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INT J TUBERC LUNG DIS 19(11):1276–1289 STATE OF THE ART Q 2015 The Union http://dx.doi.org/10.5588/ijtld.15.0389

Mechanisms of drug resistance in : 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 multi- resistance. However, further research is needed to and extensively drug-resistant TB, is posing a significant address the significance of newly discovered gene challenge to effective treatment and TB control world- mutations in causing drug resistance. Improved knowl- wide. New progress has been made in our understanding edge of drug resistance mechanisms will help understand of the mechanisms of resistance to anti-tuberculosis the mechanisms of action of the drugs, devise better drugs. This review provides an update on the major molecular diagnostic tests for more effective DR-TB advances in drug resistance mechanisms since the management (and for personalised treatment), and previous publication in 2009, as well as added informa- facilitate the development of new drugs to improve the tion on mechanisms of resistance to new drugs and treatment of this disease. repurposed agents. The recent application of whole KEY WORDS: ; drug resistance; mechanisms; genome sequencing technologies has provided new molecular diagnostics; new drugs insight into the mechanisms and complexity of drug

The use of multiple-drug therapy, although defi- drug-resistant TB (DR-TB) epidemic thus remains nitely beneficial, is not an absolute guarantee an alarming problem, and is further aggravated by against the emergence of drug-resistant in- human immunodeficiency virus (HIV) coinfection.3 fections. . . Consequently, we cannot have confi- The present review is aimed at updating readers on dence that drug-resistant tubercle bacilli will not major advances in drug resistance mechanisms in emerge simply because multidrug therapy is Mycobacterium tuberculosis since the publication of employed.1 the previous article in 2009.4 Additional information ACCORDING TO the World Health Organization’s pertaining to newly developed drugs and repurposed (WHO’s) 2014 global tuberculosis report,2 there were agents have also been included. about 9.0 million new tuberculosis (TB) patients and 1.5 million deaths in 2013; 3.5% of newly diagnosed BASIC CONCEPTS IN THE DEVELOPMENT OF and 20.5% of previously treated patients had DRUG-RESISTANT TUBERCULOSIS multidrug-resistant TB (MDR-TB, defined as bacil- lary resistance to at least [RMP] and There are two types of drug resistance in M. (INH]). The highest levels of MDR-TB were tuberculosis: genetic resistance and phenotypic resis- found in Eastern Europe and Central Asia, with rates tance. Genetic drug resistance is due to mutations in reaching 20% and 50%, respectively. At least one chromosomal genes in growing bacteria, while case of extensively drug-resistant TB (XDR-TB, phenotypic resistance or drug tolerance is due to defined as MDR-TB with additional resistance to epigenetic changes in gene expression and protein fluoroquinolone[s] [FQs] and one or more of three modification that cause tolerance to drugs in non- second-line injectable drugs [SLIDs], namely capreo- growing persister bacteria. The two types of resis- mycin [CPM], kanamycin [KM] and tance have been responsible for a number of problems [AMK]) had been reported to the WHO from 92 in effective TB control, with genetic resistance (Yang countries by the end of 2012. An estimated 9% of resistance), as present in MDR-/XDR-TB, causing MDR-TB patients had XDR-TB. The worldwide 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) 410 614 2975. e-mail: [email protected] Article submitted 5 May 2015. Final version accepted 18 June 2015. [A version in French of this article is available from the Editorial Office in Paris and from the Union website www.theunion.org] Mechanisms of drug resistance: update 2015 1277 typic drug resistance, or tolerance (Yin resistance), as tant findings.17 Among over 1200 patients, 43.7% present in persisters, entails prolonged treatment and showed bacillary resistance to at least one SLD, 20% risk of post-treatment relapse.5,6 The situation in vivo to at least one SLID and 12.9% to at least one FQ, appears more complex, and the two types of with 6.7% of cases meeting the definition for XDR- resistance can overlap and interconvert. Prior stress TB. Previous treatment with SLDs was consistently or subinhibitory concentration of drugs may induce the strongest risk factor for resistance to these drugs, efflux pump expression,7,8 which causes phenotypic with a four-fold increased risk for XDR-TB. Bacillary resistance and may in turn facilitate the development resistance to FQs and XDR-TB were found more of more stable genetic drug resistance,8 while genetic frequently in women than men. Unemployment, resistance in growing organisms can develop persis- alcohol abuse and smoking were associated with tence or phenotypic resistance. There is increasing mycobacterial resistance to SLIDs across countries. In interest in understanding the biology of mycobacte- addition, in a study in the United States, the risk rial persisters and developing anti-tuberculosis drugs factors for bacillary acquired resistance to SLIDs that target them.5 included age 25–44 years, positive HIV status, MDR- M. tuberculosis drug-resistant strains develop TB at treatment initiation and treatment with any largely through the selection of genetic mutants. This SLD. However, the only predictor for bacillary is almost a wholly man-made phenomenon, resulting acquired resistance to FQs was MDR-TB at treatment from suboptimal physician prescription and/or poor initiation.18 A recent expanded analysis of these data patient adherence. However, there is some recent from the same group of researchers further showed evidence that pharmacokinetic-pharmacodynamic that mortality was significantly higher among TB variability scenarios related to the induction of the patients with bacillary acquired resistance to SLDs, mycobacterial drug efflux pump may also facilitate after controlling for age. MDR-TB at treatment the development of genetic mutations in M. tubercu- initiation, positive HIV status and extra-pulmonary losis.9 disease were also significantly associated with mor- The development of drug resistance as a result of tality.19 A meta-analysis on treatment outcomes of mutations in drug resistance genes in M. tuberculosis MDR-TB was undertaken in about 6700 patients may incur a cost in terms of fitness and virulence of from 26 centres using individual patient data the organism. Acquired resistance can also be analysis.20 Compared with treatment failure, relapse compounded by transmitted resistance. Recent sys- and death, treatment success was higher in MDR-TB tematic reviews have alluded to an association patients carrying bacilli without additional resistance between primary resistance in M. tuberculosis and or with additional resistance to SLIDs only than in HIV coinfection, suggesting transmitted DR-TB, as a those with resistance to FQs alone or to FQs plus significant challenge to the management of this SLIDs. In XDR-TB patients, treatment success was patient population.10,11 Furthermore, recent studies highest if at least six drugs were used in the intensive from China have indicated that a significant number phase and four in the continuation phase (odds ratios of MDR- and XDR-TB cases are due to the active 4.9 and 6.1, respectively). Likewise, in another transmission of (mainly) the Beijing genotype,12 and similar study focusing on XDR-TB, the odds of cure the same is true for Europe13 and Africa.14 This is a were significantly lower in XDR-TB patients with worrying development that requires more studies to additional bacillary resistance to CPM and KM/AMK better understand how such apparently virulent DR- than patients with only XDR-TB. The odds of failure TB strains evolve and adapt in the host, and and death were found to be higher in all XDR-TB highlights the need for more effective means to curtail patients with additional bacillary resistance, inclusive transmission. of groups with bacillary resistance to oral bacterio- static agents, with or without (EMB) plus Clinical relevance of anti-tuberculosis drug resistance PZA.21 It was recently noted that combined resis- The clinical relevance of resistance to RMP and INH tance to FQ (high-level) and PZA was associated with has been amply discussed.4 Besides the known a poor treatment outcome in some MDR-TB patients negative impact of bacillary resistance to pyrazin- treated with the 9-month Bangladesh regimen.22 amide (PZA) on treatment outcomes among MDR- TB patients, including PZA in the treatment regimen MECHANISMS OF RESISTANCE TO FIRST- AND for MDR-TB as guided by drug susceptibility testing SECOND-LINE ANTI-TUBERCULOSIS DRUGS (DST) might substantially improve early sputum culture conversion and subsequent cure or treatment The mechanisms of resistance of the various drugs are completion.15,16 discussed below. Table 1 summarises the genetic basis A prospective cohort study conducted in eight and related information on resistance in M. tubercu- countries that addressed the prevalence of and risk losis to first-line drugs and SLDs. The resistance factors for resistance to second-line drugs (SLDs) in mutation profile for each drug is not listed due to MDR-TB patients has yielded interesting and impor- space limitations. Readers may refer to drug resis- 1278 h nentoa ora fTbruoi n ugDisease Lung and Tuberculosis of Journal International The

