Germ Tube Formation Research Articles

Synergistic potential of essential oils with antibiotics to combat fungal pathogens: Present status and future perspectives.

The steady rise in the emergence of antibiotic-resistant fungal pathogens has rendered most of the clinical antibiotics available in the market to be ineffective. Therefore, alternative strategies are required to tackle drug-resistant fungal infections. An effective solution is to combine the available antibiotics with adjuvants such as phytochemicals or essential oils to enhance the efficacy and activity of antibiotics. The present review aims to summarize the studies on synergistic combinations of essential oils and anti-fungal antibiotics. The current findings, methods used for measuring synergistic effects, possible mechanisms of synergism, and future perspectives for developing synergistic EO-antibiotic therapeutic formulations are discussed in this study. Several essential oils exhibit synergistic effect in combination with antibiotics against human fungal pathogens such as Candida albicans. The possible mechanisms of synergy exhibited by essential oil- antibiotic combinations in fungi include disruption of cell wall structure/ ergosterol biosynthesis pathway, enhanced transdermal penetration of antibiotics, alterations in membrane permeability, intracellular leakage of cellular contents, inhibition of germ tube formation or fungal biofilm formation, and competition for a primary target. Synergistic combination of essential oils and antibiotics can prove to be a valid and pragmatic alternative to develop drugs with increased drug-efficacy, and low toxicity.

Embryonic Stem Cell-Derived Exosomes Attenuate Transverse Aortic Constriction Induced Heart Failure by Increasing Angiogenesis.

Background: Although there are concerns regarding their clinical use, embryonic stem cells (ESCs) hold a great promise for cardiac repair. Exosomes deriving from ESCs constitute a promising alternative for heart restoration. However, their effects in hypertension-induced heart failure are still unknown.Objective and Methods: To investigate the effects of ESCs-derived exosomes on hypertension-induced heart failure and the underlying mechanisms, sustained transverse aortic constriction (TAC) was performed on 8-week-old C57BL/6 male mice. After 1 months, ESCs-derived exosomes were isolated and injected intravenously once a week for 6 weeks. Echocardiography, wheat germ agglutinin (WGA), Masson staining, immunohistochemistry, and tube formation assays were all involved in our study.Results: Proteomics analyses revealed that ESC-derived exosomes contain FGF2 protein. Tube formation induced by these exosomes could be inhibited by FGF2R siRNA interference. ESCs-derived exosomes evidently attenuated TAC-induced heart failure, improving cardiac function and promoting myocardial angiogenesis which can be attenuated by selective FGF2 inhibitor AZD4547.Conclusions: ESC-derived exosomes attenuate TAC-induced heart failure mostly by promoting myocardial angiogenesis. FGF2 signaling plays a vital role in the myocardial angiogenesis induced by ESC-derived exosomes.

Differential Physiological Prerequisites and Gene Expression Profiles of Conidial Anastomosis Tube and Germ Tube Formation in Colletotrichum gloeosporioides

The conidia of a hemibiotrophic fungus, Colletotrichum gloeosporioides, can conventionally form a germ tube (GT) and develop into a fungal colony. Under certain conditions, they tend to get connected through a conidial anastomosis tube (CAT) to share the nutrients. CAT fusion is believed to be responsible for the generation of genetic variations in few asexual fungi, which appears problematic for effective fungal disease management. The physiological and molecular requirements underlying the GT formation versus CAT fusion remained underexplored. In the present study, we have deciphered the physiological prerequisites for GT formation versus CAT fusion in C. gloeosporioides. GT formation occurred at a high frequency in the presence of nutrients, while CAT fusion was found to be higher in the absence of nutrients. Younger conidia were found to form GT efficiently, while older conidia preferentially formed CAT. Whole transcriptome analysis of GT and CAT revealed highly differential gene expression profiles, wherein 11,050 and 9786 genes were differentially expressed during GT formation and CAT fusion, respectively. A total of 1567 effector candidates were identified; out of them, 102 and 100 were uniquely expressed during GT formation and CAT fusion, respectively. Genes coding for cell wall degrading enzymes, germination, hyphal growth, host-fungus interaction, and virulence were highly upregulated during GT formation. Meanwhile, genes involved in stress response, cell wall remodeling, membrane transport, cytoskeleton, cell cycle, and cell rescue were highly upregulated during CAT fusion. To conclude, the GT formation and CAT fusion were found to be mutually exclusive processes, requiring differential physiological conditions and sets of DEGs in C. gloeosporioides. This study will help in understanding the basic CAT biology in emerging fungal model species of the genus Colletotrichum.

