Detection of Virulence Factors from Candida spp. Isolated from Oral and Vaginal Candidiasis in Iraqi Patients

1. Introduction

Yeast-like fungi (YLF) of the genus Candida are unicellular microorganisms of relatively large size and rounded shape, aerobes, and belong to conditionally pathogenic microorganisms. The genus Candida includes approximately 150 species, which are classified as Deuteromycetes due to the absence of a sexual stage of development. From a medical point of view, seven of them are recognized as the most important disease-causing species: C. albicans, C. tropicalis, C. glabrata, C. krusei, C. guilliermondii, C. parapsilosis, and C. Kefir ( 1 ). Approximately 30 container species cause injury to humans. C. albicans is the most common species, then other pathological species containing C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. lusitaniae, C. guilliiermondii, C. kefyr, C. rugosa, C. inconspicua, C. dubliniensis, C. famata, C. lipolytica, C. sake, C. apicola, C. pulcherriima, C. zeylanoides, C. pelliculosa, C. valida, C. intermedia, C. haemulonii, C. humicola, C. utilis, C. ciferrii, C. lambica, C. holmii, C. marina, C. humicola, C. sphaerica and C. stellatoidea, C. colliculosa ect. Candida colonies appear small, creamy, or whitish colonies with a characteristic yeast odor on SDA; on CHROM agar, Candida appears as glistening green color colonies for C. alibicans and another color for each Candida species. As well, under the microscopic, all Candida spp. showed that all species produce blastoconidia. Blastoconidia may be round or elongate; most species produce pseudohyphae which may be extended, branched, or curved.

Candida spp. infections are caused by several virulence mechanisms, including adhesion to the host and abiotic medical surfaces, biofilm formation, and the production of hydrolytic enzymes. Furthermore, Candida albicans resistance to antimicrobials, particularly azoles, is widely documented, particularly when Candida cells are in biofilm form. The role of Candida virulence factors that mediate their success are as follows: pathogens, such as membrane and cell wall barriers, dimorphism, biofilm formation, signal transduction pathway, proteins related to stress tolerance, hydrolytic enzymes, e.g., proteases, lipases, hemolysins and toxin production ( 2 ). Phospholipases are the enzymes that can hydrolyze the ester bonds in glycerophospholipids. These enzymes cause cell membrane breakdown; conversely, proteases can break down various host cell proteins, including collagen, keratin, mucus, antibodies, complements, and cytokines are all broken down by these enzymes ( 3 ). Esterases are enzymes that help to break down an ester group. Esterase synthesis was detected in Candida albicans, Candida tropicalis, Candida parapsilosis, and Candida lipolytica, as well as their low activity in C. parapsilosis ( 4 ). Hemolysins are involved in Candida pathogenicity because they are responsible for red blood cell destruction and iron uptake. Iron and other inorganic elements are required to develop Candida cells and create the infection process ( 5 ). Biofilms are complex microbial populations that are very heterogenous and provide their residents with increased chances of survival ( 2 ). Therefore, this study aimed to detect virulence factors from Candida spp. isolated from oral and vaginal candidiasis.

2. Materials and Methods

2.1. Isolation of Candida Isolates

From May 2021 to the end of June 2021, fifty-eight specimens were collected from patients, including twenty-eight oral swabs from children in AL-elwia pediatric teaching hospital and thirty vaginal swabs from infected women in Imamein kadhimen medical city in Baghdad and transported under aseptic conditions to the laboratory for Candida spp. isolation. The samples were streaked directly onto Sabouraud Dextrose agar (SDA) enriched with chloramphenicol (250 mg/liter) and chromogenic candida agar and incubated aerobically at 37°C for 48 hours ( 6 ).

2.2. Identification of Candida Isolates

2.2.1. Cultural Examination

Candida spp. were identified based on morphological characteristics of sabouraud dextrose agar and chromogenic candida agar, which included colony shape, color, texture, size, opacity, and margin. The isolates were subcultured on chromogenic candida agar for 48 hours at 37°C after being obtained from sabouraud dextrose agar (SDA) at 37°C for 48 hours. Chromogenic candida agar can be used as a selective or differential media for Candida spp. After incubation, yeast identification was done based on colony color.

