Clofazimine as a comparator for preclinical efficacy evaluations of experimental therapeutics against pulmonary M. abscessus infection in mice.

Mycobacteroides abscessus (Mab, also known as Mycobacterium abscessus) can cause chronic pulmonary disease in the setting of structural lung conditions. Current treatment recommendations require at least one year of daily therapy with repurposed antibiotics. Yet these therapies are often ineffective and associated with significant adverse events. To address this challenge, research efforts are underway to develop new antibiotics and regimens. During the preclinical phase of treatment development, experimental agents require testing and comparison alongside positive controls that are known agents with clinical history. As there are no FDA approved treatments for this indication, here, we have considered repurposed antibiotics currently included in the recommendation for treating Mab disease as candidates for selection of an ideal standard comparator that can serve as a positive control in preclinical studies. Clofazimine meets the criteria for an ideal positive control as it can be administered via the least invasive route, requires only once-daily dosing, is well tolerated, and is widely available in high purity from independent sources. Using a mouse model of pulmonary Mab disease, we assessed for ideal dosages of clofazimine in C3HeB/FeJ and BALB/c mice in a six-week treatment window. Clofazimine, 25 mg/kg, once daily, produced desired reduction in Mab burden in the lungs of C3HeB/FeJ and BALB/c mice. Based on these findings, we conclude that clofazimine meets the criteria for a positive control comparator in mice for use in preclinical efficacy assessments of agents for treatment of Mab pulmonary disease. Although not included in the current standard-of-care for treating Mab disease, rifabutin, 20 mg/kg, also produced desired reduction in Mab lung burden in C3HeB/FeJ mice but not in BALB/c mice. IMPORTANCE: Mycobacteroides abscessus can cause life-threatening infections in patients with chronic lung conditions. New treatments are needed as cure rate using existing drugs is low. During pre-clinical phase of treatment development, it is important to compare the efficacy of the experimental drug against existing ones with known history. Here, we demonstrate that clofazimine, one of the antibiotics repurposed for treating Mab disease, can serve as a positive control comparator for efficacy assessments of experimental drugs and regimens to treat M. abscessus disease in mice.

Structure-Activity Relationship of Penem Antibiotic Side Chains Used against Mycobacteria Reveals Highly Active Compounds.

The rise of antibiotic-resistant Mycobacterium tuberculosis and non-tuberculous mycobacterial infections has placed ever-increasing importance on discovering new antibiotics to treat these diseases. Recently, a new penem, T405, was discovered to have strong antimicrobial activity against M. tuberculosis and Mycobacteroides abscessus. Here, a penem library of C2 side-chain variants was synthesized, and their antimicrobial activities were evaluated against M. tuberculosis H37Rv and M. abscessus ATCC 19977. Several new penems with antimicrobial activity stronger than the standard-of-care carbapenem antibiotics were identified with some candidates improving on the activity of the lead compound, T405. Moreover, many candidates showed little or no increase in the minimum inhibitory concentration in the presence of serum compared to the highly protein-bound T405. The penems with the strongest activity identified in this study were then biochemically characterized by reaction with the representative l,d-transpeptidase LdtMt2 and the representative penicillin-binding protein d,d-carboxypeptidase DacB2.

Glby, Encoded by MAB_3167c, Is Required for In Vivo Growth of Mycobacteroides abscessus and Exhibits Mild ß-Lactamase Activity.

Mycobacteroides abscessus (Mab; also known as Mycobacterium abscessus) is an emerging opportunistic pathogen. Patients with structural lung conditions such as bronchiectasis, cystic fibrosis, and chronic obstructive pulmonary disease are at high risk of developing pulmonary Mab disease. This disease is often chronic as the current treatment regimens are sub-efficacious. Here, we characterize the phenotype of a Mab strain lacking the MAB_3167c locus, which encodes a protein hereafter referred to as Glby. We demonstrate that the loss of Glby impairs normal planktonic growth in liquid broth, results in longer average cell length, and a melding of surfaces between cells. Glby also exhibits a mild ß-lactamase activity. We also present evidence that amino acid substitutions that potentially alter Glby function are not favored. Lastly, we demonstrate that, in a mouse model of pulmonary Mab infection, the mutant lacking Glby was unable to proliferate, gradually cleared, and was undetectable after 3 weeks. These data suggest that an agent that inhibits Glby in vivo may be an efficacious treatment against Mab disease. IMPORTANCE Mycobacteroides abscessus can cause chronic pulmonary infections requiring administration of multiple antibiotics, still resulting in a low cure rate. The incidence of M. abscessus disease is increasing in the United States and the developed regions of the world. We show for the first time that a protein, Glby, affects growth of this bacterium. Using a mouse model of lung M. abscessus disease, we demonstrate that Glby is required for this bacterium to cause disease.

Potency of Omadacycline against Mycobacteroides abscessus Clinical Isolates In Vitro and in a Mouse Model of Pulmonary Infection.

