Bionanocomposites comprising mesoporous metal organic framework (ZIF-8) phytofabricated with Allium sativum as alternative nanomaterials to combat antimicrobial drug resistance.

Green nanotechnology is one of the most expanding fields that provides numerous novel nanoparticle drug formulations with enhanced bioactivity performance. This study aims to synthesize mesoporous metal organic framework (ZIF-8) phytofabricated with the herb Allium sativum (As) as an indicator system for its antibacterial and antifungal impact. The successful synthesis of ZIF-8 as nanocomposite was characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and scanning coupled with energy-dispersive X-ray spectroscopy and transmission electron microscopy (SEM-EDX and TEM) that showed the textural retainment of ZIF-8 on composite formation with A. sativum. The nanocomposite, A. sativum extract, and ZIF-8 were subjected to antimicrobial assays against Shigella flexneri, Candida albicans, and Candida parapsilosis. The comparative results indicated the potential action of nanocomposite against the bacteria and both the Candida sps; however, the antifungal action against the Candida sps was more effective than the bacterium S. flexneri. The findings suggest that plants, being an important component of ecosystems, could be further explored for the novel drug discovery using green nanotechnology to enhance their impact on the drug-resistant pathogens.

Abrogation of efflux pump activity, biofilm formation, and immune escape by candidacidal geraniol in emerging superbug, Candida auris.

During the last decade, Candida auris emerged as a threatening human fungal pathogen that notably caused outbreaks around the globe with high mortality. Considering C. auris species as newly discovered fungi, the evolutionary features remain elusive. The antifungal resistance which is a norm in C. auris underlines the need for innovative therapeutic options. ATP Binding Cassette (ABC) superfamily efflux pumps overexpression and biofilms are known to be major contributors to multidrug resistance (MDR) in C. auris. Therefore, herein, we investigated the antifungal potential of geraniol (Ger) as a promising natural compound in the fight against MDR C. auris. Our experiments proved that Ger was fungicidal in nature and impaired rhodamine 6G (R6G) efflux, confirming the specific effect on ABC transporters. Kinetic studies unravelled the competitive mode of inhibition by Ger for R6G efflux since the apparent Km increased with no change in Vmax value. Mechanistic insights also revealed that Ger depleted ergosterol content in C. auris. Furthermore, Ger led to inhibition in biofilm formation as evident from crystal violet staining, biofilm metabolic and biomass measurements. Additionally, enhanced survival of Caenorhabditis elegans model after C. auris infection demonstrated the in vivo efficacy of Ger. Lastly, the in vivo efficacy was confirmed from a THP-1 cell line model which depicted enhanced macrophage-mediated killing in the presence of Ger. Modulation of C. auris efflux pump activity and biofilm formation by Ger represents a promising approach to combat MDR. Together, this study demonstrated the potential therapeutic insights of Ger as a promising addition to the antifungal armamentarium required to treat emerging and resistant C. auris.

Unique roles of aminophospholipid translocase Drs2p in governing efflux pump activity, ergosterol level, virulence traits, and host-pathogen interaction in Candida albicans.

Infections caused by Candida albicans are rising due to increment in drug resistance and a limited arsenal of conventional antifungal drugs. Thus, elucidating the novel antifungal targets still represent an alternative that could overcome the problem of multidrug resistance (MDR). In this study, we have uncovered the distinctive effect of aminophospholipid translocase (Drs2p) deletion on major MDR mechanisms of C. albicans. We determined that efflux activity was diminished in Δdrs2 mutant as revealed by extracellular rhodamine 6G (R6G) efflux and flow cytometry. Moreover, we further unveiled that Δdrs2 mutant displayed decreased ergosterol content and increased membrane fluidity. Furthermore, Drs2p deletion affects the virulence attributes and led to inhibited hyphal growth and reduced biofilm formation. Additionally, THP-1 cell lines' mediated host-pathogen interaction studies revealed that Δdrs2 mutant displayed enhanced phagocytosis and altered cytokine production leading to increased IL-6 and decreased IL-10 production. Taken together, the present study demonstrates the relevance of Drs2p in C. albicans and consequently disrupting pathways known for mediating drug resistance and immune recognition. Comprehensive studies are further required to authenticate Drs2p as a novel antifungal drug target.