2.2.2. Microscopical Examination

The isolates were stained with gram stain after placing the specimen on a clean slide with a drop of 10% (KOH) and examining it under a microscope. Furthermore, a fragment of the colony was extracted with a sterile stick and placed on a clean slide, which was then combined with a drop of lactophenol cotton blue, covered with a cover-slip, and viewed under a light microscope to look for blue budding Candida cells ( 7 ).

2.3. Germ Tube Formation (GT)

Transferring a tiny piece of isolated colonies using a sterile stick, mixing them with 0.5 ml of human serum, and incubating them at 37°C for two and a half hours, the generation of germ tubes by Candida spp. isolates were investigated. Due to the propensity of other yeast species to create germ tubes, the incubation period must not exceed 3 hours. Using a Pasteur pipette, a drop of the cultured serum was deposited on a slide, covered with a cover - slip, and examined under the microscope for the presence of germ tubes ( 8 ). In this test, serum was prepared according to Yin, Peter ( 9 ). According to the method previously described by Mariano, Gonçalves ( 10 ), the distinction and detection of Candida spp., performed by Growth at 45°C for 48 h.

2.4. Identification Using Vitek 2 Compact System

The Vitek 2 Compact device is committed to clinically confirming important Candida organisms' detection using the manufacturer’s protocols (bioMerieux Inc., Durham, NC 27712, USA).

2.5. Detection of Some Virulence Factors of Candida spp. Isolated from Vagina and Oral Cavity

2.5.1. Extracellular Phospholipase Production

The egg yolk agar plate method was used to determine extracellular phospholipase production. The modified egg yolk agar was inoculated with ten microliters of newly generated yeast inoculum (consisting of a 24-hour-old Candida spp. colony suspended in 5 ml of sterile normal saline) equivalent to McFarland (1.5×10⁸ CFU/ml) and incubated at 37°C for 48 hours. The result was achieved by measuring the colonies' diameter and the opacity zone's diameter and calculating the Pz value as follows.

$\mathrm{Ez}=\frac{\mathrm{Thecolonydiameter}}{\mathrm{Thediameterofcolony\; +\; precipitationzone\; (b)}}$

Pz value = 1 indicates that the test isolate is negative for phospholipase, 0.80-0.89 indicates low phospholipase activity, 0.70 - 0.79 indicates moderate phospholipase activity, and 0.70 - 0.79 indicates high phospholipase activity ( 11 ).

2.5.2. Esterase Activity

This investigation assessed the esterase activity of Candida isolates using the Tween 80 opacity test. An aliquot of 10 ul of the yeast suspension was incubated at 37°C for 5 days after being inoculated on the test medium. The precipitation zone around the colonies was used to determine the esterase activity. To evaluate esterase activity, the colony diameter (a) and the colony plus precipitation-zone diameter (b) were measured ( 12 ).

2.5.3. Proteinase activity

In terms of bovine serum albumin (BSA) degradation, An aliquot 10ul of the yeasts suspension was inoculated on the test medium and incubated at 37°C for 7 days; the proteinase activity was measured as the diameter of clear zones around the colonies, proteinase activity was determined using the amido black staining solution. The ratio of the colony to the diameter of the proteolytic unstained zone was used to calculate the value of proteinase activity (Prz). A Prz value of 1 showed no proteinase was produced, whereas a Prz value of 1 indicated that proteinase activity was present ( 13 ).

$\mathrm{Przvalue}=\frac{\mathrm{Colonydiameter}}{\mathrm{proteolyticunstaionedzonediameter}}$

2.5.4. Coagulase Activity

Candida isolates were tested for coagulase production using the traditional test tube method ( 14 ). In a tube containing 500ul of EDTA rabbit plasma, 0.1 ml of an overnight Candida spp. the culture was aseptically introduced. For 4 hours, the tubes were incubated at 35 °C. A positive coagulase test revealed the existence of a clot that could not be resuspended by gentle shaking. The tube was reincubated and reexamined if no clot formed after 24 hours.

2.6. Haemolysin Production

2.6.1. PlateMethod(agarmedium)

A loop - full of pure C. albicans culture was inoculated into sabouraud dextrose agar and incubated at 37ºC for 24 h. This Growth was used to prepare a suspension compared to McFarland 0.5. Ten microliters of this suspension were placed on sabouraud dextrose blood agar medium (with 3% glucose), and then the plates were incubated at 37°C for 48 h. A translucent halo around the colony indicates positive hemolysin production. Hemolytic index, which represented the intensity of hemolysin production, was estimated by dividing the colony diameter by the diameter of the colony plus the hemolysis zone; based on the Hi index, hemolytic activity recorded to the scores: Hi 1 means not a hemolytic activity, 0.64 - 0.99 indicate moderate hemolytic activity, 0.63 ≤ indicates vigorous hemolytic activity ( 15 ).

