|Year : 2017 | Volume
| Issue : 3 | Page : 213-221
Fighting tuberculosis by drugs targeting nonreplicating Mycobacterium tuberculosis bacilli
Angelo Iacobino1, Giovanni Piccaro2, Federico Giannoni1, Alessandro Mustazzolu1, Lanfranco Fattorini1
1 Dipartimento di Malattie Infettive, Istituto Superiore di Sanità, Rome, Italy
2 Organismo Notificato Unificato, Istituto Superiore di Sanità, Rome, Italy
|Date of Web Publication||31-Jul-2017|
Dipartimento di Malattie Infettive, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome
Source of Support: None, Conflict of Interest: None
Current tuberculosis (TB) treatment requires 6 months of combination therapy with isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol for active TB and 9 months of INH or 3 months of rifapentine (RFP) + INH for latent TB. The lungs of patients with active and latent TB contain heterogeneous mixtures of cellular and caseous granulomas harboring Mycobacterium tuberculosis bacilli ranging from actively replicating (AR) to nonreplicating (NR), phenotypically drug-resistant stages. Several in vitro models to obtain NR cells were reported, including exposure to hypoxia, nutrient starvation, acid + nitric oxide, and stationary phase. Overall, these models showed that RIF, RFP, PA-824 (PA), metronidazole (MZ), bedaquiline (BQ), and fluoroquinolones were the most active drugs against NR M. tuberculosis. In hypoxia at pH 5.8, some combinations killed AR plus NR cells, as shown by lack of regrowth in liquid media, whereas in hypoxia at pH 7.3 (the pH of the caseum), only RIF and RFP efficiently killed NR bacilli while several other drugs showed little effect. In conventional mouse models, combinations containing RFP, BQ, PA, PZA, moxifloxacin, sutezolid, linezolid, and clofazimine sterilized animals in ≤2 months, as shown by lack of viable bacilli in lung homogenates after 3 months without therapy. Drugs were less effective in C3HeB/FeJ mice forming caseous granulomas. Overall, in vitro observations and in vivo studies suggest that the search for new TB drugs could be addressed to low lipophilic molecules (e.g., new rpoB inhibitors with clogP < 3) killing NR M. tuberculosis in hypoxia at neutral pH and reaching high rates of unbound drug in the caseum.
Keywords: Dormant, drugs, Mycobacterium tuberculosis, nonreplicating, tuberculosis
|How to cite this article:|
Iacobino A, Piccaro G, Giannoni F, Mustazzolu A, Fattorini L. Fighting tuberculosis by drugs targeting nonreplicating Mycobacterium tuberculosis bacilli. Int J Mycobacteriol 2017;6:213-21
|How to cite this URL:|
Iacobino A, Piccaro G, Giannoni F, Mustazzolu A, Fattorini L. Fighting tuberculosis by drugs targeting nonreplicating Mycobacterium tuberculosis bacilli. Int J Mycobacteriol [serial online] 2017 [cited 2021 Jan 16];6:213-21. Available from: https://www.ijmyco.org/text.asp?2017/6/3/213/211934
| Introduction|| |
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, a microorganism that usually attacks not only lungs but also other parts of the body such as spine, kidney, and brain. After more than one century from discovery of the tubercle bacillus by Robert Koch in 1882, the disease has not been eliminated, and the World Health Organization (WHO) estimates that in 2015, there were 10.4 million new TB cases worldwide, of which 56% involved men, 34% women, and 10% children. In the last years, several efforts have been done by WHO in the commitment of high-income countries and of public, private, and philanthropic donors to increase investments in multidisciplinary TB research to accelerate TB elimination.
The current antibiotic treatment of active TB consists in the administration of the first-line drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) for 2 months, followed by RIF and INH for 4 months. Poor adherence to long-term therapy increases the number of patients harboring multidrug-resistant (MDR) M. tuberculosis strains (i.e., resistant at least to INH and RIF) and extensively drug-resistant strains (i.e., MDR strains resistant to any fluoroquinolone (FQ) and to at least one injectable second-line drug, kanamycin, amikacin (AK), or capreomycin (CP).
Besides active TB, an estimated 2 billion people in the world have latent TB infection (LTBI) i.e., they harbor M. tuberculosis in a nonreplicating (NR) (dormant) stage in their tissues, with 10% of persons reactivating to active TB lifetime., As similar to active TB, the treatment of latent TB is also very long, including 9 months of INH or 3 months of RFP plus INH.,,
The lungs of patients with active and latent TB contain heterogeneous mixtures of cellular and caseous granulomas harboring an array of tubercle bacilli ranging from actively replicating (AR) to NR drug refractory stages.,, While in cellular granulomas, AR bacilli are killed by current therapy, in low-vascularized caseous granulomas, the low oxygen tension stimulates aerobic/microaerophilic AR bacilli to transit into a dormant state in their hypoxic centers. Here, the NR bacilli survive extracellularly in a solid necrotic material (caseum) but are unable to multiply because of anoxic conditions and pH changes.,, Analysis of the lipid composition of human caseum revealed that it contains cholesterol, cholesteryl esters, triacylglycerols, and lactosylceramide, and its formation correlated with M. tuberculosis- mediated dysregulation of host lipid metabolism. For unknown reasons, in about 10% of persons with LTBI, the solid caseous material softens and the granulomas expand to meet the bronchial tree., Expanding lesions fuse with the airway structure and form cavities, in which the caseum liquefies. In the liquefied material, the NR bacilli rapidly multiply on contact with air and are released into the airways as a mixture of AR and NR cells in the sputum of highly contagious pulmonary TB patients.
