INTRODUCTION C. tropicalis and C. krusei have emerged as virulent species causing oral infections. Both have developed resistance to commonly prescribed azole antifungal agents. OBJECTIVE The study aimed to determine the effect of mouth rinses containing chlorhexidine digluconate (CHX), cetylpyridinium chloride (CPC) and their combination (CHX-CPC) on the growth of these strains. METHODS The minimal inhibition concentrations (MIC) of the mouth rinses were determined. The growth curves of the strains produced under the mouth rinse-treated and untreated conditions, as well as alterations to the morphology of the growth colonies and cells following the treatments were compared and analysed. RESULTS The MICs of CPC compared to CHX mouth rinses were found to be lower for both Candida sp. In the mixed formulation, CPC doubled the inhibitory effect of CHX towards both Candida sp., while CHX quadrupled the activity of CPC towards C. tropicalis. The growth colonies also appeared coarse, wrinkled and dried. CONCLUSION The profound effects shown may suggest the fungicidal activities of the mouth rinses incorporated with CHX, CPC or their combination on both C. tropicalis and C. krusei. Gargling using mouth rinses with such fungicidal activity would enhance a rapid reduction in the candidal population of patients with fungal infection.

Continued research toward the development of new antifungals that act via inhibition of glycosylphosphatidylinositol (GPI) biosynthesis led to the design of E1210. In this study, we assessed the selectivity of the inhibitory activity of E1210 against Candida albicans GWT1 (Orf19.6884) protein, Aspergillus fumigatus GWT1 (AFUA_1G14870) protein, and human PIG-W protein, which can catalyze the inositol acylation of GPI early in the GPI biosynthesis pathway, and then we assessed the effects of E1210 on key C. albicans virulence factors. E1210 inhibited the inositol acylation activity of C. albicans Gwt1p and A. fumigatus Gwt1p with 50% inhibitory concentrations (IC(50)s) of 0.3 to 0.6 μM but had no inhibitory activity against human Pig-Wp even at concentrations as high as 100 μM. To confirm the inhibition of fungal GPI biosynthesis, expression of ALS1 protein, a GPI-anchored protein, on the surfaces of C. albicans cells treated with E1210 was studied and shown to be significantly lower than that on untreated cells. However, the ALS1 protein levels in the crude extract and the RHO1 protein levels on the cell surface were found to be almost the same. Furthermore, E1210 inhibited germ tube formation, adherence to polystyrene surfaces, and biofilm formation of C. albicans at concentrations above its MIC. These results suggested that E1210 selectively inhibited inositol acylation of fungus-specific GPI which would be catalyzed by Gwt1p, leading to the inhibition of GPI-anchored protein maturation, and also that E1210 suppressed the expression of some important virulence factors of C. albicans, through its GPI biosynthesis inhibition.

Candida spp. are commensal microorganisms that are part of the microflora of different sites within the oral cavity. In healthy subjects, who have an unaltered immunological status, these yeasts do not cause disease. However, in immunosuppressed individuals whose condition may have been caused by diabetes mellitus, Candida spp. can express different virulence factors and may consequently become pathogenic. Studies have detected the presence of Candida spp. in periodontal sites of patients with chronic periodontitis, especially those that are immunologically compromised. However, the role of these microorganisms in the pathogenesis of periodontal disease is still unknown. The objectives of this study were:(1) to isolate and identify Candida albicans strains from subgingival sites of diabetic patients with chronic periodontitis;(2) to evaluate the following virulence factors; colony morphology, proteinase, phospholipase and hemolysin activities and cell surface hydrophobicity (CSH) under different atmospheric conditions; and (3) to determine the genetic patterns of these C. albicans isolates. Microbial samples were collected from subgingival sites and seeded on CHROMagar for subsequent identification of C. albicans by polymerase chain reaction (PCR). For the phenotypic tests, all strains of C. albicans were grown under reduced oxygen (RO) and anaerobiosis (ANA) conditions. Genotypes were defined by the identification through PCR of the transposable introns in the 25S rDNA. The results obtained relative to virulence factors were analyzed according to the atmospheric condition or genetic group, using Chi-square and Wilcoxon non-parametric tests. In this study, 128 strains were identified as C. albicans and of these, 51.6% were genotype B, 48.4% were genotype A and Genotype C was not found. Most of the strains were alpha-hemolytic in both atmospheric conditions, without a statistical difference. However, when comparing the genotypes, 46.1% of the genotype A strains were beta-hemolytic. In relation to colony morphology, 100% of the strains under ANA showed rough colonies, which were especially prevalent in genotype A isolates. In contrast, most of the colonies were smooth under RO. C. albicans strains did not produce proteinase and phospholipase activity in the total absence of oxygen. In RO, most strains had high proteinase activity and were positive by phospholipase tests (P < 0.05). Hydrophobicity was higher in anaerobiosis and was noted mainly for genotype A isolates. In conclusion, environmental oxygen concentration influenced the virulence factors of C. albicans strains isolated from subgingival sites of diabetic and periodontal patients. In addition, genotype A seems to be more virulent based on the phenotypic tests evaluated in this study.

