Kazumi Hayama

Effect of Streptococcus salivarius K12 on the In Vitro Growth of Candida albicans and Its Protective Effect in an Oral Candidiasis Model

ABSTRACT Oral candidiasis is often accompanied by severe inflammation, resulting in a decline in the quality of life of immunosuppressed individuals and elderly people. To develop a new oral therapeutic option for candidiasis, a nonpathogenic commensal oral probiotic microorganism, Streptococcus salivarius K12, was evaluated for its ability to modulate Candida albicans growth in vitro, and its therapeutic activity in an experimental oral candidiasis model was tested. In vitro inhibition of mycelial growth of C. albicans was determined by plate assay and fluorescence microscopy. Addition of S. salivarius K12 to modified RPMI 1640 culture medium inhibited the adherence of C. albicans to the plastic petri dish in a dose-dependent manner. Preculture of S. salivarius K12 potentiated its inhibitory activity for adherence of C. albicans. Interestingly, S. salivarius K12 was not directly fungicidal but appeared to inhibit Candida adhesion to the substratum by preferentially binding to hyphae rather than yeast. To determine the potentially anti-infective attributes of S. salivarius K12 in oral candidiasis, the probiotic was administered to mice with orally induced candidiasis. Oral treatment with S. salivarius K12 significantly protected the mice from severe candidiasis. These findings suggest that S. salivarius K12 may inhibit the process of invasion of C. albicans into mucous surfaces or its adhesion to denture acrylic resins by mechanisms not associated with the antimicrobial activity of the bacteriocin. S. salivarius K12 may be useful as a probiotic as a protective tool for oral care, especially with regard to candidiasis.

Suppression of tumor necrosis factor-alpha-induced neutrophil adherence responses by essential oils

BACKGROUND In aromatherapy, essential oils are used as anti-inflammatory remedies, but experimental studies on their action mechanisms are very limited. AIMS To assess their anti-inflammatory activities, effects of essential oils on neutrophil activation were examined in vitro. METHODS Neutrophil activation was measured by tumor necrosis factor-alpha (TNF-alpha)-induced adherence reaction of human peripheral neutrophils. RESULTS All essential oils tested at 0.1% concentration suppressed TNF-alpha-induced neutrophil adherence,and, in particular, lemongrass, geranium and spearmint oils clearly lowered the reaction even at 0.0125%. Similar inhibitory activities for the neutrophil adherence were obtained by their major constituent terpenoids: citral, geraniol, citronellol and carvone. In contrast, very popular essential oils, tea tree oil and lavender oil, did not display the inhibitory activity at the concentration. CONCLUSION Thus, some essential oils used as antiinflammatory remedies suppress neutrophil activation by TNF-alpha at a low concentration (0.0125-0.025 %) in vitro.

Suppression of neutrophil recruitment in mice by geranium essential oil.

BACKGROUND: In aromatherapy, essential oils are used as anti-inflammatory remedies, but experimental studies on their action mechanisms are very limited. AIMS OF THE STUDY: To assess their anti-inflammatory activities, the effects of essential oils on neutrophil recruitment in mice were examined in vivo. METHOD: The effect of essential oils on leukocyte and neutrophil recruitment induced 6 h after intraperitoneal injection of casein in mice was examined. RESULTS: Leukocyte recruitment into the peritoneal cavity in mice was suppressed by intraperitoneal injections of geranium, lemongrass and spearmint oils at the dose of 5 microl/mouse, but was not by tea tree oil. This recruitment was inhibited dose-dependently by geranium oil. The suppression of leukocyte recruitment resulted from inhibition of neutrophil accumulation. CONCLUSION: Some essential oils used as anti-inflammatory remedies suppress neutrophil recruitment into the peritoneal cavity in mice.