In the last years, unprecedented efforts have been done in TB drug discovery in the attempt to eradicate M. tuberculosis from humans, but the battle is still long because drugs do not penetrate well both the complex lung TB lesions and the cell wall of NR bacilli living in low-vascularized necrotic granulomas.,,,, Understanding more in depth the biology of NR bacilli and set up new in vitro and in vivo models to measure NR killing by drugs/drug combinations may be important to find new, shorter, anti-TB therapies.
| The Nonreplicating Mycobacterium Tuberculosis|| |
M. tuberculosis slows down its metabolism when unfavorable conditions in the tissues of TB patients occur, including low oxygen tension and nutrient shortage or a combination of these and other stresses. This is supported by physiological studies of culture models by exposing bacilli to various adverse conditions. Several models to obtain NR cells were reported together with description of their metabolic and molecular characteristics. The most commonly used stressing conditions were hypoxia,,, nutrient starvation, acids and/or nitric oxide (NO),, stationary phase; an NR model using the streptomycin-starved strain 18b (SS18b) was also described. Other models and reviews on NR cells were also published.,,,,
Given the multiplicity of models, different NR subpopulations were generated, implying some uncertainty in the nomenclature and the need to define the phenotypic/molecular characteristics of different NR bacilli. In general, the term “dormant” was associated with NR cells generated in the hypoxic model of Wayne with early and late gene cascades being reported, such as the DosR Regulon and the enduring hypoxic response, respectively.,, In the last years, a number of investigators used the term “persisters” to indicate phenotypically drug-resistant (drug-tolerant) NR bacilli., Low numbers of drug-tolerant persisters were shown to be present in the early exponential phase of M. tuberculosis, followed by an increase up to ~ 1% of stationary phase cells. Persister transcriptome analysis of cells obtained by several in vitro dormancy models showed overexpression of toxin-antitoxin systems and identified a small number of genes upregulated in all cases, likely representing a core dormancy response., The dormant state of persisters was believed to be a stochastic phenomenon independent of genetic mutations, but a recent study showed that after exposure of M. tuberculosis to RIF, persister cells with high levels of hydroxyl radical generated genetically resistant mutants., Overall, these observations may shed a new light on the interplay between genotypic (chromosomal mutations) and phenotypic (dormancy) drug resistance.
| In Vitro Activity of Drugs Against Nonreplicating Mycobacterium Tuberculosis|| |
It is commonly thought that TB lesions contain coexisting M. tuberculosis bacilli at different physiological stages. Several in vitro models have been proposed to study activity of drugs against NR bacilli. Although none of them is truly representative of microenvironments met by M. tuberculosis in the tissues of LTBI- and TB-patients, the models have great value to understand differences in the activity of drugs against NR bacilli.
In this study, to compare killing activity (log10 CFU reduction) of drugs against NR M. tuberculosis, we performed a literature search using the terms “persistent, Mycobacterium tuberculosis, drug,” “dormant, Mycobacterium tuberculosis, drug,” and “nonreplicating, Mycobacterium tuberculosis, drug” in the PubMed database. After reviewing abstracts and articles, we decided to restrict the study to marketed drugs and new drugs in phases 1–3 of clinical development (www.newtbdrugs.org). CFU reductions were determined after subtracting drug-treated CFUs from drug-untreated CFUs in tables and graphs of selected papers. Activity against NR M. tuberculosis of molecules recently discovered or under preclinical development was reviewed elsewhere.
A rapid comparison of NR CFU reductions generated by 25 drugs tested at a total of 53 concentrations is represented as bars in [Figure 1]. The bars in the first three columns represent CFU reductions after 7 days of drug exposure in Wayne hypoxic models (18–21-day-old cells) at pH 5.8 (mimicking the environment of cellular granulomas),,, pH 6.6 (the pH of most mycobacterial media),,,, and pH 7.3 (mimicking the environment of caseous granulomas. The bars in the other five columns of [Figure 1] represent CFU reductions after 6 ± 1 days of drug exposure under the following conditions: pH 5 + NO, 7 days; 42-day nutrient starvation; 28-day stationary phase; ≥60-day stationary phase;,,,, SS18b.,,, Overall, the most active drugs against NR M. tuberculosis were the rifamycins, RIF and RFP. Indeed, 1–10 μg/ml of RIF reduced CFUs by 4.5–6.3-log10 in the pH 5.8, 6.6, and 7.3 Wayne models (19-day-old cells),,, and by 3.3–6.0-log10 in the 42-day starvation, 28-day stationary phase, and SS18b model.,,, Furthermore, 10 μg/ml of RFP reduced CFUs by 3.3–4.9-log10 in the pH 5.8 and 7.3 Wayne models (19-day-old cells).,
|Figure 1: Culture models reported to generate nonreplicating Mycobacterium tuberculosis cells. The length of the bars corresponds to the log10CFU reduction caused by the drugs. D, days of stress exposure; d, days of drug exposure|
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Other effective agents were the new anti-TB drugs PA-824 (PA) (pretomanid), FQs, and metronidazole (MZ), as shown by ≥2.0-log10 CFU reduction in at least one model. Indeed, PA-824 reduced CFUs by 2.1-log10 in the pH 6.6 Wayne model (21-day-old cells) and by 2.3–4.2-log10 in the pH 5 plus NO model, ≥60-day stationary phase, and SS18b model. As to the FQ, 5 μg/ml of moxifloxacin (MX) and 4 μg/ml of gatifloxacin (GF) reduced CFUs by 3-log10 in the 28-day stationary phase  and in the SS18b model. MZ (8 μg/ml) reduced CFUs by 2.2-log10 in the pH 6.6 Wayne model (19-day-old cells).
