Acid proteinase,phospholipase, and biofilm production of <Emphasis Type="Italic">Candida</Emphasis> species isolated from blood cultures

Three virulence factors comprising proteinase, phospholipase, and biofilm among 68 Candida albicans and 31 non-albicans Candida strains (11 C. tropicalis, 8 C. parapsilosis, 6 C. glabrata, 4 C. guillermondii, 2 C. krusei) isolated from blood cultures were analyzed. In total, 61 (89.7%) C. albicans strains were detected as proteinase positive whereas eight (25.8%) non-albicans Candida strains were proteinase positive (P < 0.05). Phospholipase production was detected in 41 (60.3%) C. albicans strains. All non-albicans Candida strains were phospholipase negative. Biofilm production was determined by both visual and spectrophotometric methods. Eight (11.8%) of C. albicans strains and 13 (41.93%) of 31 non-albicans Candida strains were biofilm positive with two of the methods (P < 0.05). According to our results, we may suggest that detection of hydrolytic enzyme and biofilm production abilities of the Candida isolates in clinical mycology laboratories may warn the clinican for a possible hematogenous infection.     

Hypertonic Sabouraud Dextrose Agar as a Substrate for Differentiation of Candida dubliniensis

In this study, we aimed to detect the proportion of Candida dubliniensis among yeast strains previously identified as C. albicans by using several phenotypic methods and PCR. For this purpose, we screened 300 strains by using phenotypic tests suggested for the identification of C. dubliniensis in the literature, but we detected high proportion of false-positive reactions. Only two strains (0.6%) were detected as true C. dubliniensis by PCR and API ID 32C methods. Moreover, these two strains gave the expected results with all the phenotypic tests, including modified salt tolerance test for C. dubliniensis. In conclusion, none of the phenotypic methods, except for the modified salt tolerance test, revealed 100% successful results in discrimination of C. albicans and C. dubliniensis species. However, in the tobacco agar test, the rate of false positivity was as low as 0.6%. We suggest that in the case of absence of PCR and other automatized identification systems, these two phenotypic tests can be used in routine laboratories to obtain a presumptive result.     

Genetic diversity amongFrankia isolated from Casuarina nodules

The nodulation ability of variousFrankia strains isolated from the nodules of Casuarina were tested on two Casuarina species (C. equisetifolia andC. glauca), and onHippophaë rhamnoides. We found that the isolates could be separated into two groups, some of them being unable to reinfect the Casuarina host-plant but infective onH. rhamnoides. Other isolates effectively nodulated the original Casuarina host-plant. The second purpose of this study was to examine the genetic diversity among the Casuarina-isolated strains using well-characterized symbiotic genes as hybridization probes. We found a relationship between nodulation characteristics and hybridization patterns.     

Differentiation of Candida stellatoidea from C. albicans and C. tropicalis by temperature-dependent growth responses on defined media

C. stellatoidea differs from both C. albicans and C. tropicalis in its i) much greater growth differential on minimal and amino acid enriched media and ii) unique inability to grow on minimal medium containing glycerol as carbon source at 37C. The relative responses to amino acid enrichment occur on media containing either fermentative or oxidative carbon sources, at 25C or 37C. Under any given conditions of carbon source and temperature, different assortments of individual amino acids are stimulatory for each of the three species. All assortments include one or more members of the glutamic acid family. However, sulfur amino acids stimulate only C. stellatoidea on all three carbon sources. On minimal-glycerol medium, wild type strains of C. stellatoidea grow prototrophically at 25C but are auxotrophic for amino acids at 37C; the particular auxotrophies expressed vary from strain to strain. Slow growing, mycelial mutants, prototrophic on glycerol at 37C arise spontaneously in wild type strains at frequencies indicating nuclear gene mutation. Such mutants can be induced by both transition and frame shift mutagens. The implications of these observations for the taxonomic relationships between the three Candida species and for identification of C. stellatoidea in particular are discussed.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Genetic relatedness of commensal strains of <Emphasis Type="Italic">Candida albicans</Emphasis> carried in the oral cavity of patients’ dental prosthesis users in Brazil

