Spectinamide MBX-4888A exhibits favorable lesion and tissue distribution and promotes treatment shortening in advanced murine models of tuberculosis.

The spectinamides are novel, narrow-spectrum semisynthetic analogs of spectinomycin, modified to avoid intrinsic efflux by Mycobacterium tuberculosis. Spectinamides, including lead MBX-4888A (Lee-1810), exhibit promising therapeutic profiles in mice, as single drugs and as partner agents with other anti-tuberculosis antibiotics including rifampin and/or pyrazinamide. Here, we show that MBX-4888A, given by injection with the front-line standard of care regimen, is treatment shortening in multiple murine tuberculosis infection models. The positive treatment responses to MBX-4888A combination therapy in multiple mouse models, including mice exhibiting advanced pulmonary disease, can be attributed to favorable distribution in tissues and lesions, retention in caseum, along with favorable effects with rifampin and pyrazinamide under conditions achieved in necrotic lesions. This study also provides an additional data point regarding the safety and tolerability of spectinamide MBX-4888A in long-term murine efficacy studies.

Role of DNA Double-Strand Break Formation in Gyrase Inhibitor-Mediated Killing of Nonreplicating Persistent Mycobacterium tuberculosis in Caseum.

Tuberculosis is the leading cause of mortality by infectious agents worldwide. The necrotic debris, known as caseum, which accumulates in the center of pulmonary lesions and cavities is home to nonreplicating drug-tolerant Mycobacterium tuberculosis that presents a significant hurdle to achieving a fast and durable cure. Fluoroquinolones such as moxifloxacin are highly effective at killing this nonreplicating persistent bacterial population and boosting TB lesion sterilization. Fluoroquinolones target bacterial DNA gyrase, which catalyzes the negative supercoiling of DNA and relaxes supercoils ahead of replication forks. In this study, we investigated the potency of several other classes of gyrase inhibitors against M. tuberculosis in different states of replication. In contrast to fluoroquinolones, many other gyrase inhibitors kill only replicating bacterial cultures but produce negligible cidal activity against M. tuberculosis in ex vivo rabbit caseum. We demonstrate that while these inhibitors are capable of inhibiting M. tuberculosis gyrase DNA supercoiling activity, fluoroquinolones are unique in their ability to cleave double-stranded DNA at low micromolar concentrations. We hypothesize that double-strand break formation is an important driver of gyrase inhibitor-mediated bactericidal potency against nonreplicating persistent M. tuberculosis populations in the host. This study provides general insight into the lesion sterilization potential of different gyrase inhibitor classes and informs the development of more effective chemotherapeutic options against persistent mycobacterial infections.

ADP-ribosylation-resistant rifabutin analogs show improved bactericidal activity against drug-tolerant M. abscessus in caseum surrogate.

Necrotic lesions and cavities filled with caseum are a hallmark of mycobacterial pulmonary disease. Bronchocavitary Mycobacterium abscessus disease is associated with poor treatment outcomes. In caseum surrogate, M. abscessus entered an extended stationary phase showing tolerance to killing by most current antibiotics, suggesting that caseum persisters contribute to the poor performance of available treatments. Novel ADP-ribosylation-resistant rifabutin analogs exhibited bactericidal activity against these M. abscessus persisters at concentrations achievable by rifamycins in caseum.

A Novel Tool to Identify Bactericidal Compounds against Vulnerable Targets in Drug-Tolerant M. tuberculosis found in Caseum.