Mass spectrometry-based untargeted lipidomics reveals new compositional insights into membrane dynamics of Candida albicans under magnesium deprivation.

AIMS: There is growing appreciation in adopting new approaches to disrupt multidrug resistance in human fungal pathogen, Candida albicans. The plasma membrane of C. albicans comprises potential lipid moieties that contribute towards the survival of pathogen and could be utilized as antifungal targets. Considering promising applications of developments in mass spectrometry (MS)-based lipidomics technology, the aim of the study was to analyse lipidome profile and expose lipid-dependent changes in response to Mg deprivation. METHODS AND RESULTS: We found that both phosphatidylcholine (PC) and lysophosphatidylcholine (LysoPC) were decreased. Increased flip (inward translocation) in the fluorophore labelled NBD-PC was ascribed to enhanced PC-specific flippase activity. Furthermore, a decrease in phosphatidylethanolamine (PE) leading to altered membrane fluidity and loss of cellular material was prominent. Additionally, we observed decreased phosphatidylglycerol (PG) and phosphatidylinositol (PI) leading to genotoxic stress. Besides, we could detect enhanced levels of phosphatidylserine (PS), diacylglycerol (DAG) and triacylglycerides (TAG). The altered gene expressions of lipid biosynthetic pathway by RT-PCR correlated with the lipidome profile. Lastly, we explored abrogated ionic (Na+ and K+ ) transport across the plasma membrane. CONCLUSIONS: We propose that C. albicans exposed to Mg deprivation could reorganize plasma membrane (lipid species, membrane fluidity and ionic transport), and possibly redirected carbon flux to store energy in TAGs as an adaptive stress response. This work unravels several vulnerable targets governing lipid metabolism in C. albicans and pave way for better antifungal strategies. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that magnesium availability is important when one considers dissecting drug resistance mechanisms in Candida albicans. Through mass spectrometry (MS)-based lipidomics technology, the study analyses lipidome profile and exposes lipid-dependent changes that are vulnerable to magnesium availability and presents an opportunity to employ this new information in improving treatment strategies.

CLEVER assay: A visual and rapid RNA extraction-free detection of SARS-CoV-2 based on CRISPR-Cas integrated RT-LAMP technology.

AIM: The current scenario of COVID-19 pandemic has presented an almost insurmountable challenge even for the most sophisticated hospitals equipped with modern biomedical technology. There is an urgency to develop simple, fast and highly accurate methods for the rapid identification and isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. To address the ongoing challenge, the present study offers a CLEVER assay (CRISPR-Cas integrated RT-LAMP Easy, Visual and Extraction-free RNA) which will allow RNA extraction-free method to visually diagnose COVID-19. RNA extraction is a major hurdle in preventing rapid and large-scale screening of samples particularly in low-resource regions because of the logistics and costs involved. METHOD AND RESULT: Herein, the visual SARS-CoV-2 detection method consists of RNA extraction-free method directly utilizing the patient's nasopharyngeal and oropharyngeal samples for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12-based system using different guide RNAs of N, E and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dip sticks with unaided eye (~100 min). Overall, the clinical sensitivity and specificity of the CLEVER assay were 89.6% and 100%, respectively. CONCLUSION: Together, our CLEVER assay offers a point-of-care tool with no equipment dependency and minimum technical expertise requirement for COVID-19 diagnosis. SIGNIFICANCE AND IMPACT OF THE STUDY: To address the challenges associated with COVID-19 diagnosis, we need a faster, direct and more versatile detection method for an efficient epidemiological management of the COVID-19 outbreak. The present study involves developing a method for detection of SARS-CoV-2 in human body without RNA isolation step that can visually be detected with unaided eye. Taken together, our assay offers to overcome one major defect of the prior art, that is, RNA extraction step, which could limit the deployment of the previous assays in a testing site having limited lab infrastructure.

Insights into the modulatory effect of magnesium on efflux mechanisms of Candida albicans reveal inhibition of ATP binding cassette multidrug transporters and dysfunctional mitochondria.