Detection of als1 and hwp1 genes involved in biofilm formation in Candida albicans isolated from catheter associated candiduria.

Background: Candida species are opportunistic fungal pathogens that can affect immune compromised patients and may cause life threatening infections. Biofilm formation is an important virulence factor that enables Candida species to invade the host tissues. This study aimed to assess the ability of Candida albicans to form biofilm in patients with catheter associated candiduria and to determine the presence of some genes involved in biofilm formation at the molecular level. Methods: This cross-sectional descriptive study was carried out from June 2019 to June 2020 on 93 catheterized patients admitted to different wards at SCUHs, Ismailia. Candida albicans strains were identified phenotypically by their growth on sabouraud dextrose agar, Gram staining, growth on hypertonic sabouraud broth, chlamydospore formation and germ tube formation test. Biofilm formation was detected by tissue culture plate method. Detection of the genes involved in biofilm formation was done by PCR. Results: Twenty eight Candida albicans strains wereisolated from 36 Candida species. 9 isolates (32.1%) were strong biofilm producers, 6 isolates (21.4%) were moderate biofilm producers and 13 isolates (46.4%) were weak or non-biofilm producers. ALS1 gene was detected in 13 isolates (46.4%), while HWP1 gene was detected in 16 isolates (57.1 %). Conclusion: This study documented the prevalence of candiduria in catheterized patients and found a statistically significant higher prevalence of the tested biofilm forming genes among biofilm forming isolates as compared to the weak or non-biofilm forming ones; but additional studies should be carried out as the management of catheter associated caniduria is still controversial.

Evaluation of the antifungal photodynamic activity of Thymophylla pentachaeta extracts against Candida albicans and its virulence factors

BackgroundCandida albicans is one of the most common causative of opportunistic infections. Treatment of candidiasis is challenging considering the few antifungal drugs available and the increase in resistance. Antimicrobial photodynamic therapy (aPDT) is a recently developed therapeutic option that combines a non-toxic photosensitizer (PS) and light to kill the microbial pathogens. Targeting virulence, defined as the ability of a pathogen to cause overt disease, represents another attractive target for the development of novel antifungal agents. Thymophylla pentachaeta (DC.) Small var. belenidium (DC.) is an endemic plant from Argentina in which the presence of thiophenes, biologically active compounds whose antifungal activity is enhanced by irradiation with Ultraviolet A (UVA), have been already described. PurposeThe purpose of this study was to evaluate the photodynamic antifungal activity of hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) extracts from T. pentachaeta var. belenidium and their inhibitory effects on C. albicans virulence factors as well as biofilm formation and eradication. Study Design/MethodsAntifungal photodynamic activity of Hex, DCM, EtOAc and MeOH extracts from different parts of the plant were assessed with the microbroth dilution, bioautography and the time-kill assays, under light and darkness conditions. The capacities of the most active extracts of inhibiting Candida virulence factors (adherence to epithelial cells, germ tube and pseudomycelium formation and hydrolytic enzyme secretion) were assessed. In addition, the activity against biofilm formation and eradication has been investigated by reaction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) that quantifies living cells in these structures. ResultsHex and DCM extracts from T. pentachaeta roots exhibited high photodynamic antifungal activity against C. albicans [Minimal fungicide concentrations (MFCs)= 7.8 µg/ml] under UVA light irradiation. Chemical analysis of active extracts (Hex and DCM from roots) revealed the presence of photoactive thiophenes. Both extracts generate reactive oxygen species through type I and II mechanisms. These extracts, at sub-inhibitory concentrations, under light conditions decreased the adherence of C. albicans to Buccal Epithelial Cells (BEC), inhibited germ tube formation and reduced esterase production. Finally, they demonstrated activity against preformed biofilms submitted to irradiation (MFCs= 3.91 µg/ml and 15.63 µg/ml for Hex and DCM extracts, respectively). ConclusionTaking together, results demonstrated the strong photodynamic effects of T. pentachaeta root extracts under UVA irradiation, making them valuable alternatives to the already established antifungal drugs against C. albicans.

