The quantity of initiatives in antimicrobial drug development has significantly diminished since the remarkable era of antibiotic discovery, and several factors contribute to this decline. Firstly, the increasing prevalence of drug-resistant bacteria limits the effectiveness of new antibiotics, making it challenging to recoup investments in antibiotic development. Secondly, antibiotics are typically prescribed for short durations, in contrast to drugs for chronic conditions like hypertension or diabetes, which may render antibiotics less financially appealing to pharmaceutical companies. Thirdly, novel effective drugs are often preserved as last-resort treatments for highly-resistant bacterial infections. The goal is to mitigate the development of further resistance by limiting their widespread use. Overuse or misuse of these potent drugs can accelerate the emergence of resistant strains, making them ineffective sooner. Overexposure can diminish their efficacy over time, making it crucial to reserve them for cases where no other options are viable (Baker et al., 2017). Lastly, the discovery of new antibiotics presents scientific challenges (Ventola, 2015; Quang and Jang, 2021). Finding compounds that are both effective against bacteria and safe for humans is a complex process, and success is not guaranteed (Chopra et al., 2011). The identification and development of innovative and potent medications for combating NTMs are of paramount importance in the medical field. In comparison to the tuberculosis drug pipeline, which features a significant number of compounds undergoing clinical trials, the NTMs drug pipeline lags considerably (Wu et al., 2018). Notably, there is a critical need for the development of new treatments targeting Mab as there are currently no antibiotics approved by the Food and Drug Administration (FDA) for Mab infection.

Two primary strategies exist to bolster the development of effective Mab-targeting medicines. The first strategy follows the conventional drug development process, which encompasses the identification of novel chemical compounds. This process commences with drug screening using chemical and natural product libraries, progressing through hit identification, lead optimization, target identification, comprehensive preclinical investigations, and ultimately clinical trials (Egorova et al., 2021). Various screening methods have been employed in this pursuit for Mab drug discovery, including reporter-based assays, resazurin-based microplate assays, and image-based phenotypic screens (Gupta et al., 2017; Jeong et al., 2018; Richter et al., 2018; Kim et al., 2019; Malin et al., 2019; Hanh et al., 2020a,b). Nevertheless, despite these efforts, promising new chemical leads ready for clinical trials and market release remain scarce (Hanh et al., 2020a). This challenge may be attributed to the intrinsic drug-resistant mechanisms of Mab, resulting in a low hit rate for compounds targeting this bacterium (Malin et al., 2019). It’s noteworthy that the hit rate achieved in Mab screens is significantly lower than what is typically observed in screens targeting Mtb (Malin et al., 2019; Hanh et al., 2020a). Moreover, recent Mab drug screens have relied on conventional libraries composed of known antimycobacterial or antibacterial agents (Malin et al., 2019), diminishing the likelihood of identifying novel compounds targeting new mechanisms of action. Hence, there is an urgent need to develop new libraries with expanded chemical diversity to discover unique compounds. Additionally, reliable cell-based or in vivo assessment/screening methods that accurately mimic the host environment infected with Mab are imperative to advance our understanding and discovery of effective treatments for Mab-induced human infections (Carvalho et al., 2011; Bernut et al., 2014, 2015). The second strategy involves repurposing or repositioning existing medications for novel therapeutic indications. Most contemporary antibiotics and potential prospects against Mab have origins in the repurposing of pre-existing drugs or the cross-testing of compounds with activity and various mechanism of action against Mtb (Egorova et al., 2021). This method is particularly intriguing in the field of antibacterials, as the rapid evolution of resistance often outpaces the pace of medication development (Egorova et al., 2021). Repurposing previously approved pharmaceuticals can expedite the development process and reduce expenses (Egorova et al., 2021). Regrettably, the Mab drug pipeline remains underpopulated (Ganapathy and Dick, 2022). Currently, there are four recruiting, four completed, one terminated, and two non-recruiting clinical trials evaluating drug efficacy in Mab infection (NIH ClinicalTrials, 2023). However, these clinical trials have primarily utilized existing antibiotics through various drug delivery methods, including inhalation, novel drug encapsulation using biocompatible liposomes, and the exploration of new drug combinations (Quang and Jang, 2021). The primary reason for the limited success in anti-Mab drug discovery is the remarkable intrinsic resistance capabilities of Mab and its rapid acquired resistance against currently available active drugs (Wu et al., 2018).

