The impact of BCG dose and revaccination on trained immunity.

The innate immune system can display heterologous memory-like responses termed trained immunity after stimulation by certain vaccinations or infections. In this randomized, placebo-controlled trial, we investigated the modulation of Bacille Calmette-Guérin (BCG)-induced trained immunity by BCG revaccination or high-dose BCG administration, in comparison to a standard dose. We show that monocytes from all groups of BCG-vaccinated individuals exerted increased TNFα production after ex-vivo stimulation with various unrelated pathogens. Similarly, we observed increased amounts of T-cell-derived IFNγ after M. tuberculosis exposure, regardless of the BCG intervention. NK cell cytokine production, especially after heterologous stimulation with the fungal pathogen Candida albicans, was predominantly boosted after high dose BCG administration. Cytokine production capacity before vaccination was inversely correlated with trained immunity. While the induction of a trained immunity profile is largely dose- or frequency independent, baseline cytokine production capacity is associated with the magnitude of the innate immune memory response after BCG vaccination.

An In Vitro Perspective on What Individual Antimicrobials Add to Mycobacterium avium Complex Therapies.

For Mycobacterium avium complex pulmonary disease (MAC-PD), current treatment regimens yield low cure rates. To obtain an evidence-based combination therapy, we assessed the in vitro activity of six drugs, namely, clarithromycin (CLR), rifampin (RIF), ethambutol (EMB), amikacin (AMK), clofazimine (CLO), and minocycline (MIN), alone and in combination, against Mycobacterium avium and studied the contributions of individual antibiotics to efficacy. The MICs of all antibiotics against M. avium ATCC 700898 were determined by broth microdilution. We performed kinetic time-kill assays of all single drugs and clinically relevant two-, three-, four-, and five-drug combinations against M. avium. Pharmacodynamic interactions of these combinations were assessed using area under the time-kill curve-derived effect size and Bliss independence. Adding a second drug yielded an average increase of the effect size (E) of 18.7% ± 32.9%, although antagonism was seen in some combinations. Adding a third drug showed a smaller increase in effect size (+12.2% ± 11.5%). The RIF-CLO-CLR (E of 102 log10 CFU/ml · day), RIF-AMK-CLR (E of 101 log10 CFU/ml · day), and AMK-MIN-EMB (E of 97.8 log10 CFU/ml · day) regimens proved more active than the recommended RIF-EMB-CLR regimen (E of 89.1 log10 CFU/ml · day). The addition of a fourth drug had little impact on effect size (+4.54% ± 3.08%). In vitro, several two- and three-drug regimens are as effective as the currently recommended regimen for MAC-PD. Adding a fourth drug to any regimen had little additional effect. In vitro, the most promising regimen would be RIF-AMK-macrolide or RIF-CLO-macrolide.

The Benzimidazole SPR719 Shows Promising Concentration-Dependent Activity and Synergy against Nontuberculous Mycobacteria.

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is emerging worldwide. Currently recommended multidrug treatment regimens yield poor outcomes, and new drugs and regimens are direly needed. SPR719, the active moiety of SPR720, is a new benzimidazole antibiotic with limited data on antimycobacterial activity. We determined MICs and MBCs against 138 clinical and reference strains of M. avium complex (MAC), M. kansasii, M. abscessus, M. xenopi, M. malmoense, and M. simiae and determined synergy with antimycobacterial drugs by checkerboard titrations. To study pharmacodynamics, we performed time-kill kinetics assays of SPR719 alone and in combinations against M. avium, M. kansasii, and M. abscessus and assessed synergy by response surface analysis according to Bliss independence. SPR719 showed potent activity against MAC (MIC90, 2 mg/liter) and M. kansasii (MIC90, 0.125 mg/liter) and modest activity against M. abscessus (MIC90, 8 mg/liter); its activity is bacteriostatic and concentration-dependent. We recorded a potential for combination therapy with ethambutol against M. kansasii and M. avium and synergy with clarithromycin against M. abscessus Ethambutol increased the SPR719 kill rate against M. kansasii but only prevented SPR719 resistance in M. avium SPR719 is active in vitro against NTM; its activity is strongest against M. kansasii, followed by MAC and M. abscessus SPR719 shows promise for combination therapy with ethambutol against MAC and M. kansasii and synergy with clarithromycin against M. abscessus The parent drug SPR720 could have a role especially in MAC pulmonary disease treatment. Further studies in dynamic models and trials are ongoing to advance clinical development.

