Adherence and biofilm formation of non-Candida albicans Candida species

Most cases of candidosis have been attributed to Candida albicans, but recently non-C. albicans Candida species have been identified as frequent human pathogens. Candida pathogenicity has been attributed to several factors, including adhesion to medical devices and/or host cells, biofilm formation, and secretion of hydrolytic enzymes (proteases, phospholipases and haemolysins). Although 'new'Candida species are emerging, there is still a lack of information about their pathogenicity. This review discusses recent advances in our knowledge of Candida glabrata, Candida parapsilosis and Candida tropicalis virulence factors, specifically those of adhesion and biofilm formation, which are key components in Candida pathogenicity.     

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.     

Characterization of Candida Species from Different Populations in Taiwan

The opportunistic Candida species existing as part of commensal microbiota in humans are usually the etiological agents causing infections. We investigated whether isolates collected from different age groups, hospital units, and sources have distinct characteristics. A total of 913 isolates comprising 395 Candida albicans, 230 Candida tropicalis, 202 Candida glabrata, 62 Candida parapsilosis, 13 Candida krusei, and 11 of other six species were analyzed. Urine was the most common source (41.2%), followed by sputum (16.3%), blood (15.2%), and others (27.3%). Candida albicans and C. parapsilosis were more prevalent in the working group [from 19 to 65 years], whereas C. tropicalis and C. glabrata were more prevalent in the elder one (≥ 66 years). We found that the age of patients and the source of isolates affect the distribution of species. On the other hand, the drug susceptibility of isolates was associated with fungal species and whether patients were hospitalized.     

Phenotypic and genotypic identification of Candida spp. isolated from hospitalized patients

As candidosis incidence continue to rise, quick laboratory identification of Candida species is becoming increasingly important for a growing population of patients at-risk. RAPD techniques were used on samples of Candida obtained from patients hospitalized at Santa Casa de Misericordia in Belo Horizonte (SCMBH) Brazil, from March 1998 to December 2000 and then compared with the results of phenotypic identification techniques. Two hundred and forty two yeasts were isolated and phenotypically identified as follows: Candida albicans (105), Candida tropicalis (62), Candida parapsilosis (28), Candida glabrata (19), Candida krusei (8), Candida guilliermondii (5) and Candida spp. (15). Samples from the three most frequent species isolated were selected randomly in order to compare the phenotypic and genotypic analyses. Genotypic analysis using RAPD primer M13 (F/R) displayed the best results of all test samples. There was both agreement and consistency between phenotypic and genotypic analysis using RAPD, demonstrating that is possible to apply this method for the identification of Candida species.     

Candida proteinases and candidosis

Infections caused by the opportunistic yeast pathogen, Candida albicans, are becoming increasingly important. Superficial Candida infections, particularly those of the mouth and vagina, are very common; for example, candidal vaginitis plaques millions of women worldwide, often proving refractory to treatment. Systemic candidosis is much rarer, but it is an important hazard of modern medical procedures such as transplant surgery, i.v. hyperalimentation, and immunosuppressive therapy. One significant virulence factor of C. albicans is its ability to secrete extracellular acid proteinase. This attribute is shared by C. tropicalis and C. parapsilosis, but not by other less pathogenic Candida species. The enzymes produced by these yeasts are all carboxyl proteinases capable of degrading secretory IgA, the major immunoglobulin of mucous membranes. Some have keratino- or collagenolytic activity. Two secretory proteinases of C. albicans have been purified and characterized; their properties are reviewed. Possible applications of this work to the treatment and diagnosis of candidosis are discussed.     

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.     

Secretion of inducible proteinase by pathogenic Candida species

The ability of three isolates each of seven pathogenic Candida species to grow in a liquid medium containing bovine serum albumin (BSA) as a nitrogen source was determined. All three strains of C. albicans, two strains of C. guilliermondii and one strain of C. tropicalis grew well. At any time proteinase activity was detected in the culture filtrates of only the most virulent species--C. albicans, C. tropicalis and C. parapsilosis and this observation was related to complete hydrolysis of BSA. Serologically, cross reactions were demonstrated between anti-proteinase antiserum and C. albicans and C. tropicalis culture filtrates. These results further emphasise the role of the inducible proteinase of Candida in the pathogenesis of candidosis.     

