The Evaluation of the virulence factors of clinical Candida isolates and the anti-biofilm activity of Elettaria cardamomum against multi-drug resistant Candida albicans

Authors

1 Vivekanandha College of Arts and Sciences for Women (Autonomous), Tamil Nadu, India

2 Department of Medical Laboratory Sciences, College of Medicine and Health Sciences, Hawassa University, Ethiopia

Abstract

Background and Purpose: Today, treatment of life-threatening fungal infections, caused by Candida species, has become a major problem. In the present study, we aimed to evaluate the antifungal susceptibility patterns of different clinical Candida isolates, determine the virulence factors in multi-drug resistant (MDR) Candida species, and assess the anti-biofilm activity of Elettaria cardamomum against MDR Candida species.
Materials and Methods: A total of 202 isolates from different Candida species were obtained from three governmental hospitals in Senthamangalam, Tiruchengode, and Namakkal, Tamil Nadu, India. The isolates were identified, using conventional methods. Candida species were tested for virulence factors such as biofilm, protease, and phospholipase activity. The minimum inhibitory concentration (MIC) of Elettaria cardamomum against MDR biofilm-forming C. albicans was determined, using plate and tube methods.
Results:The identified Candida isolates (n=202) were C. albicans (74/202), C. glabrata (53/202), C. parapsilosis (44/202), C. tropicalis (15/202), and C. dubliniensis (16/202). The isolates were subjected to antifungal susceptibility testing and the virulence factors were determined. In terms of biofilm production, non-C. albicans species such as C. dubliniensis showed 75% activity. Also, regarding protease activity, C. parapsilosis (75%) showed the highest percentage of protease production. In addition, Candida species showed strong positivity for phospholipase activity (62.87%). In the MIC method, the acetonic extract completely inhibited biofilm production at a concentration of 125 μl (56.25 μg). In comparison with the ethanolic extract, the acetonic extract showed major activity against biofilm production.
Conclusion: Based on the findings, pathogenic C. albicans species were inhibited by the ethanolic and acetonic extracts of
E. cardamomum. In recent years, MDR and biofilm-forming pathogenic Candida species have been increasingly detected
in clinical settings. Therefore, herbal derivatives might contribute to the treatment of infections without causing any sideeffects
and prevent the associated mortality.

Keywords


Introduction

Today, Candida species are emerging as the major agents of hospital-acquired infections. These species are ranked as the third or fourth most commonly isolated bloodstream pathogens. Although C. albicans is recognized as the predominant etiological agent of candidiasis, other Candida species such as C. krusei, C. glabrata, C. lusitaniae, and C. dubliniensis (the most recently identified Candida species), which tend to be less susceptible to common antifungal drugs, have also emerged as substantial opportunistic pathogens [1, 2].

Infectious diseases account for various health problems in developing countries such as India. Microorganisms have developed resistance to many antibiotics, and as a result, immense clinical problems have emerged in the treatment of these diseases [3]. The resistance of organisms has increased due to the indiscriminate use of commercial antimicrobial drugs, commonly used for the treatment of infectious diseases. Consequently, researchers have been encouraged to seek new antimicrobial compounds from various sources, including medicinal plants [4].

Candida strains possess a number of virulence factors (such as slime production), which enable the organisms to cause hematogenously disseminated infections in susceptible hosts. Biofilms are structured microbial communities, which are attached and encased in a matrix of exopolymeric materials [5] and are essential to the development of clinical infections.

A typical laboratory fungal model of biofilm formation involves two operational steps: 1) adhesion, and 2) biofilm growth and maturation [6]. The presence of virulence factors determines the pathogenic potential of any microorganism. Candida species have been shown to possess virulence factors such as haemolysin, coagulase, and biofilm formation, phospholipase activity, phenotypic switching, surface hydrophobicity, adherence to vaginal epithelial cells, and proteinase production [7, 8].

Elettaria cardamomum has been traditionally used in the treatment of various gastrointestinal, cardiovascular, and neural disorders. This herb is used as a powerful aromatic, carminative, diuretic, and stimulant agent. In India, E. cardamomum is used for many conditions, including asthma, bronchitis, kidney stones, anorexia, general debility, and urinary tract disorders. Previous studies have revealed the effectiveness of this herb as an anti-carcinogenic, anti-ulcerogenic, antimicrobial, and anticonvulsant agent [9-11].

