Tuberculosis (TB) along with acquired immune deficiency syndrome (AIDS) and malaria rank among the top three fatal infectious diseases which pose threat to global public health, especially in middle and low income countries. TB caused by Mycobacterium tuberculosis (Mtb) is an airborne infectious disease and one third of the world’s population gets infected with TB leading to nearly 1.6 million deaths annually. TB drugs are administered in different combinations of four first line drugs (rifampicin, isoniazid, pyrazinamide and ethambutol) which form the core of treatment regimens in the initial treatment phase of 6 to 9 months. Several reasons account for the failure of TB therapy such as i) late diagnosis, ii) lack of timely and proper administration of effective drugs, iii) lower availability of less toxic, inexpensive and effective drugs, iv) long treatment duration, v) non‐adherence to drug regimen and vi) evolution of drug resistant TB strains. Drug resistant TB poses a significant challenge to TB therapy and control programs. In the background of worldwide emergence of 558,000 new TB cases with resistance to rifampicin in the year 2017 and of them, 82 % becoming MDR‐TB, it is essential to continuously update the knowledge on the mechanisms and molecular basis for evolution of Mtb drug resistance. This narrative and traditional review summarizes the progress on the anti‐tubercular agents, their mode of action and drug resistance mechanisms in Mtb. The aim of this review is to provide recent updates on drug resistance mechanisms, newly developed/ repurposed anti‐TB agents in pipeline and international recommendations to manage MDR‐TB. It is based on recent literature and WHO guidelines and aims to facilitate better understanding of drug resistance for effective TB therapy and clinical management.

Silver nanoparticles induced alterations in multiple cellular targets, which are critical for drug susceptibilities and pathogenicity in fungal pathogen (Candida albicans) Venkatraman Srinivasan Radhakrishnan,1,2 Mohana Krishna Reddy Mudiam,3 Manish Kumar,1,2 Surya Prakash Dwivedi,4 Surinder Pal Singh,5 Tulika Prasad1,2 1Advanced Instrumentation Research and Facility (AIRF), Jawaharlal Nehru University (JNU), New Delhi, Delhi, India; 2Special Centre for Nano Sciences (SCNS), Jawaharlal Nehru University (JNU), New Delhi, Delhi, India; 3Analytical Chemistry Lab, Council for Scientific and Industrial Research (CSIR)-Indian Institute of Toxicology Research (IITR), Lucknow, Uttar Pradesh, India; 4School of Biotechnology, IFTM University, Moradabad, Uttar Pradesh, India; 5CSIR-National Physical Laboratory (NPL), New Delhi, Delhi, India Purpose: A significant increase in the incidence of fungal infections and drug resistance has been observed in the past decades due to limited availability of broad-spectrum antifungal drugs. Nanomedicines have shown significant antimicrobial potential against various drug-resistant microbes. Silver nanoparticles (AgNps) are known for their antimicrobial properties and lower host toxicity; however, for clinical applications, evaluation of their impact at cellular and molecular levels is essential. The present study aims to understand the cellular and molecular mechanisms of AgNp-induced toxicity in a common fungal pathogen, Candida albicans. Methods: AgNps were synthesized by chemical reduction method and characterized using UV–visible spectroscopy, X-ray powder diffraction, transmission electron microscopy, scanning electron microscopy–energy dispersive X-ray spectroscopy, energy dispersive X-ray fluorescence, and zeta potential. The anti-Candida activity of AgNps was assessed by broth microdilution and spot assays. Effects of AgNps on cellular and molecular targets were assessed by monitoring the intracellular reactive oxygen species (ROS) production in the absence and presence of natural antioxidant, changes in surface morphology, cellular ultrastructure, membrane microenvironment, membrane fluidity, membrane ergosterol, and fatty acids. Results: Spherical AgNps (10–30 nm) showed minimum inhibitory concentration (minimum concentration required to inhibit the growth of 90% of organisms) at 40 µg/mL. Our results demonstrated that AgNps induced dose-dependent intracellular ROS which exerted antifungal effects; however, even scavenging ROS by antioxidant could not offer protection from AgNp mediated killing. Treatment with AgNps altered surface morphology, cellular ultrastructure, membrane microenvironment, membrane fluidity, ergosterol content, and fatty acid composition, especially oleic acid. Conclusion: To summarize, AgNps affected multiple cellular targets crucial for drug resistance and pathogenicity in the fungal cells. The study revealed new cellular targets of AgNps which include fatty acids like oleic acid, vital for hyphal morphogenesis (a pathogenic trait of Candida). Yeast to hypha transition being pivotal for virulence and biofilm formation, targeting virulence might emerge as a new paradigm for developing nano silver-based therapy for clinical applications in fungal therapeutics. Keywords: AgNps, nanomedicine, drug resistance, membrane fluidity, antifungals, reactive oxygen species, ROS

