Minimal effective dose of bedaquiline administered orally and activity of a long acting formulation of bedaquiline in the murine model of leprosy.

BACKGROUND: Bedaquiline (BDQ), by targeting the electron transport chain and having a long half-life, is a good candidate to simplify leprosy treatment. Our objectives were to (i) determine the minimal effective dose (MED) of BDQ administered orally, (ii) evaluate the benefit of combining two inhibitors of the respiratory chain, BDQ administered orally and clofazimine (CFZ)) and (iii) evaluate the benefit of an intramuscular injectable long-acting formulation of BDQ (intramuscular BDQ, BDQ-LA IM), in a murine model of leprosy. METHODOLOGY/PRINCIPAL FINDINGS: To determine the MED of BDQ administered orally and the benefit of adding CFZ, 100 four-week-old female nude mice were inoculated in the footpads with 5x103 bacilli of M. leprae strain THAI53. Mice were randomly allocated into: 1 untreated group, 5 groups treated with BDQ administered orally (0.10 to 25 mg/kg), 3 groups treated with CFZ 20 mg/kg alone or combined with BDQ administered orally 0.10 or 0.33 mg/kg, and 1 group treated with rifampicin (RIF) 10 mg/kg. Mice were treated 5 days a week during 24 weeks. To evaluate the benefit of the BDQ-LA IM, 340 four-week-old female swiss mice were inoculated in the footpads with 5x103 to 5x101 bacilli (or 5x100 for the untreated control group) of M. leprae strain THAI53. Mice were randomly allocated into the following 11 groups treated with a single dose (SD) or 3 doses (3D) 24h after the inoculation: 1 untreated group, 2 treated with RIF 10 mg/kg SD or 3D, 8 treated with BDQ administered orally or BDQ-LA IM 2 or 20 mg/kg, SD or 3D. Twelve months later, mice were sacrificed and M. leprae bacilli enumerated in the footpad. All the footpads became negative with BDQ at 3.3 mg/kg. The MED of BDQ administered orally against M. leprae in this model is therefore 3.3 mg/kg. The combination of CFZ and BDQ 10-fold lower than this MED did not significantly increase the bactericidal activity of CFZ. The BDQ-LA IM displayed similar or lower bactericidal activity than the BDQ administered orally. CONCLUSION: We demonstrated that the MED of BDQ administered orally against M. leprae was 3.3 mg/kg in mice and BDQ did not add significantly to the efficacy of CFZ at the doses tested. BDQ-LA IM was similar or less active than BDQ administered orally at equivalent dosing and frequency but should be tested at higher dosing in order to reach equivalent exposure in further experiments.

Lipophilic quinolone derivatives: Synthesis and in vitro antibacterial evaluation.

This paper reports on the design of a series of 10 novel lipophilic piperazinyl derivatives of the 1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, their synthesis, their characterisation by 1H, 13C and 19F NMR, IR spectroscopy and HRMS, as well as their biological activity against bacteria of medical interest. Among these derivatives, 2 were as potent as the parent quinolone against Neisseriagonorrhoeae whereas all the compounds displayed lower activity than the parent quinolone against other bacteria of medical interest. Our results showing that the increased lipophilicity was deleterious for antibacterial activity may help to design new quinolone derivatives in the future, especially lipophilic quinolones which have been poorly investigated previously.

A systematic review of Mycobacterium leprae DNA gyrase mutations and their impact on fluoroquinolone resistance.

