Abstract

Anti-microbial treatment is extensively used in conventional tuberculosis treatment, leading to resistance development. In this review, we summarized the mode of action and susceptibility protocols of anti-Tubercular drugs. An effort to elucidate the role of genetic variations, cell membrane adaptions, and efflux pump modalities in treatment failure will be an asset in devising prospective strategies.

Key Words:

Tuberculosis, Drug resistance, Mechanism of Action, Mutations

Introduction

Despite being a preventable and curable disease, TB remains on top of the infectious killing disease, claiming 1.5 million lives every year. Young people between the ages of 15-34 are carrying the heaviest burden of this disease. Tuberculosis is the principal reason for anti-microbial resistance and fatalities among people suffering from HIV. In 2020, WHO reveals the 30% global decline in deaths caused by Tuberculosis, which shifts its place from 7th to 13th in 2019. But still, this communicable disease is a major challenge in developing countries. Several treatment regimens have been implicated against Tuberculosis and MDRTB. XDRTB is treated with repurposed drugs like Phenothiazines and Novel drugs, including Bedaquiline, Delamanid, or Pretomanid. Mainly genetic mutation confers to resistance and now has also been shown by Novel drugs, indicating an alarming situation that needs utmost attention.

First-Line Drug Resistance Isoniazid

Isoniazid (INH) was presented in 1952 (Johnnson et al., 1997) and is deemed to be one of the potent prodrugs (Zhang and Yew, 2009) that exist as first-line antibiotics (Raghunandanan et al., 2018). It is triggered by the catalase/peroxidase katG enzyme (Boellela et al., 2016) and becomes a robust bacteriostatic agent (Kendler et al., 2018) against Mycobacterium Tuberculosis (Mtb) (Lentz et al., 2018). It produces its activity by impeding mycolic acid production in the bacterial outer membrane while obstructing reductase enzymes, which is encoded by inhA (Rawat et al., 2003) and has MIC of [0.02microg/ml to 0.06microg/ml] (Lempens et al., 2018).

The missense, mutation, implantation, redundancy, or even complete omission of genes (Vilcheze and Jacobs, 2007) can originate in ndh, kasA, katG, along with ahpC and inhA (Almeida and Palomino, 2011; Larsen et al., 2002). As previously discussed, it most frequently happens in S315T of katG, evolving substantial decrease or permanent failure of catalase/peroxidase function (Zhang et al., 1992). Structural reforms of the active domain and genetic variation in 215CT promoter region emerges in inhA  (Leung et al., 2006), whereas hyper-expression of ahpC (Sherman et al., 1996) and silent genetic changes of mabAg609a (Ando et al., 2014) confers to resistance (Seifert et al., 2015). 64% katG S315T mutation have a significant decrease in susceptibility with MIC >1microgram/ml, while 19 % mutation in inhA promoter has mild resistance with MIC < 1microgram/ml (Riviere et al., 2020; Ayanwale et al., 2020). Scientific investigations have revealed a 2-fold increase in resistance by inhibition of dihydrofolate reductase in Mtb due to the INH-NADP 4R isomer (Wang et al., 2010).

Rifampicin

Rifampicin, amongst the broad-spectrum antibiotics, is used against bacterial pathogens (Sensi et al., 1960). Rifampicin, possessing exceptional sterilizing activity (Rattan and Musser, 1998). RIF adheres to RNA polymerase enzyme, in turn, hinders mRNA production resulting in organism destruction.

Resistance in rifampicin is correlated with a minimum of 10 genetic mutations (Sensi, 1983). The amino acid substitution in the rpoB gene is the major cause of resistance of RIF (Herrera et al., 2003). The 51 bp RRDR of the rpoB gene mainly contributes to mutations in codon 516, 526, and 531 (Ramaswamy, 1998; Herrera, 2003). Non-compliance drug resistance occurs. The modifications in several codons contribute to minor drug resistance (Heil and Zillig, 1970). Primary codon positions include 511,516 along with 518. Various factors not limited to age, HIV infection prevalence, and geography may also account for resistance. Despite the rare occurrence of RIF resistance, studies have deduced concomitant resistance in RIF and isoniazid (Cohn et al, 1997).

