A heterocyclic thiazole derivate, benzothiazinone BTZ043 was reported to be one of the most potent inhibitors of M. tuberculosis yet described and displayed bactericidal activity both in vitro and in ex vivo models of TB [28-30]. Previously, similar heterocyclic phenothiazine derivates such as thioridazine and toluidine blue were investigated and reported to be effective for treatment of pulmonary tuberculosis; however, we could not find any studies investigating the inhibitory effects of MB (another phenothiazine derivate) on M. tuberculosis complex clinical isolates [31, 32]. Another similar phenothiazine compound chlorpromazine was shown to be effective on M. tuberculosis complex isolates; however, it was later understood that the inhibitory concentration of chlorpromazine was many times higher than the safely achievable dose in the patient [33, 34]. Based upon the data collected for thioridazine, after novel investigations, it was reported that there could be at least four mechanisms by which this phenothiazine compound acts on Mycobacterium tuberculosis. The effect may occur by increasing the killing activity of human macrophages, by inhibiting over-expressed efflux pump and oxygen consumption of the pathogen, and by reaching higher concentrations of thioridazine than MIC or MBC of the bacterium [32]. Since methylene blue is a similar heterocyclic phenothiazine derivate, several studies investigating the antimicrobial effect of this compound against Mycobacteria other than tuberculosis have been performed by scientists. Shim et al. reported the inhibitory effect of methylene blue-mediated photodynamic therapy on Mycobacterium smegmatis in 2016 [35]. One year later, Pal et al. conducted research on Candida albicans and Mycobacterium smegmatis and stated that MB alone inhibited the growth of Mycobacterium smegmatis at 15.62 µg ml-1 in a bacteriostatic manner similar to its fungistatic characteristic (Myco-bacteria means fungus-like bacteria in Greek) [36]. They also reported that MB was leading to impaired cell surface phenotypes, altered colony morphologies, and DNA damage in Mycobacteria. They suggested performing further investigations on Mycobacterium tuberculosis, since this pathogen contains unique cell envelope components with complex lipids providing pathogenicity [36]. However, in the literature, there are no reports investigating the effects of MB on M. tuberculosis complex clinical isolates. Our study might be the first in vitro non-photodynamic research investigating the antimicrobial effect of MB compound on Mycobacterium tuberculosis complex clinical isolates and showing the potential of MB alone for treatment of tuberculosis infection.
In our study, we investigated four different critical concentrations of MB because MB can be used as a drug in different forms for different indications (topical, oral or intravenous) and varied concentrations of MB can be found in several drug types. In a previous study, Walter-Sac et al. reported that MB plasma concentration reached 2 µg ml-1 after oral intake of 500 mg MB and remained above this level for more than five hours in healthy individuals. They also showed that MB plasma concentration reached 0.2 µg ml-1 after intravenous injection of 50 mg MB and remained above this level for more than seven hours [37]. In two other studies investigating the bioavailability of MB tablets, the areas under curve 0-t [AUC (0-t)] were found to be about 25 and 33 µg ml-1 x hour after intake of 200 mg MB [38,39]. In another study investigating the pharmacokinetics and organ distributions of intravenous and oral methylene blue, it was shown that much higher concentrations of MB were reached in some organs than in blood. In the animal models, twenty-fold higher concentrations than in blood were found in the brain following administration of intravenous MB [40].
When we compare these studies with the findings of our research, the critical concentration of MB at 2 µg ml-1 which inhibited 35% of our study isolates could have potential as an alternative anti-tuberculosis drug. When we accept 20 µg ml-1 as a target critical MB concentration, 53% of the isolates could be inhibited by administration of MB. If we take three isolates (isolate no: 7, 14, and 16) with borderline inhibition (103, 103, and 102 GU) into consideration, the rate of susceptibility rises to 70% at the 20 µg ml-1 critical concentration. Since the proportion method accepts the resistance breakpoint as 1% (equal to 400 GU) of the control population, these borderline values may also be accepted as susceptible [41, 42]. Secondly, the ability of MB to cross the blood-brain barrier and diffuse inside the brain at higher concentrations than blood could make MB attractive as a potential therapeutic agent for central nervous system infection such as TB meningitis and TB encephalitis [40].
In the literature, there are some clinical studies investigating the effects of the topical form of MB against dermatological diseases such as fungal infections, acne vulgaris, psoriasis, and lichen planus [20, 21, 25, 26]. In these studies, the cream, hydro-gel and solution forms of MB were prepared at the concentrations of 500, 1000 and 200,000 µg ml-1 for photodynamic therapy (not for tuberculosis) and they were found to be effective for treating patients, without any significant side effects. Comparing the MB doses of these topical preparations with our results, 1000 µg ml-1 concentration of MB inhibited 82% of the Mycobacterium tuberculosis complex isolates in our study. If the two borderline resistant clinical isolates (<400 GU and >100 GU) are accepted as susceptible, the rate of inhibition rises to 94% at 1000 µg ml-1 concentration. We know that Mycobacterium tuberculosis complex may also cause mucosal and cutaneous infections. So, topical drugs containing much higher MB concentrations (e.g. at 1000 µg ml-1) may be used to treat local cutaneous infections due to Mycobacterium tuberculosis complex [43]. In our study only one (4%) MDR isolate (No.:5) was found resistant to MB at all concentrations and second MDR isolate (No.:3) was resistant to MB at 0.2, 2, and 20 µg ml-1 critical concentrations.
The agar proportion method is the accepted “gold” standard for first-line antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates. But this method is labour intensive and requires a calculation by counting colonies. In this method, if there is ≥ 1% growth on the drug-containing medium as compared to the drug-free medium, the organism is considered resistant [41, 42]. The MGIT 960 AMDS uses a similar broth proportion method and the system is currently recommended by the WHO as the gold standard for second-line drug susceptibility testing [44]. Since the aim of this study is to investigate MB as a novel and alternative (second-line) anti-tuberculosis drug, MGIT 960 AMDS was included in the study. In a multicentre study, levofloxacin, amikacin, capreomycin, and ethionamide drugs were investigated and the overall agreement between the agar proportion method and the MGIT 960 AMDS was found to be 96.4% [45]. Thus, the AMDS system is not only practical but also has a good agreement with the proportion method.