Mechanisms of drug resistance in Mycobacterium tuberculosis: update 2015

Transcription

1 INT J TUBERC LUNG DIS 19(11): Q 2015 The Union STATE OF THE ART Mechanisms of drug resistance in Mycobacterium tuberculosis: update 2015 Y. Zhang,* W-W. Yew *Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA; Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University of Hong Kong, Hong Kong SAR, China SUMMARY Drug-resistant tuberculosis (DR-TB), including multiand extensively drug-resistant TB, is posing a significant challenge to effective treatment and TB control worldwide. New progress has been made in our understanding of the mechanisms of resistance to anti-tuberculosis drugs. This review provides an update on the major advances in drug resistance mechanisms since the previous publication in 2009, as well as added information on mechanisms of resistance to new drugs and repurposed agents. The recent application of whole genome sequencing technologies has provided new insight into the mechanisms and complexity of drug resistance. However, further research is needed to address the significance of newly discovered gene mutations in causing drug resistance. Improved knowledge of drug resistance mechanisms will help understand the mechanisms of action of the drugs, devise better molecular diagnostic tests for more effective DR-TB management (and for personalised treatment), and facilitate the development of new drugs to improve the treatment of this disease. KEY WORDS: antibiotics; drug resistance; mechanisms; molecular diagnostics; new drugs The use of multiple-drug therapy, although definitely beneficial, is not an absolute guarantee against the emergence of drug-resistant infections... Consequently, we cannot have confidence that drug-resistant tubercle bacilli will not emerge simply because multidrug therapy is employed. 1 ACCORDING TO the World Health Organization s (WHO s) 2014 global tuberculosis report, 2 there were about 9.0 million new tuberculosis (TB) patients and 1.5 million deaths in 2013; 3.5% of newly diagnosed and 20.5% of previously treated patients had multidrug-resistant TB (MDR-TB, defined as bacillary resistance to at least rifampicin [RMP] and isoniazid (INH]). The highest levels of MDR-TB were found in Eastern Europe and Central Asia, with rates reaching 20% and 50%, respectively. At least one case of extensively drug-resistant TB (XDR-TB, defined as MDR-TB with additional resistance to fluoroquinolone[s] [FQs] and one or more of three second-line injectable drugs [SLIDs], namely capreomycin [CPM], kanamycin [KM] and amikacin [AMK]) had been reported to the WHO from 92 countries by the end of An estimated 9% of MDR-TB patients had XDR-TB. The worldwide drug-resistant TB (DR-TB) epidemic thus remains an alarming problem, and is further aggravated by human immunodeficiency virus (HIV) coinfection. 3 The present review is aimed at updating readers on major advances in drug resistance mechanisms in Mycobacterium tuberculosis since the publication of the previous article in Additional information pertaining to newly developed drugs and repurposed agents have also been included. BASIC CONCEPTS IN THE DEVELOPMENT OF DRUG-RESISTANT TUBERCULOSIS There are two types of drug resistance in M. tuberculosis: genetic resistance and phenotypic resistance. Genetic drug resistance is due to mutations in chromosomal genes in growing bacteria, while phenotypic resistance or drug tolerance is due to epigenetic changes in gene expression and protein modification that cause tolerance to drugs in nongrowing persister bacteria. The two types of resistance have been responsible for a number of problems in effective TB control, with genetic resistance (Yang resistance), as present in MDR-/XDR-TB, causing problems worldwide, while the more subtle pheno- Correspondence to: Ying Zhang, Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, 615 N Wolfe Street, Baltimore, MD 21205, USA. Tel: (þ1) yzhang@jhsph.edu Article submitted 5 May Final version accepted 18 June [A version in French of this article is available from the Editorial Office in Paris and from the Union website

2 Mechanisms of drug resistance: update typic drug resistance, or tolerance (Yin resistance), as present in persisters, entails prolonged treatment and risk of post-treatment relapse. 5,6 The situation in vivo appears more complex, and the two types of resistance can overlap and interconvert. Prior stress or subinhibitory concentration of drugs may induce efflux pump expression, 7,8 which causes phenotypic resistance and may in turn facilitate the development of more stable genetic drug resistance, 8 while genetic resistance in growing organisms can develop persistence or phenotypic resistance. There is increasing interest in understanding the biology of mycobacterial persisters and developing anti-tuberculosis drugs that target them. 5 M. tuberculosis drug-resistant strains develop largely through the selection of genetic mutants. This is almost a wholly man-made phenomenon, resulting from suboptimal physician prescription and/or poor patient adherence. However, there is some recent evidence that pharmacokinetic-pharmacodynamic variability scenarios related to the induction of the mycobacterial drug efflux pump may also facilitate the development of genetic mutations in M. tuberculosis. 9 The development of drug resistance as a result of mutations in drug resistance genes in M. tuberculosis may incur a cost in terms of fitness and virulence of the organism. Acquired resistance can also be compounded by transmitted resistance. Recent systematic reviews have alluded to an association between primary resistance in M. tuberculosis and HIV coinfection, suggesting transmitted DR-TB, as a significant challenge to the management of this patient population. 