Prophylaxis of Antifungal Drugs against Systemic Fungemia induced by Oral Candidiasis in Mice.

Oral mucositis is highly prevalent among the elderly, for whom oral care is often difficult. Oral mucositis, such as candidiasis, can induce systemic fungemia. Antifungal prophylaxis may be useful in such cases to prevent systemic fungemia; however, studies on this are limited. The objective of this study was to demonstrate the effectiveness of antifungal prophylaxis to prevent systemic Candida dissemination compared to oral care using a mice model. Oral candidiasis was induced using chemotherapy and inoculation with C. albicans in 8-week-old male mice. Group A was given oral care, Group B was orally administered an antifungal drug, Group C was intravenously administered an antifungal drug, and Group D was used as the negative control group. Macroscopic features of the tongue surface, colony forming units (CFU) on the tongue, and blood culture for C. albicans were evaluated. CFU was significantly higher in Group A than in Groups B and C. The oral care group, but not the groups administered antifungal agents, showed significantly higher positive numbers of animals with C. albicans in the blood as compared to the control group, indicating the effectiveness of antifungal prophylaxis over oral care. Antifungal prophylaxis may be an option for the prevention of systemic fungemia in individuals with difficulty in oral care.

Effect of Systemic Administration of Amitriptyline on Oral Microbes in Rats.

BACKGROUND/AIM: Amitriptyline is a major tricyclic antidepressant that is also used to relieve chronic orofacial pain. Recently, alterations in gut flora due to various antidepressants have been demonstrated. However, it remains unknown how antidepressants affect the oral environment, including microbiota and innate immunity. The aim of this study was to investigate the effects of amitriptyline on oral microflora and antimicrobial peptides. MATERIALS AND METHODS: Sprague-Dawley rats were intraperitoneally injected with amitriptyline for 2 weeks. The DNA extracted from the oral swabs were used to perform 16SrRNA sequencing to evaluate the oral microbiome. Quantitative RT-PCR was performed to evaluate the mRNA levels of antimicrobial peptides in the buccal tissues. RESULTS: No significant differences in salivary flow rates were observed between the amitriptyline and control groups. Taxonomic analysis showed significant alterations in bacteria such as Corynebacterium, Rothia, and Porphyromonas due to amitriptyline administration. The beta diversity showed significant differences between the amitriptyline and control groups. Additionally, the predicted metagenome functions were significantly different between the two groups. The mRNA expression levels of antimicrobial peptides in the amitriptyline group were significantly higher as compared to controls. CONCLUSION: Systemic administration of amitriptyline may affect the oral environment, including oral microbes and innate immunity in the oral mucosa.

White-to-opaque switching is involved in the phospholipase B production of Candida dubliniensis on Price's egg yolk agar.

Measuring the production of Candida dubliniensis (C. dubliniensis) phospholipase B (PLase B) by the Price's method has long been considered to be unattainable because the levels of PLase produced are undetectable. In this study, C. dubliniensis, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis and C. tropicalis were shown to produce PLase B and form clear white zones around their colonies when peptone, a component of the original Price's egg yolk (OP) agar, is replaced with a yeast nitrogen base (YNB). This new medium is named modified Price's (MP) agar. Based on this finding, we propose a new modified Price's (NMP) agar containing 0.75% peptone and 0.25% YNB, which enabled measurement of PLase B production by C. dubliniensis and C. albicans with results consistent with those obtained for C. albicans grown on OP agar. We strongly believe that the MP and NMP agars are very useful for screening PLase B production by C. dubliniensis and non-albicans Candida spp. Moreover, the addition of several bioactive agents (the proteinase inhibitors pepstatin A and saquinavir, the calcineurin inhibitors cyclosporine A and tacrolimus, the cell-permeable cAMP analog dBcAMP, and the quorum-sensing molecule farnesol) to the OP agar enhanced PLase B production by C. dubliniensis. During the course of our study to clarify the reason why PLase B was not produced, we found that C. dubliniensis cells grown on OP agar undergo a white-to-opaque transition, which may explain why they showed minimal production of PLase B on this medium.

Systemic hematogenous dissemination of mouse oral candidiasis is induced by oral mucositis.