Ethambutol

Introduced in the 1960s (Lee and Neguyen, 2020), Ethambutol (EMB) is a first-Line Drug (Sreevatsan et al., 1997) with bacteriostatic action (Thomas et al., 1961), prescribed for the treatment of Tuberculosis since 1966 (Goude, 2009), only effective D-form (Lee and Neguyen, 2020). EMB is not given on its own, but in Quadruple following combinational therapy (Jeong et al., 2015) generating its effect by more than just interfering with the core polymer, Arabinogalactan AG (Takayama and Kilburn, 1989), but also hindering the synthesis of lipoarabinomannan LAM of the Mycobacterium cell wall (Dengg et al., 1995). Scholars demonstrated enhanced action of INH when it binds to transcriptional regulator TerR (Zhy, 2018).

Almost 4% of the clinical isolates displayed resistance (Wright and Zignol, 2008) mainly driven by genetic substitution in Rv3806c and Rv3792 (pathway genes), decaprenylphosphoryl-B-D-arabinose (DPA) (Safi et al., 2013), arabinosyl transferase emb operon counting embB codon 289, 292 and 306 (Lety, 1997; Starks et al., 2019). Moreover, embC and embA (Ramaswamy et al., 2000) interferes with outer membranes permeability (Bakula et al., 2013). Current reviews managed to show no mutagenesis in embB, raising the question of having other mechanisms involved (Zhang and Yew, 2009). Analyses of the allelic exchange implied substitution of the amino acid (Sreevatsan et al., 1997) in which the most common shift was Gly406Ala at nucleotide position 1217 by the transformation of G ?C (Bakula et al., 2013) Mild, moderate, and major-level substitution have EMB MIC 20, 100, and >256microgram/ml respectively (Telanti et al., 1997). 

Pyrazinamide

Pyrazinamide, like isoniazid and ethionamide, is a prodrug that requires mycobacterial enzyme pyrazinamidase for its conversion to pyrazinoic acid (Konno et al., 1967; Scorpio and zhang, 1996). Pyrazinamide introduction has shortened the TB treatment to 6 months (Mitchison, 1985). Various novel drug candidates are used together with pyrazinamide, in the mouse TB infection model, for optimal efficacy (K. Andries et al., 2005; Nuermberger et al., 2008). Postulated PAO action mechanism includes retardation in the kinetics of membrane, inhibition of membrane transmission, the production of Co-enzyme A, and increase in acidity of cell plasma (Zhang et al., 2003; Njire et al., 2016). Various studies propose fatty-acid synthase enzyme Type-I as a Pyrazinamide target (Zimhony et al., 2007; Zimhony et al., 2000). Pyrazinamide encoding pncA gene mutation is majorly linked to decreased PZA susceptibility (Scorpio et al., 1997; Palomino et al., 2014). Unlike isoniazid, the domain for mutation of  PZA is significant (Pym et al.,  2002). However, few PZA resistant strains possess no pncA mutations (Cheng et al., 2000; Smith et al., 2013), which hints at an alternative mechanism. Several studies reveal the overexpression of the rpsA gene accounts for PZA resistance (Shi et al., 2011). Detailed analysis of rpsA gene, in resistant strains without pncA, indicated deletion of 3 base pairs GCC, leading to the exit of Ala 438 (Boni et al., 2000 ). Concerns exist over the contribution of rpsA in PZA resistance, which may conclude, need for further studies.