The drugs with <2 log10 CFU reduction were bedaquiline (BQ) (1.8–1.9-log10 reduction in the pH 6.6 Wayne model [18-day-old-cells] and in the SS18b model ); the protein synthesis inhibitors AK, sutezolid (SZ), linezolid (LZ), CP (1–1.8-log10 reduction in the pH 6.6 Wayne model , and the SS18b model ); thioridazine (TH), nitazoxanide (NZ), clofazimine (CL), and meropenem + clavulanic acid (0.9–1.4-log10 reduction in the pH 5.8 Wayne model , or the SS18b model ,,). In all models examined, the least active drugs were INH, EMB, PZA, niclosamide (NC), PBTZ169, and SQ109.
However, results obtained by different 7-day-drug exposure models need some analysis. For instance, in the Wayne model at pH 5.8 (19-day-old cells), when exposure was prolonged from 7 to 14 or 21 days, RFP, PA, BQ, MZ, CL, NC, NZ increased their activity by >100 times., Furthermore, in this model, PZA showed a time-dependent killing, reaching a 1.4-log10 CFU reduction on day 21. In contrast, CFU reduction by MX, AK, and LZ was similar on day 7, 14, or 21., The activity of RIF, BQ, and PA in the Wayne model at pH 6.6 also increased by >100 times when incubation was extended from 7 to 14 or 21 days.,, In general, in the same model, a dose–response increase in CFU reduction of NR M. tuberculosis was observed. In the pH 5 + NO model, dose–response CFU assays showed that the rank of activity at 10 μg/ml was PA > RIF > BQ > MX. Dose–response studies in 42-day nutrient starvation  and/or ≥60-day stationary phase models ,,,, showed that 10 μg/ml RIF and PA were very active while FQ showed low activity. High killing by 1 μg/ml of RIF and 4 μg/ml of GF in the 28-day stationary phase model  was possibly due to the presence of semidormant cells in the cultures, in comparison with ≥60-day stationary phase models. The SS18b model ,,,, showed that RIF, PA, and BQ were active against NR M. tuberculosis; however, MX, SZ, and LZ efficiently reduced CFUs (from 1.8 to 3-log10). After comparison of all these models, the most evident conclusion was that RIF, RFP, and PA were the most active drugs in vitro against NR M. tuberculosis. However, in the Wayne model at pH 7.3 mimicking environment of caseum, only RIF and RFP efficiently killed NR bacilli, while other drugs (PZA, PA, BQ, CL, NC, NZ, TH, INH, AK, MX) had no or little effect. This novel model may be important for testing activity of drugs against NR M. tuberculosis.
The results of a battery of potency assays in microplates against NR M. tuberculosis are shown in [Table 1], including the Wayne cidal concentrations (WCC90) and the Loebel cidal concentrations (LCC90),,,, the minimum inhibitory concentrations (MIC90) obtained by the luminescence-based low oxygen recovery assay (LORA) and at pH 5+NO,,, minimum bactericidal concentrations (MBC ≥99) obtained at pH 5 + NO by the normal agar resazurin assay (NARA), and charcoal agar resazurin assay (CARA). These assays showed that, in general, the MIC90 obtained in hypoxia (WCC90 and LORA MIC90) and at pH 5 + NO were lower than those obtained by starvation (LCC90) and that carryover of compounds from the NR stage (liquid media) can be affected in the replicating stage of the assay (agar and CARA plates) when drugs were removed by activated charcoal (NARA MBC ≥99< CARA MBC ≥99).
|Table 1: Inhibitory (MIC90) and bactericidal (WCC90, MBC≤99, LCC90) concentrations of drugs (μg/ml) against nonreplicating M. tuberculosis*|
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Overall, as judged from the MICs, the rifamycins (RIF, RFP) were the most active drugs against the hypoxic (“Wayne” and “LORA”), “pH 5 + NO,” and “Loebel” bacilli, with MIC90 ranging from 0.31 to 1.25 μg/ml), followed by BQ, MX, PA, depending on the test. For instance, BQ and MX showed MIC90 ranging from ≤0.2 to 4 μg/ml in three assays (WCC90, LORA MIC90, pH 5 + NO MIC90) while PA MIC90 ranged from 0.4 to 4.3 μg/ml in two assays (LORA MIC90 and pH 5 + NO MIC90). However, as to RIF, BQ, MX, PA, and several other drugs, the carryover effect, i.e., the percentage of drug removed by activated charcoal, was very high (99.9%) showing the limitations of liquid-based MICs for assessing drug activity under NR and AR conditions. Due to the good activity of RIF, BQ, MX, and PA, both in vitro (against NR and AR M. tuberculosis) and in animal models, these four molecules were recently named “dual active molecules.”