A number of studies have been conducted on the chemical and antimicrobial properties of E. cardamomum [12, 13]. However, the present study focuses on the antimicrobial activity of different solvent E. cardamomum extracts against infection-causing pathogens. The objective of our study was to evaluate the virulence factors and antifungal susceptibility patterns of different Candida species and validate the antimicrobial potential of E. cardamomum extracts against Candida species causing infection.

Materials and Methods

Study design

The present study was performed to analyze pathogenic Candida species and the virulence factors. The devices were collected from patients who were infected and admitted to the intensive care unit. After the Candida species were isolated from the devices, they were identified using selective media, standard biochemical tests, and microscopic observation.

Then, the identified Candida species were assessed in terms of their antibiogram activity against azoles and amines via disc diffusion method. Multi-drug resistant (MDR) Candida species were evaluated in terms of virulence factors such as protease, hemolysin, biofilm, and phospholipase. Considering the presence of different biotype characteristics, Candida species were confirmed. Then, MDR biofilm-forming Candida species were inactivated, using E. cardamomum extracts through disc diffusion and tube methods.

Collection of Candida species

A total of 202 Candida isolates were obtained from three governmental hospitals in Senthamangalam, Tiruchengode, and Namakkal, Tamil Nadu, India during August 2011 and June 2012.

Identification of Candida species

All the isolates were streaked on Bromocresol Green Agar (Hi-Media, Mumbai, India) for genus-level identification and further streaked on HiCrome Candida Differential Agar (Hi-Media, Mumbai, India) for species-level confirmation. Biochemical tests were performed via sugar fermentation and assimilation methods. Among Candida species, C. albicans showed the ability to produce germ tubes in human serum.

Antifungal susceptibility test

Antifungal susceptibility test was performed via disc diffusion method. The used discs included amphotericin B (100 units), fluconazole (10 µg), clotrimazole (10 µg), nystatin (100 µg), itraconazole (10 µg), and ketoconazole (10 µg). The inhibition zone was measured and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [14]. The control C. albicans ATCC 10231, C. glabrata ATCC 15126, and C. tropicalis ATCC 10610 strains were also used in this study.

Determination of virulence factors Biofilm production

Biofilm production was determined by visual methods. A loopful of colonies from the surface of Sabouraud Dextrose Agar (SDA) plate was inoculated into a polystyrene tube (Falcon conical tube with a screw cap), containing 10 ml of Sabouraud Dextrose Broth (SDB), supplemented with glucose (final concentration of 8%). After incubation at 35°C for 48 h, broth in the tubes was gently aspirated. The tubes were washed with distilled water twice and then stained with safranin 2% for 10 min. Afterwards, they were tested to determine the presence of an adherent layer. With respect to adherence to the surface of the tube, biofilm production was scored as strong (+++), moderate (++), and weak (+) [15].

Determination of protease activity

Protease activity of Candida species was determined in the present study. A loopful of colonies was introduced to a sterile filter paper disc and placed on the surface of bovine serum albumin agar (BSA; pH: 5.0). The inoculated plates were incubated at 37°C for two days, and the diameter of the inhibition zone around the discs was measured to determine protease activity, using the standard formula [16]:

Pz= Colony diameter/(colony diameter + zone of precipitation)

Phospholipase production

The phospholipase activity of Candida species was determined, using egg yolk agar medium. Approximately 5µl of the standard inoculum of the test strain, containing 108 cells/ml Candida, was aseptically inoculated onto the egg yolk agar. The plates were dried at room temperature and then incubated at 37°C for 48 h. The plates were examined in terms of the presence of precipitation zone around the colony. The presence of precipitation zone indicated phospholipase enzyme production [17].

Adherence of Candida species by cell surface hydrophobicity test

The hydrophobicity of Candida isolates was measured by assessing the adhesion of the yeast to hydrocarbons, such as cyclohexane or xylene. The tested strains were grown overnight in 5 ml of yeast peptone dextrose or yeast nitrogen base broth at 28°C. The cells were washed with phosphate-buffered saline (PBS) and concentrated to obtain a solution corresponding to an optical density of 1 in 600.

