Efficacy and Mechanisms of Flavonoids against the Emerging Opportunistic Nontuberculous Mycobacteria

Nontuberculous mycobacteria (NTM) are the causative agent of severe chronic pulmonary diseases and is accountable for post-traumatic wound infections, lymphadenitis, endometritis, cutaneous, eye infections and disseminated diseases. These infections are extremely challenging to treat due to multidrug resistance, which encompasses the classical and existing antituberculosis agents. Hence, current studies are aimed to appraise the antimycobacterial activity of flavonoids against NTM, their capacity to synergize with pharmacological agents and their ability to block virulence. Flavonoids have potential antimycobacterial effects at minor quantities by themselves or in synergistic combinations. A cocktail of flavonoids used with existing antimycobacterial agents is a strategy to lessen side effects. The present review focuses on recent studies on naturally occurring flavonoids and their antimycobacterial effects, underlying mechanisms and synergistic effects in a cocktail with traditional agents.


Introduction
Mycobacteria belong to Mycobacteriaceae and genus Actinobacteria, are slow-growing, immobile, Gram-neutral or weakly Gram-positive thin rod-shaped to filamentous bacteria and can be categorized into three key groups for the determination of diagnosis and therapy. They do not cause TB; however, they can produce pulmonary infections, lymphadenitis, skin disease, endometritis and disseminated disease. Thus, NTM are denoted by other names such as environmental mycobacteria or mycobacteria other than tuberculosis (MOTT) and atypical mycobacteria (ATM) [1][2][3][4].
More than 200 different species of NTM have been identified in nature (https://www.bacterio. net/genus/mycobacterium), and among them; about 95% are environmental bacteria with maximum existence as saprophytes, opportunistic pathogens or nonpathogenic to humans and animals [5]. NTM are generally found in the environment, mostly in wet soil, rivers, streams, estuaries, marshland and hospital settings. They are less pathogenic when compared to tuberculous mycobacteria, however they can cause illness to immunocompromised or pulmonary infected individuals [6]. Among NTM streams, estuaries, marshland and hospital settings. They are less pathogenic when compared to tuberculous mycobacteria, however they can cause illness to immunocompromised or pulmonary infected individuals [6]. Among NTM pathogens, M. avium complex are the most significant and recurrent pathogenic organisms that causes pulmonary and extrapulmonary infections. In addition, M. xenopi, M. kansasii, M. malmoense are the most causative agents for pulmonary infections. Skin and cutaneous tissue infections are also caused by M. ulcerans and M. marinum [7]. M. abscessus, M. fortuitum, M. chelonae, M. chimaera are the infectious agents accountable for most soft tissue infections [8].
According to the Runyon classification (Figure 1), mycobacteria have broad categories based on phenotypic factors including pigmentation and the frequency of bacterial growth [9]. They are classified as rapidly growing mycobacteria-RGM (visible colonies appear within seven days) and slow-growing mycobacteria-SGM (visible colonies appear after seven days). Most pathogenic mycobacteria are associated with the SGM, due to their virulence and growth rate. The members of the M. chelonae-M. abscessus complex and M. fortuitum complex are classified under the RGM family ( Figure 1). The classification of mycobacteria remains greatly active and is continually developing, owing to the available technological progressions including sequencing of bacterial isolates. However, this improvement provides only taxonomy of the evolving novel mycobacteria, and still, their documentation remains uncertain and is obligatory to find the potential phenotypic and genetic polymorphisms of the M. abscessus complex. RGM, M. chelonei, M. fortuitum and M. abscessus complex are well-renowned pathogens that often occur in cutaneous infections related to plastic surgery and cosmetic techniques. They appear widely in different pathologic conditions viz., cellulitis, superficial lymphadenitis, chronic nodular lesions, abscesses, nonhealing ulcers, verrucous lesions and commonly occur in the subcutaneous tissue and skin [10].
M. abscessus is often misidentified as M. chelonae. It is documented that M. chelonae is seldom accountable for lung disease [11]. In addition, M. chelonae fails to develop in the culture at 37 °C when compared to M. abscessus. M. chelonae is abundant in aquatic systems that can cause infection in immunocompromised hosts [12]. Hence, this inappropriate identification of M. abscessus is highly possible in several pilot trials specifically in pulmonary contagions, consequently flouting the significance of this mycobacterium. Notably, the augmented occurrence of M. abscessus in the individual with cystic fibrosis directs that this pathogenic organism has developed progressively to become widespread in the past decade [13,14]. The cultures of M. abscessus grow in less than seven days using agar medium (the combination of Bactec 12B and Middlebrook 7H10/ 7H11) and the strains of M. chelonae can be cultivated at 30 °C. Most of the NTM species can grow in the RGM culture medium at 30 °C, and M. xenopi can grow in the Lowenstein-Jensen (LJ) medium at 36 °C [15]. RGM, M. chelonei, M. fortuitum and M. abscessus complex are well-renowned pathogens that often occur in cutaneous infections related to plastic surgery and cosmetic techniques. They appear widely in different pathologic conditions viz., cellulitis, superficial lymphadenitis, chronic nodular lesions, abscesses, nonhealing ulcers, verrucous lesions and commonly occur in the subcutaneous tissue and skin [10].