Table 1 Mechanisms of resistance in M. tuberculosis against first- and second-line drugs

Drug MIC Gene involved Mutation frequency (year of discovery) lg/ml in resistance Gene function Mechanism of action % Isoniazid (1952) 0.02–0.2 katG, inhA Catalase-peroxidase, Inhibition of synthesis and other multiple effects 50–95 enoyl ACP reductase 8–43

Pyrazinamide (1952) 16–100 pncA, rpsA, panD Nicotinamidase/pyrazinamidase, ribosomal Depletion of membrane energy; inhibition of trans-translation; 72–99 S1 protein, aspartate decarboxylase inhibition of pantothenate and CoA synthesis Rifampin (1966) 0.05–1 rpoB ß-subunit of RNA polymerase Inhibition of RNA synthesis 95 Ethambutol (1961) 1–5 embB Arabinosyl transferase, *DPPR synthase Inhibition of arabinogalactan synthesis 47–65 ubiA (1943) 2–8 rpsL, rrs, gidB S12 ribosomal protein, Inhibition of protein synthesis 52–59 16S rRNA, 8–21 16S rRNA methyltransferase (G527 in 530 loop) Amikacin/kanamycin (1957) 2–4 rrs, eis, whiB7 16S rRNA, Inhibition of protein synthesis 76 acetyltransferase, transcriptional regulator (1960) 2–4 rrs 16S rRNA, 20-O-methyltransferase Inhibition of protein synthesis 85 tlyA Quinolones (1963) 0.5–2.5 gyrA DNA gyrase subunit A, Inhibition of DNA synthesis 75–94 gyrB DNA gyrase subunit B (1956) 2.5–10 etaA/ethA Flavin monooxygenase, Inhibition of mycolic acid synthesis 37 ethR transcription repressor, 56 inhA enoyl ACP reductase

PAS (1946) 1–8 thyA Thymidylate synthase, Inhibition of folic acid and thymine nucleotide metabolism 37 dfrA dihydrofolate reductase, folC dihydrofolate synthase, ribD enzyme in riboflavin (1955) 10–40 alr racemase, Inhibition of synthesis To be determined ddl D-alanine-D-alanine ligase, cycA D-serine proton symporter

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. Mechanisms of drug resistance: update 2015 1279 tance mutation databases for major frontline and completely lose catalase activity, have a limited effect SLDs at https://tbdreamdb.ki.se/Info/, https:// on fitness or virulence. In contrast, mutations in inhA, umr5558-bibiserv.univ-lyon1.fr/mubii/mubii-select. which cause low-level resistance, do not cause loss of cgi or http://pathogenseq.lshtm.ac.uk/rapiddrdata. virulence.34

