Jussevania Pereira Santos¹, Natalia de Souza Botelho¹, Ludmilla Gomes Pereira¹, Célia Roman², Katia Roberta Fávaro², Alexandre Tadachi Morey³, Eliandro Reis Tavares¹, Sueli Fumie Yamada-Ogatta¹, Lucy Megumi Yamauchi^1*

¹Universidade Estadual de Londrina, Departamento de Microbiologia, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid s/n, km 380 PR445 Campus Universitário, Londrina, PR, 86057-970, Brazil
²Unidade de Ensino Superior Ingá, Centro Universitário Ingá, Gleba Ribeirão Morangueiro, 21, Maringá, PR, 87035-510, Brazil
³Instituto Federal de Educação Ciência e Tecnologia do Rio Grande do Sul, R. Dra. Maria Zélia Carneiro de Figueiredo, 870, Igara, Canoas, RS, 92412-240, Brazil

*Corresponding Author: Lucy Megumi Yamauchi, Universidade Estadual de Londrina, Departamento de Microbiologia, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid s/n, km 380 PR445 Campus Universitário, Londrina, PR, 86057-970, Brazil; Email: [email protected]

Published Date: 23-12-2022

Copyright^© 2022 by Santos JP, et al. All rights reserved. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Background: Candida species colonize human microbiota and some conditions, such as immunosuppression or chronic illness, predispose the individual to fungal infections; among them, diabetes mellitus, a metabolic disorder frequently associated with higher rates of yeast infections.

Material and Methods: The prevalence of Candida species in the oral cavity of patients with diabetes mellitus was evaluated and the carriage was compared between type 1 and type 2 diabetic groups. In addition, in vitro susceptibility to antifungals, biofilm formation, cell surface hydrophobicity, and the production of hydrolytic enzymes were tested.

Results: The results demonstrated the presence of different Candida species in the oral cavity of diabetic patients; and, also showed that type 1 diabetic patients are more susceptible to Candida colonization. Almost all isolates produce virulence factors such as proteases, phospholipases, or form biofilm; and they are sensitive to fluconazole and nystatin.

Conclusion: Colonization of Candida spp. oral isolates from type 1 and type 2 diabetic patients were similar; however, type 1 presented a higher colony-forming unit counting. Overall, Candida isolates from the oral cavity of diabetic patients are potential pathogens of candidiasis.

Keywords

Candida Species; Oral Isolates; Diabetes Mellitus; Antifungal Susceptibility; Virulence Factors

Abbreviations

DM: Diabetes Mellitus; T1DM: Type 1 Diabetes; T2DM: Type 2 Diabetes; PBS: Phosphate-Buffered Saline; SD: Sabouraud Dextrose; CFU: Colony-Forming Units; PCR: Polymerase Chain Reaction; MIC: Minimum Inhibitory Concentration; CLSI: Clinical and Laboratory Standards Institute; BSA: Bovine Serum Albumin, DZ: Degradation Zone; PZ: Precipitation Zone; CSH: Cell Surface Hydrophobicity Assay

Introduction

Worldwide, Diabetes Mellitus (DM) is recognized as one of the major health problems, mainly in low and middle-income countries and it is known as a disease and one of the key causes of other diseases. Autoimmune destruction of pancreatic beta cells causes Type 1 Diabetes (T1DM), and Type 2 Diabetes (T2DM) are triggered by resistance to insulin; both types result in almost 500 million adults living with diabetes worldwide [1]. These numbers are increasing every year, reaching 1/3 of the United States of America or the Canadian population, leading these patients to start insulin therapy [2]. DM is one of the largest risks to public health, boosting the susceptibility of patients to get infected, in addition, they also have a 2 folds risk of premature non-communicable diseases death [1].

DM patients, due to a deficiency in the functions of some immune response cells, are frequently colonized with Candida yeast [3]. Candida species are members of the human microbiota; however, they may become pathogenic during the impairment of the host immune response. Some conditions of DM patients, such as adhesion to epithelial cell surfaces, salivary flow and salivary glucose levels, facilitate Candida infection [4]. In addition to host factors, Candida yeasts can express virulence factors, such as hydrolytic enzymes and biofilm formation that contribute to the pathogenesis of candidiasis [5-8]. We address here, Candida’s carriage of the oral cavity of diabetic patients seen at the primary healthcare unit in Maringá city, Paraná, Brazil, their characteristics, and the antifungal susceptibility.

