Influence of artificial saliva in biofilm formation of Candida albicans in vitro

Abstract

Due to the increase in life expectancy, new treatments have emerged which, although palliative, provide individuals with a better quality of life. Artificial saliva is a solution that contains substances that moisten a dry mouth, thus mimicking the role of saliva in lubricating the oral cavity and controlling the existing normal oral microbiota. This study aimed to assess the influence of commercially available artificial saliva on biofilm formation by Candida albicans. Artificial saliva I consists of carboxymethylcellulose, while artificial saliva II is composed of glucose oxidase, lactoferrin, lysozyme and lactoperoxidase. A control group used sterile distilled water. Microorganisms from the oral cavity were transferred to Sabouraud Dextrose Agar and incubated at 37°C for 24 hours. Colonies of Candida albicans were suspended in a sterile solution of NaCl 0.9%, and standardisation of the suspension to 106 cells/mL was achieved. The acrylic discs, immersed in artificial saliva and sterile distilled water, were placed in a 24-well plate containing 2 mL of Sabouraud Dextrose Broth plus 5% sucrose and 0.1 mL aliquot of the Candida albicans suspension. The plates were incubated at 37°C for 5 days, the discs were washed in 2 mL of 0.9% NaCl and placed into a tube containing 10 mL of 0.9% NaCl. After decimal dilutions, aliquots of 0.1 mL were seeded on Sabouraud Dextrose Agar and incubated at 37°C for 48 hours. Counts were reported as CFU/mL (Log10). A statistically significant reduction of 29.89% (1.45 CFU/mL) of Candida albicans was observed in saliva I when compared to saliva II (p = 0.002, considering p≤0.05).

Saliva; Artificial; Candida albicans; Biofilm

MICROBIOLOGY

Influence of artificial saliva in biofilm formation of Candida albicans in vitro

Michelle Peneluppi Silva; José Chibebe Junior; Adeline Lacerda Jorjão; Ana Karina da Silva Machado; Luciane Dias de Oliveira; Juliana Campos Junqueira; Antonio Olavo Cardoso Jorge

Department of Biosciences and Oral Diagnosis, School of Dentistry of São José dos Campos, UNESP - Univ. Estadual Paulista, São José dos Campos, SP, Brazil.

^{Corresponding author}

ABSTRACT

Due to the increase in life expectancy, new treatments have emerged which, although palliative, provide individuals with a better quality of life. Artificial saliva is a solution that contains substances that moisten a dry mouth, thus mimicking the role of saliva in lubricating the oral cavity and controlling the existing normal oral microbiota. This study aimed to assess the influence of commercially available artificial saliva on biofilm formation by Candida albicans. Artificial saliva I consists of carboxymethylcellulose, while artificial saliva II is composed of glucose oxidase, lactoferrin, lysozyme and lactoperoxidase. A control group used sterile distilled water. Microorganisms from the oral cavity were transferred to Sabouraud Dextrose Agar and incubated at 37 °C for 24 hours. Colonies of Candida albicans were suspended in a sterile solution of NaCl 0.9%, and standardisation of the suspension to 106 cells/mL was achieved. The acrylic discs, immersed in artificial saliva and sterile distilled water, were placed in a 24-well plate containing 2 mL of Sabouraud Dextrose Broth plus 5% sucrose and 0.1 mL aliquot of the Candida albicans suspension. The plates were incubated at 37 °C for 5 days, the discs were washed in 2 mL of 0.9% NaCl and placed into a tube containing 10 mL of 0.9% NaCl. After decimal dilutions, aliquots of 0.1 mL were seeded on Sabouraud Dextrose Agar and incubated at 37 °C for 48 hours. Counts were reported as CFU/mL (Log10). A statistically significant reduction of 29.89% (1.45 CFU/mL) of Candida albicans was observed in saliva I when compared to saliva II (p = 0.002, considering p≤ 0.05).

Descriptors: Saliva, Artificial; Candida albicans; Biofilm.

INTRODUCTION

Humans produce and secrete between 1200 to 1500 mL of saliva per day. Approximately 90% of this volume is produced by the salivary glands, and the remaining 10% are produced by numerous minor salivary glands present in the submucosa of the oral and oropharyngeal cavity.¹

Saliva is essential for protection of the mucosa as well as structures of the oral cavity and adjacent gastrointestinal epithelium.¹ In the mouth, saliva participates in the activities of chewing, speaking and swallowing, as well as taste sensitivity, lubrication of tissues, mucosal protection against penetration of various substances, regulation of pH and the formation of an oral biofilm. Further, saliva helps control oral microbiota, protects teeth against harmful substances and injuries and participates in the remineralisation process.²

Hyposalivation, which is characterised by decreased salivary flow, and xerostomia, the subjective feeling of a dry mouth, may or may not be associated.^3,4