Isoniazid INH, a pro-drug that is activated by the catalase- PZA is a paradoxical drug with unique sterilising peroxidase enzyme (KatG) encoded by the katG activity in anti-tuberculosis treatment.35 In striking gene23 to generate highly reactive species, is capable contrast to common antibiotics that kill or inhibit of attacking multiple targets in M. tuberculosis,24 the growing bacteria, PZA only kills non-growing primary one being the InhA enzyme (enoyl acyl persister bacteria and has poor activity against carrier protein reductase). The active species (iso- growing bacteria.36 PZA is a pro-drug that has to nicotinic acyl radical or anion) reacts with nicotin- be converted to its active form, pyrazinoic acid amide adenine dinucleotide (H), forming INH-NAD (POA), for activity by the pyrazinamidase/nicotina- adduct, which then inhibits InhA, causing inhibition midase enzyme encoded by the pncA gene of M. of cell wall mycolic acid synthesis.25 INH is active tuberculosis.37 PZA has multiple effects on M. only against growing M. tuberculosis but not against tuberculosis, including interference with membrane non-growing bacilli (persisters). INH tolerance in energy production,38 and inhibition of RpsA (ribo- non-growing organisms may be caused by mycobac- somal protein S1), which is involved in trans- terial DNA-binding protein 1 (MDP1), a histone-like translation,39 and PanD, which is involved in protein, which downregulates katG transcription and pantothenate and co-enzyme A synthesis.40,41 Over- could lead to tolerance to INH.26 expression of RpsA renders M. tuberculosis more Mutations in katG are the major mechanism of resistant to PZA.39 In addition, a low-level PZA- INH resistance (Table 1).4,23,27 The KatG S315 resistant clinical strain, DHM444, without pncA mutation is the most common mutation in INH- mutations,42 was found to contain a deletion of resistant strains, accounting for 50–95% of INH- alanine at the 438th residue (438 DA) due to a 3 base resistant clinical isolates.4,24 KatG S315 mutations pair (bp) GCC deletion in the C-terminus of RpsA.39 usually do not completely eliminate catalase activity, POA bound to the wt RpsA but not the mutant and such strains may still retain fitness and virulence, RspADA438 from the PZA-resistant strain, and which may explain its frequent occurrence among specifically inhibited the trans-translation of M. clinical isolates. Mutations in the katG promoter tuberculosis but not its canonical translation.39 More region furA-katG intergenic region that affect KatG recently, panD-encoding aspartate decarboxylase, expression were occasionally found to cause INH involved in the synthesis of ß-alanine, which is a resistance in some strains.28,29 Resistance to INH can precursor for pantothenate and co-enzyme A biosyn- also occur due to mutations in the promoter region of thesis, has been shown to serve as a new target of mabA(fabG1)/inhA operon causing overexpression of PZA.41 InhA or by mutations at the InhA active site.25,30 In Mutations in the pncA gene are the main mecha- contrast to katG mutations, which usually cause nism of PZA resistance in M. tuberculosis.37,42–47 high-level resistance, mutations in inhAorits pncA mutations are highly diverse and scattered promoter region are usually associated with low- along the gene, which is unique to PZA resistance. level resistance (minimum inhibitory concentration Most PZA-resistant M. tuberculosis strains (72–99%, [MIC] 0.2–1 lg/ml) and are less frequent than katG average 85%) have mutations in pncA,42–46 but some mutations (Table 1).4,24 Mutations in inhA not only do not. Those that do not have mutations in the drug cause INH resistance, they also confer cross-resis- target RpsA.39,48 RpsA target mutations are usually tance to the structurally related drug ethionamide associated with low-level PZA resistance (MIC 200– (ETH).30 A small percentage of low-level INH- 300 lg/ml PZA). More recently, panD was found to resistant strains do not have mutations in katGor be involved in PZA resistance.40 panD mutations inhA,31–33 which may be due to new mechanism(s) of were identified in naturally PZA-resistant M. canettii resistance. However, an alternative explanation is strains and some PZA-resistant MDR-TB strains. M. heteroresistance or mixed population, where purifi- canettii, a member of the M. tuberculosis complex, is cation of single clones shows a small proportion of intrinsically resistant to PZA,48,49 and has mutations resistant clones containing katG mutations among in both rpsA48 and panD.40 sensitive clones with wild-type (wt) katG (Y Zhang, Culture-based DST of PZA is difficult and unreli- unpublished observations). INH-resistant strains due able, with frequent problems of false resis- to katG deletion or mutations that lead to complete tance.36,50,51 The very high correlation between loss of catalase activity causing high-level resistance PZA resistance and pncA mutations provides the may cause loss of virulence. Strains with point opportunity to rapidly detect PZA resistance using mutations in katG, such as KatG315, which do not pncAsequencing.42,52–54 Dividing MDR-TB into 1280 The International Journal of Tuberculosis and Lung Disease