2.6.2. SpectrophotometricMethod(Liquidmedium)

The hemolysin production in a liquid medium was determined using a spectrophotometric method reported by Lee, Kim ( 16 ), in which ( 20 ) µl of Candida spp. suspension compared to (0.5) McFarland was added to (180) µl of sabouraud dextrose broth and incubated at 37° C for 24 hours. Human red blood cells were prepared by centrifugation at 3000 rpm for 5 minutes and washed three times with PBS buffer. The pellet was diluted in PBS buffer (330 µl red blood cells were added to 10 ml PBS buffer), and 100 µl candidal culture was added to 10 ml red blood cells previously prepared and incubated at 37° C for 1 hour. After centrifugation at 12,000 rpm for 10 minutes, the supernatants were collected, and the amount of hemoglobin released from lysed erythrocytes was measured using a spectrophotometer at 543 nm. Blood cells with only a medium served as the experiment's control.

2.6.3. Biofilm Formation

The biofilm formation was determined using polystyrene 96-well microplates, following the methods reported by Deepa, Jeevitha ( 17 ) with some modifications. Candida spp. were inoculated into a tube containing 2 ml of Yeast extract peptone dextrose (YPD) broth and incubated at 37°C for 24 hours. All tubes were diluted at a ratio of 1:20 with freshly prepared YPD broth, and a 96 flat bottom well microtiter plate was filled with 200 μlof this final solution, while the control wells were filled with 200 μlof YPD. Microtiter plates were sealed with lids and incubated at 37°C for 24 hours; after that, the media in the wells was removed and washed twice with sterile phosphate buffer solution (PBS) to remove any unattached Candida spp. cells then dried at room temperature for 15 minutes. After that, each well was filled with 200 liters of crystal violet and left for 20 minutes. The crystal violet was taken from the wells and washed three times with PBS (pH 7.2) to remove the unbound dye, then dried at room temperature. Finally, a 200 μl combination of acetone and ethanol (20:80 v/v) was added to each well, and O.D was read at 450 nm by an ELISA reader. These OD values were used to determine if Candida could form biofilms ( 18 ) (Table 1).

3. Results and Discussion

3.1. Cultural Characteristics

The morphology of C. albicans colonieson sabouraud dextrose agar was white colored, smooth, andyeast-like, as indicated in figure 1, C. parapsilosis colonies were white to creamy, shiny, and smooth, and C. glabrata colonies were smooth and cream colored.

Figure 1.Candida albicans growth on sabouraud dextrose agar (SDA) (24 h/ 37°C)

Chromogenic Candida agar is a differential and selective media that promotes the rapid isolation of Candida from mixed culture sand can differentiate Candida species from other organisms based on colony morphology, as indicated in figure 2. Chromogenic Candida agar has a unique reactive substrate with specific enzymic belonging to Candida spp. specifically C. albicans and C. glabrata. The current study's results agree with previously published work. The color of colonies showed various species of Candida, which were as. C. albicans or C. dubliniensis orgreen color, Candida dubliniensis isolates grow well at 30°C and 37°C on commonly used culture media for Candida spp. It is impossible to differentiate between C. albicans and C. dubliniensis colonies on conventional solid media. Colonies of the two species can be distinguished following primary isolation from clinical specimens using the newly developed Chromogenic Candida agar. The results showed that the colonies of C. dubliniensis are pale green and easily distinguished from those of other Candida spp. The recorded data in the current study agreed with. C. glabrata for pink-purple, C. parapsilosis for white to pale pink, and the other species developing colonies from white to rose. These results agreed with a study by Hospenthal, Beckius ( 19 ).

Figure 2.Candida spp. growth on CHROM agar Candida (CaC) (24 h / 37 °C)

3.2. Microscopical Examination

After staining with Methylene blue dye, the Candida spp. cell shape and size were identified. Candida spp. isolated appeared oval, spherical cell shape with budding yeast-like cells.

