Cytokine responses of intestinal epithelial-like Caco-2 cells to non-pathogenic and opportunistic pathogenic yeasts in the presence of butyric acid

Candida albicans, Saccharomyces cerevisiae and their cell wall components, zymosan and glucan, have been shown to stimulate interleukin-8 (IL-8/CXCL-8) production by intestinal epithelial cell-like Caco-2 cells pre-cultured with 10 mM butyric acid. We examined in this study whether these yeasts also altered the production of other cytokines and cyclooxygenases (COXs) by Caco-2 cells. Culturing Caco-2 cells with 10 mM butyric acid and 15% FBS for 4 days enhanced the basal levels of mRNA encoding IL-6, IL-8, IL-18, monocyte chemoattractant protein (MCP)-1, stem cell factor, transforming growth factor (TGF)-beta1, TGF-beta3, tumor necrosis factor (TNF)-alpha, COX-1, and COX-2, but not of granulocyte-macrophage colony-stimulating factor (GM-CSF) and TGF-beta2. The inclusion of live S. cerevisiae or C. albicans further enhanced the production of IL-8, but not of the other cytokines and COXs. The non-pathogenic yeasts, C. kefyr, C. utilis, C. versatilis, Kluyveromyces lactis, K. marxianus, Schizosaccharomyces pombe and Zygosaccharomyces rouxii, used for the production of fermented foods and probiotics, and the opportunistic pathogens, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis, isolated from human tissue samples also enhanced IL-8 secretion by Caco-2 cells.     

Alveolar macrophage reaction to candida species

Candida species are increasingly important fungal pathogens. The reaction of rat alveolar macrophages (AM) to Candida albicans was compared with that to C. glabrata and C. krusei . Phagocytosis of C. glabrata was similar to that of C. albicans , but significantly slower for C. krusei due to reduced attachment. After opsonization, attachment of C. albicans and C. krusei to AM was significantly increased and there was no significant difference between the two species. The oxidative metabolism of AM with candida species was two to three times higher than that of the resting AM both during and 24 h after the phagocytosis. All three species showed a considerable fraction (5–10%) of phagolysosomes with pH ≥ 6·5 after 3 h and a smaller percentage (1%) after 24 h.     

Optically active antifungal azoles: synthesis and antifungal activity of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol

A series of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (11a-n) and (2R,3S)-2-(2,4-difluorophenyl)-3-(5-[2-[4-aryl-piperazin-1-yl]-ethyl]-tetrazole-1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol (12a-n) has been synthesized. The antifungal activity of compounds was evaluated by in vitro agar diffusion and broth dilution assay. Compounds 11d and its positional isomer 12d having 3-trifluoromethyl substitution on the phenyl ring of piperazine demonstrated significant antifungal activity against variety of fungal cultures (Candida spp. C. neoformans and Aspergillus spp.). The compound 12d showed MIC value of 0.12 microg/mL for C. albicans, C. albicans V-01-191A-261 (resistant strain); 0.25 microg/mL for C. tropicalis, C. parapsilosis ATCC 22019 and C. krusei and MIC value of 0.5 microg/mL for C. glabrata, C. krusei ATCC 6258, which is comparable to itraconazole and better than fluconazole. Further, compound 11d showed significant activity (MIC; 0.25-0.5 microg/mL) against Candida spp. and strong anticryptococcal activity (MIC; 0.25 microg/mL) against C. neoformans.     