PZA-susceptible and PZA-resistant MDR-TB for suggest that the molecular test may not be able to improved treatment of MDR-TB has thus been completely replace phenotypic DST. However, a proposed.15 However, some PZA-susceptible strains recent study showed that disputed rpoB mutations were reported to have non-synonymous mutations in may be responsible for RMP resistance among new pncA.31,53 These mutations could cause low-level cases and may lead to adverse treatment outcomes resistance close to the MIC cut-off for resistance that with first-line agents.64 is not easily detected by current DST for PZA, and Reports of RMP-dependent or enhanced strains of may be mistaken for PZA susceptibility. Further M. tuberculosis in some MDR-TB strains65–67 is studies are required to address this issue. PZA- potentially worrying and less appreciated. RMP- resistant strains with diverse pncA mutations do not dependent strains may be more prevalent than appear to have fitness cost or loss of virulence.43 currently realised, as the current diagnostic practice Interestingly, a recent study found that patients uses only drug-free media and may simply discount or infected with PZA-monoresistant strains had worse discard strains that grow poorly in normal culture clinical outcomes than those with drug-susceptible media without RMP. A recent study from China strains.55 suggests that RMP-dependent/enhanced strains are common, with as many as 39% (18/46) of MDR-TB Rifampicin strains having RMP dependence or enhancement RMP is highly bactericidal and sterilising for M. phenomenon.67 The circumstances under which these tuberculosis. Although RMP is known to interfere strains arise remain unclear, but they often occur as with RNA synthesis by binding to the ß subunit of the MDR-TB, and seem to develop upon repeated RNA polymerase, a recent study showed that RMP treatment with in retreatment patients,66 binding to the RpoB target induces hydroxyl radical and could be a result of overlapping genetic and formation in susceptible but not resistant bacilli, and phenotypic resistance. RBT has been suggested for may contribute to the killing effect of RMP.56 RMP treating RMP-resistant strains.68 However, this might resistance occurs at a frequency of 107/8, but a be a risky practice, as more powerful rifamycins such recent study showed a much higher mutation as RBT could lead to the development of RMP frequency, at 103, for the Beijing genotype strain dependence or enhancement and potentially worsen than with the EAI (East African Indian) genotype the disease,66 possibly as a result of genetic and strain, at 106 for RMP resistance, but no difference epigenetic changes that enhance the fitness and for other drugs, including INH and virulence of the organism. The impact of rpoB (MFX).57 This high mutation frequency is intriguing mutations on the fitness and pathogenesis of the and may be related to the lower RMP concentration organism has recently been reviewed,69 and is worth used or previous exposure to RMP before selection paying attention to, as RMP resistance may carry for RMP resistance.58 Mutations in a defined region more severe consequences, with enhanced virulence of the 81-bp region of rpoB are found in about 96% and pathogenesis. It would be of interest to determine of RMP-resistant M. tuberculosis isolates.59 Muta- the frequency of RMP dependence/enhancement, the tions at positions 531, 526 and 516 are among the mechanisms involved, and the contribution of such most frequent mutations in RMP-resistant strains. strains to treatment failure. RMP resistance due to Mutations in rpoB generally result in cross-resistance rpoB mutations could alter M. tuberculosis fitness; to all rifamycins, including (RBT), but some however, compensatory mutations in rpoCorrpoA RMP-resistant strains are RBT-susceptible.60 Cross- could occur.70–72 resistance to RMP and RBT seems to involve both the common 531 and 526 sites and also the beginning of Ethambutol the RpoB region61 and the D516A-R529Q double EMB interferes with the biosynthesis of cell wall mutations.60 Mutations at F514FF, D516V and arabinogalactan.73 Arabinosyl transferase, encoded S522L are associated with resistance to RMP but by embB, an enzyme involved in the synthesis of susceptibility to RBT.60 However, not all mutations in arabinogalactan, is the target of EMB in M. rpoB are associated with RMP resistance.60,62 For tuberculosis.74 Mutations in embCAB operon, in example, mutations at E510H, L511P, D516Y, particular embB, and occasionally embC, are respon- N518D, H526N and L533P are not associated with sible for resistance to EMB.74 embB codon 306 RMP resistance and are found in RMP-susceptible mutation is most frequent in clinical isolates resistant strains.60,63 In a separate study, about 10.5% (16/ to EMB, accounting for as much as 68% of resistant 133) of the strains with rpoB mutations identified by strains.75,76 While mutations at EmbB306 leading to Xpertw MTB/RIF (Cepheid, Sunnyvale, CA, USA) as certain amino acid changes caused EMB resistance, RMP-resistant were found to be RMP-susceptible on other amino acid substitutions had little effect on phenotypic MGITe test (BD, Sparks, MD, USA).62 EMB resistance.77 However, about 35% of EMB- These findings caution us against relying solely on the resistant strains (MIC , 10 lg/ml) do not have embB molecular test for detecting RMP resistance, and mutations,78 suggesting other