BACKGROUND: The fact that Mycobacterium leprae does not grow in vitro remains a challenge in the survey of its antimicrobial resistance (AMR). Mainly molecular methods are used to diagnose AMR in M. leprae to provide reliable data concerning mutations and their impact. Fluoroquinolones (FQs) are efficient for the treatment of leprosy and the main second-line drugs in case of multidrug resistance. OBJECTIVES: This study aimed at performing a systematic review (a) to characterize all DNA gyrase gene mutations described in clinical isolates of M. leprae, (b) to distinguish between those associated with FQ resistance or susceptibility and (c) to delineate a consensus numbering system for M. leprae GyrA and GyrB. DATA SOURCES: Data source was PubMed. STUDY ELIGIBILITY CRITERIA: Publications reporting genotypic susceptibility-testing methods and gyrase gene mutations in M. leprae clinical strains. RESULTS: In 25 studies meeting our inclusion criteria, 2884 M. leprae isolates were analysed (2236 for gyrA only (77%) and 755 for both gyrA and gyrB (26%)): 3.8% of isolates had gyrA mutations (n = 110), mostly at position 91 (n = 75, 68%) and 0.8% gyrB mutations (n = 6). Since we found discrepancies regarding the location of substitutions associated with FQ resistance, we established a consensus numbering system to properly number the mutations. We also designed a 3D model of the M. leprae DNA gyrase to predict the impact of mutations whose role in FQ-susceptibility has not been demonstrated previously. CONCLUSIONS: Mutations in DNA gyrase are observed in 4% of the M. leprae clinical isolates. To solve discrepancies among publications and to distinguish between mutations associated with FQ resistance or susceptibility, the consensus numbering system we proposed as well as the 3D model of the M. leprae gyrase for the evaluation of the impact of unknown mutations in FQ resistance, will provide help for resistance surveillance.

Telacebec (Q203)-containing intermittent oral regimens sterilized mice infected with Mycobacterium ulcerans after only 16 doses.

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is currently treated with a daily combination of rifampin and either injectable streptomycin or oral clarithromycin. An intermittent oral regimen would facilitate treatment supervision. We first evaluated the bactericidal activity of newer antimicrobials against M. ulcerans using a BU animal model. The imidazopyridine amine telacebec (Q203) exhibited high bactericidal activity whereas tedizolid (an oxazolidinone closely related to linezolid), selamectin and ivermectin (two avermectine compounds) and the benzothiazinone PBTZ169 were not active. Consequently, telacebec was evaluated for its bactericidal and sterilizing activities in combined intermittent regimens. Telacebec given twice a week in combination with a long-half-life compound, either rifapentine or bedaquiline, sterilized mouse footpads in 8 weeks, i.e. after a total of only 16 doses, and prevented relapse during a period of 20 weeks after the end of treatment. These results are very promising for future intermittent oral regimens which would greatly simplify BU treatment in the field.

Population Genomics of Mycobacterium leprae Reveals a New Genotype in Madagascar and the Comoros.

Human settlement of Madagascar traces back to the beginning of the first millennium with the arrival of Austronesians from Southeast Asia, followed by migrations from Africa and the Middle East. Remains of these different cultural, genetic, and linguistic legacies are still present in Madagascar and other islands of the Indian Ocean. The close relationship between human migration and the introduction and spread of infectious diseases, a well-documented phenomenon, is particularly evident for the causative agent of leprosy, Mycobacterium leprae. In this study, we used whole-genome sequencing (WGS) and molecular dating to characterize the genetic background and retrace the origin of the M. leprae strains circulating in Madagascar (n = 30) and the Comoros (n = 3), two islands where leprosy is still considered a public health problem and monitored as part of a drug resistance surveillance program. Most M. leprae strains (97%) from Madagascar and Comoros belonged to a new genotype as part of branch 1, closely related to single nucleotide polymorphism (SNP) type 1D, named 1D-Malagasy. Other strains belonged to the genotype 1A (3%). We sequenced 39 strains from nine other countries, which, together with previously published genomes, amounted to 242 genomes that were used for molecular dating. Specific SNP markers for the new 1D-Malagasy genotype were used to screen samples from 11 countries and revealed this genotype to be restricted to Madagascar, with the sole exception being a strain from Malawi. The overall analysis thus ruled out a possible introduction of leprosy by the Austronesian settlers and suggests a later origin from East Africa, the Middle East, or South Asia.

Fully weekly antituberculosis regimen: a proof-of-concept study.