焦性没食子酸联合唑类药物对念珠菌的抑菌作用

目的了解光滑念珠菌临床菌株的药敏情况以及中药单体焦性没食子酸联合唑类药物对念珠菌的抑菌作用。方法收集临床分离的光滑念珠菌116株、白念珠菌49株、热带念珠菌42株、克柔念珠菌4株和近平滑念珠菌13株,采用ATB FUNGUS3药敏试条检测光滑念珠菌的药敏情况;同时采用棋盘肉汤稀释法检测焦性没食子酸联合唑类药物对念珠菌的抑菌情况。结果116株光滑念珠菌中,14.66%(17株)的菌株对氟康唑耐药,22.41%(26株)对伊曲康唑表现为非野生型和81.03%(94株)对伏立康唑表现为非野生型。焦性没食子酸对5种念珠菌的抑菌情况,46.55%光滑念珠菌的MIC值为64μg/mL;34.69%白念珠菌的MIC值为64μg/mL;59.52%热带念珠菌的MIC值为64μg/mL;25%克柔念珠菌的MIC值为128μg/mL;46.15%近平滑念珠菌的MIC值为128μg/mL。唑类药物与焦性没食子酸联合用药时,100%、99.14%、99.14%的光滑念珠菌分别对氟康唑、伊曲康唑和伏立康唑表现为协同作用或相加作用,差异无统计学意义(P>0.05);而白念珠菌、热带念珠菌、克柔念珠菌和近平滑念珠菌均表现为无关作用和拮抗作用。与单独用药相比,联合用药时81.03%、68.1%、77.59%的光滑念珠菌分别对氟康唑、伊曲康唑、伏立康唑的MIC值降低2~3个浓度梯度,且耐药组与非耐药组之间差异具有统计学意义(P<0.05)。结论光滑念珠菌对唑类药物具有耐药性,伏立康唑非野生型菌株所占比例最高。焦性没食子酸单独用药时,5组念珠菌中对光滑念珠菌的抑菌效果最好,且敏感组比耐药组的抑菌效果更加显著。焦性没食子酸联合唑类药物显著降低了光滑念珠菌唑类药物的MIC值,且耐药组比非耐药组的效果更加显著,为中西医结合治疗临床光滑念珠菌感染提供实验依据。     

Evaluation of a new chromogenic medium (Candida ID) for the isolation and presumptive identification of Candida albicans and other medically important yeasts

Candidiasis is a frequent human infection caused mainly by Candida albicans. However, other species are emerging as important pathogens, as Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei or Candida guilliermondii. Rapid identification of clinical isolates could facilitate diagnosis and treatment. Candida ID (bioMerieux, Spain) is a new medium for the isolation and presumptive identification of yeasts: C. albicans grows as blue colonies, and C. tropicalis, C. guilliermondii, Candida kefyr and Candida lusitaniae as pink ones. The utility of Candida ID was evaluated with more than 700 clinical isolates and type culture collection strains from different genera including Candida, Cryptococcus, Saccharomyces, and Rhodotorula. Presumptive identification was confirmed by germ tube test, microscopic morphology and chlamydoconidia production on corn meal agar and carbohydrate assimilation on API-ATB ID 32C or Vitek (bioMerieux). Growth on Candida ID was rapid (18-24 h) for most of the yeast strains tested. Sensitivity and specificity of identification of C. albicans was significantly high (>98%), since a very low number of isolates were found to be false negative or false positive. A better result was obtained for species growing as pink colonies (>99.5%). Detection of different species of medical important yeasts was easy with Candida ID, as perfectly distinct colors and textures of colonies were observed on this medium. Candida ID allowed the discrimination between C. glabrata (creamy and smooth) and C. krusei (rough and white) colonies. Other species showed different colony textures and colours, white being the predominant colour. Candida ID was very useful for the presumptive identification C. albicans isolates.     

Distribution and susceptibility of Candida species isolated in the Medical University of Debrecen

Data of Candida albicans and non-albicans Candida species isolated during the 1997-2000 period in the Medical and Health Science Center of the University of Debrecen are analysed. The number of yeast isolates increased from 408 to 1213 per year during this period. Dominance of C. albicans has been persistent, but a slight increase of C. glabrata and C. krusei could be observed. Distribution of different Candida species isolated from 16 body sites indicates that C. albicans seems to be still the most aggressive Candida species. Investigation of 244 urinary Candida isolates (parallel with bacterial cultures) suggests that tha aetiological role of Candida species in the pathogenesis of urinary tract infections can be hypothesized if colony forming unit (CFU) number of yeasts is higher than 10(4)/ml and bacteria are present in low CFU number or are absent. Antifungal susceptibility testing of C. albicans, C. glabrata, C. tropicalis and C. krusei against Flucytosine, Amphotericin-B, Miconazole, Ketoconazole and Fluconazole suggests that Amphotericin-B is still the most effective antifungal agent. Finally, the problems in judging the aetiological role of isolated Candida species in the pathogenesis of different types of diseases are critically discussed.     

In Vitro Evaluation of Putative Virulence Attributes of Oral Isolates of <Emphasis Type="Italic">Candida</Emphasis> spp. Obtained from Elderly Healthy Individuals

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.     