Thioridazine Is an Efflux Pump Inhibitor in Mycobacterium avium Complex but of Limited Clinical Relevance.

Treatment of Mycobacterium avium complex pulmonary disease (MAC-PD) is challenging partly due to high efflux pump expression. Thioridazine might block these efflux pumps. We explore the efficacy of thioridazine against M. avium isolates using MICs, time-kill combination assays, ex vivo macrophage infection assays, and efflux assays. Thioridazine is bactericidal against M. avium, inhibits intracellular growth at 2× MIC, and blocks ethidium bromide efflux. However, its toxicity and low plasma concentrations make it unlikely to add efficacy to MAC-PD therapy.

Inhaled tigecycline is effective against Mycobacterium abscessus in vitro and in vivo.

BACKGROUND: Mycobacterium abscessus causes chronic pulmonary infections. Owing to its resistance to most classes of antibiotics, treatment is complex and cure rates are only 45%. Tigecycline is active against M. abscessus, but severe toxicity and the need for IV administration limit its use. OBJECTIVES: To assess the potential of inhaled tigecycline as a treatment for M. abscessus pulmonary disease, by measuring its efficacy in a mouse model of chronic M. abscessus pulmonary disease, establishing the intracellular activity of tigecycline against M. abscessus in human macrophages and measuring the activity of tigecycline in the sputum of cystic fibrosis patients. METHODS: We infected GM-CSF knockout mice with M. abscessus by intrapulmonary aerosol. Infected mice were treated with tigecycline in 0.25, 1.25 and 2.5 mg doses, by inhalation, or untreated, for 28 days. Tigecycline was added to human peripheral blood-derived macrophages infected with M. abscessus to assess its intracellular activity. We performed a time-kill kinetics experiment of tigecycline against M. abscessus with and without sputum of cystic fibrosis patients. RESULTS: Inhaled tigecycline proved highly effective against M. abscessus in GM-CSF knockout mice. The effect was dose dependent. Tigecycline showed potent activity against M. abscessus in macrophages and retained most of its activity in the presence of sputum of cystic fibrosis patients. CONCLUSIONS: Inhaled tigecycline may represent a viable treatment option for M. abscessus pulmonary disease, where treatment outcomes are currently very poor. A stable and safe formulation is required to proceed to further pharmacodynamic studies and ultimately clinical trials.

Is there a role for tedizolid in the treatment of non-tuberculous mycobacterial disease?

BACKGROUND: Pulmonary infections caused by non-tuberculous mycobacteria (NTM) are hard to treat and have low cure rates despite intensive multidrug therapy. OBJECTIVES: To assess the feasibility of tedizolid, a new oxazolidinone, for the treatment of Mycobacterium avium and Mycobacterium abscessus. METHODS: We determined MICs of tedizolid for 113 isolates of NTM. Synergy with key antimycobacterial drugs was assessed using the chequerboard method and calculation of the FIC index (FICI). We performed time-kill kinetics assays of tedizolid alone and combined with amikacin for M. abscessus and with ethambutol for M. avium. Human macrophages were infected with M. abscessus and M. avium and subsequently treated with tedizolid; intracellular and extracellular cfu were quantified over time. RESULTS: NTM isolates generally had a lower MIC of tedizolid than of linezolid. FICIs were lowest between tedizolid and amikacin for M. abscessus (FICI = 0.75) and between tedizolid and ethambutol for M. avium (FICI = 0.72). Clarithromycin and tedizolid showed initial synergy, which was abrogated by erm(41)-induced macrolide resistance (FICI = 0.53). Tedizolid had a weak bacteriostatic effect on M. abscessus and combination with amikacin slightly prolonged its effect. Tedizolid had concentration-dependent activity against M. avium and its efficacy was enhanced by ethambutol. Both combinations had a concentration-dependent synergistic effect. Tedizolid could inhibit the intracellular bacterial population of both M. avium and M. abscessus. CONCLUSIONS: Tedizolid should be further investigated in pharmacodynamic studies and clinical trials for M. avium complex pulmonary disease. It is less active against M. abscessus, but still promising.