The aim of this study is to describe the degree of yeast-colonization in diabetic and hemodialysed-users of dental prostheses. Individuals (306) were examined using an oral rinse technique in order to evaluate the incidence of yeast-carriage, and genotype of C. albicans. Yeasts were isolated from 68.4% (91/133) individual’s dental prostheses users. Dental prostheses were found to be a significant factor for the yeast colonization (P < 0.05). Overall, the intensity of carriage was higher in diabetic patients as compared with health and hemodialysed individuals (P < 0.05). The isolation rates were: C. albicans (51.7%), C. parapsilosis (20.9%), C. tropicalis (14.3%), C. glabrata (6.6%), C. krusei (3.3%), C. rugosa (1.1%), and Pichia (Pichia ohmeri, 2.2%). Ready-To-Go RAPD Analysis Beads were used and primer OPJ 6 distinguished the C. albicans isolates found in prostheses users. All the isolates were grouped into 11 RAPD profiles in four main clusters and, the average S _AB for the entire collection of 47 C.␣albicans isolates were 0.779 ± 0.178. Over 85% of isolates had a similarity level higher than or equal to 0.8 reinforcing the idea that the use of dental prostheses, independently of the host’s clinical condition, probably provides the necessary conditions for these strains to gain a growth-specific advantage over others.     

Effect of Candida albicans cell wall components on the adhesion of the fungus to human and murine vaginal mucosa

In this study, cell walls from Candida albicans were separated and chitin was isolated from these cell walls. A chitin soluble extract (CSE) prepared from the chitin inhibited in vitro adhesion of C. albicans to human epithelial vaginal cells (VEC), and blocked in vivo attachment to murine vaginal mucosa, thereby preventing candidal infection in these animals. These findings suggest that the CSE acts as an adhesin-like substance.Fractionation of CSE yielded two fractions: FI and FII, of which only FI exhibited inhibitory activity. Chemical analysis of CSE and its two fractions revealed that CSE contains over 70% of proteins, most of which were found in the non-active fraction. In addition, 3% of amino-sugars were found in the FI active fraction. Lipids were also detected in the unfractionated CSE and in both fractions.Experiments to further characterize the component(s) in the CSE inhibiting the attachment of C. albicans are in progress in our laboratory.     

Adhesion of Candida albicans to epithelial cells effect of polyoxin D

Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.     

Restriction fragment length polymorphisms distinguish among accessions ofCeratopteris thalictroides andC. richardii (Parkeriaceae)

We have used cDNA clones as probes on Southern blots to detect restriction fragment length polymorphisms among sevenCeratopteris thalictroides accessions, threeC. richardii accessions, and one putative interspecific hybrid. We found that the stringency of post-hybridization washes was a critical parameter affecting the quality of our blots; even with homologous cDNA sequences low stringency conditions resulted in a smear of signal, but high stringency washes gave blots with distinct bands. Most probes showed hybridization with four or more genomic fragments. Similarities in the number and size of fragments between and within species indicated that (i)C. richardii shows limited polymorphism among accessions tested, (ii)C. thalictroides is highly polymorphic, and (iii) Hawaiian accessions ofC. thalictroides are divergent relative to their continental cohorts and among themselves. The putative interspecific hybrid did not group closely with either of these species.     

Extreme diversification driven by parallel events of massive loss of heterozygosity in the hybrid lineage of Candida albicans

Candida albicans is the most commonly reported species causing candidiasis. The taxonomic classification of C. albicans and related lineages is controversial, with Candida africana (syn. C. albicans var. africana) and Candida stellatoidea (syn. C. albicans var. stellatoidea) being considered different species or C. albicans varieties depending on the authors. Moreover, recent genomic analyses have suggested a shared hybrid origin of C. albicans and C. africana, but the potential parental lineages remain unidentified. Although the genomes of C. albicans and C. africana have been extensively studied, the genome of C. stellatoidea has not been sequenced so far. In order to get a better understanding of the evolution of the C. albicans clade, and to assess whether C. stellatoidea could represent one of the unknown C. albicans parental lineages, we sequenced C. stellatoidea type strain (CBS 1905). This genome was compared to that of C. albicans and of the closely related lineage C. africana. Our results show that, similarly to C. africana, C. stellatoidea descends from the same hybrid ancestor as other C. albicans strains and that it has undergone a parallel massive loss of heterozygosity.     