10,11 Furthermore, recent studies from China have indicated that a significant number of MDR- and XDR-TB cases are due to the active transmission of (mainly) the Beijing genotype, 12 and the same is true for Europe 13 and Africa. 14 This is a worrying development that requires more studies to better understand how such apparently virulent DR- TB strains evolve and adapt in the host, and highlights the need for more effective means to curtail transmission. Clinical relevance of anti-tuberculosis drug resistance The clinical relevance of resistance to RMP and INH has been amply discussed. 4 Besides the known negative impact of bacillary resistance to pyrazinamide (PZA) on treatment outcomes among MDR- TB patients, including PZA in the treatment regimen for MDR-TB as guided by drug susceptibility testing (DST) might substantially improve early sputum culture conversion and subsequent cure or treatment completion. 15,16 A prospective cohort study conducted in eight countries that addressed the prevalence of and risk factors for resistance to second-line drugs (SLDs) in MDR-TB patients has yielded interesting and important findings. 17 Among over 1200 patients, 43.7% showed bacillary resistance to at least one SLD, 20% to at least one SLID and 12.9% to at least one FQ, with 6.7% of cases meeting the definition for XDR- TB. Previous treatment with SLDs was consistently the strongest risk factor for resistance to these drugs, with a four-fold increased risk for XDR-TB. Bacillary resistance to FQs and XDR-TB were found more frequently in women than men. Unemployment, alcohol abuse and smoking were associated with mycobacterial resistance to SLIDs across countries. In addition, in a study in the United States, the risk factors for bacillary acquired resistance to SLIDs included age years, positive HIV status, MDR- TB at treatment initiation and treatment with any SLD. However, the only predictor for bacillary acquired resistance to FQs was MDR-TB at treatment initiation. 18 A recent expanded analysis of these data from the same group of researchers further showed that mortality was significantly higher among TB patients with bacillary acquired resistance to SLDs, after controlling for age. MDR-TB at treatment initiation, positive HIV status and extra-pulmonary disease were also significantly associated with mortality. 19 A meta-analysis on treatment outcomes of MDR-TB was undertaken in about 6700 patients from 26 centres using individual patient data analysis. 20 Compared with treatment failure, relapse and death, treatment success was higher in MDR-TB patients carrying bacilli without additional resistance or with additional resistance to SLIDs only than in those with resistance to FQs alone or to FQs plus SLIDs. In XDR-TB patients, treatment success was highest if at least six drugs were used in the intensive phase and four in the continuation phase (odds ratios 4.9 and 6.1, respectively). Likewise, in another similar study focusing on XDR-TB, the odds of cure were significantly lower in XDR-TB patients with additional bacillary resistance to CPM and KM/AMK than patients with only XDR-TB. The odds of failure and death were found to be higher in all XDR-TB patients with additional bacillary resistance, inclusive of groups with bacillary resistance to oral bacteriostatic agents, with or without ethambutol (EMB) plus PZA. 21 It was recently noted that combined resistance to FQ (high-level) and PZA was associated with a poor treatment outcome in some MDR-TB patients treated with the 9-month Bangladesh regimen. 22 MECHANISMS OF RESISTANCE TO FIRST- AND SECOND-LINE ANTI-TUBERCULOSIS DRUGS The mechanisms of resistance of the various drugs are discussed below. Table 1 summarises the genetic basis and related information on resistance in M. tuberculosis to first-line drugs and SLDs. The resistance mutation profile for each drug is not listed due to space limitations. Readers may refer to drug resis-

3 1278 The International Journal of Tuberculosis and Lung Disease Table 1 Mechanisms of resistance in M. tuberculosis against first- and second-line drugs Drug (year of discovery) MIC lg/ml Gene involved in resistance Gene function Mechanism of action Mutation frequency % Isoniazid (1952) katg, inha Catalase-peroxidase, enoyl ACP reductase Inhibition of mycolic acid synthesis and other multiple effects Pyrazinamide (1952) pnca, rpsa, pand Nicotinamidase/pyrazinamidase, ribosomal S1 protein, aspartate decarboxylase Depletion of membrane energy; inhibition of trans-translation; inhibition of pantothenate and CoA synthesis Rifampin (1966) rpob ß-subunit of RNA polymerase Inhibition of RNA synthesis 95 Ethambutol (1961) 1 5 embb Arabinosyl transferase, *DPPR synthase Inhibition of arabinogalactan synthesis ubia Streptomycin (1943) 2 8 rpsl, rrs, gidb S12 ribosomal protein, Inhibition of protein synthesis S rrna, S rrna methyltransferase (G527 in 530 loop) Amikacin/kanamycin (1957) 2 4 rrs, eis, whib7 16S rrna, aminoglycoside acetyltransferase, transcriptional regulator Capreomycin (1960) 2 4 rrs tlya Quinolones (1963) gyra gyrb Ethionamide (1956) etaa/etha ethr inha Inhibition of protein synthesis 76 16S rrna, 2 0 -O-methyltransferase Inhibition of protein synthesis 85 DNA gyrase subunit A, DNA gyrase subunit B Flavin monooxygenase, transcription repressor, enoyl ACP reductase Inhibition of DNA synthesis Inhibition of mycolic acid synthesis PAS (1946) 1 8 thya dfra folc ribd Cycloserine (1955) alr ddl cyca Thymidylate synthase, dihydrofolate reductase, dihydrofolate synthase, enzyme in riboflavin biosynthesis Alanine racemase, D-alanine-D-alanine ligase, D-serine proton symporter Inhibition of folic acid and thymine nucleotide metabolism 37 Inhibition of cell wall peptidoglycan synthesis To be determined MIC ¼ minimum inhibitory concentration; ACP ¼ acyl-carrier-protein; CoA ¼ coenzyme A; DPPR ¼ 5-phospho-a-d-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase.