mechanisms of resis- Mechanisms of drug resistance: update 2015 1281 tance. Mutations in ubiA, encoding the DPPR sary for FQ activity. These approaches underscore the synthase involved in cell-wall synthesis, have recently relevance of genetic mutations to FQ resistance. been found to cause higher-level EMB resistance in Aside from the mycobacterial pentapeptide MfpA conjunction with embB mutations.79,80 and ATPase complex Rv2686c-Rv2687c-Rv2688c operon discussed, other efflux pumps that might be Fluoroquinolones at play in FQ resistance include antiporters LfrA and The well-known dominant role of gyrA mutations in Tap.94 A recent study in FQ-monoresistant clinical FQ-resistant TB remains unchanged. In a systematic isolates of M. tuberculosis revealed high levels of review of gyr mutations,81 of 1220 FQ-resistant M. efflux pump pstB transcripts in a few of these tuberculosis isolates that were subjected to sequenc- isolates,95 suggesting a contribution of the pump to ing of the quinolone-resistance-determining region resistance. In the presence of unstable drug exposure (QRDR) of gyrA, 780 (64%) were found to have for the bacilli, facilitation in acquiring additional mutations. The QRDR of gyrB was sequenced in 534 genetic resistance through gyr mutations could resistant isolates, only 17 (3%) of which had markedly worsen the scenario.9 Likewise, a recent mutations. Of the gyrA mutations, 81% were inside study has shown a worsening of resistance of M. the QRDR and 19% outside. Mutations at gyrA tuberculosis to FQ with the concomitant administra- codons 90, 91 and 94 were present in 54% of the FQ- tion of RMP, perhaps related to the induction of the resistant isolates (substitutions at amino acid 94 efflux pump.96 accounted for 37%). Of the gyrB mutations, only 44% were inside the QRDR. Such findings may have (streptomycin, kanamycin/amikacin) implications for the currently available genotypic and capreomycin diagnostic tests for FQ resistance in M. tuberculo- Streptomycin (SM) inhibits protein synthesis by sis.82 Less common genetic mutations in gyrA are binding to the 30S subunit of bacterial ribosome, associated with amino acid positions 7483 and 88.84 causing misreadings of the mRNA message. Resis- Those associated with amino acid 80 might not tance to SM is caused by mutations in the S12 protein confer FQ resistance,85 but may represent non- encoded by the rpsL gene and 16S rRNA encoded by functional polymorphisms similar to mutations in- the rrs gene.97 Mutations in rpsL and rrs are the volving codon 95.4 principal mechanism of SM resistance,97,98 account- Heteroresistance generally refers to the coexistence ing for respectively about 50% and 20% of SM- of genetic populations/clones with differing nucleo- resistant strains.97,98 However, about 20–30% of tides at a drug resistance locus in a sample of SM-resistant strains with low-level resistance (MICs organisms. FQ-resistant M. tuberculosis isolates were , 32 lg/ml) do not have mutations in rpsLorrrs,99 recently found to have a distinct proportion of and may have mutations in gidB encoding a heteroresistance, and heteroresistant isolates fre- conserved 7-methylguanosine (m(7)G) methyltrans- quently demonstrated multiple mutations.86 ferase specific for 16S rRNA.100 It has been shown On the whole, codons 94, 90 and 88 of gyrA were recently that mutations in the promoter region of demonstrated to confer high-level FQ resistance,87–89 whiB7 contribute to cross-resistance to SM and KM as did multiple mutations. On the other hand, the due to increased expression of the tap efflux gene and much less common gyrB mutations (up to a total of eis controlled by whiB7.101 Mutations at the 16S 10–15%) were generally, but not consistently, asso- rRNA (rrs) position 1400 cause high-level resistance ciated with lower levels of FQ resistance;81,89 to KM and AMK.102,103 SM-resistant strains usually however, combined gyrA and gyrB mutations could remain susceptible to KM and AMK. Mutations in result in a much higher level of resistance.90 Some the promoter region of the eis gene, encoding notable examples included Asn538lle (GyrB)-As- aminoglycoside acetyltransferase, cause low-level p94Ala (GyrA) and Ala543Val (GyrB)-Asp94Asn resistance to KM but not to AMK.104 (GyrA). Furthermore, mutations in gyrA and As- Mutations in tlyA encoding rRNA methyltransfer- n538Asp as well as Asp500His substitutions in gyrB ase105 and the 23S rRNA gene rrs (A1401G and were linked to cross-resistance of M. tuberculosis to G1484T) are involved in CPM resistance. rRNA FQs, whereas Arg485His in gyrB did not result in methyltransferase modifies the C1409 nucleotide in such resistance.91 helix 44 of 16S rRNA and the C1920 nucleotide in Some studies have been undertaken in recent years helix 69 of 23S rRNA.106 Mutants with A1401G in to address the functional genetic analysis of gyrA and the rrs gene could cause resistance to KM and CPM gyrB mutations.83,92 In a study on the structural but not viomycin, while C1402T or G1484T could modelling of the interaction between cause resistance to CPM, KM or viomycin.107 (LVX) and the M. tuberculosis gyrase catalytic site,93 Multiple mutations may occur in the rrs gene in one the loss of an acetyl group in the Asp94Gly mutation strain, conferring cross-resistance among these removes the acid-base interaction with LVX neces- agents.107 1282 The International Journal of Tuberculosis and Lung Disease

