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.     

The influence of post-filtration washing on the in vitro assay ofCandida albicans adherence to human buccal epithelial cells

A simple in vitro assay technique was used to determine the effect of post-filtration washing on the adherence ofC. albicans (NCPF 3736) to human buccal epithelial cells (BEC). Washing was carried out with a range of volumes of phosphate buffered saline (PBS), viz. 0, 5, 10 and 20 ml, at a standard flow rate. Both the number ofC. albicans adherent to BEC and the percentage of BEC with adherentC. albicans were significantly decreased (p<0.001 for each of these measures) after washing with 5 ml PBS. Further increases in the volume of PBS did not significantly decrease either measure of adherence. These data indicate that only a small volume of PBS, 5 ml, is required to achieve the removal of non-adherentC. albicans from the surface of BEC. The result of the adherence assay is not significantly affected by increasing the volume of PBS used. It is concluded that considerable savings in time may be made through using only a small (5 ml) volume of washing buffer at a standard flow rate.Abbreviations BEC buccal epithelial cells - PBS phosphate buffered saline - MEM Eagle's minimum essential medium - NCPF National collection of pathogenic fungi     

Disruption of the GPI Protein-Encoding Gene <Emphasis Type="Italic">IFF4</Emphasis> of <Emphasis Type="Italic">Candida</Emphasis> <Emphasis Type="Italic">albicans</Emphasis> Results in Decreased Adherence and Virulence

Candida albicans is the most important cause of systemic fungal infection in immunocompromised humans. Candidiasis is often initiated by the adherence and the colonization of inert surfaces such as peripheral venous catheters, central catheters, prosthetic cardiac valves, and other prostheses. We have studied the early stage of adherence and have shown that the disruption of C. albicans IFF4 gene encoding a GPI-anchor protein, led to a decrease of adherence of the germ tubes to plastic. Here, we demonstrated the role of the IFF4 gene in adherence to silicone catheter, as well as in virulence using a murine model of disseminated candidiasis. The iff4 Δ null mutant showed both a decrease of adherence to silicone catheter and a reduction of virulence. This work presents evidence for the importance of the IFF4 gene in host-fungal interaction.     

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.     

Oral yeast flora and its ITS sequence diversity among a large cohort of medical students in Hainan,China

The most prevalent fungal infection of humans is candidiasis which is caused by species of Candida that are typical members of the commensal microbial flora of the oral mucosa and other body surfaces. Since species of Candida differ in virulence properties and susceptibilities to anti-fungal drugs, understanding the human commensal yeast flora will have a significant impact on designing treatment and prevention strategies against yeast infections. However, although there is a global interest in Candida species, the global distributions of Candida species remain largely unknown, especially among healthy hosts. Here we report the oral yeast flora from the surveys of over 1,000 medical students in China. Our results showed that this population had a yeast carriage rate (4.5%) much lower than other population samples reported previously from Mainland China (40–70%). In addition, C. albicans was isolated at a much higher frequency than those from other Chinese samples, with a frequency (80.9%) more similar to those in developed regions such as North America. The oral yeast carriage rates and yeast species compositions were similar between male and female students and between the hosts borne and raised on Hainan Island and those borne and raised on Mainland China. Furthermore, the sequence variation at the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene cluster was analyzed for strains of the dominant species, C. albicans. Our analysis identified 14 ITS types among the 41 Hainan isolates of C. albicans. However, only four of the 14 ITS types were identical to those in reference strains from Europe and North America. Taken together, our analyses suggest that the oral yeast flora among host populations in China is highly heterogeneous and that there is a high ITS sequence diversity in the Hainan population of C. albicans.     

Detection of Candida sp. mannan antigen by indirect ELISA-inhibition

Summary We have obtained mannans from four Candida species: C. albicans A, C. albicans B and C. tropicalis; antimannan sera against C. albicans A, C. albicans B and C. tropicalis were obtained by immunizing rabbits sub-cutaneously with the respective yeast extract. The efficacy of these sera in reacting with mannans obtained from three Candida sp. has been proven by indirect ELISA-inhibition.Any of three immune sera can be used to detect mannan antigen from the three Candida sp. tested. This confirms the existence of crossed reactivity and the possibility of detecting mannan antigen in serum from patients infected by different Candida sp., although we had only one immune serum and one Candida mannan.     