| In Vitro Activity of Drug Combinations Against Nonreplicating Mycobacterium Tuberculosis|| |
The in vitro activity of drug combinations against NR M. tuberculosis is depicted in [Figure 2]. Shown are log10 CFU reductions after 7 ± 1 days of drug exposure in the Wayne model (19-day-old cells) at pH 5.8 (panel A) and at pH 6.6 (panel B), in the 28-day stationary phase ,, (panels C and D), and in the SS18b model  (panel E). CFUs were reduced by 0.5 log10 at 0.1 μg/ml of RIF or by >5 log10 at 1 or 8 μg/ml of RIF, and addition of various agents in 2-, 3- or 4-drug combinations usually increased RIF activity, but CFUs were often below the limit of detection. In the stationary phase, RIF activity was increased by MZ. In the Wayne model, to overcome the limit of CFU detection, the killing of NR M. tuberculosis was demonstrated as lack of regrowth of drug-exposed cells in MGIT 960 tubes after >100 days of incubation. This parameter (day-to-positivity after >100 days, DTP >100) was much more sensitive than CFUs and demonstrated that RIF + MX + AK + PA sterilized both 19-day-old NR and 5-day-old AR bacilli in 14 days and that several RIF + MX-containing combinations sterilized 19-day-old NR bacilli in 21 days., This suggests that the stringent DTP >100 assay may be used in place of CFU counts when studying the sterilizing activity of new drugs or combinations. In the SS18b model, the cell wall inhibitor in phase 1 trial PBTZ169 improved the NR killing of BQ and CF. Again, these observations are in keeping with the knowledge that RIF, BQ, MX, and PA are pivotal drugs in the killing of NR M. tuberculosis.
|Figure 2: In vitro activity of drug combinations against nonreplicating Mycobacterium tuberculosis. Shown are log10CFU reduction (length of the bars) after 7 ± 1 days of drug exposure in the Wayne model at pH 5.8 (panel A) and at pH 6.6 (panel B), in the 28-day stationary phase,, (panels C and D), and in the SS18b model (panel E). The acronyms of drugs are indicated in the text|
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| In Vivo Sterilizing Activity of Drug Combinations Against Mycobacterium Tuberculosis|| |
Besides in vitro tests, the sterilizing activity of a drug combination can be determined in animal models reaching different stages of granuloma formation, ranging from conventional mouse models, which do not develop caseating granuloma and cavitary lesions (e.g., the BALB/c mice), to guinea pig, rabbit, and macaque models, all showing necrosis, caseation, liquefaction, and cavity formation. In the low-cost mouse models, drug efficacy is assessed by CFU counts in organs at selected time points during treatment (a measure of bactericidal activity) and by the proportion of mice with culture-positive relapse 3 months after discontinuation of treatment (a measure of sterilizing activity). Relapse was defined as the presence of M. tuberculosis colonies upon plating of entire undiluted lung homogenate.
The number of months of treatment necessary to sterilize mice by various regimens (lack of colonies after completion of treatment plus 3 months without treatment) is shown in [Table 2]. In BALB/c mice, the RIF-containing combinations RIF10 + INH10 + PZA150 and RIF40 + INH10 + PZA150 sterilized animals in 6 and 3 months, respectively., Furthermore, the RFP-containing combinations RFP10 + INH10 + PZA150 and RFP20 + INH10 + PZA150 sterilized BALB/c mice in 3 and 2.5 months, respectively. This indicated that increased doses of RIF and of the long-lasting rifamycin RFP (recently suggested for short treatment of LTBI),, consistently shortened the duration of sterilizing therapy in BALB/c mice. In the combination RIF10 + INH10 + EMB100 + PZA150, substitution of MX for INH reduced the sterilizing time from 4 to 3 months. BALB/c mice were sterilized in 2 months by BQ25 + RFP10 + PZA150 and BQ25 + CL20 + PZA150, BQ25 + PZA150, BQ25 + TBA-35450 + PZA150, and SZ50 + PA50 and in 1.5 months by BQ25 + RFP10 + CL20 + PZA150, SZ50 + TB-35425 and SZ50 + TB-35450, and by 1 month of BQ25 + PA100 + LZ100 + PZA150 followed by 1 month of BQ25 + PA100 + PZA150. These observations also show that besides RIF and RFP, other drugs such as BQ, PA, SZ, LZ, CL, MX, TBA-354, and PZA are promising to shorten anti-TB therapy in the future, and all but TBA-354 are presently tested in combination in human trials (www.newtbdrugs.org). Indeed, because of side effects in phase 1 studies, clinical development of TBA-354 was discontinued in 2016 (https://www.tballiance.org/news/phase-1-clinical-trial-tb-drug-candidate-tba-354 discontinued).
|Table 2: Sterilizing activity of drug combinations against M. tuberculosis-infected BALB/c and C3HeB/FeJ mice|
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Recently, M. tuberculosis-infected C3HeB/FeJ mice were reported to develop necrotic lung granulomas with abundant extracellular bacilli, so they are becoming a candidate to supplement, or even replace, conventional mouse strains for evaluation of TB drugs.,, As expected, drug treatments were less effective than in BALB/c mice; however, 2 months of RIF10 + INH10 + EMB100 + PZA150 followed by 4 months of RIF10 + INH10 + PZA150 sterilized C3HeB/FeJ mice in 4.5 months [Table 2].
| Targeting Nonreplicating Mycobacterium Tuberculosis in the Caseum|| |
In conventional mouse models, M. tuberculosis bacilli were found primarily intracellularly, whereas in guinea pigs, the majority of bacteria in the necrotic lesions of the lungs were extracellular. Following drug treatment, a homogenous bacillary reduction across granulomas was observed in mice, whereas in guinea pigs, the NR extracellular bacilli persisted in lesions with residual necrosis. These observations indicated that drug development should be designed to target NR populations and that drug regimens should be evaluated in appropriate animal models.