A d-octapeptide drug efflux pump inhibitor acts synergistically with azoles in a murine oral candidiasis infection model

Clinical management of patients undergoing treatment of oropharyngeal candidiasis with azole antifungals can be impaired by azole resistance. High-level azole resistance is often caused by the overexpression of Candida albicans efflux pump Cdr1p. Inhibition of this pump therefore represents a target for combination therapies that reverse azole resistance. We assessed the therapeutic potential of the D-octapeptide derivative RC21v3, a Cdr1p inhibitor, in the treatment of murine oral candidiasis caused by either the azole-resistant C. albicans clinical isolate MML611 or its azole-susceptible parental strain MML610. RC21v3, fluconazole (FLC), or a combination of both drugs were administered orally to immunosuppressed ICR mice at 3, 24, and 27 h after oral inoculation with C. albicans. FLC protected the mice inoculated with MML610 from oral candidiasis, but was only partially effective in MML611-infected mice. The co-application of RC21v3 (0.02 μmol per dose) potentiated the therapeutic performance of FLC for mice infected with either strain. It caused a statistically significant decrease in C. albicans cfu isolated from the oral cavity of the infected mice and reduced oral lesions. RC21v3 also enhanced the therapeutic activity of itraconazole against MML611 infection. These results indicate that RC21v3 in combination with azoles has potential as a therapy against azole-resistant oral candidiasis.

Therapeutic Effects of Cinnamaldehyde and Potentiation of its Efficacy in Combination with Methylcellulose on Murine Oral Candidiasis

We examined the therapeutic effects of cinnamaldehyde and the potentiation of those effects with cassia and cinnamaldehyde when combined with the food additive methylcellulose against murine oral candidiasis. When 19.5mg/ml of cinnamaldehyde was administered in the oral cavity of Candida infected mice, the oral symptoms were improved. Furthermore, when either a cassia or a cinnamaldehyde preparation in combination with methylcellulose was administered to oral candidiasis-inflicted mice, the therapeutic effects of cassia or cinnamaldehyde potentiated. Methylcellulose itself did not affect the oral symptoms or the viable number of C. albicans cells. GC/MS analysis showed that the dose of cinnamaldehyde remaining in the tongue tissue of mice treated with the cinnamaldehyde-methylcellulose mixture was higher than that in mice administered cinnamaldehyde alone, and also showed that cinnamaldehyde was not detected in the blood of any of the tested mice. These findings suggested that the combination of cassia or cinnamaldehyde and methylcellulose may be a useful prophylactic or therapeutic tool against oral candidiasis.

N-acetylglucosamine increases symptoms and fungal burden in a murine model of oral candidiasis

The amino sugar N-acetylglucosamine (GlcNAc) is an in vitro inducer of the hyphal mode of growth of the opportunistic pathogen Candida albicans. The development of hyphae by C. albicans is considered to contribute to the pathogenesis of mucosal oral candidiasis. GlcNAc is also a commonly used nutritional supplement for the self-treatment of conditions such as arthritis. To date, no study has investigated whether ingestion of GlcNAc has an effect on the in vivo growth of C. albicans or the pathogenesis of a C. albicans infection. Using a murine model of oral candidiasis, we have found that administration of GlcNAc, but not glucose, increased oral symptoms of candidiasis and fungal burden. Groups of mice were given GlcNAc in either water or in a viscous carrier, i.e., 1% methylcellulose. There was a dose-dependent relationship between GlcNAc concentration and the severity of oral symptoms. Mice given the highest dose of GlcNAc, 45.2 mM, also showed a significant increase in fungal burden, and increased histological evidence of infection compared to controls given water alone. We propose that ingestion of GlcNAc, as a nutritional supplement, may have an impact on oral health in people susceptible to oral candidiasis.

