The Many Hosts of Mycobacteria 9 (MHM9): A conference report.

The Many Hosts of Mycobacteria (MHM) meeting series brings together basic scientists, clinicians and veterinarians to promote robust discussion and dissemination of recent advances in our knowledge of numerous mycobacterial diseases, including human and bovine tuberculosis (TB), nontuberculous mycobacteria (NTM) infection, Hansen's disease (leprosy), Buruli ulcer and Johne's disease. The 9th MHM conference (MHM9) was held in July 2022 at The Ohio State University (OSU) and centered around the theme of "Confounders of Mycobacterial Disease." Confounders can and often do drive the transmission of mycobacterial diseases, as well as impact surveillance and treatment outcomes. Various confounders were presented and discussed at MHM9 including those that originate from the host (comorbidities and coinfections) as well as those arising from the environment (e.g., zoonotic exposures), economic inequality (e.g. healthcare disparities), stigma (a confounder of leprosy and TB for millennia), and historical neglect (a confounder in Native American Nations). This conference report summarizes select talks given at MHM9 highlighting recent research advances, as well as talks regarding the historic and ongoing impact of TB and other infectious diseases on Native American Nations, including those in Southwestern Alaska where the regional TB incidence rate is among the highest in the Western hemisphere.

Quantitative Analysis of the Phase Transition Mechanism Underpinning the Systemic Self-Assembly of a Mechanopharmaceutical Device.

Clofazimine (CFZ) is a poorly soluble, weakly basic, small molecule antibiotic clinically used to treat leprosy and is now in clinical trials as a treatment for multidrug resistant tuberculosis and COVID-19. CFZ exhibits complex, context-dependent pharmacokinetics that are characterized by an increasing half-life in long term treatment regimens. The systemic pharmacokinetics of CFZ have been previously represented by a nonlinear, 2-compartment model incorporating an expanding volume of distribution. This expansion reflects the soluble-to-insoluble phase transition that the drug undergoes as it precipitates out and accumulates within macrophages disseminated throughout the organism. Using mice as a model organism, we studied the mechanistic underpinnings of this increasing half-life and how the systemic pharmacokinetics of CFZ are altered with continued dosing. To this end, M. tuberculosis infection status and multiple dosing schemes were studied alongside a parameter sensitivity analysis (PSA) to further understanding of systemic drug distribution. Parameter values governing the sigmoidal expansion function that captures the phase transition were methodically varied, and in turn, the systemic concentrations of the drug were calculated and compared to the experimentally measured concentrations of drug in serum and spleen. The resulting amounts of drug sequestered were dependent on the total mass of CFZ administered and the duration of drug loading. This phenomenon can be captured by altering three different parameters of an expansion function corresponding to key biological determinants responsible for the precipitation and the accumulation of the insoluble drug mass in macrophages. Through this analysis of the context dependent pharmacokinetics of CFZ, a predictive framework for projecting the systemic distribution and self-assembly of precipitated drug complexes as intracellular mechanopharmaceutical devices of this and other drugs exhibiting similarly complex pharmacokinetics can be constructed.

An Adaptive Biosystems Engineering Approach towards Modeling the Soluble-to-Insoluble Phase Transition of Clofazimine.

Clofazimine (CFZ) is a weakly basic, small-molecule antibiotic used for the treatment of mycobacterial infections including leprosy and multidrug-resistant tuberculosis. Upon prolonged oral administration, CFZ precipitates and accumulates within macrophages throughout the host. To model the pharmacokinetics of CFZ, the volume of distribution (Vd) was considered as a varying parameter that increases with continuous drug loading. Fitting the time-dependent change in drug mass and concentration data obtained from CFZ-treated mice, we performed a quantitative analysis of the systemic disposition of the drug over a 20-week treatment period. The pharmacokinetics data were fitted using various classical compartmental models sampling serum and spleen concentration data into separate matrices. The models were constructed in NONMEM together with linear and nonlinear sigmoidal expansion functions to the spleen compartment to capture the phase transition in Vd. The different modeling approaches were compared by Akaike information criteria, observed and predicted concentration correlations, and graphically. Using the composite analysis of the modeling predictions, adaptive fractional CFZ sequestration, Vd and half-life were evaluated. When compared to standard compartmental models, an adaptive Vd model yielded a more accurate data fit of the drug concentrations in both the serum and spleen. Including a nonlinear sigmoidal equation into compartmental models captures the phase transition of drugs such as CFZ, greatly improving the prediction of population pharmacokinetics and yielding further insight into the mechanisms of drug disposition.

Clofazimine has delayed antimicrobial activity against Mycobacterium tuberculosis both in vitro and in vivo.

OBJECTIVES: The anti-leprosy drug clofazimine has been shown to have antimicrobial activity against Mycobacterium tuberculosis and has been associated with treatment-shortening activity in both clinical and preclinical studies of TB chemotherapy. However, a reported lack of early bactericidal activity (EBA) in TB patients has raised questions regarding the usefulness of clofazimine as an anti-TB drug. Our objective was to systematically evaluate the EBA of clofazimine in vitro and in vivo to provide insight into how and when this drug exerts its antimicrobial activity against M. tuberculosis. METHODS: We evaluated the 14 day EBA of clofazimine (i) in vitro at concentrations ranging from 4 times below to 4 times above the MIC for M. tuberculosis and (ii) in vivo in infected BALB/c mice at doses ranging from 1.5 to 100 mg/kg/day, and serum clofazimine levels were measured. In both experiments, isoniazid was used as the positive control. RESULTS: In vitro, clofazimine, at any concentration tested, did not exhibit bactericidal activity during the first week of exposure; however, in the second week, it exhibited concentration-dependent antimicrobial activity. In vivo, clofazimine, at any dose administered, did not exhibit bactericidal activity during the first week, and limited antimicrobial activity was observed during the second week of administration. While serum clofazimine levels were clearly dose dependent, the antimicrobial activity was not significantly related to the dose administered. CONCLUSIONS: Our data suggest that clofazimine's delayed antimicrobial activity may be due more to its mechanism of action rather than to host-related factors.

