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Cell morphology variations and budding patterns in Candida isolates.

Introduction

Candida species are yeast and members of the Fungi Imperfecti. They are also widely distributed and often considered to be unicellular organisms, even though they are, in fact polymorphic and can form variety of additional morphotypes, (Grigoriu et al., 1987; Abaitua et al., 1999). Candida occurs as a harmless endosaprophyte or commensal in man but can become pathogenic when the microflora balance in the body is disrupted, causing a disease called candidiasis Genitourinary Candida infections have become increasingly common in clinical practice and are having a major impact on public health causing life-threatening disorders (Stafford, 2000; Carlsen, 2001). Also, Candida infections are becoming difficult to treat due to the organism's resistance to commonly used antimycotic drugs (Ako-Nai et al., 1993).

Figuring out exactly how the transformation of Candida from a non-pathogenic to a pathogenic state and how it can be interrupted is a challenge for mycologists, if this pathogen must be brought under control. Budding (reproduction) is an essential part of the origin and spread of a disease. Candida species are diverse (Okungbowa et al., 2003; Vivier et al., 1997). Therefore, a study to investigate cell properties is appropriate. The aim of this study was, therefore, to find out and document information on cell morphology variations and budding patterns, in Candida species, which will bring to light the diversity in Candida species and pave way for future research especially in the understanding of its morphogenesis, pathogenicity and search for new drugs.

Materials and Methods

Candida-positive genital specimens comprising high vaginal swabs (480), endocervical swabs (22) and urine (15) were collected from symptomatic female patients who reported to hospitals and medical diagnostic laboratories in seven cities located between latitude 5-7.5[degrees]N and longitude 2.5-7[degrees]N in the southern rainforest and middle-belt region of Nigeria, from March 2000-August 2001. Each sample was cultured on Sabouraud Glucose Agar, SGA (SIFIN, Berlin) at 37[degrees]C for 48 hours. Wet preparations of pure cultures (without staining) were observed under the X40 power of an optical microscope.

Subcultures were made on SGA slants, in McCartney bottles, and incubated for 24 hours at 37[degrees]C. Each stored culture was revived on SGA every two weeks.

Candida species were identified by the CHROM-Agar and API 20C System (Analytab Products, Plainview, USA) as used by Houang et al. (1997) and Rex et al. (1995), respectively.

Results and Discussion

Four categories of cell shapes were observed, namely, spherical, oval, oval/spherical and elongated (Tab. 1; Fig. 1). Where both oval and spherical cells were present in about the same number such cells were described as oval/spherical; these cells had the highest frequency of 48.7 % while elongated cells were few (9.0 %). The cell shapes were variously distributed among the different Candida species. About 98.4 % of C. guilliermondii and 1.7 % C. glabrata were spherical while 77.1% C. albicans and 33.3 % C. pseudotropicalis were oval. Oval/spherical cells were present in all species except C. albicans and C. parapsilosis. Elongate cells were found in C. parapsilosis (100 %) and C. albicans (22.9 %). There were three distinct budding types (Tab. 2 and Fig. 2). Bipolar/Multipolar/Random budding was most frequent being present in all species (Tab. 3). The three budding types were present in all cell shapes, except spherical which exhibited only terminal budding (Tab.4).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Discussion:

Candida albicans and C. parapsilosis had no oval/spherical cells at all. This shape may be inherently absent in these species (strains in the case of C. albicans). Stafford (2000) described C. albicans cells as oval or spherical. Although all the C. stellatoidea cells were oval/spherical, the small number was a hindrance in determining if this was the only shape characteristic of this species. Several factors, such as cultural and environmental factors, could affect cell shape (Abaitua et al, 1999; Rycovska et al., 2000; Szabo 1999). There was no correlation between cell shape and species type. There is no documented report of any correlation between cell shape and virulence but it would be of interest to investigate if there is a link between cell shape and ability of Candida cell to attach itself to host tissue.

The site for bud formation is determined in a genetically programmed manner that depends on cell type (Gausmann et al., 1999; Park et al., 1999). These reports and others (Chant, 1994; Lew and Reed 1995; Yang et al. 1997) all point to the distribution of the substance, actin, and bud site proteins as the determinants of bud site. According to these workers, when a cell reaches the threshold size there is redistribution of the actin cytoskeleton with a high concentration at the selected site, followed by cytokinesis. The localization of actin and bud site proteins at more than one site leads to bipolar and multipolar budding. Budding type may not be directly related to pathogen virulence but multipolar budding may lead to faster proliferation of pathogen since the buds (which later form daughter cells) appear at various points on the reproducing parent cell. Table 4 shows the relationship between cell shape and budding type. Regression ANOVA showed that budding type was determined by cell shape (p< 0.05). The implication of this is that in the light of pathogen control, the multipolar type of budding, and the spherical shape (in which only terminal budding was observed), might be important for consideration as the former will lead to faster, and the latter, slower reproduction. Two pertinent questions to be addressed in future research are the factors that determine the shape of a cell, and how cell shape determines pattern of budding, in Candida species.

Acknowledgment

Authors are grateful to the Third World Academy of Science (TWAS), Italy and the Council of Scientific and Industrial Research (CSIR), India for fellowship to F.I.O.

References

Abaitua, F., A. Rementeria, R.S. Millan, Eguzikiza A., J.A. Rodriguez, J. Ponton and M.J. Sevilla, 1999. In vitro survival and germination of Candida albicans in the presence of nitrogen compounds. Microbiology, 145: 1641-1647.