Mycobacteria’s cell wall is primarily composed of lipids, specifically mycolic acids, constituting a significant portion (up to 60%) of the bacteria’s overall dry mass (Brennan and Nikaido, 1995). This cell wall features a waxy composition that serves as a physical barrier (Nessar et al., 2012), rendering mycobacteria less permeable than the outer membranes of gram-negative bacteria. In more detail, the mycobacterial envelope consists of three distinct layers: a typical plasma membrane, a complex cell wall, and an outer layer. The cell wall, notably, comprises a thick peptidoglycan layer covalently linked to arabinogalactan, which is esterified by mycolic acids, forming the inner leaflet of the mycomembrane (Figure 2). This unique structure inherently makes mycobacteria resistant to many antimicrobials. Hydrophilic drugs penetrate the mycobacterial cell wall slowly due to the inefficiency of mycobacterial porin in allowing antibiotic permeation, resulting in low antibiotic concentrations within the bacteria. The dense mycobacterial cell wall not only shields the bacterium from stressors but also poses challenges in nutrient uptake from the environment. To address this, mycobacteria often produce porins, proteins that create limited pathways for nutrient absorption. The expression of these porins is closely tied to the growth rate of NTMs, and they provide a conduit for certain antimicrobial agents to enter the mycobacterial cell (Saxena et al., 2021). Lipophilic agents may be hindered by the lipid bilayer, which has unusually low fluidity and thickness (Jarlier and Nikaido, 1994; Gutiérrez et al., 2018). Intriguingly, Mycobacterium chelonae, a species closely related to Mab due to its nearly identical biochemical features, has a cell envelope that is about 10–20 times less permeable than that of Mtb (Jarlier and Nikaido, 1994). Similar to Mtb, Mab possesses a mycobacterial cell wall with low permeability, which contributes to its drug resistance. Notably, like Mtb, Mab is thought to regulate cell wall structure and homeostasis through lipoprotein glycosylation. For example, the absence of protein-O-mannosyltransferase Pmt (MAB_1122c) in Mab leads to increased cell wall permeability and greater susceptibility to antibiotics such as RFP (Ganapathy et al., 2019). Furthermore, glycosylation of lipoproteins limits cell wall permeability to antibiotics like β-lactam agents that inhibit peptidoglycan synthesis. In β-lactam drug resistance, mycobacterial porins also play a role by facilitating the transport of small hydrophilic drugs across the membrane. Once antibiotics are internalized, they can reach their target in the cytoplasm and activate potential internal drug resistance mechanisms, collectively known as the “intrinsic resistome.” This resistome includes efflux pumps, antibiotic-modifying/inactivating enzymes, target-modifying enzymes, and genes conferring metal resistance (Nguyen and Thompson, 2006; Nessar et al., 2012).

Remarkably, unlike Mtb, Mab exhibits two distinct colony morphotypes: smooth non-cording (S) and rough cording morphotype (R). These differences in morphotypes depend on the presence or absence of cell surface-associated glycopeptidolipids (GPL), respectively (Howard et al., 2006). This distinctive property, associated with GPL status, affects sliding motility, biofilm formation, and drug susceptibility. For example, S morphotype strains that contain GPL, such as M. abscessus and M. bolletii, facilitate sliding across the surface and biofilm formation. Indeed, the Mab growing inside biofilms become tolerant to antibiotics due to physical barrier that can prevent the intracellular penetration of compounds. In fact, in vitro biofilm models of Mab have been exhibited to have decreased susceptibility to several first-line antibiotics, such as cefoxitin, amikacin, and clarithromycin (Greendyke and Byrd, 2008; Marrakchi et al., 2014). Conversely, R morphotype strains exhibit aggregation and cording. Recent studies suggest that Mab R strains are capable of growing in biofilm-like structures, which, similar to S biofilms, show greater tolerance than planktonic cultures to acidic pH, hydrogen peroxide, and treatment with antibiotics like AMK, AZM, and β-lactams (Gutiérrez et al., 2018; Story-Roller et al., 2018; Daher et al., 2022; López-Roa et al., 2022). Furthermore, biofilms formed by R colony types display higher mechanical resistance compared to those formed by S colony types (López-Roa et al., 2022). Biofilms also inhibit oxygen and nutrients from entering the cell, which causes a reduction Mab metabolism and, consequently, an increased tolerance to a harsh environment such as antibiotics treatment. Therefore, compounds that specifically target biofilm formation during antibiotic therapy are a new therapeutic strategy for clearance of Mab.