A comparison of experimental pathogenicity of Candida species in cyclophosphamide-immunodepressed mice

The experimental pathogenicity of Candida albicans, C. krusei, C. guilliermondii, C. parapsilosis, C. tropicalis and C. viswanathii was tested in normal and in cyclophosphamide-(Cy) immunodepressed mice. In unpretreated CD1 mice only C. albicans, C. tropicalis and C. viswanathii were pathogenic on intravenous challenge, with LD50 of 1.0 X 10(6), 4.8 X 10(6), 7.2 X 10(8) cells, respectively, per kg. Three days after a single intraperitoneal injection of Cy (150 mg kg-1) mice had a marked decrease in spleen weight and cellularity as well as reduced numbers of circulating leukocytes. Under these conditions, there was a significant, proportional increase in pathogenicity of C. albicans, C. tropicalis and C. viswanathii but the animals were still resistant to challenge with C. krusei, C. guilliermondii and C. parapsilosis. This pattern of susceptibility was not influenced by higher doses of Cy. Only C. albicans and C. tropicalis were capable of rapid and extensive multiplication in target organs such as kidney and brain in normal and Cy-treated mice and for both these species of Candida, there was a 'rebound' effect of increased resistance to experimental infection after 12 days from Cy administration. This study shows that the strong immunodepression provoked by Cy does not modify significantly the susceptibility of the animal to those species of Candida which were endowed with low or no pathogenicity for normal mice, but it greatly increases the susceptibility to those species of Candida that are already pathogenic for unmodified host.     

Saliva promotes survival and even proliferation of Candida species in tap water

Candida yeasts colonize the human oral cavity as commensals or opportunistic pathogens. They may be isolated from water circulating in dental unit waterlines mixed with traces of saliva mainly because of the dysfunction of antiretraction valves. This study deals with the growth ability of Candida albicans, Candida glabrata and Candida parapsilosis in tap water with saliva (0-20% v/v). Results show that C. glabrata is the most susceptible species in tap water. Furthermore, saliva promotes both survival and proliferation of the three studied Candida species in tap water.     

Susceptibility to fluconazole and amphotericin B in clinical Candida isolates.

Susceptibility to fluconazole and amphotericin B in 84 clinical isolates of Candida was determined by a macrodilution method (NCCLS). Amphotericin B was very active (CMI < 1.25 microg/ml) against C. tropicalis and C. parapsilosis. Less than 5% of C. albicans and/or C. glabrata isolates presented low susceptibility to the drug (CMI 80 > 2.50 microg/ml). Fluconazole was less active against C. glabrata and C. krusei (CMI 80 > 100 microg/ml). The susceptibility profile for fluconazole indicated the importance to the treatment of identification to species level.     

Interactions of Candida albicans with other Candida spp. and bacteria in the biofilms

AIMS: To study the interactions between Candida albicans and 12 other species of Candida and bacteria in biofilms. METHODS AND RESULTS: The number of cells within growing biofilms in a polystyrene tube model was measured after adding C. albicans to preformed biofilms of other micro-organisms and vice versa. It was also measured after simultaneous biofilm formation of C. albicans and other micro-organisms. The number of cells of C. albicans within the growing biofilms decreased significantly (P < 0.05) when the fungus was added to preformed biofilms of Candida spp. and bacteria except, with C. parapsilosis, Torulopsis glabrata and the glycocalyx producer Pseudomonas aeruginosa. When C. parapsilosis, Staphylococcus epidermidis (nonglycocalyx producer) or Serratia marcescens was added to preformed biofilms of C. albicans, the number of cells of these micro-organisms increased in the growing biofilms. CONCLUSIONS: Biofilms of C. albicans are capable of holding other micro-organisms and more likely to be heterogeneous with other bacteria and fungi in the environment and on medical devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Recognition of the heterogeneity of biofilm-associated organisms can influence treatment decisions, particularly in patients who do not respond to initial appropriate therapy.     