Ako-Nai, A.K., O.O. Kassim, O.O. Adeniran and O. Taiwo, 1993. A study of urinary tract infections at Ile-Ife, Nigeria. East African Medical Journal, 70: 10-14.

Carlsen, G., 2001. The Candida Yeast Answer, Candida Wellness Center, Provo.

Chant, J., 1994. Cell polarity in yeast. Trends in Genetics, 10: 328-333.

Gausmann, U., E. Franzil, and C. Kurischoko, 1999. Distribution of the actin cytoskeleton during the cell cycle of Yarrowia lipolytica and the visualization of the tubulin cytoskeleton by immuno-fluorescence. Yeast, 15: 1079-1086.

Grigoriu D., J. Delacretaz, D. Borelli , 1987. Medical Mycology, Hans Huber Publishers, Toronto.

Houang, E.T., K.C. Chu, and A.T.F. Chen, 1997. Use of CHROM-Agar Candida for genital specimens in the diagnostic laboratory. Journal Clinical Pathology, 50: 563-565.

Lew, D.J. and S.I. Reed, 1995. Cell cycle control of morphogenesis in budding yeast. Current Opinions in Genetics and Development, 18: 1001-1006.

Okungbowa, F.I., O.S. Isikhuemhen and A.P.O. Dede, 2003. The distribution frequency of Candida species in the genitourinary tract among symptomatic individuals in Nigerian cities. Revista Iberoammericana de Micologia, 20: 60-63.

Park, H.O., A. Sanson and I. Herskowitz, 1999. Localization of Bud 2p, a GTPase--activating protein necessary for programming cell polarity in yeast to the presumptive bud site. Genes and Development, 13: 1912-1917.

Rex, J.H., M.A. Pfaller, A.L. Barr, P.W. Nelson and C.D.Webb, 1995. Antifungal susceptibilities testing of isolates from a randomized multicenter trial of fluconazole versus Amphotericin B as treatment of nonneutropenic patients with candidemia. Antimicrobial Agents and Chemotherapy, 39: 40-44.

Rycovska, A., R. Szabo, L. Tomaska and J. Nosek, 2000. The respiratory complex 1 in yeast: isolation of a gene NUO51 coding for the nucleotide binding subunit of NADH Ubiquinone Oxidoreductase from the obligately aerobic yeast Yarrowia lipolytica. Folia Microbiologica, 44: 19-24.

Stafford, P., 2000. Dimorphism in Candida albicans Part 1. Educational Instructional Library, American Society for Microbiology, Washington D.C.

Szabo, R., 1999. Dimorphism in Yarrowia lipolytica: filament formation is suppressed by nitrogen starvation and inhibition of respiration. Folia Microbiologica, 44:19-24.

Vivier, M.A., M.G. Lambreclits and I.S. Pretorius, 1997. Coregulation of starch degradation and dimorphism in the yeast Saccharomyces cerevisiae Critical Reviews in Biochemistry and Molecular Biology, 32: 405-435.

Yang, S., K.R. Ayscough and D.G. Drubin, 1997. A role for the actin cytoskeleton of Saccharomyces cerevisiae in bipolar bud-site selection. Journal of Cell Biology, 136: 121-123.

(1) Okungbowa (1) F.I, Dede (2) A.P.O. Isikhuemhen O.S.

(1) Department of Botany, University of Benin, P.M.B. 1154, Benin City, Nigeria;

(2) Department of Natural Resources & Environmental Design, North Carolina A & T State University, Greensboro, NC 27411, USA.

Corresponding Author: Dr Francisca Iziegbe Okungbowa, Department of Botany, Faculty of Life Sciences University of Benin, PMB 1154, Ugbowo Benin City, Nigeria. Phone: 234-8055376204. E-mail: fiokun2002@yahoo.com
Table 1: Percentage distribution of cell shapes among Candida species.

Cell shape   Distribution among Candida species (%)

             C. glab.   C. trop.   C. guill.   C. alb.

Spherical    1.7        21.3       98.4        --
Oval         --         --         --          77.1
Oval/Spher   98.3       78.7       1.5         --
Elongate     --         --         --          22.9

Cell shape   Distribution among Candida species (%)

             C. pseud.   C. para.   C. stell.   % Total

Spherical    --          --         --          27.1
Oval         33.3        --         --          14.8
Oval/Spher   --          --         100         48.7
Elongate     66.7        100        --          9.4

* (--) = Absent.

Table 2: Description of budding in Candida.

Budding type          Position of bud

Terminal              Any of the two polar ends
Subterminal           Skewed away from polar end
Bipolar/Multipolar/
  Random              Both polar ends plus any other position

Table 3: Distribution of budding types among Candida species

Candida species   Distribution (%) of budding types

                  Terminal   Subterminal   Bipolar/ Multipolar /Random

C. glab.          2.8        --            97.2
C. trop.          21.3       --            78.7
C.guill.          98.5                     1.5
C.alb.            --         --            100
C. pseu.          --         --            100
C. para.          --         61.1          38.9
C. stell.         --         --            100
% Total           27.4       1.8           70.8

* (--) = Absent.

Table 4: Relationship between cell shape and budding
type in Candida species

Cell shape       Type of budding associated with it

Spherical        Terminal
Oval/Spherical   Terminal, Subterminal, Bipolar/Multipolar/Random
Oval             Terminal, Subterminal, Bipolar/MultipolarRandom
Elongated        Terminal, Subterminal, Bipolar/Multipolar/Random
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Title Annotation:Original Article
Author:Okungbowa, F.I.; Dede, A.P.O.; Isikhuemhen, O.S.
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:6NIGR
Date:May 1, 2009
Words:1699
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