OBJECTIVE The objective of this study was to compare the antimicrobial effect of mouthwashes containing Calendula officinalis L., Camellia sinensis (L.) Kuntze and 0.12% chlorhexidine digluconate on the adherence of microorganisms to suture materials after extraction of unerupted third molars. MATERIAL AND METHODS Eighteen patients with unerupted maxillary third molars indicated for extraction were selected (n=6 per mouthwash). First, the patients were subjected to extraction of the left tooth and instructed not to use any type of antiseptic solution at the site of surgery (control group). After 15 days, the right tooth was extracted and the patients were instructed to use the Calendula officinalis, Camellia sinensis or chlorhexidine mouthwash during 1 week (experimental group). For each surgery, the sutures were removed on postoperative day 7 and placed in sterile phosphate-buffered saline. Next, serial dilutions were prepared and seeded onto different culture media for the growth of the following microorganisms: blood agar for total microorganism growth; Mitis Salivarius bacitracin sucrose agar for mutans group streptococci; mannitol agar for Staphylococcus spp.; MacConkey agar for enterobacteria and Pseudomonas spp., and Sabouraud dextrose agar containing chloramphenicol for Candida spp. The plates were incubated during 24-48 h at 37ºC for microorganism count (CFU/mL). RESULTS The three mouthwashes tested reduced the number of microorganisms adhered to the sutures compared to the control group. However, significant differences between the control and experimental groups were only observed for the mouthwash containing 0.12% chlorhexidine digluconate. CONCLUSIONS Calendula officinalis L. and Camellia sinensis (L.) Kuntze presented antimicrobial activity against the adherence of microorganisms to sutures but were not as efficient as chlorhexidine digluconate.

[(n-Bu)Sn(2Ac4oClPh)Cl2](1),[(n-Bu)Sn(2Ac4oFPh)Cl2](2),[(n-Bu)Sn(2Ac4oNO2Ph)Cl2](3),[(n-Bu)Sn(2Bz4oClPh)Cl2](4),[(n-Bu)Sn(2Bz4oFPh)Cl2](5) and [(n-Bu)Sn(2Bz4oNO2Ph)Cl2](6) were obtained by reacting [(n-Bu)SnCl3] with 2-acetylpyridine-N4-orthochlorophenyl thiosemicarbazone (H2Ac4oClPh), 2-acetylpyridine-N4-orthofluorphenyl thiosemicarbazone (H2Ac4oFPh), 2-acetylpyridine-N4-orthonitrophenyl thiosemicarbazone (H2Ac4oNO2Ph), and with the corresponding 2-benzoylpyridine-derived thiosemicarbazones (H2Bz4oClPh, H2ABz4oFPh and H2Bz4oNO2Ph). The antifungal activity of the studied compounds was evaluated against several Candida species. Upon coordination of H2Bz4oNO2Ph to tin in complex (6) the antifungal activity increased three times against Candida albicans and Candida krusei and six times against Candida glabrata and Candida parapsilosis. The minimum inhibitory concentration (MIC) values of H2Ac4oNO2Ph and its complex (3) against C. albicans, C. parapsilosis and C. glabrata are similar to that of fluconazole. All studied compounds were more active than fluconazole against C. krusei.

The in vitro susceptibilities of 140 laboratory reference strains of fungi, including type strains, and 165 clinical yeast isolates from Japan towards isavuconazole compared with fluconazole (FLC), itraconazole (ITC), voriconazole and amphotericin B were measured. Broth microdilution methods based on Clinical and Laboratory Standards Institute (CLSI) methods were used for yeasts, and RPMI-MOPS medium semi-solidified with 0.2% low-melting-point agarose based on CLSI guidelines was used for moulds. The range of isavuconazole minimum inhibitory concentrations (MICs) was 0.0004-0.21 mg/L for Candida albicans, 0.0036-0.4 mg/L for Candida glabrata, 0.023-0.058 mg/L for Candida krusei, 0.0026-0.032 mg/L for Cryptococcus neoformans, 0.1-0.39mg/L for Aspergillus fumigatus and 0.2-0.39 mg/L for Aspergillus terreus. Isavuconazole was as active as ITC against the dimorphic true pathogenic fungi, with a range of MICs from <0.0004 mg/L to 0.0063 mg/L for Blastomyces dermatitidis and Histoplasma capsulatum. It was also active against uncommon dematiaceous fungi such as Exophiala spp. and Phialophora spp. as well as against dermatophytic species. Isavuconazole showed very good in vitro antifungal activity with a broad spectrum, including against FLC-resistant Candida spp., Aspergillus spp. and uncommon opportunistic fungal species. This is the first report of the in vitro susceptibility of Japanese clinical yeast isolates to isavuconazole. No cross-resistance was found to isavuconazole amongst FLC-resistant strains.