Methodology

Ethics Statement and Study Population

The study was carried out on diabetic patients seen in 2011 at a primary healthcare unit in Maringá city, Paraná, Brazil. One hundred and twenty patients were enrolled and signed a written informed consent form to participate in this study, agreeing with the publication of this report and any accompanying images. The Ethics Committee approved the study protocol of Uningá (CEP no. 0017/11). A medical record of each patient was available, and a history of the previous diagnosis determined the diabetes type. According to the treatment they were taking for diabetes, they were subdivided into Type 1 Diabetes Mellitus (T1DM) included those patients who were on insulin and Type 2 Diabetes Mellitus (T2DM) included those patients who were on oral medication and diet control.

Sample Collection and Identification

Samples were collected using the oral rinse method according to Samaranayake et al. [9], with some modifications. Briefly, patients rinsed the oral cavity using 10 mL of sterile physiological solution (NaCl 0,85%) for 30 seconds and spit it out into a clean container. Samples were centrifuged at 1,500 x g for 10 min at 4 ºC, supernatants were discarded, and the pellets were resuspended in 0.5 mL of 50 mM Phosphate-Buffered Saline (PBS), pH 7.4 containing 0.15 M NaCl. 100 µL sample was spread on Sabouraud Dextrose (SD) agar (Himedia, India) supplemented with 0.1 mg/mL chloramphenicol (Inlab, Brazil) and in CHROMagar Candida medium (Himedia, India). The cultures were incubated at 37 ºC for at least 48 h and the colonies were counted and expressed as the number of Colony-Forming Units (CFU). The presence of Candida yeasts was confirmed by Gram staining and pigmentation characteristics on chromogenic agar. Each isolate was identified at the species level by standard mycological methods [10]. Species identification was also confirmed by molecular identification methods based on PCR [11]. All yeasts were maintained at -80 ºC in SD broth containing 30 % glycerol.

Antifungal Susceptibility Testing

The Minimum Inhibitory Concentration (MIC) of fluconazole (Sigma) and nystatin (Sigma) for all isolates was determined by the broth microdilution assay for yeasts according to the Clinical and Laboratory Standards Institute guidelines, using M27-S4 recommendations [12]. Candida parapsilosis ATCC 22019 was included in each assay as quality control. The results were presented as a range of variations of minimum and maximum minimum inhibitory concentration values obtained for all Candida species. The antifungal concentration tested ranged from 128 to 0.25 µg/mL for fluconazole and 64 to 0.125 µg/mL for nystatin. Two wells were also included as growth and sterility controls. Interpretative criteria for susceptibility to fluconazole were those published in the CLSI [12]. For nystatin, MIC was defined as the value in which 100% growth inhibition was observed [13].

Determination of Protease and Phospholipase Activities

Candida isolates were assayed on minimal medium (55 µmol/L glucose, 11 mol/L KH2PO4, 6 mol/l KCl, 2 mol/L MgSO₄.7H₂O, 65 mol/L FeSO₄, 61 mol/L ZnSO₄) supplemented with 0.5% Bovine Serum Albumin (BSA, Sigma) and SD agar plates containing 4.0% egg yolk as protease and phospholipase substrate, respectively. To determine the protease activity [14], the isolates were previously cultured at 37 ºC for 18 h in minimal medium broth supplemented with 0.5% BSA, pH 4.0, to induce enzyme secretion. To determine phospholipase activity, the cell suspensions were obtained from a 24 h SD broth yeast culture, and the assay was carried out in SD agar supplemented with 4% egg yolk, 350 µmol/L NaCl, and 6.5 µmol/L CaCl₂, pH 4.5. For both assays, cells were counted in a hemocytometer, and a 10 µL suspension of 1×10⁶ yeasts was spotted on the surface of the agar medium. Cultures were incubated at 37 ºC for 96 h, afterwards, the diameter of the degradation (protease activity – Dz) or precipitation (phospholipase activity – Pz) zone around the colony was determined. The enzyme activity was determined by the ratio between colony diameter and colony diameter plus the degradation/precipitation zone. The isolates were classified according to Price, et al., where Dz/Pz values of 1 indicate no detectable protease or phospholipase activity, respectively. All Candida isolates were tested in triplicate and the experiment was carried out on three independent experiments [15].

