Molecular Identification, Dimorphism and Virulence of C. albicans


Mohsen A. Sayed, Gihad A. Sayed*, Eman Abdullah M. Ali

Botany and Microbiology Department, Faculty of Science, Cairo University, 12613, Giza, Egypt.

*Corresponding Author E-mail:



C. albicans causes human diseases, especially in immune-compromised patients. The current study aimed to identify Candida albicans using different techniques. Dimorphism and virulence behaviour were also studied. A Candida albicans strain was firstly identified by biochemical methods using VITEK 2 Compact automated technique and chromogenically using CHROMagar differential media that differentiate between Candida spp. Based on an enzymatic reaction. Molecular identification using ITS primers was also used to confirm Candida albicans identification. Accession number of the identified C. albicans was obtained as OK104215. The enhancement of dimorphism was studied using RPMI 1640 media (Roswell Park Memorial Institute Medium), while monitoring growth at different time intervals under microscope to investigate dimorphic changes. C. albicans showed its optimum dimorphism after 36-66 hours at 37C. HPLC analysis for the enzyme product S-adenosylmethionine (SAM) was carried out at different time intervals. By increasing time, SAM production increased until optimum production reached after 72h of incubation on RPMI 1640. After that the production of SAM began to decrease.


KEYWORDS: Candida albicans, Dimorphism, Virulence, Molecular identification, HPLC.





C. albicans causes candidiasis or oral thrush in human, especially in immune-compromised patients1,2. It frequently colonizes skin and mucous membranes; it causes nosocomial bloodstream infections worldwide3. C. albicans is a dimorphic fungus has the ability to change its morphology from single yeast cell to filamentous form4. The morphogenic switch between hyphae and yeast form is associated with different factors, that is environmental (pH, temperature, oxygen and glucose concentration) or biochemical (enzyme activity and protein biosynthesis)5,6. Dimorphism is the key of virulence behavior in C. albicans7. Chromogenic Candida agar is used in identification of Candida8,9.


Parka et al.10 used biochemical kits such as API 20 C AUX and Vitek-2C in identification of Candida spp. in addition to molecular identification. Identification of Fungi on DNA bases is gaining more importance11. Many identification systems depend on sequencing of DNA to accurately identify organisms12.


However, global system for fungal identification is a need13. Barcoding of DNA is an important method applied for species identification depending on sequences of DNA14. Region of internal transcribed spacer (ITS) is the common sequenced region of DNA used in fungal identification15 and is recommended as a global fungal sequence of barcoding16. Morphogenesis of some eukaryotic organisms is affected by some enzymes which are specified for fungus, some enzymes can affect virulence and pathogenic capacity of the microorganisms such as phospholipases, lipases, proteinases, and dimorphic growth in some Candida       sp. 1,17. Genes that initiate C. albicans dimorphism are regulated by a network of key signaling pathway and transcriptional factors18. S-adenosylmethionine synthetase (SAMs), also named as methionine adenosyltranferase (MAT), controls methylation reactions, and speeds up methylation of L-methionine (non-polar amino acid) to form S-adenosylmethionine (SAM)19 which is a methyl donor that facilitates methylation of DNA and once DNA is methylated it switch the genes off; therefore SAM can be considered to control gene expression, and that’s why it play an important role in yeast dimorphism process18,20,21.


The main objectives of this study were to identify Candida albicans using chromogenic, biochemical and molecular techniques. Dimorphism and virulence behavior were also studied using HPLC analysis for SAM product.




C. albicans:

C. albicans was kindly provided by microbiology laboratory, Micro-Analytical Centre, Faculty of Science, Cairo University, Giza, Egypt.


Cultivation of Candida:

Candida was cultivated on the following media: Yeast malt extract agar (g/l): Glucose, 10; Yeast extract, 3; Malt extract, 3; agar, 20. (pH 7.4). Sabouraud’s Dextrose agar (g/l): Dextrose, 40; Peptone, 10; Agar, 15. (pH 7.4). Sabouraud’s glucose broth (g/l) Glucose 40; Peptone; (pH 7.4). Candida was incubated at 37C for 48 hours.