Candida infections pose a serious hazard to public health followed by widespread and prolonged deployment of antifungal drugs has which has led multidrug resistance (MDR) progress in prevalent human fungal pathogen, Candida albicans. Despite the fact that MDR is multifactorial phenomenon govern by several mechanisms in C. albicans, overexpression of drug efflux transporters by far remains the leading cause of MDR govern by ATP Binding Cassette (ABC) or major facilitator superfamily (MFS) transporters. Hence searching for strategies to target efflux pumps transporter still signifies a promising approach. In this study we analyzed the effect of magnesium (Mg) deprivation, on efflux pump action of C. albicans. We explored that Mg deprivation specially inhibits efflux of transporters (CaCdr1p and CaCdr2p) belonging to ABC superfamily as revealed by rhodamine 6G and Nile red accumulation. Furthermore, Mg deprivation causes mislocalization of CaCdr1p and CaCdr2p and reduced transcripts of CDR1 and CDR2 with no effect on CaMdr1p. Additionally, Mg deprivation causes depletion of ergosterol content in azole sensitive and resistant clinical matched pair of isolates Gu4/Gu5 and F2/F5 of C. albicans. Lastly, we observed that Mg deprivation impairs mitochondrial potential which could be the causal reason for abrogated efflux activity. With growing appreciation of manipulating metal homeostasis to combat MDR, inhibition of efflux activity under Mg deprivation warrants further studies to be utilized as an effective antifungal strategy.

Rec A disruption unveils cross talk between DNA repair and membrane damage, efflux pump activity, biofilm formation in Mycobacterium smegmatis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has emerged in recent decades as one of the leading causes of mortality worldwide. The burden of TB is alarmingly high, with one third affected global population as reported by WHO. Short-course treatment with an antibiotic is a powerful weapon to treat infection of susceptible MTB strain, however; MTB has developed resistance to anti-TB drugs, which is an escalating global health crisis. Thus there is urgent need to identify new drug targets. RecA is a 38 kilodalton protein required for the repair and maintenance of DNA and regulation of the SOS response. The objective of this study is to understand the effect of disruption of RecA gene (deletion mutant ΔdisA from previous study) in a surrogate model for MTB, Mycobacterium smegmatis. This study demonstrated that disruption of RecA causes enhanced susceptibility towards rifampicin and generation of ROS leading to lipid peroxidation and impaired membrane homeostasis as depicted by altered cell membrane permeability and efflux pump activity. Mass spectrometry based lipidomic analysis revealed decreased mycolic acid moieties, phosphatidylinositol mannosides (PIM), Phthiocerol dimycocerosate (DIM). Furthermore, biofilm formation was considerably reduced. Additionally, we have validated all the disrupted phenotypes by RT-PCR which showed a good correlation with the biochemical assays. Lastly, RecA mutant displayed reduced infectivity in Caenorhabditis elegans illustrating its vulnerability as antimycobacterial target. Together, present study establishes a link between DNA repair, drug efflux and biofilm formation and validates RecA as an effective drug target. Intricate studies are needed to further understand and exploit this therapeutic opportunity.

Lipidomic insights to understand membrane dynamics in response to vanillin in Mycobacterium smegmatis.

Considering the emergence of multidrug resistance (MDR) in prevalent human pathogen, Mycobacterium tuberculosis (MTB), there is parallel spurt in development of novel strategies aimed to disrupt MDR. The cell envelope of MTB comprises a wealth of lipid moieties contributing towards long-term survival of pathogen that could be exploited as efficient antitubercular target owing to advancements made in mass spectrometry-based lipidomics technology. This study aimed to utilize the lipidomics approach to unveil several lipid associated changes in response to natural antimycobacterial compound vanillin (Van) in Mycobacterium smegmatis, a surrogate for MTB. Lipidomic analyses revealed that that Van alters the composition of fatty acid (FA), glycerolipid (GL), glycerophospholipid (GP), and saccharolipids (SL). Furthermore, Van leads to potentiation of ampicillin and displayed additive effect. The differential expressions of various lipid biosynthetic pathway genes by RT-PCR corroborated with the lipidomics data. Lastly, we demonstrated enhanced survival of Mycobacterium-infected Caenorhabditis elegans model in presence of Van. Thus, lipidomics approach provided detailed insight into mechanisms of membrane disruption by Van in Mycobacterium smegmatis. Our work offers the basis of further understanding the regulation of lipid homeostasis in MTB so that better therapeutic targets could be identified to combat MDR.