Species diversity of yeast in oral colonization of insulin-treated diabetes mellitus patients

The aim of this study was to investigate oral yeast colonization, antifungal susceptibility and strain diversity in insulin-dependent diabetes mellitus patients (175), as well as to evaluate the influence of dental prostheses. Oral rinse samples were cultured on selective media, in order to isolate, count and identify the yeasts recovered. More than half of the diabetic subjects (53%) carried significant amounts of Candida cells in the buccal cavity and these organisms were recovered at higher densities in diabetics wearing dentures. A total of 93 yeast strains were isolated from these patients, including: Candida spp. (n = 89); Pichia (n = 02); Trichosporon (n = 1), and Geotrichum (n = 1). C. albicans represented 56% of these strains, non-albicans Candida 39.8%, and other genera of yeast 4.3%. C. albicans was prevalent, followed by C. parapsilosis, C. tropicalis, C. glabrata, C. krusei, C. rugosa and C. guilliermondii. Agar disk-diffusion tests of the susceptibility of non-albicans Candida and other genera of yeast to fluconazole showed resistance in 21.9%, mainly in C. rugosa (100%), C. glabrata (57%) and C. krusei (50%). Local oral factors, such as the presence of dentures, in association with diabetes, seemed to have the effect of increasing the amount and variety of Candida species in the oral cavities, mainly those with lower drug susceptibilities.     

The impact of polyene,azole, and DNA analogue antimycotics on the cell surface hydrophobicity of Candida albicans and Candida tropicalis in HIV infection

Oral candidiasis is the most common opportunistic infection in individuals infected with the human immunodeficiency virus. Though Candida albicans is the major aetiological agent, non-albicans species such Candida tropicalis are now emerging as important agents of such infection. The Candida cell surface hydrophobicity (CSH) is considered a critical factor contributing to its colonization potential and virulence. It is also known that brief exposure to sub-cidal concentrations of antifungal agents is a likely scenario in the oral environment where the administered drugs are diluted continuously due to the flushing action of saliva. Hence the objective of the present study was to compare the CSH of 10 isolates each of C. albicans and C. tropicalis from HIV-infected individuals following brief exposure (1hour) of isolates to sub-therapeutic concentrations of nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine. The CSH was assessed by a previously described biphasic aqueous-hydrocarbon assay. The mean percentage reduction of CSH of C. albicans following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was27.33 (p < 0.001), 21.34 (p < 0.05), 11.74 (p > 0.05), 18.4 (p > 0.05) and 14.64 (p > 0.05) respectively. The mean percentage reduction of CSH of C. tropicalis following brief exposure to nystatin, amphotericin B, ketoconazole, fluconazole and 5-flurocytosine was 33.81 (p < 0.01), 28.88 (p < 0.01), 12.6 (p > 0.05), 21.53(p > 0.05) and 17.68 (p > 0.05) respectively. A significant inter-species variation in CSH was observed for nystatin and amphoterecin B. Overall the results reveal that the CSH of C. albicans is affected to a significantly lesser degree compared with C. tropicalis when exposed to the antifungals. These data further illustrate another mode of action of antifungals on Candida leading to a reduction in the CSH and thereby the yeast adherence to host tissues. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Isolation of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> for the First Time in Cali,Colombia, and its Identification with Phenotyping Methods

Summary   Candida dubliniensis is an emerging pathogenic yeast isolated mainly from the oral cavity of HIV-infected patients. The close phenotypic and genotypic relationship between C. albicans and C. dubliniensis has led to incorrectly identifying isolates of C. dubliniensis as C. albicans. The oral cavities of 107 diabetic patients were studied in Cali, Colombia, and 72 colonies of Candida, with shades of green on CHROMagar Candida culture media, were obtained. Various phenotypic tests were carried out, which included germ tube formation and production of chlamydospores on corn meal Agar. Additionally, growth studies were carried out at 42°C and 45°C and on Sabouraud agar with 6.5%, sodium chloride. Identification of C. dubliniensis with these tests was confirmed with API 20C Aux. We identified 65 and 7 colonies of C. albicans and C. dubliniensis, respectively. This is the first time that C. dubliniensis is identified with phenotypic methods in Colombia.     

Influence of mucosal cell origin on the in vitro adherence of Candida albicans: Are mucosal cells from different sources equivalent?