BACKGROUND: The World Health Organization recommends supervising the treatment of tuberculosis. Intermittent regimens have the potential to simplify the supervision and improve compliance. Our objective was to analyse the sterilising activity of once-weekly regimens based on drugs with a long half-life, bedaquiline and rifapentine, in a murine model of tuberculosis. METHODS: 300 Swiss mice were infected intravenously infected with ×10-6 CFU Mycobacterium tuberculosis H37Rv. Mice were treated once weekly with regimens containing: 1) bedaquiline, rifapentine and pyrazinamide (BPZ); 2) BPZ plus moxifloxacin (BPZM); 3) BPZM plus clofazimine (BPZMC); 4) the standard daily regimen of tuberculosis. All regimens were given for 4 or 6 months. Bactericidal and sterilising activity were assessed. RESULTS: After 2 months of treatment, the mean count in lungs was 0.76±0.60 log10 CFU in mice treated with the daily control regimen and negative in all mice treated with once-weekly regimens (p<0.05 compared to the daily control). All mice had negative lung cultures on completion of either 4 or 6 months of treatment, whereas 3 months after 4 and 6 months of treatment, respectively, the relapse rate was 64% and 13% in the standard daily regimen, 5% and 0% in BPZ, 0% and 0% in BPMZ and 0% and 5% in BPMZC (p<0.05 for all once-weekly regimens versus 4-month daily control; p>0.05 for all once-weekly regimens versus 6-month daily control). CONCLUSIONS: BPZ-based once-weekly regimens have higher sterilising activity than the standard daily regimen and could greatly simplify treatment administration and possibly shorten the duration of tuberculosis treatment.

Targeting the Mycobacterium ulcerans cytochrome bc1:aa3 for the treatment of Buruli ulcer.

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a neglected tropical skin disease that is most commonly found in children from West and Central Africa. Despite the severity of the infection, therapeutic options are limited to antibiotics with severe side effects. Here, we show that M. ulcerans is susceptible to the anti-tubercular drug Q203 and related compounds targeting the respiratory cytochrome bc1:aa3. While the cytochrome bc1:aa3 is the primary terminal oxidase in Mycobacterium tuberculosis, the presence of an alternate bd-type terminal oxidase limits the bactericidal and sterilizing potency of Q203 against this bacterium. M. ulcerans strains found in Buruli ulcer patients from Africa and Australia lost all alternate terminal electron acceptors and rely exclusively on the cytochrome bc1:aa3 to respire. As a result, Q203 is bactericidal at low dose against M. ulcerans replicating in vitro and in mice, making the drug a promising candidate for Buruli ulcer treatment.

Are moxifloxacin and levofloxacin equally effective to treat XDR tuberculosis?

Background: Moxifloxacin retains partial activity against some fluoroquinolone-resistant mutants of Mycobacterium tuberculosis . Levofloxacin is presumed to be as active as moxifloxacin against drug-susceptible tuberculosis and to have a better safety profile. Objectives: To compare the in vivo activity of levofloxacin and moxifloxacin against M. tuberculosis strains with various levels of fluoroquinolone resistance. Methods: BALB/c mice were intravenously infected with 10 6 M. tuberculosis H37Rv and three isogenic mutants: GyrA A90V, GyrB E540A and GyrB A543V. Treatment with 50 or 100 mg/kg levofloxacin and 60 or 66 mg/kg moxifloxacin was given orally every 6 h, for 4 weeks. Results: Levofloxacin 50 and 100 mg/kg q6h and moxifloxacin 60 and 66 mg/kg q6h generated AUCs in mice equivalent to those of levofloxacin 750 and 1000 mg/day and moxifloxacin 400 and 800 mg/day, respectively, in humans. Moxifloxacin 60 and 66 mg/kg q6h had bactericidal activity against strain H37Rv (MIC ≤ 0.25 mg/L) and mutants GyrB E540A and GyrB A543V (MIC = 0.5 mg/L). Against mutant GyrA A90V (MIC = 2 mg/L), moxifloxacin 60 mg/kg q6h did not prevent bacillary growth, whereas 66 mg/kg q6h had bacteriostatic activity. Levofloxacin 50 mg/kg q6h had bactericidal activity against H37Rv (MIC ≤ 0.25 mg/L) but not against the mutant strains. Levofloxacin 100 mg/kg q6h had bactericidal activity against H37Rv and mutants GyrB E540A (MIC = 0.5 mg/L) and GyrB A543V (MIC= 1 mg/L) but not against mutant GyrA A90V (MIC = 4 mg/L). Conclusions: All mutations reduced fluoroquinolone activity, even those classified as susceptible according to phenotypic tests. High-dose levofloxacin is less effective than high-dose moxifloxacin against both fluoroquinolone-resistant and -susceptible M. tuberculosis strains in mice.