中鎖脂肪酸カプリン酸の Candida 菌糸形発育阻止作用と口腔カンジダ症治療効果

We assessed anti-C. albicans activities of the 4 fatty acids : caproic acid, caprylic acid, capric acid and lauric acid in vitro. All four inhibited not only the mycelial but also the yeast-form growth of Candida albicans. In particular, capric acid and caprylic acid inhibited Candida mycelia growth at very low concentrations. The effects of treatment of these two fatty acids on oral candidiasis were examined using a murine model. When 50 µl of capric acid (more than 48.8 µM) was administered three times into the oral cavity of Candida-infected mice, symptom scores of tongues of the mice were significantly improved. Histological studies of the capric acid-treated animals indicated that the fatty acid suppressed mycelial growth of the fungus on the tongue surface. These results suggest that all four fatty acids, and especially capric acid, have potential as substances supporting anti-Candida treatment.

テルピネン-4-オールと中鎖脂肪酸カプリン酸の併用によるCandida albicansの菌糸形発育阻止作用とマウス口腔カンジダ症への治療効果

The combined effect of terpinen-4-ol, the main component of tea tree oil, and capric acid against mycelial growth of Candida albicans and murine oral candidiasis was evaluated in vitro and in vivo. Mycelial growth of C. albicans was estimated by the Cristal violet method. Combination of these compounds revealed a potent synergistic inhibition of growth. Therapeutic efficacy of the combination was evaluated microbiologically in murine oral candidiasis, and its application of the compounds clearly demonstrated therapeutic activity. Based on these results, the combined agent of terpinen-4-ol and capric acid was discussed as a possible candidate for oral candidiasis therapy.

Antifungal activity in vitro and in vivo of a salmon protamine peptide and its derived cyclic peptide against Candida albicans.

Abstract Protamine peptide (PP) derived from salmon is a 14‐mer with 10 arginine residues. We investigated the in vitro and in vivo antifungal activity of PP against Candida albicans. PP showed a concentration‐dependent dual mode of action, with fungicidal activity and inhibitory activity for hyphal development in vitro. At lethal concentrations of PP, intracellular accumulation of PP was energy‐dependent but independent of endocytosis, and resulted in ATP efflux and the generation of reactive oxygen species in the cells. PP at sublethal concentrations inhibited hyphal development in C. albicans by binding to the cell surface. Though antifungal activity of PP was inactivated by high concentrations of NaCl, the antifungal activity of the synthetic cyclic (via a disulfide bond) form of PP (cyclic PP) was not. Cyclic PP also showed the concentration‐dependent dual mode of action, and had five‐fold greater antifungal activity than PP. The advantage of antifungal activity of cyclic PP compared with PP in vitro resulted in a high in vivo efficacy in a murine oral candidiasis model. Oral treatment with cyclic PP inhibited hyphal development of C. albicans on mouse tongues and protected against the development of severe candidiasis. This study shows the therapeutic potential of cyclic PP as an antifungal peptide against C. albicans.

Cell preparation method with trypsin digestion for counting of colony forming units in Candida albicans-infected mucosal tissues

Colony forming units (CFU) of Candida albicans in cell suspensions or homogenates prepared from C. albicans-infected tissues are not always accurate indicators of the severity of infection in mucosal tissues. In order to improve the reliability of CFU counts in the murine oral candidiasis model, we developed a new cell preparation method in which a dispersal process involving trypsin digestion was included in the processing of Candida albicans-infected tongue tissues. Trypsin digestion, which was added to the conventional method for preparing Candida suspension from tongue homogenates, improved the recovery yield as evidenced by an increase in Candida CFUs. This method also increased the number of planktonic Candida cells which could pass through a mesh filter, perhaps because the trypsin contributed to the break up of the complex mass of tissue-debris and C.albicans cells. Using this trypsin digestion technique, we confirmed the protective action of farnesol by a relative decrease in the number of viable Candida cells in homogenates of infected tongues, which was correlated with improvement of the symptoms of oral candidiasis. These results indicate that our new method of trypsin digestion is valuable in that it reflects the protective activity of some bioactive substances against mucosal candidiasis.