M. abscessus is often misidentified as M. chelonae. It is documented that M. chelonae is seldom accountable for lung disease [11]. In addition, M. chelonae fails to develop in the culture at 37 • C when compared to M. abscessus. M. chelonae is abundant in aquatic systems that can cause infection in immunocompromised hosts [12]. Hence, this inappropriate identification of M. abscessus is highly possible in several pilot trials specifically in pulmonary contagions, consequently flouting the significance of this mycobacterium. Notably, the augmented occurrence of M. abscessus in the individual with cystic fibrosis directs that this pathogenic organism has developed progressively to become widespread in the past decade [13,14]. The cultures of M. abscessus grow in less than seven days using agar medium (the combination of Bactec 12B and Middlebrook 7H10/7H11) and the strains of M. chelonae can be cultivated at 30 • C. Most of the NTM species can grow in the RGM culture medium at 30 • C, and M. xenopi can grow in the Lowenstein-Jensen (LJ) medium at 36 • C [15].

Challenges in Diagnosing and Treatment of NTM Diseases
RGM are usually isolated from blood, sputum or tissues for diagnosis and are often misidentified as diphtheroids. RGM species normally cultivate as routine culture in liquid broth blood culture medium or on solid agars that can grow quickly within seven days. These strains relatively stain with Gram stain not with Ziehl-Neelsen stain to demonstrate the acid-fast characteristics. A fresh young culture of RGM may not constantly show branching or beaded structures and exhibit weakly Gram-positive bacilli, thus misleading the diagnosis and often incorrectly concluded as diphtheroids [46]. NTM in tissue specimens can also be identified based on the molecular method of determination, which includes, 16S rRNA gene sequencing, PCR analysis and HPLC. The diagnosis of NTM often fails to recognize the species and subspecies of the different samples from the affected individual. Most NTM microscopically appears similar to Mycobacterium tuberculosis (MTB), and the colony morphology varies in culture. The culture difference and microscopic appearance are shown in Figure 2. A total of 16S ribosomal RNA sequencing aids in individual NTM species identification [20]. Diagnosis is generally completed by recurrent isolation accompanied by certain clinical and radiological features. There is no explicit treatment of NTM infections and therapy depends upon the particular species and its resistance to antibiotics [47].  In addition, using these chemical agents produce various complications including, diarrhea, headache, renal failure and colitis. Mycobacteriosis is an acute/chronic, systemic, granulomatous disease caused by NTM, which is extremely challenging in selecting effective antimicrobial therapy based on the antimicrobial resistance [53]. The RGM involves individualized treatment according to the outcomes found in vitro vulnerability tests for cefoxitin, amikacin, clarithromycin, The diagnosis of NTM are difficult to confirm using acid-fast microscopy, which is the primary diagnostic tool for TB in numerous developing nations. As an outcome, most cases of NTM causing pulmonary infections are not recognized and eventually treated with traditional anti-TB medications. These treatments often fail because NTM are mostly resistant to anti-TB therapy [48]. Hence, in developed nations, caseloads of 8.6/100,000 total population and 20.4/100,000 population over 50 years old are typical [49]. In developing nations, the occurrence rate and diagnosis of NTM cannot be observed due to the lack of laboratory arrangement and identification of mycobacteria. Hence, the escalating rate of pathogenic NTM in developing nations has been greater particularly with the advent of HIV/AIDS patients. Normally, HIV/AIDS individuals with severe immunosuppression are at high risk of NTM infections, which often cause localized or disseminated infections [50]. In addition, the failure of NTM treatment can frequently occur due to resistance to some of the available antibiotics (Table 2).