The causes of fungemia include immunosuppression and neutropenia stemming from diverse factors as well as the placement of central venous catheters. However, the relationship between fungemia and the oral cavity has not been substantiated. In this study, we explored the pathological conditions of Candida albicans-derived oral candidiasis in a mouse model, which always develops oral mucositis as a complication. In oral candidiasis, the hyphae of C. albicans are believed to primarily invade the stratum granulosum, but not the subepithelium, of the mucous membrane. We provide histological evidence that in concomitant oral mucositis, the hyphae infiltrate the subepithelium and blood vessels. Blood cultures and tissue samples revealed the onset of fungemia only in the mucositis-induced groups. Positive numbers of colony-forming units were found in groups A (chemotherapy), B (chemotherapy + mucositis) and C (mucositis), but were highest in group B. Some organs revealed positive CFU in groups B and C. The presence of fungal DNA in blood plasma and tissue was confirmed by PCR. The fungal DNA frequency was significantly higher in the mucositis group when compared with the non-mucositis group. The results suggest that fungi first invade the subepithelium and then the blood vessels, from which they disseminate throughout the body, and that oral mucositis is an important risk factor for fungemia. This study clearly demonstrates the relationship between oral mucositis, fungemia, and the potential systemic fungal dissemination, which has not been previously proven. Our findings highlight the importance of oral care for patients at risk of fungemia.

[Virulence of Candida dubliniensis in comparison with Candida albicans using an experimental model of mouse oral candiddiasis].

Certain species of Candida are known as opportunistic fungal pathogens and Candida albicans has especially been isolated oral candidiasis patients at high frequency as a result of its strong pathogenicity. Recently C. dubliniensis is isolated mainly from immunocompromised patients, but is also detected from healthy persons. C. dubliniensis has similar cell morphology and molecular biological properties to C. albicans. Thus, in order to clarify the pathogenicity of C. dubliniensis, the activities of two extracellular enzymes, phospholipase (PL) and proteinase (PT), were measured, and pathological features were compared using mice. PL activity was examined in the improved Price's PL activity assay. In brief, the white precipitation zone was detected by spraying NaCl on egg yold plates without NaCl after colonies had grown. PL activity was no detected in any of the 31 C. dubliniensis strains tested. On the other hand, PT acitivty of C. dubliniensis was almost equivalent to that of C. albicans. Although we attempted to make an experimental model of mouse oral candidiasis using C. dubliniensis in yeast form as an inoculum following the conventional method, oral candidiasis did not develop in any mice. Thrush was successfully developed after inoculation with mycelial form cells, and there was no significant difference in histopathological findings of the thrush in comparison with C. albicans. These results strongly suggest that the two enzymes, PT and PL, do not play a crusial role in the establishment of mouse oral experimental candidiasis by C. dubliniensis.

Abc1p is a multidrug efflux transporter that tips the balance in favor of innate azole resistance in Candida krusei.

Most Candida krusei strains are innately resistant to fluconazole (FLC) and can cause breakthrough candidemia in immunocompromised individuals receiving long-term prophylactic FLC treatment. Although the azole drug target, Erg11p, of C. krusei has a relatively low affinity for FLC, drug efflux pumps are also believed to be involved in its innate FLC resistance. We describe here the isolation and characterization of Abc1p, a constitutively expressed multidrug efflux pump, and investigate ERG11 and ABC1 expression in C. krusei. Examination of the ERG11 promoter revealed a conserved azole responsive element that has been shown to be necessary for the transcription factor Upc2p mediated upregulation by azoles in related yeast. Extensive cloning and sequencing identified three distinct ERG11 alleles in one of two C. krusei strains. Functional overexpression of ERG11 and ABC1 in Saccharomyces cerevisiae conferred high levels of resistance to azoles and a range of unrelated Abc1p pump substrates, while small molecule inhibitors of Abc1p chemosensitized C. krusei to azole antifungals. Our data show that despite the presence of multiple alleles of ERG11 in some, likely aneuploid, C. krusei strains, it is mainly the low affinity of Erg11p for FLC, together with the constitutive but low level of expression of the multidrug efflux pump Abc1p, that are responsible for the innate FLC resistance of C. krusei.