Differential adhesion of pathogenic Candida species to epithelial and inert surfaces

Abstract Growth in medium containing 500 mM galactose is known to promote the adhesion of Candida albicans to buccal epithelial cells or to acrylic in vitro. Of 5 other Candida species tested, only C. tropicalis (one strain) showed substantially increased adhesion to buccal cells (but not to acrylic) after growth under these conditions. A second strain of C. tropicalis as well as C. stellatoidea, C. parapsilosis, C. pseudotropicalis, C. guilliermondii and Saccharomyces cerevisiae showed little or no increased adhesion to either surface. However, after growth in medium containing 50 mM glucose, C. tropicalis and C. parapsilosis were significantly more adherent to acrylic than glucose-grown yeasts of the other species, including C. albicans . These results are discussed in relation to the colonization and infection potential of the pathogenic Candida species.     

Purification and characterization of secretory proteinase of Candida albicans.

Proteolytic activity of medically important yeasts was tested in both YCB-BSA agar and medium. All of 134 strains of Candida albicans, 13 of 18 strains of Candida tropicalis and 11 of 18 strains of Candida parapsilosis had this activity, while none of 52 Candida glabrata strains or of 11 Cryptococcus neoformans strains tested had proteolytic activity. Strains of C. albicans fell into five groups based on the level and time-course of in vitro proteinase productivity. Five strains randomly selected from each group were tested for pathogenicity in mice. The strain possessing the strongest pathogenicity was used to purify proteinase. The molecular weight of the proteinase was approximately 44,000 daltons and its isoelectric point was pH 4.2. Optimal pH of the proteinase was 3.2 and the enzyme was stable below pH 7.0 and lost its activity above pH 8.0 at 37 C in a 60-min incubation. The 23 amino acid sequence of the proteinase N-terminus was determined.     

Genetic speciation of Candida isolates by arbitrarily primed polymerase chain reaction

Abstract Candida species are opportunistic human pathogens capable of causing a variety of clinical diseases. Rapid and precise identification of Candida to species level is essential for effective treatment and management strategies. Conventional diagnosis of candidiasis is sometimes slow and variable. By using a random primer 5'-ACGGGCCAGT-3' in arbitrarily primed polymerase chain reaction, seven common Candida species (i.e. C. albicans, C. guilliermondii, C. parapsilosis, C. krusei, C. lipolytica, C. tropicalis and C. (Torulopsis) glabrata ) produced characteristic DNA band patterns, which enabled rapid determination to species level among them. Further analysis of the nucleotide sequences of Candida specific fragments should improve our understanding of the molecular structures and possible functions of the gene regions involved.     

近平滑念珠菌ERG 11基因编码区的克隆及生物信息学分析

目的克隆、测序近平滑念珠菌ERG11基因的编码区序列并进行生物信息学分析。方法运用生物信息学的方法 ,通过与白念珠菌ERG11基因碱基序列同源性比对,在近平滑念珠菌基因组(www.sanger.ac.uk/sequencing/Candida/pa-rapsilosis/)中寻找可能的ERG11基因序列(CpERG11),并据此序列设计引物,经PCR扩增近平滑念珠菌标准株(ATCC22019)的ERG11基因片段,产物经电泳、纯化、克隆到质粒prG-AMAI-NotI中,转染DH10B大肠杆菌细胞,并酶切鉴定筛选阳性克隆测序分析。结果近平滑念珠菌ERG11编码区由1569个碱基组成,编码一段含522个氨基酸的多肽。近平滑念珠菌ERG11的编码区序列与白念珠菌、热带念珠菌、光滑念珠菌、酿酒酵母菌ERG11基因的同源性分别为74%、75%、65%、64%。该近平滑念珠菌ERG11的编码区为唑类药物作用靶酶基因。结论成功克隆、测序、并生物信息学分析近平滑念珠菌ERG11基因的编码区序列,为进一步的功能研究奠定基础。     

Rapid identification of Candida species by using nuclear magnetic resonance spectroscopy and a statistical classification strategy

Nuclear magnetic resonance (NMR) spectra were acquired from suspensions of clinically important yeast species of the genus Candida to characterize the relationship between metabolite profiles and species identification. Major metabolites were identified by using two-dimensional correlation NMR spectroscopy. One-dimensional proton NMR spectra were analyzed by using a staged statistical classification strategy. Analysis of NMR spectra from 442 isolates of Candida albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis resulted in rapid, accurate identification when compared with conventional and DNA-based identification. Spectral regions used for the classification of the five yeast species revealed species-specific differences in relative amounts of lipids, trehalose, polyols, and other metabolites. Isolates of C. parapsilosis and C. glabrata with unusual PCR fingerprinting patterns also generated atypical NMR spectra, suggesting the possibility of intraspecies discontinuity. We conclude that NMR spectroscopy combined with a statistical classification strategy is a rapid, nondestructive, and potentially valuable method for identification and chemotaxonomic characterization that may be broadly applicable to fungi and other microorganisms.     