DNA-DNA hybridization of Rhodopseudomonas capsulata,Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains

The genetic relatedness of 21 Rhodopseudomonas strains has been studied by means of DNA-DNA hybridization. All strains included in the study belonged to the subgroup of the genus Rhodopseudomonas which is characterized by a short-rod to coccus morphology, a vesicular intracytoplasmic membrane system and carotenoids of the spheroidene group. Mol percentages guanine + cytosine ranged from 64 to 73, most strains having values between 68 and 72. With few exceptions, the hybridization data obtained were in agreement with the subdivision in three (or possibly four) species on the basis of classical taxonomy. Strain SCJ, formerly considered to be a somewhat atypical R. capsulata strain, is most probably a R. sphaeroides strain and two out of seven strains that were received as R. sulfidophila did not fit in this species on the basis of the hybridization data. The results also showed that two undesignated strains that were previously thought to be related to R. capsulata (Hansen et al. 1975) cannot be assigned to this species and may be representatives of another species. The seven strains that required approximately 2.5% NaCl in the medium and that had been designated R. sulfidophila were found to synthesize far higher levels of bacteriochlorophyll during fully aerobic growth in the dark than the purple bacteria studied thus far.Abbreviations GC guanosine + cytosine - SSC standard saline citrate buffer     

Molecular cloning and analysis of CDC28 and cyclin homologues from the human fungal pathogen Candida albicans

In the budding yeast Saccharomyces cerevisiae, progress of the cell cycle beyond the major control point in G1 phase, termed START, requires activation of the evolutionarily conserved Cdc28 protein kinase by direct association with GI cyclins. We have used a conditional lethal mutation in CDC28 of S. cerevisiae to clone a functional homologue from the human fungal pathogen Candida albicans. The protein sequence, deduced from the nucleotide sequence, is 79% identical to that of S. cerevisiae Cdc28 and as such is the most closely related protein yet identified. We have also isolated from C. albicans two genes encoding putative G1 cyclins, by their ability to rescue a conditional GI cyclin defect in S. cerevisiae; one of these genes encodes a protein of 697 amino acids and is identical to the product of the previously described CCN1 gene. The second gene codes for a protein of 465 residues, which has significant homology to S. cerevisiae Cln3. These data suggest that the events and regulatory mechanisms operating at START are highly conserved between these two organisms.     

Genomic studies on killer yeasts belonging to the genusPichia

Twenty-four species belonging to the genusPichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA.Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii andCandida albicans were also included in this study. The RFLP patterns were obtained from digestion of yeast DNA with several restriction endonuclease enzymes, and showed various bands with different mobility; in most isolates, the more deeply stained bands were species-specific. This observation was confirmed by the results obtained from Southern blot hybridization of theEcoRI andXhoI RFLP patterns withP. anomala UCSC 25F DNA, digested with the same enzymes, used as probes. These bands are likely to be ribosomal DNA as shown by hybridization of digested DNA from unrelated yeast species (S. cerevisiae, K. lactis andC. albicans). However, one hybridized band, located at 3.9–4.1 Kb, seems to be peculiar to thePichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.     

Phospholipase activity in Cryptococcus neoformans

Phospholipases have only been detected in a few fungi and yeasts, in particular in Candida albicans. Secreted phospholipases are considered by some researchers to be a potential factor of virulence and pathogenicity in C. albicans. Twenty-three Cryptococcus neoformans strains were tested in order to observe phospholipase production. Twenty-two of the 23 strains tested were able to produce phospholipases, and the ratio diameter of the colony to total diameter of the colony plus zone of precipitation (Pz) ranged between 0.271 and 0.949. C. neoformans, just like C. albicans, can be divided on the basis of the Pz into different strains according to their virulence and pathogenicity. There also appeared to be a correlation between the phospholipase production and the size of the capsule in the strains isolated from AIDS patients. For this reason, further studies on C. neoformans phospholipase activity would be useful in evaluating the virulence of different strains.     