3.3. Germ Tube Formation

The results indicated that isolates of C. albicans generate germ tubes in the form of long tube-like projections extending from the yeast cells. Within two hours of incubation with human serum, germ tubes formed. The findings of this investigation corroborated with those of Matare, Nziramasanga ( 8 ), who indicated that all C. albicans isolates could form germ tubes when examined directly from the colony. Developing germ tubes in human serum allows for the quick detection of C. albicans. Conversion of C. albicans from yeast to filamentous or mycelial Growth occurs due to the production of germ tubes in human serum. These morphological changes frequently reflect the fungus's response to changing environmental conditions and may enable the fungus to adapt to different biological factors ( 20 ). At the same time, the other species from non C. albicans showed a negative result except C. dublinienesis. These results are in agreement with. C. albicans and other candida spp. could differentiate between them by germ tube formation. Candida albicans formatted germ tube, but others have not formatted ( 8 )

All C. albicans isolates grew well at 45°C, while the others failed to grow. This result was in agreement with Kalaiarasan, Singh ( 21 ), who mentioned that the Growth at 45° C was deemed a viable method for differentiating C. albicans from C. dubliniensis. Moreover, indicated that fungi growth at 45° C was performed on all isolated fungi. The C. albicans have grown, but others have not.

3.4. Identification of Candida spp. by Vitek 2 Compact System

The Vitek 2 system is fully automated equipment for the susceptibility and identification of microorganisms. It appears to be a superior alternative detection method for clinical laboratories performing fungal diagnostics. The isolates were finally identified using the bioMerieux (USA) Vitek 2 compact technology. Identifications of isolates based on 64 biochemical tests for an unknown biopattern were compared to a database of reactions for each taxon, followed by a numerical likelihood estimate.

3.5. Detection of Virulence Factor for Candida Species

Extracellular phospholipase activity of pathogenic Candida spp. Isolated from the oral cavity and vaginal infection were tested using the egg yolk agar plate method. In 21 isolates of Candida spp.isolated from the oral cavity, the results showed that 8 isolates were vigorous phospholipase activity with a Pz value of < 0.70, while 4isolates were moderate phospholipase activity with a Pz value of (0.70 - 0.79), and two isolates were weak phospholipase activity with Pz value of (0.80- 0.89) (Table 2).As for these 7 isolates, they do not produce a phospholipase enzyme. In ten isolates of Candida spp. isolated from vaginal infections, results showed that 2 isolates were strong phospholipase activity, while 2 isolates had moderate phospholipase activity, and only one isolate had weak phospholipase activity. As for these 5 isolates, they do not produce a phospholipase enzyme. Phospholipases are enzymes that hydrolyze the ester bonds in glycerophospholipids, causing cell membrane breakdown ( 3 ). Hence, phospholipases could bind to the cell surface or be released into the environment ( 22 ). Secreted phospholipases are extracellular hydrolytic enzymes ( 23 ). According to the study conducted by Lan, Hou ( 13 ), phospholipase synthesis is strain-dependent. de Paula Menezes, de Melo Riceto ( 24 ) found that phospholipase activity was identified in 99% of C. albicans isolates and another research the production of these enzymes was observed among 38% ( 21 ), 69% ( 25 ) or 81% ( 11 ) of C. albicans isolates. On the other hand, ( 24 ), Abi-Chacra, Souza ( 26 ) showed that Phospholipase activity was not detected within C. parapsilosis isolates.

3.6. Production of Proteinase Activity

Proteinase activity of pathogenic Candida spp. isolated from the oral cavity, and vaginal infections were tested using the Agar plate method. In 21 isolates of Candida spp. isolated from the oral cavity. The results shown in table 2 revealed that 12 isolates were strong proteinase activity with a Prz value of < 0.70, while 2 isolates were moderate proteinase activity with a Prz value of (0.70 - 0.79), and 3isolates were weak proteinase activity with Prz value of (0.80-0.89 ). These 4 isolates were negative Proteinase activity. In ten isolates of Candida spp. isolated from vaginal infections, results showed that 5 isolates had strong proteinase activity, while 2isolates were moderate proteinase activity, and 2 isolates had weak proteinase activity. As for these, only one isolate was negative proteinase activity; proteinase is a crucial factor contributing to Candida infection that facilitates microbial invasion by damaging mucosa ( 28 ). On the other hand, these enzymes down protease breakdown in various host cell proteins, including collagen, keratin, mucus, antibodies, complement, and cytokines ( 3 ).