Streptomycin

Streptomycin, classified as a glycoside anti-microbial

agent, was the initial curative agent for TB (Tasha et al., 2013), introduced in 1942 (Dookie et al., 2018). Streptomycin adheres irreversibly to s12 protein in ribosomes and 16s rRNA (Moazed and Noller 1987; Finken et al., 1993), leading to inhibition of translation (Ruusala and Kurland, 1984), and interference with ribosomal proofreading (Winder, 1982). Initially, mycobacterium tuberculosis was susceptible to SM. Gradually, resistance evolved due to mono drug therapy (Crofton and Mitchison, 1948). Mutations in rrs or rpsL genes account majorly for SM resistance. Simultaneous genetic variation in rrs or rpsL genes are rarely observed. Predominantly, rpsL gene alterations were at codon 43 and 88 while, codon 513 and 516 were observed for rrs gene (Betzaida et al., 2013). Investigation deduced the participation of gidB gene, in mild resistance (Verma et al., 2014; Spies et al., 2008; Okamoto et al., 2007). Further, mechanisms, including efflux pumps and cell membrane disruptions, may also confer resistance. 

Second-Line Drugs 

Injectables Aminoglycosides 

WHO (World Health Organization) reported 480,000 cases of MDR (Multi-Drug Resistance) Tuberculosis across the globe in 2014 (Zulma et al., 2015). Currently, Aminoglycoside Injectables KAN, AMK, (Johansen et al., 2006) and Tuberactinomycin CAP and viomycin (Akbergenov et al., 2011) are being used as potential drugs against MDRT (Reeves et al., 2013). Therapy through these medications is complicated, expensive, and hazardous due to its lengthy timeframe (Quenard et al., 2017; Zimen et al., 2013).

Kanamycin (KAN) was discovered in 1957 and clinically used in 1958 (WHO, 2009), producing its remarkable bactericidal effects (Hota et al., 2018; WHO, 2009). The action mechanism of aminoglycoside includes hampering of protein synthesis when it adheres with the 30S subunit in the ribosomes (Zaunbrecher et al., 2009). A research study found three rss gene mutations appearing in A140G, G1484T and C1402T (Jugheli et al., 2009; Maus et al., 2005) inevitably results in cross-resistance amongst Capreomycin, Kanamycin and Amykacin (Campbell et al., 2011). Further investigations reported conformational changes and mutation in eis gene, resulting in accelerated expression of eis, which inactivates the KAN (Reeves et al., 2013; Abraham et al., 2020) but not AMK (Zaunbrecher et al., 2009). The same author suggests substitution in whiB7 of transcriptional activator provoking enhanced whiB7 transcripts, which ultimately leads to increased expressions of eis (Rv2416c) and tap (Rv1258c) (Reeves et al., 2013). 

Capreomycin (CAP) is being extensively used against XDRT since 2006 after replacing KAN and AMK (Georghiou et al., 2012). CAP and Viomycin are bacteriostatic anti-microbial agents that bind to 50S subunits interfering with the translation process through intersubunit bridge B2a (Stanley et al., 2010) but do not interfere with mRNA (Tsukamura, 1969). Resistance transpired due to genetic variation in the tlyA gene (Brossier et al., 2017), causing the absence of the methylation process in rRNA (Johansen et al., 2006).

Fluoroquinolones

Fluoroquinolones, possessing robust bactericidal activity, are classified amongst second-line drug therapy for TB (Almeida et al., 2011). Nalidixic acid derivatives include ciprofloxacin, ofloxacin, and a few novel compounds such as gatifloxacin and moxifloxacin (Dookie et al., 2018). Majorly FQ’s inhibit topoisomerase II (Aubry et. al 2004) and IV (Zhang and Yew, 2005; Bernard et al., 2015), resulting in DNA breakdown and microbial fatality (Andriole, 2005). FQ’s decrease susceptibility is associated with aminoacid switching of gyrA and gyrB genes (Takiff et al., 1994; Che et al., 2017; Smith et al., 2013). Studies reveal the predominant role gyrA gene over gyrB (Smith et al., 2013). Alanine 90 and Aspartate 94 account majorly in gyrA gene mutation (Sun et al., 2008). Contrarily, codon 74, 88, and 91 role is limited (Maruri et al., 2012; Aubry et al., 2006; Matrat et al., 2006). For significant FQ’s resistance, double amino acid substitution in gyrA or co-occurring mutations in gyr A and gyr B are prerequisites (Takiff et al., 1994; Kocagöz et al., 1996). Alteration in cell membrane permeability to the drug (Almeida et al., 2011) and efflux-pump is significant in mediating resistance (Escribano et al., 2007; Takiff et al., 1996; Cambau et al., 1996; Jarlier and Nikaido 1994). Further, MfpA protein, homologous to DNA structure, binds to topoisomerase II and inturn inhibits its action (Hegde et al., 2005), resulting in minor resistance (Smith et al., 2013; Ginsburg et al., 2003).