Octyl gallate triggers dysfunctional mitochondria leading to ROS driven membrane damage and metabolic inflexibility along with attenuated virulence in Candida albicans.

Recently the high incidence of worldwide Candida infections has substantially increased. The growing problem about toxicity of antifungal drugs and multidrug resistance aggravates the need for the development of new effective strategies. Natural compounds in this context represent promising alternatives having potential to be exploited for improving human health. The present study was therefore designed to evaluate the antifungal effect of a naturally occurring phenolic, octyl gallate (OG), on Candida albicans and to investigate the underlying mechanisms involved. We demonstrated that OG at 25 µg/ml could effectively inhibit C. albicans. Mechanistic insights revealed that OG affects mitochondrial functioning as Candida cells exposed to OG did not grow on non-fermentable carbon sources. Dysfunctional mitochondria triggered generation of reactive oxygen species (ROS), which led to membrane damage mediated by lipid peroxidation. We explored that OG inhibited glucose-induced reduction in external pH and causes decrement in ergosterol levels by 45%. Furthermore, OG impedes the metabolic flexibility of C. albicans by inhibiting the glyoxylate enzyme isocitrate lyase, which was also confirmed by docking analysis. Additionally, OG affected virulence traits such as morphological transition and cell adherence. Furthermore, we depicted that OG not only prevented biofilm formation but eliminates the preformed biofilms. In vivo studies with Caenorhabditis elegans nematode model confirmed that OG could enhance the survival of C. elegans after infection with Candida. Toxicity assay using red blood cells showed only 27.5% haemolytic activity. Taken together, OG is a potent inhibitor of C. albicans that warrants further structural optimization and pharmacological investigations.

Altered drug efflux under iron deprivation unveils abrogated MmpL3 driven mycolic acid transport and fluidity in mycobacteria.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is a global threat to human health hence better understanding of the MTB pathogenesis for improved therapeutics requires immediate attention. Emergence of drug-resistant strains has stimulated an urgent need for adopting new strategies that could be implemented to control TB. One of the contributing mechanisms by which MTB evades drug doses is overexpression of drug efflux pumps. Thus blocking or modulating the functionality of efflux pumps represents an attractive approach to combat drug resistance. Iron is a critical micronutrient required for MTB survival and not freely available inside the host. In this study, we demonstrated that iron deprivation impairs drug efflux pump activity and confers synergism for anti-TB drugs in presence of efflux pump inhibitors against MTB. Mechanistic insights revealed that iron deprivation inhibit resistance nodulation division superfamily transporter activity. This was evident from enhanced Nile red accumulation and reduced expression of MmpL3, a transmembrane promising target involved in mycolic acid transport across membrane. Furthermore, iron deprivation led to abrogated MA transport particularly of class methoxy-MA which was confirmed by TLC and mass spectrometry based lipidome analysis. Additionally, iron deprivation leads to enhanced membrane fluidity in MTB. Together, MmpL3 being a promiscuous anti-TB target, metal chelation strategy could be adopted to boost the effectiveness of current anti-TB drug regimes to combat drug resistance TB.

Monoterpenoid perillyl alcohol impairs metabolic flexibility of Candida albicans by inhibiting glyoxylate cycle.