In addition, using these chemical agents produce various complications including, diarrhea, headache, renal failure and colitis. Mycobacteriosis is an acute/chronic, systemic, granulomatous disease caused by NTM, which is extremely challenging in selecting effective antimicrobial therapy based on the antimicrobial resistance [53]. The RGM involves individualized treatment according to the outcomes found in vitro vulnerability tests for cefoxitin, amikacin, clarithromycin, sulfamethoxazole, ciprofloxacin, imipenem and doxycycline [54]. The M. fortuitum and M. chelonae are members of M. abscessus complex and M. massiliense, M. abscessus and M. bolletii are subspecies, which are the chief NTM related to cutaneous tissue involvement [55]. All these mycobacteria are regularly found with several skin lesions, however M. fortuitum is often found in a sole lesion [33]. The susceptibility to antimicrobials generally depends upon the individual species. M. abscessus complex is likely to be vulnerable to the cocktail of amikacin, azithromycin, imipenem and cefoxitin, since, it is known that clarithromycin resistance due to the occurrence of the erm41 gene [56]. In vivo study demonstrates that NTM isolates show resistance to azithromycin or clarithromycin [56,57]. Azithromycin is normally the desired antibiotic for M. abscessus infections, while azithromycin or clarithromycin is highly efficient in the cases of M. massiliense [56,57]. M. fortuitum, M. abscessus and M. chelonae are resistant to all of the existing anti-TB agents [10,56,57]. M. fortuitum is highly susceptible to amikacin, trimethoprim-sulfamethoxazole, azithromycin or clarithromycin, fluoroquinolones and doxycycline. M. chelonae is also often susceptible to azithromycin or clarithromycin, tobramycin, fluoroquinolones and cefoxitin [55]. The guideline of the therapy recommends performing susceptibility testing of NTM to enhance the option of a cocktail of the antimycobacterial drug relates clinically in vivo trials to antimicrobial treatment for various species of NTM. From the microbiologic perspective, heterogeneity of NTM needs sophisticated and rapid laboratory techniques. Since the present pharmacological treatment of NTM diseases are tricky, and often fails to scope the long-term removal of pathogens. Moreover, it is obligatory to hunt novel agents or treatment and dosage regimens for effective treatment of these NTM diseases, specifically serious in immunocompromised individuals. Hence, it is necessary to find alternative remedial regimens. One of the alternative resources is traditional medicinal plants or their derivatives, which are well-known for their therapeutic properties. Most of the researchers have a positive approach toward natural products due to their natural origin and low noxious with fewer side effects [3,[58][59][60][61][62][63][64][65][66]. A trial of anti-Mycobacterial effects of these medicinal plants, particularly those that are conventionally used for pulmonary infections is significant.
Natural products as a source of medicine are potentially valuable due to their natural origin and low toxicity with lesser side effects. Medicinal herbs with the traditional practice of crude extracts or active principles have been widely used for treating and averting human illnesses for many centuries. These ethnopharmacological techniques have been reinforced to yield bioactive compounds that support to improve modern medicine as beneficial tools [67][68][69][70]. Bioactive compounds often contribute a noteworthy function in drug finding by helping as a novel drug of interest and templates for synthetic agents [71][72][73]. Copious investigations have established that natural bioactive compounds have possible antimycobacterial activities [2,60,74,75]. The single-handed practice of bioactive compounds or cocktails with classical antibiotics signifies a greater alternative treatment. Additionally, the cocktails of those antimicrobial agents often require only a minor amount. Therefore, this smaller amount may provide less toxicity to the host, ensuring great lenience to the antibacterial drugs. Grounded on the existing information, there has been inadequate literature regarding antimycobacterial phytocompounds [76][77][78][79]. Thus, the present review aims to emphasize the antimycobacterial effects of flavonoids and their underlying mechanisms.
The literature of flavonoids and antimycobacterial effects were obtained in electronic search using Google Scholar, Science Direct and PubMed The following keywords were used in the Title/Abstract/Keywords: "flavonoids" and "antimycobacterial" or "Nontuberculous mycobacteria" or "M. fortuitum or M. abscessus or M. chelonae," and checking all available findings of clinical, in vivo and in vitro connection among flavonoids and their antimycobacterial effects. The underlying antimycobacterial mechanism was composed and organized in a suitable place.

Flavonoids
Most commonly the flavonoids are the secondary metabolites of the plant kingdom with well-known wide-ranging classes of polyphenols. They normally exist in all kinds of vegetables, fruits and beverages [80][81][82][83][84]. WHO estimated that 25% of existing drugs are derived from plants used in folk medicine [85,86]. Besides the long-established clinical use, the plant-derived compounds display good tolerance and acceptance among patients and seem like a credible source of antimicrobial compounds. Among 109 new antibacterial drugs, approved in the period 1981-2006, 69% originated from natural products [87]. One of the major groups of phytochemicals that has been studied extensively for their antimicrobial properties are flavonoids [66,88]. Flavonoids are organized with the structure of two phenyl rings fixed with the heterocyclic ring as C6-C3-C6 and arranged up to a skeleton of 15-carbon. They are classified into many subclasses based on variation in the central carbon ring viz., flavanones, flavonols, flavones, flavan, isoflavones and anthocyanidins [89]. There has been accumulating scientific interest in the study range of flavonoids that demonstrate the following pharmacological functions: antioxidant [90,91], antidiabetic and anti-obesity [92,93], hypolipidemic [94], anti-inflammatory [95], antimicrobial [96][97][98], anticancer [99][100][101], anti-aging [102], antiallergic and antithrombotic [103], hepatoprotective [104][105][106][107], cardioprotective [108], neuroprotective [109], nephroprotective [110], protect from lung injury [111] and improving endothelial function, adjourning age-related cognitive and neurodegenerative diseases [112,113]. The evidence has validated that the prolonged consumption of dietary flavonoids at higher quantity has also produced minor side effects, which may arise due to the shortage of bioavailability and gut permeability as well as the greater metabolic rate [114]. Moreover, the intake of flavonoids produces a poor absorption coefficient, which may cause only minor toxicity to animals and humans [115,116]. All of these data support investigations to discover and inspect the attractive healing indices of Flavonoids concerning human wellbeing. The daily intake of dietary flavonoids is estimated to be about 1-2.5 g; flavonols and flavones have been found to be 23 mg [114,117]. Hence, regular intake of flavonoids could be favorable in preventing or treating various illnesses and improving health outcomes.

