G Luo

Candida Species Exhibit Differential In Vitro Hemolytic Activities

ABSTRACT A total of 80 Candida isolates representing 14 species were examined for their respective responses to an in vitro hemolytic test. A modification of a previously described plate assay system where the yeasts are incubated on glucose (3%)-enriched sheep blood agar in a carbon dioxide (5%)-rich environment for 48 h was used to evaluate the hemolytic activity. A group of eight Candidaspecies which included Candida albicans (15 isolates),C. dubliniensis (2), C. kefyr(2), C. krusei (4), C. zeylanoides (1), C. glabrata(34), C. tropicalis (5), andC. lusitaniae (2) demonstrated both alpha and beta hemolysis at 48 h postinoculation. Only alpha hemolysis was detectable in four Candida species, viz., C. famata (3), C. guilliermondii(4), C. rugosa (1), and C. utilis (1), while C. parapsilosis (5) and C. pelliculosa (1) failed to demonstrate any hemolytic activity after incubation for 48 h or longer. This is the first study to demonstrate the variable expression profiles of hemolysins by different Candida species.

Candida glabrata, an emerging fungal pathogen, exhibits superior relative cell surface hydrophobicity and adhesion to denture acrylic surfaces compared with Candida albicans.

Oral candidosis is a common opportunistic infection in debilitated individuals and Candida glabrata is the second most frequently isolated species from this condition, after Candida albicans. Candidal adherence to various biological or non‐biological surfaces is considered a prerequisite for colonization, and pathogenesis of candidal infections, and their relative cell surface hydrophobicity (CSH) is likely to be a possible contributory force involved in this process. Whereas a large body of data on the latter features of C. albicans is available, there is surprisingly little information on C. glabrata. As a comprehensive database on the relative adhesion and CSH of Candida spp. is instructive and useful, we investigated in vitro the latter attributes of 34 oral isolates of C. glabrata and 15 isolates of C. albicans. There were remarkable intraspecies differences in both the CSH and the adhesive ability of C. glabrata strains (p<0.001). Compared with C. albicans, C. glabrata demonstrated a four‐fold greater CSH value (30.63±11.20% vs 7.23±3.56%, p<0.0001) and a two‐fold greater tendency to adhere to denture acrylic surfaces (75.18±39.96 vs 30.36±9.21, p<0.0001). A significant positive correlation between CSH and adhesion was also noted for both C. glabrata (r=0.674, p<0.0001) and C. albicans (r=0.636, p<0.05). When the effect of different incubation conditions on the relative CSH and adherence of C. glabrata was examined, CSH and the adherence to acrylic surfaces of four of six C. glabrata isolates were significantly affected by a reduction of the culture temperature (from 37 °C to 25 °C). A positive relationship also emerged when the temperature‐induced variations in the adherence values were correlated with their relative CSH. These data provide hitherto unavailable archival information on important pathogenic attributes of the two most common oral Candida species that may help explain their predominance in this milieu.

Reverse transcriptase polymerase chain reaction (RT-PCR) detection of HLP gene expression in Candida glabrata and its possible role in in vitro haemolysin production

Although haemolysins are known to be putative virulence factors contributing to pathogenicity in Candida species, the haemolytic activity of Candida glabrata and its genetic expression is ill understood at present. Thus, we studied a total of 34 Candida glabrata isolates for their in vitro haemolytic activity using a previously described plate assay system. The mRNA expression of HLP, a putative haemolysin gene, of these isolates was also evaluated using a semi‐quantitative, non‐competitive RT‐PCR assay. All 34 C. glabrata isolates exhibited both partial (α) and complete (β) haemolytic activity to varying degrees. In parallel with the haemolytic activity, all isolates were also positive for HLP mRNA expression. The expression levels of HLP mRNA (as relative units) ranged from 1.01 to 1.82, with a mean value of 1.32. On regression analysis of latter values and the haemolytic activity (in terms of the dimension of the haemolytic zone in the plate assay) of the C. glabrata isolates a highly significant positive correlation was noted (r=0.759, p<0.0001). Taken together, our data illustrate not only the phenotypic characteristics of haemolysin(s) and HLP expression of a battery of C. glabrata clinical isolates, but also, for the first time, evidence for a role of HLP in haemolysis.

Up-regulation of Fas ligand and down-regulation of Fas expression in oral carcinogenesis

An important molecule involved in delivering the death signal that initiates apoptosis is called Fas, or Apo-1, which sits on the cell surface. When another molecule called the Fas ligand (FasL) binds to it, Fas triggers a series of events inside the cell that leads to apoptosis. In order to investigate the mechanism of immune escape and the expression of Fas and FasL in oral premalignant lesions (OPLs) and oral squamous cell carcinomas (OSCCs), a total of 64 samples were evaluated by an immunohistochemical method using a labelled streptavidin-biotin assay. These samples comprised nine hyperkeratotic and 24 oral premalignant lesions (nine of mild, moderate, and six of severe dysplastic lesions), and 24 OSCCs, together with seven healthy controls. The results demonstrated that the majority of invasive OSCCs showed down-regulation of Fas expression but up-regulation of FasL expression. These phenomena were also detected in OPLs. The results indicate that the expression of Fas and FasL is involved in oral carcinogenesis and this may be a mechanism by which the cancer cells evade the host immune assault. Perhaps, in future, Fas/FasL system may be used as a prognostic biomarker in predicting the behavior of oral premalignant lesions.

Actinomyces spp. in supragingival plaque of ethnic Chinese preschool children with and without active dental caries

Very limited molecular epidemiological data are available on the role of Actinomyces spp. in the pathogenesis of caries in the primary dentition. Therefore, we investigated their distribution in supragingival plaque of ethnic Chinese preschool children from Singapore and Hong Kong, either with or without active caries. Plaque samples were taken from intact interproximal enamel areas using dental floss. Bacterial genomic DNA of each sample was extracted and variable regions of 16S ribosomal DNA amplified and labelled with digoxigenin. Oligonucleotide probes specific for Actinomyces bovis, Actinomyces gerencseriae, Actinomyces israelii, Actinomyces meyeri, Actinomyces odontolyticus, catalase-negative Actinomyces naeslundii (genospecies 1 and 2) and catalase-positive Actinomyces naeslundii genospecies 2 (previously Actinomyces viscosus serotype II) were used to detect these species using Southern hybridization with a Minislot and Miniblotter system. A. odontolyticus, A. gerencseriae and A. meyeri were detected with similar frequency in both Singapore and Hong Kong samples or in those with and without active caries. However, the prevalence of A. naeslundii was significantly different in the two locales (p < 0.05). A. odontolyticus (88.7%), A. gerencseriae (56.6%) and A. naeslundii (50.9%) were detected in a majority of the samples and the positive hybridization signals of A. gerencseriae in the caries-active group were stronger than from the caries-free group. A. bovis and A. israelii were undetectable in any of the samples. These data imply that A. odontolyticus, A. naeslundii and A. gerencseriae may play an important role in supragingival plaque formation on primary teeth in ethnic Chinese, with others such as A. meyeri contributing.