Caseous necrosis is a hallmark of tuberculosis (TB) pathology and creates a niche for drug-tolerant persisters within the host. Cavitary TB and high bacterial burden in caseum require longer treatment duration. An in vitro model that recapitulates the major features of Mycobacterium tuberculosis (Mtb) in caseum would accelerate the identification of compounds with treatment-shortening potential. We have developed a caseum surrogate model consisting of lysed and denatured foamy macrophages. Upon inoculation of Mtb from replicating cultures, the pathogen adapts to the lipid-rich matrix and gradually adopts a nonreplicating state. We determined that the lipid composition of ex vivo caseum and the surrogate matrix are similar. We also observed that Mtb in caseum surrogate accumulates intracellular lipophilic inclusions (ILI), a distinctive characteristic of quiescent and drug-tolerant Mtb. Expression profiling of a representative gene subset revealed common signatures between the models. Comparison of Mtb drug susceptibility in caseum and caseum surrogate revealed that both populations are similarly tolerant to a panel of TB drugs. By screening drug candidates in the surrogate model, we determined that the bedaquiline analogs TBAJ876 and TBAJ587, currently in clinical development, exhibit superior bactericidal against caseum-resident Mtb, both alone and as substitutions for bedaquiline in the bedaquiline-pretomanid-linezolid regimen approved for the treatment of multidrug-resistant TB. In summary, we have developed a physiologically relevant nonreplicating persistence model that reflects the distinct metabolic and drug-tolerant state of Mtb in caseum. IMPORTANCE M. tuberculosis (Mtb) within the caseous core of necrotic granulomas and cavities is extremely drug tolerant and presents a significant hurdle to treatment success and relapse prevention. Many in vitro models of nonreplicating persistence have been developed to characterize the physiologic and metabolic adaptations of Mtb and identify compounds active against this treatment-recalcitrant population. However, there is little consensus on their relevance to in vivo infection. Using lipid-laden macrophage lysates, we have designed and validated a surrogate matrix that closely mimics caseum and in which Mtb develops a phenotype similar to that of nonreplicating bacilli in vivo. The assay is well suited to screen for bactericidal compounds against caseum-resident Mtb in a medium-throughput format, allowing for reduced reliance on resource intensive animal models that present large necrotic lesions and cavities. Importantly, this approach will aid the identification of vulnerable targets in caseum Mtb and can accelerate the development of novel TB drugs with treatment-shortening potential.

10-DEBC Hydrochloride as a Promising New Agent against Infection of Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) causes chronic pulmonary infections. Its resistance to current antimicrobial drugs makes it the most difficult non-tuberculous mycobacteria (NTM) to treat with a treatment success rate of 45.6%. Therefore, there is a need for new therapeutic agents against M. abscessus. We identified 10-DEBC hydrochloride (10-DEBC), a selective AKT inhibitor that exhibits inhibitory activity against M. abscessus. To evaluate the potential of 10-DEBC as a treatment for lung disease caused by M. abscessus, we measured its effectiveness in vitro. We established the intracellular activity of 10-DEBC against M. abscessus in human macrophages and human embryonic cell-derived macrophages (iMACs). 10-DEBC significantly inhibited the growth of wild-type M. abscessus and clinical isolates and clarithromycin (CLR)-resistant M. abscessus strains. 10-DEBC's drug efficacy did not have cytotoxicity in the infected macrophages. In addition, 10-DEBC operates under anaerobic conditions without replication as well as in the presence of biofilms. The alternative caseum binding assay is a unique tool for evaluating drug efficacy against slow and nonreplicating bacilli in their native caseum media. In the surrogate caseum, the mean undiluted fraction unbound (fu) for 10-DEBC is 5.696. The results of an in vitro study on the activity of M. abscessus suggest that 10-DEBC is a potential new drug for treating M. abscessus infections.

Tonsillotomy by a Fractional Carbon Dioxide Laser: A New Technique in the Treatment of Chronic Tonsillitis.

Introduction: Chronic tonsillitis has a global prevalence, ranging from 5% to 12%. Its clinical manifestations, like recurrent acute tonsillitis, tonsils hypertrophy, caseum and halitosis, can lead adult patients to be submitted to palatine tonsillectomy, surgery that has morbidity and the potential risk of complications, including severe bleeding. This article proposes a new therapy for chronic tonsillitis in adult patients using a fractional carbon dioxide (CO2) laser, which is a fast, minimally invasive procedure capable of removing the need for the traditional tonsillectomy in many patients. The present research aimed to verify the efficacy of tonsillotomy by fractional ablation using the CO2 laser by comparing the number of bacterial infections, tonsils hypertrophy, halitosis and caseum; it is also aimed at analyzing the benefits, risks and complications of the technique. Methods: In this clinical prospective study, 20 patients were subjected to one session of tonsillotomy by fractional ablation and were followed up for a year. The control group was formed by the same patients in the pre-procedure period (one year) without treatment. Statistical analysis: The Wilcoxon paired test, Friedman tests, and multiple non-parametric comparisons were utilized to analyze the data (significance level of 5%). Results: No complications occurred, and the procedure was fast (30 seconds), safe and tolerated well without general anesthesia. After 1 year, there was a total remission of recurrent acute tonsillitis in 95% of the patients, and after 6 months there was a statistically significant improvement in halitosis and caseum, and tonsils size reduction (P<0.05). The level of satisfaction average was 10 after 3 months and 8 after one year. Conclusion: tonsillotomy by fractional ablation using the CO2 laser is a safe, efficient procedure for chronic tonsillitis in adults, and it can be incorporated into daily clinical practice.