Anti-Nontuberculous Mycobacterial Efficacy and Mechanisms
Flavonoids have been used in the treatment of the wide spectrum of human illnesses since time immemorial [118][119][120]. Flavonoids may inhibit NTM growth with various underlying mechanisms, including inhibiting cell wall formation, biofilm formation, bacterial DNA synthesis and efflux mediated pumping systems. In addition, the mixture of flavonoids with antimycobacterial agents may be a greater approach to combat mycobacterial infections and microbial resistance.

Inhibition of Cell Wall Formation
Flavonoids inhibit bacterial growth, microbial adhesions and cell wall or transport proteins [121]. Some anti-NTM drugs normally damage the cell membrane's integrity that leads to the leakage of intracellular components, which leads to alterations in membrane permeability. Flavonoids can also damage the cell wall of bacteria [121]. Body cells and tissues are continuously threatened by the injury caused by free radicals and reactive oxygen species (ROS) which are produced during normal oxygen metabolism or are induced by exogenous damage [122]. Eventually, these excess ROS can produce unadorned oxidative stress to the bacterial cell membrane leads to increased permeability, nucleic acid damage and oxidation of protein and fatty acids in the membrane ( Figure 3) [123][124][125]. Unfortunately, these free radicals can attract various inflammatory mediators in the host, contributing to a general inflammatory response and host tissue damage. These elevated ROS species cause depletion of the endogenous scavenging compounds and reduced the levels of antioxidant equilibrium. Flavonoids may have an additive effect on the endogenous scavenging compounds and abolish the effect of the free radical causing inflammatory response and combat to regulate antioxidant levels in the host [126]. Flavonoids are measured as effective ROS scavengers however, the level of flavonoid in human plasma and most tissues is too little to effectively reduce ROS [127]. Moreover, flavonoid as ROS scavenger usage should be carefully measured, since low levels of ROS are, on the contrary, beneficial for bacteria and can persuade resistance. Therefore, the function of flavonoids as an antimicrobial potentiator should rather be related to the regulation of the activities of different proteins and molecular processes, and there is a need for further investigations, specifically regarding their synergistic action.
Antibiotics 2020, 9, x FOR PEER REVIEW 9 of 35 [162,163]. This enzyme is normally found in the outer membrane of pathogenic mycobacteria that crucially involves in the synthesis of poly-L-glutamate-glutamine. quercetin plays a key function in regulating the cellular levels of NH3 in the infected host and eliminate the pathogen through phagosome acidification and phagosome-lysosome fusion [161]. Fatty acid synthase II (FAS-II) is a key enzyme, requires endogenous fatty acid synthesis in the bacterial membrane, represents a possible target for novel antimycobacterial agents [164]. FAS-I is accountable for de novo fatty acid (FA) synthesis to form FA chain elongation (16-24 carbons) and then lengthened by the FAS-II monofunctional enzymes to yield long-chain fatty acids (36-48 carbons) and mycolic acids. Mutation of monofunctional enzymes often provides drug resistance to the mycobacteria [165]. Flavonoids such as isoliquiritigenin, butein, fisetin and 2,2′,4′trihydroxychalcone prevent the growth of M. smegmatis by targeting the dehydratase enzyme of FAS-II [164]. D-alanine-d-alanine ligase is an enzyme involved in cell wall synthesis. Another study has also confirmed that quercetin and apigenin (4′,5,7-trihydroxyflavone) inhibit ATP binding pocket of D-alanine-D-alanine ligase and prevent bacterial peptidoglycan synthesis [166].

