Search

Telacebec (Q203) is a new antituberculosis drug in clinical development that has extremely potent activity against Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU). The potency of Q203 has prompted investigation of its potential role in ultrashort, even single-dose, treatment regimens for BU in mouse models. However, the relationships of Q203 dose, dose schedule, duration, and host immune status to treatment outcomes remain unclear, as does the risk of emergence of drug resistance with Q203 monotherapy. Here, we used mouse footpad infection models in immunocompetent BALB/c and immunocompromised SCID-beige mice to compare different Q203 doses, different dosing schedules, and treatment durations ranging from 1 day to 2 weeks, on long-term outcomes. We also tested whether combining Q203 with a second drug can increase efficacy. Overall, efficacy depended on total dose more than on duration. Total doses of 5 to 20 mg/kg rendered nearly all BALB/c mice culture negative by 13 to 14 weeks posttreatment, without selection of Q203-resistant bacteria. Addition of a second drug did not significantly increase efficacy. Although less potent in SCID-beige mice, Q203 still rendered the majority of footpads culture negative at total doses of 10 to 20 mg/kg. Q203 resistance was identified in relapse isolates from some SCID-beige mice receiving monotherapy but not in isolates from those receiving Q203 combined with bedaquiline or clofazimine. Overall, these results support the potential of Q203 monotherapy for single-dose or other ultrashort therapy for BU, although highly immunocompromised hosts may require higher doses or durations and/or combination therapy.

Great progress has been made in understanding the pathogenesis and treatment of Buruli ulcer over the last 20 years. The rediscovery of the mouse footpad model of the disease with translation to clinical practice has changed treatment of this infectious disease, caused by Mycobacterium ulcerans, from surgery and skin grafting to the administration of antibiotics for 8 weeks or less with superior cure rates. Here we describe the development and enhancement of the mouse model during the last two decades.

As acknowledged by the Clinical and Laboratory Standards Institute (CLSI), there is an insufficient evidence base on which to recommend a standard method for antimicrobial susceptibility testing against M. ulcerans. The agar proportion method has been recognized as the standard method for susceptibility testing against Mycobacterium tuberculosis complex (MTBC) isolates for decades (Woods GL, Engenack NL, Lin G, Turnidge JD (2018) CLSI standards: guidelines for health care excellence. Susceptibility testing of mycobacteria, Nocardia spp., and other aerobic Actinomycetes, 3rd edn. Clinical and Laboratory Standards Institute Copyright©2018 Clinical and Laboratory Standards Institute, Wayne (PA)). While it is more labor-intensive and requires larger amounts of drug or compound than broth-based testing, we recommend the agar proportion method for determination of minimum inhibitory concentrations against M. ulcerans. Herewith we present the method we implemented in our laboratory over the last 2 decades.

Telacebec (Q203) is a new anti-tuberculosis drug in clinical development with extremely potent activity against Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU). The potency of Q203 has prompted investigation of its potential role in ultra-short, even single-dose, treatment regimens for BU in mouse models. However, the relationships of Q203 dose and duration and host immune status to treatment outcomes remain unclear, as does the risk of emergence of drug resistance with Q203 monotherapy. In the present study, immunocompetent BALB/c and immunocompromised SCID-beige mice were infected in both hind footpads and treated eight weeks later. In both mouse strains, controls received rifampin-clarithromycin; others received Q203 at 0.5 or 2 mg/kg/d for 5 or 10 days. Additionally, BALB/c mice received a single dose of 2.5 or 10 mg/kg or 3.3 mg/kg/d for 3 days. Treatment response was based on changes in footpad swelling and CFU counts at the end of treatment as well as 4 and 13 weeks after stopping treatment. Efficacy depended on total dose more than duration. Total doses of 5-20 mg/kg rendered nearly all BALB/c mice culture-negative 13 weeks post-treatment without selection of Q203-resistant bacteria. Although less potent in SCID-beige mice, Q203 still rendered the majority of footpads culture-negative at total doses of 10-20 mg/kg. However, Q203 resistance was identified in relapse isolates from some SCID-beige mice. Overall, these results support the potential of Q203 monotherapy for single-dose or other ultra-short therapy for BU, although highly immunocompromised hosts may require higher doses or durations and/or combination therapy.

The antimicrobial treatment of Mycobacterium ulcerans infection, or Buruli ulcer (BU), has a long duration and is therefore burdensome and linked to indirect costs for affected patients. The new antimycobacterial drug telacebec (Q203) has previously shown promising treatment-shortening potential in mouse models of BU. In the present study, we investigated the potential of Q203 to reduce the treatment duration further. The first experiment investigated the possibility of cure by one, three or five doses of Q203 (2 mg/kg) with or without a companion drug (bedaquiline, BDQ, clofazimine, CFZ, or clarithromycin, CLR) in immunocompetent BALB/c mice. The second experiment assessed the effect of five doses of Q203 with or without BDQ or CFZ on Mycobacterium ulcerans infection of immunocompromised SCID-beige mice with the aim to evaluate the contribution of host immunity to treatment efficacy. In BALB/c mice, a treatment duration as short as 3 days was sufficient to prevent relapse in nearly all footpads and a single dose of Q203 with or without BDQ or CFZ prevented relapse in approximately 50% of footpads. Unlike in BALB/c mice, a small percentage of SCID-beige mouse footpads were culture-positive after a treatment duration of five days, highlighting an important role of host immunity for M. ulcerans clearance. Our results confirm the marked potency and prolonged bactericidal and sterilizing effects of Q203, even in immunocompromised SCID-beige mice.