Coblation cryptolysis method in treatment of tonsil caseum-induced halitosis.

PURPOSE: Halitosis, is a social problem affecting many patients seeking help from clinicians. Tonsil stones can cause halitosis and especially occur in crypts of palatine tonsils. Coblation cryptolysis is an alternative method for tonsil caseum treatment. The coblation technology includes passing a radiofrequency bipolar electrical current through a medium of normal saline which results in the production of a plasma field of sodium ions. In this study, our aim was to investigate the effectiveness of coblator cryptolysis treatment method in chronic caseous tonsillitis-induced halitosis. METHODS: We included in our study 28 patients who underwent coblator cryptolysis surgery for halitosis due to chronic caseous tonsillitis. The efficacy of treatment and the presence of caseoma were evaluated with the Finkelstein test, organoleptic test and VAS before the procedure and at the 6th month control after the treatment was completed. RESULTS: At the 6th month follow-up after the procedure (a single coblation cryptolysis) we found that 23 of the patients (82.1%) had no caseum. There was a statistically significant change in Finkelstein measurements before and after the procedure (p < 0.001). Organoleptic measurements demonstrated that 21 patients had no halitosis postoperatively and the mean organoleptic test score was calculated as 0.39 ± 0.79 after the procedure. The recovery was statistically significant (p < 0.001). The mean VAS score before coblation cryptolysis was 8.0 ± 1.33 (range 5-10). On the other hand 6 months after a single coblation cryptolysis session, the mean VAS score was 1.25 ± 1.78 (range: 0-6). This difference was statistically significant (p < 0.001). CONCLUSIONS: Our results suggest that coblation crptolysis is an effective, safe, minimally invasive and practical alternative method in treatment of halitosis due to tonsil caseums. We did not observe any complication after the procedure.

Caseum: a Niche for Mycobacterium tuberculosis Drug-Tolerant Persisters.

Caseum, the central necrotic material of tuberculous lesions, is a reservoir of drug-recalcitrant persisting mycobacteria. Caseum is found in closed nodules and in open cavities connecting with an airway. Several commonly accepted characteristics of caseum were established during the preantibiotic era, when autopsies of deceased tuberculosis (TB) patients were common but methodologies were limited. These pioneering studies generated concepts such as acidic pH, low oxygen tension, and paucity of nutrients being the drivers of nonreplication and persistence in caseum. Here we review widely accepted beliefs about the caseum-specific stress factors thought to trigger the shift of Mycobacterium tuberculosis to drug tolerance. Our current state of knowledge reveals that M. tuberculosis is faced with a lipid-rich diet rather than nutrient deprivation in caseum. Variable caseum pH is seen across lesions, possibly transiently acidic in young lesions but overall near neutral in most mature lesions. Oxygen tension is low in the avascular caseum of closed nodules and high at the cavity surface, and a gradient of decreasing oxygen tension likely forms toward the cavity wall. Since caseum is largely made of infected and necrotized macrophages filled with lipid droplets, the microenvironmental conditions encountered by M. tuberculosis in foamy macrophages and in caseum bear many similarities. While there remain a few knowledge gaps, these findings constitute a solid starting point to develop high-throughput drug discovery assays that combine the right balance of oxygen tension, pH, lipid abundance, and lipid species to model the profound drug tolerance of M. tuberculosis in caseum.

Efficacy and Improved Resistance Potential of a Cofactor-Independent InhA Inhibitor of Mycobacterium tuberculosis in the C3HeB/FeJ Mouse Model.

AN12855 is a direct, cofactor-independent inhibitor of InhA in Mycobacterium tuberculosis In the C3HeB/FeJ mouse model with caseous necrotic lung lesions, AN12855 proved efficacious with a significantly lower resistance frequency than isoniazid. AN12855 drug levels were better retained in necrotic lesions and caseum where the majority of hard to treat, extracellular bacilli reside. Owing to these combined attributes, AN12855 represents a promising alternative to the frontline antituberculosis agent isoniazid.

Proteomic Profiling Reveals the Architecture of Granulomatous Lesions Caused by Tuberculosis and Mycobacterium avium Complex Lung Disease.