Inhibition of Biofilm Formation
The biofilm formation is normally associated with virulence, pathogenicity, resistance to antibacterial substances and survival in the environment [167]. Antibacterial resistance of biofilmdeveloping mycobacteria may cause the failure of the treatment, and biofilms must be materially exterminated to resolve the infection. The formation of biofilms provides relationships among microbial populations with a high spectrum of colonization and functional activities. They form on Fathima and Rao [128] described that the flavonoid catechin plays a bactericidal action through the oxidative burst and generation of ROS that causes a change in the membrane permeability and membrane injury. Similarly, liposome studies also confirmed membrane disruption during oxidative stress which occurs only at high concentrations of epigallocatechin gallate [129]. Quercetin from propolis (natural resinous mixture produced by honey bees, that have potential antimicrobial applications: upper respiratory tract infections, common cold, wound healing, treatment of burns, acne, herpes simplex and genitalis and neurodermatitis) causes a decrease of proton-motive force and increased membrane permeability in the bacterium which has been employed by the synergistic activity of quercetin with antibiotics, including ampicillin and tetracycline [130,131]. Additionally, flavones-acacetin and apigenin, as well as flavonols morin and rhamnetin caused destabilization of the membrane structure by disordering and disorientation of the membrane lipids and induced leakage from the vesicle [132]. Lipid peroxidation has been shown to destroy the bacterial cell wall and alter membrane potential, ensuing augmented permeability, decreased fluidity and disruption Antibiotics 2020, 9, 450 9 of 34 of phospholipids [76]. The connection between the lipid bilayer and production of ROS is often linked in the malondialdehyde production that is the key marker of lipid peroxidation. This lipid peroxidation is not only harmful to the bacterial lipid bilayer, but also affects the host cell membrane. Ethyl acetate leaves extract of Aegle tamilnadensis and Schkuhria pinnata and their active principles of flavonoids have exerted antioxidant and antimycobacterial activity against M. smegmatis with MIC range of 0.01 to 2.50 mg/mL [76,133]. Four well-known testing systems were carried out in this study to assess the antioxidant potential viz., lipid peroxidation inhibition, nitric oxide radical inhibition, ferric thiocyanate and ABTS radical scavenging assay. Based on the findings, ethyl acetate extract demonstrated a noteworthy antioxidant activity and significant antimycobacterial activity [76,133].
Heritiera littoralis Dryand mangrove flora produces novel flavonoids; tribuloside, afzelin, and astilbin that were revealed to possess antimycobacterial activity against the various species of NTM with a minimum inhibitory concentration (MIC) of 5.0 mg/mL. All these flavonoids exhibited growth inhibition of NTM while co-administered with standard anti-TB drugs [153].  [155]. Lipophilic flavonoids which are highly hydroxylated can be more disruptive for membrane structure [156,157]. Hence, it is worth observing that the flavonoids decrease the bacterial toxin secretion by damaging the membrane [158,159].
Amikacin is a semi-synthetic aminoglycoside extensively used to treat disease caused by NTM and gentamicin resistant Gram-negative bacterium. Conversely, the clinical use of drugs regularly causes ototoxicity due to the generation of ROS. A natural flavonoid, galangin pretreatment demonstrated to provide defensive functions against amikacin-provoked mitochondrial dysfunction by decreasing ROS generation [160]. The antioxidant properties of quercetin-3-O-β-d-glucoside prevent the formation of biofilm and encourage membrane disturbances, ensuing shrinkage of size and outflow of intracellular constituents of M. smegmatis [161]. In addition, quercetin accelerates the inhibition of mycobacterial glutamine synthetase. Glutamine synthetase is the key enzyme involved in virulence factors, as well as pathogenesis that had been recognized as a possible antibiotic target [162,163]. This enzyme is normally found in the outer membrane of pathogenic mycobacteria that crucially involves in the synthesis of poly-l-glutamate-glutamine. quercetin plays a key function in regulating the cellular levels of NH 3 in the infected host and eliminate the pathogen through phagosome acidification and phagosome-lysosome fusion [161].
Fatty acid synthase II (FAS-II) is a key enzyme, requires endogenous fatty acid synthesis in the bacterial membrane, represents a possible target for novel antimycobacterial agents [164]. FAS-I is accountable for de novo fatty acid (FA) synthesis to form FA chain elongation (16-24 carbons) and then lengthened by the FAS-II monofunctional enzymes to yield long-chain fatty acids (36-48 carbons) and mycolic acids. Mutation of monofunctional enzymes often provides drug resistance to the mycobacteria [165]. Flavonoids such as isoliquiritigenin, butein, fisetin and 2,2 ,4 -trihydroxychalcone prevent the growth of M. smegmatis by targeting the dehydratase enzyme of FAS-II [164]. d-alanine-d-alanine ligase is an enzyme involved in cell wall synthesis. Another study has also confirmed that quercetin and apigenin (4 ,5,7-trihydroxyflavone) inhibit ATP binding pocket of d-alanine-d-alanine ligase and prevent bacterial peptidoglycan synthesis [166].

