Preclinical murine models for the testing of antimicrobials against Mycobacterium abscessus pulmonary infections: Current practices and recommendations.

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, is increasingly recognized as an important pathogen of the human lung, disproportionally affecting people with cystic fibrosis (CF) and other susceptible individuals with non-CF bronchiectasis and compromised immune functions. M. abscessus infections are extremely difficult to treat due to intrinsic resistance to many antibiotics, including most anti-tuberculous drugs. Current standard-of-care chemotherapy is long, includes multiple oral and parenteral repurposed drugs, and is associated with significant toxicity. The development of more effective oral antibiotics to treat M. abscessus infections has thus emerged as a high priority. While murine models have proven instrumental in predicting the efficacy of therapeutic treatments for M. tuberculosis infections, the preclinical evaluation of drugs against M. abscessus infections has proven more challenging due to the difficulty of establishing a progressive, sustained, pulmonary infection with this pathogen in mice. To address this issue, a series of three workshops were hosted in 2023 by the Cystic Fibrosis Foundation (CFF) and the National Institute of Allergy and Infectious Diseases (NIAID) to review the current murine models of M. abscessus infections, discuss current challenges and identify priorities toward establishing validated and globally harmonized preclinical models. This paper summarizes the key points from these workshops. The hope is that the recommendations that emerged from this exercise will facilitate the implementation of informative murine models of therapeutic efficacy testing across laboratories, improve reproducibility from lab-to-lab and accelerate preclinical-to-clinical translation.

Efficacies of three drug regimens containing omadacycline to treat Mycobacteroides abscessus disease.

Mycobacteroides abscessus (Mab, also known as Mycobacterium abscessus) causes opportunistic pulmonary and soft tissue infections that are difficult to cure with existing treatments. Omadacycline, a new tetracycline antibiotic, exhibits potent in vitro and in vivo activity against Mab. As regimens containing multiple antibiotics are required to produce a durable cure for Mab disease, we assessed efficacies of three three-drug combinations in a pre-clinical mouse model of pulmonary Mab disease to identify companion drugs with which omadacycline exhibits the highest efficacy. Additionally, we assessed the susceptibility of Mab recovered from mouse lungs after four weeks of exposure to the three triple-drug regimens. Among the three-drug regimens, omadacycline + imipenem + amikacin produced the largest reduction in Mab burden, whereas omadacycline + imipenem + linezolid exhibited the most effective early bactericidal activity. Omadacycline + linezolid + clofazimine, a regimen that can be administered orally, lacked early bactericidal activity but produced a gradual reduction in the lung Mab burden over time. The robust efficacy exhibited by these three regimens in the mouse model supports their further evaluation in patients with Mab lung disease. As we were unable to isolate drug-resistant Mab mutants at the completion of four weeks of treatment, these triple-drug combinations show promise of producing durable cure and minimizing selection of resistant mutants.

A strip-based assay for detection of CrfA enzyme activity to differentiate Mycobacterium tuberculosis and non-tuberculous mycobacteria.

There is an unmet need for tools that permit diagnosis of Tuberculosis (TB) that are affordable, low-tech, and can differentiate Mycobacterium tuberculosis (M.tb) from non-tuberculous mycobacteria (NTM). In this study, we have developed a strip-based assay to detect the activity of a unique Carbapenem Resistance Factor A (CrfA) enzyme present only in M.tb. The strip comprises of PVDF (Polyvinylidene fluoride) membrane that has an immobilized anti-CrfA antibody to capture the CrfA enzyme from M.tb lysate. Lysate of mycobacteria is applied to the strip, washed, and incubated in the presence of chromogenic reporter dye which is a substrate for CrfA. A change in the color of the dye that is readily visible to the naked eye is the readout. We evaluated lysates from M.tb and various NTMs namely, M. abscessus, M. chelonae, M. avium, M. obuense, M. paraintracellulare, M. kansasi, including the patient-derived sputum samples. The strip assay selectively identified only those samples containing M.tb. Based on this evidence, this new assay enables the identification and differentiation of M.tb from NTMs in patient sputum samples. As this tool can be simple to use, therefore has the potential to serve the unmet need for diagnosis of TB and NTM infections in resource-limited settings.