4 Mechanisms of drug resistance: update tance mutation databases for major frontline and SLDs at umr5558-bibiserv.univ-lyon1.fr/mubii/mubii-select. cgi or Isoniazid INH, a pro-drug that is activated by the catalaseperoxidase enzyme (KatG) encoded by the katg gene 23 to generate highly reactive species, is capable of attacking multiple targets in M. tuberculosis, 24 the primary one being the InhA enzyme (enoyl acyl carrier protein reductase). The active species (isonicotinic acyl radical or anion) reacts with nicotinamide adenine dinucleotide (H), forming INH-NAD adduct, which then inhibits InhA, causing inhibition of cell wall mycolic acid synthesis. 25 INH is active only against growing M. tuberculosis but not against non-growing bacilli (persisters). INH tolerance in non-growing organisms may be caused by mycobacterial DNA-binding protein 1 (MDP1), a histone-like protein, which downregulates katg transcription and could lead to tolerance to INH. 26 Mutations in katg are the major mechanism of INH resistance (Table 1). 4,23,27 The KatG S315 mutation is the most common mutation in INHresistant strains, accounting for 50 95% of INHresistant clinical isolates. 4,24 KatG S315 mutations usually do not completely eliminate catalase activity, and such strains may still retain fitness and virulence, which may explain its frequent occurrence among clinical isolates. Mutations in the katg promoter region fura-katg intergenic region that affect KatG expression were occasionally found to cause INH resistance in some strains. 28,29 Resistance to INH can also occur due to mutations in the promoter region of maba(fabg1)/inha operon causing overexpression of InhA or by mutations at the InhA active site. 25,30 In contrast to katg mutations, which usually cause high-level resistance, mutations in inha or its promoter region are usually associated with lowlevel resistance (minimum inhibitory concentration [MIC] lg/ml) and are less frequent than katg mutations (Table 1). 4,24 Mutations in inha not only cause INH resistance, they also confer cross-resistance to the structurally related drug ethionamide (ETH). 30 A small percentage of low-level INHresistant strains do not have mutations in katg or inha, which may be due to new mechanism(s) of resistance. However, an alternative explanation is heteroresistance or mixed population, where purification of single clones shows a small proportion of resistant clones containing katg mutations among sensitive clones with wild-type (wt) katg (Y Zhang, unpublished observations). INH-resistant strains due to katg deletion or mutations that lead to complete loss of catalase activity causing high-level resistance may cause loss of virulence. Strains with point mutations in katg, such as KatG315, which do not completely lose catalase activity, have a limited effect on fitness or virulence. In contrast, mutations in inha, which cause low-level resistance, do not cause loss of virulence. 34 Pyrazinamide PZA is a paradoxical drug with unique sterilising activity in anti-tuberculosis treatment. 35 In striking contrast to common antibiotics that kill or inhibit growing bacteria, PZA only kills non-growing persister bacteria and has poor activity against growing bacteria. 36 PZA is a pro-drug that has to be converted to its active form, pyrazinoic acid (POA), for activity by the pyrazinamidase/nicotinamidase enzyme encoded by the pnca gene of M. tuberculosis. 37 PZA has multiple effects on M. tuberculosis, including interference with membrane energy production, 38 and inhibition of RpsA (ribosomal protein S1), which is involved in transtranslation, 39 and PanD, which is involved in pantothenate and co-enzyme A synthesis. 40,41 Overexpression of RpsA renders M. tuberculosis more resistant to PZA. 39 In addition, a low-level PZAresistant clinical strain, DHM444, without pnca mutations, 42 was found to contain a deletion of alanine at the 438 th residue (438 DA) due to a 3 base pair (bp) GCC deletion in the C-terminus of RpsA. 39 POA bound to the wt RpsA but not the mutant RspADA438 from the PZA-resistant strain, and specifically inhibited the trans-translation of M. tuberculosis but not its canonical translation. 39 More recently, pand-encoding aspartate decarboxylase, involved in the synthesis of ß-alanine, which is a precursor for pantothenate and co-enzyme A biosynthesis, has been shown to serve as a new target of PZA. 41 Mutations in the pnca gene are the main mechanism of PZA resistance in M. tuberculosis. 37,42 47 pnca mutations are highly diverse and scattered along the gene, which is unique to PZA resistance. Most PZA-resistant M. tuberculosis strains (72 99%, average 85%) have mutations in pnca, but some do not. Those that do not have mutations in the drug target RpsA. 39,48 RpsA target mutations are usually associated with low-level PZA resistance (MIC lg/ml PZA). More recently, pand was found to be involved in PZA resistance. 40 pand mutations were identified in naturally PZA-resistant M. canettii strains and some PZA-resistant MDR-TB strains. M. canettii, a member of the M. tuberculosis complex, is intrinsically resistant to PZA, 48,49 and has mutations in both rpsa 48 and pand. 40 Culture-based DST of PZA is difficult and unreliable, with frequent problems of false resistance. 36,50,51 The very high correlation between PZA resistance and pnca mutations provides the opportunity to rapidly detect PZA resistance using pnca sequencing. 42,52 54 Dividing MDR-TB into