The differential effect of clarithromycin and azithromycin on induction of macrolide resistance in Mycobacterium abscessus.

Aim: Antibiotic resistance in Mycobacterium abscessus renders treatment poorly effective. Despite erm(41)-gene-mediated macrolide resistance, treatment with azithromycin or clarithromycin is recommended. It is contested whether macrolides differ in erm(41) induction. We determine whether this is the case. Methods:M. abscessus CIP104536 was used. Minimum inhibitory concentrations of clarithromycin and azithromycin were determined. Time-kill kinetics of M. abscessus exposed to azithromycin or clarithromycin were performed and RNA was isolated at predetermined intervals for erm(41) quantification. Results: Minimum inhibitory concentrations increased >30-fold. Time-kill kinetics showed a temporary bacteriostatic effect, abrogated by induced resistance. Erm(41) expression was increased following exposure to either macrolide for 7 days. Conclusion: Both macrolides induce resistance similarly, and this should not be an argument in choosing either macrolide for therapy.

Auranofin Activity Exposes Thioredoxin Reductase as a Viable Drug Target in Mycobacterium abscessus.

Nontuberculous mycobacteria (NTM) are highly drug-resistant, opportunistic pathogens that can cause pulmonary disease. The outcomes of the currently recommended treatment regimens are poor, especially for Mycobacterium abscessus New or repurposed drugs are direly needed. Auranofin, a gold-based antirheumatic agent, was investigated for Mycobacterium tuberculosis Here, we test auranofin against NTM in vitro and ex vivo We tested the susceptibility of 63 NTM isolates to auranofin using broth microdilution. Next, we assessed synergy between auranofin and antimycobacterial drugs using the checkerboard method and calculated the fractional inhibition concentration index (FICI). Using time-kill kinetics assays (TK), we assessed pharmacodynamics of auranofin alone and in combination with drug combinations showing the lowest FICIs for M. abscessus CIP 104536. A response surface analysis was used to assess synergistic interactions over time in TKs. Primary isolated macrophages were infected with M. abscessus and treated with auranofin. Finally, using KEGG Orthology, we looked for orthologues to auranofins drug target in M. tuberculosisM. abscessus had the lowest auranofin MIC50 (2 µg/ml) among the tested NTM. The lowest average FICIs were observed between auranofin and amikacin (0.45) and linezolid (0.50). Auranofin exhibited concentration-dependent killing of M. abscessus, with >1-log killing at concentrations of >2× MIC. Only amikacin was synergistic with auranofin according to Bliss independence. Auranofin could not lower the intracellular bacterial load in macrophages. Auranofin itself may not be feasible for M. abscessus treatment, but these data point toward a promising, unutilized drug target.

Stable pantothenamide bioisosteres: novel antibiotics for Gram-positive bacteria.

The emergence of multidrug resistant bacteria has prioritized the development of new antibiotics. N-substituted pantothenamides, analogs of the natural compound pantetheine, were reported to target bacterial coenzyme A biosynthesis, but these compounds have never reached the clinic due to their instability in biological fluids. Plasma-stable pantothenamide analogs could overcome these issues. We first synthesized a number of bioisosteres of the prototypic pantothenamide N7-Pan. A compound with an inverted amide bond (CXP18.6-012) was found to provide plasma-stability with minimal loss of activity compared to the parent compound N7-Pan. Next, we synthesized inverted pantothenamides with a large variety of side chains. Among these we identified a number of novel stable inverted pantothenamides with selective activity against Gram-positive bacteria such as staphylococci and streptococci, at low micromolar concentrations. These data provide future direction for the development of pantothenamides with clinical potential.

Clofazimine inhalation suspension for the aerosol treatment of pulmonary nontuberculous mycobacterial infections.