Efficacy of Omadacycline-Containing Regimen in a Mouse Model of Pulmonary Mycobacteroides abscessus Disease.

Mycobacteroides abscessus is an opportunistic pathogen in people with structural lung conditions such as bronchiectasis, chronic obstructive pulmonary disease, and cystic fibrosis. Pulmonary M. abscessus infection causes progressive symptomatic and functional decline as well as diminished lung function and is often incurable with existing antibiotics. We investigated the efficacy of a new tetracycline, omadacycline, in combination with existing antibiotics recommended to treat this indication, in a mouse model of M. abscessus lung disease. Amikacin, azithromycin, bedaquiline, biapenem, cefoxitin, clofazimine, imipenem, linezolid, and rifabutin were selected as companions to omadacycline. M. abscessus burden in the lungs of mice over a 4-week treatment duration was considered the endpoint. Omadacycline in combination with linezolid, imipenem, cefoxitin, biapenem, or rifabutin exhibited early bactericidal activity compared to any single drug. Using three M. abscessus isolates, we also determined the in vitro frequency of spontaneous resistance against omadacycline to be between 1.9 × 10-10 and 6.2 × 10-10 and the frequency of persistence against omadacycline to be between 5.3 × 10-6 and 1.3 × 10-5. Based on these findings, the combination of omadacycline and select drugs that are included in the recent treatment guidelines may exhibit improved potency to treat M. abscessus lung disease. IMPORTANCE M. abscessus disease incidence is increasing in the United States. This disease is difficult to cure with existing antibiotics. In this study, we describe the efficacy of a new tetracycline antibiotic, omadacycline, in combination with an existing antibiotic to treat this disease. A mouse model of M. abscessus lung disease was used to assess the efficacies of these experimental treatment regimens. Omadacycline in combination with select existing antibiotics exhibited bactericidal activity during the early phase of treatment.

Microbiological profile, preclinical pharmacokinetics and efficacy of CRS0393, a novel antimycobacterial agent targeting MmpL3.

The benzothiazole amide CRS0393 demonstrated excellent in vitro activity against nontuberculous mycobacteria (NTM), including M. abscessus isolates from cystic fibrosis (CF) patients, with minimum inhibitory concentrations (MICs) of ≤0.03-0.5 µg/mL. The essential transport protein MmpL3 was confirmed as the target via analysis of spontaneous resistant mutants and further biological profiling. In mouse pharmacokinetic studies, intratracheal instillation of a single dose of CRS0393 resulted in high concentrations of drug in epithelial lining fluid (ELF) and lung tissue, which remained above the M. abscessus MIC for at least 9 hours post-dose. This exposure resulted in a penetration ratio of 261 for ELF and 54 for lung tissue relative to plasma. CRS0393 showed good oral bioavailability, particularly when formulated in kolliphor oil, with a lung-to-plasma penetration ratio ranging from 0.5 to 4. CRS0393 demonstrated concentration-dependent reduction of intracellular M. abscessus in a THP-1 macrophage infection model. CRS0393 was well tolerated following intranasal administration (8 mg/kg) or oral dosing (25 mg/kg) once daily for 28 days in dexamethasone-treated C3HeB/FeJ mice. Efficacy against M. abscessus strain 103 was achieved via the intranasal route, while oral dosing will need further optimization. CRS0393 holds promise for development as a novel agent with broad antimycobacterial activity.

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.

Peptidoglycan compositional analysis of Mycobacterium smegmatis using high-resolution LC-MS.