The metabolic pathway such as glyoxylate cycle (GC) enables Candida albicans, to survive under glucose deficient conditions prevalent in the hostile niche. Thus its key enzymes (Isocitrate lyase; ICL and malate synthase; MLS) represent attractive targets against C. albicans. We have previously reported the antifungal potential of a natural monoterpenoid perillyl alcohol (PA). The present study uncovers additional role of PA as a potent GC inhibitor. We explored that PA phenocopied ICL1 deletion mutant and were hypersensitive under low carbon utilizing conditions. The effect of PA on GC was substantiated by molecular docking analyses, which reveals the in-silico binding affinity of PA with ICL and MLS and explored that PA binds to the active sites of both proteins with better binding energy in comparison to their known inhibitors 3-nitropropionate and bromopyruvate respectively. Enzyme kinetics by Lineweaver-Burk plot unravels that PA inhibits ICL and MLS enzymes in competitive and non-competitive manner respectively. Moreover, semi-quantitative RT-PCR indicated that PA inhibits ICL1 and MLS1 mRNA expressions. Lastly, we demonstrated the antifungal efficacy of PA by enhanced survival of Caenorhabditis elegans model and less hemolytic activity (10.6%) on human blood cells. Further studies are warranted for PA to be considered as viable drug candidate.

Mechanistic insights into the mode of action of anticandidal sesamol.

Previously we have deciphered the antifungal effect of sesamol (Ses), a phenolic compound obtained from sesame oil, against human fungal pathogen Candida albicans. To gain deeper insights into the possible mechanisms involved, transcription profiling was done in presence of Ses which revealed various targets through which Ses was barricading the growth of C. albicans. We observed that Ses perturbs membrane integrity confirming our previous observations and displayed disrupted plasma membrane ATPase activity. We further investigated that Ses leads to inhibited morphological transition, biofilm formation and epithelial cell adhesion which are significant virulence attributes required for pathogenesis. Interestingly, Ses also causes amendment in iron homeostasis as revealed by hypersensitivity under iron deprivation, ferroxidase assay to estimate iron levels and concomitant upregulation of FTR2, a high affinity iron transporter. Finally we assessed that Ses causes defect in mitochondrial functioning and DNA repair mechanism. Together, being source of consumable natural product, further studies on Ses are warranted so that it can be exploited as effective antifungal agent.

Insights into the mode of action of anticandidal herbal monoterpenoid geraniol reveal disruption of multiple MDR mechanisms and virulence attributes in Candida albicans.

The anticandidal potential of Geraniol (Ger) against Candida albicans has already been established. The present study reveals deeper insights into the mechanisms of action of Ger. We observed that the repertoire of antifungal activity was not only limited to C. albicans and its clinical isolates but also against non-albicans species of Candida. The membrane tampering effect was visualized through transmission electron micrographs, depleted ergosterol levels and altered plasma membrane ATPase activity. Ger also affects cell wall as revealed by spot assays with cell wall-perturbing agents and scanning electron micrographs. Functional calcineurin pathway seems to be indispensable for the antifungal effect of Ger as calcineurin signaling mutant was hypersensitive to Ger while calcineurin overexpressing strain remained resistant. Ger also causes mitochondrial dysfunction, impaired iron homeostasis and genotoxicity. Furthermore, Ger inhibits both virulence attributes of hyphal morphogenesis and biofilm formation. Taken together, our results suggest that Ger is potential antifungal agent that warrants further investigation in clinical applications so that it could be competently employed in therapeutic strategies to treat Candida infections.

Efficacy of LAMP assay for Mycobacterial spp. detection to prevent treatment delays and onset of drug resistance: a systematic review and meta-analysis.

Background: Tuberculosis (TB) remains a deadly disease affecting one-third population globally. Long turnaround time and poor sensitivity of the conventional diagnostics are the major impediments for faster diagnosis of Mycobacterial spp to prevent drug resistance. To overcome these issues, molecular diagnostics have been developed. They offer enhanced sensitivity but require sophisticated infrastructure, skilled manpower and remain expensive. Methods: In that context, loop-mediated isothermal amplification (LAMP) assay, recommended by the WHO in 2016 for TB diagnosis, sounds as a promising alternative that facilitates visual read outs. Therefore, the aim of the present study is to conduct a meta-analysis to assess the diagnostic efficiency of LAMP for the detection of a panel of Mycobacterium spp. following PRISMA guidelines using scientific databases. From 1600 studies reported on the diagnosis of Mycobacterium spp., a selection of 30 articles were identified as eligible to meet the criteria of LAMP based diagnosis. Results: It was found that most of the studies were conducted in high disease burden nations such as India, Thailand, and Japan with sputum as the most common specimen to be used for LAMP assay. Furthermore, IS6110 gene and fluorescence-based detections ranked as the most used target and method respectively. The accuracy and precision rates mostly varied between 79.2% to 99.3% and 73.9% to 100%, respectively. Lastly, a quality assessment based on QUADAS-2 of bias and applicability was conducted. Conclusion: LAMP technology could be considered as a feasible alternative to current diagnostics considering high burden for rapid testing in low resource regions.