The C3HeB/FeJ mice showed heterogeneity in the size and degree of liquefaction of caseous lesions between mice and between lesions within the same mouse. Furthermore, unlike the common belief that the pH of caseum was acidic, the pH of liquefied caseous material from C3HeB/FeJ mice was found to be 7.4 (range 7.2–7.5). This value was similar to that found in lesions of M. tuberculosis infected guinea pigs (pH 7.2, range 7.0–7.5), rabbits (pH 6.4–7.4), and humans (pH 6.1–7.4). Overall, C3HeB/FeJ mice may be a good model for mimic human TB. Due to the variety of granulomatous lesions in these mice, PZA had no or little activity in a subset of animals with large caseous lesions, where the pH approached 7.4. Indeed, sterilization occurred when PZA was administered in combination with the first-line drugs in mice with less extensive disease. A similar dichotomous activity was seen in BQ-treated C3HeB/FeJ mice.
A limited activity of CL, a poorly soluble, lipophilic drug, was reported in C3HeB/FeJ mice. The lipophilic character of a drug is mostly expressed as hydrophobicity (calculated octanol/water partitioning coefficient, clogP). Drugs with clogP <0 are hydrophilic. Compounds with moderate hydrophobicity (clogP between 0 and 3) are optimal for oral administration owing to a good balance of solubility and permeability. In the current TB therapy, only RIF is lipophilic (clogP of 3.85) while the other three drugs (INH, EMB, PZA: clogP of −0.71, −0.12, −0.71, respectively) (http://www.drugbank.ca) are hydrophilic.
The caseum is devoid of vascular supply, and in M. tuberculosis-infected rabbits, it was found that only the free drug fraction (fraction unbound, fu) can penetrate this matrix through passive diffusion. Hydrophobicity and aromatic ring count of a drug were shown to be proportional to caseum binding and compounds with clogP <1 had a high chance of achieving fu >10%. Lipophilic drugs nonspecifically bind to caseum macromolecules at the outer edge of the caseum core, preventing further passive diffusion toward the center of the necrotic core.
Indeed, the fu of the highly lipophilic agents CL and BQ (clogP of 7.39 and 6.37, respectively) was <0.01%, while that of hydrophilic molecules such as INH and PZA was >99.9%. Drugs with intermediate clogP values such as RFP, RIF, PA, and SZ (clogP of 4.83, 3.85, 2.8, and 1.22, respectively) showed increasing free fraction (fu of 0.5, 5.1, 7.3, and 30.1%, respectively)., Other drugs including LZ and MX (clogP of 0.61 and 0.01, respectively) showed fu of 29.3 and 13.5%, respectively.
In summary, to combat the battle against TB, we should know more in depth the biology of the caseum and of NR bacilli , and find new tools for the search of novel sterilizing combinations. In the Wayne model at pH 5.8, it was reported that lipophilic drugs were more active than hydrophilic agents against NR M. tuberculosis. However, in the Wayne model at pH 7.3, only RIF and RFP (inhibitors of the subunit beta of RNA polymerase, rpoB) efficiently killed NR bacilli, while several other drugs had no or little effect irrespective of being lipophilic or hydrophilic. It is known that RIF accumulates and maintains therapeutic levels in the caseum, where dormant M. tuberculosis resides  and that high-dose treatments with RIF in TB patients reduced the time to culture conversion. In addition, there is increasing evidence that RFP + INH regimen for the treatment of latent TB is as effective, better tolerated, and more likely to be completed compared to INH.,, In this respect, novel combinations containing optimized rifamycins dosages might shorten treatments of active and latent TB in the future.
In summary, on the basis of in vitro observations on NR bacilli and recent studies on drug penetration in caseum, we suggest that the search for new TB drugs could be addressed to molecules with low lipophilicity (e.g., new rpoB inhibitors with clogP < 3) killing NR M. tuberculosis in hypoxia at neutral pH (e.g., at around pH 7.3).
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| References|| |
World Health Organization. Global Tuberculosis Report. WHO/HTM/TB/2016.13. Geneva, Switzerland: World Health Organization; 2016.
World Health Organization. A Global Action Framework for TB Research in Support of the Third Pillar of WHO's End TB Strategy. WHO/HTM/TB/2015.26. Geneva, Switzerland: World Health Organization; 2015.
Zumla A, Chakaya J, Centis R, D'Ambrosio L, Mwaba P, Bates M, et al.
Tuberculosis treatment and management-an update on treatment regimens, trials, new drugs, and adjunct therapies. Lancet Respir Med 2015;3:220-34.
Pai M, Behr MA, Dowdy D, Dheda K, Divangahi M, Boehme CC, et al.
Tuberculosis. Nat Rev Dis Primers 2016;2:16076.
Dutta NK, Karakousis PC. Latent tuberculosis infection: Myths, models, and molecular mechanisms. Microbiol Mol Biol Rev 2014;78:343-71.
Getahun H, Matteelli A, Chaisson RE, Raviglione M. Latent Mycobacterium tuberculosis
infection. N Engl J Med 2015;372:2127-35.
Kahwati LC, Feltner C, Halpern M, Woodell CL, Boland E, Amick HR, et al.
Primary care screening and treatment for latent tuberculosis infection in adults: Evidence Report and Systematic Review for the US Preventive Services Task Force. JAMA 2016;316:970-83.
Barry CE 3rd
, Boshoff HI, Dartois V, Dick T, Ehrt S, Flynn J, et al.
The spectrum of latent tuberculosis: Rethinking the biology and intervention strategies. Nat Rev Microbiol 2009;7:845-55.
Lenaerts A, Barry CE 3rd
, Dartois V. Heterogeneity in tuberculosis pathology, microenvironments and therapeutic responses. Immunol Rev 2015;264:288-307.
