Phospholipase activity of Candida species isolated from diabetic patients
Negar Amini1, Rasoul Mohammadi2
1 Department of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences; Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
|Date of Submission||12-Mar-2022|
|Date of Acceptance||26-Apr-2022|
|Date of Web Publication||27-Jan-2023|
Dr. Rasoul Mohammadi
Associate Professor of Medical Mycology, Department of Medical Parasitology and Mycology, School of Medicine Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
Background: Candidiasis is one of the prevalent fungal infections caused by the genus Candida. The clinical manifestation ranges from mucocutaneous colonization to disseminated and fatal infections such as candidemia. Diabetes mellitus is one of the significant predisposing factors for this fungal infection. Candida spp. may release many exoenzymes such as phospholipase to debilitate the immune system and facilitate adherence and invasion of the fungus to the host cells. The aim of the present study is evaluation of phospholipase activity of Candida species isolated from candidemia and gastroesophageal candidiasis (GEC) among diabetic patients.
Materials and Methods: Eighty-three Candida isolates were evaluated for enzyme activity by phenotypic (the precipitation zone around the colonies) and molecular methods (detection of phospholipase genes using duplex polymerase chain reaction with specific primers).
Results: Eight out of eighty-three clinical isolates (9.6%) were negative for phospholipase production. All phospholipase producers among candidemia and GEC isolates were categorized in high production group.
Conclusions: Our findings revealed no differences in phospholipase activity among isolates obtained from different body sites (blood, oesophagus and stomach); however, non-albicans Candida species had less phospholipase activity.
Keywords: Candida species, candidemia, diabetes mellitus, gastroesophageal candidiasis, phospholipase activity
|How to cite this article:|
Amini N, Mohammadi R. Phospholipase activity of Candida species isolated from diabetic patients. Adv Biomed Res 2023;12:19
| Introduction|| |
Candida species are opportunistic yeasts that cause a wide spectrum of infections in immunocompromised patients, which range from superficial to disseminated infections. Diabetes mellitus is one of the main risk factors for this fungal infection. Elevated serum glucose level can impair neutrophil and monocyte adhesion and movement, phagocytosis and killing of pathogens. The increased glucose level in involved tissues increments Candida colonization and invasion. Candida species may release many exoenzymes such as phospholipase to impair of cellular membranes, which facilitate adherence and invasion of the fungus to the host cells. In the present study, we examined the in vitro phospholipase activity of Candida species isolated from candidemia and gastroesophageal candidiasis.
| Materials and Methods|| |
The protocol of the present study was approved by the Ethics Committee of Isfahan University of Medical Science (no. IR.MUI.MED.REC.1399.316). Eighty-three Candida isolates were tested among which, 37 and 46 strains were related to candidemia and GEC, respectively. Thirty-four and 12 isolates were obtained from the stomach and oesophagus, respectively. With HpaII restriction enzyme, clinical isolates were previously identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method as follows: Candida albicans (n = 67; 80.7%), Candida parapsilosis (n = 8; 9.6%), Candida glabrata (n = 6; 7.2%) and Candida krusei (n = 2; 0.25%).
Detection of phospholipase activity
This test was performed according to the method of Price et al. Briefly, Sabouraud Dextrose Agar (SDA) was supplemented with 0.005 mol/L CaCl2, 1 mol/L NaCl, and 8% sterile egg yolk emulsion and was poured (about 23 mL) in 90 mm Petri dishes. Ten microliter of the yeasts suspension was inoculated on the test medium, and plates were incubated at 37°C for 5 days. Phospholipase activity (Pz) was measured according to the precipitation zone around the colonies. The value of Pz was determined as the growth diameter ratio to the colony's total diameter plus the precipitation zone. It was scored as follows: negative (–), low production (0.75–0.9), moderate production (0.51–0.74) and high production (0.35–0.5).
Detection of Candida phospholipase genes
Detection of phospholipase genes (PLB1 and PLB2) were performed by duplex PCR (dPCR) and specific primers containing: PLB1 (forward: 5′-CCT ATT GCC AAA CAA GCA TTG TC-3′ and reverse: 5′-CCA AGC TAC TGA TTT CAC CTG CTC C-3′) and PLB2 (forward: 5′-GTG GGA TCT TGC AGA GTT CAA GC-3′ and reverse: 5′-CTC AAA GCT CTC CCA TAG ACA TCT G-3′). The size of amplicons for PLB1 and PLB2 were 179 bp and 270 bp, respectively.
SPSS Statistics 25 (IBM, Chicago, USA) was used for data analysis. Kolmogorov–Smirnov test was applied for normal data distribution, the independent t-test and Chi-square test were used to compare quantitative and qualitative data between the two groups, respectively. A P value of < 0.05 was considered significant.
| Results|| |
Among blood isolates, 4 out of 37 (10.8%) Candida spp. (C. glabrata = 2 and C. parapsilosis = 2) were negative for phospholipase production. In GEC group, 4 out of 46 (8.7%) isolates (C. glabrata = 1 and C. albicans = 3) didn't secrete phospholipase. All phospholipase producers among candidemia and GEC isolates were categorized in high production group (0.35–0.5) [Figure 1]. The PLB1 and PLB2 gene expression percentage in the candidemia and GEC groups were 76.5% and 64.7%, and 86.4% and 77.3%, respectively [Figure 2]. There was no statistically significant difference in the expression of both phospholipase genes between the two groups (p-value = 0.15).
|Figure 1: Enzymatic activity of Candida spp. on SDA supplemented with CaCl2, NaCl and egg yolk emulsion|
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|Figure 2: Amplification of PLB1and PLB2 genes by dPCR, lane M is 100 bp DNA size marker, lanes 1, 2 are C. albicans isolates that show both PLB1 (179 bp) and PLB2 (270 bp) amplicons, and N is negative control|
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| Discussion|| |
Virulence factors in microorganisms may vary depending on the type of infection, different species, the site of infection, geographical origin and host response. Pathogenesis of Candida spp. closely related to these virulence factors such as phospholipase, proteinase and haemolysin. Detection of pathogenicity factors may help new drug discovery to improve therapeutic regimens. Pakshir et al. reported that among 84 clinical Candida spp., C. parapsilosis strains had less phospholipase activity. In agreement, C. parapsilosis and C. glabrata isolates had the lowest phospholipase activity in the present study. In line with our findings, Kantarcioglu and Yucel did not show any discrepancy in enzyme production by C. albicans from various anatomical sites. Although no remarkable differences of phospholipase activity among the Candida spp. from different sites have been found in the current study, we declared that catheter isolates presented lower phospholipase activity than other isolates obtained from candidemia. The phospholipase family contains four distinct classes (A, B, C and D); however, only the products of the PLB1 and PLB2 genes have been detected extracellularly, and this is the reason to select these two genes in the present investigation. Bassyouni et al. indicated that the PLB1 and PLB2 genes were positive in 87.5% -and 45% of Candida isolates of diabetic patients, but these percentages were 82.1% and 71.8% in our study.
| Conclusion|| |
The results of the present investigation showed no differences in phospholipase activity in different sites of infection (blood, oesophagus and stomach); however, non-albicans Candida species had less phospholipase activity. This study suggests that the pathogenicity of genus Candida can be related to the fungal species.
We appreciate Mrs. Ranjbar and Mrs. Mottaghi for their cooperation to collect the clinical isolates.
Financial support and sponsorship
This study was funded by Isfahan University of Medical Sciences (Grant No. 399273).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]