Telacebec (Q203) is a new antitubercular drug with extremely potent activity against Mycobacterium ulcerans. Here, we explored the treatment-shortening potential of Q203 alone or in combination with rifampin (RIF) in a mouse footpad infection model. The first study compared Q203 at 5 and 10 mg/kg doses alone and with rifampin. Q203 alone rendered most mouse footpads culture negative in 2 weeks. Combining Q203 with rifampin resulted in a relapse-free cure 24 weeks after completing 2 weeks of treatment, compared to a 25% relapse rate in mice receiving RIF with clarithromycin, the current standard of care, for 4 weeks. The second study explored the dose-ranging activity of Q203 alone and with RIF, including the extended activity of Q203 after treatment discontinuation. The bactericidal activity of Q203 persisted for > 4 weeks beyond the last dose. All mice receiving just 1 week of Q203 at 2 to 10 mg/kg were culture negative 4 weeks after stopping treatment. Mice receiving 2 weeks of Q203 at 0.5, 2, and 10 mg/kg were culture negative 4 weeks after treatment. RIF did not increase the efficacy of Q203. A pharmacokinetics substudy revealed that Q203 doses of 2 to 10 mg/kg in mice produce plasma concentrations similar to those produced by 100 to 300 mg doses in humans, with no adverse effect of RIF on Q203 concentrations. These results indicate the extraordinary potential of Q203 to reduce the duration of treatment necessary for a cure to < 1 week (or 5 doses of 2 to 10 mg/kg) in our mouse footpad infection model and warrant further evaluation of Q203 in clinical trials.

Telacebec (Q203) is a new anti-tubercular drug with extremely potent activity against Mycobacterium ulcerans. Here, we explored the treatment-shortening potential of Q203 alone or in combination with rifampin (RIF) in a mouse footpad infection model. The first study compared Q203 at 5 and 10 mg/kg doses alone and with rifampin. Q203 alone rendered most mouse footpads culture-negative in 2 weeks. Combining Q203 with rifampin resulted in relapse-free cure 24 weeks after completing 2 weeks of treatment, compared to a 25% relapse rate in mice receiving RIF+clarithromycin, the current standard of care, for 4 weeks.The second study explored the dose-ranging activity of Q203 alone and with RIF, including the extended activity of Q203 after treatment discontinuation. The bactericidal activity of Q203 persisted for ≥ 4 weeks beyond the last dose. All mice receiving just 1 week of Q203 at 2-10 mg/kg were culture-negative 4 weeks after stopping treatment. Mice receiving 2 weeks of Q203 at 0.5, 2 and 10 mg/kg were culture-negative 4 weeks after treatment. RIF did not increase the efficacy of Q203. A pharmacokinetics sub-study revealed that Q203 doses of 2-10 mg/kg in mice produce plasma concentrations similar to those produced by 100-300 mg doses in humans, with no adverse effect of RIF on Q203 concentrations.These results indicate the extraordinary potential of Q203 to reduce the duration of treatment necessary for cure to ≤ 1 week (or 5 doses of 2-10 mg/kg) in our mouse footpad infection model and warrant further evaluation of Q203 in clinical trials.

Buruli ulcer is treatable with antibiotics. An 8-week course of rifampin (RIF) and either streptomycin (STR) or clarithromycin (CLR) cures over 90% of patients. However, STR requires injections and may be toxic, and CLR shares an adverse drug-drug interaction with RIF and may be poorly tolerated. Studies in a mouse footpad infection model showed that increasing the dose of RIF or using the long-acting rifamycin rifapentine (RPT), in combination with clofazimine (CFZ), a relatively well-tolerated antibiotic, can shorten treatment to 4 weeks. CFZ is reduced by a component of the electron transport chain (ETC) to produce reactive oxygen species toxic to bacteria. Synergistic activity of CFZ with other ETC-targeting drugs, the ATP synthase inhibitor bedaquiline (BDQ) and the bc1:aa3 oxidase inhibitor Q203 (now named telacebec), was recently described against Mycobacterium tuberculosis. Recognizing that M. tuberculosis mutants lacking the alternative bd oxidase are hypersusceptible to Q203 and that Mycobacterium ulcerans is a natural bd oxidase-deficient mutant, we tested the in vitro susceptibility of M. ulcerans to Q203 and evaluated the treatment-shortening potential of novel 3- and 4-drug regimens combining RPT, CFZ, Q203, and/or BDQ in a mouse footpad model. The MIC of Q203 was extremely low (0.000075 to 0.00015 μg/ml). Footpad swelling decreased more rapidly in mice treated with Q203-containing regimens than in mice treated with RIF and STR (RIF+STR) and RPT and CFZ (RPT+CFZ). Nearly all footpads were culture negative after only 2 weeks of treatment with regimens containing RPT, CFZ, and Q203. No relapse was detected after only 2 weeks of treatment in mice treated with any of the Q203-containing regimens. In contrast, 15% of mice receiving RIF+STR for 4 weeks relapsed. We conclude that it may be possible to cure patients with Buruli ulcer in 14 days or less using Q203-containing regimens rather than currently recommended 56-day regimens.

Our inability to predict whether certain mutations will confer antibiotic resistance has made it difficult to rapidly detect the emergence of resistance, identify pre-existing resistant populations and manage our use of antibiotics to effective treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is important in aerobic respiration and essential for emergence from dormancy, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, even though it was never exposed to pretomanid. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.

The novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, ≥4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis : loss-of-function mutations in pepQ ( Rv2535c ), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678 , mmpS5 , or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene.

Experimental and clinical studies have indicated that the antileprosy drug clofazimine may contribute treatment-shortening activity when included in tuberculosis treatment regimens. Clofazimine accumulates to high levels in tissues, has a long half-life, and remains in the body for months after administration is stopped. We hypothesized that in tuberculosis treatment, accumulated clofazimine may contribute sustained antimicrobial activity after treatment cessation, and we used the BALB/c mouse model of chronic tuberculosis chemotherapy to address this hypothesis. Mycobacterium tuberculosis -infected mice were treated for 4 weeks or 8 weeks with either isoniazid alone, clofazimine alone, the first-line regimen rifampin-isoniazid-pyrazinamide-ethambutol, or a first-line regimen where clofazimine was administered in place of ethambutol. To evaluate posttreatment antimicrobial activity, bacterial regrowth in the lungs and spleens was assessed at the day of treatment cessation and 2, 4, 6, and 8 weeks after treatment was stopped. Bacterial regrowth was delayed in all mice receiving clofazimine, either alone or in combination, compared to the mice that did not receive clofazimine. This effect was especially evident in mice receiving multidrug therapy. In mice not receiving clofazimine, bacterial regrowth began almost immediately after treatment was stopped, while in mice receiving clofazimine, bacterial regrowth was delayed for up to 6 weeks, with the duration of sustained antimicrobial activity being positively associated with the time that serum clofazimine levels remained at or above the 0.25-μg/ml MIC for M. tuberculosis . Thus, sustained activity of clofazimine may be important in the treatment-shortening effect associated with this drug.