Diagnostic Efficacy of LAMP Assay for Human Fungal Pathogens: a Systematic Review and Meta-analysis.

Purpose: Human fungal infections particularly caused by Candida and Aspergillus have emerged as major public health burden. Long turnaround time and poor sensitivity of the conventional diagnostics are the major impediments for faster diagnosis of human fungal pathogens. Recent Findings: To overcome these issues, molecular-based diagnostics have been developed. They offer enhanced sensitivity but require sophisticated infrastructure, skilled manpower, and remained expensive. In that context, loop-mediated isothermal amplification (LAMP) assay represents a promising alternative that facilitates visual read outs. However, to eradicate fungal infections, all forms of fungi must be accurately detected. Thus, a need for alternative testing methodologies is imperative that should be rapid, accurate and facilitate widespread adoption. Therefore, the aim of the present study is to conduct a meta-analysis to assess the diagnostic efficiency of LAMP in the detection of a panel of human fungal pathogens following PRISMA guidelines using scientific databases viz. PubMed, Google Scholar, Science Direct, Scopus, BioRxiv, and MedRxiv. Summary: From various studies reported on the diagnosis of fungi, only 9 articles were identified as eligible to meet the criteria of LAMP based diagnosis. Through this meta-analysis, it was found that most of the studies were conducted in China and Japan with sputum and blood as the most common specimens to be used for LAMP assay. The collected data underlined that ITS gene and fluorescence-based detections ranked as the most used target and method. The pooled sensitivity values of meta-analysis ranged between 0.71 and 1.0 and forest plot and SROC (summary receiver operating characteristic) curve revealed a pooled specificity values between 0.13 and 1.0 with the confidence interval of 95%, respectively. The accuracy and precision rates of eligible studies mostly varied between 70 to 100% and 68 to 100%, respectively. A quality assessment based on QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies) of bias and applicability was conducted which depicted low risk of bias and applicability concerns. Together, LAMP technology could be considered as a feasible alternative to current diagnostics considering high fungal burden for rapid testing in low resource regions.

Current Understanding of the Molecular Basis of Spices for the Development of Potential Antimicrobial Medicine.

Antimicrobial resistance increases day by day around the world. To overcome this situation new antimicrobial agents are needed. Spices such as clove, ginger, coriander, garlic, and turmeric have the potential to fight resistant microbes. Due to their therapeutic properties, medicinal herbs and spices have been utilized as herbal medicines since antiquity. They are important sources of organic antibacterial substances that are employed in treating infectious disorders caused by pathogens such as bacteria. The main focus of the study is the bioactivity of the active ingredients present in different kinds of naturally available spices. We conducted a thorough search of PubMed, Google Scholar, and Research Gate for this review. We have read many kinds of available literature, and in this paper, we conclude that many different kinds of naturally available spices perform some form of bioactivity. After reading several papers, we found that some spices have good antimicrobial and antifungal properties, which may help in controlling the emerging antimicrobial resistance and improving human health. Spices have many phytochemicals, which show good antimicrobial and antifungal effects. This review of the literature concludes that the natural bioactivate compounds present in spices can be used as a drug to overcome antimicrobial resistance in human beings.

4-Allyl-2-methoxyphenol modulates the expression of genes involved in efflux pump, biofilm formation and sterol biosynthesis in azole resistant Aspergillus fumigatus.