Molecular typing of Candida spp. by random amplification of polymorphic DNA and analysis of restriction fragment length polymorphism of ribosomal DNA repeats

Random amplification of polymorphic DNA (RAPD) using an arbitrary oligonucleotide primer (5'-CGGTGCGACG) and analysis of restriction fragment length polymorphism of ribosomal DNA (rDNA-RFLP) after digestion of genomic DNA with restriction endonuclease EcoRI were investigated as tools for genotypic delineation beyond the species level of 91 Candida clinical isolates and four reference strains including 33 Candida albicans, 19 Candida tropicalis, 22 Candida krusei and 21 Candida (Torulopsis) glabrata. Results indicated that both techniques can be useful for typing isolates of the above species, although showing a variable discriminative potential with different species. As compared to RAPD fingerprinting, the discriminative potential of rDNA-RFLP appeared to be highest for C. albicans and lowest for C. glabrata, being overall similar for C. krusei and identical for C. tropicalis. A comparative analysis of the results obtained with the two typing techniques showed that, except for C. tropicalis, they were able to provide non-redundant information, and that their use in combination could enhance the discriminative potential for delineation among C. glabrata and C. krusei isolates.     

Candida biotypes isolated from clinical specimens in Malaysia

The distribution of Candida species was examined using 1114 yeasts isolated from various clinical specimens. The isolates were identified by germ tube test, hyphal/pseudohyphae and chlamydoconidia production and carbohydrate assimilation test using ten carbohydrates (glucose, sucrose, trehalose, cellobiose, arabinose, galactose, mannitol, raffinose, lactose and maltose). Among the 1114 isolates studied, 9 species of Candida were identified and the relative frequency of isolation was C. albicans (44.2%), C. parapsilosis (26.0%), C. tropicalis (17.7%), C. glabrata (9.6%), C. krusei (1.2%), C. rugosa (0.6%), C. guilliermondii (0.2%), C. lusitaniae (0.08%) and C. kefyr (0.08%). Non- C. albicans was the most common Candida species isolated from blood, respiratory system, urine and skin. The isolate from vaginal swabs was predominantly C. albicans. 82.2% of C. glabrata and 64.2% of C. krusei isolated in this study were from vaginal swabs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cell-surface hydrophobicity of Candida species as determined by the contact-angle and hydrocarbon-adherence methods

Cell-surface hydrophobicities of six Candida species were studied by two methods: measurement of the contact angle, and partitioning with aqueous-hydrocarbon (n-octane, n-hexadecane and p-xylene) mixtures. C. tropicalis, C. glabrata and C. krusei adhered better to the hydrocarbons than did C. albicans, C. stellatoidea and C. parapsilosis. Contact angles for the less adherent species were smaller than those for the more adherent species. Thus the two methods gave results that were similar overall and indicated that C. tropicalis, C. glabrata and C. krusei have greater cell-surface hydrophobicities than C. albicans, C. stellatoidea and C. parapsilosis.     

Identification and susceptibility against fluconazole and albaconazole of 100 yeasts' strains isolated from vaginal discharge

Vulvovaginal candidiasis is a condition that affects a great number of fertile women. It is considered the second cause of genital infection after vaginosis due to GAM complex. Candida albicans is the most frequent isolated species from vaginal discharge. However, sometimes more than one yeast species could be found in the same clinical sample that are more resistant to antifungal drugs. Nowadays, it is necessary to identify properly up to species level the isolated microorganism and to determine the antifungal susceptibility profile. One hundred strains obtained from vaginal discharge of 94 patients suffering acute vulvovaginal candidiasis were studied. The identification of the isolates showed: C. albicans 86%, Candida glabrata 6%, Candida inconspicua 3%, Candida krusei 2% and Candida intermedia, Candida holmii and Trichosporon asahii one case each. Minimal inhibitory concentrations (MIC) of all the yeasts against fluconazole and albaconazole were performed. C. glabrata, C. krusei and C. inconspicua were the most resistant against fluconazole, on the other hand albicans was susceptible to this drug. All the isolates presented MIC against albaconazole much lower than fluconazole.     

Increased susceptibility of TNF-alpha lymphotoxin-alpha double knockout mice to systemic candidiasis through impaired recruitment of neutrophils and phagocytosis of Candida albicans.

