Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus

An effective regimen for treatment of tuberculosis (TB) is comprised of multiple drugs that inhibit a range of essential cellular activities in Mycobacterium tuberculosis. The effectiveness of a regimen is further enhanced if constituent drugs act with synergy. Here, we report that faropenem (a penem) or biapenem, doripenem, or meropenem (carbapenems), which belong to the β-lactam class of antibiotics, and rifampin, one of the drugs that forms the backbone of TB treatment, act with synergy when combined. One of the reasons (carba)penems are seldom used for treatment of TB is the high dosage levels required, often at the therapeutic limits. The synergistic combination of rifampin and these (carba)penems indicates that (carba)penems can be administered at dosages that are therapeutically relevant. The combination of faropenem and rifampin also limits the frequency of resistant mutants, as we were unable to obtain spontaneous mutants in the presence of these two drugs. The combinations of rifampin and (carba)penems were effective not only against drug-sensitive Mycobacterium tuberculosis but also against drug-resistant clinical isolates that are otherwise resistant to rifampin. A combination of doripenem or biapenem and rifampin also exhibited synergistic activity against Mycobacterium abscessus. Although the MICs of these three drugs alone against M. abscessus are too high to be of clinical relevance, their concentrations in combinations are therapeutically relevant; therefore, they warrant further evaluation for clinical utility to treat Mycobacterium abscessus infection, especially in cystic fibrosis patients.     

Lack of Antimicrobial Bactericidal Activity in Mycobacterium abscessus

Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organism''s natural resistance toward most clinically available antimicrobials. We investigated the bactericidal activity of antibiotics commonly administered in M. abscessus infections in order to better understand the poor therapeutic outcome. Time-kill curves were generated for clinical M. abscessus isolates, Mycobacterium smegmatis, and Escherichia coli by using antibiotics commonly categorized as bactericidal (amikacin and moxifloxacin) or bacteriostatic (tigecycline and linezolid). In addition, the impact of aminoglycoside-modifying enzymes on the mode of action of substrate and nonsubstrate aminoglycosides was studied by using M. smegmatis as a model organism. While amikacin and moxifloxacin were bactericidal against E. coli, none of the tested compounds showed bactericidal activity against M. abscessus. Further mechanistic investigations of the mode of action of aminoglycosides in M. smegmatis revealed that the bactericidal activity of tobramycin and gentamicin was restored by disruption of the chromosomal aac(2′) gene in the mycobacterial genome. The lack of bactericidal antibiotics in currently recommended treatment regimens provides a reasonable explanation for the poor therapeutic outcome in M. abscessus infection. Our findings suggest that chromosomally encoded drug-modifying enzymes play an important role in the lack of aminoglycoside bactericidal activity against rapidly growing mycobacteria.     

Tigecycline Is Highly Efficacious against Mycobacterium abscessus Pulmonary Disease

Mycobacterium abscessus causes chronic pulmonary infections that are extremely difficult to cure. The currently recommended combination therapy is associated with high failure rates and relapse. Tigecycline has been explored in salvage regimens, with a response rate of 43% in those who received at least a month of therapy. We performed a dose-response study in a hollow-fiber system model of pulmonary M. abscessus infection in which we recapitulated tigecycline human pulmonary concentration-time profiles of 8 different doses for 21 days. We identified the maximal kill or efficacy in CFU per milliliter and the ratio of the 0- to 24-h area under the concentration-time curve to MIC (AUC/MIC) associated with 80% efficacy (EC₈₀). The tigecycline efficacy was 5.38 ± 2.35 log₁₀ CFU/ml, and the drug achieved the unprecedented feat of a bacterial level of 1.0 log₁₀ CFU/ml below the pretreatment inoculum (1-log kill) of M. abscessus in the hollow-fiber system. The EC₈₀ AUC/MIC ratio was 36.65, while that for a 1-log kill was 44.6. Monte Carlo experiments with 10,000 patients were used to identify the clinical dose best able to achieve the EC₈₀ or 1-log kill. The standard dose of 100 mg/day had a cumulative fraction of response of 51% for the EC₈₀ and 46% for 1-log kill. For both the EC₈₀ target and 1-log kill, the optimal tigecycline clinical dose was identified as 200 mg/day. The susceptibility breakpoint was ≤0.5 mg/liter. Tigecycline is the most active single agent evaluated to date, and we propose that 200 mg/day be examined as the backbone of new combination therapy regimens to replace current treatment.     