BACKGROUND: Nontuberculous mycobacteria are recognized as a concern for cystic fibrosis (CF) patients due to increasing disease prevalence and the potential for detrimental effects on pulmonary function and mortality. Current standard of care involves prolonged systemic antibiotics, which often leads to severe side effects and poor treatment outcomes. In this study, we investigated the tolerability and efficacy of a novel inhaled therapeutic in various mouse models of NTM disease. METHODS: We developed clofazimine inhalation suspension (CIS), a novel formulation of clofazimine developed for inhaled administration. To determine the efficacy, minimum inhibitory concentrations were evaluated in vitro, and tolerability of CIS was determined in naïve mouse models over various durations. After establishing tolerability, CIS efficacy was tested in in vivo infection models of both Mycobacterium avium and M. abscessus. Lung and plasma clofazimine levels after chronic treatments were evaluated. RESULTS: Clofazimine inhalation suspension demonstrated antimycobacterial activity in vitro, with MIC values between 0.125 and 2 µg/ml for M. avium complex and M. abscessus. Administration into naïve mice showed that CIS was well tolerated at doses up to 28 mg/kg over 28 consecutive treatments. In vivo, CIS was shown to significantly improve bacterial elimination from the lungs of both acute and chronic NTM-infected mouse models compared to negative controls and oral clofazimine administration. Clofazimine concentrations in lung tissue were approximately four times higher than the concentrations achieved by oral dosing. CONCLUSION: Clofazimine inhalation suspension is a well tolerated and effective novel therapeutic candidate for the treatment of NTM infections in mouse models.

A bedaquiline/clofazimine combination regimen might add activity to the treatment of clinically relevant non-tuberculous mycobacteria.

BACKGROUND: Non-tuberculous mycobacteria (NTM) infections are hard to treat. New antimicrobial drugs and smarter combination regimens are needed. OBJECTIVES: Our aim was to determine the in vitro activity of bedaquiline against NTM and assess its synergy with established antimycobacterials. METHODS: We determined MICs of bedaquiline for clinically relevant NTM species and Mycobacterium tuberculosis by broth microdilution for 30 isolates. Synergy testing was performed using the chequerboard method for 22 reference strains and clinical isolates of Mycobacterium abscessus (MAB) and Mycobacterium avium complex (MAC). Time-kill kinetics (TK) assays with resistance monitoring of bedaquiline alone and combined with clofazimine were performed for MAB CIP 104536 and M. avium ATCC 700898; bedaquiline/clarithromycin combinations were evaluated against M. avium ATCC 700898. Interactions were assessed for TK experiments based on Bliss independence. RESULTS: Bedaquiline had modest activity against tested NTM, with MICs between <0.007 and 1 mg/L. Bedaquiline showed no interaction with tested drugs against MAB or MAC. Lowest mean fractional inhibitory concentration index (FICI) values were 0.79 with clofazimine for MAB and 0.97 with clofazimine and 0.82 with clarithromycin for MAC. In TK assays, bedaquiline showed a bacteriostatic effect. Clofazimine extended the bacteriostatic activity of bedaquiline against MAB and yielded a slight bactericidal effect against M. avium. The bedaquiline/clofazimine combination slowed emergence of bedaquiline resistance for M. avium but promoted it for MAB. Relative to Bliss independence, bedaquiline/clofazimine showed synergistic interaction over time for MAB and no interaction for M. avium and bedaquiline/clarithromycin showed antagonistic interaction for M. avium. CONCLUSIONS: Following these in vitro data, a bedaquiline/clofazimine combination might add activity to MAB and MAC treatment. The bedaquiline/clarithromycin combination might have lower activity compared with bedaquiline alone for MAC treatment.

Minocycline Has No Clear Role in the Treatment of Mycobacterium abscessus Disease.

Mycobacterium abscessus causes a difficult-to-treat pulmonary disease (MAb-PD). After initial intravenous treatment, minocycline is recommended in the oral continuation phase of treatment. We determined the MICs, synergy, and time-kill kinetics of minocycline against M. abscessus With MICs of 8 to 512 mg/liter, rapid emergence of tolerance in time-kill assays, and no synergy with other drugs used to treat MAb-PD, minocycline appears ineffective against M. abscessus These in vitro data question its role as a MAb-PD treatment modality.

Mycobacterium avium complex bacteria remain viable in sputum during storage and refrigeration.

Diagnostic mycobacteriology often involves shipping of samples to centralized laboratories. Using two quantitative culture techniques, we show that 7 days storage of sputum samples at room temperature or 4 °C does not affect the number of viable Mycobacterium avium complex bacteria. Storage at room temperature increases the chance of contamination.