The influence of collecting mucosal cells from various anatomical sites, and varying the date of collection and cell donor on adhesion of Candida albicans to human epithelial cells was examined by using an in vitro adherence assay. Examination of buccal mucosal cells from twenty-four donors showed statistically significant differences in the number of attached yeasts between individuals. Sex did not exert a significant influence on adhesion. Examination of buccal mucosal cells from ten donors collected on five different dates revealed that yeast attachment to mucosal epithelial cells varied significantly within subjects across time. Epithelial cells from some donors manifested greater date-to-date variations in yeast adhesion than others. Adherence of Candida to mucosal cells from three anatomical sites (mouth, vagina and urinary tract) collected from ten different donors was also tested. Yeast adherence to buccal cells was highest, lowest using urinary tract cells, while vaginal epithelium was intermediate. Adherence to mucosal cells from three sites was significantly different both within and between individuals although some subjects manifested larger variations than others. These data suggest that the in vitro adherence of Candida albicans is influenced by mucosal cell donor, date of collection and body site of origin. Mucosal cells from different sources do not appear to be equivalent in receptiveness to C. albicans and this might explain some of the discrepancies observed when adhesion studies performed by different investigators are compared. The existing need for a more uniform methodology with which to pursue studies on fungal attachment to mucosal surfaces is emphasized.     

Multiple mechanisms may contribute to the adherence ofCandida yeasts to living cells

The adherence ofCandida yeasts to monolayers of human intestinal epithelium was studied in order to determine the specific and nonspecific mechanisms that might contribute to yeast adherence. Multiple factors were shown to significantly affect the adherence of yeasts to intestinal cells. It was demonstrated that hydrophobic yeasts adhered two times greater than normal yeasts, and positively charged yeasts adhered ten times greater than normal yeasts to monolayers of intestinal epithelium. The binding of yeasts to the intestinal cells was saturable and was most effectively blocked by mucin, which caused an 83% reduction in adherence, whereas the addition ofd-glucose caused a 41% reduction in adherence. Aggregation or coadherence of yeasts occurred as the yeast inocula were increased.Candida appears to possess the ability to adhere to living tissue by several mechanisms, such as adhesin-receptor interactions, nonspecific hydrophobic and ionic bonding, and aggregation or coadherence. This is the first demonstration of multiple forces that may act simultaneously in the process of adherence of yeasts to living cells.     

Good response in a patient with deep-seated subcutaneous ulcer due to <Emphasis Type="Italic">Candida</Emphasis> species

Deep-seated subcutaneous ulcers infected with Candida species are rare. We are reporting a 51-year-old Cantonese woman who had a large, deep-seated subcutaneous ulcer on her right shoulder for more than a year. Direct smears of the purulent extrusion revealed many pseudohyphae and yeast cells. Candida species were isolated from the purulent extrusion and further identified as Candida albicans and C. parapsilosis. A skin lesion biopsy contained yeast cells and pseudohyphae. C. parapsilosis were once isolated from the biopsy specimen. Total healing was obtained with itraconazole (200 mg twice daily for 16 days and then 100 mg twice daily for 14 days) combined with phototherapy.     

Incidence and significance of opportunistic fungi in leukemia patients in India

Twenty-four patients with acute leukemia were investigated for the incidence of opportunistic fungi. Culture isolations of the sputum and urine samples revealed significant levels of Candida in 14 patients; Candida albicans, C. tropicalis and C. pseudotropicalis were the predominant ones isolated. Aspergillus flavus was isolated from blood in two cases and C. albicans and a black yeast from the blood of another two. Serological studies showed fungal antibodies in seven patients; precipitins against Candida were detected in five and Aspergillus in two. Both of the Aspergillus positive cases and two patients who had rising antibodies against Candida died during the course of investigation. In this study 13 of 24 patients developed oral candidiasis.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

Species Distribution and Antifungal Susceptibility Profile of Oral Candida Isolates from HIV-infected Patients in the Antiretroviral Therapy Era

In this study, we investigated the yeasts colonization of genus Candida, including C. dubliniensis, isolated of HIV-infected patients oral cavities and we accessed in vitro susceptibility pattern of the Candida isolates to four antifungal agents. Out of 99 patients investigated, 62 (62.6%) were colonized with yeasts. C. albicans was the prevailing species (50%). C. dubliniensis isolates were not recovered in our study. We verified that 8.1% of the yeasts isolated were resistant to fluconazole, 8.1% to itraconazole and 3.2% to voriconazole. The isolates demonstrated very low voriconazole MICs, in which 79% (49/62) presented values of 0.015 μg/ml. All Candida isolates were susceptible to amphotericin B. The results reported here showed that although C. albicans continues to be present in one-half of oral Candida carriage of HIV-infected patients, Candida non-albicans species are increasing among these patients. Besides, the findings of resistant isolates endorse the role of antifungal susceptibility testing whenever antifungal treatment with azoles is planned.     