The ability of Candida albicans to act as an opportunistic fungal pathogen is linked to its ability to switch among different morphological forms. This conversion is an important feature of C. albicans and is correlated with its pathogenesis. Many conserved positive and negative transcription factors regulate morphogenetic transition of C. albicans. Here, we show the results of functional analysis of CaAFT2, which is an orthologue of the Saccharomyces cerevisiae AFT2 gene. We have cloned CaAFT2 which has an ability to complement the S. cerevisiae aft1Δ mutant strain growth defect. Interestingly, although disruption of the AFT2 gene did not affect cell growth in solid and liquid iron-limited conditions, the cell surface ferric reductase activity was significantly decreased. Importantly, deletion of AFT2 in C. albicans led to growth of a smooth colony with no peripheral hyphae. Moreover, virulence of an aft2Δ/aft2Δ mutant was markedly attenuated in a mouse model. Our results suggest that CaAft2p represents a novel activator and that it functions in ferric reductase activity, morphogenesis and virulence in C. albicans.

Summary The aim of the present study was to characterise phospholipase and proteinase activities of oral Candida isolates from 100 denture wearers and to study the relationship of these activities with denture stomatitis. Of 100 patients studied, 44 suffered from denture stomatitis. Specimens were collected by swabbing the denture and underlying mucosa. Isolates were previously identified by conventional mycological and genotypic methods. The phospholipase and proteinase activities were evaluated by agar plate methods. A total of 152 isolates were recovered from denture and underlying mucosa, including 101 Candida albicans, 18 Candida tropicalis, 14 Candida glabrata, 11 Candida guilliermondii, four Candida parapsilosis, two Saccharomyces cerevisiae and one isolate each of Candida dubliniensis and Candida krusei. Most C. albicans (97%) showed phospholipase activity; furthermore, the unique C. dubliniensis isolate showed a moderate phospholipase activity. The isolation of C. albicans (chi-square test, P = 0.0016) and phospholipase production by Candida spp.(chi-square test, P = 0.0213) was found to be significantly associated with denture stomatitis. Proteinase production was observed in <30% of isolates, and it was not related to the presence of denture stomatitis (P = 0.7675). Candida albicans isolates may produce both virulence factors, although the proteinase production was only observed in <30% of the isolates. Phospholipase production was exclusive of C. albicans and C. dubliniensis.

In this study, we measured time-kills and post-antifungal effects (PAFEs) for micafungin against Candida albicans (n=4), Candida glabrata (n=3), Candida parapsilosis (n=3) and Candida krusei (n=2) isolates and further characterised the PAFEs. Minimum inhibitory concentrations (MICs) were 0.5-1.0 mg/L against C. parapsilosis and 0.008-0.125 mg/L against the other species. Micafungin caused kills >1 log at 1 x MIC, 4 x MIC (range 1.19-3.10 log) and 16 x MIC (2.27-3.68 log), achieving fungicidal levels (> or = 3 log) against nine isolates. One-hour drug exposure during PAFE experiments resulted in kills of 0.73-2.88 log and 1.72-3.55 log at 4 x and 16 x MIC, respectively, achieving fungicidal levels against five isolates. Isolates of each species collected 8 h after a 1-h exposure to micafungin (4 x and 16 x MIC) were hypersusceptible to sodium dodecyl sulphate (SDS) and Calcofluor White. Cells tested during the PAFE period demonstrated cell wall disturbances as evident on electron micrographs as well as significant reductions in adherence to epithelial cells. Phagocytosis by J774 macrophages was significantly enhanced for three PAFE isolates tested. Micafungin is fungicidal and exerts PAFEs that kill diverse Candida spp., disturb cell walls of viable organisms, reduce adherence and enhance susceptibility to phagocytosis.

This study has been conducted to asses the in vitro activity of the novel triazole antifungal agent posaconazole against 123 clinically important isolates of yeasts. Susceptibility was tested using the Sensititre YeastOne microdilution commercial method. Minimun inhibitory concentrations (MICs) were determined at the recommended endpoints and time intervals. The activity of posaconazole against Candida glabrata was compared with those of fluconazole, itraconazole, ketoconazole and voriconazole. The most susceptible species to posaconazole were C. albicans, C. parapsilosis, C. tropicalis and C. dubliniensis. Candida glabrata was the least susceptible. The percentage of strains with MIC for posaconazole >/= 1 mg/L was 9%, all of them were C. glabrata. The species with MIC for itraconazole >/= 0.5 mg/L were 36%(41 C. glabrata, 1 C. krusei, 1 C. guilliermondii, 1 C. ciferrii). Candida glabrata strains resistant to fluconazole, ketoconazole and voriconazole were 8%, 4% and 4%, respectively. Posaconazole exhibited good activity to the majority of Candida species. However, it was similar to itraconazole and less active than ketoconazole and voriconazole against C. glabrata. Key words: Posaconazole. Yeasts. Candida glabrata. Rev Esp Quimioter 2009;22(3):139-143.