A rapid PCR-based method for identification of four important Candida species

The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidosis. Currently available culture and biochemical methods for detection and identification of Candida species are time-consuming. This study describes the use of a simple and rapid PCR method using species-specific oligonucleotides for the detection of clinical isolates of Candida species. These species-specific oligonucleotides are complementary to unique sequences within the intergenic transcribed spacer 2, located in between the 5.8S and 28S ribosomal DNA, and generated DNA fragments by both the conventional and hemi-nested PCR reactions. Conventional PCR produced a single DNA fragment of variable size in all isolates, while the hemi-nested PCR produced two discrete DNA fragments, both with the expected sizes of 111bp/57bp (C. albicans), 84bp/42bp (C. glabrata), 94bp/45bp (C. krusei) and 95bp/49bp (C. parapsilosis). In conclusion, the PCR-based method described in this study is fast and specific for the identification of clinically important Candida species.     

The distribution frequency of Candida species in the genitourinary tract among symptomatic individuals in Nigerian cities

A clinical survey was carried out in seven cities in the southern part of Nigeria to determine the relative distribution of genitourinary Candida species in symptomatic patients reporting for diagnosis and treatment. Seven Candida species were identified using the CHROMagar Candida method and the API 20C System. Candida species were represented by Candida glabrata (33.7%), Candida albicans (20.1%), Candida tropicalis (18%), Candida guilliermondii (17.8%) Candida pseudotropicalis (5%), Candida parapsilosis (5%), and C. albicans var.stellatoidea (1.2%). The distribution of these species among the various age groups (15-20, 21-25, 26-30, 31-35, 36-40 and 41 plus years) was statistically insignificant. Out of the 517 positive samples, 182 (35%) were found in the age group 26-30 years, while age 41 plus had the lowest frequency (1.2%). The results presented show that C. albicans, usually reported to be the most frequently isolated species, is not the main species in the cities studied. With C. glabrata in preponderance, the finding supports recent studies reporting that several pathogenic non-C. albicans species are now being frequently isolated. The level of social activities, such as drug abuse and sexual promiscuity, may be important in the distribution frequency of Candida species in different age groups and locations.     

Candida species: new insights into biofilm formation

Biofilms of Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis are associated with high indices of hospital morbidity and mortality. Major factors involved in the formation and growth of Candida biofilms are the chemical composition of the medical implant and the cell wall adhesins responsible for mediating Candida-Candida, Candida-human host cell and Candida-medical device adhesion. Strategies for elucidating the mechanisms that regulate the formation of Candida biofilms combine tools from biology, chemistry, nanoscience, material science and physics. This review proposes the use of new technologies, such as synchrotron radiation, to study the mechanisms of biofilm formation. In the future, this information is expected to facilitate the design of new materials and antifungal compounds that can eradicate nosocomial Candida infections due to biofilm formation on medical implants. This will reduce dissemination of candidiasis and hopefully improve the quality of life of patients.     

Evolutionary relationships among pathogenic Candida species and relatives.

Small subunit rRNA sequences have been determined for 10 of the most clinically important pathogenic species of the yeast genus Candida (including Torulopsis [Candida] glabrata and Yarrowia [Candida] lipolytica) and for Hansenula polymorpha. Phylogenetic analyses of these sequences and those of Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, and Aspergillus fumigatus indicate that Candida albicans, C. tropicalis, C. parapsilosis, and C. viswanathii form a subgroup within the genus. The remaining significant pathogen, T. glabrata, falls into a second, distinct subgroup and is specifically related to S. cerevisiae and more distantly related to C. kefyr (psuedotropicalis) and K. marxianus var. lactis. The 18S rRNA sequence of Y. lipolytica has evolved rapidly in relation to the other Candida sequences examined and appears to be only distantly related to them. As anticipated, species of several other genera appear to bear specific relationships to members of the genus Candida.