Functional analysis through site-directed mutations and phylogeny of the Candida albicans LYS1-encoded saccharopine dehydrogenase

Candida albicans LYS1-encoded saccharopine dehydrogenase (CaLys1p, SDH) catalyzes the final biosynthetic step (saccharopine to lysine + α-ketoglutarate) of the novel α-aminoadipate pathway for lysine synthesis in fungi. The reverse reaction catalyzed by lysine-α-ketoglutarate reductase (LKR) is used exclusively in animals and plants for the catabolism of excess lysine. The 1,146 bp C. albicans LYS1 ORF encodes a 382 amino acid SDH. In the present investigation, we have used E. coli-expressed recombinant C. albicans Lys1p for the determination of both forward and reverse SDH activities in vitro, compared the sequence identity of C. albicans Lys1p with other known SDHs and LKRs, performed extensive site-directed mutational analyses of conserved amino acid residues and analyzed the phylogenetic relationship of C. albicans Lys1p to other known SDHs and LKRs. We have identified 14 of the 68 amino acid substitutions as essential for C. albicans Lys1p SDH activity, including two highly conserved functional motifs, H93XXF96XH98 and G138XXXG142XXG145. These results provided new insight into the functional and phylogenetic characteristics of the distinct biosynthetic SDH in fungi and catabolic LKR in higher eukaryotes.     

Induction of secretory aspartyl proteinase of <Emphasis Type="Italic">Candida albicans </Emphasis>by HIV-1 but not HSV-2 or some other microorganisms associated with vaginal environment

The most common type of candidiasis involves mucosal sites such as the oral cavity, the gastrointestinal tract and the vagina. Among many of virulence factors, the production of secretory aspartyl proteinase (Sap) by Candida albicans (C. albicans) has gained much attention, and factors leading to Sap induction are thus under intense study. The aim of this study was to examine whether some microorganisms such as Lactobacillus, Gardnerella vaginalis (G. vaginalis), human immunodeficiency virus type-1 (HIV-1) and human herpes simplex virus type-2 (HSV-2) had any Sap inducing effect on C. albicans. Here we showed that among the microorganisms tested in vitro only HIV-1 induced Sap production from C. albicans.     

Nosocomial Candidemia in a tertiary care hospital in Saudi Arabia

Demographic information, risk factors, therapy, and outcome for all patients who had candidemia at King Fahad teaching hospital Al-khobar, between January 1995 and January 2000 were retrospectively reviewed. Thirty-two candidemic patients were identified. Candida parapsilosis was the most frequently isolated species (44%), followed by Candida tropicalis (25%), Candida albicans (19%), Candida krusei (6%), Candida glabrat a (3%), and Candida guilliermondi (3%). Risk factors included recent broad-spectrum antibiotics use (100%), ICU residency (71%), central venous catheters (66%), recent surgery (56%), total parenteral nutrition (43%), and immunosuppressive therapy (31%). Fluconazole was used before the onset of candidemia in only two patients. The overall mortality rate was 44%. Eight (25%) episodes of candidemia were not diagnosed and treated before the patient's demise. In view of the high mortality rate associated with hematogenous candidiasis, and lack of sensitive and specific laboratory tests necessary for the premortem diagnosis of infection, empirical antifungal therapy is recommended for high-risk patients. This revised version was published online in June 2006 with corrections to the Cover Date.     

Psychrotrophic,lactic acid-producing bacteria from anoxic waters in Ace Lake,Antarctica; Carnobacterium funditum sp. nov. and Carnobacterium alterfunditum sp. nov.

Heterofermentative, lactic acid-producing, gram-positive, motile bacteria were isolated from the waters of Ace Lake, Antarctica. All strains produced virtually only l(+)lactic acid from d(+)glucose. d(–)ribose was fermented to lactic, acetic, and formic acids, and ethanol. Cell walls contained meso-diaminopimaleic acid. The strains did not grow at 30°C and were psychrotrophic. Whole cells contained 18:1cis 9 as a major component of their fatty acids. At 20°C, the strains grew better anaerobically than aerobically and all strains lacked catalase, oxidase and respiratory lipoquinones. DNA that coded for most of the 16S rRNA gene of one of the strains was amplified by the polymerase chain reaction and sequenced. The strain was phylogenetically most closely related to Carnobacterium mobile (K_nuc=0.0214). The isolates separated into two phenotypes. DNA/DNA homology studies determined on a representative from each phenotype showed low homology between the phenotypes (38±8%), and with Carnobacterium mobile (26±2%, 34±2%). Carnobacterium funditum sp. nov. produced acid from mannitol, trehalose, but not amygdalin. The G+C content of the DNA was 32–34%, and the Type strain is DSM 5970 (=ACAM 312). Carnobacterium alterfunditum sp. nov. produced acid weakly from amygdalin but not from mannitol or trehalose. The G+C content was 33–34%, and the Type strain is DSM 5972 (=ACAM 313).     