A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic second-line drugs like D-cycloserine. Here, we used whole-genome sequences from 498 strains of M. tuberculosis to identify novel resistance-conferring genotypes. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we found that loss-of-function mutations in ald (Rv2780), encoding L-alanine dehydrogenase, were associated with unexplained drug resistance. Convergent evolution of this loss-of-function was observed exclusively among multidrug-resistant strains. Drug susceptibility testing established that ald loss-of-function conferred resistance to D-cycloserine, and susceptibility to the drug was partially restored by complementation of ald. Clinical strains with mutations in ald and alr exhibited increased resistance to D-cycloserine when cultured in vitro. Incorporation of D-cycloserine resistance in novel molecular diagnostics could allow for targeted utilization of this toxic drug among patients with susceptible infections.

Novel regimens combining bedaquiline and pretomanid with either linezolid (BPaL regimen) or moxifloxacin and pyrazinamide (BPaMZ regimen) shorten the treatment duration needed to cure TB in BALB/c mice compared to the first-line regimen and have yielded promising results in initial clinical trials. However, the independent contribution of the investigational new drug pretomanid to the efficacy of BPaMZ has not been examined and its contribution to BPaL has been examined only over the first 2 months of treatment. In the present study, the addition of pretomanid to BL increased bactericidal activity, prevented emergence of bedaquiline resistance, and shortened the duration needed to prevent relapse with drug-susceptible isolates by at least 2 months in BALB/c mice. Addition of pretomanid to BMZ resulted in a 1 log10 greater CFU reduction after 1 month of treatment and/or reduced the number of mice relapsing in each of 2 experiments in BALB/c mice and in immunocompromised nude mice. Bedaquiline-resistant isolates were found at relapse in only one BMZ-treated nude mouse. Treatment of infection with a pyrazinamide-resistant mutant in BALB/c mice with BPaMZ prevented selection of bedaquiline-resistant mutants and reduced the proportion of mice relapsing compared to BMZ alone. Among severely ill C3HeB/FeJ mice with caseous pneumonia and cavitation, BPaMZ increased median survival (≥60 vs. 21 days) and reduced median lung CFU by 2.4 log10 at 1 month compared to BMZ. In conclusion, in 3 different mouse models, pretomanid contributed significantly to the efficacy of the BPaMZ and BPaL regimens, including restricting the selection of bedaquiline-resistant mutants.

Novel regimens combining bedaquiline and pretomanid with either linezolid (BPaL regimen) or moxifloxacin and pyrazinamide (BPaMZ regimen) shorten the treatment duration needed to cure tuberculosis (TB) in BALB/c mice compared to that of the first-line regimen and have yielded promising results in initial clinical trials. However, the independent contribution of the investigational new drug pretomanid to the efficacy of BPaMZ has not been examined, and its contribution to BPaL has been examined only over the first 2 months of treatment., Novel regimens combining bedaquiline and pretomanid with either linezolid (BPaL regimen) or moxifloxacin and pyrazinamide (BPaMZ regimen) shorten the treatment duration needed to cure tuberculosis (TB) in BALB/c mice compared to that of the first-line regimen and have yielded promising results in initial clinical trials. However, the independent contribution of the investigational new drug pretomanid to the efficacy of BPaMZ has not been examined, and its contribution to BPaL has been examined only over the first 2 months of treatment. In the present study, the addition of pretomanid to BL increased bactericidal activity, prevented emergence of bedaquiline resistance, and shortened the duration needed to prevent relapse with drug-susceptible isolates by at least 2 months in BALB/c mice. Addition of pretomanid to bedaquiline, moxifloxacin, and pyrazinamide (BMZ) resulted in a 1-log10 greater CFU reduction after 1 month of treatment and/or reduced the number of mice relapsing in each of 2 experiments in BALB/c mice and in immunocompromised nude mice. Bedaquiline-resistant isolates were found at relapse in only one BMZ-treated nude mouse. Treatment of infection with a pyrazinamide-resistant mutant in BALB/c mice with BPaMZ prevented selection of bedaquiline-resistant mutants and reduced the proportion of mice relapsing compared to that for BMZ treatment alone. Among severely ill C3HeB/FeJ mice with caseous pneumonia and cavitation, BPaMZ increased median survival (≥60 versus 21 days) and reduced median lung CFU by 2.4 log10 at 1 month compared to the level for BMZ. In conclusion, in 3 different mouse models, pretomanid contributed significantly to the efficacy of the BPaMZ and BPaL regimens, including restricting the selection of bedaquiline-resistant mutants.

Rifampin (RIF) plus clarithromycin (CLR) for 8 weeks is now the standard of care for Buruli ulcer (BU) treatment, but CLR may not be an ideal companion for rifamycins due to bidirectional drug-drug interactions. The oxazolidinone linezolid (LZD) was previously shown to be active against Mycobacterium ulcerans infection in mice but has dose- and duration-dependent toxicity in humans. Sutezolid (SZD) and tedizolid (TZD) may be safer than LZD. Here, we evaluated the efficacy of these oxazolidinones in combination with rifampin in a murine BU model. Mice with M. ulcerans-infected footpads received control regimens of RIF plus either streptomycin (STR) or CLR or test regimens of RIF plus either LZD (1 of 2 doses), SZD, or TZD for up to 8 weeks. All combination regimens reduced the swelling and bacterial burden in footpads after two weeks of treatment compared with RIF alone. RIF+SZD was the most active test regimen, while RIF+LZD was also no less active than RIF+CLR. After 4 and 6 weeks of treatment, neither CLR nor the oxazolidinones added significant bactericidal activity to RIF alone. By the end of 8 weeks of treatment, all regimens rendered footpads culture negative. We conclude that SZD and LZD warrant consideration as alternative companion agents to CLR in combination with RIF to treat BU, especially when CLR is contraindicated, intolerable, or unavailable. Further evaluation could prove SZD superior to CLR in this combination.

