Molecular Identification of Candida albicans and C. dubliniensis Using Small Subunit rRNA Gene Sequence in Kerbala, Iraq

1. Introduction

Candida is found as normal flora in the skin and mucosal surface of the intestine, vagina, respiratory system, and oral cavity; however, under some conditions, such as poor oral hygiene, diabetes, random use of antibiotics, and tumors, it transforms from a commensal organism to a pathogen able to cause infection ( 1 , 2 ). Candida has 200 species, 30% of which have been isolated from human diseases, which is increasing ( 3 ). Until recently, Candida albicans (C. albicans) was considered the most frequent opportunistic pathogen in this genus; however, other non-albicans, such as Candida dubliniensis (C. dubliniensis), Candida tropicalis, Candida parapsilosis, and Candida glabrata, were also recorded as causative agents of infection ( 4 , 5 ). Over the past few years, there has been an increase in the incidence of infections caused by C. dubliniensis isolated from the mouth of patients infected with AIDS, those who frequently use antibiotics, and cases of tooth decay. It shares many phenotypic characteristics with C. albicans, such as the production of germ tubes in human serum and chlamydospores in the Cornmeal Agar medium. Therefore, using traditional methods to distinguish between them leads to a false identification in fungi laboratories ( 6 ). However, many genotypic methods have been used to differentiate between these two species, such as the conventional polymerase chain reaction (PCR), Nested PCR, Multiplex PCR, RT PCR, qPCR, FISH, Microarray, Lamp, RNA interference, and RNA-seq ( 7 ). This study aimed to draw a molecular distinction between C. albicans and C. dubliniensis using the 18 SrRNA.

2. Materials and Methods

2.1. Candida Isolates

Samples taken from oral candidiasis cases were cultured on the Sabouraud Dextrose Agar medium supplemented with amoxicillin (250 mg/L) and incubated at 37°C for 24-48 h. Candida species were identified by germ tube production in human serum, chlamydospores, and pseudohyphae formation in the Cornmeal Agar supplemented with tween 80, and they were grown at 45°C ( 8 ).

2.2. DNA Extraction and Purification

The process of extracting and purifying the DNA from the growing colonies of Candida was conducted using the DNeasy Plant Kit (QIAGEN, Germany) following a few steps. First, a sample was taken from pure colonies and filled with the loop. It was then transferred to a 1.5 mL Eppendorf tube, and 400 μL of AP1 cell lysis was added. The tube was incubated in a water bath at 65°C for 10 min. Afterward, 130 μL of buffer P3 was added to the tube containing the mixture, then it was mixed and incubated for 5 min on ice. This step precipitates the detergents for buffer solutions, proteins, and polysaccharides. The tube was centrifuged at 14000 rpm for 5 min, the supernatant solution was transferred to a purple-colored QIAshredder Mini spin column tube, and a centrifugal process was conducted at the same speed (mentioned above) for 2 min. The filtrate was transferred to a new tube, 700 μL of Purification buffer (AW1) was added, and a micropipette was mixed with the contents. Subsequently, 650 μL of the mixture was transferred by a small pipette to the white DNeasy Mini spin column tube containing a special filter for purification. The DNA was centrifuged at 8000 rpm for 1 min, and the filtrate was discarded. After that, 500 μL of AW2 solution was added to the tube and centrifuged at 8000 rpm for 1 min, and the filtrate was disposed of. A total of 500 μL of AW2 solution was added to the tube and centrifuged at 14000 rpm for 2 min.

The filtrate was then disposed of. The DNeasy Mini spin column tube was placed inside a 2 mL Eppendorf tube, and 100 μL of Elution Buffer TE solution was added directly to it and incubated for 5 min at room temperature. A centrifugal process was then conducted at 8000 rpm for 1 min to obtain the filtrate containing the DNA. The tube was kept at -20°C until use.

2.3. Polymerase Chain Reaction

2.3.1. Preparation of the Stock Primers Solution

The primers (QIAGEN, Germany), shown in table 1, were used and prepared according to Sarhan ( 8 ) and the manufacturer’s instructions to obtain a final concentration of 100 picomol/µl. The working solution was prepared for each initiator by taking 10 µl of the stock solution each separately and adding 90 µl to it with 1 µl of deionized water to get a concentration of 10 picomol/µl.

2.3.2. Preparation of the Ready-To-Go PCR Beads Kit

The PCR test was carried out using the Ready-To-Go PCR Beads kit (GE Healthcare, UK), which included 2.5 units of PuRE Taq DNA polymerase, 10 mM Tris HCI(PH9), 50 mM KCL, 1.5 mM MgCl2, 200 µM of each dNTP, stabilizers, and BSA. The final volume of the reaction was 25 μL, so 2 μL of the DNA was added to each tube, 1 μL from the front and 1 μL from the back primer. The volume was supplemented to 25 μL by adding deionized water.