Cell Surface Hydrophobicity Assay

The biphasic aqueous-hydrocarbon assay was used to assess Cell Surface Hydrophobicity Assay (CSH) on Candida isolates [16]. Briefly, all isolates were grown at 37 ºC for 24 h in SD broth. Yeasts were centrifuged, washed twice, and the cell densities were adjusted to an absorbance of 0.4 at 660 nm in 5 mL of PBS. A volume of 2.5 mL of cell suspension was added to two sterile glass test tubes, in addition, 0.5 mL of xylene (Merck) was added to one tube. After 10 min incubation in the water bath at 37 ºC, the test tube was vigorously mixed for 1 min and incubated for an additional 30 min under the same conditions. The aqueous phase was carefully removed, and the absorbance was measured at 520 nm. CSH was expressed as the percentage decrease in the optical density of the test compared with the control. Thus, the more significant the change in absorbance of the aqueous phase, the more hydrophobic the yeast sample. Each assay was performed on two separate occasions with duplicate determinations each time.

Biofilm Formation

Biofilm production was determined on polystyrene flat-bottomed 96-well microtiter plates (Techno Plastic Products, Switzerland) according to Shin, et al., with modifications [17]. Briefly, Candida isolate was grown at 37ºC for 24 h in SD broth and a 20 µL SD-suspension of 6 x 105 yeasts was placed in each well containing 180 µL SD broth. The plates were incubated at 37ºC for 24 h and washed once with sterile distilled water. Crystal violet staining was used to quantify bulk biofilm production [18]. Briefly, after biofilm formation, each well was washed twice with PBS and the plate was dried for 20 min at 35 °C. Biofilms were stained with 110 μL of 0.4% aqueous crystal violet solution for 45 min, washed three times with ultra-pure sterile water, and discolored with 200 μL of 95% ethanol. After 45 min, 100 μL of distaining solution from each sample was transferred to a new plate and measured with a spectrophotometer plate reader (Universal Microplate Reader ELx 800; Bio-Tek Instruments, USA) at 595 nm. Samples exhibiting a very intense blue color were diluted 1:4 with sterile water before performing a second absorbance reading. The absorbance values of the negative controls (containing no cells) were subtracted from the values of the test wells to minimize background interference. Experiments were carried out in triplicate on two different occasions.

Statistical Analysis

Demographic data were reported as mean values or percentages of the total. The differences between group rates were assessed by Student’s t-test, p<0.05 was considered statistically significant. All statistical analyses were performed with the software Graphpad prism 4.0 for windows (Prism Graphpad software, San Diego CA, USA).

Results and Discussion

T1DM Patients Present an Increased Counting of Candida albicans in Oral Yeast Carriage Analyses

One hundred and twenty patients were enrolled and signed a written informed consent form to participate in this study. The subjects consisted of 49 males (34.2%) and 79 females (65.8%), and most of them were elderly; the age mean was 67.8±13.9 years (ranging from 5 to 93 years). A medical record of each patient was available, and a history of the previous diagnosis determined the diabetes category: T1DM included those patients who were on insulin treatment and T2DM included those patients who were on oral medication and diet control. Among them, 35% were T1DM patients and 65% were T2DM patients, with no differences between their ages mean, 68.3±10.2 and 65.5±19.1, respectively.

Identification and Characterization of Candida spp Isolated in Oral Yeast Carriage Analyses

A total of 66 patients were positive for Candida carriage (Table 1). The T1DM group presented a higher Candida carriage than T2DM patients, 32 and 34 individuals, respectively (Table 1). The colony counts ranged from 5 to up 103 CFU (Fig. 1), and interestingly, T1MD patients had a significant increase in CFU compared to T2DM patients (p<0.05).

Table 1: Distribution type of diabetes and frequency of yeast culture and species found in the saliva of diabetic patients.

Figure 1: Candida colony counting from samples of oral cavities of diabetes Mellitus type 1 and 2 patients (n = 66 patients). *𝑃 < 0.05, for a comparison between T1DM and T2DM.

Each Candida isolate was identified at the species level (Table 1), in both groups, C. albicans represented the most frequently isolated species followed by C. tropicalis, C. glabrata, C. dubliniensis and C. parapsilosis (Table 1). The distribution of Candida species is close to the Brazilian epidemiology, with a predominance of C. albicans, C. tropicalis and followed by others [19].

Candida ability to produce hydrolytic enzymes used in the invasion of host tissue and liberation of nutrients is related to its overgrowth [20]. The enzyme activity was determined by the ratio between colony diameter and colony diameter plus the degradation/precipitation zone [15]. The results showed that some Candida isolates presented protease or phospholipase activities (Table 2). These activities have seemed in a lower percentage of samples [21, 22].

Table 2: Protease, phospholipase activities, cell surface hydrophobicity, biofilm formation, and in-vitro susceptibility to fluconazole and nystatin of Candida isolates from diabetic patients.