TNF-alpha and lymphotoxin-alpha (LT) are members of the TNF family, and these cytokines play crucial roles in the defense against infection with Candida albicans. The aim of the present study was to investigate the role of endogenous TNF and LT during disseminated candidiasis in TNF-/-LT-/- knockout mice. The TNF- and LT-deficient animals had a significantly increased mortality following C. albicans infection compared with control mice, and this was due to a 10- to 1000-fold increased outgrowth of the yeast in their organs. No differences between TNF-/-LT-/- mice and TNF+/+LT+/+ were observed when mice were rendered neutropenic, suggesting that activation of neutrophils mediates the beneficial effects of endogenous TNF and LT. Histopathology of the organs, combined with neutrophil recruitment experiments, showed a dramatic delay in the neutrophil recruitment at the sites of Candida infection in the TNF-/-LT-/- mice. Moreover, the neutrophils of deficient animals were less potent to phagocytize Candida blastospores than control neutrophils. In contrast, the killing of Candida and the oxygen radical production did not differ between neutrophils of TNF-/-LT-/- and TNF+/+LT+/+ mice. Peak circulating IL-6 was significantly higher in TNF-/-LT-/- mice during infection. Peritoneal macrophages of TNF-/-LT-/- mice did not produce TNF, and synthesized significantly lower amounts of IL-1alpha, IL-1beta, IL-6, and macrophage-inflammatory protein-1alpha than macrophages of TNF+/+LT+/+ animals did. In conclusion, endogenous TNF and/or LT contribute to host resistance to disseminated candidiasis, and their absence in TNF-/-LT-/- mice renders the animals susceptible through impaired recruitment of neutrophils and impaired phagocytosis of C. albicans.     

Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells

Toll-like receptor (TLR) 2 and TLR4 play a pivotal role in recognition of Candida albicans. We demonstrate that TLR2(-/-) mice are more resistant to disseminated Candida infection, and this is associated with increased chemotaxis and enhanced candidacidal capacity of TLR2(-/-) macrophages. Although production of the proinflammatory cytokines TNF, IL-1alpha, and IL-1beta is normal, IL-10 release is severely impaired in the TLR2(-/-) mice. This is accompanied by a 50% decrease in the CD4+CD25+ regulatory T (Treg) cell population in TLR2(-/-) mice. In vitro studies confirmed that enhanced survival of Treg cells was induced by TLR2 agonists. The deleterious role of Treg cells on the innate immune response during disseminated candidiasis was underscored by the improved resistance to this infection after depletion of Treg cells. In conclusion, C. albicans induces immunosuppression through TLR2-derived signals that mediate increased IL-10 production and survival of Treg cells. This represents a novel mechanism in the pathogenesis of fungal infections.     

Pathogenicity of various species of Candida in an experimental murine system

The systemic infection induced by Candida albicans, Candida krusei and Candida viswanathii was studied in an experimental murine system. Candida albicans is able to kill outbred CD1 mice within a few days and at a very low concentration; C. krusei is not pathogenic not even when inoculated at a higher concentration; C. viswanathii is able to kill animals only a a higher concentration. The different resistances do not seem to be under genic control, in as much as the different strains of mice used (hybrid CD2F1 and B6C3HF1, inbred Balb/c) show the same degree of resistance as the CD1 mice to the three species of Candida. The colony forming units (CFU) in the kidneys of CD1 mice inoculated intravenously with 10(5) cells of the three species of Candida, collected at various intervals showed a good correlation with the median survival times: a rapid moltiplication of the C. albicans is evident in the kidneys of the animals 24 hours after the inoculation, while the C. krusei and the C. viswanathii do not moltiply.     

Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species.

Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.     

米诺环素对临床几种常见假丝酵母菌体外药敏的初步探究

探究米诺环素在体外对常见酵母菌的药敏特点。采用Rosco纸片扩散法对168株常见的几种假丝酵母菌进行米诺环素的药敏试验。米诺环素有抑菌环的比率:白假丝酵母菌41%,光滑假丝酵母菌1.2%,热带假丝酵母菌和克柔假丝酵母菌没有抑菌环。在常见几种酵母菌中,米诺环素几乎只对白假丝酵母菌在体外有抗菌活性。     

In vitro antiproliferative and antifungal activity of essential oils from Erigeron acris L. and Erigeron annuus (L.) Pers