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is a neglected tropical skin and soft tissue infection that is associated with disability and social stigma. The mainstay of BU treatment is an eight-week course of 10 mg/kg rifampin (RIF) and 150 mg/kg streptomycin (STR). Recently, the injectable STR has been shown to be replaceable with oral clarithromycin (CLR) for smaller lesions for the last four weeks of treatment. A shorter, all-oral, highly efficient regimen for BU is needed, as the long treatment duration and indirect costs currently burden patients and health systems. Increasing the dose of RIF or replacing it with the more potent rifamycin drug rifapentine (RPT) could provide such a regimen. Here, we performed a dose-ranging experiment of RIF and RPT in combination with CLR over four weeks of treatment in a mouse model of M. ulcerans disease. A clear dose-dependent effect of RIF on both clinical and microbiological outcomes was found, with no ceiling effect observed with tested doses up to 40 mg/kg. RPT-containing regimens were more effective on M. ulcerans. All RPT-containing regimens achieved culture negativity after only four weeks while only the regimen with the highest RIF dose (40 mg/kg) did so. We conclude that there is dose-dependent efficacy of both RIF and RPT and that a ceiling effect is not reached with the current standard regimen used in the clinic. A regimen based on higher rifamycin doses that are currently being evaluated against tuberculosis in clinical trials could shorten and improve therapy of Buruli ulcer.

Starting in 2004, the standard regimen for treatment of Buruli ulcer (BU) recommended by the World Health Organization has been daily treatment for eight weeks with rifampin (RIF) and streptomycin. Based on recent clinical trials, treatment with an all-oral regimen of RIF and clarithromycin (CLR) may be an effective alternative. With the achievement of an all-oral regimen, a new goal is to find a regimen that can shorten the duration of treatment without compromising efficacy. We recently observed that increasing the dose of RIF from the standard 10 mg/kg dose to 20 or 40 mg/kg, or replacing RIF with the more potent long-acting rifamycin, rifapentine (RPT) at 10 mg/kg or 20 mg/kg increased the bactericidal activity of the RIF+CLR regimen in a mouse model of BU. We also recently showed that replacing CLR with clofazimine(CFZ) at 25 mg/kg may have greater sterilizing activity than the RIF+CLR regimen. Here, we demonstrate that combining high-dose rifamycins with CFZ at a lower dose of 12.5 mg/kg results in similar reductions in swelling, bacterial burden and mycolactone concentrations in mouse footpads compared to the standard regimens and more rapid sterilization of footpads as determined by the proportions of footpads harboring viable bacteria three months after completion of treatment. The potential of these high-dose rifamycin and CFZ combinations to shorten BU treatment to four weeks warrants evaluation in a clinical trial. Trial registration ClinicalTrials.gov NCT03474198, NCT01659437, Author summary Buruli ulcer, a neglected tropical skin disease caused by Mycobacterium ulcerans, is treatable since 2004 with antibiotics instead of surgery. Treatment with either rifampin plus streptomycin or, more recently, rifampin plus clarithromycin requires taking the drugs daily for 8 weeks. Streptomycin is administered by injection and may result in hearing loss. Clarithromycin often causes gastrointestinal discomfort. Our goal is to identify a regimen that is both shorter and associated with fewer side effects. Rifampin, previously an expensive drug, is well tolerated not only at the standard dose of 10 mg/kg but at doses of 20 and 40 mg/kg. The related rifamycin, rifapentine, has a longer half-life and is also well tolerated. We tested in a mouse model of Buruli ulcer whether higher doses of these rifamycins together with clofazimine, a drug that has transient skin pigmentation side effects but no toxicities, could effectively reduce lesion size, the number of bacteria, and production of the mycolactone toxin, in a shorter time than that for the existing drug regimens. We found that treatment for 4 weeks with a high dose rifamycin plus clofazimine is as effective as 8 weeks of the current standard regimens of rifampin plus streptomycin or rifampin plus clarithromycin.

The antileprosy drug clofazimine was recently repurposed as part of a newly endorsed short-course regimen for multidrug-resistant tuberculosis. It also enables significant treatment shortening when added to the first-line regimen for drug-susceptible tuberculosis in a mouse model. However, clofazimine causes dose- and duration-dependent skin discoloration in patients, and the optimal clofazimine dosing strategy in the context of the first-line regimen is unknown. We utilized a well-established mouse model to systematically address the impacts of duration, dose, and companion drugs on the treatment-shortening activity of clofazimine in the first-line regimen. In all studies, the primary outcome was relapse-free cure (culture-negative lungs) 6 months after stopping treatment, and the secondary outcome was bactericidal activity, i.e., the decline in the lung bacterial burden during treatment. Our findings indicate that clofazimine activity is most potent when coadministered with first-line drugs continuously throughout treatment and that equivalent treatment-shortening results are obtained with half the dose commonly used in mice. However, our studies also suggest that clofazimine at low exposures may have negative impacts on treatment outcomes, an effect that was evident only after the first 3 months of treatment. These data provide a sound evidence base to inform clofazimine dosing strategies to optimize the antituberculosis effect while minimizing skin discoloration. The results also underscore the importance of conducting long-term studies to allow the full evaluation of drugs administered in combination over long durations.