To colonize and cause disease, Candida species must evade the host immune response and multiply, therefore, yeast adhesion on host surfaces is essential for the establishment of colonization. This mechanism involves many biological factors such as the presence of adhesins and cell surface hydrophobicity resulting in invasion and biofilm formation, and this is strongly related to growth on tissues or implanted medical devices [4]. Cell Surface Hydrophobicity assay (CSH) on Candida isolates was assessed using a biphasic aqueous-hydrocarbon test [16]. The mean relative CSH of all isolates was 47.40±26.58, ranging from 1.16 to 93.50 (Table 2), these data showed that Candida isolates have the potential of yeast adhesion to epithelial cells and extracellular matrix proteins increasing the potential to form biofilm.

Biofilm production was determined on polystyrene flat-bottomed 96-well microtiter plates [22]. Our results showed that the majority of Candida isolates formed a biofilm (Table 2). The main concern is that C. albicans is the leading fungal microorganism found in implanted medical devices and biofilm increases resistance to antifungal drugs, which makes it a challenge and leads to many clinical complications [23,24].

Finally, the Minimum Inhibitory Concentration (MIC) of fluconazole and nystatin for all isolates was determined by the broth microdilution assay for yeasts. Most Candida isolates were susceptible to fluconazole (MIC ranging from 0.125-32 µg/mL), 8 isolates were susceptible dose-dependent and one C. albicans isolate was resistant to fluconazole (Table 2). Regarding nystatin (Table 2), all isolates were susceptible (MIC ranging from 0.125 to 2 µg/mL).

Overall, Candida isolates from oral cavities of diabetes patients are potentially pathogenic, capable of forming a biofilm. T1DM patients are more susceptible to Candida colonization and indeed, they presented higher CFU counting. A study of the oropharyngeal microbiome of elderly individuals showed several microorganisms such as bacteria and yeast that can become opportunistic pathogens in these individuals with debilitated immune response or health problems; its colonization appears to be associated with some physiological changes as salivary pH and loss of glycemic control [3]. Our study found that C. albicans is the most common species of the oral cavity of diabetes patients, followed by C. tropicalis, C. glabrata, C. dubliniensis and C. parapsilosis similar to another study [25]. An increase in the frequency of different Candida species isolation has clinical importance since they may have a reduced susceptibility to antifungals compared to C. albicans, although most Candida isolates were susceptible to antifungals tested [26]. Fluconazole was established as one of the main drugs to treat most cases of candidiasis, in addition, it is the choice of initial treatment for suspected fungal infections. The main concern is that although most isolates showed susceptibility to fluconazole and nystatin in-vitro, we detected a reduced susceptibility in some isolates, especially in reading after 24 hours. Monitoring antifungal resistance among Candida species will help in empirical treatment due to the knowledge of what is happening in the community, especially among diabetes patients.

Conclusion

Type 1 and type 2 diabetic patients present similar rates of Candida spp. oral carriage colonization; however, type 1 presented a higher colony-forming unit counting. These isolates are potential pathogens of candidiasis, presenting several virulence factors to enhance Candida pathogenesis, such as phospholipase and protease activity. In addition, they are capable to form biofilm, and some isolates present fluconazole resistance.

Acknowledgment

The authors thank the technician Ediel C. Costa for his help with laboratory maintenance. Natalia de S. Botelho, Alexandre T. Morey, Eliandro R. Tavares, received fellowships from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, Finance Code 001). Sueli F. Yamada-Ogatta and Lucy M. Yamauchi received PQ fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.

Ethical Approval

This study was previously approved by the Ethics Committee of Uningá (CEP no. 0017/11). All procedures performed in studies involving human participants were following the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Conflict of Interest

The authors declare that have no competing interest and not any conflict of interest.

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Article Type

Research Article

Publication History

Received Date: 29-11-2022
Accepted Date: 15-12-2022
Published Date: 23-12-2022

Copyright© 2022 by Santos JP, et al. All rights reserved. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Yamauchi LM, et al. Biofilm Formation, Virulence Factors and Antifungal Susceptibility of Candida spp. Isolated From the Oral Cavity of Diabetes Mellitus Patients.  J Clin Immunol Microbiol. 2022;3(3):1-12.

Figure 1: Candida colony counting from samples of oral cavities of diabetes Mellitus type 1 and 2 patients (n = 66 patients). *𝑃 < 0.05, for a comparison between T1DM and T2DM.

Table 1: Distribution type of diabetes and frequency of yeast culture and species found in the saliva of diabetic patients.

Table 2: Protease, phospholipase activities, cell surface hydrophobicity, biofilm formation, and in-vitro susceptibility to fluconazole and nystatin of Candida isolates from diabetic patients.