For adhesion assays, 3 ml of the cell suspension was mixed with 150 μl of cyclohexane or xylene in acid-washed glass tubes. The sample was vigorously mixed, using a vortex mixer for 1 min. After being placed at room temperature for 20-60 min, absorbance at 600 nm in the aqueous phase (A1) was measured and compared with the value obtained prior to the mixing procedure (A0). A percentage of cells in the cyclohexane/xylene layer (adhered cells) was used to estimate hydrophobicity, using the following formula [18]:

Percentage of cell adhesion= (A1/A0) × 100

Preparation of ethanolic and acetonic extracts of E. cardamomum

For this purpose, 10 g of each powdered plant sample was mixed with 250 ml of 96% ethanol and 100% acetone in a separate conical flask. The mixture was kept for 2-5 days in tightly sealed containers at room temperature and shaken several times every day. This mixture was filtered through a filter paper to remove the coarse plant materials. Further extraction of the residue was repeated 3-5 times until a clear supernatant liquid was obtained. The filtrates of each tested plant were evaporated to dryness, using a rotary evaporator at 40°C. The final dried samples were weighed and stored at -20°C until further use. The MIC of E. cardamomum was determined using the following two methods:

Plate method

The wells were punctured in plates containing SDA, and the ethanolic and acetonic extracts were loaded in different concentrations, i.e., 20, 50, 70, and 100 µl. Afterwards, the plate was incubated and the results were reported [19].

Tube method

A loopful of organisms from the surface of SDA plates was inoculated into the tubes (tubes were taken in pairs for each Candida strain), containing 6 ml of Sabouraud broth, supplemented with 8% glucose to the final concentration. Also, a control tube with broth, without cell suspension, was used for the comparisons. The tubes were incubated at 37°C for 48 h. Afterwards, various concentrations of acetonic and ethanolic E. cardamomum extracts (25-300 µl) were added to Candida broth tubes. They were maintained for 30 min, and then, the cell suspension in the tubes was poured or aspirated out. The walls of the tubes were stained with safranin 1%, and the adhesive layer, produced on the tube walls, was interpreted for biofilm or slime inhibition [20].

Candida species Fluconazole (%) Clotrimazole (%) Amphotericin (%) Nystatin (%) Ketoconazole (%) Itraconazole (%)
Candida albicans 95.94 94.59 68.91 89.18 90.54 93.24
Candida glabrata 94.33 94.33 73.58 90.56 94.33 88.67
Candida parapsilosis 95.45 95.45 72.72 81.81 86.36 93.18
Candida dubliniensis 87.50 100 00.00 87.50 93.75 100
Candida tropicalis 100 80 60 93.33 93.33 100
Table 1. In vitro antifungal susceptibility of 202 clinical Candida isolates against six antifungal agents
Candida species A1/A0×100 range
From To
Candida albicans 0.0 90.90
Candida glabrata 0.0 98.33
Candida parapsilosis 0.0 90.00
Candida dubliniensis 0.0 51.28
Candida tropicalis 30.00 51.28
Table 2.Adherence percentage of multi-drug resistant biofilm-producing Candida species

Figure 1.Bromocresol Green Agar medium

Figure 2.Sabouraud Dextrose Agar medium

Figure 3. Candida Differential Agar medium

Figure 4.Tube biofilm formation

Figure 5.Protease activity

Figure 6.Phospholipase activity

Figure 7.Anti-biofilm activity of E. cardamomum against MDR Candida albicans

Results

Identification of Candida species

In SDA, the organisms produced smooth, diploid colonies and were confirmed by the Bromocresol Green Agar medium. Chlamydospore production was confirmed by using corn meal agar. Species were distinguished by germ tube production. C. albicans showed a light green color in HiCrome Candida Differential Agar. Also, C. Glabrata, C. parapsilosis, C. tropicalis, and C. dubliniensis showed pink, cream, metallic blue, and dark green colors, respectively. Biochemical characteristics were confirmed by sugar fermentation. Sugar assimilation test results are presented in Figures 1, 2, and 3. A total of 202 isolates were identified as C. albicans (n=74), C. glabrata (n=53), C. parapsilosis (n=44), C. tropicalis (n=15), and C. dubliniensis (n=16).

