Cryptococcosis is an emerging and recalcitrant systemic infection occurring in immunocompromised patients. This invasive fungal infection is difficult to treat due to the ability of Cryptococcus neoformans and Cryptococcus laurentii to form biofilms resistant to standard antifungal treatment. The toxicity concern of these drugs has stimulated the search for natural therapeutic alternatives. Essential oil and their active components (EO-ACs) have shown to possess the variety of biological and pharmacological properties. In the present investigation the effect of six (EO-ACs) sourced from Oregano oil (Carvacrol), Cinnamon oil (Cinnamaldehyde), Lemongrass oil (Citral), Clove oil (Eugenol), Peppermint oil (Menthol) and Thyme oil (thymol) against three infectious forms; planktonic cells, biofilm formation and preformed biofilm of C. neoformans and C. laurentii were evaluated as compared to standard drugs. Data showed that antibiofilm activity of the tested EO-ACs were in the order: thymol>carvacrol>citral>eugenol=cinnamaldehyde>menthol respectively. The three most potent EO-ACs, thymol, carvacrol, and citral showed excellent antibiofilm activity at a much lower concentration against C. laurentii in comparison to C. neoformans indicating the resistant nature of the latter. Effect of the potent EO-ACs on the biofilm morphology was visualized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), which revealed the absence of extracellular polymeric matrix (EPM), reduction in cellular density and alteration in the surface morphology of biofilm cells. Further, to realize the efficacy of the EO-ACs in terms of human safety, cytotoxicity assays and co-culture model were evaluated. Thymol and carvacrol as compared to citral were the most efficient in terms of human safety in keratinocyte- Cryptococcus sp. co-culture infection model suggesting that these two can be further exploited as cost-effective and non-toxic anti-cryptococcal drugs.

The recalcitrant biofilm formed by fungus Cryptococcus neoformans is a life-threatening pathogenic condition responsible for further intensifying cryptococcosis. Considering the enhanced biofilm resistance and toxicity of synthetic antifungal drugs, the search for efficient, nontoxic, and cost-effective natural therapeutics has received a major boost. Phenolic (thymol and carvacrol) and aldehydic (citral) terpenes are natural and safe alternatives capable of efficient microbial biofilm inhibition. However, the biofilm inhibition mechanism of these terpenes still remains unclear. In this study, we adopted an integrative biophysical and biochemical approach to elucidate the hierarchy of their action against C. neoformans biofilm cells. The microscopic analysis revealed disruption of the biofilm cell surface with elevation in surface roughness and reduction in cell height. Although all terpenes acted through ergosterol biosynthesis inhibition, the phenolic terpenes also selectively interacted via ergosterol binding. Further, the alterations in the fatty acid profile in response to terpenes attenuated the cell membrane fluidity with enhanced permeability, resulting in pore formation and efflux of the K+/intracellular content. Additionally, mitochondrial depolarization caused higher levels of reactive oxygen species, which led to increased lipid peroxidation and activation of the antioxidant defense system. Indeed, the oxidative stress caused a significant decline in the amount of extracellular polymeric matrix and capsule sugars (mannose, xylose, and glucuronic acid), leading to a reduced capsule size and an overall negative charge on the cell surface. This comprehensive data revealed the mechanistic insights into the mode of action of terpenes on biofilm inhibition, which could be exploited for formulating novel anti-biofilm agents.

BackgroundHarnessing the halotolerant characteristics of microalgae provides a viable alternative for sustainable biomass and triacylglyceride (TAG) production. Scenedesmus sp. IITRIND2 is a fast growing fresh water microalga that has the capability to thrive in high saline environments. To understand the microalga’s adaptability, we studied its physiological and metabolic flexibility by studying differential protein, metabolite and lipid expression profiles using metabolomics, proteomics, real-time polymerase chain reaction, and lipidomics under high salinity conditions.ResultsOn exposure to salinity, the microalga rewired its cellular reserves and ultrastructure, restricted the ions channels, and modulated its surface potential along with secretion of extrapolysaccharide to maintain homeostasis and resolve the cellular damage. The algal-omics studies suggested a well-organized salinity-driven metabolic adjustment by the microalga starting from increasing the negatively charged lipids, up regulation of proline and sugars accumulation, followed by direction of carbon and energy flux towards TAG synthesis. Furthermore, the omics studies indicated both de-novo and lipid cycling pathways at work for increasing the overall TAG accumulation inside the microalgal cells.ConclusionThe salt response observed here is unique and is different from the well-known halotolerant microalga; Dunaliella salina, implying diversity in algal response with species. Based on the integrated algal-omics studies, four potential genetic targets belonging to two different metabolic pathways (salt tolerance and lipid production) were identified, which can be further tested in non-halotolerant algal strains.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1343-1) contains supplementary material, which is available to authorized users.

Candida tropicalis is an emerging non-albicans Candida species which is pathogenic to the immune-compromised humans, especially in tropical countries, including India. The acquired resistance of Candida species towards antifungal therapies is of major concern. Moreover, limited efficacy and dosage constraint of synthetic drugs have indicated the prerequisite of finding new and natural drugs for treatment. In the present study, we have compared the influence of citral and thymol on C. tropicalis and its biofilm along with expression levels of certain antifungal tolerance genes. The antifungal and anti-biofilm activities of the both were studied using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt (XTT) reduction assay, field emission scanning electron microscope (FE-SEM) and confocal laser scanning microscope (CLSM) and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. Citral and thymol have damaged the cells with distorted surface and less viability. Quantitative real-time PCR analysis showed augmented expression of the cell membrane biosynthesis genes including ERG11/CYT450 against citral and the cell wall related tolerance genes involving CNB1 against thymol thus, depicting their differential mode of actions.

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