紫草素对白色念珠菌的抑制作用机制

【目的】研究紫草素抑制白色念珠菌的作用机制。【方法】通过微量稀释法测定紫草素对白色念珠菌的最低抑菌浓度(MIC)和最低杀菌浓度(MFC);紫外分光光度法测定紫草素对白色念珠菌细胞膜渗透性的影响;扫描电镜观察紫草素对菌体形态的影响;激光共聚焦显微镜测定紫草素对白色念珠菌细胞内钙离子浓度的影响;卵黄平板培养基法检测紫草素对白色念珠菌的细胞膜磷脂酶活性的影响;RT-PCR检测紫草素对白色念珠菌PLB1和PLB2基因表达量的影响。【结果】紫草素对白色念珠菌有较强的抑制作用,其对白色念珠菌的MIC和MFC分别为16μg/m L和32μg/mL。紫草素能破坏白色念珠菌细胞膜的完整性,使细胞膜的通透性增加,导致细胞内DNA和RNA等大分子物质的泄漏和细胞内钙离子的流失。其中MIC的紫草素作用菌体16 h后,上清液中的DNA和RNA等大分子含量与对照组相比增加了117.32%(P0.01);细胞内的[Ca~(2+)]降低了72.02%(P0.01)。扫描电镜结果也证明了紫草素对白色念珠菌细胞膜的破坏作用。紫草素也能抑制白色念珠菌分泌磷脂酶,且呈浓度剂量依赖。其中,与对照组相比,MIC的紫草素能使白色念珠菌分泌磷脂酶的量下降56.3%(P0.01)。RT-PCR结果显示,紫草素能抑制编码磷脂酶B的基因PLB1和PLB2的表达量,其中1/2 MIC的紫草素作用白色念珠菌16 h后,与对照组相比,PLB1和PLB2基因的相对表达量分别降低了56.4%和61.4%(P0.01)。【结论】紫草素对白色念珠菌有较强的抑杀作用,其作用机制是通过破坏白色念珠菌细胞膜的完整性,增加菌体细胞膜的通透性,导致细胞内DNA和RNA等大分子的泄漏和细胞内[Ca~(2+)]的流失,最终引起菌体的死亡。而紫草素对白色念珠菌磷脂酶分泌的抑制作用,致使其不能及时维护和修复由紫草素造成的细胞膜的破坏和损伤,也是导致菌体死亡的原因。     

The attachment to human buccal epithelial cells by Candida albicans: An in vitro kinetic study using concanavalin A

The early in vitro kinetics of Candida albicans attachment to human buccal epithelial cells was studied with the aid of an adhesion assay and solutions of concanavalin A (Con A), a lectin which is capable of inhibiting yeast adhesion. Various saccharides and putative receptor analogues were also tested. Solutions of each single reagent were added to tubes containing aliquots of mucosal cells and germinated yeasts at the beginning of a 1-hour incubation period (time O) or at 10 minute intervals during the assay. The number of yeasts attached to 200 mucosal cells was subsequently determined microscopically. Yeast adhesion remained constant following addition of phosphate-buffered saline (PBS) at time 0 or at any time thereafter. However, addition of Con A at 0, 10 or 20 minutes of incubation decreased adhesion significantly to 38%, 45% and 63% of control values. This inhibitory effect dwindled as time of incubation prior to lectin addition increased and Con A could not inhibit adhesion significantly after twenty minutes. Results obtained with Con A using live germinated yeasts were similar to those obtained with formalin-killed C. albicans. The other reagents tested failed to decrease adhesion significantly. These included the putative receptor analogues fibronectin, N-acetyl-d-glucosamine and d-galactose, and several non-specific saccharides such as -d-methylglucopyranoside, d-ribose and d-xylose. It is suggested that in vitro attachment to human mucosal cells by C. albicans is inhibitable up to a defined point in time by a lectin with affinity for mannosecontaining surface moieties, but becomes non-reversible thereafter. This experimentally-observed irreversibility is independent of yeast cell viability.