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Updated: Dec 22, 2025
Assay for Adhesion and Agar Invasion in S. cerevisiae
Published on: November 8, 2006
Distribution of Pathogenic Yeasts in Different Clinical Samples: Their Identification, Antifungal Susceptibility
Satish T Pote1,2, Mahesh S Sonawane1, Praveen Rahi1
1National Centre for Microbial Resource (NCMR), National Centre for Cell Science, NCCS Complex, S.P. Pune University, Pune 411 007, Maharashtra, India.
Introduction:
Species of genus Candida are part of the common microbiota of humans; however, some of the Candida species are known opportunistic pathogens. Formation of biofilms, resistance to antifungal drugs, and increase in asymptomatic infections demands more studies on isolation, identification and characterization of Candida from clinical samples.
Methods:
The present manuscript deals with assessment of authentic yeast identification by three methods viz., DNA sequencing of 28S rRNA gene, protein profiles using MALDI-TOF MS, and colony coloration on chromogenic media. Antifungal susceptibility and in vitro cell invasion assays were performed to further characterize these isolates.
Results:
Comparison of three methods showed that DNA sequence analysis correctly identified more than 99.4% of the isolates up to species level as compared to 89% by MALDI-TOF MS. In this study, we isolated a total of 176 yeasts from clinical samples and preliminary morphological characters indicated that these yeast isolates belong to the genus Candida. The species distribution of isolates was as follows: 75 isolates of Candida albicans (42.61%), 50 of C. tropicalis (28.40%), 22 of C. glabrata (12.5%), 14 of C. parapsilosis (7.95%) and 4 of Clavispora lusitaniae (2.27%). Other species like Cyberlindnera fabianii, Issatchenkia orientalis, Kluyveromyces marxianus, Kodamaea ohmeri, Lodderomyces sp., and Trichosporon asahii were less than 2%. Antifungal susceptibility assay performed with 157 isolates showed that most of the isolates were resistant to the four azoles viz., clotrimazole, fluconazole, itraconazole, and ketoconazole, and the frequency of resistance was more in non-albicans Candida isolates. The susceptibility to azole drugs ranged from 7% to 48%, while 75% of the tested yeasts were susceptible to nystatin. Moreover, 88 isolates were also tested for their capacity to invade human cells using HeLa cells. In vitro invasion assay showed that most of the C. albicans isolates showed epithelial cell invasion as compared to isolates belonging to C. glabrata, C. parapsilosis and C. tropicalis.
Discussion:
The identification of yeasts of clinical origin by sequencing of 28S rRNA gene performed better than MALDI-TOF MS. The present study reiterates the world scenario wherein there is a shift from Candida strains to emerging opportunistic pathogens which were earlier regarded as environmental strains. The present study enlightens the current understanding of identification methods for clinical yeast isolates, increased antifungal drug resistance, epithelial cell invasion as a virulence factor, and diversity of yeasts in Indian clinical samples.
Insights
DNA sequencing of the 28S rRNA gene accurately identified clinical yeast isolates, outperforming MALDI-TOF MS. This study highlights increasing antifungal resistance and the emergence of opportunistic Candida pathogens.
Area of Science:
- Medical Mycology
- Clinical Microbiology
- Molecular Diagnostics
Background:
- Candida species are common human microbiota, with some acting as opportunistic pathogens.
- Biofilm formation, antifungal resistance, and asymptomatic infections necessitate further Candida research.
- Accurate identification and characterization of Candida from clinical samples are crucial.
Purpose of the Study:
- To evaluate and compare DNA sequencing, MALDI-TOF MS, and chromogenic media for identifying clinical yeast isolates.
- To characterize the antifungal susceptibility and in vitro cell invasion capabilities of isolated Candida species.
- To assess the prevalence and characteristics of Candida species in Indian clinical samples.
Main Methods:
- DNA sequencing of the 28S rRNA gene for yeast identification.
- Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) for protein profiling.
- Chromogenic media for colony coloration and preliminary identification.
- Antifungal susceptibility testing against azoles and nystatin.
- In vitro epithelial cell invasion assays using HeLa cells.
Main Results:
- DNA sequencing achieved >99.4% accurate species-level identification, surpassing MALDI-TOF MS (89%).
- Seventeen Candida species were identified from 176 clinical isolates, with Candida albicans (42.61%) being most prevalent, followed by C. tropicalis (28.40%) and C. glabrata (12.5%).
- Most isolates exhibited azole resistance (7-48% susceptibility), while nystatin showed higher efficacy (75% susceptibility). C. albicans demonstrated greater epithelial cell invasion than non-albicans species.
Conclusions:
- 28S rRNA gene sequencing is a superior method for identifying clinical yeast isolates compared to MALDI-TOF MS.
- There is a discernible shift towards emerging opportunistic pathogens among clinical Candida isolates.
- The study underscores the importance of accurate identification, rising antifungal resistance, and epithelial cell invasion as a virulence factor in clinical yeast isolates from India.
