The host-directed therapeutic imatinib mesylate accelerates immune responses to Mycobacterium marinum infection and limits pathology associated with granulomas.

Infections caused by members of the mycobacterium tuberculosis complex [MTC] and nontuberculous mycobacteria [NTM] can induce widespread morbidity and mortality in people. Mycobacterial infections cause both a delayed immune response, which limits rate of bacterial clearance, and formation of granulomas, which contain bacterial spread, but also contribute to lung damage, fibrosis, and morbidity. Granulomas also limit access of antibiotics to bacteria, which may facilitate development of resistance. Bacteria resistant to some or all antibiotics cause significant morbidity and mortality, and newly developed antibiotics readily engender resistance, highlighting the need for new therapeutic approaches. Imatinib mesylate, a cancer drug used to treat chronic myelogenous leukemia [CML] that targets Abl and related tyrosine kinases, is a possible host-directed therapeutic [HDT] for mycobacterial infections, including those causing TB. Here, we use the murine Mycobacterium marinum [Mm] infection model, which induces granulomatous tail lesions. Based on histological measurements, imatinib reduces both lesion size and inflammation of surrounding tissue. Transcriptomic analysis of tail lesions indicates that imatinib induces gene signatures indicative of immune activation and regulation at early time points post infection that resemble those seen at later ones, suggesting that imatinib accelerates but does not substantially alter anti-mycobacterial immune responses. Imatinib likewise induces signatures associated with cell death and promotes survival of bone marrow-derived macrophages [BMDMs] in culture following infection with Mm. Notably, the capacity of imatinib to limit formation and growth of granulomas in vivo and to promote survival of BMDMs in vitro depends upon caspase 8, a key regulator of cell survival and death. These data provide evidence for the utility of imatinib as an HDT for mycobacterial infections in accelerating and regulating immune responses, and limiting pathology associated with granulomas, which may mitigate post-treatment morbidity.

Small-Molecule Inhibitors Targeting Topoisomerase I as Novel Antituberculosis Agents.

Bacterial topoisomerase functions are required for regulation of DNA supercoiling and overcoming the DNA topological barriers that are encountered during many vital cellular processes. DNA gyrase and topoisomerase IV of the type IIA bacterial topoisomerase family are important clinical targets for antibacterial therapy. Topoisomerase I, belonging to the type IA topoisomerase family, has recently been validated as a potential antitubercular target. The topoisomerase I activity has been shown to be essential for bacterial viability and infection in a murine model of tuberculosis. Mixture-based combinatorial libraries were screened in this study to identify novel bacterial topoisomerase I inhibitors. Using positional-scanning deconvolution, selective small-molecule inhibitors of bacterial topoisomerase I were identified starting from a polyamine scaffold. Antibacterial assays demonstrated that four of these small-molecule inhibitors of bacterial topoisomerase I are bactericidal against Mycobacterium smegmatis and Mycobacterium tuberculosis The MICs for growth inhibition of M. smegmatis increased with overexpression of recombinant M. tuberculosis topoisomerase I, consistent with inhibition of intracellular topoisomerase I activity being involved in the antimycobacterial mode of action.

Predictive value of molecular drug resistance testing of Mycobacterium tuberculosis isolates in Valle del Cauca, Colombia.

Previous evaluations of the molecular GenoType tests have promoted their use to detect resistance to first- and second-line antituberculosis drugs in different geographical regions. However, there are known geographic variations in the mutations associated with drug resistance in Mycobacterium tuberculosis, and especially in South America, there is a paucity of information regarding the frequencies and types of mutations associated with resistance to first- and second-line antituberculosis drugs. We therefore evaluated the performance of the GenoType kits in this region by testing 228 M. tuberculosis isolates in Colombia, including 134 resistant and 94 pansusceptible strains. Overall, the sensitivity and specificity of the GenoType MTBDRplus test ranged from 92 to 96% and 97 to 100%, respectively; the agreement index was optimal (Cohen's kappa, >0.8). The sensitivity of the GenoType MTBDRsl test ranged from 84 to 100% and the specificity from 88 to 100%. The most common mutations were katG S315T1, rpoB S531L, embB M306V, gyrA D94G, and rrs A1401G. Our results reflect the utility of the GenoType tests in Colombia; however, as some discordance still exists between the conventional and molecular approaches in resistance testing, we adhere to the recommendation that the GenoType tests serve as early guides for therapy, followed by phenotypic drug susceptibility testing for all cases.