Ultrasonic Synthesis and Biomedical Application of Mn0.5Zn0.5ErxYxFe2-2xO4 Nanoparticles.

In the present study, biocompatible manganese nanoparticles have been linked with zinc and iron molecules to prepare different derivatives of Mn0.5Zn0.5ErxYxFe2−2xO4 NPs (x = 0.02, 0.04, 0.06, 0.08, 0.10), using an ultrasonication approach. The structure, surface morphology, and chemical compositions of Mn0.5Zn0.5ErxYxFe2−2xO4 NPs were elucidated by X-ray diffractometer (XRD), High-resolution transmission electron microscopy (HR-TEM), scanning electron microscope (SEM), and Energy Dispersive X-Ray Analysis (EDX) techniques. The bioactivity of Mn0.5Zn0.5ErxYxFe2−2xO4 NPs on normal (HEK-293) and (HCT-116) colon cancer cell line was evaluated. The Mn0.5Zn0.5ErxYxFe2−2xO4 NPs treatment post 48 h resulted in a significant reduction in cells (via MTT assay, having an IC50 value between 0.88 µg/mL and 2.40 µg/mL). The specificity of Mn0.5Zn0.5ErxYxFe2−2xO4 NPs were studied by treating them on normal cells line (HEK-293). The results showed that Mn0.5Zn0.5ErxYxFe2−2xO4 NPs did not incur any effect on HEK-293, which suggests that Mn0.5Zn0.5ErxYxFe2−2xO4 NPs selectively targeted the colon cancerous cells. Using Candida albicans, antifungal activity was also studied by evaluating minimum inhibitory/fungicidal concentration (MIC/MFC) and the effect of nanomaterial on the germ tube formation, which exhibited that NPs significantly inhibited the growth and germ tube formation. The obtained results hold the potential to design nanoparticles that lead to efficient bioactivity.

Protective Effects of the Probiotic Bacterium Streptococcus thermophilus on Candida albicans Morphogenesis and a Murine Model of Oral Candidiasis.

Background: Oral candidiasis is a frequent form of candidiasis, caused by Candida species, in particular, Candida albicans (C. albicans). The transition of C. albicans from yeast to hyphae allows its attachment to epithelial cells, followed by biofilm formation, invasion, and tissue damage. Hence, we investigated the effect of Streptococcus salivarius subspecies thermophilus (S thermophilus) on the growth as well as biofilm and germ-tube formation of C. albicans both in vitro and in vivo in a murine model.Methods: This experimental study was performed in the Department of Medical Mycology and Parasitology, School of Medicine, in collaboration with the Central Research Laboratory and the Comparative Biomedical Center, Shiraz University of Medical Sciences, Shiraz, Iran (2017 to 2018). The inhibitory activity of S. thermophilus against Candida species growth was evaluated using the broth microdilution method, and the inhibition of C. albicans biofilm formation was measured using the XTT assay. The inhibition of C. albicans germ-tube formation by S. thermophilus was evaluated using the plate assay and fluorescence microscopy. The experimental activity of the probiotic bacterium was assessed by culture and histopathological methods in six groups of five mice, comprising those treated with four concentrations of probiotics, fluconazole, and distilled water. The one-way analysis of variance, followed by a Tukey post hoc test, was used and a P value of less than 0.05 was considered significant.Results: S. thermophilus inhibited Candida species growth at concentrations of 16 to 512 µg/mL. This probiotic inhibited the formation of C. albicans biofilms and germ tubes in a dose-dependent manner. S. thermophilus significantly reduced the colony-forming units in the mice receiving 30 mg/mL of this probiotic treatment compared with the control group (P=0.024). The histopathological analysis showed that Candida colonization was diminished in the mice following the administration of the probiotic. Conclusion: Given the inhibitory activity of S. thermophilus against the growth, transition, and biofilm formation of C. albicans, it could be used in the management of oral candidiasis.