Inhibition of Biofilm Formation
The biofilm formation is normally associated with virulence, pathogenicity, resistance to antibacterial substances and survival in the environment [167]. Antibacterial resistance of biofilm-developing mycobacteria may cause the failure of the treatment, and biofilms must be materially exterminated to resolve the infection. The formation of biofilms provides relationships among microbial populations with a high spectrum of colonization and functional activities. They form on many surfaces including, human tissue, medical equipment, plumbing pipes and drinking water systems [168]. In hospital wards, the development of biofilms on ventilators and hospital apparatus that permits pathogens to continue as pools which may freely spread to patients. After invading into the host, these biofilms let pathogens disrupt the host immune systems and can persist for a long-time [169]. Studies have also supported that tap water functions as a primary source for human colonization and/or infection outbreak of NTM [169,170]. The developed biofilms often contain M. fortuitum, which produce biofilm-dispersing agents such as biosurfactant. Moreover, M. chelonae and M. fortuitum developed thick biofilms with asymmetrical forms that were comparatively resistant to available antibiotics even at 10× MIC [169].
The hydrophobicity and metal resistance of mycobacteria often permits adhesion of cells and the successive development of biofilms on aquatic surface later. In addition, NTM in tap water are normally able to survive and are often resistant to the chemicals glutaraldehyde and chlorine [169,170]. The proliferation of these NTM from standing biofilms that can aid the spread of infections to individuals, demonstrates a noteworthy health risk in hospital environments [171]. Novel approaches with potential antibiofilm agents that improve treatment efficacy must be developed which is urgently necessary for the suitable therapy of NTM infected patients.
Flavonoids are well recognized as anti-NTM agents and prevent biofilm developments. Research in this area has generated interest in the ability of flavonoids to enhance the outcomes of untreatable infections, especially on antibiotic-resistant bacteria like NTM. Several researchers have confirmed that the structure-relationship of flavonoids enhances the bactericidal actions and demonstrated as antibacterial agents [141,146,[172][173][174]. The anti-NTM activity and inhibition of biofilm effects of flavones and flavanones are usually based on the hydrophobic compounds on one aromatic ring and a hydrogen-bonding group on another aromatic ring [175]. These biofilm developments can be inhibited by the hydrophobic substituents of flavonoids, which comprises various heterocyclic moieties including, alkyl, prenyl, nitrogen or oxygen-containing heterocyclic and alkylamino chains [141,172]. This structural activation of flavonoids can directly kill the bacteria in the biofilm formation, synergistically activate with the antibiotics and weaken the bacterial pathogenic effects [141,172]. Few recent studies showed a series of flavonoid derivatives significantly exhibited their antimycobacterial activity against various NTM species through inhibition of biofilm formation [176,177]. Apigenin normally has a cyclic or aliphatic chain at the 8-C position that enhanced the antimycobacterial activities and prevents biofilm formation [178]. Few supporting studies demonstrated that C-benzylated dihydrochalcone and the dihydrochalcone dimer have shown significant antibacterial activity against M. chelonae and M. fortuitum [179]. An active flavanone compound, Platyisoflavanone obtained from Platycelphium voense revealed antimycobacterial activity using microplate alamar blue assay against M. chelonae with MIC of 23.7 mmol/L [180].
Another study demonstrates that synergistic combinations of amikacin and curcumin (compound isolated from Curcuma longa), employs antimycobacterial activity against M. abscessus clinical strain with MIC of 128 mg/L. Furthermore, curcumin induced an over-all decrease in microbial masses in the biofilm and considerable loss in cell viability [123]. Two methoxylated flavonoids, flavonoid 7-methylquercetagetin and 7-methylquercetagetin-4 -O-β-d-glucopyranoside were extracted from Paepalanthus latipes which showed significant antimycobacterial activity against NTM species with MIC ranged from 1-2 mg/L [181].

Inhibition of Efflux Mediated Pumping System
Efflux pumps are well-recognized proteins and protein complexes that provide antibiotic resistance in bacteria, including mycobacteria [182]. Hence, the finding of efflux pump inhibitors is a fascinating target in antimycobacterial treatment. Plant-derived natural bioactive compounds are potent inhibitors of an efflux pump that may capable adjunct to traditional chemotherapy by improving mycobacterial vulnerability to antibiotics. Flavonoids exert noteworthy antimycobacterial activities and exhibited considerable outcomes as antimycobacterial agents [183]. A study showed that the inhibition of the efflux pump has been performed using flavonoid, pinocembrin isolated from Alpinia katsumadai, which showed antimycobacterial activities against M. smegmatis using MIC: 64 mg/L, further the antimycobacterial activity was synergistically significant in combination with rifampicin [184]. Similarly, the isoflavone biochanin A exhibited significant efflux pump inhibiting activity against M. smegmatis that has evoked much attention as promising novel targets in antimycobacterial treatment [144].