New for old—TB drug development Tuberculosis (TB) is a global health threat for which there is only lengthy drug treatment. Patients need to consume multiple tablets over several months and frequently fail to complete their treatment. Consequently, drug-resistant strains of the pathogen have emerged, which add to the threat. Kling et al. revisited a natural product called griselimycin, extracted from the same organism that produced the prototype anti-TB drug, streptomycin. Unmodified griselimycin has poor pharmacological properties. However, one synthetic derivative had improved oral uptake and penetrated cells of the immune system that harbor the TB mycobacterium. In combination with other drugs, the griselimycin derivative showed high potency in mice with TB. Science , this issue p. 1106

Improved biomarkers are needed for tuberculosis. To develop tests based on products secreted by tubercle bacilli that are strictly associated with viability, we evaluated 3 bacterial-derived, species-specific, small molecules as biomarkers: 2 mycobactin siderophores and tuberculosinyladenosine. Using liquid chromatography–tandem mass spectrometry, we demonstrated the presence of 1 or both mycobactins and/or tuberculosinyladenosine in serum and whole lung tissues from infected mice and sputum, cerebrospinal fluid (CSF), or lymph nodes from infected patients but not uninfected controls. Detection of the target molecules distinguished host infection status in 100% of mice with both serum and lung as the target sample. In human subjects, we evaluated detection of the bacterial small molecules (BSMs) in multiple body compartments in 3 patient cohorts corresponding to different forms of tuberculosis. We detected at least 1 of the 3 molecules in 90%, 71%, and 40% of tuberculosis patients' sputum, CSF, and lymph node samples, respectively. In paucibacillary forms of human tuberculosis, which are difficult to diagnose even with culture, detection of 1 or more BSM was rapid and compared favorably to polymerase chain reaction–based detection. Secreted BSMs, detectable in serum, warrant further investigation as a means for diagnosis and therapeutic monitoring in patients with tuberculosis.

Drug efflux pumps play important roles in intrinsic and acquired drug resistance. Verapamil, an efflux inhibitor that enhances the activity of bedaquiline, clofazimine, and other drugs against Mycobacterium tuberculosis , has been proposed as a potential adjunctive agent for treatment of tuberculosis (TB). However, the extent to which verapamil enhances in vivo efficacy by inhibiting bacterial efflux pumps versus inhibiting mammalian drug transporters to improve oral bioavailability has not been delineated. We found that verapamil potentiated the in vitro activity of bedaquiline and clofazimine against M. tuberculosis clinical isolates, including those harboring rv0678 mutations. Verapamil increased the efficacy of bedaquiline in a murine TB model by the same extent to which it increased systemic bedaquiline exposure. However, verapamil showed no effect on the oral bioavailability or efficacy of clofazimine in mice. The addition of verapamil increased the sterilizing activity of a regimen composed of bedaquiline, clofazimine, and pyrazinamide. These results confirm that verapamil has adjunctive activity in vivo , but they also demonstrate that the adjunctive effect is likely due to enhanced systemic exposure to companion drugs via effects on mammalian transporters, rather than inhibition of bacterial pumps. Therefore, there may be no advantage to administering verapamil versus increasing the doses of companion drugs.

Rationale: A major priority in tuberculosis (TB) is to reduce effective treatment times and emergence of resistance. Recent studies in macrophages and zebrafish show that inhibition of mycobacterial efflux pumps with verapamil reduces the bacterial drug tolerance and may enhance drug efficacy. Objectives: Using mice, a mammalian model known to predict human treatment responses, and selecting conservative human bioequivalent doses, we tested verapamil as an adjunctive drug together with standard TB chemotherapy. As verapamil is a substrate for CYP3A4, which is induced by rifampin, we evaluated the pharmacokinetic/pharmacodynamic relationships of verapamil and rifampin coadministration in mice. Methods: Using doses that achieve human bioequivalent levels matched to those of standard verapamil, but lower than those of extended release verapamil, we evaluated the activity of verapamil added to standard chemotherapy in both C3HeB/FeJ (which produce necrotic granulomas) and the wild-type background C3H/HeJ mouse strains. Relapse rates were assessed after 16, 20, and 24 weeks of treatment in mice. Measurements and Main Results: We determined that a dose adjustment of verapamil by 1.5-fold is required to compensate for concurrent use of rifampin during TB treatment. We found that standard TB chemotherapy plus verapamil accelerates bacterial clearance in C3HeB/FeJ mice with near sterilization, and significantly lowers relapse rates in just 4 months of treatment when compared with mice receiving standard therapy alone. Conclusions: These data demonstrate treatment shortening by verapamil adjunctive therapy in mice, and strongly support further study of verapamil and other efflux pump inhibitors in human TB.

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.

In previous experiments, replacing the 10-mg/kg of body weight daily dose of rifampin with 7.5 to 10 mg/kg of rifapentine in combinations containing isoniazid and pyrazinamide reduced the duration of treatment needed to cure tuberculosis in BALB/c mice by approximately 50% due to rifapentine's more potent activity and greater drug exposures obtained. In the present study, we performed dose-ranging comparisons of the bactericidal and sterilizing activities of rifampin and rifapentine, alone and in combination with isoniazid and pyrazinamide with or without ethambutol, in BALB/c mice and in C3HeB/FeJ mice, which develop necrotic lung granulomas after infection with Mycobacterium tuberculosis . Each rifamycin demonstrated a significant increase in sterilizing activity with increasing dose. Rifapentine was roughly 4 times more potent in both mouse strains. These results reinforce the rationale for ongoing clinical trials to ascertain the highest well-tolerated doses of rifampin and rifapentine. This study also provides an important benchmark for the efficacy of the first-line regimen in C3HeB/FeJ mice, a strain in which the lung lesions observed after M. tuberculosis infection may better represent the pathology of human tuberculosis.

Mycobacterium ulcerans causes Buruli ulcer, a potentially disabling ulcerative skin disease. Only recently was antimicrobial therapy proven effective. Treatment for 2 months with rifampin plus streptomycin was first proposed after experiments in the mouse footpad model demonstrated bactericidal activity. This treatment is now considered the treatment of choice, although larger ulcers may require adjunctive surgery. Shorter, oral regimens are desired, but evaluating drug activity in mice is hampered by the very slow growth of M. ulcerans , which takes 3 months to produce countable colonies. We created a recombinant bioluminescent M. ulcerans strain expressing luxAB from Vibrio harveyi for real-time evaluation of antimicrobial effects in vivo . Mouse footpads were injected with wild-type M. ulcerans 1059 (WtMu) or the recombinant bioluminescent strain (rMu). Two weeks later, mice received rifampin plus streptomycin, kanamycin alone (to which rMu is resistant), or streptomycin alone for 4 weeks and were observed for footpad swelling (preventive model). Untreated controls and kanamycin-treated rMu-infected mice received rifampin plus streptomycin for 4 weeks after developing footpad swelling (curative model). Compared to WtMu, rMu exhibited similar growth and virulence in vivo and similar drug susceptibility. A good correlation was observed between luminescence (measured as relative light units) and number of viable bacteria (measured by CFU) in footpad homogenates. Proof of concept was also shown for serial real-time evaluation of drug activity in live mice. These results indicate the potential of bioluminescence as a real-time surrogate marker for viable bacteria in mouse footpads to accelerate the identification of new treatments for Buruli ulcer.