Tuberculosis (TB) and Mycobacterium avium complex lung disease (MAC-LD) are both characterized pathologically by granuloma lesions, which are typically composed of a necrotic caseum at the center surrounded by fibrotic cells and lymphocytes. Although the histological characterization of TB and MAC-LD granulomas has been well-documented, their molecular signatures have not been fully evaluated. In this research we applied mass spectrometry-based proteomics combined with laser microdissection to investigate the unique protein markers in human mycobacterial granulomatous lesions. Comparing the protein abundance between caseous and cellular sub-compartments of mycobacterial granulomas, we found distinct differences. Proteins involved in cellular metabolism in transcription and translation were abundant in cellular regions, while in caseous regions proteins related to antimicrobial response accumulated. To investigate the determinants of their heterogeneity, we compared the protein abundance in caseous regions between TB and MAC-LD granulomas. We found that several proteins were significantly abundant in the MAC-LD caseum of which proteomic profiles were different from those of the TB caseum. Immunohistochemistry demonstrated that one of these proteins, Angiogenin, specifically localized to the caseous regions of selected MAC-LD granulomas. We also detected peptides derived from mycobacterial proteins in the granulomas of both diseases. This study provides new insights into the architecture of granulomatous lesions in TB and MAC-LD.

Extreme Drug Tolerance of Mycobacterium tuberculosis in Caseum.

Tuberculosis (TB) recently became the leading infectious cause of death in adults, while attempts to shorten therapy have largely failed. Dormancy, persistence, and drug tolerance are among the factors driving the long therapy duration. Assays to measure in situ drug susceptibility of Mycobacterium tuberculosis bacteria in pulmonary lesions are needed if we are to discover new fast-acting regimens and address the global TB threat. Here we take a first step toward this goal and describe an ex vivo assay developed to measure the cidal activity of anti-TB drugs against M. tuberculosis bacilli present in cavity caseum obtained from rabbits with active TB. We show that caseum M. tuberculosis bacilli are largely nonreplicating, maintain viability over the course of the assay, and exhibit extreme tolerance to many first- and second-line TB drugs. Among the drugs tested, only the rifamycins fully sterilized caseum. A similar trend of phenotypic drug resistance was observed in the hypoxia- and starvation-induced nonreplicating models, but with notable qualitative and quantitative differences: (i) caseum M. tuberculosis exhibits higher drug tolerance than nonreplicating M. tuberculosis in the Wayne and Loebel models, and (ii) pyrazinamide is cidal in caseum but has no detectable activity in these classic nonreplicating assays. Thus, ex vivo caseum constitutes a unique tool to evaluate drug potency against slowly replicating or nonreplicating bacilli in their native caseous environment. Intracaseum cidal concentrations can now be related to the concentrations achieved in the necrotic foci of granulomas and cavities to establish correlations between clinical outcome and lesion-centered pharmacokinetics-pharmacodynamics (PK-PD) parameters.

Prediction of Drug Penetration in Tuberculosis Lesions.

The penetration of antibiotics in necrotic tuberculosis lesions is heterogeneous and drug-specific, but the factors underlying such differential partitioning are unknown. We hypothesized that drug binding to macromolecules in necrotic foci (or caseum) prevents passive drug diffusion through avascular caseum, a critical site of infection. Using a caseum binding assay and MALDI mass spectrometry imaging of tuberculosis drugs, we showed that binding to caseum inversely correlates with passive diffusion into the necrotic core. We developed a high-throughput assay relying on rapid equilibrium dialysis and a caseum surrogate designed to mimic the composition of native caseum. A set of 279 compounds was profiled in this assay to generate a large data set and explore the physicochemical drivers of free diffusion into caseum. Principle component analysis and modeling of the data set delivered an in silico signature predictive of caseum binding, combining 69 molecular descriptors. Among the major positive drivers of binding were high lipophilicity and poor solubility. Determinants of molecular shape such as the number of rings, particularly aromatic rings, number of sp(2) carbon counts, and volume-to-surface ratio negatively correlated with the free fraction, indicating that low-molecular-weight nonflat compounds are more likely to exhibit low caseum binding properties and diffuse effectively through caseum. To provide simple guidance in the property-based design of new compounds, a rule of thumb was derived whereby the sum of the hydrophobicity (clogP) and aromatic ring count is proportional to caseum binding. These tools can be used to ensure desirable lesion partitioning and guide the selection of optimal regimens against tuberculosis.