5 1280 The International Journal of Tuberculosis and Lung Disease PZA-susceptible and PZA-resistant MDR-TB for improved treatment of MDR-TB has thus been proposed. 15 However, some PZA-susceptible strains were reported to have non-synonymous mutations in pnca. 31,53 These mutations could cause low-level resistance close to the MIC cut-off for resistance that is not easily detected by current DST for PZA, and may be mistaken for PZA susceptibility. Further studies are required to address this issue. PZAresistant strains with diverse pnca mutations do not appear to have fitness cost or loss of virulence. 43 Interestingly, a recent study found that patients infected with PZA-monoresistant strains had worse clinical outcomes than those with drug-susceptible strains. 55 Rifampicin RMP is highly bactericidal and sterilising for M. tuberculosis. Although RMP is known to interfere with RNA synthesis by binding to the ß subunit of the RNA polymerase, a recent study showed that RMP binding to the RpoB target induces hydroxyl radical formation in susceptible but not resistant bacilli, and may contribute to the killing effect of RMP. 56 RMP resistance occurs at a frequency of 10 7/ 8, but a recent study showed a much higher mutation frequency, at 10 3, for the Beijing genotype strain than with the EAI (East African Indian) genotype strain, at 10 6 for RMP resistance, but no difference for other drugs, including INH and moxifloxacin (MFX). 57 This high mutation frequency is intriguing and may be related to the lower RMP concentration used or previous exposure to RMP before selection for RMP resistance. 58 Mutations in a defined region of the 81-bp region of rpob are found in about 96% of RMP-resistant M. tuberculosis isolates. 59 Mutations at positions 531, 526 and 516 are among the most frequent mutations in RMP-resistant strains. Mutations in rpob generally result in cross-resistance to all rifamycins, including rifabutin (RBT), but some RMP-resistant strains are RBT-susceptible. 60 Crossresistance to RMP and RBT seems to involve both the common 531 and 526 sites and also the beginning of the RpoB region 61 and the D516A-R529Q double mutations. 60 Mutations at F514FF, D516V and S522L are associated with resistance to RMP but susceptibility to RBT. 60 However, not all mutations in rpob are associated with RMP resistance. 60,62 For example, mutations at E510H, L511P, D516Y, N518D, H526N and L533P are not associated with RMP resistance and are found in RMP-susceptible strains. 60,63 In a separate study, about 10.5% (16/ 133) of the strains with rpob mutations identified by Xpert w MTB/RIF (Cepheid, Sunnyvale, CA, USA) as RMP-resistant were found to be RMP-susceptible on phenotypic MGITe test (BD, Sparks, MD, USA). 62 These findings caution us against relying solely on the molecular test for detecting RMP resistance, and suggest that the molecular test may not be able to completely replace phenotypic DST. However, a recent study showed that disputed rpob mutations may be responsible for RMP resistance among new cases and may lead to adverse treatment outcomes with first-line agents. 64 Reports of RMP-dependent or enhanced strains of M. tuberculosis in some MDR-TB strains is potentially worrying and less appreciated. RMPdependent strains may be more prevalent than currently realised, as the current diagnostic practice uses only drug-free media and may simply discount or discard strains that grow poorly in normal culture media without RMP. A recent study from China suggests that RMP-dependent/enhanced strains are common, with as many as 39% (18/46) of MDR-TB strains having RMP dependence or enhancement phenomenon. 67 The circumstances under which these strains arise remain unclear, but they often occur as MDR-TB, and seem to develop upon repeated treatment with rifamycins in retreatment patients, 66 and could be a result of overlapping genetic and phenotypic resistance. RBT has been suggested for treating RMP-resistant strains. 68 However, this might be a risky practice, as more powerful rifamycins such as RBT could lead to the development of RMP dependence or enhancement and potentially worsen the disease, 66 possibly as a result of genetic and epigenetic changes that enhance the fitness and virulence of the organism. The impact of rpob mutations on the fitness and pathogenesis of the organism has recently been reviewed, 69 and is worth paying attention to, as RMP resistance may carry more severe consequences, with enhanced virulence and pathogenesis. It would be of interest to determine the frequency of RMP dependence/enhancement, the mechanisms involved, and the contribution of such strains to treatment failure. RMP resistance due to rpob mutations could alter M. tuberculosis fitness; however, compensatory mutations in rpoc orrpoa could occur Ethambutol EMB interferes with the biosynthesis of cell wall arabinogalactan. 73 Arabinosyl transferase, encoded by embb, an enzyme involved in the synthesis of arabinogalactan, is the target of EMB in M. tuberculosis. 74 Mutations in embcab operon, in particular embb, and occasionally embc, are responsible for resistance to EMB. 74 embb codon 306 mutation is most frequent in clinical isolates resistant to EMB, accounting for as much as 68% of resistant strains. 75,76 While mutations at EmbB306 leading to certain amino acid changes caused EMB resistance, other amino acid substitutions had little effect on EMB resistance. 77 However, about 35% of EMBresistant strains (MIC, 10 lg/ml) do not have embb mutations, 78 suggesting other mechanisms of resis-