Sterilizing Activity of Fully Oral Intermittent Regimens against Mycobacterium Ulcerans Infection in Mice.

BACKGROUND: The treatment of Buruli ulcer (BU) that is caused by Mycobacterium ulcerans, is currently based on a daily administration of rifampin and streptomycin (RIF-STR). A fully oral intermittent regimen would greatly simplify its treatment on the field. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this study was to assess the bactericidal and sterilizing activities of intermittent oral regimens in a murine model of established M. ulcerans infection. Regimens combining rifapentine (RFP 20 mg/kg) with either moxifloxacin (MXF 200 mg/kg), clarithromycin (CLR 100 mg/kg) or bedaquiline (BDQ 25 mg/kg) were administrated twice (2/7) or three (only for RFP-CLR 3/7) times weekly during 8 weeks. The bactericidal but also the sterilizing activities of these four intermittent oral regimens were at least as good as those obtained with control weekdays regimens, i.e. RFP-CLR 5/7 or RIF-STR 5/7. A single mouse from the RFP-MFX 2/7 group had culture-positive relapse at the end of the 28 weeks following treatment completion among the 157 mice treated with one of the four intermittent regimens (40 RFP-CLR 2/7, 39 RFP-CLR 3/7, 39 RFP-MXF 2/7, 39 RFP-BDQ 2/7). CONCLUSIONS/SIGNIFICANCE: These results open the door for a fully intermittent oral drug regimen for BU treatment avoiding intramuscular injections and facilitating supervision by health care workers.

In vivo Mycobacterium tuberculosis fluoroquinolone resistance emergence: a complex phenomenon poorly detected by current diagnostic tests.

OBJECTIVES: Heteroresistance, described both in terms of various point mutations resulting in different levels of resistance and in terms of a mixture of mutant and WT bacilli, is identified in up to one-third of fluoroquinolone (FQ)-resistant Mycobacterium tuberculosis isolates. Heteroresistance is a challenge for current phenotypic and genotypic susceptibility testing (DST) regimes. We aimed to compare the performances of different phenotypic and genotypic DST in the context of FQ heteroresistance by mimicking, in a murine model, the course of selection of FQ resistance during treatment. METHODS: The capacity of different phenotypic DST [Lowenstein-Jensen (LJ) medium containing either 2 mg/L ofloxacin or 0.5, 1 or 2 mg/L moxifloxacin] and genotypic DST (gyrA/B Sanger sequencing) to detect FQ resistance was analysed. RESULTS: Ninety-seven percent of mice harboured a heterogeneous population. The proportion of mice in which FQ resistance was detected varied according to the medium used (97% for 0.5 mg/L moxifloxacin, 80% for 2 mg/L ofloxacin, 47% for 1 mg/L moxifloxacin and 25% for 2 mg/L moxifloxacin). Compared with phenotypic DST, genotypic DST had a low sensitivity for detection of resistance (33%). CONCLUSIONS: Our study shows the in vivo complexity of FQ resistance emergence and the poor sensitivity of Sanger DNA sequencing for detection of heteroresistance. Our data support the use of 0.5 mg/L moxifloxacin in LJ for detection of FQ resistance, but not the recent increase in the ofloxacin critical concentration from 2 to 4 mg/L given in the WHO recommendations.

New Insights into the Geographic Distribution of Mycobacterium leprae SNP Genotypes Determined for Isolates from Leprosy Cases Diagnosed in Metropolitan France and French Territories.