Clofazimine shortens the duration of the first-line treatment regimen for experimental chemotherapy of tuberculosis.

A key drug for the treatment of leprosy, clofazimine has recently been associated with highly effective and significantly shortened regimens for the treatment of multidrug-resistant tuberculosis (TB). Consequently, we hypothesized that clofazimine may also shorten the duration of treatment for drug-susceptible TB. We conducted a controlled trial in the mouse model of TB chemotherapy comparing the activity of the 6-mo standard regimen for TB treatment, i.e., 2 mo of daily rifampin, isoniazid, pyrazinamide, and ethambutol followed by 4 mo of rifampin and isoniazid, with a 4-mo clofazimine-containing regimen: 2 mo of daily rifampin, isoniazid, pyrazinamide, and clofazimine followed by 2 mo of rifampin, isoniazid, and clofazimine. Treatment efficacy was assessed on the basis of Mycobacterium tuberculosis colony counts in the lungs and spleens during treatment and on the proportion of mice with culture-positive relapse 6 mo after treatment cessation. No additive effect of clofazimine was observed after the first week of treatment, but, by the second week of treatment, the colony counts were significantly lower in the clofazimine-treated mice than in the mice receiving the standard regimen. Lung culture conversion was obtained after 3 and 5 mo in mice treated with the clofazimine-containing and standard regimens, respectively, and relapse-free cure was obtained after 3 and 6 mo of treatment with the clofazimine-containing and standard regimens, respectively. Thus, clofazimine is a promising anti-TB drug with the potential to shorten the duration of TB chemotherapy by at least half (3 mo vs. 6 mo) in the mouse model of TB.

Microbiological, histological, immunological, and toxin response to antibiotic treatment in the mouse model of Mycobacterium ulcerans disease.

Mycobacterium ulcerans infection causes a neglected tropical disease known as Buruli ulcer that is now found in poor rural areas of West Africa in numbers that sometimes exceed those reported for another significant mycobacterial disease, leprosy, caused by M. leprae. Unique among mycobacterial diseases, M. ulcerans produces a plasmid-encoded toxin called mycolactone (ML), which is the principal virulence factor and destroys fat cells in subcutaneous tissue. Disease is typically first manifested by the appearance of a nodule that eventually ulcerates and the lesions may continue to spread over limbs or occasionally the trunk. The current standard treatment is 8 weeks of daily rifampin and injections of streptomycin (RS). The treatment kills bacilli and wounds gradually heal. Whether RS treatment actually stops mycolactone production before killing bacilli has been suggested by histopathological analyses of patient lesions. Using a mouse footpad model of M. ulcerans infection where the time of infection and development of lesions can be followed in a controlled manner before and after antibiotic treatment, we have evaluated the progress of infection by assessing bacterial numbers, mycolactone production, the immune response, and lesion histopathology at regular intervals after infection and after antibiotic therapy. We found that RS treatment rapidly reduced gross lesions, bacterial numbers, and ML production as assessed by cytotoxicity assays and mass spectrometric analysis. Histopathological analysis revealed that RS treatment maintained the association of the bacilli with (or within) host cells where they were destroyed whereas lack of treatment resulted in extracellular infection, destruction of host cells, and ultimately lesion ulceration. We propose that RS treatment promotes healing in the host by blocking mycolactone production, which favors the survival of host cells, and by killing M. ulcerans bacilli.

BCG-mediated protection against Mycobacterium ulcerans infection in the mouse.

BACKGROUND: Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) is widely used to reduce the risk of childhood tuberculosis and has been reported to have efficacy against two other mycobacterial diseases, leprosy and Buruli ulcer caused by M. ulcerans (Mu). Studies in experimental models have also shown some efficacy against infection caused by Mu. In mice, most studies use the C57BL/6 strain that is known to develop good cell-mediated protective immunity. We hypothesized that there may be differences in vaccination efficacy between C57BL/6 and the less resistant BALB/c strain. METHODS: We evaluated BCG vaccine efficacy against challenge with ∼3×10(5)M. ulcerans in the right hind footpad using three strains: initially, the Australian type strain, designated Mu1617, then, a Malaysian strain, Mu1615, and a recent Ghanaian isolate, Mu1059. The latter two strains both produce mycolactone while the Australian strain has lost that capacity. CFU of both BCG and Mu and splenocyte cytokine production were determined at intervals after infection. Time to footpad swelling was assessed weekly. PRINCIPAL FINDINGS: BCG injection induced visible scars in 95.5% of BALB/c mice but only 43.4% of C57BL/6 mice. BCG persisted at higher levels in spleens of BALB/c than C57BL/6 mice. Vaccination delayed swelling and reduced Mu CFU in BALB/c mice, regardless of challenge strain. However, vaccination was only protective against Mu1615 and Mu1617 in C57BL/6 mice. Possible correlates of the better protection of BALB/c mice included 1) the near universal development of BCG scars in these mice compared to less frequent and smaller scars observed in C57BL/6 mice and 2) the induction of sustained cytokine, e.g., IL17, production as detected in the spleens of BALB/c mice whereas cytokine production was significantly reduced, e.g., IL17, or transient, e.g., Ifnγ, in the spleens of C57BL/6 mice. CONCLUSIONS: The efficacy of BCG against M. ulcerans, in particular, and possibly mycobacteria in general, may vary due to differences in both host and pathogen.