Dartois V. The path of anti-tuberculosis drugs: From blood to lesions to mycobacterial cells. Nat Rev Microbiol 2014;12:159-67.
Grosset J. Mycobacterium tuberculosis
in the extracellular compartment: An underestimated adversary. Antimicrob Agents Chemother 2003;47:833-6.
Kim MJ, Wainwright HC, Locketz M, Bekker LG, Walther GB, Dittrich C, et al.
Caseation of human tuberculosis granulomas correlates with elevated host lipid metabolism. EMBO Mol Med 2010;2:258-74.
Fattorini L, Piccaro G, Mustazzolu A, Giannoni F. Targeting dormant bacilli to fight tuberculosis. Mediterr J Hematol Infect Dis 2013;5:e2013072.
Cadena AM, Flynn JL, Fortune SM. The importance of first impressions: Early events in Mycobacterium tuberculosis
infection influence outcome. MBio 2016;7:e00342-16.
Garton NJ, Waddell SJ, Sherratt AL, Lee SM, Smith RJ, Senner C, et al.
Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum. PLoS Med 2008;5:e75.
Sarathy J, Dartois V, Dick T, Gengenbacher M. Reduced drug uptake in phenotypically resistant nutrient-starved nonreplicating Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2013;57:1648-53.
Coates AR, Hu Y. New strategies for antibacterial drug design: Targeting non-multiplying latent bacteria. Drugs R D 2006;7:133-51.
Prideaux B, Via LE, Zimmerman MD, Eum S, Sarathy J, O'Brien P, et al.
The association between sterilizing activity and drug distribution into tuberculosis lesions. Nat Med 2015;21:1223-7.
Sarathy JP, Zuccotto F, Hsinpin H, Sandberg L, Via LE, Marriner GA, et al.
Prediction of Drug Penetration in Tuberculosis Lesions. ACS Infect Dis 2016;2:552-63.
Wayne LG, Hayes LG. An in vitro
model for sequential study of shiftdown of Mycobacterium tuberculosis
through two stages of nonreplicating persistence. Infect Immun 1996;64:2062-9.
Sohaskey CD, Voskuil MI.In vitro
models that utilize hypoxia to induce non-replicating persistence in Mycobacteria. Methods Mol Biol 2015;1285:201-13.
Park HD, Guinn KM, Harrell MI, Liao R, Voskuil MI, Tompa M, et al.
Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis
. Mol Microbiol 2003;48:833-43.
Betts JC, Lukey PT, Robb LC, McAdam RA, Duncan K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis
persistence by gene and protein expression profiling. Mol Microbiol 2002;43:717-31.
Voskuil MI, Schnappinger D, Visconti KC, Harrell MI, Dolganov GM, Sherman DR, et al.
Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis
dormancy program. J Exp Med 2003;198:705-13.
de Carvalho LP, Lin G, Jiang X, Nathan C. Nitazoxanide kills replicating and nonreplicating Mycobacterium tuberculosis
and evades resistance. J Med Chem 2009;52:5789-92.
Hu Y, Mangan JA, Dhillon J, Sole KM, Mitchison DA, Butcher PD, et al.
Detection of mRNA transcripts and active transcription in persistent Mycobacterium tuberculosis
induced by exposure to rifampin or pyrazinamide. J Bacteriol 2000;182:6358-65.
Sala C, Dhar N, Hartkoorn RC, Zhang M, Ha YH, Schneider P, et al.
Simple model for testing drugs against nonreplicating Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2010;54:4150-8.
Salina EG, Waddell SJ, Hoffmann N, Rosenkrands I, Butcher PD, Kaprelyants AS. Potassium availability triggers Mycobacterium tuberculosis
transition to, and resuscitation from, non-culturable (dormant) states. Open Biol 2014;4. pii: 140106.
Lipworth S, Hammond RJ, Baron VO, Hu Y, Coates A, Gillespie SH. Defining dormancy in mycobacterial disease. Tuberculosis (Edinb) 2016;99:131-42.
Alnimr AM. Dormancy models for Mycobacterium tuberculosis
: A minireview. Braz J Microbiol 2015;46:641-7.
Pasipanodya JG, Nuermberger E, Romero K, Hanna D, Gumbo T. Systematic analysis of hollow fiber model of tuberculosis experiments. Clin Infect Dis 2015;61 Suppl 1:S10-7.
Franzblau SG, DeGroote MA, Cho SH, Andries K, Nuermberger E, Orme IM, et al.
Comprehensive analysis of methods used for the evaluation of compounds against Mycobacterium tuberculosis
. Tuberculosis (Edinb) 2012;92:453-88.
Rustad TR, Harrell MI, Liao R, Sherman DR. The enduring hypoxic response of Mycobacterium tuberculosis
. PLoS One 2008;3:e1502.
Iona E, Pardini M, Mustazzolu A, Piccaro G, Nisini R, Fattorini L, et al. Mycobacterium tuberculosis
gene expression at different stages of hypoxia-induced dormancy and upon resuscitation. J Microbiol 2016;54:565-72.
Van den Bergh B, Fauvart M, Michiels J. Formation, physiology, ecology, evolution and clinical importance of bacterial persisters. FEMS Microbiol Rev 2017;41:219-251.
Gold B, Nathan C. 2017. Targeting phenotypically tolerant Mycobacterium tuberculosis
. Microbiol Spectrum 5:TBTB2-0031-2016. doi:10.1128/microbiolspec.TBTB2-0031-2016.
Keren I, Minami S, Rubin E, Lewis K. Characterization and transcriptome analysis of Mycobacterium tuberculosis
persisters. MBio 2011;2:e00100-11.