In a randomized controlled trial in Ghana, treatment of Mycobacterium ulcerans infection with streptomycin (SM)-rifampin (RIF) for 8 weeks was compared with treatment with SM-RIF for 4 weeks followed by treatment with RIF-clarithromycin (CLA) for 4 weeks. The extent of the interaction of RIF and CLA combined on the pharmacokinetics of the two compounds is unknown in this population and was therefore studied in a subset of patients. Patients received CLA at a dose of 7.5 mg/kg of body weight once daily, rounded to the nearest 125 mg. RIF was administered at a dose of 10 mg/kg, rounded to the nearest 150 mg. SM was given at a dose of 15 mg/kg once daily as an intramuscular injection. Plasma samples were drawn at steady state and analyzed by liquid chromatography-tandem mass spectroscopy. Pharmacokinetic parameters were calculated with the MW/Pharm (version 3.60) program. Comedication with CLA resulted in a 60% statistically nonsignificant increase in the area under the plasma concentration-time curve (AUC) for RIF of 25.8 mg·h/liter (interquartile ratio [IQR], 21.7 to 31.5 mg·h/liter), whereas the AUC of RIF was 15.2 mg·h/liter (IQR, 15.0 to 17.5 mg·h/liter) in patients comedicated with SM ( P = 0.09). The median AUCs of CLA and 14-hydroxyclarithromycin (14OH-CLA) were 2.9 mg·h/liter (IQR, 1.5 to 3.8 mg·h/liter) and 8.0 mg·h/liter (IQR, 6.7 to 8.6 mg·h/liter), respectively. The median concentration of CLA was above the MIC of M. ulcerans , but that of 14OH-CLA was not. In further clinical studies, a dose of CLA of 7.5 mg/kg twice daily should be used (or with an extended-release formulation, 15 mg/kg should be used) to ensure higher levels of exposure to CLA and an increase in the time above the MIC compared to those achieved with the currently used dose of 7.5 mg/kg once daily.

Pyrazinamide (PZA) is a key antituberculosis drug, yet no rapid susceptibility test is commercially available. PZA drug susceptibility testing (DST) was performed directly on sputum samples from 327 patients and compared with the indirect method by using the Bactec MGIT 960 system in the context of patient screening for participation in a drug trial. Compared to standard indirect PZA DST, direct DST was successful in only 59% of cases, but results obtained were highly accurate and available faster. Agreement between the direct and indirect methods varied from 90 to 100% in each laboratory. The median times for obtaining PZA results from the time when the specimen was collected ranged from 11 to 16 days for the direct test and 18 to 95 days for the indirect test across laboratories. The direct method is accurate and reproducible across laboratories. It can be expected to accelerate results in >50% of cases, but it cannot replace indirect DST for PZA. Phenotypic methods remain the gold standard for DST in drug trials. If future studies can optimize the method to decrease the number of uninterpretable results, direct MGIT DST could be the new phenotypic DST standard for clinical trials, providing more rapid detection of resistance to new drugs in experimental regimens.

The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.

The creation of new chemotherapeutic regimens that permit shortening the duration of treatment is a major priority for antituberculosis drug development. In this study, we used the murine model of experimental tuberculosis therapy to determine whether incorporation of the investigational new nitroimidazopyran PA-824 into the standard first-line regimen has the potential to shorten the 6-month duration of treatment. As demonstrated previously, PA-824 alone had significant bactericidal activity over the first 2 months of treatment. Moreover, the substitution of PA-824 for isoniazid led to significantly lower lung CFU counts after 2 months of treatment and to more rapid culture-negative conversion compared to the standard regimen of rifampin, isoniazid, and pyrazinamide. Despite this, there was no difference in the proportion of mice relapsing after completing 6 months of therapy (2 of 19 mice treated with PA-824 in place of isoniazid relapsed versus 0 of 46 mice treated with the standard regimen). Meanwhile, no other PA-824-containing regimen tested was superior to the standard regimen on any assessment. Thus, we were unable to establish a clear role for PA-824 in a treatment-shortening regimen that includes two or more of the current first-line drugs. Future preclinical studies should include the evaluation of novel combinations of PA-824 with new drug candidates in addition to existing antituberculosis drugs for their potential to substantially improve the treatment of both drug-susceptible and multidrug-resistant tuberculosis.

The antileprosy drug clofazimine has shown potential for shortening tuberculosis treatment; however, the current dosing of the drug is not evidence based, and the optimal dosing is unknown. Our objective was to conduct a preclinical evaluation of the pharmacokinetics and pharmacodynamics of clofazimine in the mouse model of tuberculosis, with the goal of providing useful information on dosing for future studies. Pharmacokinetic parameters were evaluated in infected and uninfected BALB/c mice. Pharmacodynamic parameters were evaluated in Mycobacterium tuberculosis -infected mice that were treated for 12 weeks with one of six different clofazimine dosing regimens, i.e., doses of 6.25, 12.5, and 25 mg/kg of body weight/day and 3 regimens with loading doses. Clofazimine progressively accumulated in the lungs, livers, and spleens of the mice, reaching levels of greater than 50 μg/g in all tissues by 4 weeks of administration, while serum drug levels remained low at 1 to 2 μg/ml. Elimination of clofazimine was extremely slow, and the half-life was dependent on the duration of drug administration. Clofazimine exhibited dose-dependent tissue and serum concentrations. At any dose, clofazimine did not have bactericidal activity during the first 2 weeks of administration but subsequently demonstrated potent, dose-independent bactericidal activity. The antituberculosis activity of clofazimine was dependent on neither the dose administered nor the drug concentrations in the tissues, suggesting that much lower doses could be effectively used for tuberculosis treatment.