Ethionamide

Ethionamide, a second line structural analogue of isoniazid, is mainly utilized in multi-drug resistant tuberculosis therapy (Engohang-Ndong et al., 2004). Similar to isoniazid, ETH is a prodrug and possesses a common pathway, which may result in cross-resistance (Morlock et al., 2003). ETH may get activated by enzymatic action and bacterial metabolism (alian et al., 2017). Obstruction in the production of mycolic acid results in the breakdown of cell wall biosynthesis by activated drug (Morlock et al., 2003).

ETH resistance may result from ethA and inhA mutations (Jacob et al., 1994; Clifton et al., 2000). Structural variations in C15–T of inhA are predominantly responsible for reduced susceptibility to ethionamide (Vannelli et al., 2002). Additionally, inhA-based ETH resistance may also conform to the cross-resistance of isoniazid (Diana et al., 2013). Studies demonstrate ethA expression is opposed by neighboring ethR gene (Engohang-Ndong et al., 2004).

Para-Amino Salicylic Acid

In combinational therapy against Tuberculosis, second-line Para aminosalicylic acid (PAS) has been functioning since 1940 (Sumit et al., 2013; Lehmann, 1946; Dye et al., 2002). Being less tolerated and toxic, its consumption was decreased dramatically (Iwainsky, 1988). 

Constrained growth of tubercle bacilli, attributed to the bacteriostatic drug disposition, is expected to be driven through impeding the synthesis of folic acid (Dye et al., 2002) and cell wall component mycobactin. (Vanessa et al., 2009).

The primary explanation for the susceptibility against PAS takes place on account of mutagenesis of thyA and drfA coding region, pertaining to the biosynthesis of thymine nucleotide (Pablo et al., 2009; Mitnick et al. 2003; sumit Chakraborty et al., 2013). Maximum gene variations in thyA were recorded in TB patients from China (Bharti et al., 2019).

Novel or Repurposed Drugs

Bedaquiline

Bedaquiline, a diarylquinoline drug, seems to possess an unorthodox action mechanism against TB (Hendrik et al., 2014). WHO warned of deliberate drug administration, encouraging the emergence of resistance (Kenny et al., 2014). BDQ resistance, typically attributed to the mutations in atpE, Rv0678, intergenic region between Rv0678 and Rv0677c, and pepQ (Rv2435c) genes (N. Engyl et al., 2015; Kenny et al., 2014). In addition, the substitution of Rv0678 gene, acting as an inhibitor of the efflux pump, results in minute resistance (Amber, 2017). Polymorphisms in the Rv1979c and PepQ (Rv2535c) genome is affiliated with concomitant resistance in clofazimine (CFZ) (Deepak et al., 2016).