Heteroduplex mobility assay of the 26S rDNA D1/D2 region for differentiation of clinically relevant Candida species

The Heteroduplex Mobility Assay (HMA) method using the PCR amplified D1/D2 region of the 26S rDNA was tested for the differentiation of clinically relevant Candida species. Strains belonging to the same species are not expected to form heteroduplexes in this assay when their PCR products are mixed. D1/D2 HMA experiments between all Candida type strains tested showed heteroduplex formation, including Candida albicans and Candida dubliniensis. There was no heteroduplex formation when most clinical and non-type strains were tested against the type strain of their presumptive species, except when C. albicans WVE and C.␣dubliniensis TAI were analysed. Additional HMA experiments, phenotypic characterisation, and D1/D2 sequencing identified these isolates as Candida tropicalis and Candida parapsilosis, respectively. HMA provides a rapid and relatively simple molecular tool for the differentiation of potentially pathogenic Candida species.     

Systemic Candida infection in University Hospital 1997–1999: the distribution of Candida biotypes and antifungal susceptibility patterns

A total of 102 Candida species were isolated from blood cultures from January 1997 to October 1999. Using assimilation of carbohydrate test, 52 (51.0%) of the Candida sp. were identified as C. parapsilosis, 25.5% (26) were C. tropicalis. C. albicans made up 11.8% (12), 6.9% (7) were C. rugosa, 3.8% (4) C. glabrata and 1% (1) C. guilliermondii. No C. dubliniensis was found in the study. In vitro antifungal susceptibility tests showed that all Candida species were sensitive to nystatin, amphotericin B and ketoconazole. Although all isolates remained sensitive to fluconazole, intermediate susceptibility was found in 3 C. rugosa isolates. Antifungal agents with high frequency of resistance were econazole, clotrimazole, miconazole and 5-fluorocytosine. Candida species found to have resistance to these antifungal agents were non-C. albicans. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distribution and phospholipase activity of Candida species in different denture stomatitis types

The aim of this study was to evaluate the correlation between frequency and phospholipase activity of Candida species and denture stomatitis according to Newton’s classification. Seventy-five complete denture wearers were evaluated for the presence of yeasts on the palatal mucosa by culture method. In addition, the number of yeast isolates producing phospholipase and amount of this enzyme were determined using egg yolk agar plate method. According to Newton’s classification, 25 denture wearers were with healthy palatal mucosa while 50 were with any types of denture stomatitis. The frequency of yeasts was linked to whether subjects had Type II or Type III, but not Type I denture stomatitis. Candida albicans was the most frequently isolated species in denture wearers with and without clinical signs of denture stomatitis and it was the only species produced phospholipase. Although the amount of phospholipase produced by the C. albicans isolates from denture wearers in control and Type II and III DS groups was not significantly different, there was statistically significant difference in the number of C. albicans isolates producing phospholipase between patients with and without clinical signs of DS.     

Mannan and D-arabinitol concentrations in serum from a patient with Candida albicans endocarditis

In an attempt to clarify the comparative values of serological and microbiological examinations for the early diagnosis of systemic candidiasis, antibodies against Candida albicans, serum mannan, and the D-arabinitol creatinine ratio were investigated in a patient with aortic valve endocarditis associated with carcinoma of the bile duct. Candida precipitins and the antibody titer against Candida cell wall mannan were examined by an immunodiffusion technique and hemagglutination test, respectively. Serum mannan was tested by enzyme-linked immunosorbent assay (ELISA) using the biotin-streptavidin procedure. The upper limit of negativity of the assay was determined by adding 0.06 to the absorbance of pooled serum from healthy laboratory workers. This value ws about 0.8 ng/ml with ELISA. The D-arabinitol concentration in serum was examined by an enzymatic fluorometric method. Rising antibody titers against C. albicans, mannan antigenemia, and an elevated D-arabinitol creatinine ratio were first observed between the 11th and 12th hospital days. Blood cultures obtained on 8th, 9th, and 11th hospital days grew C. albicans after 3 to 4 days of incubation. Of 11 serum samples, 5 were positive for mannan, whereas D-arabinitol creatinine ratio was positive in 7 of 9 samples. Blood cultures was the earliest evidence of Candida infections in our cases. However, because of saprophytic nature of Candida species, tests for antibodies, antigenemia, and the D-arabinitol creatinine ratio in combination with blood cultures are necessary to confirm systemic candidiasis at an early stage of infection.Abbreviations ELISA enzyme-linked immunosorbent assay