Here, we describe the synthesis of 2–3 nm, hydrophilic, blue fluorescence-emitting carbon dots (C-Dots, made using a DNA precursor) by the hydrothermal route from the gelling concentration of 2% (w/v) DNA. These dots exhibited highly efficient internalization in pathogenic fungal cells, negligible cytotoxicity, good PL stability, and high biocompatibility, thus demonstrating their potential as nanotrackers in microbial studies. Bioimaging was performed using Candida albicans as the representative for microbial pathogens. The novelty of these dots is that they formed fluorescent nanocomposite hydrogels with the same DNA much below the gelation concentration (1% w/v) and the tunable gels possessed strength between 20 and 80 Pa with the corresponding gelation temperature T_gel between 40 to 50 °C. The network density and gelation free energy data supported the superior crosslinking ability of these dots. The as-prepared hydrogels can replace the existing toxic quantum dot-based hydrogels for drug delivery. We also demonstrated the use of a DNA hydrogel-fabricated working electrode (DNA-C-Dot/ITO electrode) for the biosensing of dopamine. Our electrochemical biosensor had a detection limit of 5 × 10⁻³ mM for dopamine. These multifunctional, fluorescent C-Dots and hydrogel after suitable conjugation or loading with molecules and drugs hold promising potential for further exploitation in bioimaging, targeted drug delivery, wound healing, and biosensing applications.

Herein, we report a facile microwave-assisted synthesis of cadmium-free water-soluble silver indium sulfide (AgInS₂ or AIS) and AgInS@ZnS (or AIS@ZnS) core-shell quantum dots (QDs) using glutathione (GSH) as stabilizer. The core and core-shell nanocrystals exhibit tunable bandgap ranging of 2.3–3.1 and 2.4–3.5 eV, mean particle size of 2.5 and 3.25 nm, quantum yield of 26% and 49%, and fluorescence lifetimes of 326 and 438 ns, respectively. The core-shell QDs exhibit color-tunable emission in the visible region (500 to 600 nm), where the tunability was achieved by varying the molar ratio of Ag:In in the precursors. In vitro evaluation of antifungal activity of these water/ buffer stable QDs against the fungal pathogen, Candida albicans demonstrated that these were not toxic to the fungal cells upto a concentration of 100 µg/ml for 16 hours of incubation. Confocal imaging and spectrofluorometric studies showed enhanced fluorescence inside the microbial cells suggesting that AIS@ZnS particles had the capability to easily penetrate the cells. The increased generation of reactive oxygen species was evaluated for the core-shell QDs (photosensitizers) by using 9, 10-anthracenediyl-bis(methylene)dimalonic acid (ABMDMA) as singlet oxygen (¹O₂) scavenger molecule. These QDs have the potential for use as high contrast cell imaging, photodynamic and antifungal agents.

In vitro studies on oxidative stress-independent, Ag nanoparticles-induced cell toxicity of Candida albicans, an opportunistic pathogen Venkatraman Srinivasan Radhakrishnan,1 Surya Prakash Dwivedi,2 Mohammed Haris Siddiqui,3 Tulika Prasad1 1Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi, 2School of Biotechnology, IFTM University, Moradabad, 3Department of Bioengineering, Integral University, Lucknow, Uttar Pradesh, India Silver nanoparticles (AgNps) have attracted maximal attention among all metal nanoparticles, and the study of their biological properties has gained impetus for further medical adoption. This study evaluated the cellular and molecular mechanisms associated with the action of AgNps against an opportunistic pathogen, Candida albicans. Spherical, stable AgNp (average size 21.6 nm) prepared by a chemical reduction method showed minimum inhibitory concentration (required to inhibit the growth of 90% of organisms) at 40 µg/mL. AgNps have been reported to induce oxidative stress-mediated programmed cell death through the accumulation of intracellular reactive oxygen species (ROS). However, this study demonstrated that intracellular levels of AgNp-induced ROS could be reversed by using antioxidant ascorbic acid, but the sensitivity of AgNp-treated Candida cells could not be completely reversed. Moreover, in addition to the generation of ROS, the AgNps were found to affect other cellular targets resulting in altered membrane fluidity, membrane microenvironment, ergosterol content, cellular morphology, and ultrastructure. Thus, the generation of ROS does not seem to be the sole major cause of AgNp-mediated cell toxicity in Candida. Rather, the multitargeted action of AgNps, generation of ROS, alterations in ergosterol content, and membrane fluidity together seem to have potentiated anti-Candida action. Thus, this “nano-based drug therapy” is likely to favor broad-spectrum activity, multiple cellular targets, and minimum host toxicity. AgNps, therefore, appear to have the potential to address the challenges in multidrug resistance and fungal therapeutics. Keywords: antifungal, nanomedicine, ROS, drug resistance

Purpose: Immuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen. Methods: GNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR.
Results: Transmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 10⁵-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6. Conclusion: Unlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis.