Inhibited Germ Tube Formation Research Articles

Farnesol Concentrations Required To Block Germ Tube Formation in Candida albicans in the Presence and Absence of Serum

Concentrations of (E,E)-farnesol needed to inhibit germ tube formation were determined for Candida albicans strains A72 and SC5314 by using six different conditions known to trigger germination. For defined media, 1 to 2 microM farnesol was sufficient. However, with serum at 2 to 20%, up to 250 microM farnesol was required. Farnesol blocked germ tube formation but did not block elongation of existing germ tubes.

Antifungal activity ofLavandula angustifoliaessential oil againstCandida albicansyeast and mycelial form

The antifungal activity of the essential oil of Lavandula angustifolia Mill. (lavender oil) and its main components, linalool and linalyl acetate, was investigated against 50 clinical isolates of Candida albicans (28 oropharyngeal strains, 22 vaginal strains) and C. albicans ATCC 3153. Growth inhibition, killing time and inhibition of germ tube formation were evaluated. The chemical composition of the essential oil was determined by gas chromatography and mass spectrometry. Lavender oil inhibited C. albicans growth: mean minimum inhibitory concentration (MIC) of 0.69% (vol./vol.) (vaginal strains) and 1.04% (oropharyngeal strains); mean MFC of 1.1% (vaginal strains) and 1.8% (oropharyngeal strains). Linalool was more effective than essential oil: mean MIC of 0.09% (vaginal strains) and 0.29% (oropharyngeal strains); mean MFC of 0.1% (vaginal strains) and 0.3% (oropharyngeal strains). Linalyl acetate was almost ineffective. Lavender oil (2%) killed 100% of the C. albicans ATCC 3153 cells within 15 min; linalool (0.5%) killed 100% of the cells within 30 s. The essential oil inhibited germ tube formation (mean MIC of 0.09%), as did the main components (MIC of 0.11% for linalool and 0.08% for linalyl acetate). Both the essential oil and its main components inhibited hyphal elongation of C. albicans ATCC 3153 (about 50% inhibition at 0.016% with each substance). Lavender oil shows both fungistatic and fungicidal activity against C. albicans strains. At lower concentrations, it inhibits germ tube formation and hyphal elongation, indicating that it is effective against C. albicans dimorphism and may thus reduce fungal progression and the spread of infection in host tissues.

In vitro inhibitory effect of Streblus asper leaf-extract on adhesion of Candida albicans to human buccal epithelial cells

This in vitro study aimed at determining the effects of a sublethal concentration of Streblus asper Lour (Moraceae) leaf ethanolic extract on adherence of Candida albicans to human buccal epithelial cells (HBEC). The minimum concentration of Streblus asper leaf ethanolic extract (SAE) that significantly reduced adherence ( P < 0.05) after a 1-h exposure was 15.6 mg/ml. However, there was a significant reduction ( P < 0.05) of candidal adhesion to HBEC after 1-min exposure to 125 mg/ml of SAE. Pre-treatment of either Candida or HBEC, or both, with 125 mg/ml of SAE for 1 h resulted in reduced adherence. SAE at concentrations of 125 and 250 mg/ml also showed 41 and 61% inhibition of germ tube formation, respectively, which might affect adherence. These findings indicate that the sublethal concentration of SAE may modulate candidal colonization of the oral mucosa thereby suppressing the invasive potential of the pathogen.

Regulation ofCandida albicansMorphogenesis by Fatty Acid Metabolites

Candida albicans is an opportunistic dimorphic fungus that inhabits various host mucosal sites. Conversion from the yeast to the hyphal form has been associated with increased virulence and mucosal invasiveness. C. albicans morphogenesis is regulated by multiple signals and signaling pathways. However, signals that control morphogenesis in vivo are unknown. We investigated the effects of host long chain fatty acids, eicosanoids, and bacterial short chain fatty acids on control of germination. None of the C18 or C20 fatty acids tested had an effect on enhancing germ tube formation (arachidonic acid, oleic acid, linolenic acid, or gamma-linolenic acid). Among the different eicosanoids, both prostaglandin E2 and thromboxane B2 significantly enhanced serum-induced germination by C. albicans. Addition of antiprostaglandin or antithromboxane antibodies to serum alone inhibited germ tube formation by almost 30%, while control antibody had no effect, indicating that these eicosanoids are major morphogenic factors in the serum. Since these molecules also bind to albumin, this may also explain the hyphal transforming activity in serum that associates with albumin. Interestingly, short chain fatty acids (butyric acid), the product of lactic acid bacteria (LAB), inhibited germination. In addition, LAB culture supernatants as well as live LAB also inhibited C. albicans morphogenesis. Overall, these results indicate that fatty acid metabolites and fatty acid pathways can up-regulate and down-regulate germination in C. albicans.

Antifungal activity of Thymus oils and their major compounds.

The increasing recognition and importance of fungal infections, the difficulties encountered in their treatment and the increase in resistance to antifungals have stimulated the search for therapeutic alternatives. Essential oils have been used empirically. The essential oils of Thymus (Thymus vulgaris, T. zygis subspecies zygis and T. mastichina subspecies mastichina) have often been used in folk medicine. The aim of the present study was to evaluate objectively the antifungal activity of Thymus oils according to classical bacteriological methodologies - determination of the minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) - as well as flow cytometric evaluation. The effect of essential oils upon germ tube formation, an important virulence factor, was also studied. The mechanism of action was studied by flow cytometry, after staining with propidium iodide. The chemical composition of the essential oils was investigated by gas chromatography (GC) and gas chromatography/mass spectroscopy (GC/MS). The antifungal activity of the major components (carvacrol, thymol, p-cymene and 1,8-cineole) and also possible interactions between them were also investigated. The essential oils of T. vulgaris and T. zygis showed similar antifungal activity, which was greater than T. mastichina. MIC and MLC values were similar for all the compounds tested. At MIC values of the essential oils, propidium iodide rapidly penetrated the majority of the yeast cells, indicating that the fungicidal effect resulted primarily from an extensive lesion of the cell membrane. Concentrations below the MIC values significantly inhibited germ tube formation. This study describes the potent antifungal activity of the essential oils of Thymus on Candida spp., warranting future therapeutical trials on mucocutaneous candidosis.

Sensitive assay for antifungal activity of glucan synthase inhibitors that uses germ tube formation in Candida albicans as an end point.

We implemented a simple, sensitive, objective, and rapid cellular assay to reveal the antifungal activity of a novel class of glucan synthase inhibitors. The assay, especially useful for early drug discovery, measures the transformation of Candida albicans from the yeast form to the hyphal form. Test compounds were ranked by potency (50% inhibitory concentration) and efficacy (percent inhibition of germ tube formation); the intra-assay coefficients of variation for these parameters were 17 and 5%, respectively. The germ tube formation assay proved useful for the early-stage antifungal characterization of a novel class of glucan synthase inhibitors discovered at Pharmacia. Drug concentrations required in this assay to inhibit germ tube formation were lower for 90% of the novel compounds than the concentrations required to determine MICs. The method may have utility for other mechanistic classes of antifungal compounds during the hit-to-lead transition of drug discovery.

Chemical Composition and Antifungal Activity of the Essential Oil ofOriganum virensonCandidaSpecies

The composition and the antifungal activity of the essential oil of Origanum virens on Candida species were studied. The essential oil was obtained from the aerial parts of the plant by hydrodistillation and analyzed by GC and GC-MS. The oil was characterized by its high content of carvacrol (68.1 %) and its biogenetic precursors, gamma-terpinene (9.9 %) and p-cymene (4.5 %). The minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) were used to evaluate the antifungal activity against Candida strains (7 clinical isolates and 3 ATCC type strains). The inhibition of germ tube formation and flow cytometry, using the fluorescent probe propidium iodide (PI), were used to evaluate their mechanisms of action. MIC and MLC values were similar for most tested strains, ranging from 0.16 to 0.32 microL/mL. Concentrations lower than MIC values strongly prevent germ tube formation. The fungicidal effect is primarily due to an extensive lesion of the membrane.

Inhibition of hyphal growth of Candida albicans by activated lansoprazole, a novel benzimidazole proton pump inhibitor.

The effect of activated lansoprazole (AG 2000), a novel benzimidazole proton pump inhibitor, against hypha formation of Candida albicans was examined in hypha-forming medium pH 7 (HFM7) after 20 h. AG 2000, at 50-800 microM, did not inhibit germ tube formation. However, it inhibited elongation of germ tubes to form hyphae and favored conversion of germ tubes to resume yeast growth at concentrations of > or =200 microM. Pre-treatment of AG 2000 with a sulfhydryl reagent (1:1), such as 2-mercaptoethanol. blocked the inhibitory property of AG 2000 on hypha formation.

Effect of pH, carbon source and K+ on the Na+ -inhibited germ tube formation of Candida albicans

Effect of pH, carbon source and K+ on the Na+ -inhibited germ tube formation of Candida albicans

Inhibition of germ tube formation by Candida albicans by local anesthetics: an effect related to ionic channel blockade.

Formation of germ tubes by Candida albicans has been assumed as a putative virulence factor. Local anesthetics (LAs), e.g., lidocaine and bupivacaine, are known to inhibit germ tube formation. The study confirmed this observation for the novel drug ropivacaine, although it was less potent than the former two drugs. Hypothesizing that the effect is due to blockading ionic channels, we exposed Candida albicans to selective calcium blockers, i.e., nifedipine and verapamil, and to a general blocker of ionic channels, i.e., lanthanum. All blockers inhibited germ tube formation. The effect was dose-dependent and pH-independent. Addition of calcium reverted the effect of the blockers as well as the effect of lidocaine and ropivacaine. The study suggests that the inhibitory effect of LAs on germ tube formation by C. albicans is due to blockade of ionic channels, particularly calcium channels. Therefore, LAs can affect morphology and probably also the pathogenesis of C. albicans.

Anticandidal activity of SPA-S-843, a new polyenic drug

The activity of a new, soluble and stable polyene (SPA-S-843) against Candida albicans was assessed by contact and culture tests and by inhibition of germ-tube formation. The drug demonstrated a higher contact activity and lower MICs than amphotericin B. This antimicrobial activity was more evident under acid pH and low ionic strength. In addition, the ability of SPA-S-843 to inhibit Candida sp. conversion from yeast to mycelial form was evident at low drug concentrations (0.25-0.62 mg/L).

Effect of pH, carbon source and K+ on the Na+-inhibited germ tube formation of Candida albicans.

The effect of pH, carbon source and K+ on the Na+ -inhibited germ tube formation of the pathogenic fungus Candida albicans was examined in the arginine-phosphate modified (APM) medium. All C. albicans cells formed germ tubes in APM medium at pH 5.0-9.0. Na+ inhibited germ tube formation in a concentration dependent manner ranging from 0.2 to 1.0 M, and was further influenced by the pH of the medium. The inhibitory effect of Na+ was lowest at pH 8.0, and germ tube formation ceased at 1.0 M Na+ for any pH (4.0-9.0). At pH > or = 6.0, non-germ tube-forming cells did not show yeast growth; whereas at pH < or = 5.0, Na+ inhibited only germ tube formation but did not inhibit yeast growth. The inhibitory effect of Na+ was stronger in glucose medium than in galactose medium as carbon source. K+, at 0-0.8 M, had almost no effect on germ tube formation. However, in the presence of Na+, a very low concentration of K+ (0.5 mM) was able to release the cells from Na+ arrest and produced an increase in the rate as well as the percentage of germ tube formation. Intracellular Na+/K+ ratios increased with the increase in extracellular Na+ concentration, whereas the ratios decreased and remained within nontoxic levels when the extracellular K+ concentration was increased.

Further characterization of human salivary anticandidal activities in a human immunodeficiency virus-positive cohort by use of microassays.

