|Year : 2015 | Volume
| Issue : 1 | Page : 52-55
Investigation of possible virulence factors in Candida strains isolated from blood cultures
MA Atalay, AN Koc, G Demir, H Sav
Department of Clinical Microbiology, Faculty of Medicine, Erciyes University, Kayseri, Turkey
|Date of Submission||16-Jun-2014|
|Date of Web Publication||15-Dec-2014|
M A Atalay
Erciyes University Faculty of Medicine, Department of Medical Microbiology, 38039 Melikgazi, Kayseri
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The Candida species, which are one of the most common causes of nosocomial bloodstream infections, present with high mortality and morbidity rates. This study aims to investigate the production of esterase, phospholipase, proteinase, and biofilm formation ability of the Candida strains isolated from the blood cultures.
Materials and Methods: Between June 2011 and July 2012, the Candida strains, which were isolated from blood cultures of a total of 50 patients, were studied. The esterase activity was analyzed in the Tween-80 agar, while phospholipase activity was studied in the egg yolk agar. The proteinase activity and biofilm formation were identified by using the petri dish method and microplate method, respectively.
Results: Of 50 specimens obtained from individual patients, 17 (34%) were identified as C. albicans, 14 (28%) as C. glabrata, 9 (18%) as C. parapsilosis, 5 (10%) as C. krusei, 4 (8%) as C. kefyr, and 1 (2%) as C. tropicalis. The rate of proteinase, phospholipase, and esterase positivity was higher in the C. albicans isolates. Biofilm formation was the highest in the C. parapsilosis strains.
Conclusions: Higher rate of virulence factors in the most commonly isolated Candida species than other species indicates that these virulence factors play a crucial role in the pathogenesis.
Keywords: Biofilm, blood culture, candidemia, esterase, phospholipase, proteinase
|How to cite this article:|
Atalay M A, Koc A N, Demir G, Sav H. Investigation of possible virulence factors in Candida strains isolated from blood cultures. Niger J Clin Pract 2015;18:52-5
|How to cite this URL:|
Atalay M A, Koc A N, Demir G, Sav H. Investigation of possible virulence factors in Candida strains isolated from blood cultures. Niger J Clin Pract [serial online] 2015 [cited 2019 Oct 18];18:52-5. Available from: http://www.njcponline.com/text.asp?2015/18/1/52/146979
| Introduction|| |
In the recent years, the incidence of serious fungal infections, invasive Candida infections particularly, has been increasing due to an increased number of patients receiving immunosuppressive therapy, increased major surgeries and broad-spectrum antibiotherapy, hyperalimentation, prolonged intensive care unit stay for patients with poor health status, and increased use of artificial prostheses. , The Candida species are the fourth most common cause of nosocomial bloodstream infections, accounting for nearly 10% of all. It follows coagulase-negative staphylococci, Staphylococcus aureus, and enterococci with high morbidity and mortality rate. ,, Virulence factors as well as attenuated defense mechanism of the host play a critical role in the development of these infections. Extra-cellular hydrolytic enzymes of Candida species facilitate adherence and tissue penetration, and therefore invasion of the host.  However, biofilm producing Candida species are known to be more resistant to immune response and antimicrobial agents, which lead to treatment failure.  Although there is a growing number of studies reporting virulence factors of other strains, C. albicans is the most commonly analyzed strain in which several virulence factors including phenotypic changes, morphological dimorphism, adhesion molecules, hydrolytic enzymes (e.g. phospholipase, lipase, and aspartic proteinases), catalase, superoxide dismutase, and heat-shock proteins have been identified. ,
In this study, we aimed to investigate the production of esterase, phospholipase, proteinase, and biofilm formation ability of the Candida strains isolated from the blood cultures.
| Materials and Methods|| |
Definition of species
Between June 2011 and July 2012, the Candida strains, which were isolated from blood cultures of a total of 50 patients, were studied. The blood samples which were sent from various departments of our hospital to the central laboratory were incubated in the BacT/Alert 3D Automation System (Biomerieux, France). Some preparations were done using the blood samples with a hint for bacterial production, and those with yeast cells as confirmed by Gram-staining were passaged to the CHROMagar Candida (BBL, France) and Sabouraud dextrose agar (Oxoid, UK). The isolates were identified by the germ tube test results; morphological images obtained from the Tween-80 corn-meal agar, and API 20C AUX system (Biomeriuex, France).