An in vitro study on the antifungal and antibiofilm activities of probiotic bacteria against Candida species isolated from orthodontic appliances and dental caries

Candida species are opportunistic pathogens that may cause infections in predisposed persons. This work aimed to detect the antifungal and antibiofilm potentials of Lactobacillus acidophilus and Lactobacillus plantarum supernatants on Candida spp. About 60 % and 80 % of Candida isolates were recovered from saliva samples of 20 patients with fixed orthodontic appliances, and 20 children having dental caries, respectively. The antifungal susceptibility of Candida spp. was investigated using disk diffusion assay. C. albicans strains showed low resistance to fluconazole (15 %) and amphotericin B (10 %). Using the agar well diffusion assay, both L. acidophilus and L. plantarum supernatants inhibited the in vitro growth of all tested Candida spp. with inhibition zone diameters of (11-19 mm) and (7-16 mm), respectively. On observing the effects of L. plantarum and L. acidophilus supernatants on Candida cells’ morphology; the light microscopic examination demonstrated the inhibition in germ tube formation of all tested C. albicans with percentages of 68 % and 53 %, and for C. krusei with inhibition percentages of 69 % and 59 %, respectively. L. acidophilus and L. plantarum strains showed high co-aggregation ability with C. albicans strains with ranges of 42-49 % and 30-35 %, respectively. The antibiofilm activities of the two Lactobacillus supernatants were determined using the tissue culture plate assays. Significant inhibition of biofilms formation by Candida spp. was recorded on treatment with L. plantarum and L. acidophilus supernatants, with reduction percentages of 50-72 % and 74-85 %, respectively.

Distribution and antifungal susceptibility of candida species isolated from blood cultures.

To determine the distribution of samples exhibiting candida growth among the blood cultures, and the antifungal susceptibility. The retrospective study was conducted at Samsun Training and Research Hospital, Samsun, Turkey, and comprised data of immunosuppressed patients with sepsis from January to December 2018. Yeast growth was typed by means of colony morphology, germ tube formation and the VITEK 2 system. The susceptibility was determined using the same automated system. Resistant strains were also tested using the dilution method. Data was analysed using SPSS 25. Of the 50 patients, 31(62%) were males and 19(38%) were females. The overall mean age was 60.84±22.05 years. Of the total samples, 31(62%) were received from intensive care units, 14(28%) from the palliative care unit, and 5(10%) from inpatients. The most common isolate was candida albicans 26(52%), while candida parapsiosis was the most common among non-albicans species 13(26%). Resistance to antifungals was observed in 13(26%) isolates; 5(10) to fluconazole, 4(8%) to voriconazole, 2(4%) each to flucytosine and amphotericin B. The findings obtained through both the dilution method and the automated system were consistent. The resistance to antifungal drugs varied among different candida species.

Bursting the Virulence Traits of MDR Strain of Candida albicans Using Sodium Alginate-based Microspheres Containing Nystatin-loaded MgO/CuO Nanocomposites.