6 Mechanisms of drug resistance: update tance. Mutations in ubia, encoding the DPPR synthase involved in cell-wall synthesis, have recently been found to cause higher-level EMB resistance in conjunction with embb mutations. 79,80 Fluoroquinolones The well-known dominant role of gyra mutations in FQ-resistant TB remains unchanged. In a systematic review of gyr mutations, 81 of 1220 FQ-resistant M. tuberculosis isolates that were subjected to sequencing of the quinolone-resistance-determining region (QRDR) of gyra, 780 (64%) were found to have mutations. The QRDR of gyrb was sequenced in 534 resistant isolates, only 17 (3%) of which had mutations. Of the gyra mutations, 81% were inside the QRDR and 19% outside. Mutations at gyra codons 90, 91 and 94 were present in 54% of the FQresistant isolates (substitutions at amino acid 94 accounted for 37%). Of the gyrb mutations, only 44% were inside the QRDR. Such findings may have implications for the currently available genotypic diagnostic tests for FQ resistance in M. tuberculosis. 82 Less common genetic mutations in gyra are associated with amino acid positions and Those associated with amino acid 80 might not confer FQ resistance, 85 but may represent nonfunctional polymorphisms similar to mutations involving codon Heteroresistance generally refers to the coexistence of genetic populations/clones with differing nucleotides at a drug resistance locus in a sample of organisms. FQ-resistant M. tuberculosis isolates were recently found to have a distinct proportion of heteroresistance, and heteroresistant isolates frequently demonstrated multiple mutations. 86 On the whole, codons 94, 90 and 88 of gyra were demonstrated to confer high-level FQ resistance, as did multiple mutations. On the other hand, the much less common gyrb mutations (up to a total of 10 15%) were generally, but not consistently, associated with lower levels of FQ resistance; 81,89 however, combined gyra and gyrb mutations could result in a much higher level of resistance. 90 Some notable examples included Asn538lle (GyrB)-Asp94Ala (GyrA) and Ala543Val (GyrB)-Asp94Asn (GyrA). Furthermore, mutations in gyra and Asn538Asp as well as Asp500His substitutions in gyrb were linked to cross-resistance of M. tuberculosis to FQs, whereas Arg485His in gyrb did not result in such resistance. 91 Some studies have been undertaken in recent years to address the functional genetic analysis of gyra and gyrb mutations. 83,92 In a study on the structural modelling of the interaction between levofloxacin (LVX) and the M. tuberculosis gyrase catalytic site, 93 the loss of an acetyl group in the Asp94Gly mutation removes the acid-base interaction with LVX necessary for FQ activity. These approaches underscore the relevance of genetic mutations to FQ resistance. Aside from the mycobacterial pentapeptide MfpA and ATPase complex Rv2686c-Rv2687c-Rv2688c operon discussed, other efflux pumps that might be at play in FQ resistance include antiporters LfrA and Tap. 94 A recent study in FQ-monoresistant clinical isolates of M. tuberculosis revealed high levels of efflux pump pstb transcripts in a few of these isolates, 95 suggesting a contribution of the pump to resistance. In the presence of unstable drug exposure for the bacilli, facilitation in acquiring additional genetic resistance through gyr mutations could markedly worsen the scenario. 9 Likewise, a recent study has shown a worsening of resistance of M. tuberculosis to FQ with the concomitant administration of RMP, perhaps related to the induction of the efflux pump. 96 Aminoglycosides (streptomycin, kanamycin/amikacin) and capreomycin Streptomycin (SM) inhibits protein synthesis by binding to the 30S subunit of bacterial ribosome, causing misreadings of the mrna message. Resistance to SM is caused by mutations in the S12 protein encoded by the rpsl gene and 16S rrna encoded by the rrs gene. 97 Mutations in rpsl and rrs are the principal mechanism of SM resistance, 97,98 accounting for respectively about 50% and 20% of SMresistant strains. 97,98 However, about 20 30% of SM-resistant strains with low-level resistance (MICs, 32 lg/ml) do not have mutations in rpsl orrrs, 99 and may have mutations in gidb encoding a conserved 7-methylguanosine (m(7)g) methyltransferase specific for 16S rrna. 100 It has been shown recently that mutations in the promoter region of whib7 contribute to cross-resistance to SM and KM due to increased expression of the tap efflux gene and eis controlled by whib Mutations at the 16S rrna (rrs) position 1400 cause high-level resistance to KM and AMK. 102,103 SM-resistant strains usually remain susceptible to KM and AMK. Mutations in the promoter region of the eis gene, encoding aminoglycoside acetyltransferase, cause low-level resistance to KM but not to AMK. 104 Mutations in tlya encoding rrna methyltransferase 105 and the 23S rrna gene rrs (A1401G and G1484T) are involved in CPM resistance. rrna methyltransferase modifies the C1409 nucleotide in helix 44 of 16S rrna and the C1920 nucleotide in helix 69 of 23S rrna. 106 Mutants with A1401G in the rrs gene could cause resistance to KM and CPM but not viomycin, while C1402T or G1484T could cause resistance to CPM, KM or viomycin. 107 Multiple mutations may occur in the rrs gene in one strain, conferring cross-resistance among these agents. 107