Similarly, the function of efflux pumps in clarithromycin resistance with nine clinical isolates of M. abscessus subsp. abscessus or bolletii complex was studied. Based on the findings, the team has highlighted the requirement for additional investigation on M. abscessus efflux response to implement more efficient alternative antimicrobial beneficial regimens and direction in the improvement of novel drugs against mycobacterium [77]. In search of efflux pump inhibitors, flavonoids are a promising therapy for potent antimycobacterial activity and antibiotic resistance modulating activities (Figure 3).

Inhibition of Bacterial DNA Synthesis
Flavonoids are well-known topoisomerases inhibitors, contributes to antimycobacterial activity. DNA topoisomerase is a key enzyme for DNA replication that contribute to a central target for antimycobacterial agents [78]. Earlier, in silico analysis study has confirmed that quercetin is a significant DNA topoisomerase inhibitor at B subunit of the enzyme and prevents the growth of M. smegmatis [188]. This statement was further established using different DNA topoisomerase subunits that also showed quercetin binding to the B subunit of topoisomerase and parallel obstruction of ATP binding pocket by the development of H-bonds in the amino acid residues of DNA topoisomerase [78]. Previously, several molecular docking studies suggested that quercetin inhibits DNA topoisomerase and DNA supercoiling, which competitively interacts with the ATP binding site in the B subunit of DNA topoisomerase [78,189,190]. Finally, quercetin binds with DNA that alleviates the DNA topoisomerase complex leads to the breakdown of bacterial DNA [189]. The binding of flavonoids with DNA topoisomerase usually favored by the active groups positioned in the flavonoids viz., 4-carbonyl, 3-hydroxyl, 5-hydroxyl and 7-hydroxyl groups [78,189].

Synergistic Action of Flavonoids with Antimycobacterial Agents
This synergistic effect of flavonoids with conventional agents is often effective and beneficial for both the proportion and degree of bacterial destructions and microbial resistance modulating activities [191]. The available conventional agents have a spectrum of underlying modes of action, and the combination of two or more agents can contribute diverse targets, ensuing multi-targeting. The implementation of the multi-targeting policy usually eases drug resistance [192]. These synergistic approaches largely evade toxicity and intolerance of the drug [79]. Previously, various in vitro investigations have been studied and reduce the minimum inhibitory concentration of bioactive compounds with conventional antimycobacterial agents (Table 3) [123][124][125]144,153,181,193,194].
Several studies have demonstrated that the bactericidal antibiotics such as β-lactams, aminoglycosides, and fluoroquinolones induced oxidative stress, regardless of their specific targets, and involved in the ROS-antibiotic bacteria-killing [195,196]. Conversely, other reports failed to indicate the connection between ROS and antibiotic-mediated killing [197]. These varying data may have resulted from the generation of ROS, which is produced through the hyperactivation of normal cell metabolism, as well as the related difficulty or even the impossibility to completely separate the effects of reduced levels of ROS and ROS production as a consequence of the action of antibiotics [195][196][197]. Flavonoids are synergistic potentiators with conventional agents in improving the antibiotic efficiency against NTM [194]. Flavonoids generally protect the cells from the harmful effects of ROS generation [198,199]. Markedly, Brynildsen et al. [200] suggested that to enhance the antibiotic efficiency not by damaging the bacterial ROS defense systems by flavonoids, but by increasing the endogenous ROS generation in the host, which could negate its capacity to manage with oxidative stress from the available antibiotics. Bactericidal antibiotics such as quinolones, β-lactams and aminoglycosides often induced Fenton reaction resulting in the production of OH• radical [201]. These OH• radicals lead to bactericidal antibiotic-mediated cell loss. Flavonoids play as iron-chelating agents and quenching the hydroxyl radical that attenuate killing by bactericidal drugs [201]. Additionally, the practice of aminoglycoside antibiotics (AGs) such as amikacin, gentamycin, spectinomycin, neomycin, streptomycin and tobramycin, which is driven through the proton motive force and abolished as soon as ROS levels are augmented [202,203]. Flavonoids are iron chelators that protect against AGs by blocking the intake of AGs through the damage of Fe-S cluster synthesis ensuring the impendence of the proton motive force [202]. Co-administration of inhibitory concentrations of resveratrol increased the activity of aminoglycosides, including gentamicin, kanamycin, neomycin, streptomycin and tobramycin, up to 32-fold against various Gram-positive pathogens. Eventually, resveratrol increases the efficacy of aminoglycosides appears to be unrelated to membrane hyperpolarization and disruption of membrane integrity, which have been related with increased aminoglycoside susceptibility [204].