The incidence of nontuberculous mycobacterial diseases in the United States is rising and has surpassed that of tuberculosis. Most notable among the nontuberculous mycobacteria is Mycobacteroides abscessus, an emerging environmental opportunistic pathogen capable of causing chronic infections. M. abscessus disease is difficult to treat, and the current treatment recommendations include repurposed antibiotics, several of which are associated with undesirable side effects. In this study, we have evaluated the activity of omadacycline, a new tetracycline derivative, against M. abscessus using in vitro and in vivo approaches. Omadacycline exhibited an MIC90 of 0.5 µg/mL against a panel of 32 contemporary M. abscessus clinical isolates, several of which were resistant to antibiotics that are commonly used for treatment of M. abscessus disease. Omadacycline combined with clarithromycin, azithromycin, cefdinir, rifabutin, or linezolid also exhibited synergism against several M. abscessus strains and did not exhibit antagonism when combined with an additional nine antibiotics also commonly considered to treat M. abscessus disease. Concentration-dependent activity of omadacycline was observed in time-kill assessments. Efficacy of omadacycline was evaluated in a mouse model of lung infection against four M. abscessus strains. A dose equivalent to the 300-mg standard oral human dose was used. Compared to the untreated control group, within 4 weeks of treatment, 1 to 3 log10 fewer M. abscessus CFU were observed in the lungs of mice treated with omadacycline. Treatment outcome was biphasic, with bactericidal activity observed after the first 2 weeks of treatment against all four M. abscessus strains.

Development of a penem antibiotic against Mycobacteroides abscessus.

ß-lactams are the most widely used antibiotic class to treat bacterial infections in humans. Mycobacteroides abscessus is an emerging pulmonary pathogen resistant to most antibiotics, including penicillins and cephalosporins. With no current FDA-approved treatment and cure rates <50%, there is a pressing need for effective therapies. Here we report T405, a new ß-lactam of the penem subclass that exhibits potent activity against M. abscessus and a panel of drug-resistant strains isolated from cystic fibrosis patients. Additionally, in combination with the ß-lactamase inhibitor avibactam, the rate of spontaneous resistance of M. abscessus to T405 approached the limit of detection. Lastly, we show the favorable pharmacokinetic profile of T405 in mice and the absence of toxicity at elevated dosage, which support the clinical potential of this compound.

A mouse model of pulmonary Mycobacteroides abscessus infection.

There is no preclinical mouse model to investigate pulmonary Mycobacteroides abscessus (formerly Mycobacterium abscessus) infection in an immunocompetent mouse strain, especially in the context of antibiotic testing and regimen development. We developed a mouse model of pulmonary M. abscessus infection using the aerosolized route of infection that leads to an increase in bacterial burden post- implantation and develops pathology as a result. In this mouse model, treatment with corticosteroid allows for initial proliferation and sustained M. abscessus pulmonary infection and permits evaluation of efficacies of antibiotics. Administration of corticosteroids that permitted higher levels of bacterial burden in the lungs were more likely to have pathology. Treatment of mice with antibiotics administered intranasally or subcutaneously significantly reduced lung M. abscessus burden. In addition to the reference strain, independent clinical isolates of M. abscessus also readily establish infection and proliferate in the lungs of mice in this model.

Synergistic Efficacy of ß-Lactam Combinations against Mycobacterium abscessus Pulmonary Infection in Mice.

Mycobacterium abscessus is an emerging pathogen capable of causing invasive pulmonary infections in patients with chronic lung diseases. These infections are difficult to treat, necessitating prolonged multidrug therapy, which is further complicated by extensive intrinsic and acquired resistance exhibited by clinical M. abscessus isolates. Therefore, development of novel treatment regimens effective against drug-resistant strains is crucial. Prior studies have demonstrated synergistic efficacy of several ß-lactams against M. abscessusin vitro; however, these combinations have never been tested in an animal model of M. abscessus pulmonary disease. We utilized a recently developed murine system of sustained M. abscessus lung infection delivered via an aerosol route to test the bactericidal efficacy of four novel dual ß-lactam combinations and one ß-lactam/ß-lactamase inhibitor combination. All five of the novel combinations exhibited synergy and resulted in at least 6-log10 reductions in bacterial burden in the lungs of mice at 4 weeks compared to untreated controls (P = 0.038).

Select ß-Lactam Combinations Exhibit Synergy against Mycobacterium abscessus In Vitro.

Mycobacterium abscessus is a nontuberculous mycobacterium that causes invasive pulmonary infections in patients with structural lung disease. M. abscessus is intrinsically resistant to several classes of antibiotics, and an increasing number of strains isolated from patients exhibit resistance to most antibiotics considered for treatment of infections by this mycobacterium. Therefore, there is an unmet need for new regimens with improved efficacy to treat this disease. Synthesis of the essential cell wall peptidoglycan in M. abscessus is achieved via two enzyme classes, l,d- and d,d-transpeptidases, with each class preferentially inhibited by different subclasses of ß-lactam antibiotics. We hypothesized that a combination of two ß-lactams that comprehensively inhibit the two enzyme classes will exhibit synergy in killing M. abscessus Paired combinations of antibiotics tested for in vitro synergy against M. abscessus included dual ß-lactams, a ß-lactam and a ß-lactamase inhibitor, and a ß-lactam and a rifamycin. Of the initial 206 combinations screened, 24 pairs exhibited synergy. A total of 13/24 pairs were combinations of two ß-lactams, and 12/24 pairs brought the MICs of both drugs to within the therapeutic range. Additionally, synergistic drug pairs significantly reduced the frequency of selection of spontaneous resistant mutants. These novel combinations of currently available antibiotics may offer viable immediate treatment options against highly-resistant M. abscessus infections.

Mycobacterium abscessus and ß-Lactams: Emerging Insights and Potential Opportunities.

ß-lactams, the most widely used class of antibiotics, are well-tolerated, and their molecular mechanisms of action against many bacteria are well-documented. Mycobacterium abscessus (Mab) is a highly drug-resistant rapidly-growing nontuberculous mycobacteria (NTM). Only in recent years have we started to gain insight into the unique relationship between ß-lactams and their targets in Mab. In this mini-review, we summarize recent findings that have begun to unravel the molecular basis for overall efficacy of ß-lactams against Mab and discuss emerging evidence that indicates that we have yet to harness the full potential of this antibiotic class to treat Mab infections.