Introduction: Antifungal therapy for aspergillosis is becoming problematic because of the toxicity of currently available drugs, biofilm formation on host surface, and increasing prevalence of azole resistance in Aspergillus fumigatus. Plants are rich source of bioactive molecules and antimicrobial activity of aromatic bioactive compounds draws attention because of its promising biological properties. The present study elucidated the antibiofilm activity of 4-allyl-2-methoxyphenol (eugenol) against azole-resistant environmental A. fumigatus isolates. Methods: Soil samples were collected from agricultural fields across India; azole-resistant A. fumigatus (ARAF) were isolated followed by their molecular identification. Antibiofilm activity of eugenol was calculated via tetrazolium based-MTT assay. The expression of the multidrug efflux pumps genes MDR1, MDR4, transporters of the MFS gene, erg11A gene encoding 14α demethylase, and transcription regulatory genes, MedA, SomA and SrbA, involved in biofilm formation of A. fumigatus were calculated by quantitative real time PCR. Results: Out of 89 A. fumigatus isolates, 10 were identified as azole resistant. Eugenol exhibited antibiofilm activity against ARAF isolates, ranging from 312 to 500 µg/mL. Confocal laser scanning microscopy analysis revealed absence of extracellular matrix of ARAF biofilm after eugenol treatment. The gene expression indicated significantly low expression of efflux pumps genes MDR1, MDR4, erg11A and MedA in eugenol treated ARAF isolates when compared with untreated isolates. Conclusions: Our results demonstrate that eugenol effects the expression of efflux pump and biofilm associated genes as well as inhibits biofilm formation in azole resistant isolates of A. fumigatus.

Mechanistic insights into the antimycobacterial action of unani formulation, Qurs Sartan Kafoori.

Background and aim: Tuberculosis (TBC) is a deadly disease and major health issue in the world. Emergence of drug resistant strains further worsens the efficiency of available anti-TBC drugs. Natural compounds and particularly traditional medicines such as Unani drugs are one of the promising alternatives that have been widely used nowadays. This study aims to evaluate the efficacy of unani drug Qurs-e-Sartan Kafoori (QSK) on Mycobacterium tuberculosis (MTB). Experimental procedures: Drug susceptibilities were estimated by broth microdilution assay. Cell surface integrity was assessed by ZN staining, colony morphology and nitrocefin hydrolysis. Biofilms were visualized by crystal violet staining and measurement of metabolic activity and biomass. Lipidomics analysis was performed using mass spectrometry. Host pathogen interaction studies were accomplished using THP-1 cell lines to estimate cytokines by ELISA kit, apoptosis and ROS by flow cytometry. Results: QSK enhanced the susceptibilities of isoniazid and rifampicin and impaired membrane homeostasis as depicted by altered cell surface properties and enhanced membrane permeability. In addition, virulence factor, biofilm formation was considerably reduced in presence of QSK. Lipidomic analysis revealed extensive lipid remodeling. Furthermore, we used a THP-1 cell line model, and investigated the immunomodulatory effect by estimating cytokine profile and found change in expressions of TNF-α, IL-6 and IL-10. Additionally, we uncover reduced THP-1 apoptosis and enhanced ROS production in presence of QSK. Conclusion: Together, this study validates the potential of unani formulation (QSK) with its mechanism of action and attempts to highlight its significance in MDR reversal.

An Inventory of Diagnostic Tools for Detection of COVID-19.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by SARS-COV-2 has afflicted millions of lives globally and disrupted almost all the activities of mankind. Under such pressing circumstances when no effective therapeutics are available, a fast and accurate diagnosis of the coronavirus is the only way out to limit the transmission. Since the inception of COVID-19, the demand for diagnostic tests has increased day by day and RT-PCR is the commonly used screening test that is not only time-consuming but requires sophisticated resources. To address the increasing rate of spread of COVID-19, there is an urgent need for more diagnostic tools as the research on vaccines is still at a rudimentary level. This review summarizes an inventory of the diverse and currently available diagnostic methods based on nucleic acid and serology along with some of those working on novel principles viz. CRISPR, biosensors, and NGS. Additionally, accessible diagnostic kits that are already approved by the US and European authorities for the diagnosis of COVID-19 are also suggested that will help in selecting the most effective tests under the given scenario. Taken together, this review will pave way for further strengthening the research on the rapid and safer diagnostics of SARS-COV-2.