MAP Drug Susceptibility Testing: Where Are We in 2019?

One of the key advances related to DST was the development of a reliable method to quantify MAP burden using a modified MGIT™ system. This nonradiometric BACTEC™ MGIT™ 960 system, which detects mycobacterial species commonly found in patient samples, was adapt­ed for use with a culture medium specific for MAP (MGIT™ Para-TB medium).

In 2008, Beckler and colleagues under­took the challenge of establishing a correlation between gene mutations and phenotypic an­tibiotic resistance (Beckler et al., 2008). They proposed that the most direct route to MAP DST is a molecular typing method rather than rely­ing on the slow-growing nature of MAP (dou­bling time >24 h). Frontline anti-TB drugs RIF and rifabutin (RFB), with the well-established corresponding target RpoB, were used to il­lustrate correlation between single nucleotide polymorphisms (SNPs) and clinical significance. A molecular-based polymerase chain reaction (PCR) method was designed to probe the 81- bp rifampicin resistance-determining region (RRDR) in rpoB of the ATCC MAP strain, nu­merous human and cattle isolates, and three external species. MAP strains that harboured mutations in the RRDR displayed a marked in­crease in MIC to RIF (Table 10.1). Additionally, differing effects on MIC to RIF were observed de­pending on the mutations detected.

Table 10.1. rpoB mutations and clinical significance. (Adapted from Beckler et al., 2008.)

Rifampicin minimum inhibitory % Inhibited at

Microorganism Strain concentration (mg/l) 1 mg/l of RIF rpoB gene ∆Amino acid

MAP, Mycobacterium avium subsp.

alternative to classical DST, but further studies would be required to evaluate the DST prediction of clinical outcomes for MAP. Molecular typing results are categorical (wild type vs mutant) yet the clinical outcome is determined by the phe­notype of the bacteria, which is a quantitative evaluation (see Box 10.2). Moreover, molecular DST loses sensitivity when mutations fall outside of the target region, illustrated by the clinical isolate MAP185. This isolate contained a silent mutation in the RRDR (i.e. no amino acid substi­tution in RpoB) yet still manifested an increased MIC to RIF, suggesting that RIF resistance in MAP, as in M. tuberculosis and many other bac­terial species, may result from mutations outside of the RRDR.

According to a review by Gisbert and Panes in 2009, up to 40% of naive Crohn's disease patients fail both traditional and biological therapies, specifically tumour necrosis factor (TNF) monoclonal antibody inhibitor inflixi­mab (Gisbert and Panes, 2009). Authors of an Expert Opinion on Biological Therapy article pro­pose that the 40% of patients who display pri­mary refractoriness should be investigated for MAP infection and might benefit from treatment with antimicrobials for mycobacteria upon con­firmation (Savarino et al., 2019). Several ran­domized controlled trials (Borody et al., 2002; Selby et al., 2007; Alcedo et al., 2016) and case series (Chamberlin et al., 2007b; Alcedo et al., 2016) reported promising results from Crohn's disease patients on anti-MAP regimens and complete healing of Crohn's disease ulcers over a 6-month period when RIF and clarithromy­cin (CLA) were used as a dual therapy, respec­tively. Building upon these findings, RedHill Biopharma created a triple antibiotic combina­tion therapy in one pill that they call RHB-104: 95mg CLA (0.63%), 10mg clofazimine (CLO) (0.067%) and 45 mg RIF (0.30%), with an in­tended adult dose of five pills twice daily (Alcedo et al., 2016).

2020.1 Patients were stratified for concomitant use of anti-TNF agents and treated with either RHB-104 or placebo as an add-on to standard of care for 52 weeks in an open-label regimen. Primary outcome was remission after 16 weeks with RHB-104. Secondary outcomes include time of response, response duration, time to remission and remission duration. Early data suggest RHB-104 is a potentially new treatment for Crohn's disease.² Remission rates and safety analysis in patients with anti-TNF use indicate that RHB-104 may be effective and safe as an adjunct treatment to standard of care therapy for Crohn's disease.

In 2011, Fecteau et al. postulated that similar trivalent metal ions to iron, like gallium, could interfere with iron homeostasis and cause bacterial cell death (Fecteau et al., 2011a). They evaluated the efficacy of gallium nitrate as a growth inhibitor against MAP isolates from cat­tle, bison, alpaca and humans. They reported MIC₉₀ values ranging from 200 to 700 μM. The authors abandoned gallium nitrate studies when it failed to provide sufficient protection to calves from MAP infection (Fecteau et al., 2011b). The same authors turned to gallium maltolate, a metal-organic coordination complex with improved lipid solubility, as a potential MAP growth inhibitor and compared antimicrobial activities with the original metal salt, gallium nitrate. MIC₉₀ values for two bovine isolates were reported as the following: 190 μM (164-220 95% CI) and 140 μM (102-194 95% CI) for gal­lium nitrate, and 94 μM (74-122) (95% CI) and 132 μM (99-179) (95%CI) for gallium malto­late (Fecteau et al., 2014). Gallium maltolate dis­played a slight increase in antimicrobial activity with greater lipophilicity. Despite similarities to iron, gallium appears to be a poor antimicrobial agent whether as a salt (nitrate) or coordination complex (maltolate).

Most antimicrobial agents tested against MAP have been small inorganic molecules (see gallium study) or repurposed TB drugs. In 2017, one of the first studies by Gonec et al. (2017) examined the antimicrobial activity of novel, fully synthetic n-arylpiperazines containing an ethane-1,2-diyl linker. Compounds 8a-c and 8e­h exhibited MIC₉₀ >500 μM. Notwithstanding adequate lipophilic properties, the n- arylpiperazine series with ethane-1,2-diyl linker appeared to be a poor chemical scaffold against MAP.

Perhaps the most promising study of MAP drug discovery was from 2018. Quinolones have been recognized as a prominent class of fully synthetic antimicrobials since 1962 when Lesher et al. made their serendipitous discovery of nalidixic acid (Andriole, 2005). Then in 1980, the class of quinolones was revolutionized by the addition of fluorine, and the first generation of fluoroquinolones was born. The effective use of fluoroquinolones prompted medicinal chemists to probe derivatives like quinolines. Indeed, the most impactful study on drug discovery in the field of mycobacteria in recent years was the revealing of bedaquiline, a diarylquinoline anti­biotic approved for the treatment of multidrug­resistant TB in 2012. This novel quinoline backbone created sparks in the medicinal chem­istry community, and Lougheed and collabora­tors discovered anti-TB activity from primaquine (PQ), an 8-aminoquinoline anti-malarial drug (Lougheed et al., 2009). Pavic et al synthesized four novel series consisting of amides, ureas, semicarbazides or bis-ureas as the linkers with the main primaquine pharmacophore and ex­amined their antimycobacterial activity on MAP (Pavic et al., 2018). They observed that hydroxyl, halogen and trifluoromethyl substi­tuted benzene rings belonging to the urea series were universally active against all mycobacteria tested, while amides exhibited weak activity. The semicarbazide series showed equipotent activity to the parent compound PQ. Four promising can­didates were discovered in the bis-urea series. In the end, compounds 2 m, 4 p and 4 u (MAP MIC: 2μg∕ml, 2μg∕ml, 4μg∕ml, respectively) were selected as the most potent antimycobacterial agents and well tolerated in an L6 cytotoxicity cell model (cytotoxicity: 150μg∕ml, 30μg∕ml, 81.6μg∕ml). The MIC against MAP and L6 cy­totoxicity of PQ is 32 μg∕ml and 2 7.1 ug∕ml, re­spectively. Thus, Pavic et al. (2018) were able to build upon the primaquine pharmacophore and improve the activity of the parent compound while minimizing cytotoxic effects.

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