2.3.3. Polymerase Chain Reaction Conditions

The DNA of the isolated fungi was doubled using the steps shown in table 2.

2.4. Electrophoresis

Agarose gel was prepared by dissolving 1.5 g in 100 mL of buffer solution (10×TBE prepared by adding 90 mL of sterile distilled water to 10 mL of TBE solution) and mixing until a clear solution was formed. After that, 5 μL of ethidium bromide dye solution was added after decreasing the temperature of the solution to 40-45°C. The mold was prepared for pouring the agarose gel containing a comb to make holes inside the agarose layer. The dissolved agarose containing the dye was poured and left at room temperature to solidify. The replication products were detected by loading 5 µL of the replicated DNA into each hole of the agarose gel layer. In addition, 5 μL of the volume guide (2000 bp DNA ladder) was added to the left side of the added samples to determine the size of the replicated DNA. The electrophoresis of the samples was carried out, and the reaction results were examined under ultraviolet rays.

The DNA replication products isolated from the PCR were sent to the Korean company Macrogen to determine the sequence of nitrogenous bases. The nitrogenous sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) program to find the similarity between the studied fungi and those registered globally. They were also compared with the data recorded in the National Center for Technical Information (NCBI).

3. Results and Discussion

The results of the phenotypic and physiological diagnosis of the study samples revealed the diagnosis of two Candida species, which were C. albicans and C. dubliniensis. A PCR operation was performed to distinguish between them.

The results of the PCR showed the possibility of complete duplication of the 18 SrRNA region, which is vital in the diagnosis of fungi. This result agrees with what was mentioned by Ban Taha, Zakra Mohammad Kazem ( 9 ), ( 10 - 12 ) The selection of the 18 SrRNA in the diagnosis of fungi to the level of genus and species is due to several reasons, including the presence of many universal primers for fungi based on the conserved regions of the 18 SrRNA gene, which makes it possible to obtain the PCR products. In addition, the deposit of a large number of 18 SrRNA sequences in the Genbank database makes the similarity search convenient, and the repetitive nature of this gene has more than 100 copies per fungi genome, which makes rRNA-dependent amplification easier. Therefore, this gene is suitable for finding diverse and conserved regions within a group of fungi to establish phylogenetic relationships between closely related fungi ( 10 ).

In this study, replications were obtained with sizes ranging from 1639 bp for C. albicans to 1643-1645 bp for C. dubliniensis after DNA extraction from the fungi in the presence of the forward and reverse primers, NS1 and NS8 ( 13 ) (Figure 1). It was deposited in the GenBank database of the NCBI. The isolate was identified as C. albicans with the sequence code MZ574137.1 and C. dubliniensis with the sequence code MZ574410.1.

Figure 1. Electrophoresis of the PCR product of C. albicans and C. dubliniensis. M=2000bp DNA ladder, 2= C. albicans, 6= C. dubliniensis, 1,3,4,5 = other yeasts. 7,8 = no addition.

The analysis of the nitrogenous bases of the DNA replication products using the BLAST program proved that the studied strains belong to C. albicans and C. dubliniensis. The results also showed variations between the two diagnosed species, as C. albicans was distinguished by the presence of two mutations (Transition G/A at sites 995 and 1026 and Transition (C/G) at site 1040). On the other hand, C. dubliniensis showed two mutations of Transition (C/G) at sites 1079 and 1084 (Table 3).

The comparison of the sequence of nitrogenous bases for the study strains showed a 99% match between the isolates of our study and the global ones. The MEGA evolutionary genetic analysis program (version 7) was used. One of the programs was designed to analyze and compare the sequences of identical genes, evolutionary relationships, as well as the pattern of DNA and protein evolution. The program also provides many facilities through serial data, which can be presented as an evolutionary tree ( 14 ). The studied strain of C. albicans showed a similarity rate of 99% with global isolates of C. albicans from Japan, Brazil, the United States, Germany, India, China, Pakistan, and Egypt. The studied strain also showed a genetic similarity rate of 98% with isolates from Iraq (Wasit) and Australia and a genetic similarity rate of 97% with the isolates from Holland (Table 4).

The results of the phylogenetic analysis confirmed this diagnosis by grouping the fungal isolate within groups of isolates belonging to the same species shown in figure 2. The genetic distance was calculated using the neighbor-joining method.