7 1282 The International Journal of Tuberculosis and Lung Disease Table 2 Some newly developed drugs and repurposed agents of focus Drug Drug class New drug Repurposed agent Bedaquiline (TMC207) Diarylquinoline Yes Pretomanid (PA-824) Nitroimidazopyran Yes Delamanid (OPC-67683) Nitrodihydroimidazooxazole Yes SQ109 Ethylenediamine Yes Linezolid Oxazolidinone Yes Clofazimine Riminophenazine Yes Ethionamide/prothionamide Ethionamide (ETH) is a derivative of isonicotinic acid, and is a bactericidal agent only against M. tuberculosis. The MICs of ETH for M. tuberculosis are lg/ml in liquid media, lg/ml in 7H11 agar and 5 20 lg/ml in Löwenstein-Jensen medium. Like INH, ETH is also a prodrug that is activated by EtaA/EthA (a mono-oxygenase) 108,109 and inhibits the same target as INH, i.e., InhA of the mycolic acid synthesis pathway. 30 The structure and activity of prothionamide are almost identical to those of ETH. EthA is an FAD-containing enzyme that oxidises ETH to the corresponding S-oxide, which is further oxidised to 2-ethyl-4-amidopyridine, presumably via the unstable oxidised sulfinic acid intermediate. 110 EthA also activates thioacetazone, thiocarlide, thiobenzamide and perhaps other thioamide drugs, 110 which explains the cross-resistance between these agents (e.g., Isoxyl). In addition, mutations in the target InhA confer resistance to both ETH and INH. D-cycloserine/terizidone D-cycloserine (DCS) is a bacteriostatic agent used in the treatment of MDR-TB. Terizidone is a combination of two molecules of DCS. The MIC of DCS for M. tuberculosis ranges widely from 1.5 to 30 lg/ml, depending on the medium of culture used. DCS inhibits the synthesis of cell wall peptidoglycan by blocking the action of D-alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl). 111 Alr is involved in the conversion of L-alanine to D-alanine, which then serves as a substrate for Ddl. Overexpression of alra encoding D-alanine racemase from M. smegmatis causes resistance to DCS in M. bovis bacille Calmette- Guérin (BCG). 112 Overexpression of Alr confers higher resistance to DCS than Ddl overexpression in M. smegmatis, suggesting that Alr might be the primary target of DCS. 113 However, a recent study suggests that the primary target of DCS in M. tuberculosis is Ddl. 114 It was recently reported that cyca encoding D-serine, L- and D-alanine and glycine transporter involved in the uptake of D-cycloserine was defective in M. bovis BCG, which could be related to its natural resistance to DCS. 115 However, the mechanism of DCS resistance in M. tuberculosis remains to be established. Para-aminosalicylic acid Para-aminosalicylic acid (PAS) is a bacteriostatic agent with an MIC of lg/ml for M. tuberculosis. 4 Interference with folic acid biosynthesis 116 and inhibition of iron uptake 117 have been proposed as two possible mechanisms of action. 118 PAS was recently shown to be a prodrug that inhibits folic acid metabolism by incorporation into the folate pathway through activation by dihydropteroate synthase (DHPS, FolP) and dihydrofolate synthase (DHFS, FolC) to generate a toxic hydroxydihydrofolate antimetabolite which inhibits dihydrofolate reductase (DHFR, encoded by dfra [Rv2763c]). 119,120 Mutations causing PAS resistance occur at a frequency of 10 5 to The mechanism of PAS resistance is not well understood. Mutations in thya encoding thymidylate synthase, which reduce the utilisation of tetrahydrofolate, were responsible for resistance in about 37% of PAS-resistant clinical isolates. 116,121 Mutations in folc and dfra have also been found in PAS-resistant strains. 121,122 Overexpression of the PAS drug-activating enzyme DHFS (FolC) restored sensitivity to PAS in the resistant strain, 122 while overexpression of the target DHFR caused PAS resistance. 119 More studies are needed to validate the PAS resistance genes identified in clinical isolates. RESISTANCE TO NEWLY DEVELOPED DRUGS AND REPURPOSED AGENTS FOR TB Table 2 depicts the drugs and repurposed agents for TB discussed below. Table 3 summarises the genetic basis and associated information regarding M. tuberculosis resistance to these listed drugs. Bedaquiline Bedaquiline (TMC207), which is highly active against M. tuberculosis (MIC 0.03 lg/ml), 123 inhibits mycobacterial F1F0 proton adenosine triphosphate (ATP) synthase, a novel target, 123 leading to ATP depletion. Bedaquiline is active against both growing and non-growing mycobacterial populations, 124 and is also active against MDR-TB strains in vitro and in mice. 125 Of particular interest is the synergy between bedaquiline and PZA, and this combination provides the highest sterilising effects in the mouse TB model. 123 The finding is consistent with the previous

8 Mechanisms of drug resistance: update Table 3 Mechanisms of resistance against newly developed drugs and repurposed agents in M. tuberculosis Drug (year of discovery) Pretomanid (PA-824) (2000) Delamanid (OPC-67683) (2006) Bedaquiline (TMC207) (2005) SQ109 (2005) Clofazimine (1956) Linezolid (1996) MIC lg/ml ddn fdg ddn fdg1 Gene involved in resistance Gene function Mechanism of action Deazaflavin-dependent nitroreductase, F420-dependent glucose-6-phosphate dehydrogenase Deazaflavin-dependent nitroreductase, F420-dependent glucose-6-phosphate dehydrogenase ATP synthase c chain, transcription repressor for transporter MmpL5 Inhibition of mycolic acid synthesis, production of reactive nitrogen species Inhibition of mycolic acid synthesis, production of reactive nitrogen species Inhibition of ATP production atpe rv mmpl3 Membrane transporter Inhibition of mycolic acid synthesis rv0678 Transcription repressor for transporter MmpL5 Production of reactive oxygen species, inhibition of energy production, membrane disruption rrn 23S rrna Inhibition of protein synthesis MIC ¼ minimum inhibitory concentration; ATP ¼ adenosine triphosphate. observation pertaining to N,N -dicyclohexyl carbodiimide (DCCD) with the same drug target, as synergised with PZA. 38 Resistance to bedaquiline is caused by mutations in the subunit c encoded by atpe in the F0 moiety of the mycobacterial F1F0 proton ATP synthase, which is a key enzyme in ATP synthesis and membrane potential generation. 