IntroductionCandida albicans is a major opportunistic pathogen that causes a wide range of human infections. Currently available therapeutic agents are limited for treating these fungal infections due to multidrug resistance as well as their nonbiodegradability, poor biocompatibility and toxicity. In order to battle these limitations, we have synthesized a polymeric system as microcarriers to deliver the antifungal drug. The objective of the present study was to immobilize MgO/CuO nanocomposite and nystatin-loaded MgO/CuO nanocomposites in nontoxic, nonimmunogenic, biodegradable and biocompatible sodium alginate microspheres for the first time.Materials and MethodsNanoparticle-loaded sodium alginate microspheres were prepared by ionotropic gelation technique using calcium chloride as a cross-linker. Synthesized microspheres were characterized using standard characterization techniques and were evaluated for biological activity against MDR strain of C. albicans.ResultsCharacterization of microspheres by Fourier-transform infrared spectroscopy confirmed loading of Nys-MgO/CuO NPs, scanning electron microscopy (SEM) revealed rough spherical beads with a highly porous surface having an average size in the range of 8–10 µm. X-ray diffraction (XRD) analyzed its semicrystalline structure. Entrapment efficiency of Nys-MgO/CuO NPs was 80% and release kinetic study revealed sustained and prolonged release of drug in pH 5.5. Flow cytometry analysis showed yeast cell death caused by Nys-MgO/CuO MS exhibits late apoptotic features. In cytotoxicity assay 5–14 mg of microspheres did not cause hemolysis. Microspheres reduced virulence traits of C. albicans such as germ tube and biofilm formation were compromised at concentration of 5 mg/mL. Antimicrobial assessment results revealed a pronounced inhibitory effect against C. albicans.ConclusionThe in vitro experiments have shown promising results based on good stability, Nys-MgO/CuO NP-encapsulated microspheres can be used as a prolonged controlled release system against MDR pathogenic C. albicans.

Biologically rapid synthesized silver nanoparticles from aqueous Eucalyptus camaldulensis leaf extract: Effects on hyphal growth, hydrolytic enzymes, and biofilm formation in Candida albicans.

Bionanotechnology has increasingly gained attention in biomedical fields as antifungal and antibiofilm agents. In this study, biosynthesized silver nanoparticles (bio-AgNPs) using aqueous Eucalyptus camaldulensis leaf extract were successfully performed by a one-step green approach. Spherical-shaped nanoparticles, approximately8.65 nm, exhibited noncytotoxicity to erythrocytes, HeLa, and HaCaT cells. The synthesized nanoparticles showed strong fungicidal activity ranging from 0.5 to 1 µg/ml. The nanoparticles affected Candida adhesion and invasion into host cells by reduced germ tube formation and hydrolytic enzyme secretion. Inhibitory effects of bio-AgNPs on Candida biofilms were evaluated by the prevention of yeast-to-hyphal transition. A decrease in cell viability within mature biofilm demonstrated the ability of bio-AgNPs to penetrate into the extracellular matrix and destroy yeast cell morphology, leading to cell death. Molecular biology study on biofilms confirmed downregulation in the expression of genes ALS3, HWP1, ECE1, EFG1, TEC1, ZAP1, encoding hyphal growth and biofilm development and PLB2, LIP9, SAP4, involved in hydrolytic enzymes. In addition to candida treatment, the bio-AgNPs could be applied as an antioxidant to protect against oxidative stress-related human diseases. The findings concluded thatbio-AgNPs could be used as an antifungal agent for candida treatment, as well as be incorporated in medical devices to prevent biofilm formation.

Non-antifungal drugs inhibit growth, morphogenesis and biofilm formation in Candida albicans.

The increased resistance/tolerance of Candida infections to antimicrobial treatment can be attributed to biofilm-associated cells. A way to overcome this situation is to re-purpose non-anti-fungal drugs that could be active against fungi. We have explored the potential of a small library of eighteen non-antifungal drugs used in different human diseases. Candida albicans was cultured in the presence and absence of different concentrations of these drugs. Subsequently, inhibition of growth, germ tube formation, adhesion, and biofilm development were studied. Out of eighteen drug molecules, six showed a reduction in planktonic and biofilm growth in a dose-dependent manner and three drugs inhibited germ tube formation. This study shows the potential of non-antifungal drugs for the development of new anti-Candida agents.