Figure 2. The genetic tree of Candida albicans (marked with a purple triangle) showing the relationship with global fungal strains

In addition, the comparison of the sequence of the nitrogenous bases of the doubled NS1 and NS8 regions of C. dubliniensis isolated in this study revealed that it has a genetic similarity rate of 99% with C. dubliniensis isolates from the United States, Holland, France, and Germany, as well as a 98% similarity with those from Ireland, Holland, and the United States, while its percentage of similarity with the Thai isolates was 96% (Table 5 and Figure 3).

Figure 3. The genetic tree of Candida dubliniensis (marked in a red triangle) showing the relationship with global fungal strains

Despite the importance of phenotypic diagnosis in limiting the fungi under study into smaller groups, many problems accompany this diagnosis, including the researcher’s need for high expertise in the diagnostic process, especially in diagnosing closely related species, as well as the need for significant time and effort. On the other hand, the diagnosis of fungi using interaction technology sequential polymerization is characterized by high accuracy, which helps the researcher to overcome diagnostic problems typical of traditional methods ( 7 ). Khot, Ko ( 14 ) indicated that fungal infections, including candidiasis, change with the increase in the use of medicine. It also changes with the increase in the use of antifungal drugs, so it is necessary to use diagnostic methods capable of diagnosing pathogens, including molecular methods, which increase the chance of success in choosing the appropriate antifungal treatment.

Authors' Contribution

Study concept and design: M. T. A.

Acquisition of data: B. M. H.

Analysis and interpretation of data: S. A. A.

Drafting of the manuscript: B. M. H.

Critical revision of the manuscript for important intellectual content: B. M. H.

Statistical analysis: M. T. A.

Administrative, technical, and material support: M. T. A.

Conflict of Interest

The authors declare that they have no conflict of interest.

References

1. Mayer FL, Wilson D, Hube B. Candida albicans pathogenicity mechanisms. Virulence. 2013; 4(2):119-28.
2. Miceli MH, Diaz JA, Lee SA. Emerging opportunistic yeast infections. Lancet Infect Dis. 2011; 11(2):142-51.
3. Wu J-Q, Zhu L-P, Ou X-T, Xu B, Hu X-P, Wang X, et al. Epidemiology and risk factors for non-Candida albicans candidemia in non-neutropenic patients at a Chinese teaching hospital. Med Mycol. 2011; 49(5):552-5.
4. Mirhendi H, Makimura K, Zomorodian K, Maeda N, Ohshima T, Yamaguchi H. Differentiation of Candida albicans and Candida dubliniensis using a single-enzyme PCR-RFLP method. Jpn J Infect Dis. 2005; 58(4):235.
5. Shariff FM. Classification and division of fungi. Memory for puplishing and distribution. Iraq. 2012;278.
6. Asadzadeh M, Ahmad S, Al-Sweih N, Khan Z. Rapid and Accurate Identification of Candida albicans and Candida dubliniensis by Real-Time PCR and Melting Curve Analysis. Med Princ Pract. 2018; 27(6):543-8.
7. Aslam S, Tahir A, Aslam MF, Alam MW, Shedayi AA, Sadia S. Recent advances in molecular techniques for the identification of phytopathogenic fungi – a mini review. J Plant Interact. 2017; 12(1):493-504.
8. Sarhan ART. Practical mycology. Baghdad. 2012.
9. Ban Taha M, Zakra Mohammad Kazem A-M, Kansa Abdolhossein EA-A. Morphological and molecular isolation and diagnosis of Candida albicans from mouth and dipers of in fants in hospital of children. J Kerbala Univ. 2016; 14(4)
10. Chan GF, Sinniah S, Idris TI, Puad MS, Abd Rahman AZ. Multiple rare opportunistic and pathogenic fungi in persistent foot skin infection. Pak J Biol Sci. 2013; 16(5):208-18.
11. Haase G, Sonntag L, van de Peer Y, Uijthof JM, Podbielski A, Melzer-Krick B. Phylogenetic analysis of ten black yeast species using nuclear small subunit rRNA gene sequences. Antonie Van Leeuwenhoek. 1995; 68(1):19-33.
12. Imran ZK, Al-Asadi YF. Comparison of 25S and 18S primers for estimating clinical isolates of Candida diversity and evaluate their virulence in eyes infections in Iraq. Appl Sci Report. 2015; 9:166-71.
13. White TJ, Bruns T, Lee S, Taylor J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods applications. 1990; 18(1):315-22.
14. Khot PD, Ko DL, Fredricks DN. Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays. Appl Environ Microbiol. 2009; 75(6):1559-65.