123 Mutations in the transcriptional regulator Rv0678, leading to upregulation of efflux pump MmpL5, were recently found to cause cross-resistance involving both clofazimine (CFZ) and bedaquiline. 126,127 Pretomanid and delamanid Pretomanid (PA-824) is highly active against both growing and non-growing M. tuberculosis (MIC lg/ml). The drug was initially thought to inhibit cell-wall lipid biosynthesis. 128 It is a prodrug activated by a deazaflavin-dependent nitroreductase (Ddn)(Rv3547) to form three primary metabolites, the main one being the des-nitroimidazole (des-nitro). 129,130 Des-nitro compounds generate reactive nitrogen species, including nitric oxide (NO), and are responsible for the anaerobic activity of these compounds and may synergise the mycobacterial killing with the host-derived NO produced by macrophages. Mutations in ddn (Rv3547) encoding deazaflavin-dependent nitroreductase and fgd1 (Rv0407) encoding F420-dependent glucose-6-phosphate dehydrogenase, both of which are involved in F420 coenzyme biosynthesis, are found in mutants resistant to pretomanid and complementation restored susceptibility. 130 Mutations in fgd1 and ddn found in 65 M. tuberculosis complex strains representing the main phylogenetic lineages do not appear to cause resistance to pretomanid (MIC, 0.25 lg/ml). Interestingly, M. canetti, a member of the M. tuberculosis complex, was found to be intrinsically resistant to pretomanid, with an MIC of 8 lg/ml; 131 however, the basis of its resistance is unknown. Because of the intrinsic resistance of M. canetti to both pretomanid 131 and PZA, 48 the pretomanid-mfx-pza regimen that is currently in Phase 3 trial for shortening antituberculosis treatment may not work for disease caused by M. canetti. Delamanid (OPC-67683) is a new drug that has recently received approval from the European Union and Japan for the treatment of MDR-TB in combination with other drugs. Delamanid inhibits mycolic acid synthesis of M. tuberculosis. It has an MIC of lg/ml, and thus appears to be 20 times more active than pretomanid. Delamanid has activity against non-replicating persister bacteria. 132 Similar to pretomanid, it is a prodrug that is activated by Ddn. Mutations in one of the five coenzyme F420 genes (fgd1, ddn, fbia, fbib and fbic) are associated with resistance to delamanid. SQ109 SQ109 (N-geranyl-N -(2-adamantyl)ethane-1,2-diamine) is a new compound derived from high throughput screening of EMB analogues; however, its mode of action is distinct from that of EMB. 133,134 SQ109 is active against M. tuberculosis, with an MIC of 0.5 lg/ml. SQ109 inhibits cell-wall synthesis and is active against drug-susceptible, EMB-resistant and MDR-TB strains. SQ109 targets MmpL3, a transporter of trehalose monomycolate involved in mycolic acid incorporation to the cell-wall core. 135 An alternative mechanism of action of SQ109 is the disruption of the membrane potential required for the transport of lipids and other substances. 136 The activity on the membrane suggests that SQ109 is active against both growing and non-replicating bacilli. 136 Mutations in the mmpl3 gene, encoding the transmembrane transporter, are associated with SQ109 resistance. 135

9 1284 The International Journal of Tuberculosis and Lung Disease Linezolid Oxazolidinones are originally a class of antibiotics approved for the treatment of drug-resistant Grampositive bacterial infections. 137 Oxazolidinones have significant activity against M. tuberculosis, with an MIC of lg/ml. 138 The congeners inhibit an early step of protein synthesis by binding to ribosomal 50S subunits, most likely within domain V of the 23S rrna peptidyl transferase, and forming a secondary interaction with the 30S subunit. 137 Mutations at G2061T and G2576T in the 23S rrna of M. tuberculosis can cause high levels of resistance to linezolid, an early member of the oxazolidinones, with MICs of 32 lg/ml and 16 lg/ml, respectively, but low-level resistance mutants (MIC 4 8 lg/ml) have no mutations in 23S rrna. 139 Mutations in the rplc encoding ribosomal protein L3 (T460C mutation) were putatively involved in linezolid resistance in M. tuberculosis, 140 largely accounting for low-level resistance phenotypes. 141 A recent study from China suggests that resistance to linezolid was already seen in about 11% of MDR-TB strains; however, only 30% of these strains had mutations in the 23S rrna gene or the rplc gene, 141 suggesting other, currently unknown possible mechanisms. Linezolid has a recognised role in the treatment of complicated MDR- and XDR-TB. 142,143 However, significant side effects such as peripheral neuropathy, optic neuropathy and anaemia occur with high frequency. 142,144 Clofazimine CFZ, which has good activity against mycobacteria, including M. tuberculosis (MIC lg/ml), 145 is conventionally used for leprosy treatment; 146 however, emergence of MDR-TB led to renewed interest in the use of CFZ for the treatment of this disease. 145 The Bangladesh regimen, a 9 12-month standardised regimen for treating MDR-TB, comprising high-dose gatifloxacin, alongside CFZ, EMB and PZA throughout, supplemented by KM, PTH and medium high dose INH during the intensive phase of a minimum of 4 months, achieved a high relapse-free cure rate (88%). 147,148 The mechanisms of action of CFZ are poorly understood, and may include the production of reactive oxygen species, 149 the inhibition of energy production through inhibiting NDH-2 (NADH dehydrogenase) and membrane disruption, which could lead to the inhibition of Kþ uptake and subsequent reduction in ATP production. 