Factors associated with unsuccessful treatment outcome for tuberculosis in previously treated patients in Cali, Colombia, during the period 2015-2019 / Factores asociados al tratamiento no exitoso para tuberculosis en pacientes previamente tratados en Cali, Colombia, en el periodo 2015-2019

Introduction. The success rates in the treatment of tuberculosis are suboptimal. Objective. To identify associated factors with the lack of success of antituberculosis treatment in patients with a tuberculosis treatment history. Materials and methods. We performed a retrospective, analytical, observational, and cohort study of patients reentering the Mycobacterium program in Cali, Colombia. We included patients over 15 years old with pulmonary tuberculosis between 2015 and 2019 and a history of tuberculosis treatment. Patients with drug-resistant tuberculosis were excluded. Results. A total of 605 patients with a treatment history were included, 60% due to unfinished treatment and 40% due to relapse. Compared to patients reentering due to relapse (ORa=2.34, CI=1.62-3.38), the independent variables associated with treatment failure at discharge were homelessness (ORa=2.45, CI=1.54-3.89), substance dependence (ORa=1.95, CI=1.24-3.05), tuberculosis/HIV coinfection (ORa=1.69, CI=1.00-2.86), diabetes (ORa=1.89, CI=1.29-2.77), and unfinished previous tuberculosis treatment due to follow-up loss, abandonment, or other causes. Programmatic variables favoring treatment success were voluntary HIV testing counseling (p<0.001) and HIV testing (p<0.001). Conclusion. Homelessness, substance dependence, tuberculosis/HIV coinfection, diabetes, and incomplete previous treatment due to loss to follow-up, abandonment, or treatment failure hindered the success of antituberculosis. These characteristics should be identified and addressed during the initial care of patients reentering treatment for tuberculosis.

Introducción. Las tasas de éxito del tratamiento de la tuberculosis continúan siendo subóptimas. Objetivo. Identificar los factores asociados al tratamiento no exitoso para tuberculosis en pacientes con antecedentes de tratamiento para la tuberculosis. Materiales y métodos. Se realizó un estudio observacional retrospectivo, analítico, de cohorte de pacientes que reingresaron a un programa de micobacterias en Cali, Colombia. Se incluyeron mayores de 15 años con tuberculosis pulmonar entre el 2015 y el 2019 con antecedentes de tratamiento para la tuberculosis. Se excluyeron los pacientes con tuberculosis resistente. Resultados. Ingresaron 605 pacientes con antecedentes de tratamiento, 60 % por tratamiento inconcluso y 40 % por recaída. En comparación con los pacientes que reingresaron por recaída (ORa= 2,34; IC=1,62-3,38), las variables que explicaron de manera independiente el no tener éxito con el tratamiento para la tuberculosis al egreso fueron: estar en situación de calle (ORa = 2,45; IC = 1,54-3,89), ser farmacodependiente (ORa = 1,95; IC=1,24-3,05), tener coinfección tuberculosis/VIH (ORa = 1,69; IC =1,00-2,86) o diabetes (ORa =1,89; IC=1,29-2,77), y el incumplimiento de un tratamiento previo por pérdida de seguimiento, abandono u otras causas. Las variables programáticas que favorecieron el éxito del tratamiento fueron la asesoría de la prueba voluntaria de VIH (p <0,001) y la realización de la prueba de VIH (p < 0,001). Conclusión. Estar en situación de calle, ser farmacodependiente, tener coinfección de tuberculosis y VIH, o diabetes, así como el incumplimiento del tratamiento previo por pérdida del seguimiento, abandono o fracaso del mismo, dificultaron el éxito del tratamiento antituberculoso. En la primera atención al reingreso de los pacientes con tuberculosis se deben identificar y abordar estas características.

Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4.

