Search

As a result of a high-throughput compound screening campaign using Mycobacterium tuberculosis -infected macrophages, a new drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (50% inhibitory concentration [IC 50 ] = 0.07 μM), is active against extracellular bacteria in cholesterol-containing culture medium, and exhibits no cross-resistance with known antitubercular drugs.

Bedaquiline (BDQ, B) is the first-in-class diarylquinoline to be approved for treatment of tuberculosis (TB). Recent guidelines recommend its use in treatment of multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB).

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.

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.

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 8-week course of rifampin (RIF) at 10 mg/kg of body weight 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 4 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 4 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 4 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 than are currently being evaluated against tuberculosis in clinical trials could shorten and improve therapy of Buruli ulcer.

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.

New regimens based on 2 or more novel agents are sought to shorten or to simplify treatment of tuberculosis (TB), including drug-resistant forms. Prior studies showed that the novel combinations of bedaquiline (BDQ) plus pretomanid (PMD) plus pyrazinamide (PZA) and PMD plus moxifloxacin (MXF) plus PZA shortened the treatment duration necessary to prevent relapse by 2 to 3 months and 1 to 2 months, respectively, compared with the current first-line regimen, in a murine TB model. These 3-drug combinations are now being studied in clinical trials. Here, the 4-drug combination of BDQ+PMD+MXF+PZA was compared to its 3-drug component regimens and different treatment durations of PZA and MXF were explored, to identify the optimal regimens and treatment times and to estimate the likelihood of success against drug-resistant strains. BDQ+PMD+MXF+PZA rendered all mice relapse-free after 2 months of treatment. PZA administration could be discontinued after the first month of treatment without worsening outcomes, whereas the absence of MXF, PZA, or BDQ administration from the beginning necessitated approximately 0.5, 1, or 2 months, respectively, of additional treatment to attain the same outcome.

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.

New regimens based on two or more novel agents are sought to shorten or simplify treatment of tuberculosis (TB). Pretomanid (PMD) is a nitroimidazole in phase 3 trials that has significant bactericidal activity alone and in combination with bedaquiline (BDQ) and/or pyrazinamide (PZA). We previously showed that the novel combination of BDQ+PMD plus the oxazolidinone sutezolid (SZD) had sterilizing activity superior to that of the first-line regimen in a murine model of TB. The present experiments compared the activity of different oxazolidinones in combination with BDQ+PMD with or without PZA in the same model. The 3-drug regimen of BDQ+PMD plus linezolid (LZD) had sterilizing activity approaching that of BDQ+PMD+SZD and superior to that of the first-line regimen. The addition of PZA further enhanced activity. Reducing the duration of LZD to 1 month did not significantly affect the activity of the regimen. Halving the LZD dose or replacing LZD with RWJ-416457 modestly reduced activity over the first month but not after 2 months. AZD5847 and tedizolid also increased the bactericidal activity of BDQ+PMD, but they were less effective than the other oxazolidinones. These results provide optimism for safe, short-course oral regimens for drug-resistant TB that may also be superior to the current first-line regimen for drug-susceptible TB.

A multidrug resistance gene, cfr , and a phenicol resistance gene, fexA , were detected in a Bacillus strain, BS-01, isolated from swine feces. The cfr gene was carried on a novel 16.5-kb plasmid, designated pBS-01. A complete Tn 917 structure, which harbors the macrolide-lincosamide-streptogramin B resistance gene erm (B), was located downstream of the cfr gene. The fexA gene was discovered in the chromosomal DNA of the BS-01 strain and identified in a Tn 558 variant.