Diversity of laccase among Cryptococcus neoformans serotypes.

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra-serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper-binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.

Clinical safety of licorice flavonoid oil (LFO) and pharmacokinetics of glabridin in healthy humans.

OBJECTIVE: Licorice flavonoids have various physiological activities such as abdominal fat-lowering, hypoglycemic and antioxidant effects. Licorice flavonoid oil (LFO: Kaneka Glavonoid Rich Oil) is a new dietary ingredient containing licorice flavonoids dissolved in medium-chain triglycerides (MCT). Glabridin is one of the bioactive flavonoids included specifically in licorice Glycyrrhiza glabra L. and is the most abundant flavonoid in LFO. In this study, we assessed the safety of LFO in healthy humans and determined the plasma concentration profile of glabridin as a marker compound. METHODS: A single-dose and two multiple-dose studies at low (300 mg), moderate (600 mg) and high (1200 mg) daily doses of LFO were carried out using a placebo-controlled single-blind design. In each study the safety of LFO and the pharmacokinetics of glabridin were assessed. RESULTS: Pharmacokinetic analysis in the single-dose study with healthy male subjects (n = 5) showed that glabridin was absorbed and reached the maximum concentration (Cmax) after approximately 4 h (Tmax), and then eliminated relatively slowly in a single phase with a T1/2 of approximately 10 h at all doses. The Cmax and AUC(0-24 h) increased almost linearly with dose. The multiple-dose studies with healthy male and female subjects for 1 week and 4 weeks suggested that plasma glabridin reached steady state levels within 2 weeks with a single daily administration of 300 to 1200 mg/day LFO. In these human studies at three dose levels, there were no clinically noteworthy changes in hematological or related biochemical parameters. All clinical events observed were mild and considered to be unrelated to LFO administration even after repeated administration for 4 weeks. CONCLUSION: These studies demonstrated that LFO is safe when administered once daily up to 1200 mg/day. This is the first report on the safety of licorice flavonoids in an oil preparation and the first report on the pharmacokinetics of glabridin in human subjects.

[Cell biology of respiration-deficient mutants of Candida albicans].

Respiration-deficient (petite) mutation is caused by hereditary impairment in mitochondrial functions. Yeasts have been grouped into "petite-positive" and "petite-negative" yeasts. Candida albicans has been regarded as a member of the petite-negative yeasts in which the respiration deficiency cannot be easily induced. We have succeeded in inducing the petite mutation in C. albicans by culturing in the presence of a chemical mutagen, acriflavine, at an elevated temperature. In the present review, we describe the cell biology of C. albicans petite mutants on the basis of experiments performed by our research group: namely, on respiratory activity and cytochrome composition, fine structures of cells and mitochondria, mitochondrial DNA structure, pathogenicity, oxidative stress sensitivity, generation of reactive oxygen species (ROS) and the roles of ROS in antifungal actions. We discuss also the usefulness of petite mutants in Candida research.

Comparisons of the laccase gene among serotypes and melanin-deficient variants of Cryptococcus neoformans.

Cryptococcus neoformans is a fungus causing life-threatening infections in immunocompromised hosts. Melanin production is a major virulence factor of this fungus and the initial steps of dihydroxyphenylalanine (DOPA)-melanin biosynthesis pathways are catalyzed by laccase. To understand phylogenetic relationships among serotypes of three varieties, partial sequences (about 600 bases) of the laccase gene (CNLAC1) were determined in a total of 64 strains, including 10 melanin-deficient variants. The phylogenetic tree constructed from the nucleotide sequence grouped the 64 strains into the clusters corresponding to the three varieties. The diversity of the fragment sequences was very minor among strains of each of var. grubii and var. neoformans. Strains in var. gattii, however, were subdivided into two groups, although differences between serotypes B and C were not large. The sequences of the melanin-deficient variants were almost completely homologous to those of the melanin-producing strains in the same serotype. Results of laccase assay and northern blot analysis suggested that the lower melanin production in the variants was associated with lower transcription of the laccase gene.

Differences in extracellular enzymatic activity between Candida dubliniensis and Candida albicans isolates.

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.