BACKGROUND: Between 20 and 30 bacteriologically confirmed cases of leprosy are diagnosed each year at the French National Reference Center for mycobacteria. Patients are mainly immigrants from various endemic countries or living in French overseas territories. We aimed at expanding data regarding the geographical distribution of the SNP genotypes of the M. leprae isolates from these patients. METHODOLOGY/PRINCIPAL FINDINGS: Skin biopsies were obtained from 71 leprosy patients diagnosed between January 2009 and December 2013. Data regarding age, sex and place of birth and residence were also collected. Diagnosis of leprosy was confirmed by microscopic detection of acid-fast bacilli and/or amplification by PCR of the M. leprae-specific RLEP region. Single nucleotide polymorphisms (SNP), present in the M. leprae genome at positions 14 676, 1 642 875 and 2 935 685, were determined with an efficiency of 94% (67/71). Almost all patients were from countries other than France where leprosy is still prevalent (n = 31) or from French overseas territories (n = 36) where leprosy is not totally eradicated, while only a minority (n = 4) was born in metropolitan France but have lived in other countries. SNP type 1 was predominant (n = 33), followed by type 3 (n = 17), type 4 (n = 11) and type 2 (n = 6). SNP types were concordant with those previously reported as prevalent in the patients' countries of birth. SNP types found in patients born in countries other than France (Comoros, Haiti, Benin, Congo, Sri Lanka) and French overseas territories (French Polynesia, Mayotte and La Réunion) not covered by previous work correlated well with geographical location and history of human settlements. CONCLUSIONS/SIGNIFICANCE: The phylogenic analysis of M. leprae strains isolated in France strongly suggests that French leprosy cases are caused by SNP types that are (a) concordant with the geographic origin or residence of the patients (non-French countries, French overseas territories, metropolitan France) or (b) more likely random in regions where diverse migration flows occurred.

Aedesin: structure and antimicrobial activity against multidrug resistant bacterial strains.

Multidrug resistance, which is acquired by both Gram-positive and Gram-negative bacteria, causes infections that are associated with significant morbidity and mortality in many clinical settings around the world. Because of the rapidly increasing incidence of pathogens that have become resistant to all or nearly all available antibiotics, there is a need for a new generation of antimicrobials with a broad therapeutic range for specific applications against infections. Aedesin is a cecropin-like anti-microbial peptide that was recently isolated from dengue virus-infected salivary glands of the Aedes aegypti mosquito. In the present study, we have refined the analysis of its structural characteristics and have determined its antimicrobial effects against a large panel of multidrug resistant bacterial strains, directly isolated from infected patients. Based the results from nuclear magnetic resonance spectroscopy analysis, Aedesin has a helix-bend-helix structure typical for a member of the family of α-helix anti-microbial peptides. Aedesin efficiently killed Gram-negative bacterial strains that display the most worrisome resistance mechanisms encountered in the clinic, including resistance to carbapenems, aminoglycosides, cephalosporins, 4th generation fluoroquinolones, folate inhibitors and monobactams. In contrast, Gram-positive strains were insensitive to the lytic effects of the peptide. The anti-bacterial activity of Aedesin was found to be salt-resistant, indicating that it is active under physiological conditions encountered in body fluids characterized by ionic salt concentrations. In conclusion, because of its strong lytic activity against multidrug resistant Gram-negative bacterial strains displaying all types of clinically relevant resistance mechanisms known today, Aedesin might be an interesting candidate for the development of alternative treatment for infections caused by these types of bacteria.

Resistance of M. leprae to quinolones: a question of relativity?