First total synthesis of chromanone A, preparation of related compounds and evaluation of their antifungal activity against Candida albicans, a biofilm forming agent.

A straightforward and convenient approach for the first total syntheses of chromanone A and a related 7-OMe substituted natural product is reported. These unique C-3 substituted 2-hydroxymethyl chromones were recently isolated as fungal metabolites. Chromanone A was synthesized in 25.3% overall yield from the readily available pyrocatechol, whereas the second natural product was prepared in 39.7% global yield. A small library of chromones, including both natural products and some of their synthetic heterocyclic precursors, was evaluated against Candida albicans ATCC 10231, a biofilm forming agent. It was found that 8-methoxy-3-methyl-4-oxo-4H-chromene-2-carbaldehyde, a partially oxidized form of chromanone A, exhibited a minimum inhibitory concentration of 7.8 μg mL−1 and significantly inhibited the yeast's virulence factors, including the adherence to buccal epithelial cells and the secretion of phospholipases, as well as the formation of germ tubes and the generation of the hyphal pseudomycelium. In addition, despite the heterocycle exhibiting non-significant inhibition of the formation of the Candida biofilm, it completely inhibited the growth of C. albicans in preformed biofilms at 62.5 μg mL−1.

New insights on the antifungal activity of essential oil of Salvia desoleana Atzei et Picci, an endemic plant from folk medicine of Sardinia, Italy

This work reports the results concerning the antifungal activity of the essential oil obtained from Salvia desoleana, an endemic plant from folk medicine of Sardinia Island, Italy. Chemical analysis of S. desoleana essential oil isolated by hydrodistillation was carried out by gas chromatography (GC-FID) and gas chromatography–mass spectrometry (GC-MS). The essential oil contains high amounts of oxygenated monoterpenes and sesquiterpene hydrocarbons, being linalyl acetate (21.0%), a-terpinyl acetate (17.3%), 1,8-cineole (6.7%), linalool (3.6%), sclareol (3.5%) and germacrene D (22.1%) the main compounds. The oil was more active against the yeast Cryptococcus neoformans and the dermatophyte Trichophyton rubrum with MIC values of 0.16 μL/mL and 0.32 μL/mL, respectively. The oil revealed an important inhibitory effect on the germ tube formation in C. albicans. It was able to achieve about 40% of inhibition of filamentation at concentrations as low as 0.08 µL/mL. These findings add significant information to the biological activity of the essential oil of S. desoleana, specifically to its antifungal properties, thus justifying and reinforcing the use of this plant in traditional medicine.

Antifungal Susceptibility Pattern of Candida Isolates Causing Vulvovaginitis in Reproductive Age Women

Objectives: The study was designed to isolate and identify Candida species from high vaginal swab, and to determine the antifungal susceptibility pattern of Candida spp. among women of reproductive age group i.e. 15- 50 years old.
 Methods: High vaginal swabs were processed to isolate Candida species and identified by Gram’s stain, germ tube formation test, carbohydrate (glucose, sucrose, maltose, lactose) fermentation tests and antifungal susceptibility test were performed as recommended by Clinical Laboratory Standard Institute.
 Results: Out of total 261 samples, 42.1% (110/261) were found to be culture positive for Candida spp. Candida albicans was the most common (56.4%) and among non-albicans, predominate species was C. krusei (19.1%) isolated from high vaginal swab specimens All the isolates of Candida species were sensitive to Nystatin and Miconazole and resistant to Itraconazole. Fluconazole, Itraconazole and Clotrimazole are widely used drug against vaginal candidiasis but showed high resistance which leads to treatment failure.
 Conclusion: This study on the infection rate of Candida and its antifungal susceptibility pattern may help in the choice of appropriate therapy in the clinical setting.