Table 2 Some newly developed drugs and repurposed agents of focus

New Repurposed Drug Drug class drug agent (TMC207) Diarylquinoline Yes (PA-824) Nitroimidazopyran Yes (OPC-67683) Nitrodihydroimidazooxazole Yes SQ109 Yes Oxazolidinone Yes Riminophenazine Yes

Ethionamide/ Para-aminosalicylic acid Ethionamide (ETH) is a derivative of isonicotinic Para-aminosalicylic acid (PAS) is a bacteriostatic acid, and is a bactericidal agent only against M. agent with an MIC of 0.5–2 lg/ml for M. tuberculo- tuberculosis. The MICs of ETH for M. tuberculosis sis.4 Interference with folic acid biosynthesis116 and are 0.5–2 lg/ml in liquid media, 2.5–10 lg/ml in inhibition of iron uptake117 have been proposed as 7H11 agar and 5–20 lg/ml in Lowenstein-Jensen¨ two possible mechanisms of action.118 PAS was medium. Like INH, ETH is also a prodrug that is recently shown to be a prodrug that inhibits folic acid activated by EtaA/EthA (a mono-oxygenase)108,109 metabolism by incorporation into the folate pathway and inhibits the same target as INH, i.e., InhA of the through activation by dihydropteroate synthase mycolic acid synthesis pathway.30 The structure and (DHPS, FolP) and dihydrofolate synthase (DHFS, activity of prothionamide are almost identical to FolC) to generate a toxic hydroxydihydrofolate those of ETH. EthA is an FAD-containing enzyme antimetabolite which inhibits dihydrofolate reductase that oxidises ETH to the corresponding S-oxide, (DHFR, encoded by dfrA [Rv2763c]).119,120 Muta- which is further oxidised to 2-ethyl-4-amidopyridine, tions causing PAS resistance occur at a frequency of presumably via the unstable oxidised sulfinic acid 105 to 109. The mechanism of PAS resistance is not intermediate.110 EthA also activates , well understood. Mutations in thyA encoding thymi- , thiobenzamide and perhaps other thio- dylate synthase, which reduce the utilisation of amide drugs,110 which explains the cross-resistance tetrahydrofolate, were responsible for resistance in between these agents (e.g., Isoxyl). In addition, about 37% of PAS-resistant clinical isolates.116,121 mutations in the target InhA confer resistance to Mutations in folC and dfrA have also been found in both ETH and INH. PAS-resistant strains.121,122 Overexpression of the PAS drug-activating enzyme DHFS (FolC) restored D-cycloserine/ sensitivity to PAS in the resistant strain,122 while D-cycloserine (DCS) is a bacteriostatic agent used in overexpression of the target DHFR caused PAS the treatment of MDR-TB. Terizidone is a combina- resistance.119 More studies are needed to validate tion of two molecules of DCS. The MIC of DCS for the PAS resistance genes identified in clinical isolates. M. tuberculosis ranges widely from 1.5 to 30 lg/ml, depending on the medium of culture used. DCS RESISTANCE TO NEWLY DEVELOPED DRUGS inhibits the synthesis of cell wall peptidoglycan by AND REPURPOSED AGENTS FOR TB blocking the action of D-alanine racemase (Alr) and D-alanine:D-alanine ligase (Ddl).111 Alr is involved in Table 2 depicts the drugs and repurposed agents for the conversion of L-alanine to D-alanine, which then TB discussed below. Table 3 summarises the genetic serves as a substrate for Ddl. Overexpression of alrA basis and associated information regarding M. encoding D-alanine racemase from M. smegmatis tuberculosis resistance to these listed drugs. causes resistance to DCS in M. bovis bacille Calmette- Guerin´ (BCG).112 Overexpression of Alr confers Bedaquiline higher resistance to DCS than Ddl overexpression in Bedaquiline (TMC207), which is highly active M. smegmatis, suggesting that Alr might be the against M. tuberculosis (MIC 0.03 lg/ml),123 inhibits primary target of DCS.113 However, a recent study mycobacterial F1F0 proton adenosine triphosphate suggests that the primary target of DCS in M. (ATP) synthase, a novel target,123 leading to ATP tuberculosis is Ddl.114 It was recently reported that depletion. Bedaquiline is active against both growing cycA encoding D-serine, L- and D-alanine and glycine and non-growing mycobacterial populations,124 and transporter involved in the uptake of D-cycloserine is also active against MDR-TB strains in vitro and in was defective in M. bovis BCG, which could be mice.125 Of particular interest is the synergy between related to its natural resistance to DCS.115 However, bedaquiline and PZA, and this combination provides the mechanism of DCS resistance in M. tuberculosis the highest sterilising effects in the mouse TB remains to be established. model.123 The finding is consistent with the previous Mechanisms of drug resistance: update 2015 1283

Table 3 Mechanisms of resistance against newly developed drugs and repurposed agents in M. tuberculosis

Drug MIC Gene involved (year of discovery) lg/ml in resistance Gene function Mechanism of action Pretomanid (PA-824) 0.015–0.25 ddn Deazaflavin-dependent nitroreductase, Inhibition of mycolic acid synthesis, (2000) fdg1 F420-dependent glucose-6-phosphate production of reactive nitrogen dehydrogenase species Delamanid (OPC-67683) 0.006–0.012 ddn Deazaflavin-dependent nitroreductase, Inhibition of mycolic acid synthesis, (2006) fdg1 F420-dependent glucose-6-phosphate production of reactive nitrogen dehydrogenase species Bedaquiline (TMC207) 0.06–0.12 atpE ATP synthase c chain, transcription Inhibition of ATP production (2005) rv0678 repressor for transporter MmpL5 SQ109 0.5 mmpL3 Membrane transporter Inhibition of mycolic acid synthesis (2005) Clofazimine 0.1–0.25 rv0678 Transcription repressor for transporter Production of reactive oxygen (1956) MmpL5 species, inhibition of energy production, membrane disruption Linezolid 0.25–0.5 rrn 23S rRNA Inhibition of protein synthesis (1996)