In vitro activity of tedizolid against the Mycobacterium abscessus complex

Infections due to Mycobacterium abscessus carry a poor prognosis since this rapidly growing mycobacterium is intrinsically resistant to most antibiotics. Here, we evaluate the in vitro activity of the new oxazolidinone tedizolid against a collection of 44 M. abscessus clinical isolates. The MIC₅₀s and MIC₉₀s of tedizolid (2 and 8 μg/mL, respectively) were 2- to 16-fold lower than those of linezolid. There was no difference between the 3M. abscessus subspecies. Time-kill assays did not show any bactericidal activity at 4- and 8-fold the MIC. Combination of tedizolid with clarithromycin was synergistic against 1 out of 6 isolates, while indifferent interactions were observed for tedizolid combined with tigecycline, ciprofloxacin, and amikacin.     

Activities of moxifloxacin in combination with macrolides against clinical isolates of Mycobacterium abscessus and Mycobacterium massiliense

Infections caused by Mycobacterium abscessus and Mycobacterium massiliense are on the rise among humans. Although macrolides, including clarithromycin (CLR) and azithromycin (AZM), are key antibiotics for the treatment of M. abscessus and M. massiliense infections, treatment regimens for these infections are still largely undefined. In this study, we evaluated the in vitro, ex vivo, and in vivo activities of moxifloxacin (MXF) in combination with macrolides against clinically isolated M. abscessus and M. massiliense strains. Overall, CLR, AZM, and MXF alone showed activity against both species in vitro, ex vivo, and in vivo. When MXF was combined with a macrolide against M. abscessus isolates, antagonism was observed in 65.4% (17/26) of the strains with CLR and 46.2% (12/26) of the strains with AZM in vitro as well as in 66.7% (10/15) of the strains with CLR and 40.0% (6/15) of the strains with AZM in macrophages as determined by the fractional inhibitory concentration index. In contrast, either indifferent or synergistic effects of the MXF-macrolide combinations were observed against only M. massiliense strains. Moreover, a murine infection model showed similar results. Antagonism between the MXF and macrolide combinations was observed in five out of seven M. abscessus strains, while indifferent and synergistic effects for these combinations were observed for three of the six M. massiliense strains tested, respectively. In conclusion, the activity of MXF in combination with a macrolide differed for M. abscessus and M. massiliense infections and the addition of MXF to macrolide therapy had no benefit for the treatment of M. abscessus infections.     

In vitro activity of TP-271 against Mycobacterium abscessus, Mycobacterium fortuitum, and Nocardia species

The in vitro activities of TP-271, a novel fluorocycline antimicrobial, against 22 isolates of Mycobacterium abscessus, 22 isolates of Mycobacterium fortuitum, and 19 isolates of Nocardia spp. were studied by a microtiter broth dilution method. The MIC(90)s for M. abscessus, M. fortuitum, and Nocardia spp. were 0.5 μg/ml, 0.03 μg/ml, and 8 μg/ml, respectively. TP-271 was significantly more active than the respective control drug in virtually all tests.     

Gallium Compounds Exhibit Potential as New Therapeutic Agents against Mycobacterium abscessus