Mechanistic insights into Candida biofilm eradication potential of eucalyptol.

Candida-associated fungal infections are prevalent in hospitalized and immune-compromised patients. Their biofilm architecture and high rate of antifungal resistance make treatment challenging. Eucalyptol (EPTL), a monoterpene majorly present in the essential oil of eucalyptus is well known for curing respiratory infections. Hence, the present study investigated the anti-biofilm efficacy of EPTL against the laboratory strains and clinical isolates of Candida to delineate its mode of action. The effect of EPTL on the viability, biofilm formation, and mature biofilm of Candida strains was studied. Furthermore, its effect on cell cycle arrest, mitochondrial membrane potential (MMP), ROS generation, germ tube formation, ergosterol content and transcriptional expression of selected genes was also investigated. EPTL exhibited anti-biofilm activity against mature and developing biofilm of Candida albicans and Candida glabrata along with their clinical isolates. The biochemical components and enzyme activity were differentially modulated in EPTL-treated biofilm extracellular matrix. EPTL generated ROS and arrested cell cycle at the G1 /S phase in both the species, while altered MMP was recorded in C. glabrata. Transcriptional analysis evidenced for differential gene expression of selected ABC transporters, secreted hydrolytic enzymes, and cell wall biogenesis in C. albicans/C. glabrata upon treating with EPTL. The current data on anti-biofilm activity of EPTL establish its candidacy for drug development or as an adjuvant with existing antifungal formulations. Present investigation elucidates the mode of action of Eucalyptol as antifungal agent and would stand as a candidate for management of topical fungal infection.

ASSESSMENT OF PROBIOTIC PROPERTIES OF THE STRAIN LACTOBACILLUS PARACASEI D6 AND ITS ANTICANDIDAL ACTIVITIES

Objective: The objective of the study was to determine the probiotic properties of a strain Lactobacillus paracasei D6 (GenBank Accession No. KJ867173) and to assess its antifungal activities against certain Candida strains.
 Methods: Antifungal activities of Lactobacillus paracasei D6 was tested against Candida tropicalis BSS7 and C. albicans MTCC 3017. The cell-free extract was tested against the formation of biofilm and germ tube of the aforementioned Candida strains. Different probiotic activities such as tolerance to simulated gastrointestinal fluid, adhesion to hydrocarbons, and Caco-2 cell line were evaluated for the strain Lactobacillus paracasei D6. Inhibition of Candida strains to Caco-2 cell line was also tested. The strains were identified using gene sequencing followed by phylogenetic tree construction.
 Results: The probiotic properties of Lactobacillus paracasei D6 were found to be statistically comparable with a standard Lactobacillus plantarum MTCC 1407. The biofilm and germ tube formation patterns of the food spoilage isolate C. tropicalis BSS7 were found to be similar to the strain C. albicans MTCC 3017. Cell-free extract of Lactobacillus paracasei D6 exhibited minimum biofilm inhibitory concentration of 0.438 mg/ml against C. tropicalis BSS7, which was found to be sufficient to inhibit its germ tube formation. The adhesion of C. tropicalis BSS7 to the epithelial Caco-2 cell line was also significantly reduced by the antifungal metabolites.
 Conclusion: This work gives insight on possible virulent nature of the food isolate C. tropicalis BSS7. Exometabolites produced by L. paracasei D6 were able to inhibit growth, biofilm and germ tube formation of both the strains. With this work, the authors envisage the beginning of an alternative strategy for anticandidal therapy.