MIC ¼ minimum inhibitory concentration; ATP ¼ adenosine triphosphate. observation pertaining to N,N’-dicyclohexyl carbo- of its resistance is unknown. Because of the intrinsic diimide (DCCD) with the same drug target, as resistance of M. canetti to both pretomanid131 and synergised with PZA.38 Resistance to bedaquiline is PZA,48 the pretomanid-MFX-PZA regimen that is caused by mutations in the subunit c encoded by atpE currently in Phase 3 trial for shortening anti- in the F0 moiety of the mycobacterial F1F0 proton tuberculosis treatment may not work for disease ATP synthase, which is a key enzyme in ATP synthesis caused by M. canetti. and membrane potential generation.123 Mutations in Delamanid (OPC-67683) is a new drug that has the transcriptional regulator Rv0678, leading to recently received approval from the European Union upregulation of efflux pump MmpL5, were recently and Japan for the treatment of MDR-TB in combi- found to cause cross-resistance involving both clo- nation with other drugs. Delamanid inhibits mycolic 126,127 fazimine (CFZ) and bedaquiline. acid synthesis of M. tuberculosis. It has an MIC of 0.006–0.024 lg/ml, and thus appears to be 20 times Pretomanid and delamanid more active than pretomanid. Delamanid has activity Pretomanid (PA-824) is highly active against both against non-replicating persister bacteria.132 Similar growing and non-growing M. tuberculosis (MIC to pretomanid, it is a prodrug that is activated by 0.015–0.25 lg/ml). The drug was initially thought Ddn. Mutations in one of the five coenzyme F420 to inhibit cell-wall lipid biosynthesis.128 It is a genes (fgd1, ddn, fbiA, fbiB and fbiC) are associated prodrug activated by a deazaflavin-dependent nitro- with resistance to delamanid. reductase (Ddn)(Rv3547) to form three primary metabolites, the main one being the des-nitro- SQ109 (des-nitro).129,130 Des-nitro compounds SQ109 (N-geranyl-N‘-(2-adamantyl)ethane-1,2-di- generate reactive nitrogen species, including nitric amine) is a new compound derived from high oxide (NO), and are responsible for the anaerobic activity of these compounds and may synergise the throughput screening of EMB analogues; however, 133,134 mycobacterial killing with the host-derived NO its mode of action is distinct from that of EMB. produced by macrophages. Mutations in ddn SQ109 is active against M. tuberculosis, with an MIC (Rv3547) encoding deazaflavin-dependent nitrore- of 0.5 lg/ml. SQ109 inhibits cell-wall synthesis and is ductase and fgd1 (Rv0407) encoding F420-depen- active against drug-susceptible, EMB-resistant and dent glucose-6-phosphate dehydrogenase, both of MDR-TB strains. SQ109 targets MmpL3, a trans- which are involved in F420 coenzyme biosynthesis, porter of trehalose monomycolate involved in my- are found in mutants resistant to pretomanid and colic acid incorporation to the cell-wall core.135 An complementation restored susceptibility.130 Muta- alternative mechanism of action of SQ109 is the tions in fgd1 and ddn found in 65 M. tuberculosis disruption of the membrane potential required for the complex strains representing the main phylogenetic transport of lipids and other substances.136 The lineages do not appear to cause resistance to activity on the membrane suggests that SQ109 is pretomanid (MIC , 0.25 lg/ml). Interestingly, M. active against both growing and non-replicating canetti, a member of the M. tuberculosis complex, bacilli.136 Mutations in the mmpL3 gene, encoding was found to be intrinsically resistant to pretoma- the transmembrane transporter, are associated with nid, with an MIC of 8 lg/ml;131 however, the basis SQ109 resistance.135 1284 The International Journal of Tuberculosis and Lung Disease

