Visualization of pill in Extensively and totally drug resistant Mycobacterium Tuberculosis Using Light Microscopy

With worldwide increases of rifampicin resistance, the World Health Organization (WHO) comes with a new definition of pre-extensively drug-resistant tuberculosis (XDR-TB) and XDR-TB. New definitions for pre-XDR and XDR-TB helped to define more precisely groups of TB patients who require complex treatment regimens.[1] The definition of pre-XDR-TB applied to TB caused by Mycobacterium tuberculosis strains that fulfill the definition of multidrug-resistant (MDR) and rifampicin-resistant TB (RR-TB) and which are also resistant to any fluoroquinolone.[2–4] The updated definition of XDR-TB applied to TB caused by M. tuberculosis strains that fulfill the definition of MDR/RR-TB and which are also resistant to any fluoroquinolone and at least one additional Group A drug (Group A drugs are the most potent group of drugs in the ranking of second-line medicines for the treatment of drug-resistant forms of TB using longer treatment regimens and comprise levofloxacin, moxifloxacin, bedaquiline [BDQ], and linezolid). BDQ, a diarylquinoline antimycobacterial drug that inhibits adenosine triphosphate synthase of M. tuberculosis,[5,6] is indicated as a part of combination therapy in adult and pediatric patients (≥5 years, weighing ≥15 kg) with pulmonary MDR-TB. BDQ has become central to MDR-TB therapy and is also a part of the shorter and oral regimen. Based on the result of a systematic review and a meta-analysis, the WHO revised the guidelines for the management of patients with MDR-TB by prioritizing the fluoroquinolones, BDQ, and linezolid (all classified as Group A agents) in 2019.[7] In particular, the recently introduced novel anti-TB drug BDQ raised great expectations with the potential to reduce death rates, shorten MDR-TB treatment durations, and decrease treatment failure rates, and thus has been administered to MDR-TB patients in >50 countries up to date.[8–11] However, a number of resistance-associated variants may decrease susceptibility to BDQ.[12–18] In this regard, Dr. Ismail et al. have published their ground-breaking research on the epidemiological and genetic aspects of BDQ resistance and the clinical outcomes in patients with RR-TB.[19] Ismail et al. concluded that BDQ resistance was associated with poorer treatment outcomes.[19–24] Within the past 15 years, Velayati et al. tried to explain that up to 10% of drug-resistant (DR) isolates have morphological changes at the cellular level, including significant thickness at the cell wall, shape variability, and different types of cell division.[25–30] In addition, these DR-isolates showed various protrusion appendages, which later on named as pili.[25–30] The name selected as totally DR (TDR) TB because it seems that these bacilli are adapted at the cellular and genomic level in such a way that the available antibiotics are not effective on them. After a decade, today, we have enough reports based on clinical and laboratory investigations that approved resistant even in Group A drugs proposed by the WHO. From 2005, scientists documented a large variety of adherence-mediating components and strategies in relation to M. tuberculosis and host cells. Alteri et al. documented two types of pili (Curli and IV types of pili); other in vitro studies have demonstrated the binding affinity of pili and laminin and suggest pili have adhesive properties that may be necessary for M. tuberculosis to colonize within the host.[2,31–33] Furthermore, the genomic analysis showed the Rv3312A (mtp) gene encodes the pilin subunits of pili in M. tuberculosis.[2,34] In a study by Naidoo et al., it was discovered through amplicon sequencing that almost all clinical isolates of M. tuberculosis (86 out of 88) contained a conserved sequence for the mtp gene.[35] In addition, Hosseini et al. found that the mtp and flp genes were completely conserved in clinical isolates (n = 36).[36] Recently, we had reported seven different Types of pili, of which two are seen only in XDR and TDR-TB bacilli. Here, three methods for the direct visualization of pili for XDR and TDR-TB bacilli using a light microscope are described [Figures 1-3]. Samples were subjected to electron transmission and atomic force microscopy, and the presence of pili was confirmed in the same isolates but with higher resolution. Although we cannot identify types of pili by light microscopy, its presence was confirmed in XDR and TDR-TB bacilli even under light microcopy. Not all the XDR and TDR-TB bacilli are producing pili, but this low percent (up to 10%) should not be ignored anymore. These bacilli remain alive and become active later on, when there is no treatment. In fact, it is also possible that these adaptations occur under cocktail of antibiotics, which need further investigation. However since among these adaptations, pili acts as a “double-edged Sword,” we can use them to destroy bacilli. Pili can be consider a promising approach to address drug-resistant TB bacilli, because they can very well act as an antibiotic carrier.Figure 1: Modified periodic acid staining; the dried smears were kept under 600 C overnight. The smears were first oxidized using 10% periodic acid solution for 24 h. The smears were washed using distilled water and then stained with carbol fuchsin and decolorized by 70% alcohol and rinsed with water in 5 s. Flood the slide with counter stain malachite-green for 20 s and then rinse the smear (Yellow arrow)Figure 2: Modified silver staining in for extensively drug resistant and totally drug resistant-tuberculosis isolates. The slide smear prepared from live suspension of Mycobacterium tuberculosis under biological safety cabinet (Type III) (White arrow)Preparation The A solution prepared by mixing 25 mL of a saturated aqueous solution of aluminum potassium sulfate, 50 mL of a 10% tannic acid solution, and 5 mL of a 5% ferric chloride solution. The resulting mixture turns black and is stored in the dark at 5°C. Then solution B (solution B is made by slowly adding concentrated ammonium hydroxide to 90 mL of a 5% silver nitrate solution until the brown precipitate formed just re-dissolves. More 5% silver nitrate is then added dropwise to the solution until a faint cloudiness persists; this takes from 2 to 20 mL. The stain is stored in the dark at 50 C). The smears of confirmed XDR and TDR-TB bacilli were prewared in 400 C and then stained using with solution A for 15 min. Place in distilled water and wash for three times. The solution B was added and after drying was observed under light microscopy.[37]Figure 3: The negative stain of extensively drug resistant and totally drug resistant-tuberculosis isolates by modified Burri’s technique. The dried bacterial slide (alcoholic fixation) over steamed water bath under biological safety cabinet (III) at 600 C overnight. The smears stained with malachite-green instead of Indian-ink (Mycobacterium tuberculosis isolates remained unstained against dark green background; in some experiments, the nigrosine or Congo red were also be used). Slides examine under light microscopy using oil immersion (White arrow)