In invasive infections causing pulmonary colonization, Mab R strains are accountable for producing higher levels of trehalose dimycolate, consequently leading to the formation of massive bacterial cords. With the rough variant, the entire phagosome quickly merges with the lysosome, inducing phagosomal acidification and activating apoptosis and autophagy (Roux et al., 2016; López-Roa et al., 2022). This robust apoptosis-driven cell-death activity facilitates the extracellular replication of the R variant through rapid cord formation, preventing the engulfment of bacilli by neutrophils and macrophages. This process leads to abscess formation, tissue destruction, and acute infection (Jönsson et al., 2013; Bernut et al., 2016; López-Roa et al., 2022).

Mab possesses a family of transcriptional regulators that may play a role in conferring drug resistance, particularly the WhiB gene family (Morris et al., 2005). WhiB7, a transcriptional activator belonging to the WhiB family of transcriptional regulators, is conserved in actinomycetes and regulates critical cellular processes, including cell division, pathogenesis, and oxidative stress responses. The presence of a helix-turn-helix motif indicates its DNA-binding function (Soliveri et al., 2000; Burian et al., 2012; Nessar et al., 2012). In Mab, 128 genes, including erm(41) and eis2, have been identified in the WhiB7 regulon, indicating their induction through a WhiB7-dependent mechanism. Deletion of Mab whiB7 (MAB_3508c) renders the bacteria more susceptible to antibiotics, such as erythromycin, CLR, streptomycin, spectinomycin (SPC), AMK, and tetracycline, although it does not affect resistance to RFP or INH (Hurst-Hess et al., 2017). Significantly, exposing Mab to sub-inhibitory concentrations of CLR leads to the activation of whiB7 gene expression (Pryjma et al., 2017). This activation subsequently results in the development of resistance to AMK and CLR due to increased expression of erm(41) and eis2 genes (Pryjma et al., 2017; Johansen et al., 2020b).

Tetracycline molecules feature a linear fused tetracyclic core to which several functional groups are attached. These compounds are known to inhibit bacterial growth by preventing the binding of charged aminoacyl-tRNA to the ribosomal A site (Chopra and Roberts, 2001). Tigecycline (TGC) became the first glycylcycline antibiotic approved by the US FDA (Stein and Craig, 2006). While tetracyclines have been one of the most successful classes of antibiotics, their widespread use has led to extensive drug resistance, necessitating their discontinuation in treating various bacterial infections (Rudra et al., 2018). Occasionally, tetracyclines become ineffective due to TetX enzymes, also known as tetracycline destructases. In the past, the limited tolerance of Msm and Mtb to tetracycline was attributed to the WhiB7-dependent TetV/Tap efflux pump. However, Mab exhibits a resistance level approximately 500-fold higher than that of Msm and Mtb. Recently, Rudra and colleagues revealed that this heightened resistance in Mab to the tetracycline class is conferred by a WhiB7-independent tetracycline-inactivating monooxygenase, MAB_1496c (MabTetX). Exposure to sublethal doses of tetracycline and doxycycline leads to a more than 200-fold induction of MabTetX. Conversely, an isogenic deletion strain shows high sensitivity to both antibiotics. The authors also demonstrated that MabTetX’s expression is suppressed by MabTetRx. This finding highlights the potential use of an inhibitor to potentially reinstate the effectiveness of tetracycline and doxycycline (Rudra et al., 2018). As for acquired drug resistance, mutations in the sigH-rshA genes, which regulate heat shock and oxidative-stress responses, have also been found to be involved in TGC resistance or decreased sensitivity in Mab (Ng and Ngeow, 2022). Overexpression of the sigH gene, resulting from the C51R mutation in rshA, causes resistance to or decreased susceptibility to TGC (Ng and Ngeow, 2022).

Recently, OMC, which is classified as a tetracycline antibiotic, has exhibited favorable MIC against various Mab species (Singh et al., 2023). The activity and efficacy of OMC against Mab infection have been proven in both in vitro and in vivo (Shoen et al., 2019; Watkins and Deresinski, 2019; Kaushik et al., 2019a; Brown-Elliott and Wallace, 2021; Nicklas et al., 2022). OMC, like other tetracycline derivatives via binding to the tetracycline-binding site of the bacterial 16S ribosomal RNA, inhibiting bacterial protein synthesis (Brown-Elliott and Wallace, 2021). In contrast to first-generation tetracyclines, OMC has been intentionally engineered to bypass ribosomal protection and tetracycline efflux mechanisms. In Mab, the production of a monooxygenase enzyme may degrade tetracyclines, such as minocycline and doxycycline, but does not affect OMC (Luthra et al., 2018; El Ghali et al., 2023). This feature, in part, revitalizes its activity against recalcitrant MAB and may serve as a deterrent against the development of resistance during treatment. For example, studies by Bax et al. revealed that, except for a 1.5% occurrence at an OMC concentration of 4 mg/L, no instances of drug resistance selection exceeded the spontaneous mutation frequency (Bax et al., 2019).