Minimal nutrient requirements for induction of germination of Aspergillus niger conidia

Aspergillus niger reproduces asexually by forming conidia. Here, the minimal nutrient requirements were studied that activate germination of A. niger conidia. To this end, germination was monitored in time using an oCelloScope imager. Data was used as input in an asymmetric model to describe the process of swelling and germ tube formation. The maximum number of spores (Pmax) that were activated to swell and to form germ tubes was 32.54% and 20.51%, respectively, in minimal medium with 50 mM glucose. In contrast, Pmax of swelling and germ tube formation was <1% in water or 50 mM glucose. Combining 50 mM glucose with either NaNO3, KH2PO4, or MgSO4 increased Pmax of swelling and germination up to 15.25% and 5.4%, respectively, while combining glucose with two of these inorganic components further increased these Pmax values up to 25.85% and 10.99%. Next, 10 mM amino acid was combined with a phosphate buffer and MgSO4. High (e.g. proline), intermediate and low (e.g. cysteine) inducing amino acids were distinguished. Together, a combination of an inducing carbon source with either inorganic phosphate, inorganic nitrogen or magnesium sulphate is the minimum requirement for A. niger conidia to germinate.

Tracking and assessment of Puccinia graminis f. sp. tritici colonization on rice phyllosphere by integrated fluorescence imaging and qPCR for nonhost resistance phenotyping

Fluorescence microscopy and qPCR-based pathogen tracking tools were developed to elucidate the growth and development of Puccinia graminis f. sp. tritici on leaves of nonhost plant (rice: Oryza sativa L.). Stem rust race-Puccinia graminis f. sp. tritici 40A was identified using Internal Transcribed Spacer (ITS) sequences and gene sequences coding for hypothetical protein (PGTG_08233_hypothetical protein, HP). A reliable qPCR-based quantitation assay was developed for stem rust fungus exploiting HP primers that yielded 377-bp amplicon in conventional PCR and a forma specialis-specific 146 bp in qPCR with a detection sensitivity of 250-fg genomic DNA. The fungal cell wall N-acetyl-glucosamine was green fluorescence labelled and visualized on propidium iodide-stained nonhost leaf. This technique along with scanning electron microscopy allowed imaging of various developmental structures of Puccinia graminis f. sp. tritici 40A on plant epiphytic and endophytic niches. The microscopy coupled with qPCR-based pathogen load estimation revealed that the nonhost (rice) and host (wheat) phyllosphere surface supported uredospore germination, germ tube formation, hyphal elongation, epiphytic growth, stomatal entry and endophytic growth of fungus in an identical manner. However, the nonhost plant did not show any sign of rusting caused by uredospore production, which instead displayed induced H2O2 accumulation, on leaf. Development of qPCR-based Puccinia quantitation and green fluorescent tag-based qualitative assessment of Puccini proliferation will facilitate nonhost resistance phenotyping not only in rice but also in other nonhost plants of stem rust pathogen.

Antifungal and anti-inflammatory potential of the endangered aromatic plant Thymus albicans

Thymus albicans is an endemic species of the Iberian Peninsula with a vulnerable conservation status. In an attempt to contribute to the valorization of this species, the present study brings new insights on the antifungal and anti-inflammatory mechanism of action of T. albicans essential oil. The antifungal activity of the oil and its major compounds was assessed for the first time against standard and clinically isolated strains of yeasts and filamentous fungi. The effect on the two major virulence factors of Candida albicans (germ tube formation and biofilm disruption) was considered in more detail. At 0.08 μL/mL, the oil inhibited C. albicans germ tube formation by more than 40% and decreased biofilm biomass at MIC values, thus pointing out its antivirulent potential. The anti-inflammatory activity of the essential oil was investigated on LPS-stimulated mouse macrophages (RAW 264.7) by evaluating the levels of several pro-inflammatory mediators, namely nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). T. albicans oil reduced the production of nitrites, a NO derived sub-product, at non-cytotoxic concentrations of 0.32 and 0.64 μL/mL, by 27 and 41%, respectively. In addition, the iNOS protein levels of essential oil pre-treated cells were reduced by 14%. Overall, the high essential oil yield of T. albicans as well as its bioactive effects at concentrations without cytotoxicity, encourage further studies on the potential pharmacological applications of this species. Furthermore, these results raise awareness for the need to preserve endangered species that may hold relevant medicinal value.