UNLABELLED: Multidrug resistant leprosy, defined as resistance to rifampin, dapsone and fluoroquinolones (FQ), has been described in Mycobacterium leprae. However, the in vivo impact of fluoroquinolone resistance, mainly mediated by mutations in DNA gyrase (GyrA2GyrB2), has not been precisely assessed. Our objective was to measure the impact of a DNA gyrase mutation whose implication in fluoroquinolone resistance has been previously demonstrated through biochemical studies, on the in vivo activity of 3 fluoroquinolones: ofloxacin, moxifloxacin and garenoxacin. METHODOLOGY/PRINCIPAL FINDINGS: We used the proportional bactericidal method. 210 four-week-old immunodeficient female Nude mice (NMRI-Foxn1(nu) /Foxn1(nu) ) were inoculated in the left hind footpad with 0.03 ml of bacterial suspension containing 5 × 10(3), 5 × 10(2), 5 × 10(1), and 5 × 10(0) M. leprae AFB organisms of strain Hoshizuka-4 which is a multidrug resistant strain harboring a GyrA A91V substitution. An additional subgroup of 10 mice was inoculated with 5 × 10(-1) bacilli in the untreated control group. The day after inoculation, subgroups of mice were treated with a single dose of ofloxacin, moxifloxacin, garenoxacin or clarithromycin at 150 mg/kg dosing. 12 months later mice were sacrificed and M. leprae bacilli were numbered in the footpad. The results from the untreated control group indicated that the infective inoculum contained 23% of viable M. leprae. The results from the moxifloxacin and garenoxacin groups indicated that a single dose of these drugs reduced the percentage of viable M. leprae by 90%, similarly to the reduction observed after a single dose of the positive control drug clarithromycin. Conversely, ofloxacin was less active than clarithromycin. CONCLUSION/SIGNIFICANCE: DNA gyrase mutation is not always synonymous of lack of in vivo fluoroquinolone activity in M. leprae. As for M. tuberculosis, in vivo studies allow to measure residual antibiotic activity in case of target mutations in M. leprae.

Impact of fluoroquinolone resistance on bactericidal and sterilizing activity of a moxifloxacin-containing regimen in murine tuberculosis.

It has been shown previously that fluoroquinolone resistance (defined by resistance to at least 2 mg/liter ofloxacin) has a different impact on moxifloxacin monotherapy depending on the mutation in the sole fluoroquinolone target in Mycobacterium tuberculosis, i.e., DNA gyrase. Since tuberculosis treatment relies on multidrug therapy, we wished to determine the impact of fluoroquinolone resistance on the bactericidal and sterilizing activity of a second-line antituberculous regimen containing moxifloxacin. A total of 280 mice were inoculated with the wild-type Mycobacterium tuberculosis H37Rv strain or one of 3 isogenic fluoroquinolone-resistant mutant strains with increasing moxifloxacin resistance (the GyrB D500N, GyrA A90V, and GyrA D94G strains) and then treated for 6 months with a second-line regimen containing moxifloxacin, pyrazinamide, and ethionamide supplemented with amikacin during the first 2 months. Mice were sacrificed during treatment for measurement of bactericidal activity and 3 months after treatment completion for measurement of relapse rates (sterilizing activity). The CFU counts decreased faster in mice inoculated with the wild type than in mice inoculated with the mutant strains. The relapse rate after treatment completion was different among mice inoculated with mutant strains in relation to the drug resistance level: wild type, 0%; GyrB D500N strain, 33%; GyrA A90V strain, 50%; and GyrA D94G strain, 86%. The relapse rate observed with the GyrB D500N strain was the only one not statistically different from that observed with the wild-type strain. We demonstrated that the impact on sterilizing activity of the most active second-line drug regimen containing moxifloxacin depends on the MIC of moxifloxacin. We suggest that the precise level of moxifloxacin resistance be determined for all strains resistant to 2 mg/liter ofloxacin.

New mutations in the mycobacterial ATP synthase: new insights into the binding of the diarylquinoline TMC207 to the ATP synthase C-ring structure.

TMC207 is a new antituberculous drug belonging to the diarylquinoline class which very efficiently inhibits the ATP synthase of mycobacteria such as Mycobacterium tuberculosis, one of the most important pathogens in the world. In order to map the amino acid residues involved in the binding of the drug, we have selected in vitro TMC207-resistant mutants from M. tuberculosis and diverse atypical mycobacteria. Six distinct mutations, Asp28 → Gly, Asp28 → Ala, Leu59 → Val, Glu61 → Asp, Ala63 → Pro, and Ile66 → Met, have been identified in the subunit c forming a C ring in the ATP synthase. They were studied by evaluating the levels of resistance that they confer in the selected clones and by using an isogenic complementation system in Mycobacterium smegmatis. The rates of increase of TMC207 MIC values (8- to 133-fold) were interpreted by constructing by homology modeling a structure of the mycobacterial C ring which was used for docking simulations with TMC207. Our results suggest that the residues found to be mutated in the resistant clones, together with a tyrosine specifically conserved at position 64 in mycobacteria, define a cleft located between two adjacent c subunits in the C ring. This cleft, which encompasses the proton-binding site (Glu61), is well fitted to bind TMC207 at the level of the bromoquinoline moiety, with the drug being anchored by several ionic, hydrogen, and halogen bonds with residues Glu61, Tyr64, and Asp28, respectively. These data shed light on the molecular interactions allowing TMC207 to bind specifically and efficiently at the level of the proton-binding site of the mycobacterial C ring.