Active efflux mechanisms have been identified as potential contributors to antibiotic resistance in mycobacteria. The primary role of efflux pump (EP) systems is to protect bacteria from harmful substances, maintain cellular homeostasis, and uphold physiological equilibrium by expelling toxins or metabolites into the extracellular environment (Louw et al., 2009; Remm et al., 2022). Mab possesses genetic sequences that encode protein constituents belonging to the major facilitator family ABC transporters and mycobacterial membrane protein large (MmpL) families (Ripoll et al., 2009), although the precise contribution of EPs to antibiotic resistance in Mab is not fully elucidated. ABC-type multidrug transporters utilize ATP energy to actively remove compounds from the cellular environment. MmpL transporters are multidrug EPs that play a crucial role in transporting various substrates from the periplasmic space to the extracellular environment (Kerr, 2002; Kerr et al., 2005). In further detail, the MmpL transporter family encodes proteins belonging to the resistance, nodulation, and cell division (RND) category. These proteins function as multidrug resistance pumps with the ability to transport a wide range of compounds, including cationic, anionic, and neutral substances, such as drugs, metals, and fatty acids (Domenech et al., 2005).

SPC is an aminocyclitol antibiotic that robustly inhibits bacterial protein synthesis by binding to the 30S subunit of the ribosome. However, its effectiveness against mycobacteria is restricted due to inherent resistance mechanisms (Hurst-Hess K. R. et al., 2023). Mab exhibits a notable inherent resistance to SPC, with MIC exceeding 1,000 μg/mL, rendering it unsuitable for therapeutic applications (Hurst-Hess K. R. et al., 2023). The whiB7 is responsible for Mab resistance for SPC because sublethal exposure to SPC strongly induces whiB7 and its regulon, and a ΔMab_whiB7 strain shows SPC sensitive (Hurst-Hess K. R. et al., 2023). Furthermore, MAB_2780c, a TetV-like efflux pump, provides high-level SPC resistance in Mab (Hurst-Hess K. et al., 2023). For instance, the elimination of MAB_2780c resulted in a significant enhancement of susceptibility to SPC, approximately 150 times greater than that observed in the wildtype bacteria (Hurst-Hess K. R. et al., 2023). The inclusion of the efflux pump inhibitor (EPI), verapamil, leads to a reduction in the MIC of SPC by over 100-fold in bacteria that express MAB_2780c, bringing it down to levels comparable to those observed for the deletion mutant of MAB_2780c (Hurst-Hess K. R. et al., 2023).

Genetic polymorphisms in highly conserved genes targeted by pharmaceutical medications have been associated with variations in sensitivity to pharmacological effects in Mab infections (Nessar et al., 2012).

In recent years, aminoacyl-tRNA synthetases (AARSs) have become significant targets for new antibacterial interventions. AARSs facilitate the acylation process, linking amino acids and tRNA. Inhibiting AARS activity halts protein synthesis, impeding bacterial growth. Benzoxaboroles, known as boron-heterocyclic antibiotics, inhibit leucyl-tRNA synthetase (LeuRS) (Rock et al., 2007). These drugs obstruct protein synthesis via the oxaborole tRNA-trapping mechanism, forming adducts with uncharged tRNALeu molecules that bind to the LeuRS editing domain (Rock et al., 2007). Multiple reports discuss the anti-Mab effects of LeuRS inhibitors, particularly a new class of LeuRS inhibitors such as epetraborole, DS86760016, EC/11770, MRX-6038, and GSK656 (Kim et al., 2021; Wu et al., 2022; Ganapathy et al., 2023a). High-level epetraborole resistance may be attributed to mutations in Mab at locations S303L, T322I, T323P, F321V, G393V, and Y421D within the LeuS gene (Ganapathy et al., 2021a). Among the LeuRS inhibitors, DS86760016 exhibits an improved pharmacokinetic profile, lower plasma clearance, longer plasma half-life, and higher renal excretion than epetraborole in animal models. A recent study by Nguyen et al. demonstrated that DS86760016 displayed similar activity to epetraborole treatment against Mab in vitro, intracellularly, and in zebrafish infection models, with a significantly lower mutation frequency. Laboratory-induced DS86760016-resistant strains included D284G, Q345R, Y420C, I426T, V468L, N469Y, and E524K, which were not found on the LeuS gene in epetraborole-resistant mutants (Nguyen et al., 2023).