The Mycobacterium avium complex is the most common cause of nontuberculous mycobacterial lung disease worldwide; yet, an optimal treatment regimen for M. avium complex infection has not been established. Clarithromycin is accepted as the cornerstone drug for treatment of M. avium lung disease; however, good model systems, especially animal models, are needed to evaluate the most effective companion drugs. We performed a series of experiments to evaluate and use different mouse models (comparing BALB/c, C57BL/6, nude, and beige mice) of M. avium infection and to assess the anti- M. avium activity of single and combination drug regimens, in vitro , ex vivo , and in mice. In vitro , clarithromycin and moxifloxacin were most active against M. avium , and no antagonism was observed between these two drugs. Nude mice were more susceptible to M. avium infection than the other mouse strains tested, but the impact of treatment was most clearly seen in M. avium -infected BALB/c mice. The combination of clarithromycin-ethambutol-rifampin was more effective in all infected mice than moxifloxacin-ethambutol-rifampin; the addition of moxifloxacin to the clarithromycin-containing regimen did not increase treatment efficacy. Clarithromycin-containing regimens are the most effective for M. avium infection; substitution of moxifloxacin for clarithromycin had a negative impact on treatment efficacy.

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.

PA-824 is a nitroimidazo-oxazine in clinical testing for the treatment of tuberculosis. We report that the novel combination of PA-824, moxifloxacin, and pyrazinamide cured mice more rapidly than the first-line regimen of rifampin, isoniazid, and pyrazinamide. If applicable to humans, regimens containing this combination may radically shorten the treatment of multidrug-resistant tuberculosis.

Diagnosis of the neglected tropical disease, Buruli ulcer, can be made by acid-fast smear microscopy, specimen culture on mycobacterial growth media, polymerase chain reaction (PCR), and/or histopathology. All have drawbacks, including non-specificity and requirements for prolonged culture at 32°C, relatively sophisticated laboratory facilities, and expertise, respectively. The causative organism, Mycobacterium ulcerans, produces a unique toxin, mycolactone A/B (ML) that can be detected by thin layer chromatography (TLC) or mass spectrometric analysis. Detection by the latter technique requires sophisticated facilities. TLC is relatively simple but can be complicated by the presence of other lipids in the specimen. A method using a boronate-assisted fluorogenic chemosensor in TLC can overcome this challenge by selectively detecting ML when visualized with UV light. This report describes modifications in the fluorescent TLC (F-TLC) procedure and its application to the mouse footpad model of M. ulcerans disease to determine the kinetics of mycolactone production and its correlation with footpad swelling and the number of colony forming units in the footpad. The response of all three parameters to treatment with the current standard regimen of rifampin (RIF) and streptomycin (STR) or a proposed oral regimen of RIF and clarithromycin (CLR) was also assessed. ML was detectable before the onset of footpad swelling when there were 105 CFU per footpad. Mycolactone concentrations increased as swelling increased whereas CFU levels reached a plateau. Treatment with either RIF+STR or RIF+CLR resulted in comparable reductions of mycolactone, footpad swelling, and CFU burden. Storage in absolute ethanol appears critical to successful detection of ML in footpads and would be practical for storage of clinical samples. F-TLC may offer a new tool for confirmation of suspected clinical lesions and be more specific than smear microscopy, much faster than culture, and simpler than PCR., Author Summary The diagnosis of Buruli ulcer, caused by infection with Mycobacterium ulcerans, is complicated by its resemblance to other diseases that may also cause ulcers in the skin. Clinical diagnosis can be supported by microscopic detection of acid-fast bacilli in the skin, by prolonged culture of at least 8 weeks, in a dedicated incubator set at 32°C, or by the polymerase chain reaction in a well-equipped laboratory usually far from the clinic where the patient comes for treatment. The treatment involves taking two drugs, one requiring injections, every day for two months, a burden for patients and their families. Since all drugs may have side effects, it is important that the treatment be appropriate for the patient's disease. We describe a new technique to rapidly and inexpensively detect the presence of the unique toxin produced by M. ulcerans in the mouse footpad model of Buruli ulcer. We show that the toxin can be detected in footpads before the development of signs of the disease, that more toxin is produced as the disease progresses, and that toxin levels decline in mice treated with either the current standard regimen of rifampin and streptomycin or a proposed all-oral drug regimen of rifampin and clarithromycin.

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., Author Summary Mycobacterium ulcerans infection causes Buruli ulcer (BU), a disfiguring skin disease now found principally in poor rural areas of West Africa. M. ulcerans produces a toxin called mycolactone (ML), which destroys fat cells in skin tissue. BU typically first shows as a nodule that eventually ulcerates. 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 the bacilli and wounds gradually heal. We tried to determine if RS treatment actually stops mycolactone production before killing bacilli. Using a mouse footpad model of M. ulcerans infection where the time of infection and lesion development can be followed in a controlled manner before and after antibiotic treatment, we found that RS treatment rapidly reduced footpad swelling, M. ulcerans numbers, and ML production. Microscopic analysis of footpads revealed that RS treatment resulted in bacilli being destroyed by host cells whereas lack of treatment resulted in extracellular infection, destruction of host cells, and 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.