The most common mechanism of AGs resistance is a chemical modification by bacterial aminoglycoside-modifying enzymes: phosphotransferases, acetyltransferases and nucleotidyltransferase [205]. Flavonoids are documented as aminoglycoside-modifying enzyme inhibitors. quercetin and apigenin have recommended as phosphotransferases inhibitor, which occupies the ATP binding site and interacts with the enzyme through a series of hydrogen bonds [206]. Therefore, flavonoids play as chelators that could be employed as potential inhibitors of aminoglycoside-modifying enzymes. However, such a flavonoid application still requires a prospect investigation. To date, many flavonoids were characterized by the antibacterial activities against human pathogens, which play in different mechanisms than those of conventional drugs, and thus could be of significance in the enhancement of antimycobacterial therapy [85]. Important virulence factors, such as bacterial hyaluronidases (produced by both Gram-positive and Gram-negative bacteria), directly interact with host tissues or mask the bacterial surface from host s defense mechanisms. In the bacterial pathogenesis, hyaluronidase-mediated degradation of hyaluronan increases the permeability of connective tissues and decreases the viscosity of body fluids [207]. Notably, flavonols, such as myricetin and quercetin have been identified as hyaluronic acid lyase (Hyal B) inhibitors. Plants have a limitless ability to Antibiotics 2020, 9, 450 13 of 34 synthesize aromatic substances, most of which are secondary metabolites. The inhibitory effect of the flavonoids increased with the number of hydroxyl groups present in the flavonoid structure [208].     M. chelonae 11.81 [193] Flavonoids Pelargonium reniforme (Geraniaceae)

Future Directions and Remarks
Studies on synergistic relations between natural products and synthetic drugs are very limited. Hence, urgent studies are required for a better understanding of synergistic behavior and the underlying mechanisms of action of flavonoids-drug combinations against NTM. This attempt may accelerate the discovery of novel drugs that are effective against antibiotic resistance targets of NTM and reduce the global occurrence of severe chronic pulmonary and extrapulmonary infections. To date, the favorite strategy for the treatment of multidrug resistance is to simultaneously inhibit multiple targets such as the inhibition of DNA gyrase activity and cell wall synthesis. However, in future studies on the synergistic relations between flavonoids and synthetic drugs would be greater effects than treating conventional drugs alone. There are various motives to investigate a novel class of antimicrobial drugs and the flavonoids represent a novel set of opportunities. Based on the chemical profile of the flavonoids, the outcomes can be analyzed to show the target sites of novel drugs against extensively multidrug-resistant NTM. These new classes of drugs may be effective on NTM, which brings about better understandings of flavonoids and structure-activity relationships. Therefore, these plant-derived novel compounds could be useful to cope with the resistance problem. Although these efforts are implemented earlier in the pharma industries and being conducted on NTM drug development projects, the current progress is still inadequate to overwhelm the subject of multidrug resistance. The primary reason for ineffectiveness is based on bacterial resistance and the demands which are not gratified in terms of the requirements for the combinations of novel agents. Novel targets among the bacterial resistance mechanisms and investigation on novel molecules are vital for developing innovative anti-NTM drugs. Further, in vitro, in vivo and clinical, and pharmacokinetics studies and chemical relationship are mandatory to analyze the synergistic relations between flavonoids and synthetic drugs, which may provide the state-of-the-art and translate bench to bed treatments.

Conclusions
Recently, NTM have developed into significant bacterial pathogens for both animals and humans. In particular, the concern is the high level of antimicrobial resistance displayed by these organisms, which complicates treatment and possible effective outcomes. The state of the existing antimycobacterial agents and their hitches is relatively serious. In developing nations, the incidence rate and diagnosis of NTM have often not been noticed as a deficiency of laboratory settings and mycobacteria identification. The escalating rate of pathogenic NTM in developing nations is significantly greater in HIV/AIDS patients, which leads to high levels of morbidity and mortality globally. Furthermore, there are restrictions evident by antimycobacterial drugs: the lower bactericidal ability, multidrug usage, high resistance and toxicity and organ damage. Hence, it is imperative to find new drugs as alternative therapies in which flavonoids are promising to be safe for usage, endowed with abundant pharmacological roles that are potentially active against NTM. Several flavonoids have been used in connotation with their antimycobacterial activities and can be potential and cost-effective. They have possible antimycobacterial effects at minor quantities by themselves or in synergistic combinations. A cocktail of flavonoids used with existing antimycobacterial agents is a proposal of a novel strategy to lessen side effects. They often prevent bacterial growth in several underlying mechanisms by increasing the disturbance of the plasma membrane, inhibiting cell wall development, efflux-mediated pumping system and DNA synthesis. These flavonoids are potential in synergetic combination treatment with available conservative pharmacological agents, which can be very suitable and supportive in the search for novel drug treatment against mycobacterial pathogens.