146 The molecular basis of CFZ resistance is not clearly understood. Recent studies showed that mutations in a transcriptional regulator Rv0678 led to upregulation of efflux pump MmpL5 and caused resistance to both CFZ and bedaquiline, 126,127 as well as azole drugs. 150 Mutations in rv0678 are the main mechanism of CFZ resistance. 151 Two new genes, rv1979c and rv2535c, were found to be associated with CFZ resistance. 151 Further studies are needed to identify CFZ targets. WHOLE GENOME SEQUENCING AND DRUG RESISTANCE GENE DISCOVERY As whole genome sequencing (WGS) has become more affordable in recent years, there has been significant interest and effort in using WGS to characterise drug-resistant M. tuberculosis isolates in different parts of the world to shed light on the molecular epidemiology and strain characteristics associated with the development and evolution of drug resistance and disease transmission. 72,152,153 An interesting observation that emerges from such studies is that there are over 100 genetic loci that seem to be associated with drug resistance. 152,153 This has led to the suggestion that drug resistance may be more complex than previously realised. Besides the conventional drug resistance gene mutations discussed above, there are many other mutations that may collectively contribute to the drug resistance phenotype. This is reminiscent of cancer-genomic sequencing studies, where numerous mutations are discovered as driver mutations and passenger mutations, and where it is hard to assign a causative role. Mutations associated with drug resistance are thus expected to play varying roles, from causative to compensatory or adaptive roles, to increase fitness which is only indirectly associated with drug resistance. A recent WGS study identified more than 40 genes whose mutations are associated with INH resistance. 33 In addition to MIC assessments and correlation with clinical outcomes, the roles of the mutations identified by the WGS studies need to be carefully addressed by functional assays, structural studies or allelic exchange experiments to better delineate their relevance. Although such studies are laborious and costly, they are essential to convincingly demonstrate whether the identified mutations indeed cause resistance to the drugs. CONCLUSION AND FUTURE PERSPECTIVES Improved understanding of the drug resistance mechanisms in M. tuberculosis will help develop more reliable molecular tests and increase treatment efficacy. Significant progress has been made in our understanding of the molecular basis of drug resistance in M. tuberculosis. This knowledge is beginning to impact the diagnosis and treatment of drugresistant TB. However, how drug resistance really develops in patients and the factors that facilitate resistance development are still poorly understood and merit further study. Despite ongoing advances in molecular tools for diagnosing drug resistance in M. tuberculosis, one crucial fact must be borne in mind. Resistance-conferring mutations in bacteria can

10 Mechanisms of drug resistance: update evolve dynamically over time under antibiotic pressure in patients, 154 when both genetic and epigentic (phenotypic) resistances can develop and overlap. The relationship between drug resistance and fitness and virulence of the MDR-/XDR-TB strains has to be carefully studied using appropriate molecular epidemiology and animal studies. The recent application of WGS offers promise not only in the identification of new drug resistance genes, 40,126 but also in the comprehensive molecular detection of drug resistance mutations in clinical isolates for patient care. However, the causative role of the genes identified using WGS has to be verified by tedious genetic studies to rule out phylogenetic polymorphisms that are not involved in drug resistance. 155 The current commercial molecular tests for drug-resistant TB, such as the Hain (GenoType w, Hain Lifescience, Nehren, Germany) and Xpert tests, although useful, cannot readily provide information regarding resistance to some drugs, and their utility is limited in achieving the practice of personalised (and precision) medicine for improved treatment. Rapid molecular tests (using WGS or other platforms) capable of detecting all drug resistances are likely needed to guide the treatment of difficult bacillary resistance scenarios. Finally, an understanding of the mechanisms of drug action and resistance should also facilitate the overall development of new drugs to improve the treatment of TB. Acknowledgements This work was support by National Institutes of Health (Bethesda, MD, USA) grants AI and AI An apology is owed for incomplete citation of research work due to space limitations. Conflicts of interest: WWY is a consultant with Otsuka Pharmaceutical Co, Tokyo, Japan. No other conflicts declared. References 1 McDermott W. Antimicrobial therapy of pulmonary tuberculosis. Bull World Health Organ 1960; 23: World Health Organization. Global tuberculosis report, WHO/HTM/TB/ Geneva, Switzerland: WHO, Dean A S, Zignol M, Falzon D, Getahun H, Floyd K. HIV and multidrug-resistant tuberculosis: overlapping epidemics. Eur Respir J 2014; 44: Zhang Y, Yew W W. Mechanisms of drug resistance in Mycobacterium tuberculosis. Int J Tuberc Lung Dis 2009; 13: Zhang Y, Yew W W, Barer M R. Targeting persisters for tuberculosis control. Antimicrob Agents Chemother 2012; 56: Zhang Y. Persisters, persistent infections and the Yin-Yang Model. 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