Nitroimidazole

Nitroimidazole antibiotics were discovered in the late 1950s (Ang et. al., 2017). New prodrugs, Delamanid and Pretomanid, require bioactivation of nitro group to exert its bactericidal action, against both replication and hypoxic nonreplicating Tuberculosis, by decreased production of mycolic acid during outer membrane formation (Matsumoto et al., 2006; Samuelson, 1999), and nitric oxide release (Lamprecht et al., 2016; Singh et al., 2008), causing respiratory poisoning respectively. Resistance occurs by decreased activity/expression or mutation of reductive enzyme Ddn (Haver et al., 2015), which catalyzes menaquinone, only present in Mycobacterium Tuberculosis (Ang et al., 2017). Ddn enzyme is F420H2-Dependent (Gurumurthy et al., 2013). Genomic sequence study showed 46 no-synonymous substitutions, among which several mutants were unable to activate the drug 2 and deletion of Ddn quinone reductase(Jing et al., 2019). Variation in three binding sites (S78, Y130, Y136) and polymorphism (SNP) of genes of Ddn affect the nitroimidazole activating activity and confer to the resistance of the drug (Mohamed et al., 2016; Haver et al., 2015).  The study has also shown the difference of activity between two drugs against resistant mutant. Out of 75 mutants studied, 65 did not reduce the Delamanid, while 50 were unable to decrease Pretomanid (Lee et al., 2020; Cellitti et al., 2012; Mohamed et al., 2016). It was also revealed that Mutations in Ddn also cause the transmission of disease (Ai et al., 2016).

Phenothiazine (Chloramphenicol and Thioridazine)

Phenothiazines discovered in 1883 (Masie, 1954) are tricyclic, anti-psychotic, “Non-Antibiotic” anti-microbial agents expecting broad-spectrum intervention that modifies the cell permeability, illustrating synergistic effect along with other anti-microbial agents (Amaral and Molnar, 1991; Kristiansen and Amaral, 1997; Amaral et al., 2001). Phenothiazine compounds such as Chlorpromazine (CPZ) and Thioridazine (TDZ), share the same potential activity against MDRTB and XDRTB (Ordway et al., 2003; Amaral et al. 2004; Viveiros and Amaral, 2001).

CPZ was formulated in 1950 as an anti-psychotic agent along with many nasty consequences (BMA. 2010), but reconsidered as a potential anti-MDRTB (Alsaad et al., 2014). The concentration used 15-20mg/L is higher than the clinically indicated value for a chronic patient (Molnar et al., 1997). CPZ produces its effect by preventing the growth and killing Mtb (Amaral and Viveiros, 2012).

TDZ being less noxious, replaced the CPZ (Amaral et al., 1996). When given in combination at a dose of 200mg/day, it contributes by interfering with gene expression, inhibition of efflux pump, suppression of replication, and retardation of Ca++ and K+ transport process that leads to complete extrusion and killing of bacteria (Amaral and Viveiros, 2012; Amaral and Mornal, 2012). 

According to an electronic database study, other anti-microbial agents that could be used against XDRTB include doxycycline, and Co-trimoxazole, and metronidazole which are not on the WHO list (Alsaad et al., 2014).

SQ109 (Ethambutol Analogue)

SQ109 is a potent anti-MDRTB agent (Onajole et al., 2010) having a bactericidal activity (Boeree, 2017) that entered as an improved analogue of ethambutol in the clinical trials (Lee et al., 2003). SQ109 reported low bioavailability due to the first-pass effect and boosted up to 91.4% by administering as a prodrug (Meng et al., 2009). It targets the mycobacterial cell wall by lessening the mycolic acid concentration (Tetli et al., 2020); conversely, it acts as an inhibitor of the efflux pump (Te Brake et al., 2016). Moreover, it is also associated with MmpL3 inhibition (Tetli et al., 2020). 

Susceptibility in SQ109 tends to happen by mutagenesis in MmpL3 (Umumarararngu et al., 2020). Recent research indicates impaired menaquinone synthesis, ATP synthesis, and cellular respiration on the cytoplasmic membrane (Tetli et al., 2020). The activity of SQ109 is concentration-dependent as complete extrusion happens at a concentration of 256mg/L and kills 99% Mtb when exposed to 64mg/mL within one day (de Knegt et al., 2017). Synergistic effects are seen when SQ109 is combined along with isoniazid, rifampicin, and Bediquline, while augmented effects are noted with Streptomycin and Suetozid (PNU-100480) in in-vitro studies (Umumarararngu et al., 2020).

