Genetic Analysis of Candida glabrata from Candiduric Patients Using Microsatellite Length Polymorphism, Antifungal Susceptibility, and Enzymatic Profiles

Abstract

Background: Candida glabrata is the second agent of candiduria with increased resistance to antifungals. Microsatellite length polymorphism (MLP) is one of the genotyping techniques used in the epidemiological investigation to improve clinical management. Objectives: We aimed to detect different genotypes of C. glabrata isolates using six microsatellite markers and the MLP technique. Moreover, our genotypes' association with other countries' genotypes was illustrated using a minimum spanning tree. We investigated in vitro antifungal susceptibility and enzymatic activity profiles of the isolates. Methods: Six microsatellite markers were amplified using multiplex-PCR for 22 C. glabrata strains isolated from urine in pediatric patients admitted to the Abuzar Children's Hospital in Ahvaz, Iran. The PCR products were presented for fragment analysis, and the size of the alleles was determined. Antifungal susceptibility tests and extracellular enzyme activities were also performed. Results: Nineteen multilocus genotypes were detected so that 22.7% of the strains had identical genotypes. The isolates were wild-type for amphotericin B (0.0625 - 2 µg/mL), itraconazole (0.125 - 2 µg/mL), and voriconazole (0.0078 - 0.00625 µg/mL). All the isolates were sensitive to fluconazole at the MIC range (0.0312 - 16 μg/mL), and three of them were resistant to caspofungin (MIC ≥ 0.5 μg/mL). Moreover, 72.7 and 68.2% of the isolates had no phospholipase and esterase activities. The highest potency of enzymatic activity was obtained in hemolysin and proteinase enzymes. A high genetic diversity (19 genotypes of the 22 isolates) existed among the urinary C. glabrata isolates. Based on the minimum spanning tree, two clusters of our genotypes were related to C. glabrata genotypes in a previous study in Iran, and the third cluster was entirely connected with Chinese genotypes. Conclusions: Most of the isolates were the non-wild type for posaconazole but were rarely resistant to other antifungals. Hemolysin and proteinase secreted as the main virulence factors among the urinary C. glabrata isolates.

Enzymatic and antifungal susceptibility profiles of Candida glabrata isolates from paediatric patients and their genetic diversity based on microsatellite length polymorphism

“…In the current study, the DP was 0Á936, which was highly desirable. Previous studies reported a DP between 0Á84 and 0Á978 (Abbes et al 2012;Hou et al 2017;Kiasat et al 2019;Gharaghani et al 2021). Eighteen unrelated genotypes were detected in 36 C. glabrata isolates in the current investigation.…”

“…(2019), Gharaghani et al . (2021), and Abbes et al . (2012) reported different shared and identical genotypes (60·7 and 39·3%; 10·5 and 89·5%; 69·4 and 30·6%, respectively).…”

“…2019; Gharaghani et al . 2021). Eighteen unrelated genotypes were detected in 36 C. glabrata isolates in the current investigation.…”

“…The positive hemolytic activity was demonstrated as a ring of beta hemolysis around the colonies. Proteinases medium containing 1 g of KH 2 PO 4 (Merck, Darmstadt, Germany), 0Á5 g of MgSO 4 (Merck), 0Á1 g of yeast extract (Merck), 10 g of glucose (PanReac, Barcelona, Spain) and 2 g of bovine serum albumin (Merck) that were dissolved in 100 ml distilled water, sterilized by filtration and added to pre-autoclaved 20% agar medium (Gharaghani et al 2021). The transparent zone was visualized after staining with 1% Amido black (Cellogel, Milano, Italy).…”

“…The resulting tree was viewed using iTOLv6 (Letunic and Bork 2021; available at https://itol.embl.de/). Published papers that used the same microsatellite markers on C. glabrata from China (Hou et al 2017), Iran (Kiasat et al 2019;Gharaghani et al 2021), Tunisia (Abbes et al 2012), France (Foulet et al 2005), Spain (Dur an-Valle et al 2012), Germany, Italy, Netherlands, Nigeria, Portugal, Greece, and Turkey (Chillemi et al 2016) were collected, and genotypes from different countries were compared using MStree. The MStree construction was performed in EDENetworks v2Á18 (Kivel€ a et al 2015).…”