Antiproliferative and antifungal activities of essential oils from Erigeron acris root and herb and from Erigeron annuus herb were investigated. The cell viability assay was performed in cultured fibroblasts, cancer cell lines (MCF-7 and MDA-MBA-231), and endometrial adenocarcinoma (Ishikawa) cells as well as colon adenocarcinoma (DLD-1) cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The essential oil from E. acris root showed the highest antiproliferative activity in the MCF-7 cell line with an IC50 value of 14.5 microg/mL. No effect of the essential oil on normal cells at that concentration was found. Antifungal activity against various strains of five Candida species, i.e. C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. parapsilosis, was tested by the microdilution method. It was found that all examined oils can be useful as antifungal agents against the above-mentioned species, but the essential oil of E. acris herb was the most active. Their minimum inhibitory concentrations (MIC) ranged from 30 to 0.4 microL/mL. The data presented suggest that essential oils from E. acris and E. annuus possess antifungal activity against Candida spp. and antiproliferative activity against breast cancer MCF-7 cells.     

Studies on hydrazone derivatives as antifungal agents

The increasing clinical importance of drug-resistant fungal pathogens has urged additional need to fungal research and new antifungal compound development. For this purpose, some N-(1-benzyl-2-phenylethylidene)-N'-[4-(aryl)thiazol-2-yl]hydrazone (1a-e) and N-(1-phenylbutylidene)-N'-[4-(aryl)thiazol-2-yl]hydrazone (2a-e) derivatives were synthesised and evaluated for antifungal activity. Their antifungal activities against standard and clinical strands of Candida albicans, Candida glabrata, Candida utilis, Candida tropicalis, Candida krusei, Candida zeylanoides, and Candida parapsilosis were investigated. A significant level of activity was observed.     

Candida glabrata and Candida albicans; dissimilar tissue tropism and infectivity in a gnotobiotic model of mucosal candidiasis

Germ-free transgenic epsilon 26 (Tgepsilon26) mice, deficient in both natural killer (NK)- and T-cells, were inoculated (orally) with each of two Candida glabrata (BG2 or BG1003) or Candida albicans (CAF2-1 or SC5314) strains. Candida glabrata- or C. albicans-colonized mice exhibited similar numbers of viable Candida in the alimentary tract. Neither C. glabrata nor C. albicans caused systemic candidiasis of endogenous (alimentary tract) origin. Candida albicans invaded oroesophageal (tongue, palate, esophagus) and keratinized gastric tissues, evoked hyperkeratosis and a prominent, chronic, granulocyte-dominated, inflammatory response in all infected tissues, stimulated the production of splenic granulocytes and was lethal for the mice within 3-5 weeks after oral colonization. The two C. glabrata strains colonized the alimentary tract and penetrated into the keratinized (cardia-antrum) gastric tissues, but in contrast to C. albicans, were unable to infect oroesophageal tissues. Furthermore, C. glabrata strains were not lethal for the Tgepsilon26 mice, and did not evoke an inflammatory response in colonized gastric tissues or stimulate the production of splenic granulocytes. This 'stealth-like' behavior could explain the ability of C. glabrata to persist in infected tissues and survive as a commensal in the alimentary tract.     

Phylogenetic analysis of five medically important Candida species as deduced on the basis of small ribosomal subunit RNA sequences.

The classification of species belonging to the genus Candida Berkhout is problematic. Therefore, we have determined the small ribosomal subunit RNA (srRNA) sequences of the type strains of three human pathogenic Candida species; Candida krusei, C. lusitaniae and C. tropicalis. The srRNA sequences were aligned with published eukaryotic srRNA sequences and evolutionary trees were inferred using a matrix optimization method. An evolutionary tree comprising all available eukaryotic srRNA sequences, including two other pathogenic Candida species, C. albicans and C. glabrata, showed that the yeasts diverge rather late in the course of eukaryote evolution, namely at the same depth as green plants, ciliates and some smaller taxa. The cluster of the higher fungi consists of 10 ascomycetes and ascomycete-like species with the first branches leading to Neurospora crassa, Pneumocystis carinii, Candida lusitaniae and C. krusei, in that order. Next there is a dichotomous divergence leading to a group consisting of Torulaspora delbrueckii, Saccharomyces cerevisiae, C. glabrata and Kluyveromyces lactis and a smaller group comprising C. tropicalis and C. albicans. The divergence pattern obtained on the basis of srRNA sequence data is also compared to various other chemotaxonomic data.