J Bacteriol Virol.  2019 Dec;49(4):212-220. 10.4167/jbv.2019.49.4.212.

Identification of Candida Species Using 26S Ribosomal RNA Gene Sequencing in Patients with Periodontitis

• Al-Dabbagh NN
• Al-Janabi
• Al-Shuhaib MBS

• KMID: 2468027
• DOI: http://doi.org/10.4167/jbv.2019.49.4.212

Abstract

The infection with Candida spp. for oral cavity is being increasingly reported. However, its variations have not yet been specifically described in periodontitis. The present study was conducted to use an uniplex 26S rRNA-based amplicons to detect and discriminate Candida using only one pair of ribosomal primers. A total of 50 patients with chronic periodontitis was involved in the study. Pure Candida colonies were isolated from 23 patients and genomic DNA was extracted, and PCR was conducted. Direct DNA sequencing followed by comprehensive phylogenetic analyses were performed to confirm the identity of Candida colonies. Results indicated that the ration of Candida-infected patients was 46%, with a high prevalence of C. albicans, followed by remarkably lower ratios of C. parapsilosis, C. glabrata, C. kefyr, and C. dubliniensis respectively. Phylogenetic analyses indicated obvious discrimination amongst the analyzed Candida species as each observed species occupied a distinctive phylogenetic position. The current results reported a simple, efficient, and low-cost detection of five species of Candida without the need for other costly techniques of molecular screening. The current findings may help dentists to easily take a snapshot of the patterns of Candida infection in periodontitis cases to assess the nature and grade of infection.

Keyword

26S ribosomal RNA; Candida; infection; DNA sequence analysis; Oral cavity

MeSH Terms

Candida*
Chronic Periodontitis
Dentists
Discrimination (Psychology)
DNA
Genes, rRNA*
Humans
Mass Screening
Mouth
Periodontitis*
Polymerase Chain Reaction
Prevalence
RNA, Ribosomal*
Sequence Analysis, DNA
DNA
RNA, Ribosomal

Figure

• 

  Figure 1 The main phenotype-genotype characteristics of 23 Candida colonies isolated from periodontitis patients. a) phenotypic characteristics of five Candida species after being incubated for 72hrs at 30℃ in CHROMagar media (https://www.chromagar.com/). b) Agarose gel electrophoresis profile for the observed PCR amplicons of Candida colonies. Lane “M” refers to DNA ladder marker, lanes 1 – 23 refers to 26S rRNA-based amplicons that isolated from 23 microbiologically identified Candida colonies. c) Sequencing reaction interpretation of the corresponding identity of each identified colony. Each color refers to a particular species of Candida.

• 

  Figure 2 Phylogenetic tree of Candida spp. 26S ribosomal RNA gene partial sequences and their accession numbers, obtained from Genbank database (https://www.ncbi.nlm.nih.gov). Sequences were aligned using the online Clustal omega server (https://www.ebi.ac.uk/Tools/msa/clustalo/). Aligned sequences were used for the phylogenetic analysis conducted with Figtree Software (http://tree.bio.ed.ac.uk/software/figtree/). The method used for tree constructions was based on cladogram parameters. The number 4.0 at the bottom of the tree refers to the phylogenetic distance measure by a bootstrap scale.

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