Mohd Sajjad Ahmad Khan

Antibiofilm activity of certain phytocompounds and their synergy with fluconazole against Candida albicans biofilms

OBJECTIVES The aim of this study was to evaluate four phytocompounds (cinnamaldehyde, citral, eugenol and geraniol) for their in vitro inhibitory activity against pre-formed biofilms of Candida albicans alone or in combination with fluconazole and amphotericin B. These compounds were also tested at subinhibitory concentrations for their ability to inhibit biofilm formation. METHODS The XTT reduction assay, light microscopy and scanning electron microscopy (SEM) were employed to determine the inhibitory effect of the test compounds on biofilms. A chequerboard method was used for combination studies. RESULTS Both clinical and reference strains of C. albicans (C. albicans 04 and C. albicans SC5314, respectively) displayed formation of strong biofilms. Pre-formed Candida biofilms showed ≥1024× increased resistance to antifungal drugs and 2× increased resistance to cinnamaldehyde and geraniol, but no increased tolerance of eugenol. The test compounds were more active against pre-formed biofilms than amphotericin B and fluconazole. At 0.5× MIC, eugenol and cinnamaldehyde were the most inhibitory compounds against biofilm formation. Light and electron microscopic studies revealed the deformity of three-dimensional structures of biofilms formed in the presence of sub-MICs of eugenol and cinnamaldehyde. The cell membrane appeared to be the target site of compounds in both planktonic and sessile C. albicans cells, as observed by SEM. Combination studies showed that synergy was highest between eugenol and fluconazole (fractional inhibitory concentration index = 0.14) against pre-formed biofilms of C. albicans SC5314. CONCLUSIONS Promising antibiofilm activity was displayed by eugenol and cinnamaldehyde, which also showed synergy with fluconazole in vitro. Further evaluation in in vivo systems is required to determine whether these findings can be exploited in treating biofilm-associated candidiasis.

In vitro antifungal, anti-elastase and anti-keratinase activity of essential oils of Cinnamomum-, Syzygium- and Cymbopogon-species against Aspergillus fumigatus and Trichophyton rubrum.

This study was aimed to evaluate effects of certain essential oils namely Cinnamomum verum, Syzygium aromaticum, Cymbopogon citratus, Cymbopogon martini and their major components cinnamaldehyde, eugenol, citral and geraniol respectively, on growth, hyphal ultrastructure and virulence factors of Aspergillus fumigatus and Trichophyton rubrum. The antifungal activity of essential oils and their major constituents was in the order of cinnamaldehyde>eugenol>geraniol=C. verum>citral>S. aromaticum>C. citratus>C. martini, both in liquid and solid media against T. rubrum and A. fumigatus. Based on promising antifungal activity of eugenol and cinnamaldehyde, these oils were further tested for their inhibitory activity against ungerminated and germinated conidia in test fungi. Cinnamaldehyde was found to be more active than eugenol. To assess the possible mode of action of cinnamaldehyde, electron microscopic studies were conducted. The observations revealed multiple sites of action of cinnamaldehyde mainly on cell membranes and endomembranous structures of the fungal cell. Further, test oils were also tested for their anti-virulence activity. More than 70% reduction in elastase activity was recorded in A. fumigatus by the oils of C. verum, C. martini, eugenol, cinnamaldehyde and geraniol. Similar reduction in keratinase activity in A. niger was recorded for the oils of C. martini and geraniol. Maximum reduction (96.56%) in elastase activity was produced by cinnamaldehyde whereas; geraniol caused maximum inhibition (97.31%) of keratinase activity. Our findings highlight anti-elastase and anti-keratinase activity of above mentioned essential oils as a novel property to be exploited in controlling invasive and superficial mycoses.

Antifungal activity of essential oils and their synergy with fluconazole against drug-resistant strains of Aspergillus fumigatus and Trichophyton rubrum

The aim of this study was to screen certain plant essential oils and active compounds for antifungal activity and their in vitro interaction with fluconazole against drug-resistant pathogenic fungi. The methods employed in this work included disc diffusion, broth macrodilution, time kill methods and checkerboard microtiter tests. Oil compositions were evaluated by gas chromatography-mass spectrometry (GC-MS) analysis. Transmission electron microscopy was used to assess the effect of essential oils on cellular structures of test fungi. Test fungal strains exhibited resistance to at least two drugs (fluconazole and itraconazole). Among the 21 essential oils or active compounds tested, ten showed promising antifungal activity. GC-MS analysis revealed the presence of major active compounds in the essential oils used. Cinnamaldehyde showed the most promising antifungal activity and killing potency against Aspergillus fumigatus MTCC2550 and Trichophyton rubrum IOA-9. Cinnamaldehyde showed strongest synergy with fluconazole against A. fumigatus and T. rubrum by reducing the minimum inhibitory concentration of fluconazole up to 8-fold. Zones of lysis of the cell wall and cell membrane appeared to be where cinnamaldehyde acted on fungi. This study highlights the broad spectrum antifungal activity of essential oils and active compounds and their synergy with fluconazole against drug-resistant fungi.