Salivary anticandidal activities play an important role in oral candidal infection. R. P. Santarpia et al. (Oral Microbiol. Immunol. 7:38-43, 1992) developed in vitro anticandidal assays to measure the ability of saliva to inhibit the viability of Candida albicans blastoconidia and the formation of germ tubes by C. albicans. In this report, we describe modifications of these assays for use with small volumes of saliva (50 to 100 microl). For healthy subjects, there is strong inhibition of blastoconidial viability in stimulated parotid (75%), submandibular-sublingual (74%), and whole (97%) saliva, as well as strong inhibition of germ tube formation (>80%) for all three saliva types. The susceptibility of several Candida isolates to inhibition of viability by saliva collected from healthy subjects is independent of body source of Candida isolation (blood, oral cavity, or vagina) or the susceptibility of the isolate to the antifungal drug fluconazole. Salivary anticandidal activities in human immunodeficiency virus (HIV)-infected patients were significantly lower than those in healthy controls for inhibition of blastoconidial viability (P < 0.05) and germ tube formation (P < 0. 001). Stimulated whole-saliva flow rates were also significantly lower (P < 0.05) for HIV-infected patients. These results show that saliva of healthy individuals has anticandidal activity and that this activity is reduced in the saliva of HIV-infected patients. These findings may help explain the greater incidence of oral candidal infections for individuals with AIDS.

Inhibition of germ tube formation, filamentation and ergosterol biosynthesis in Candida albicans treated with 6-amino-2-n-pentylthiobenzothiazole.

Three representative Candida albicans strains were selected out of 26 clinical isolates and strains from culture collections on the basis of their high level of conversion to germ tubes and mycelial form at mycelium-promoting culture conditions, and on their different sensitivity to 6-amino-2-n-pentylthiobenzothiazole (APB). When these strains were treated with APB at mycelium-promoting culture conditions, a concentration-dependent decrease in the proportion of germ tubes and hyphae was observed, while the proportion of the yeast from increased. When non-saponifiable lipids were extracted from these cultures and analyzed, a concentration-dependent decrease in ergosterol and an increase in 4-methylated sterols was observed. However, the sensitivity of sterol biosynthesis did not directly relate to the sensitivity of the morphological conversion, and was exhibited at higher concentrations of APB. On the basis of these results it is suggested that the inhibition of germ tube formation and filamentation is not a consequence of inhibition of ergosterol biosynthesis in APB-treated C. albicans.

Is the lack of concurrence of bacterial vaginosis and vaginal candidosis explained by the presence of bacterial amines?

Objective and STUDY DESIGN: We report for the first time an inhibitory effect on cell division and germ tube formation by Candida albicans and strains of other Candida species by putrescine and cadaverine. Results: Both bacterial amines showed a dose-dependent inhibition of germ tube formation by C albicans, as well as budding (inhibition of cell division) of strains of other Candida species (ie, C glabrata, C krusei, and C tropicalis). Conclusions: We hypothesize that the presence of these and possibly other bacterial amines produced by anaerobes in the vaginal flora and seen in bacterial vaginosis, as in the healthy gut, may explain why candidosis is rarely seen in these instances. (Am J Obstet Gynecol 1999;181:367-70.)

In vitro effect of fibrinogen on Candida Albicans germ tube formation

Pseudohyphae formation by Candida albicans blastoconidia, as seen in vaginal smears, is a phenotypical change commonly assumed to mean fungal invasiveness, i.e. not mere colonization. C. albicans forms germ tubes in vitro in the presence of serum. In our search for inhibitory components of germ tube formation, we decided to study fibrinogen. The inhibition of germ tube formation by clinical isolates of C. albicans was evaluated in the presence of serial concentrations of fraction I, type IV and fraction I, type Is of fibrinogen from bovine plasma. Fibrinogen showed a dose-dependent, pH-independent inhibitory effect on the germ tube formation by C. albicans.

The effect of limited exposure to antifungal agents on the germ tube formation of oral Candida albicans

Candidal adherence has been implicated as the first step in the pathogenesis of oral candidosis, and germ tube formation by Candida albicans has been attributed as a co-factor that promotes adherence. Oral candidosis is treated with polyenes and the azole group of antifungal agents. As the intraoral concentrations of antifungals fluctuate considerably due to the dynamics of the oral cavity, we investigated the effect of short exposure to sub-lethal concentrations of antifungals on the germ tube formation of Candida albicans. After determining the minimum inhibitory concentration (MIC) of the antifungal agents, ten oral isolates of Candida albicans were exposed to sub-lethal concentrations of nystatin (6xMIC), amphotericin B (8xMIC), 5-fluorocytosine (8xMIC), ketoconazole (4xMIC) and fluconazole (4xMIC), for 1 h. Following removal of the antifungal agent and subsequent incubation in a germ tube-inducing medium, the germ tube formation of these isolates was quantified. When compared with the controls, exposure to nystatin and amphotericin B almost completely inhibited germ tube formation of all the isolates (mean percentage reduction of 97.68 and 97.52%, respectively; P<0.0001), while ketoconazole suppressed this activity to a lesser degree (30.84%; P=0.0174). However, 5-fluorocytosine- and fluconazole-mediated germ tube suppression was minimal (12.63 and 15.93%, respectively; P=0.3255 and P=0.3791). In clinical terms, these findings indicate that short exposure to sub-therapeutic levels of commonly prescribed antifungals may modulate candidal germ tube formation, and thereby the clearance of the organisms from the oral cavity.