Only about half the multi-drug resistant tuberculosis (MDR-TB) cases are successfully cured. Thus, there is an urgent need of new TB treatment against a novel target. Mycobacterium tuberculosis (Mtb) topoisomerase I (TopA) is the only type IA topoisomerase in this organism and has been validated as an essential target for TB drug discovery. Toxin-antitoxin (TA) systems participate as gene regulators within bacteria. The TA systems contribute to the long-term dormancy of Mtb within the host-cell environment. Mtb's toxin MazF4 (Rv1495) that is part of the MazEF4 TA system has been shown to have dual activities as endoribonuclease and topoisomerase I inhibitor. We have developed a complementary assay using an Escherichia coli strain with temperature-sensitive topA mutation to provide new insights into the MazF4 action. The assay showed that E. coli is not sensitive to the endoribonuclease activity of Mtb MazF4 but became vulnerable to MazF4 growth inhibition when recombinant Mtb TopA relaxation activity is required for growth. Results from the complementation by Mtb TopA mutants with C-terminal deletions showed that the lysine-rich C-terminal tail is required for interaction with MazF4. Site-directed mutagenesis is utilized to identify two lysine residues within a conserved motif in this C-terminal tail that are critical for MazF4 inhibition. We performed molecular dynamics simulations to predict the Mtb TopA-MazF4 complex. Our simulation results show that the complex is stabilized by hydrogen bonds and electrostatic interactions established by residues in the TopA C-terminal tail including the two conserved lysines. The mechanism of Mtb TopA inhibition by MazF4 could be useful for the discovery of novel inhibitors against a new antibacterial target in pathogenic mycobacteria for treatment of both TB and diseases caused by the non-tuberculosis mycobacteria (NTM).

Identification of Nontuberculous Mycobacteria in Drinking Water in Cali, Colombia.

Nontuberculous mycobacteria (NTM) are ubiquitous microorganisms naturally resistant to antibiotics and disinfectants that can colonize drinking water supply systems. Information regarding the spread of NTM in specifically South America and Colombia is limited. We aimed to identify and characterize NTM present in tap water samples from Cali, Colombia. Drinking water samples and faucet biofilm swabs were collected in 18 places, including the city's three main water treatment plants (WTPs). Filter-trapped material and eluates (0.45 µm) from swab washes were plated in 7H11 agar plates. Suspected colonies were evaluated microscopically, and NTM species were identified based on the rpoB gene. Antibiotic susceptibility testing was also performed. Fifty percent (9/18) of sampling points were positive for NTM (including two WTPs), from which 16 different isolates were identified: Mycobacterium mucogenicum (8/16), M. phocaicum (3/16), M. chelonae (2/16), M. mageritense (2/16), and M. fortuitum (1/16), all rapidly growing mycobacteria. A susceptibility profile was obtained from 68.75% (11/16) of the isolates. M. chelonae was the most resistant species. All NTM isolated are potentially responsible for human diseases; our findings might provide a baseline for exploring NTM transmission dynamics and clinical characterization, as well as potential associations between NTM species found in drinking water and isolates from patients.

Mechanism and resistance for antimycobacterial activity of a fluoroquinophenoxazine compound.

We have previously reported the inhibition of bacterial topoisomerase I activity by a fluoroquinophenoxazine compound (FP-11g) with a 6-bipiperidinyl lipophilic side chain that exhibited promising antituberculosis activity (MIC = 2.5 µM against Mycobacterium tuberculosis, SI = 9.8). Here, we found that the compound is bactericidal towards Mycobacterium smegmatis, resulting in greater than 5 Log10 reduction in colony-forming units [cfu]/mL following a 10 h incubation at 1.25 µM (4X MIC) concentration. Growth inhibition (MIC = 50 µM) and reduction in cfu could also be observed against a clinical isolate of Mycobacterium abscessus. Stepwise isolation of resistant mutants of M. smegmatis was conducted to explore the mechanism of resistance. Mutations in the resistant isolates were identified by direct comparison of whole-genome sequencing data from mutant and wild-type isolates. These include mutations in genes likely to affect the entry and retention of the compound. FP-11g inhibits Mtb topoisomerase I and Mtb gyrase with IC50 of 0.24 and 27 µM, respectively. Biophysical analysis showed that FP-11g binds DNA as an intercalator but the IC50 for inhibition of Mtb topoisomerase I activity is >10 fold lower than the compound concentrations required for producing negatively supercoiled DNA during ligation of nicked circular DNA. Thus, the DNA-binding property of FP-11g may contribute to its antimycobacterial mechanism, but that alone cannot account for the observed inhibition of Mtb topoisomerase I.