On the antifungal sensitivity test, C. albicans showed 95.94% resistance to fluconazole, and C. glabrata showed 94.33% resistance to fluconazole, clotrimazole, and ketoconazole. Based on the findings, C. parapsilosis showed 95.45% resistance to fluconazole and clotrimazole, C. dubliniensis exhibited 100% resistance to clotrimazole and itraconazole, and C. tropicalis showed 100% resistance to fluconazole and itraconazole. The results are presented in Table 1.

In terms of biofilm production, C. dubliniensis (75%) showed strong biofilm-producing activity, followed by C. glabrata (67.9%), C. albicans (58.11%), C. tropicalis (53.33%), and C. parapsilosis (45.45%) (Figure 4). Regarding protease activity, among Candida species, C. parapsilosis (75%) showed the highest percentage of protease production, followed by C. albicans (68.92%), C. glabrata (64.15%), C. tropicalis (40.0%), and C. dubliniensis (37.5%) (Figure 5). In terms of phospholipase activity, 62.87%, 14.35%, 8.91%, 17.82%, and 0.99% of Candida species showed strong positive, moderate positive, weak positive, poor positive, and no enzymatic activity, respectively (Figure 6).

In total, 70 isolates of MDR biofilm-producing Candida species were evaluated in terms of adherence activity. The absolute adherence percentage of Candida species ranged between 20% and 100%. Among Candida species, the isolates of C. glabrata (98.33%), followed by C. albicans, C. parapsilosis, C. dubliniensis, and C. tropicalis, showed the most significant adherence (Table 2).

Based on the assessment of the anti-biofilm activity of E. cardamomum against MDR Candida species, the inhibition zone ranged between 10 and 15 mm in diameter. In the disc diffusion method, E. cardamomum extracts showed greater activity. With 100 µl of acetonic E. cardamomum extracts, more anti-biofilm activities were observed against C. albicans isolates (inhibition zone: 15 mm), compared to the ethanolic extracts (10 mm).

In the tube method, both ethanolic and acetonic extracts completely inhibited biofilm formation at 200 µl (90 µg) and 125 µl (56.25 µg) concentrations. The acetonic extract showed the greatest biofilm inhibition at a low concentration, as indicated by the reduction in biofilm production in test tubes (Figure 7).

Discussion

Over the past few years, yeasts of the genus Candida have been recognized as one of the most important etiological agents of nosocomial infections [21]. Fungal infections are most commonly caused by pathogenic Candida species, particularly C. albicans, C. tropicalis, and C. parapsilosis. These organisms are regarded as increasingly important nosocomial pathogens [22]. Based on the phenotypic, biochemical, germ tube, and microscopic observations, five different Candida species were identified in the present study.

Azoles exert direct effects on the fatty acids of cell membranes [23] and inhibit ergosterol biosynthesis through their interactions with lanosterol demethylase, which is responsible for the conversion of lanosterol to ergosterol in fungal cell membranes, leading to the depletion of ergosterol in the membrane [24, 25]. Overall, biofilm-forming Candida cells have the ability to develop resistance against azoles, especially fluconazole and clotrimazole [26].

In the present study, more than 80% of the isolates showed resistance to all drugs, except amphotericin (73.58%). In contrast, in a previous study, C. dubliniensis, C. parapsilosis, C. lusitaniae, and C. kefyr were susceptible to all the tested azoles, whereas C. tropicalis strains were susceptible to fluconazole and voriconazole [27]. In another study, resistance rates to fluconazole and itraconazole were 2.8% and 36.8%, respectively [28].

Biofilm formation is one of the most extensively investigated virulence factors of Candida species [29]. In the current study, a higher proportion of C. dubliniensis isolates was able to form biofilms (75%), followed by C. glabrata (67.9%), C. albicans (58.11%), C. tropicalis (53.33%), and C. parapsilosis (45.45%) as strong biofilm producers, respectively. Considering the biofilm-forming capacity of these isolates, their pathogenicity could result in severe co-infection with other diseases.

In the present study, biofilm production rate was high in C. dubliniensis and C. glabrata, as these isolates were obtained from immunocompromised patients. However, it should be noted that the patient’s body condition had been already adapted for these pathogens. Therefore, the virulence factors of the isolates were easily expressed, as the immunity level was very low in these patients. Nevertheless, in another study, contradictory results were reported [30].