Background Since 2004, treatment of Mycobacterium ulcerans disease, or Buruli ulcer, has shifted from surgery to daily treatment with streptomycin (STR) + rifampin (RIF) for 8 weeks. For shortening treatment duration, we tested the potential of daily rifapentine (RPT), a long-acting rifamycin derivative, as a substitute for RIF. Methodology/Principal Findings BALB/c mice were infected with M. ulcerans in the right hind footpad and treated either daily (7/7) with STR+RIF or five days/week (5/7) with STR+RIF or STR+RPT for 4 weeks, beginning 28 days after infection when CFU counts were 4.88±0.51. The relative efficacy of the drug treatments was compared by footpad CFU counts during treatment and median time to footpad swelling after treatment cessation as measure of sterilizing activity. All drug treatments were bactericidal. After 1 week of treatment, the decline in CFU counts was significantly greater in treated mice but not different between the three treated groups. After 2 weeks of treatment, the decline in CFU was greater in mice treated with STR+RPT 5/7 than in mice treated with STR+RIF 7/7 and STR+RIF 5/7. After 3 and 4 weeks of treatment, CFU counts were nil in mice treated with STR+RPT and reduced by more than 3 and 4 logs in mice treated with STR+RIF 5/7 and STR+RIF 7/7, respectively. In sharp contrast to the bactericidal activity, the sterilizing activity was not different between all drug regimens although it was in proportion to the treatment duration. Conclusions/Significance The better bactericidal activity of daily STR+RIF and especially of STR+RPT did not translate into better prevention of relapse, possibly because relapse-freecure after treatment of Buruli ulcer is more related to the reversal of mycolactone-induced local immunodeficiency by drug treatment rather than to the bactericidal potency of drugs., Author Summary Until 2004, the treatment of Buruli ulcer (BU) was surgical excision followed by skin grafting. Now an 8-week daily regimen of streptomycin and rifampin (STR+RIF) is recommended by the World Health Organization, supplemented when necessary by surgical intervention. However, such an antimicrobial treatment is still a heavy burden in settings with limited public health infrastructure. Thus it would be beneficial to shorten the duration of treatment without reducing antibacterial activity. Among the more active drugs having the potential to reduce the duration of treatment is rifapentine (RPT), a rifamycin derivative with a much longer half-life than RIF, which permitted in mice the reduction of treatment duration for tuberculosis from 6 months to 3 months when it is substituted for RIF tuberculosis. We therefore compared in the mouse daily treatment of BU with STR+RIF and with STR+RPT. As expected, treatment with RPT was much more bactericidal than treatment with RIF, but surprisingly the relapse rate and the time to relapse after stopping treatment were not different between treatment groups. Such findings raise numerous issues on the mechanisms involved in the cure of Buruli ulcer and on the impact of bactericidal activities of rifamycin derivatives in this disease.

Mycobacterium xenopi is a common agent of non-tuberculous mycobacterial lung diseases in Europe. However, an optimal treatment regimen for M. xenopi infection has not yet been established. Appropriate in vitro and in vivo model systems are needed for characterization of the activity of potential drugs and drug combinations against M. xenopi.We utilized three experimental platforms to analyse the anti-M. xenopi activity of single and combination drug regimens. First, we determined the bacteriostatic and bactericidal activities of drugs alone and in combination in vitro. Second, we used serum from treated mice to evaluate drug activities ex vivo. Third, we analysed M. xenopi growth in four strains of mice (BALB/c, C57BL/6, beige and athymic nude) and developed a mouse model of chemotherapy for this infection.Two-drug combinations of ethambutol with rifampicin, rifapentine or moxifloxacin, and of clarithromycin with moxifloxacin were bactericidal in vitro, and the combination of ethambutol and rifampicin with either clarithromycin or moxifloxacin showed significant bactericidal activity ex vivo. Nude mice were the most susceptible strain to M. xenopi infection, and in this model amikacin-containing regimens were the most effective against M. xenopi. No difference in activity was found between regimens containing clarithromycin and moxifloxacin in vivo.The ethambutol/rifampicin combination with clarithromycin or moxifloxacin had significant bactericidal activity against M. xenopi. The nude mouse, being highly susceptible to M. xenopi, can be utilized for in vivo chemotherapy studies for this infection.

Standard tuberculosis (TB) treatment includes an initial regimen containing drugs that are both rapidly bactericidal (isoniazid) and sterilizing (rifampin and pyrazinamide), and ethambutol to help prevent the emergence of drug resistance. Antagonism between isoniazid and pyrazinamide has been demonstrated in a TB treatment mouse model. Because isoniazid’s bactericidal activity is greatest during the initial two treatment days, we hypothesized that removing isoniazid after the second day would increase the effectiveness of the standard regimen. To test this hypothesis, we developed a mouse model to measure the early bactericidal activity (EBA) of drug regimens designed to analyze the essentiality of both isoniazid and pyrazinamide during the first 14 d of therapy. Our results clearly indicate that discontinuation of isoniazid after the second day of treatment increases the EBA of standard therapy in the mouse model, whereas omitting pyrazinamide during the first 14 d was detrimental. Substitution of moxifloxacin for isoniazid on day 3 did not increase the EBA compared with only removing isoniazid after day 2. Our data show that a mouse model can be used to analyze the EBA of TB drugs, and our findings support pursuing clinical trials to evaluate the possible benefit of removing isoniazid after the first 2 treatment days.

The largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) was identified in Tugela Ferry, KwaZulu-Natal (KZN), South Africa in 2005. The antecedents and timing of the emergence of drug resistance in this fatal epidemic XDR outbreak are unknown, and it is unclear whether drug resistance in this region continues to be driven by clonal spread or by the development of de novo resistance. A whole genome sequencing and drug susceptibility testing (DST) was performed on 337 clinical isolates of Mycobacterium tuberculosis (M.tb) collected in KZN from 2008 to 2013, in addition to three historical isolates, one of which was isolated during the Tugela Ferry outbreak. Using a variety of whole genome comparative approaches, 11 drug-resistant clones of M.tb circulating from 2008 to 2013 were identified, including a 50-member clone of XDR M.tb that was highly related to the Tugela Ferry XDR outbreak strain. It was calculated that the evolutionary trajectory from first-line drug resistance to XDR in this clone spanned more than four decades and began at the start of the antibiotic era. It was also observed that frequent de novo evolution of MDR and XDR was present, with 56 and 9 independent evolutions, respectively. Thus, ongoing amplification of drug-resistance in KwaZulu-Natal is driven by both clonal spread and de novo acquisition of resistance. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics that assess only rifampicin resistance.

Until 2004, the skin disease known as Buruli ulcer, caused by Mycobacterium ulcerans, could only be treated by surgery and skin grafting. Although this worked reasonably well on early lesions typically found in patients in Australia, the strategy was usually impractical on large lesions resulting from diagnostic delay in patients in rural West Africa. Based on promising preclinical studies, treatment trials in West Africa have shown that a combination of rifampin and streptomycin administered daily for 8 weeks can kill M. ulcerans bacilli, arrest the disease, and promote healing without relapse or reduce the extent of surgical excision. Improved treatment options are the focus of research that has increased tremendously since the WHO began its Global Buruli Ulcer Initiative in 1998.