Anti-candidal activity of essential oils alone and in combination with amphotericin B or fluconazole against multi-drug resistant isolates of Candida albicans

Therapy for candidiasis is becoming problematic due to the toxicities of currently available antifungal agents and the increasing prevalence of resistance among the etiologic agents. Therefore, new antifungals and alternative approaches are needed. In this study, 20 fluconazole-resistant strains of Candida albicans were found to have varying levels of resistance to other azoles, i.e., itraconazole (MIC of 4-128 μg/ml) and ketoconazole (2-256 μg/ml). In addition, 13 of these isolates appeared resistant to amphotericin B (32-128 μg/ml). A total of 21 plant essential oils were screened for their antifungal activity against these multi-drug resistant isolates. The oils of Cymbopogon martini, i.e., citral and cinnamaldehyde, exhibited strong inhibitory activity with minimum inhibitory concentrations (MIC(50)) ranging from 90-100 μg/ml. The test oils were more effective than fluconazole and amphotericin B in inhibiting azole- and amphotericin B-resistant, as well as amphotericin B-susceptible isolates. The test oils and especially eugenol, exhibited significant synergy with fluconazole or amphotericin B against the test isolates. These findings suggest the possible effective use of certain oils alone or in combination with fluconazole or amphotericin B, against multi-drug resistant isolates of C. albicans.

Biofilm inhibition by Cymbopogon citratus and Syzygium aromaticum essential oils in the strains of Candida albicans

ETHNOPHARMACOLOGICAL RELEVANCE Oils of Cymbopogon citratus and Syzygium aromaticum have been used in traditional medicine to treat fungal infections of skin, mouth, urinary and vaginal tract in Asian countries particularly India and other developing countries. AIM OF THE STUDY To evaluate essential oils of Cymbopogon citratus and Syzygium aromaticum for their anti-biofilm activity against strong biofilm forming strains of Candida albicans. MATERIALS AND METHODS XTT reduction assay, Time kill assays, light microscopy and scanning electron microscopy (SEM) were employed to determine the effect of test oils on the Candida albicans biofilms. RESULTS Most of the Candida albicans strains tested displayed formation of moderate to strong biofilms. Preformed Candida biofilms showed ≥1024 times increased resistance to antifungal drugs, 2 times to Syzygium aromaticum, but no increased tolerance for Cymbopogon citratus. Test oils were more active against preformed biofilms compared to amphotericin B and fluconazole. At 0.5× MIC, Cymbopogon citratus followed by Syzygium aromaticum were most inhibitory against biofilm formation. Light and electron microscopic studies revealed the deformity of three dimensional structures of biofilms formed in the presence of sub-MICs of Cymbopogon citratus. The cell membranes appeared to be the target site of compounds in sessile cells as displayed by SEM observations. CONCLUSION Our data had demonstrated promising in vitro anti-biofilm activity by Cymbopogon citratus and Syzygium aromaticum and confirm the ethnopharmacological use of these oils in muco-cutaneous Candida infections. Furthermore, it suggests exploitation of these oils as new anti-biofilm products to deal with the problem of drug-resistance and recurrent infection associated with biofilm mode of growth of Candida spp.

Virulence and Pathogenicity of Fungal Pathogens with Special Reference to Candida albicans

The frequency of severe systemic fungal diseases has increased in the last few decades. The clinical use of broad spectrum antibacterial drugs and immunosuppressive agents after organ transplantation, cancer chemotherapy, and advancements in surgery are associated with increasing risk of fungal infection. Despite the effectiveness of available antifungals in combating such infections, the emergence of drug resistance to antifungals, and problems of toxicity and poor delivery of drugs at the target site in systemic infections, have necessitated a systematic approach to the study of fungal pathogens, host–fungi interactions, and identification of virulence factors. Characterization of virulence factors is expected to improve understanding of fungal pathogenesis and to help explore new drug targets. In this article we discuss the process of fungal infections, virulence factors and pathogenicity of fungal pathogens, with special reference to Candida albicans. Adherence, dimorphism, phenotypic switching, secretion of hydrolytic enzymes, biofilm formation, and ability to adapt at host body temperature are some of the well-known virulence factors among pathogenic fungi and are discussed in relation to C. albicans.