Factors related to hyposalivation and xerostomia include aging, the presence of glandular or systemic diseases and the use of certain drugs (e.g., anticholinergics, anxiolytics, antihypertensives, diuretics, antihistamines, anti-reflux). In addition, it may be a side effect of head and neck radiotherapy. Xerostomia and hyposalivation can diminish quality of life by resulting in discomfort and pain when chewing, swallowing and talking. The decrease in salivary flow increases the risk of tooth decay and other oral fungal and/or bacterial infections, facilitating the emergence of opportunistic pathogens such as Candida albicans.^2,5

Control and/or management of hyposalivation are usually done with the use of commercially available artificial saliva. Because it is typically not composed of any medications, it may be administered by the patient whenever they feel that their mouth is dry.³ The composition of artificial saliva includes substances that soothe a dry mouth by lubricating the oral cavity.^6-9

The species of the genus Candida are constituents of the oral microbiota of humans and are commensal microorganisms.¹⁰ Candida albicans is considered the most common fungal pathogen in humans and is responsible for most superficial or systemic fungal infections.^11,12 Candida spp. can be easily isolated from the oral cavity in approximately 60% of healthy adults.^13,14 Candida albicans primarily colonises the tongue, flowing into the oral mucosa, tooth surfaces, the biofilm and saliva. Its pathogenic action is favoured by local or systemic changes. Of the local factors associated with risk for Candida infection of the oral cavity, poor oral hygiene, use of ill-fitting dentures and decreased salivary flow are the most common. The systemic factors include nutritional deficiencies, malignant tumours, use of broad-spectrum antibiotics, smoking, age, diabetes, disorders of the salivary glands and deficiencies in the immune response.^13,15

In immunocompromised individuals, Candida spp. can cause fatal fungaemias.¹⁶ Of the virulence factors associated with Candida spp., the most important include the ability to adhere to surfaces by producing filamentous growth and releasing hydrolytic enzymes capable of inducing damage to host cells.¹⁰ In addition, Candida species are able to form biofilms,^10,17 and this feature has been associated with an increased expression of virulence factors,^10,18 as well as a reduced susceptibility to antimicrobial agents.¹⁰

The development of effective salivary substitutes requires an understanding of the physiological and biological properties of human saliva, which depend primarily on the role of salivary proteins and glycoproteins.^19-22 The appropriate action of salivary substitutes requires the interaction between viscosity and film formation. The oral cavity provides a favourable environment for the presence of both substances in the saliva substitutes and human saliva.²² Of the salivary proteins, the mucins are the proteins primarily responsible for lubrication and film formation in human saliva.^21,23

The objective of this study was to evaluate biofilm formation of Candida albicans in an acrylic resin disc after incubation in two artificial saliva samples of distinctly different chemical composition.

METHODOLOGY

Acrylic resin disc

A colourless acrylic resin disc was created that measured 5 mm in diameter and was 2 mm thick, yielding a total of 30 units. The disc was sterilised by gamma rays (cobalt-60) with a dose of 20 kGy for 6 hours (Embrarad, São Paulo, Brazil).

Microorganism and preparation of standardised suspension

We used a standardised suspension of a standard strain of Candida albicans (ATCC 18804) from the Laboratory of Microbiology, School of Dentistry of São José dos Campos - UNESP. The microorganisms were transferred to Sabouraud Dextrose Agar (Difco, Detroit, USA) and incubated in a bacteriological incubator at 37 °C for 24 hours. Colonies of Candida albicans were suspended in a sterile solution of NaCl 0.9%, and a standardisation of the suspension to 106 cells/mL was obtained in the spectrophotometer (B582, Micronal, São Paulo, Brazil) with a wavelength of 530 nm and an optical density of 0.284.

Artificial saliva

We used two artificial salivas, artificial saliva I (AS I) - Salivan^® (Apsen, São Paulo, Brazil), which is composed of carboxymethylcellulose, and artificial saliva II (AS II), Biotène^® (GlaxoSmithKline, Moon Township, USA) consisting of glucose oxidase, lactoferrin, lysozyme and lactoperoxidase. For the control groups, we used sterile distilled water. The acrylic resin discs were immersed for 60 minutes at 37 °C in the artificial salivas tested and in the sterile distilled water, distributed in 10 units in each group. They were then washed in sterile water.

Biofilm formation of Candida albicans

The acrylic resin discs previously incubated in the saliva were inserted, with the aid of sterile forceps, in the first row of 24-well plates (Costar Corning, New York, USA) containing 2 mL of Sabouraud Dextrose broth (HiMed, Mumbai, India) plus 5% sucrose (Synth, Diadema, São Paulo). Thereafter, each well of the plate received a 0.1 mL aliquot of the suspension of Candida albicans. The plates were incubated at 37 °C for 5 days.