Chemotherapy-associated changes of histopathological features of Mycobacterium ulcerans lesions in a Buruli ulcer mouse model.

Combination chemotherapy with rifampin and streptomycin (RIF-STR) for 8 weeks is currently recommended by the WHO as the first-line treatment for Mycobacterium ulcerans infection (Buruli ulcer). To gain better insight into the mode of action of these antibiotics against established M. ulcerans infection foci and to characterize recovery of local immune responses during chemotherapy, we conducted a detailed histopathological study of M. ulcerans-infected and RIF-STR-treated mice. Mice were inoculated with M. ulcerans in the footpad and 11 weeks later treated with RIF-STR. Development of lesions during the first 11 weeks after infection and subsequent differences in disease progression between RIF-STR-treated and untreated mice were studied. Changes in histopathological features, footpad swelling, and number of CFU were analyzed. After inoculation with M. ulcerans, massive infiltrates dominated by polymorphonuclear leukocytes developed at the inoculation site but did not prevent bacterial multiplication. Huge clusters of extracellular bacteria located in large necrotic areas and surrounded by dead leukocytes developed in the untreated mice. Chemotherapy with RIF-STR led to a rapid drop in CFU associated with loss of solid Ziehl-Neelsen staining of acid-fast bacilli. Development of B-lymphocyte clusters and of macrophage accumulations surrounding the mycobacteria demonstrated the resolution of local immune suppression. Results demonstrate that the experimental M. ulcerans mouse infection model will be a valuable tool to investigate efficacy of new treatment regimens and of candidate vaccines.

Should moxifloxacin be used for the treatment of extensively drug-resistant tuberculosis? An answer from a murine model.

The prevalence of extensively drug-resistant tuberculosis (XDR-TB), defined as TB that is resistant to isoniazid, rifampin, fluoroquinolones, and aminoglycosides, is rising worldwide. The extent of Mycobacterium tuberculosis resistance to fluoroquinolones depends on the mutation in the DNA gyrase, the only target of fluoroquinolones. The MIC of moxifloxacin, the most active fluoroquinolone against M. tuberculosis, may be lower than its peak serum level for some ofloxacin-resistant strains of Mycobacterium tuberculosis. Therefore, if the MIC of moxifloxacin is lower than its peak serum level, it may be effective against XDR-TB. Our objective was to determine the efficacy of moxifloxacin in treating ofloxacin-resistant TB. We selected isogenic fluoroquinolone-resistant mutants of M. tuberculosis H37Rv in vivo. We infected Swiss mice with either wild-type H37Rv or one of three mutant strains with different MICs that are commonly seen in clinical practice. The MICs of the mutant strains ranged from below to above the peak moxifloxacin level seen in humans (3 µg/ml). Each mouse was treated with one of four moxifloxacin doses for 1 month. Moxifloxacin was effective against mutant strain GyrB D500N, with the lowest MIC (0.5 µg/ml), when the standard dose was doubled. Moxifloxacin reduced mortality in mice infected with mutant strain GyrA A90V with an intermediate MIC (2 µg/ml). However, it had no impact on the mutant strain GyrA D94G with the highest MIC (4 µg/ml). Our study underscores current WHO recommendations to use moxifloxacin when there is resistance to early-generation fluoroquinolones such as ofloxacin, restricting this recommendation to strains with moxifloxacin MICs of less than or equal to 2 µg/ml.