Modulation of quorum sensing controlled behaviour of bacteria by growing seedling, seed and seedling extracts of leguminous plants

Effect of growing seedling, seeds and seedlings extracts from seven leguminous plants (Pisum sativum, Vigna radiata, Vigna mungo, Cajanus cajan, Lentil culinaris, Cicer arietinum and Trigonella foenum graecum) were screened for their ability to influence quorum sensing controlled pigment production in Chromobacterium violaceum indicator strains (CV12472 and CVO26). Germinating seedling and seedling extracts of only P. sativum (pea) showed inhibition of violacein production. Interestingly, the T. foenum graecum (fenugreek) seed extracts enhances the pigment production. Quorum sensing regulated swarming motility in Pseudomonas aerugionsa PAO1 was reduced by pea seedling extract while enhanced by the fenugreek seed extracts. These findings suggest that plant metabolites of some legumes interact actively with bacterial quorum sensing and could modulate its associated functions.

Microbial Applications in Agriculture and the Environment: A Broad Perspective

Microbial diversity is an important component of the overall global biological diversity. Recent technological advances in exploring microbial diversity have revealed that a large proportion of microorganisms are still undiscovered, and their ecological roles are largely unknown. Careful selection of microbes and intelligent design of test assays are the key steps in developing new technologies for effective utilization of microorganisms for sustainable agriculture, environmental protection, and human and animal health. Several microbial applications are widely known in solving major agricultural (i.e., crop productivity, plant health protection, and soil health maintenance) and environmental issues (i.e., bioremediation of soil and water from organic and inorganic pollutants). Wastewater treatment and recycling of agricultural and industrial wastes are other important uses of microbial technology. It is expected that microbes in combination with developments in electronics, software, digital imaging, and nanotechnology will play a significant role in solving global problems of the twenty-first century, including climate change. These advances are expected to enhance sustainability of agriculture and the environment. This chapter provides an overview of recent trends in microbial exploitation in plant growth promotion and sustainable environment mainly through bioremediation, biodegradation, and biosorption processes. Recent uses and application of microbes such as biosensors, synthesis of nanomaterials, and probiotics are also discussed.

Carum copticum and Thymus vulgaris oils inhibit virulence in Trichophyton rubrum and Aspergillus spp

Emergence of drug-resistant strains has demanded for alternative means of combating fungal infections. Oils of Carum copticum and Thymus vulgaris have long been used in ethnomedicine for ailments of various fungal infections. Since their activity has not been reported in particular against drug-resistant fungi, this study was aimed to evaluate the effects of oils of C. copticum and T. vulgaris on the growth and virulence of drug-resistant strains of Aspergillus spp. and Trichophyton rubrum. The gas chromatography-mass spectrometry analysis revealed thymol constituting 44.71% and 22.82% of T. vulgaris and C. copticum, respectively. Inhibition of mycelial growth by essential oils was recorded in the order of thymol > T. vulgaris > C. copticum against the tested strains. RBC lysis assay showed no tested oils to be toxic even up to concentration two folds higher than their respective MFCs. Thymol exhibited highest synergy in combination with fluconazole against Aspergillus fumigatus MTCC2550 (FICI value 0.187) and T. rubrum IOA9 (0.156) as determined by checkerboard method. Thymol and T. vulgaris essential oil were equally effective against both the macro and arthroconidia growth (MIC 72 μg/mL). A > 80% reduction in elastase activity was recorded for A. fumigatus MTCC2550 by C. copticum, T. vulgaris oils and thymol. The effectiveness of these oils against arthroconidia and synergistic interaction of thymol and T. vulgaris with fluconazole can be exploited to potentiate the antifungal effects of fluconazole against drug-resistant strains of T. rubrum and Aspergillus spp.

Bacterial Quorum Sensing and Its Interference: Methods and Significance

Bacteria use the language of low-molecular-weight ligands to assess their population densities in a process called quorum sensing (QS). Different types of quorum sensing pathways are present in Gram-negative and Gram-positive bacteria. Signal molecules most commonly used in Gram-negative bacteria are acyl homoserine lactones. In recent years, a substantial amount of literature and data have been available on bacterial QS. Recently, interest in modulation of quorum sensing with different approaches has increased among scientific communities. In this chapter, we provide an updated overview on bacterial QS, assays and methods for detecting signal molecules, and various approaches to inhibit AHL-based quorum sensing. Significance of QS interference by prokaryotic and eukaryotic organisms in relation to plant health and the environment is discussed here.