Investigation of virulence factors
The phospholipase activity was studied in the egg yolk agar. 10 μ yeast suspension was added at 0.5 McFarland turbidity and incubated at 37°C for 4 days. The ratio of colony diameter to the diameter of precipitation zone was calculated (precipitation zone; Pz). With respect to the phospholipase activity, Pz: ++++ or ≤0.69 = very strong, + + + or 0.70-0.79 = strong, ++ or 0.80-0.89 = medium, + or 0.90-0.99 = weak positive. Pz ≥ 1: Negative. ,
The esterase activity was studied using an agar containing 1% peptone, 0.5% NaCl, 0.01% CaCl 2 , and 1.5%/L at 6.8 pH. When heated at 45-50°C, 5 mL Tween-80 was added. The round-shaped strains with 10-mm in diameter were incubated at 30°C for 10 days. The presence of a transparent halo around the inoculum site indicated positivity. ,
The proteinase activity was analyzed by using an agar containing 0.1% KH 2 PO 4 , 0.05% MgSO 4 , 2% agar, and 1% bovine serum albumin per liter at 4.5 pH. 10 μL yeast suspension was added and a transparent zone around the colony was investigated. 
The biofilm formation ability of the isolates was identified by using the microplate method. The brain heart infusion broth (BHIB; Oxoid, UK) containing 0.25% glucose was prepared and put into 2-mL tubes. A loop full of each isolate was added and incubated at 37°C for 24 h. Then, each tube was diluted at 1:40 with a fresh BHIB. The final solution was seeded into a 96-well polystyrene microplate (Nunc, Denmark) and incubated at 37°C for 24 h. At the end of the incubation period, it was washed with phosphate buffer solution 3 times, inverted, and dried. 200 μL of crystal violet 1% was added and standard for 15 min. At the end of the incubation period, it was washed 3 times, and 200 μL mixture of ethanol-acetone (80:20 v/v) was added to each well. It was studied by the enzyme-linked immunosorbent assay kit (BioTek, ELX 800, USA) at 450 nm wavelength. The optic density (OD) of the well was recorded. A sterile BHIB, which contained no microorganism, was identified as a negative control. The mean OD of sterile BHIB wells was calculated, and the cut-off value was estimated using + 2 standard deviations. The samples which produced OD above the cut-off value were considered as positive, while samples with OD below the cut-off value were considered as negative. 
Candida albicans ATCC 90028 was used as a control stain in the phospholipase, esterase, and proteinase tests. All tests were repeated 3 times for each strain. Statistical analysis was performed using SPSS (SPSS Inc., Chicago, IL, USA) version 11.5 software. The Chi-square test was also done. P < 0.05 was considered as statistically significant.
| Results|| |
A total of 50 patients were included in the study. Then median age of the patients was 50 years and 32 (64%) were male, and 18 (36%) were female. Most patients were from the intensive care units (27/50; 54%), 18/50 (36%) from medical wards and 5/50 (10%) from surgical wards. At least one underlying medical condition was identified in 47 cases of candidemia (94%). The most frequent underlying disease in patients with candidemia was cancer (38%), especially solid tumors (28%). Lung disease and acute/chronic kidney failure were the 2 nd and the 3 rd most frequent underlying disease, respectively (18% and 14%, respectively). Only three patients had diabetes (6%).
Of 50 specimens obtained from different patients, 17 (34%) were identified as C. albicans, 14 (28%) as C. glabrata, 9 (18%) as C. parapsilosis, 5 (10%) as C. krusei, 4 (8%) as C. kefyr, and 1 (2%) as C. tropicalis. Eleven (64.7%) of the C. albicans strains had positive esterase activity, 14 (82.4%) had positive proteinase activity, 15 (88.2%) had positive phospholipase activity, and 1 (5.9%) had positive biofilm formation. In addition, 1 (7.1%) of the C. glabrata strains had positive esterase activity, 4 (28.6%) had positive proteinase activity, and 5 (35.7%) had positive phospholipase activity. However, biofilm formation was not identified in this strain. The rate of proteinase, phospholipase, and esterase positivity was statistically higher in the C. albicans isolates than non-albicans Candida isolates (P < 0.001). Biofilm formation was the highest in the C. parapsilosis strains, indicating statistical significance (P < 0.001). However, no virulence factor was identified in the C. kefyr strains [Table 1].
|Table 1: Esterase, phospholipase, and proteinase activity and biofilm formation ability of the Candida species |
Click here to view
| Discussion|| |
In parallel to the increasing number of high-risk immunocompromised patient population, the Candida species has become particularly important as one of the most common causes of nosocomial bloodstream infections.  The C. albicans is the most isolated pathogen in the Candida species-associated bloodstream infections, followed by C. parapsilosis, C. glabrata, C. krusei, C. tropicalis with an increasing incidence. , Despite varying incidence according to the region, C. glabrata and C. parapsilosis may be a cause of candidemia. , In our study, C. albicans (34%) is the leading cause of candidemia, followed by C. glabrata (28%).