Peptidoglycan (PG) is the exoskeleton of bacterial cells and is required for their viability, growth, and cell division. Unlike most bacteria, mycobacteria possess an atypical PG characterized by a high degree of unique linkages and chemical modifications which most likely serve as important determinants of virulence and pathogenesis in mycobacterial diseases. Despite this important role, the chemical composition and molecular architecture of mycobacterial PG have yet to be fully determined. Here we determined the chemical composition of PG from Mycobacterium smegmatis using high-resolution liquid chromatography-mass spectrometry. Purified cell walls from the stationary phase were digested with mutanolysin and compositional analysis was performed on 130 muropeptide ions that were identified using an in silico PG library. The relative abundance for each muropeptide ion was measured by integrating the extracted-ion chromatogram. The percentage of crosslink per PG subunit was measured at 45%. While both 3→3 and 4→3 transpeptide cross-linkages were found in PG dimers, a high abundance of 3→3 linkages was found associated with the trimers. Approximately 43% of disaccharides in the PG of M. smegmatis showed modifications by acetylation or deacetylation. A significant number of PG trimers are found with a loss of 41.00 amu that is consistent with N-deacetylation, whereas the dimers show a gain of 42.01 amu corresponding to O-acetylation of the PG disaccharides. This suggests a possible role of PG acetylation in the regulation of cell wall homeostasis in M. smegmatis. Collectively, these data report important novel insights into the ultrastructure of mycobacterial PG.

T405, a New Penem, Exhibits In Vivo Efficacy against M. abscessus and Synergy with ß-Lactams Imipenem and Cefditoren.

Mycobacteroides abscessus (Mab) is an emerging environmental microbe that causes chronic lung disease in patients with compromised lung function such as cystic fibrosis and bronchiectasis. It is intrinsically resistant to most antibiotics, therefore there are only few antibiotics that can be repurposed to treat Mab disease. Although current recommendations require daily intake of multiple antibiotics for more than a year, cure rate is low and often associated with significant adverse events. Here, we describe in vivo efficacy of T405, a recently discovered ß-lactam antibiotic of the penem subclass, in a mouse model of pulmonary Mab infection. Imipenem, one of the standard-of-care drugs to treat Mab disease, and also a ß-lactam antibiotic from a chemical class similar to T405, was included as a comparator. Probenecid was included with both T405 and imipenem to reduce the rate of their renal clearance. T405 exhibited bactericidal activity against Mab from the onset of treatment and reduced Mab lung burden at a rate similar to that exhibited by imipenem. The MIC of T405 against Mab was unaltered after 4 weeks of exposure to T405 in the lungs of mice. Using an in vitro assay, we also demonstrate that T405 in combination with imipenem, cefditoren or avibactam exhibits synergism against Mab. Additionally, we describe a scheme for synthesis and purification of T405 on an industrial scale. These attributes make T405 a promising candidate for further preclinical assessment to treat Mab disease.

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.

Allosteric cooperation in ß-lactam binding to a non-classical transpeptidase.

L,D-transpeptidase function predominates in atypical 3 → 3 transpeptide networking of peptidoglycan (PG) layer in Mycobacterium tuberculosis. Prior studies of L,D-transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or ß-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by ß-lactams. Here, we report identification of an allosteric site at a distance of 21 Å from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second ß-lactam molecule and influences binding at the catalytic site. We provide evidence that two ß-lactam molecules bind co-operatively to this enzyme, one non-covalently at the S-pocket and one covalently at the catalytic site. This dual ß-lactam-binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new drugs against M. tuberculosis.

Early Bactericidal Activity of Meropenem plus Clavulanate (with or without Rifampin) for Tuberculosis: The COMRADE Randomized, Phase 2A Clinical Trial.