Sala A, Bordes P, Genevaux P. Multiple toxin-antitoxin systems in Mycobacterium tuberculosis
. Toxins (Basel) 2014;6:1002-20.
Sebastian J, Swaminath S, Nair RR, Jakkala K, Pradhan A, Ajitkumar P. De novo
emergence of genetically resistant mutants of Mycobacterium tuberculosis
from the persistence phase cells formed against antituberculosis drugs in vitro
. Antimicrob Agents Chemother 2017;61. pii: e01343-16.
Piccaro G, Pietraforte D, Giannoni F, Mustazzolu A, Fattorini L. Rifampin induces hydroxyl radical formation in Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2014;58:7527-33.
Piccaro G, Giannoni F, Filippini P, Mustazzolu A, Fattorini L. Activities of drug combinations against Mycobacterium tuberculosis
grown in aerobic and hypoxic acidic conditions. Antimicrob Agents Chemother 2013;57:1428-33.
Piccaro G, Poce G, Biava M, Giannoni F, Fattorini L. Activity of lipophilic and hydrophilic drugs against dormant and replicating Mycobacterium tuberculosis
. J Antibiot (Tokyo) 2015;68:711-4.
Iacobino A, Piccaro G, Giannoni F, Mustazzolu A, Fattorini L. Mycobacterium tuberculosis
is selectively killed by rifampin and rifapentine in hypoxia at neutral pH. Antimicrob Agents Chemother 2017;61. pii: E02296-16.
Iona E, Giannoni F, Pardini M, Brunori L, Orefici G, Fattorini L. Metronidazole plus rifampin sterilizes long-term dormant Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2007;51:1537-40.
Koul A, Vranckx L, Dendouga N, Balemans W, Van den Wyngaert I, Vergauwen K, et al.
Diarylquinolines are bactericidal for dormant mycobacteria as a result of disturbed ATP homeostasis. J Biol Chem 2008;283:25273-80.
Filippini P, Iona E, Piccaro G, Peyron P, Neyrolles O, Fattorini L. Activity of drug combinations against dormant Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2010;54:2712-5.
Somasundaram S, Anand RS, Venkatesan P, Paramasivan CN. Bactericidal activity of PA-824 against Mycobacterium tuberculosis
under anaerobic conditions and computational analysis of its novel analogues against mutant Ddn receptor. BMC Microbiol 2013;13:218.
Gold B, Roberts J, Ling Y, Quezada LL, Glasheen J, Ballinger E, et al.
Rapid, semiquantitative assay to discriminate among compounds with activity against replicating or nonreplicating Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2015;59:6521-38.
Xie Z, Siddiqi N, Rubin EJ. Differential antibiotic susceptibilities of starved Mycobacterium tuberculosis
isolates. Antimicrob Agents Chemother 2005;49:4778-80.
Paramasivan CN, Sulochana S, Kubendiran G, Venkatesan P, Mitchison DA. Bactericidal action of gatifloxacin, rifampin, and isoniazid on logarithmic- and stationary-phase cultures of Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2005;49:627-31.
Hu Y, Coates AR, Mitchison DA. Sterilizing activities of fluoroquinolones against rifampin-tolerant populations of Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2003;47:653-7.
Hu Y, Coates AR, Mitchison DA. Sterilising action of pyrazinamide in models of dormant and rifampicin-tolerant Mycobacterium tuberculosis
. Int J Tuberc Lung Dis 2006;10:317-22.
Hu Y, Coates AR, Mitchison DA. Comparison of the sterilising activities of the nitroimidazopyran PA-824 and moxifloxacin against persisting Mycobacterium tuberculosis
. Int J Tuberc Lung Dis 2008;12:69-73.
Hu Y, Liu A, Ortega-Muro F, Alameda-Martin L, Mitchison D, Coates A. High-dose rifampicin kills persisters, shortens treatment duration, and reduces relapse rate in vitro
and in vivo
. Front Microbiol 2015;6:641.
Zhang M, Sala C, Hartkoorn RC, Dhar N, Mendoza-Losana A, Cole ST. Streptomycin-starved Mycobacterium tuberculosis
18b, a drug discovery tool for latent tuberculosis. Antimicrob Agents Chemother 2012;56:5782-9.
Zhang M, Sala C, Dhar N, Vocat A, Sambandamurthy VK, Sharma S, et al. In vitro
and in vivo
activities of three oxazolidinones against nonreplicating Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2014;58:3217-23.
Vocat A, Hartkoorn RC, Lechartier B, Zhang M, Dhar N, Cole ST, et al.
Bioluminescence for assessing drug potency against nonreplicating Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2015;59:4012-9.
Lechartier B, Cole ST. Mode of action of clofazimine and combination therapy with benzothiazinones against Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2015;59:4457-63.
Lakshminarayana SB, Huat TB, Ho PC, Manjunatha UH, Dartois V, Dick T, et al.
Comprehensive physicochemical, pharmacokinetic and activity profiling of anti-TB agents. J Antimicrob Chemother 2015;70:857-67.
Gengenbacher M, Rao SP, Pethe K, Dick T. Nutrient-starved, non-replicating Mycobacterium tuberculosis
requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability. Microbiology 2010;156(Pt 1):81-7.
Cho SH, Warit S, Wan B, Hwang CH, Pauli GF, Franzblau SG. Low-oxygen-recovery assay for high-throughput screening of compounds against nonreplicating Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2007;51:1380-5.