Extra-cellular hydrolytic enzymes of Candida species acting as virulence factors which contribute to host-tissue invasion by digesting proteins (hemoglobin, keratin, collagen, etc.)  formation of Candida biofilms occurring on devices such as indwelling intravascular catheters, infections become more refractory to antimicrobial agents.  Earlier studies demonstrated that C. albicans produced extra-cellular phospholipase alone. However, further studies showed that non-albicans Candida produced also phospholipase but at a lesser extent.  In the present study, we found that 15 (88.2%) of 17 C. albicans strains and only 8 (24.2%) of 33 non-albicans Candida species (C. glabrata [n = 5], C. krusei [n = 2], and C. parapsilosis [n = 1]) had positive phospholipase activity. Birinci et al.  reported that 42 (67.7%) of 62 C. albicans strains isolated from the blood samples had phospholipase activity. However, the authors reported no phospholipase activity in the non-albicans Candida species. Similarly, De Luca et al.  showed that 32 (100%) of the C. albicans strains and only one of the C. parapsiolosis strains had phospholipase activity. Another study revealed that 41 of the C. glabrata strains, 50% of the C. parapsilosis strains, 70% of the C. tropicalis, 80% of the C. lusitaniae, and 10% of the C. krusei had phospholipase activity. The authors, however, concluded that relatively low rates were associated with small sample size of the Candida strains (n = 51).  In our study, using a similar sample size with the aforementioned study, we found 35.7% of the C. glabrata strains, 40% of the C. krusei strains, and 11.1% of the C. parapsilosis strains with phospholipase activity.
Furthermore, we observed that 11 (64.7%) of 17 C. albicans and 4 (12.1%) of 33 non-albicans Candida (C. glabrata [n = 1], C. krusei [n = 2], and C. tropicalis [n = 1]) had esterase activity. Review of literature has demonstrated that C. albicans and C. tropicalis are the most common strains exerting esterase activity. ,,, Yόcesoy and Marol  reported that 95% of the C. albicans, 93% of the C. tropicalis, 57% of the C. parapsilosis, and 10% of the C. glabrata had esterase activity. In addition, Aktas et al. found that 98.3% of the C. albicans, and 100% of the C. tropicalis and C. guilliermondii had esterase activity. In our study, we observed a lower rate of esterase activity in the C. albicans strains along with one of the C. tropicalis strains with positive activity. We also found esterase activity in 40% of the C. krusei and 7.1% of the C. glabrata. However, none of the C. parapsilosis and C. keyfr had esterase activity.
Moreover, we reported that 82.4% of the C. albicans and 30.3% of the non-albicans Candida had proteinase activity. Consistent with our findings, Gokce et al.  showed proteinase activity in 89.7% of the C. albicans and 25.2% of the non-albicans Candida of the isolated from blood cultures. Similarly, Bramono et al.,  reported a significantly higher rate of proteinase activity in the C. albicans strains, followed by the C. parapsilosis C. tropicalis. In our study, the C. albicans had the highest proteinase activity, followed by C. parapsilosis (44.4%), C. krusei (40%), and C. glabrata (28.6%).
Based on our study results, only 1 (5.9%) of the C. albicans and 77.7% (7/9) of the C. parapsilosis strains had the biofilm formation ability. On the other hand, Gόltekin et al. had reported that none of the C. albicans isolates had the biofilm formation ability with the highest positivity ratio of the C. parapsilosis strains. In addition, Shin et al.  also showed that the C. parapsilosis had the highest ratio for the biofilm formation, followed by the C. albicans. The authors concluded that there was a significant difference in the biofilm formation ability between the C. parapsilosis strains isolated from the blood samples and other samples.
| Conclusion|| |
We suggest that inconsistent results of previous studies in respect of the phospholipase, proteinase, and esterase activity as well as the biofilm formation ability of the Candida species can be attributed to the nonstandardized sample size including the number of strains, patient characteristics, regional variations, and methodological differences. Higher rate of virulence factors in the most commonly isolated Candida species than other species indicates that these virulence factors play a crucial role in the pathogenesis. However, further studies in a higher number of strains of a limited number of species are required to confirm these findings.
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