Rationale: Carbapenems are recommended for treatment of drug-resistant tuberculosis. Optimal dosing remains uncertain. Objectives: To evaluate the 14-day bactericidal activity of meropenem, at different doses, with or without rifampin. Methods: Individuals with drug-sensitive pulmonary tuberculosis were randomized to one of four intravenous meropenem-based arms: 2 g every 8 hours (TID) (arm C), 2 g TID plus rifampin at 20 mg/kg once daily (arm D), 1 g TID (arm E), or 3 g once daily (arm F). All participants received amoxicillin/clavulanate with each meropenem dose. Serial overnight sputum samples were collected from baseline and throughout treatment. Median daily fall in colony-forming unit (CFU) counts per milliliter of sputum (solid culture) (EBACFU0-14) and increase in time to positive culture (TTP) in liquid media were estimated with mixed-effects modeling. Serial blood samples were collected for pharmacokinetic analysis on Day 13. Measurements and Main Results: Sixty participants enrolled. Median EBACFU0-14 counts (2.5th-97.5th percentiles) were 0.22 (0.12-0.33), 0.12 (0.057-0.21), 0.059 (0.033-0.097), and 0.053 (0.035-0.081); TTP increased by 0.34 (0.21-0.75), 0.11 (0.052-0.37), 0.094 (0.034-0.23), and 0.12 (0.04-0.41) (log10 h), for arms C-F, respectively. Meropenem pharmacokinetics were not affected by rifampin coadministration. Twelve participants withdrew early, many of whom cited gastrointestinal adverse events. Conclusions: Bactericidal activity was greater with the World Health Organization-recommended total daily dose of 6 g daily than with a lower dose of 3 g daily. This difference was only detectable with solid culture. Tolerability of intravenous meropenem, with amoxicillin/clavulanate, though, was poor at all doses, calling into question the utility of this drug in second-line regimens. Clinical trial registered with www.clinicaltrials.gov (NCT03174184).

Penicillin Binding Proteins and ß-Lactamases of Mycobacterium tuberculosis: Reexamination of the Historical Paradigm.

Penicillin binding proteins (PBPs) have been extensively studied due to their importance to the physiology of bacterial cell wall peptidoglycan and as targets of the most widely used class of antibiotics, the ß-lactams. The existing paradigm asserts that PBPs catalyze the final step of peptidoglycan biosynthesis, and ß-lactams inhibit their activities. According to this paradigm, a distinct enzyme class, ß-lactamases, exists to inactivate ß-lactams. This paradigm has been the basis for how bacterial diseases are treated with ß-lactams. We tested whether this historical view accurately reflects the relationship between ß-lactams and the PBPs and the ß-lactamase, BlaC, of Mycobacterium tuberculosis. BlaC was the major inactivator of the cephalosporin subclass of ß-lactams. However, the PBPs PonA1 and PonA2 inactivated penicillins and carbapenems more effectively than BlaC. These findings demonstrate that select M. tuberculosis PBPs are effective at inactivating several ß-lactams. Lesser-known PBPs, DacB, DacB1, DacB2, and Rv2864c, a putative PBP, were comparably more resistant to inhibition by all ß-lactam subclasses. Additionally, Rv1730c exhibited low affinity to most ß-lactams. Based on these findings, we conclude that in M. tuberculosis, BlaC is not the only source of inactivation of ß-lactams. Therefore, the historical paradigm does not accurately describe the relationship between ß-lactams and M. tuberculosis. IMPORTANCE M. tuberculosis, the causative agent of tuberculosis, kills more humans than any other bacterium. ß-lactams are the most widely used class of antibiotics to treat bacterial infections. Unlike in the historical model that describes the relationship between ß-lactams and M. tuberculosis, we find that M. tuberculosis penicillin binding proteins are able to inactivate select ß-lactams with high efficiency.

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.

Mechanistic insight on the inhibition of D, D-carboxypeptidase from Mycobacterium tuberculosis by ß-lactam antibiotics: an ONIOM acylation study.

Mycobacterium tuberculosis cell wall is intricate and impermeable to many agents. A D, D-carboxypeptidase (DacB1) is one of the enzymes involved in the biosynthesis of cell wall peptidoglycan and catalyzes the terminal D-alanine cleavage from pentapeptide precursors. Catalytic activity and mechanism by which DacB1 functions is poorly understood. Herein, we investigated the acylation mechanism of DacB1 by ß-lactams using a 6-membered ring transition state model that involves a catalytic water molecule in the reaction pathway. The full transition states (TS) optimization plus frequency were achieved using the ONIOM (B3LYP/6-31 + G(d): AMBER) method. Subsequently, the activation free energies were computed via single-point calculations on fully optimized structures using B3LYP/6-311++(d,p): AMBER and M06-2X/6-311++(d,p): AMBER with an electronic embedding scheme. The 6-membered ring transition state is an effective model to examine the inactivation of DacB1 via acylation by ß-lactams antibiotics (imipenem, meropenem, and faropenem) in the presence of the catalytic water. The ΔG# values obtained suggest that the nucleophilic attack on the carbonyl carbon is the rate-limiting step with 13.62, 19.60 and 30.29 kcal mol-1 for Imi-DacB1, Mero-DacB1 and Faro-DacB1, respectively. The electrostatic potential (ESP) and natural bond orbital (NBO) analysis provided significant electronic details of the electron-rich region and charge delocalization, respectively, based on the concerted 6-membered ring transition state. The stabilization energies of charge transfer within the catalytic reaction pathway concurred with the obtained activation free energies. The outcomes of this study provide important molecular insight into the inactivation of D, D-carboxypeptidase by ß-lactams.Communicated by Ramaswamy H. Sarma.