The rapidly growing nontuberculous mycobacterial species Mycobacterium abscessus has recently emerged as an important pathogen in patients with cystic fibrosis (CF). Treatment options are limited because of the organism''s innate resistance to standard antituberculous antibiotics, as well as other currently available antibiotics. New antibiotic approaches to the treatment of M. abscessus are urgently needed. The goal of the present study was to assess the growth-inhibitory activity of different Ga compounds against an American Type Culture Collection (ATCC) strain and clinical isolates of M. abscessus obtained from CF and other patients. In our results, using Ga(NO₃)₃ and all of the other Ga compounds tested inhibited the growth of ATCC 19977 and clinical isolates of M. abscessus. Inhibition was mediated by disrupting iron uptake, as the addition of exogenous iron (Fe) restored basal growth. There were modest differences in inhibition among the isolates for the same Ga chelates, and for most Ga chelates there was only a slight difference in potency from Ga(NO₃)₃. In contrast, Ga-protoporphyrin completely and significantly inhibited the ATCC strain and clinical isolates of M. abscessus at much lower concentrations than Ga(NO₃)₃. In in vitro broth culture, Ga-protoporphyrin was more potent than Ga(NO₃)₃. When M. abscessus growth inside the human macrophage THP-1 cell line was assessed, Ga-protoporphyrin was >20 times more active than Ga(NO₃)₃. The present work suggests that Ga exhibits potent growth-inhibitory capacity against the ATCC strain, as well as against antibiotic-resistant clinical isolates of M. abscessus, including the highly antibiotic-resistant strain MC2638. Ga-based therapy offers the potential for further development as a novel therapy against M. abscessus.     

In vitro activity between linezolid and other antimicrobial agents against Mycobacterium abscessus complex

Linezolid (LZD) serves as an effective option in the treatment of Mycobacterium abscessus complex (MABC) infection. Unfortunately, the combined activities of LZD with other antimicrobial agents against MABC have not been evaluated systemically. In this study, we randomly selected 32 Mycobacterium abscessus and 32 Mycobacterium massiliense isolates for the determination of in vitro synergistic effect between LZD and other antimicrobial agents, including amikacin (AMK), moxifloxacin (MOX), cefoxitin (CFX) and tigecycline (TGC). Out of 64 MABC isolates tested, only one (1.6%, 1/64) and two (3.2%, 2/64) exhibited resistance to AMK and LZD, respectively. Statistical analysis revealed that the percentage of TGC-resistant isolates was significantly lower among M. massiliense (9.4%, 3/32) than that among M. abscessus (25.0%, 8/32, P < 0.001). In addition, LZD and AMK showed synergy for 29 MABC isolates (45.3%), whereas no antagonism was noted for this combination. The second mostly frequent synergistic effect was found in LZD plus TGC combination, and 26.6% (17/64) of the strains tested exhibited synergy. In contrast, LZD-CFX and LZD-MOX combinations appeared antagonistic for half of the isolates (48.4%, 31/64 for CFX and 51.6%, 33/64), and almost no synergistic effect was reported in any of the strains for these two combinations. In conclusion, our data reveal that LZD and AMK show the most potent activity against MABC. The frequent synergism is observed in LZD-AMK and LZD-TGC combinations, while LZD rarely exhibits in vitro synergy with MOX and CFX when tested against MABC.     

In Vivo Assessment of Drug Efficacy against Mycobacterium abscessus Using the Embryonic Zebrafish Test System

Mycobacterium abscessus is responsible for a wide spectrum of clinical syndromes and is one of the most intrinsically drug-resistant mycobacterial species. Recent evaluation of the in vivo therapeutic efficacy of the few potentially active antibiotics against M. abscessus was essentially performed using immunocompromised mice. Herein, we assessed the feasibility and sensitivity of fluorescence imaging for monitoring the in vivo activity of drugs against acute M. abscessus infection using zebrafish embryos. A protocol was developed where clarithromycin and imipenem were directly added to water containing fluorescent M. abscessus-infected embryos in a 96-well plate format. The status of the infection with increasing drug concentrations was visualized on a spatiotemporal level. Drug efficacy was assessed quantitatively by measuring the index of protection, the bacterial burden (CFU), and the number of abscesses through fluorescence measurements. Both drugs were active in infected embryos and were capable of significantly increasing embryo survival in a dose-dependent manner. Protection from bacterial killing correlated with restricted mycobacterial growth in the drug-treated larvae and with reduced pathophysiological symptoms, such as the number of abscesses within the brain. In conclusion, we present here a new and efficient method for testing and compare the in vivo activity of two clinically relevant drugs based on a fluorescent reporter strain in zebrafish embryos. This approach could be used for rapid determination of the in vivo drug susceptibility profile of clinical isolates and to assess the preclinical efficacy of new compounds against M. abscessus.