Linezolid were found to be associated with CFZ resistance.151 Oxazolidinones are originally a class of antibiotics Further studies are needed to identify CFZ targets. approved for the treatment of drug-resistant Gram- positive bacterial .137 Oxazolidinones have WHOLE GENOME SEQUENCING AND DRUG significant activity against M. tuberculosis, with an RESISTANCE GENE DISCOVERY MIC of 0.125–0.5 lg/ml.138 The congeners inhibit an early step of protein synthesis by binding to ribosom- As whole genome sequencing (WGS) has become al 50S subunits, most likely within domain V of the more affordable in recent years, there has been significant interest and effort in using WGS to 23S rRNA , and forming a characterise drug-resistant M. tuberculosis isolates secondary interaction with the 30S subunit.137 in different parts of the world to shed light on the Mutations at G2061T and G2576T in the 23S rRNA molecular epidemiology and strain characteristics of M. tuberculosis can cause high levels of resistance associated with the development and evolution of to linezolid, an early member of the oxazolidinones, drug resistance and disease transmission.72,152,153 An with MICs of 32 lg/ml and 16 lg/ml, respectively, but interesting observation that emerges from such low-level resistance mutants (MIC 4–8 lg/ml) have studies is that there are over 100 genetic loci that 139 no mutations in 23S rRNA. Mutations in the rplC seem to be associated with drug resistance.152,153 This encoding ribosomal protein L3 (T460C mutation) has led to the suggestion that drug resistance may be were putatively involved in linezolid resistance in M. more complex than previously realised. Besides the tuberculosis,140 largely accounting for low-level conventional drug resistance gene mutations dis- resistance phenotypes.141 A recent study from China cussed above, there are many other mutations that suggests that resistance to linezolid was already seen may collectively contribute to the drug resistance in about 11% of MDR-TB strains; however, only phenotype. This is reminiscent of cancer-genomic 30% of these strains had mutations in the 23S rRNA sequencing studies, where numerous mutations are gene or the rplC gene,141 suggesting other, currently discovered as driver mutations and passenger muta- unknown possible mechanisms. Linezolid has a tions, and where it is hard to assign a causative role. recognised role in the treatment of complicated Mutations associated with drug resistance are thus MDR- and XDR-TB.142,143 However, significant side expected to play varying roles, from causative to effects such as , optic neurop- compensatory or adaptive roles, to increase fitness athy and anaemia occur with high frequency.142,144 which is only indirectly associated with drug resis- tance. A recent WGS study identified more than 40 Clofazimine genes whose mutations are associated with INH CFZ, which has good activity against mycobacteria, resistance.33 In addition to MIC assessments and including M. tuberculosis (MIC 0.5–2 lg/ml),145 is correlation with clinical outcomes, the roles of the conventionally used for treatment;146 how- mutations identified by the WGS studies need to be ever, emergence of MDR-TB led to renewed interest carefully addressed by functional assays, structural in the use of CFZ for the treatment of this disease.145 studies or allelic exchange experiments to better The Bangladesh regimen, a 9–12-month standardised delineate their relevance. Although such studies are regimen for treating MDR-TB, comprising high-dose laborious and costly, they are essential to convinc- ingly demonstrate whether the identified mutations , alongside CFZ, EMB and PZA through- indeed cause resistance to the drugs. out, 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 CONCLUSION AND FUTURE PERSPECTIVES (88%).147,148 The mechanisms of action of CFZ are Improved understanding of the drug resistance poorly understood, and may include the production mechanisms in M. tuberculosis will help develop 149 of reactive oxygen species, the inhibition of energy more reliable molecular tests and increase treatment production through inhibiting NDH-2 (NADH de- efficacy. Significant progress has been made in our hydrogenase) and membrane disruption, which could understanding of the molecular basis of drug resis- lead to the inhibition of Kþ uptake and subsequent tance in M. tuberculosis. This knowledge is beginning reduction in ATP production.146 The molecular basis to impact the diagnosis and treatment of drug- of CFZ resistance is not clearly understood. Recent resistant TB. However, how drug resistance really studies showed that mutations in a transcriptional develops in patients and the factors that facilitate regulator Rv0678 led to upregulation of efflux pump resistance development are still poorly understood MmpL5 and caused resistance to both CFZ and and merit further study. Despite ongoing advances in bedaquiline,126,127 as well as azole drugs.150 Muta- molecular tools for diagnosing drug resistance in M. tions in rv0678 are the main mechanism of CFZ tuberculosis, one crucial fact must be borne in mind. resistance.151 Two new genes, rv1979c and rv2535c, Resistance-conferring mutations in bacteria can Mechanisms of drug resistance: update 2015 1285 evolve dynamically over time under pres- 9 Pasipanodya J G, Gumbo T. A new evolutionary and sure in patients,154 when both genetic and epigentic pharmacokinetic-pharmacodynamic scenario for rapid emergence of resistance to single and multiple anti- (phenotypic) resistances can develop and overlap. tuberculosis drugs. Curr Opin Pharmacol 2011; 11: 457–463. The relationship between drug resistance and fitness 10 Suchindran S, Brouwer E S, Van Rie A. Is HIV a risk and virulence of the MDR-/XDR-TB strains has to be factor for multi-drug resistant tuberculosis? 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RESUME La tuberculose (TB) pharmacor´esistante, incluant la TB fourni de nouvelles connaissances des m´ecanismes et de multi- et ultrar´esistante, pose un d´efi majeur en termes la complexit´e de la pharmacor´esistance. Cependant, de traitement efficace et de lutte contre la TB dans le d’autres recherches sont n´ecessaires pour aborder la monde. Des progr`es ontet´ ´ eaccomplisdansnotre port´ee des mutations g´en´etiques r´ecemment d´ecouvertes compr´ehension des m´ecanismes de la r´esistance aux a` l’origine de la pharmacor´esistance. Une meilleure m´edicaments antituberculeux. Cette revue offre une connaissance des m´ecanismes de la pharmacor´esistance misea ` jour des avanc´ees majeures dans la connaissance contribueraa ` comprendre les m´ecanismes d’action des des m´ecanismes de pharmacor´esistance depuis la m´edicaments,a ` concevoir de meilleurs tests publication pr´ec´edente en 2009, ainsi que des diagnostiques pour une meilleure prise en charge de la informations suppl´ementaires sur les m´ecanismes de TB pharmacor´esistante (et un traitement personnalis´e) r´esistance aux nouveaux m´edicaments etades ` eta ` faciliter l’´elaboration de nouveaux m´edicaments m´edicaments r´eadapt´es. L’application r´ecente des pour am´eliorer le traitement de cette maladie. techniques de s´equen¸cage de l’ensemble du g´enome a

RESUMEN La tuberculosis (TB) farmacorresistente, incluida la complejidad de la resistencia a los medicamentos enfermedad multi- y extremadamente drogorresistente, antituberculosos. Aun as´ı, se precisan nuevas plantea un peligro considerable al tratamiento eficaz y el investigaciones que aborden la participacion ´ de las control mundial de la TB. Se han logrado progresos mutaciones g´enicas reci´en descubiertas en la recientes en la comprension ´ de los mecanismos de farmacorresistencia. Un mayor conocimiento de los resistencia a los medicamentos antituberculosos. En el mecanismos de resistencia a los medicamentos presente art´ıculo se ofrece una actualizacion ´ de los contribuira´ a comprender mejor los mecanismos de principales avances en este campo desde la publicacion ´ accion ´ de los fa´rmacos, desarrollar pruebas moleculares anterior en el 2009; se analiza adema´s la informacion ´ diagnosticas ´ ma´s eficaces a fin de optimizar la atencion ´ sobre los mecanismos de resistencia a los nuevos de la TB farmacorresistente (y el tratamiento medicamentos y a los fa´rmacos con una nueva personalizado) y facilitara´ el desarrollo de nuevas asignacion ´ de uso. La aplicacion ´ reciente de las mol´eculas que mejoren el tratamiento de esta t´ecnicas de secuenciacion ´ del genoma completo aporto ´ enfermedad. una mejor percepcion ´ de los mecanismos y la