Impacts of dosing frequency of the combination rifampin-streptomycin on its bactericidal and sterilizing activities against Mycobacterium ulcerans in mice.

Because of operational limitations, a significant proportion of the health centers at the peripheral level are able to provide treatment to Buruli ulcer patients with the combination rifampin (rifampicin)-streptomycin (RIF-STR) only five times weekly (5/7) instead of seven times weekly (7/7), as recommended. The objective of this experiment is to assess the impacts of various dosing frequencies of the combination on its bactericidal and sterilizing activities against Mycobacterium ulcerans in mice. The results demonstrate that the bactericidal activities did not differ significantly among five dosing frequencies of the combination, ranging from seven times to twice weekly, whereas the sterilizing activities differed widely. RIF-STR 7/7 was the only regimen that was able to sterilize the infection after 4 to 8 weeks of treatment; the sterilizing activities associated with reduced dosing frequencies were significantly diminished, and 8 weeks of 5/7 administration yielded a relapse rate greater than the generally accepted level of 5%. We recommend that the duration of treatment with 5/7 administration be prolonged beyond 8 weeks and that additional experiments with mice be carried out, with sufficient statistical power to compare the relapse rates of M. ulcerans infection between 8 weeks of 7/7 administration and 10 and 12 weeks of 5/7 administration of RIF-STR.

A once-weekly R207910-containing regimen exceeds activity of the standard daily regimen in murine tuberculosis.

RATIONALE: R207910 (TMC207 or J) is a member of the diarylquinolines, a new family of antituberculous drugs with high bactericidal activity when given daily in the murine model of tuberculosis. R207910 exhibits a long half-life and thus is a good candidate for once-weekly therapy of tuberculosis. OBJECTIVES: To study the activity of once-weekly R207910 monotherapy and combinations of R207910 with other antituberculous agents (isoniazid, rifapentine, moxifloxacin, and pyrazinamide). METHODS: The established infection model of murine tuberculosis was used. Colony counts were determined in the lungs. MEASUREMENTS AND MAIN RESULTS: Eight weeks of monotherapy reduced the bacillary load by 3 to 4 log(10) for rifapentine and by 5 to 6 log(10) for R207910 (P < 0.05). The addition of rifapentine and isoniazid or moxifloxacin did not improve the bactericidal activity of R207910 monotherapy. In contrast, the triple combination of R207910 plus rifapentine plus pyrazinamide given once weekly for 2 months (i.e., a total of only eight administrations), was significantly (P < 0.05) more active than R207910 monotherapy or other R207910 combinations, and led to lung culture negativity in 9 of 10 mice, whereas all lungs were culture positive in the groups treated with other drug combinations. Moreover, R207910 plus rifapentine plus pyrazinamide given once weekly was more active than the current standard regimen of rifampin plus isoniazid plus pyrazinamide given five times per week. CONCLUSIONS: The unprecedented activity of the triple combination of R207910 plus rifapentine plus pyrazinamide suggests that it may be feasible to develop a fully intermittent once-weekly regimen.

Bactericidal and sterilizing activities of several orally administered combined regimens against Mycobacterium ulcerans in mice.

Treatment with rifampin-clarithromycin or moxifloxacin-clarithromycin for 8 weeks displayed promising bactericidal activity against Mycobacterium ulcerans in mice; none of the mice treated with rifampin-clarithromycin relapsed, whereas 59% of those treated with moxifloxacin-clarithromycin relapsed after treatment was stopped. The bactericidal and sterilizing activities of the five-times-weekly (5/7) administration of 5 mg of rifapentine/kg of body weight, either alone or in combination, were virtually identical to those of the corresponding regimens with 10 mg of rifampin/kg of body weight; however, because of the long half-life of rifapentine, accumulation of the drug after 5/7 administration is a concern. The bactericidal activity of 20 mg/kg rifapentine in monotherapy or 20 mg/kg rifapentine in combination with 150 mg/kg streptomycin or 200 mg/kg moxifloxacin administered twice weekly was as effective as the corresponding regimens containing 10 mg/kg rifampin administered 5/7, suggesting that Buruli ulcer might be treated with intermittently administered rifapentine-containing combinations.