Linezolid

Linezolid, the member of oxazolidinones, possesses

 considerable in vivo and in vitro action against TB (Alcala et al., 2003; Cynamon et al., 1999). LZD inhibits the initial phase in protein formation by binding to 50s ribosomal subunit (Zhang, 2005; Escribano et al., 2007). G2572T and G2061T genetic variations in the rrl, leads to Anti-TB drug’s resistance (Navisha Dookie, 2018). Resistance results in elevation of MIC of 4–8 mg/L to 16–32 mg/L range (Hillemann et al., 2008).C154R mutation in the rplC gene also impart LZD resistance (Bloemberg et al., 2015). The role of efflux mechanisms or other non-ribosomal modifications cannot be neglected. 

Clofazimine

Clofazimine is a drug known as riminophenazine, specifically developed for tuberculosis treatment in 1950 (Arbiser and Moschella, 1995). The precise action mechanism is uncertain, but neutrophil and monocyte appear to be the principal location of an operation where it prohibits the inflammatory action by scavenging hypochloric acid while preventing chlorination (Arbiser and Moschella, 1995). Also, bactericidal effect is produced by redox cycling of Clofazimine (Xu et al., 2017), shortening the therapy timeframe (Zhang et al., 2015)

Genetic variation in transcriptional repressor Rv0678 linked with mmpS5 and mmpL5 genes is correlated with the upregulation of efflux pumps, which tends to result in resistant strains (Yew et al., 2017; iu et al., 2020). Mutation involves the insertion and revocation of nucleotide G at 193 positions (Zhang et al., 2015). Further investigations revealed that Rv1979c involved in the transport of amino acids and, Rv2535c which encodes a proline aminopeptidase peptidase PepQ was spotted to be the risk factors of developing drug susceptibility (Zhang et al., 2015; Xu et al., 2017; Van et al., 2020). PepQ Rv3525c gene suggests mild resistance (Ameida et al., 2016; Xu et al., 2017). Research work reported 1.2microgram/ml MIC for Clofazimine. Adequate interventions can be made by constructing the MIC data to mitigate the transmission of resistant strains (Xu et al., 2017). Improved genetic knowledge can help in molecular diagnosis and monitoring of drug resistance (Kadura et al., 2020).

NAS 91 & NAS 21

In recent times, NAS 21 & NAS 91 exhibited promising anti-mycobacterial activity (Pedro, 2011). Being a dominant pharmacophore, NAS-91 acts as a

candidate for future inhibitors (Choi et al., 2000).

According to M. bovis BCG examination, FAS-II dehydratase coded by Rv0636 developed the main target for resistance (Veemal, 2009). Partial obstruction of mycolic acid biosynthesis, as well as  

the variation in oleic acid production, occurs as a consequence of upregulated Rv0636 gene analogue. The oleic acid synthesis was inhibited, as demonstrated by an assay (Eduardo et al., 2011).

Benzothiazinone

Benzothiazinone is classified amongst potential tuberculosis treatment therapies (Variam et al., 2017). DprE1 enzyme and its inhibitors are a member of newly introduced TB medications. (Inshad et al., 2016). DprE1 and DprE2 speed up the conversion of DPR to its epimer DPA. Mycobacterial outer membrane production is modulated by DPA (Inshad et al., 2016). Mechanistic studies revealed the importance of the NO2 and Sulfur groups for anti-TB activity at position 8 and 1, respectively. Further, Trifluoromethyl significant role was also highlighted against Tuberculosis (Monika et al., 2016).

Conclusion

Decreased anti-microbial drug susceptibility against M. Tuberculosis presents a great challenge to human health globally. The inception of resistance to anti-tubercular drugs is restricting the treatment options, which tends to be a great threat to human life, especially in developing countries. New therapy regimens, surveillance studies, and strategies for early diagnosis of declined drug susceptibility deemed mandatory. 

References