After incubation, the acrylic resin discs were transferred to the second row of wells containing 2 mL of sterile NaCl 0.9%, and the plates were shaken for five minutes on an orbital shaker (Solab, Piracicaba, Brazil). This procedure was performed twice with the goal of removing non-microbial cells that were adherent to the acrylic resin discs. After washing, each disc was placed in a Falcon tube containing 10 mL of sterile NaCl 0.9% and homogenised for 30 seconds in an ultrasonic homogeniser (HD 2200 Sonoplus, Bandelin Electronic, Berlin, Germany) with a power of 50 W.

The solution obtained was assumed to have a dilution factor of 10-¹. From this, decimal dilutions (10-² and 10-³) of the biofilm suspension of each acrylic resin disc were made. Aliquots of 0.1 mL were seeded in Petri dishes containing Sabouraud Dextrose Agar (Difco, Detroit, USA) and were incubated in a bacteriological incubator at 37 °C for 48 hours. Plates containing 30-300 colonies were counted for calculation of colony forming units per millilitre (CFU / mL), and the number obtained was transformed into base 10 logarithm (log10).

Statistical analysis

The results were statistically analysed using Tukey's test and Minitab software (Inc. PA, USA).

RESULT

The means and standard deviations obtained in the tests and the results of the statistical analysis of the CFU/mL (Log10) for biofilms isolated from the control, AS I and AS II groups are shown in Figure 1.

In the comparison between biofilms isolated from the AS I and control groups, the percentage reductions observed were 11.32% (0.43 CFU/mL) for the AS I group compared to the control group (p = 0.508). Comparing the biofilm from the AS II and control groups, there was a 26.48% (1.02 CFU/mL) percentage increase observed in the AS II group when compared to the control group, a statistically significant increase (p = 0.035).

When we compared the biofilms of the AS I and AS II groups, the percentage reduction in the AS I group when compared to the AS II group was 29.89% (1.45 CFU/mL), which was statistically significant (p = 0.002).

DISCUSSION

Saliva is essential for human health. When secreted at a normal volume, it has many functions and components, the primary being the lubrication of the oral cavity, as this is critical for protecting the oropharynx from infections. Much of its protective activity is mediated by its chemical makeup, which includes enzymes, minerals, proteins and substances that inhibit the growth and activity of various microorganisms.

The decrease in salivary volume within the oral cavity may cause discomfort and can cause the individual to be more susceptible to opportunistic infections. Many conditions can reduce salivary flow, e.g., the use of dental prosthetic devices, which are often made of acrylic resin and are easily colonised by microorganisms due to their surface roughness.²⁴ According to studies, Candida albicans is the primary organism identified in materials for making dentures, and the first phase of colonisation corresponds to adherence of the organism to a surface.²⁵

Health professionals usually prescribe salivary substitutes or artificial saliva as treatment for hyposalivation. Ideally, these should have the same components as natural saliva; however, most of these products have only compounds that improve lubrication and moistening.⁶ The high number of species of Candida in patients with xerostomia suggests that saliva substitutes participate in the control of oral microbiota. Since the 1990s, enzymes such as lysozyme, lactoferrin and lactoperoxidase have been proposed for use in patients with xerostomia with the goal of preventing related diseases.^26,27

The present study compared the influence of two types of artificial saliva on biofilm formation by Candida albicans on acrylic resin discs. Based on the results, the artificial saliva products compared showed a statistically significant difference with respect to biofilm formation by Candida albicans. The AS II group had higher adherence of Candida albicans to the biofilm, although its formulation contains substances present in human saliva that would function to prevent diseases related to microbial growth. This result contrasts with that of Hahnel et al.²⁶ and Tenovuo et al.²⁷

AS I resulted in a decreased development of Candida albicans biofilms. This solution is composed mainly of carboxymethylcellulose, a semisynthetic derivative of cellulose that is indigestible and is not absorbed systemically. It has a high sodium content (10.5% to 12.9%), which may interfere with the mechanisms of adhesion of oral microorganisms. The formation of a film by a cellulose-based product that is not consistently found in the oral cavity may also explain the lower adherence of Candida albicans, as this pathogen may not be expressing specific receptors for cellulose or derivatives. Thus, it is a drug that competes with the normal microbiota of the mouth. Carboxymethylcellulose is hypoallergenic, non-toxic, has high viscosity and does not interact with other medications. When tested in vitro in our study, it decreased the likelihood of Candida albicans biofilm formation; therefore, there may be a role for it in the prevention of oral candidiasis, and it may be a good option for patients who suffer with symptoms/signs of hyposalivation.