The Effect of the New Triazole, Voriconazole (UK-109,496), on the Interactions of Candida albicans and Candida krusei with Endothelial Cells

In this study, we investigated how voriconazole affects specific endothelial cell interactions utilizing both fluconazole-susceptibleS and resistantR Candida albicans strains (C. albicanss and C. albicansR, respectively) as well as Candida krusei. Our data show that exposing C. albicansS to voriconazole significantly reduced its adherence to endothelial cells (p <0.001). The adherence of C. albicansR to endothelial cells was not affected by treatment with either antifungal agent. Exposure of C. albicans to both agents inhibited germ tube formation; however, voriconazole showed higher ability in inhibiting germination as compared with fluconazole. The effect of antifungals on germination was also tested during co-incubation of yeast cells with endothelial cells. Pretreated C. albicansS cells germinated on endothelial cells in the presence of voriconazole or fluconazole. However, the degree of germination was reduced by 81% and 16%, respectively. Similar results were observed with C. albicansR. Our data demonstrate that voriconazole treatment reduced the median germ tube length of C. albicansS and C. albicansR by approximately 60%, whereas fluconazole reduced the germ tube length of these strains by 27% and 63%, respectively (P < 0.0001 for each comparison). We compared the efficacy of voriconazole and fluconazole in protecting endothelial cells against damage caused by C. albicansS, C. albicansR, and C. krusei. Voriconazole and fluconazole reduced C. albicans-mediated endothelial cell injury by about 90% and 40%, respectively (P < 0.01 for each comparison). Additionally, voriconazole treatment significantly reduced C. krusei-mediated injury to endothelial cells by 69% (P < 0.01), whereas fluconazole did not exhibit significant protection (P < 0.6). These results demonstrate that voriconazole, in addition to its direct inhibitory activity against fungi, may act against Candida spp. by interfering with critical host/parasite interactions, such as adherence and endothelial cell damage, as well as germination. Therefore, this triazole represents a new and promising agent for the treatment of disseminated candidal infections caused by both fluconazole-susceptible and-resistant species.

Regulation of chitin synthase activity in the dimorphic fungus Benjaminiella poitrasii by external osmotic pressure

The effects of changes in external osmotic pressure on chitin synthase activity of a dimorphic fungus, Benjaminiella poitrasii, have been investigated. Mycelial and yeast cells incubated in medium of low osmolality (distilled water, 0 mOsm) for 10 min had 2–3-fold higher specific activities of native chitin synthase in mixed membrane preparations than cells that had been subjected to a high osmolality medium (1.2 M sorbitol in distilled water, 1612 mOsm). Cells suspended in media of different osmolalities for 10 min were also affected in the extent of germ tube formation. Germ tube formation was highest in cells incubated in low osmolality medium. The addition of protein phosphatase inhibitors (cyclosporin A, 1.2 μg/ml; cantharidin, 20 μM) abolished the effect of hypo-osmotic stress on chitin synthase activation of yeast mixed membrane preparations. The presence of protein kinase inhibitors (genistein, 40 μg/ml; H-7, 100 μM) and a Ca 2+ channel blocker (verapamil, 50 μM) reduced chitin synthase activity to 50–60% of that observed in cells under hypo-osmotic shock. These inhibitors also inhibited germ tube formation. This suggests that chitin synthase activity and yeast hyphal morphogenesis are both subject to regulation by osmotic pressure, phosphorylation and calcium.

Changes in the cell wall glycoprotein composition of Candida albicans associated to the inhibition of germ tube formation by EDTA

Changes in the cell wall glycoprotein composition of Candida albicans associated to the inhibition of germ tube formation by EDTA