Identification of Candida:

The yeast was firstly identified biochemically by VITEK 2 Compact technique22,23,24, then the identification was confirmed chromogenically by CHROMagar media25,26, 27,28. The medium consists of (g/l): Glucose, 20; Peptone, 10; Chloramphenicol, 0.5; Agar, 15; Chromogenic mixture, 2. The plates were incubated at 37C for 48 hours.


Molecular identification of C. albicans:

DNA extraction from the fungus was performed using SDS extraction method described by Hidalgo et al.,29. Amplification of the ITS gene was performed using the primers ITS1- F (5´- TCC GTA GGT GAA CCT TGC GG-3´) and ITS4- R (5´-TCC TCC GCT TAT TGA TAT GC-3´)30,31,32. PCR amplifications were carried out in 25 µl reaction mixture containing 2µl of the primers 31. The amplified products were purified and sequenced using automated DNA sequencer. Fungal strain were identified by submitting the ITS sequences in the National Centre for Biotechnology Information (NCBI) using Basic Local Alignment Search Tool (BLAST) search program34,35,36. Accession number of the identified C. albicans was obtained.


Enhancement of Dimorphism:

Roswell Park Memorial Institute Medium (RPMI         1640) 37 that enhances virulence, and dimorphism phenomenon 38, under aseptic conditions was inoculated from Sabouraud’s agar slants, then morphologically examined under microscope every 1, 6, 12, 18, 24, 30, 36, 42, 48, 54, 66, 72, 78 and 84 hours. The time taken for best dimorphic phenomena to happen was observed. RPMI-1640 media contain the following (mg/L): Ca (NO3)2·H2O, 100; KCl, 400; MgSO4, 48.8; NaCl, 6000; Na2HPO4, 800; NaHCO3, 2000; L-arginine, 200; L-asparagine; H2O, 56.8; L-aspartic acid, 20; L-cystine. 2HCl, 65.2;  L-glutamic acid, 20; L-glutamine, 300; glycine, 10; L-histidine, 15; hydroxy-L-proline, 20; L-isoleucine, 50; L-leucine, 50; L-lysine. HCl, 40; L-methionine, 15; L-phenylalanine, 15; L-proline, 20; L-serine, 30; L-threonine, 20; L-tryptophan, 5; L-tyrosine.2Na.2H2O, 28.83; Lvaline, 20; biotin, 0.20; D-calcium pantothenate, 0.25; choline chloride, 3; folic acid, 1; i-Inositol, 35; nicotinamide, 1; p-aminobenzoic acid, 1; pyridoxine. HCl, 1; riboflavin, 0.20; thiamine. HCl, 1; vitamin B12, 0.05; D-glucose, 2000; glutathione, 1 and phenol red, 5. The final pH of the media was 7.4.


Assay of SAM synthetase activity using high performance liquid chromatography (HPLC):

Cultivated cells exposed to sonicator for cell disruption and intercellular enzyme extraction39. Standard reaction mixture (mM): tris HCl pH 8.0 (100), KCl (200), MgCl2 (20), dithiotheritol (1), ATP (5), L-Methionine (5) (pH 7.8). Standard SAM was purchased from Sigma Company for HPLC analysis (4.6mm x 250mm, 5µm) to identify samples concentration at 254nm. SAM was diluted by an equal volume of (NH4)2SO4 cold saturated solution. The solution was stirred for 20min in ice bath and then the precipitate was removed by centrifugation at -4C, 1500rpm for 10 min. 50g of (NH4)2SO4 per 100 ml of original volume was added to the supernatant over 45min. The precipitate was collected by centrifugation at -4C (1500rpm x 10 min), and then the precipitate was dissolved in 20ml of 2% Na2CO3 in 0.1 N NaOH40,41.