Mycolic acids (MA) exist in various forms, including trehalose monomycolates (TMMs), trehalose dimycolates (TDMs), and mycolates covalently linked to arabinogalactan (AG) polysaccharides (McNeil et al., 2020). In the process of MA synthesis, MmpL3 plays a crucial role by transporting MA across the inner membrane, making its contribution to cell wall production indispensable (McNeil et al., 2020). Inhibition of the MmpL3 transporter leads to the accumulation of TMM intracellularly, causing a decrease in mycolyl arabinogalactan peptidoglycan (mAGP) and TDM levels (McNeil et al., 2020). Consequently, MmpL3 represents a versatile drug target, and inhibiting MmpL3 disrupts cell wall biosynthesis (Li et al., 2014). Multiple MmpL3 inhibitors have been recently identified through phenotypic screenings of chemical libraries against Mab. Promisingly, PIPD1, a piperidinol-based molecule, has shown potent in vitro and in vivo activity against clinical Mab strains. Treatment of infected zebrafish with PIPD1 increased embryo survival and reduced bacterial burden (Dupont et al., 2016). Major resistance to PIPD1 and other MmpL3 inhibitors, such as EJMCh-6 (2-(2-cyclohexylethyl)-5,6-dimethyl-1H-benzo[d]imidazole) and BMC-2i, is attributed to mutations in the MmpL3 gene (MAB_4508) of Mab at location A309P. The overexpression of MmpL3, containing the Ala309Pro mutation in Mab wild-type bacteria, results in significant drug resistance to MmpL3 inhibitors, confirming MmpL3 as their target (Dupont et al., 2016; Kozikowski et al., 2017).

LZ, a representative oxazolidinone, disrupts protein synthesis by inhibiting the peptidyl transferase activity of the 23S rRNA in the 50S ribosomal subunit, leading to ribosome stalling. Unfortunately, most clinical Mab isolates exhibit poor susceptibility to LZ (Negatu et al., 2023). However, the effectiveness of LZ against NTMs varies among different derivatives, and its clinical use in patients with NTMs can sometimes result in adverse events, such as peripheral neuropathy and cytopenias. Recently, Kim et al. introduced a novel oxazolidinone with a cyclic amidrazone named DPZ (LCB01-0371). DPZ demonstrated effective inhibition of Mab growth, both in vitro and in mouse lungs in vivo compared to LZ. Furthermore, DPZ exhibited bactericidal activity against all bacterial strains, irrespective of their resistance to AMK, CFX, or CLR. Kim et al. generated laboratory-induced resistant mutants to DPZ and identified mutations in rplC (encoding 50S ribosomal protein L3) at T424C and G419A, along with a nucleotide insertion at position 503 through sequencing analysis (Kim et al., 2017).

The aminobenzimidazole SPR719 targets the ATPase located on Gyrase B in Mtb. SPR719 also demonstrates activity against NTM and has recently entered clinical trials for lung diseases caused by NTM (Aragaw et al., 2022). Brown-Elliott et al. (2018) demonstrated in vitro activity of SPR719 against M. abscessus, M. massiliense, and related subspecies, with an observed MIC₅₀ (MIC required to inhibit the growth of 50% of Mab) value of ~2.0 μg/mL. Additionally, Rubio et al. reported that the phosphate prodrug SPR720 (of SPR719) exhibited favorable in vivo efficacy at a dose of 100 mg/kg/day in an SCID mouse model infected with Mab (Egorova et al., 2021). To identify the molecular target for Mab, Aragaw et al. recently induced two different morphotypes of SPR719-resistant mutants on agar containing 16 x MIC of SPR719, named large and small colonies. Interestingly, the small colony phenotype reflected a lower level of resistance, while large colonies showed high-level SPR719 resistance (>16-fold MIC increase). All strains contained a single amino acid polymorphism, Thr169Asn, in the ATPase domain of Gyrase B, and non-gyrB DNA sequence polymorphisms were revealed by whole-genome sequencing. Thr169 in Mab DNA gyrase corresponds to Ser169 in Mtb GyrB, causing resistance to SPR719 in Mtb (Aragaw et al., 2022).