In general, the results of this study showed that the number of Candida spp. Isolates secreted proteinase was higher than the number that produced phospholipase. Proteinase activity is observed among 70-100% of clinical C. albicans isolates ( 24 ). While the variable numbers of clinical C. parapsilosis isolate producing proteinase were reported at 38%, 87% ( 29 ), and 100% ( 26 ). The obtained data are in accordance with previous reports demonstrating the high proteinase production among all tested isolates such as C. albicans, C. parapsilosis, and rare species.

3.8. Coagulase Activity

Coagulase activity of pathogenic Candida spp. isolated from the oral cavity, and vaginal infections were tested by using rabbit plasma. The highest coagulase activity was seen with (100%) in C. albicans, C. parapsilosis, and C. guilliermondii with (85%), (60%), and (50%) respectively. In contrast, C. dubliniensis does not produce coagulase enzyme (Table 3). In a study conducted by Yigit, Aktas ( 30 ), they found that the coagulase activities of the different species tested after 24 h incubation at 37° C were: (43%) C. albicans isolates and (37%) non- albicans Candida strains were positive for coagulase in rabbit plasma.

3.9. Haemolysin Production

Haemolysin activity of pathogenic Candida spp. isolated from the oral cavity, vaginal infections were tested using Sabouraud dextrose agar with blood. In 21 isolates of Candida spp. isolated from the oral cavity, results showed that 14 isolates were strong hemolys in production with Hi<0.63, while 7 isolates were moderate hemolysin production with Hi=0.64-0.99. In ten isolates of Candida spp. isolated from vaginal infections, the results revealed that 5 isolates were strong hemolysin production, while 5 isolates were moderate hemolysin production (Table 4).

Haemolysin activity of pathogenic Candida spp. isolated from the oralcavity, and vaginal infections were tested using the spectrophotometric method in 21 isolates of Candida spp. The results showed that 16 isolates had strong hemolysin production isolated from the oral cavity, while 5 isolates had low hemolysin production in 10 Candida spp. isolated from vaginal infections, the results showed that 5 isolates were strong hemolysin production, while 5 isolates were low hemolysin production (Table 5).

The biofilm formation of the pathogenic Candida spp. isolated from the oralcavity, and vaginal infections were tested using the microtiter plate method. Biofilm formation was foundinall Candida spp. isolates. In 21 isolates of Candida spp. isolated from oral cavity results showed in table 6 revealed that 4 isolates were strong biofilm formation with O.D values>0.320, while16isolates were moderate biofilm formation with O.D values of 0.120–0.320. In 10 isolates of Candida spp. Isolated from vaginal infections, the results showed that 2isolates were strong biofilm formation, while 8 isolates were moderate biofilm formation. The results showed that biofilm formation was changed according to the strains. However, it was found that biofilm formation was generally high.

Candida biofilm growth is the predominant reason for Candida antifungal resistance ( 31 , 32 ). Evidence suggests that Candida spp. the pathogenic potential is also influenced by their ability to form biofilms on the abiotic and biotic surfaces ( 33 ). Silva, Henriques ( 34 ) reported that Candida albicans isolates isolated from the vagina produced high levels of biofilms. Paiva, Vidigal ( 35 ) reported that C. tropicalis produced high levels of biofilm. They reported that C. parapsilosis and C. glabrata produced less biofilm than pathogenic C. albicans. Rudenko, Vatnikov ( 1 ) showed that biofilm production is considered one of the most potent pathogenic traits contributing to treatment failures and recurrent infections and described that C. tropicalis produces strongly adherent biofilm followed by C. albicans, while C. parapsilosis produce no biofilm, which is, in contrast, to a study by Tellapragada, Eshwara ( 36 ), where higher biofilm production was seen in C. albicans.

Authors' Contribution

Study concept and design: A. A. H. A.

Acquisition of data: J. A. S. S.

Analysis and interpretation of data: H. A. A.

Drafting of the manuscript: H. A. A.

Critical revision of the manuscript for important intellectual content: A. A. H. A.

Statistical analysis: J. A. S. S.

Administrative, technical, and material support: A. A. H. A.

Ethics

The study protocol was approved by the ethics committee of the Mustansiriyah University, Baghdad, Iraq.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgment

The authors would like to hank Mustansiriyah University in Iraq () for their support in the current work.

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