Identification of Candida:

C. albicans was firstly identified biochemically using VITEK 2 Compact technique (Table1). The identification of Candida was carried out after 48 hours of incubation at 37°C. The data showed 99% probability C. albicans with excellent identification confidence. Also, C. albicans was identified chromogenically using CHROMagar differential media showing green colonies after 48 hours incubation at 37(Figure1). The identification of C. albicans was finally confirmed using molecular techniques. Amplification of the ITS region was carried out during PCR reaction using ITS1 and ITS4 primers. Accession number of the identified C. albicans was obtained as OK104215. Its relation with the other related fungal species was displayed by phylogenetic tree (Figure 2).



Figure 1: Chromogenic identification of C. albicans using CHROMagar differential medium



Figure 2:  Phylogenetic tree of C. albicans (OK104215).


Table 1: Biochemical identification of C. albicans through VITEK 2 Compact automated technique.

Biochemical Details


































































































Figure 3: Microscopic examination of C. albicans growth after different time intervals (Light microscope 40 X).




Dimorphism enhancement:

Dimorphism phenomenon enhanced by sub-culturing C. albicans on RPMI 1640 media. Growth was monitored every 1, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, and 84 h to investigate dimorphic changes. Growth after one h on RPMI 1640 growth media, showed initiation of germ tubes and mycelial formation. After 6 h, a transition phase between yeast form and mycelial (filamentous) form was observed. After 30 h, the filamentation of Candida was developed. After 36 h, the filamentation and development of mycelia was completed. After 66 h, degeneration of filaments began to develop. After 72 h, degeneration of filaments was completed (Figure 3).


HPLC analysis for SAM (enzyme product):

HPLC analysis for SAM (enzyme product) after 6, 24, 48, 72, and 96 h was carried out. It was found that SAM increased with increase in time, and the optimum production of SAM was attained after 72 h of incubation on the dimorphism enhancing media (RPMI 1640). After that the production of SAM began to decrease (Figure 4).


Description: Oval:  fDescription: Oval: e






Figure 4: HPLC analysis for SAM (enzyme product) after different time intervals.





Over one million patients die every year suffering from invasive fungal infections17. Candidiasis is a very serious problem due to its highly developed resistance against many drugs42. Targeting enzymes involved in this phenomenon may weaken fungal growth in the host 7. Dimorphism of C. albicans is a serious phenomenon, and is considers the key of its virulence22,23,51,52,53.


In the present work, C. albicans was firstly identified biochemically using VITEK 2 Compact technique, and then the identification was made using chromogenic identification. The conventional methods of identification of Candida species are time consuming and difficult to perform. VITEK method is a rapid and accurate method for identification of Candida species43. All chromogenic media tested appeared to be useful in presumptive identification of Candida spp44.


The identification of C. albicans was confirmed using molecular identification technique for accurate and precise identification, as the fungal DNA is its biological stamp as mentioned by Neppelenbroek et al.45,46; Gharanfoli et al.47 and Sankari et al.26. PCR-based detection of internal transcribed spacer regions of the rRNA genes was evaluated as a means of fungal identification by using internal transcribed spacers48.


Dimorphic fungi are those fungi which exist in two forms of growth, morphologically and biochemically different (parasitic form and commensally form).Yeast form unicellular and multicellular form4. This transference process from one form to another occurs under certain conditions with the catalysis of enzymatic activity of SAMs which plays an important role in dimorphism process5.


Regarding HPLC analysis for SAM after different time intervals, it was found that SAM concentration increases with increase in filamentation of C. albicans and as the culturing media aged, it loses its nutrients so filamentation declined as well as SAM level        declined5, 49. SAM plays an important role in biological methylation, and it also linked to polyamine               metabolism 50.



C. albicans can easily be identified by various ways, biochemically using VITEK 2 Compact technique, chromogenically using CHROMagar media, and molecularly using ITS technique. The responsible factor of C. albicans virulence is the dimorphism, which shows its optimum behavior after 36-66 hours incubation on RPMI-1640 liquid medium at 37C under aseptic conditions. Maximum SAM production was attained after 72 h.



The authors have no conflicts of interest regarding this investigation.



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Received on 07.05.2022             Modified on 03.07.2022

Accepted on 10.09.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1007-1011.

DOI: 10.52711/0974-360X.2023.00168