Assessment of carbapenems in a mouse model of Mycobacterium tuberculosis infection.

We present further study of a subset of carbapenems, arising from a previously reported machine learning approach, with regard to their mouse pharmacokinetic profiling and subsequent study in a mouse model of sub-acute Mycobacterium tuberculosis infection. Pharmacokinetic metrics for such small molecules were compared to those for meropenem and biapenem, resulting in the selection of two carbapenems to be assessed for their ability to reduce M. tuberculosis bacterial loads in the lungs of infected mice. The original syntheses of these two carbapenems were optimized to provide multigram quantities of each compound. One of the two experimental carbapenems, JSF-2204, exhibited efficacy equivalent to that of meropenem, while both were inferior to rifampin. The lessons learned in this study point toward the need to further enhance the pharmacokinetic profiles of experimental carbapenems to positively impact in vivo efficacy performance.

ß-Lactam Combinations That Exhibit Synergy against Mycobacteroides abscessus Clinical Isolates.

Mycobacteroides abscessus (Mab) is an opportunistic environmental pathogen that can cause chronic pulmonary disease in the setting of structural lung conditions such as bronchiectasis, chronic obstructive pulmonary disease, and cystic fibrosis. These infections are often incurable and associated with rapid lung function decline. Mab is naturally resistant to most of the antibiotics available today, and current treatment guidelines require at least 1 year of daily multidrug therapy, which is often ineffective and is associated with significant toxicities. ß-Lactams are the most widely used class of antibiotics and have a demonstrated record of safety and tolerability. Here, using a panel of recent clinical isolates of Mab, we evaluated the in vitro activities of dual-ß-lactam combinations to identify new treatments with the potential to treat infections arising from a wide range of Mab strains. The Mab clinical isolates were heterogeneous, as reflected by the diversity of their genomes and differences in their susceptibilities to various drugs. Cefoxitin and imipenem are currently the only two ß-lactams included in the guidelines for treating Mab disease, yet they are not used concurrently in clinical practice. However, this dual-ß-lactam combination exhibited synergy against 100% of the isolates examined (n = 21). Equally surprising is the finding that the combination of two carbapenems, doripenem and imipenem, exhibited synergy against the majority of Mab isolates. In the setting of multidrug-resistant Mab disease with few therapeutic options, these combinations may offer viable immediate treatment options with efficacy against the broad spectrum of Mab strains infecting patients today.

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.

Structure and Function of L,D- and D,D-Transpeptidase Family Enzymes from Mycobacterium tuberculosis.

Peptidoglycan, the exoskeleton of bacterial cell and an essential barrier that protects the cell, is synthesized by a pathway where the final steps are catalysed by transpeptidases. Knowledge of the structure and function of these vital enzymes that generate this macromolecule in M. tuberculosis could facilitate the development of potent lead compounds against tuberculosis. This review summarizes the experimental and computational studies to date on these aspects of transpeptidases in M. tuberculosis that have been identified and validated. The reported structures of L,D- and D,D-transpeptidases, as well as their functionalities, are reviewed and the proposed enzymatic mechanisms for L,D-transpeptidases are summarized. In addition, we provide bioactivities of known tuberculosis drugs against these enzymes based on both experimental and computational approaches. Advancing knowledge about these prominent targets supports the development of new drugs with novel inhibition mechanisms overcoming the current need for new drugs against tuberculosis.