDE-Is on the duration of treatment in tuberculous mice. METHODS We analyzed the time to death in Mycobacterium tuberculosis-infected mice receiving type 4 PDE-Is (rolipram and cilomilast) and the impact on bacterial burden, time to clearance, and relapse when types 3 and 5 PDE-Is (cilostazol and sildenafil, respectively) and rolipram were added to the standard treatment. We investigated pharmacokinetic interactions between PDE-Is (cilostazol and sildenafil) and rifampin. RESULTS The type 4 PDE-Is rolipram and cilomilast accelerated the time to death in tuberculous mice. The addition of rolipram to standard tuberculosis treatment increased bacterial burden and did not decrease the time to bacterial clearance in the lung, while the addition of the cilostazol and sildenafil reduced the time to clearance by 1 month. Cilostazol and sildenafil did not have negative pharmacokinetic interactions with rifampin. CONCLUSIONS Type 4 PDE-Is may increase the severity of tuberculosis and should be carefully investigated for use in patients with latent or active tuberculosis. Cilostazol and sildenafil may benefit tuberculosis patients by shortening the duration of therapy.

Reporter Phage and Breath Tests: Emerging Phenotypic Assays for Diagnosing Active Tuberculosis, Antibiotic Resistance, and Treatment Efficacy

The rapid and accurate diagnosis of active tuberculosis (TB) and its drug susceptibility remain a challenge. Phenotypic assays allow determination of antibiotic susceptibilities even if sequence data are not available or informative. We review 2 emerging diagnostic approaches, reporter phage and breath tests, both of which assay mycobacterial metabolism. The reporter phage signal, Green fluorescent protein (GFP) or β-galactosidase, indicates transcription and translation inside the recipient bacilli and its attenuation by antibiotics. Different breath tests assay, (1) exhaled antigen 85, (2) mycobacterial urease activity, and (3) detection by trained rats of disease-specific odor in sputum, have also been developed. When compared with culture, reporter phage assays shorten the time for initial diagnosis of drug susceptibility by several days. Both reporter phage and breath tests have promise as early markers to determine the efficacy of treatment. While sputum often remains smear and Mycobacterium tuberculosis DNA positive early in the course of efficacious antituberculous treatment, we predict that both breath and phage tests will rapidly become negative. If this hypothesis proves correct, phage assays and breath tests could become important surrogate markers in early bactericidal activity (EBA) studies of new antibiotics.

Small molecule-directed immunotherapy against recurrent infection by Mycobacterium tuberculosis.

Background: M. tuberculosis evades host-immune-responses by polarizing T helper (Th)2 and regulatory T cell (Treg) responses, which diminish protective Th1 responses. Results: Mice that are unable to generate Th2 cells and Tregs are resistant to M. tuberculosis infection. Simultaneous inhibition of these T cell subsets by therapeutic compounds dramatically reduced bacterial burden. Conclusion: Inhibition of Th2 and Treg cells increases Th1 responses that protect against M. tuberculosis infection. Significance: As therapeutic agents employed here do not directly act on harbored pathogens, they should avoid generation of drug-resistant M. tuberculosis variants. Tuberculosis remains the biggest infectious threat to humanity with one-third of the population infected and 1.4 million deaths and 8.7 million new cases annually. Current tuberculosis therapy is lengthy and consists of multiple antimicrobials, which causes poor compliance and high treatment dropout, resulting in the development of drug-resistant variants of tuberculosis. Therefore, alternate methods to treat tuberculosis are urgently needed. Mycobacterium tuberculosis evades host immune responses by inducing T helper (Th)2 and regulatory T (Treg) cell responses, which diminish protective Th1 responses. Here, we show that animals (Stat-6−/−CD4-TGFβRIIDN mice) that are unable to generate both Th2 cells and Tregs are highly resistant to M. tuberculosis infection. Furthermore, simultaneous inhibition of these two subsets of Th cells by therapeutic compounds dramatically reduced bacterial burden in different organs. This treatment was associated with the generation of protective Th1 immune responses. As these therapeutic agents are not directed to the harbored organisms, they should avoid the risk of promoting the development of drug-resistant M. tuberculosis variants.

Molecular strain typing of Mycobacterium tuberculosis complex in Bamako, Mali

OBJECTIVE To identify strains of Mycobacterium tuberculosis complex (MTC) circulating in Bamako and to examine the relationship between the strains and their drug susceptibility profiles. METHODS Between 2006 and 2010, we conducted a cross-sectional study using spoligotyping to identify strains of MTC recovered from 126 tuberculosis (TB) patients under treatment in Bamako, Mali. RESULT Three members of the MTC were isolated: M. tuberculosis (71.4%), M. africanum (27.8%) and M. bovis (0.8%). Of these, three strains were found to be the most prevalent: M. tuberculosis T1 (MTB T1; 38.9%), M. africanum F2 (MAF2; 26.2%) and M. tuberculosis Latin American and Mediterranean 10 (MTB LAM 10; 10.3%). MAF2 and MTB LAM 10 strains have a lower risk of multidrug resistance (MDR) than MTB T1 (respectively OR 0.1, 95%CI 0.03-0.4 and OR 0.1, 95%CI 0.01-0.8). Age ≥ 32 years (OR 1.4, 95%CI 0.4-3.9), negative human immunodeficiency virus status (OR 0.4, 95%CI 0.1-2.5) and male sex (OR 4, 95%CI 0.9-16.5) were not associated with MDR. The prevalence of MDR among treatment and retreatment failure patients was respectively 25% and 81.8% compared to new patients (2.9%). CONCLUSION This study indicates a low level of primary drug resistance in Bamako, affirms the importance of using correct drug regimens, and suggests that the MTB T1 strain may be associated with the development of resistance.

Whole Genome Sequencing of Mycobacterium africanum Strains from Mali Provides Insights into the Mechanisms of Geographic Restriction

Background Mycobacterium africanum, made up of lineages 5 and 6 within the Mycobacterium tuberculosis complex (MTC), causes up to half of all tuberculosis cases in West Africa, but is rarely found outside of this region. The reasons for this geographical restriction remain unknown. Possible reasons include a geographically restricted animal reservoir, a unique preference for hosts of West African ethnicity, and an inability to compete with other lineages outside of West Africa. These latter two hypotheses could be caused by loss of fitness or altered interactions with the host immune system. Methodology/Principal Findings We sequenced 92 MTC clinical isolates from Mali, including two lineage 5 and 24 lineage 6 strains. Our genome sequencing assembly, alignment, phylogeny and average nucleotide identity analyses enabled us to identify features that typify lineages 5 and 6 and made clear that these lineages do not constitute a distinct species within the MTC. We found that in Mali, lineage 6 and lineage 4 strains have similar levels of diversity and evolve drug resistance through similar mechanisms. In the process, we identified a putative novel streptomycin resistance mutation. In addition, we found evidence of person-to-person transmission of lineage 6 isolates and showed that lineage 6 is not enriched for mutations in virulence-associated genes. Conclusions This is the largest collection of lineage 5 and 6 whole genome sequences to date, and our assembly and alignment data provide valuable insights into what distinguishes these lineages from other MTC lineages. Lineages 5 and 6 do not appear to be geographically restricted due to an inability to transmit between West African hosts or to an elevated number of mutations in virulence-associated genes. However, lineage-specific mutations, such as mutations in cell wall structure, secretion systems and cofactor biosynthesis, provide alternative mechanisms that may lead to host specificity.