Upton AM, Cho S, Yang TJ, Kim Y, Wang Y, Lu Y, et al. In vitro
and in vivo
activities of the nitroimidazole TBA-354 against Mycobacterium tuberculosis
. Antimicrob Agents Chemother 2015;59:136-44.
Wayne LG, Sramek HA. Metronidazole is bactericidal to dormant cells of Mycobacterium tuberculosis
. Antimicrob Agents Chemother 1994;38:2054-8.
Sulochana S, Mitchison DA, Kubendiren G, Venkatesan P, Paramasivan CN. Bactericidal activity of moxifloxacin on exponential and stationary phase cultures of Mycobacterium tuberculosis.
J Chemother 2009;21:127-34.
Tasneen R, Li SY, Peloquin CA, Taylor D, Williams KN, Andries K, et al.
Sterilizing activity of novel TMC207- and PA-824-containing regimens in a murine model of tuberculosis. Antimicrob Agents Chemother 2011;55:5485-92.
Dutta NK, Illei PB, Jain SK, Karakousis PC. Characterization of a novel necrotic granuloma model of latent tuberculosis infection and reactivation in mice. Am J Pathol 2014;184:2045-55.
Rosenthal IM, Tasneen R, Peloquin CA, Zhang M, Almeida D, Mdluli KE, et al.
Dose-ranging comparison of rifampin and rifapentine in two pathologically distinct murine models of tuberculosis. Antimicrob Agents Chemother 2012;56:4331-40.
Li SY, Irwin SM, Converse PJ, Mdluli KE, Lenaerts AJ, Nuermberger EL. Evaluation of moxifloxacin-containing regimens in pathologically distinct murine tuberculosis models. Antimicrob Agents Chemother 2015;59:4026-30.
Williams K, Minkowski A, Amoabeng O, Peloquin CA, Taylor D, Andries K, et al.
Sterilizing activities of novel combinations lacking first- and second-line drugs in a murine model of tuberculosis. Antimicrob Agents Chemother 2012;56:3114-20.
Tasneen R, Williams K, Amoabeng O, Minkowski A, Mdluli KE, Upton AM, et al.
Contribution of the nitroimidazoles PA-824 and TBA-354 to the activity of novel regimens in murine models of tuberculosis. Antimicrob Agents Chemother 2015;59:129-35.
Tasneen R, Betoudji F, Tyagi S, Li SY, Williams K, Converse PJ, et al.
Contribution of oxazolidinones to the efficacy of novel regimens containing bedaquiline and pretomanid in a mouse model of tuberculosis. Antimicrob Agents Chemother 2015;60:270-7.
Lanoix JP, Lenaerts AJ, Nuermberger EL. Heterogeneous disease progression and treatment response in a C3HeB/FeJ mouse model of tuberculosis. Dis Model Mech 2015;8:603-10.
Lanoix JP, Ioerger T, Ormond A, Kaya F, Sacchettini J, Dartois V, et al.
Selective inactivity of pyrazinamide against tuberculosis in C3HeB/FeJ mice is best explained by neutral pH of caseum. Antimicrob Agents Chemother 2015;60:735-43.
Lanoix JP, Betoudji F, Nuermberger E. Novel regimens identified in mice for treatment of latent tuberculosis infection in contacts of patients with multidrug-resistant tuberculosis. Antimicrob Agents Chemother 2014;58:2316-21.
Lanoix JP, Betoudji F, Nuermberger E. Sterilizing activity of pyrazinamide in combination with first-line drugs in a C3HeB/FeJ mouse model of tuberculosis. Antimicrob Agents Chemother 2015;60:1091-6.
Hoff DR, Ryan GJ, Driver ER, Ssemakulu CC, De Groote MA, Basaraba RJ, et al.
Location of intra- and extracellular M. tuberculosis
populations in lungs of mice and guinea pigs during disease progression and after drug treatment. PLoS One 2011;6:e17550.
Irwin SM, Prideaux B, Lyon ER, Zimmerman MD, Brooks EJ, Schrupp CA, et al.
Bedaquiline and pyrazinamide treatment responses are affected by pulmonary lesion heterogeneity in Mycobacterium tuberculosis
infected C3HeB/FeJ mice. ACS Infect Dis 2016;2:251-67.
Irwin SM, Gruppo V, Brooks E, Gilliland J, Scherman M, Reichlen MJ, et al.
Limited activity of clofazimine as a single drug in a mouse model of tuberculosis exhibiting caseous necrotic granulomas. Antimicrob Agents Chemother 2014;58:4026-34.
Velayati AA, Farnia P, Masjedi MR. Latent tuberculosis (TB) bacilli: Yes or no to preventive chemotherapy. Int J Mycobacteriol 2012;1:1-2. [Full text]
Velayati AA, Abeel T, Shea T, Konstantinovich Zhavnerko G, Birren B, Cassell GH, et al.
Populations of latent Mycobacterium tuberculosis
lack a cell wall: Isolation, visualization, and whole-genome characterization. Int J Mycobacteriol 2016;5:66-73. [Full text]
Boeree MJ, Heinrich N, Aarnoutse R, Diacon AH, Dawson R, Rehal S, et al.
High-dose rifampicin, moxifloxacin, and SQ109 for treating tuberculosis: A multi-arm, multi-stage randomised controlled trial. Lancet Infect Dis 2017;17:39-49.
Haley CA. Treatment of latent tuberculosis infection. Microbiol Spectrum 2017;5:TNMI7-0039-2016. doi:10.1128/microbiolspec.TNMI7-0039-2016.
[Figure 1], [Figure 2]
[Table 1], [Table 2]
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