In the current study, the ability of proteinase production was reported in 52.4% of Candida isolates. Maximum proteinase production was observed in 46 C. albicans isolates (56.7%), followed by non-C. albicans species (n=18, 43.9%). These results were in agreement with the proteinase-producing ability, detected in 38 (76%) Candida isolates. Maximum proteinase production was observed in 31 C. albicans isolates (79.5%), followed by non-C. albicans species (n=7, 63.63%) [31].

The mentioned results were in contrast with those reported by Dan et al. (2002) [32], who showed proteinase activity in 34 (85.0%) Candida isolates; this rate reached 100% in non-C. albicans species. In another study by Sachin et al. (2012), proteinase production was found in 65 (59.1%) Candida isolates [33]. Also, in a study by Tsang et al. (2007), maximum proteinase production was reported in C. albicans (82.1%), followed by non-C. albicans species (80%) [34].

In a previous study, phospholipase production was reported in 85 (62.9%) isolates. A total of 49 (81.6%) C. albicans isolates showed phospholipase activity. Among non-C. albicans species, maximum phospholipase activity was reported in C. tropicalis and C. glabrata. Also, 62.8%, 17.82%, and 14.35% of Candida species showed strong, weak, and moderate positivity, respectively [35]. In line with these findings, we also revealed strong positivity for phospholipase activity in C. parapsilosis (72.72%, 32 out of 44), C. albicans (69.81%, 37 out of 53), C. glabrata (64.86%, 48 out of 74), C. dubliniensis (37.50%, 6 out of 16), and C. tropicalis (33.33%, 5 out of 16).

Based on a previous study, the hydrophobicity of Candida strains constitutes the measurement of yeast adherence to hydrocarbons [36]. In this study, optimal adherence was observed in the presence of xylene (82.68% for C. albicans, 85.23% for C. parapsilosis, 76.45% for C. glabrata, 79.07% for C. kefyr, 74.3% for C. holmii, and 40.2% for C. sake). On the contrary, in the present study, C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. dubliniensis showed 89.28%, 88.88%, 62.5%, 100.0%, and 80.00% adherence in the presence of xylene, respectively.

Nosocomial fungal infections caused by Candida species are an important cause of morbidity and mortality, especially in immunocompromised patients. Use of available treatment options for invasive mycoses is limited due to their narrow spectrum of activity, drug resistance, toxicity, and drug-drug interactions [37]. Therefore, it is essential to develop more effective and less topical agents for the treatment of common, drug-resistant fungal infections.

Plants, as a source of medicinal compounds, have played a dominant role in the maintenance of human and animal health since ancient times. According to the World Health Organization reports, plant extracts or their active constituents are used in traditional medicine by 80% of the world’s population [38]. Over 50% of all modern clinical drugs have a natural origin [39]. Despite the gradually increasing popularity of medicinal plants in clinical fields, their anti-fungal properties have been reported to be minor in most previous studies; as a result, new antifungal agents should be developed in the future [40]. In the present study, we focused on the in vitro effects of a number of local medicinal plants against the growth of C. albicans and compared their results with the antifungal activity of standard anti-Candida drugs (clotrimazole and nystatin) in the culture media.

In a previous study, the greatest antimicrobial activity of E. cardamomum was reported against S. aureus (19.3 mm) in the acetonic extracts with an MIC of 25 mg/ml. This study revealed that the organic fruit extract of E. cardamomum showed good antimicrobial activity and could be used in the development of novel herbal ear drops [41]. In the present study, similar results were reported. Compared to the ethanolic extract, the acetonic extract majorly inactivated pathogenic C. albicans. Overall, E. cardamomum is an alternative therapeutic option for the inhibition of biofilm-forming Candida species.

Conclusion

Candida species, which are normally found in human hosts, can cause infections in case the immune system is compromised; as a result, they are known as opportunistic pathogens. In addition, these species can survive the hospital environment. In general, those who continuously use antifungal drugs for infection become resistant to them. Therefore, a new generation of more potent drugs needs to be developed to prevent the increasing threat of emerging azole and amine-resistant Candida species. Overall, a better understanding of the mechanism of Candida species, resistant to azoles and amines, is essential. Consequently, modified medicines, such as plant materials and dry fruits, are used for the treatment of these infections. E. cardomomum is a dry fruit, used as an alternative medicine for the treatment of infections and biofilm inhibition. Therefore, use of alternate medicines is required to treat infections and reduce the associated side-effects.

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