There have been studies that have studied the antimicrobial effect of commercially available artificial saliva that consists of lactoferrin, glucose oxidase, lactoperoxidase, lysozyme and immunoglobulins against Streptococcus mutans, Lactobacillus acidophilus and Candida albicans. These studies found that none of the evaluated compounds exerted anticandidal effects.²⁸

Salivary substitutes are capable of forming films on the surfaces of teeth, tissue, mucous membranes and dental restorations, thus they are able to influence the initial adhesion of oral microorganisms.26 In this in vitro study, we found that the initial adhesion of Candida albicans to the tested surface in the presence of artificial saliva varied depending on the components in the saliva. More specifically, AS II resulted in an increase in biofilm, while AS I decreased the amount of biofilm present.

CONCLUSION

The components in the formulation of artificial saliva II provided greater adherence of Candida albicans biofilms when compared to artificial saliva I, which contained carboxymethylcellulose.

ACKNOWLEDGMENT

The authors are grateful to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for the scholarship provided (Process 2010/14434-4).

Received for publication on Oct 05, 2011

Accepted for publication on Dec 01, 2011

Corresponding author:

• 1. Napeñas JJ, Brennan MT, Fox PC. Diagnosis and treatment of xerostomia (dry mouth). Odontology. 2009 Jul;97(2):76-83.
• 2. Gupta A, Epstein JB, Sroussi H. Hyposalivation in elderly patients. J Can Dent Assoc. 2006 Nov;72(9):841-6.
• 3. Hahnel S, Behr M, Handel G, Bürgers R. Saliva substitutes for the treatment of radiation-induced xerostomia - a review. Support Care Cancer. 2009 Nov;17(11):1331-43.
• 4. Davies, AN. A comparison of artificial saliva and chewing gum in the management of xerostomia in patients with advanced cancer. Palliat Med. 2000 May;14(3):197-203.
• 5. Dirix P, Nuyts S, Vander Poorten V, Delaere P, Van den Bogaert W. Efficacy of the BioXtra dry mouth care system in the treatment of radiotherapy-induced xerostomia. Support Care Cancer. 2007 Dec;15(12):1429-36.
• 6. Oh DJ, Lee JY, Kim YK, Kho HS. Effects of carboxymethylcellulose (CMC)-based artificial saliva in patients with xerostomia. Int J Oral Maxillofac Surg. 2008 Nov;37(11):1027-31.
• 7. Epstein JB, Emerton S, Le ND, Stevenson-Moore P. A double-blind crossover trial of Oral Balance gel and Biotene toothpaste versus placebo in patients with xerostomia following radiation therapy. Oral Oncol. 1999 Mar;35(2):132-7.
• 8. Porter SR, Scully C, Hegarty AM. An update of the etiology and management of xerostomia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004 Jan;97(1):28-46.
• 9. Valdez IH, Fox PC. Diagnosis and management of salivary dysfunction. Crit Rev Oral Biol Med. 1993;4(3-4):271-7.
• 10. Williams D, Lewis M. Pathogenesis and Treatment of oral candidiasis. J Oral Microbiol. 2011 Jan 28;3. doi: 10.3402/jom.v3i0.5771.
• 11. Thein ZM, Samaranayake YH, Samaranayake LP. Effect of oral bacteria on growth and survival of Candida albicans biofilms. Arch Oral Biol. 2006 Aug;51(8):672-80.
• 12. Pukkila-Worley R, Peleg AY, Tampakakis E, Mylonakis E. Candida albicans hyphal formation and virulence assessed using a Caenorhabditis elegans infection model. Eukaryot Cell. 2009 Nov;8(11):1750-8.
• 13. Pereira-Cenci T, Deng DM, Kraneveld EA, Manders EM, Del Bel Cury AA, Ten Cate JM, et al. The effect of Streptococcus mutans and Candida glabrata on Candida albicans biofilms formed on different surfaces. Arch Oral Biol. 2008 Aug;53(8):755-64.
• 14. Jarosz LM, Deng DM, van der Mei HC, Crielaard W, Krom BP. Streptococcus mutans competence-stimulating peptide inhibits Candida albicans hypha formation. Eukaryot Cell. 2009 Nov;8(11):1658-64.
• 15. Rowan R, Moran C, McCann M, Kavanagh K. Use of Galleria mellonella larvae to evaluate the in vivo anti-fungal activity of [Ag2(mal)(phen)3]. Biometals. 2009 Jun;22(3):461-7.
• 16. Lim CS, Rosli R, Seow HF, Chong PP. Candida and invasive candidiasis: back to basics. Eur J Clin Microbiol Infect Dis. 2012 Jan;31(1):21-31. Epub 2011 May 5.
• 17. Silva S, Henriques M, Oliveira R, Williams D, Azeredo J. In vitro biofilm activity of non-Candida albicans Candida species. Curr Microbiol. 2010 Dec;61(6):534-40.
• 18. Rajendran R, Robertson DP, Hodge PJ, Lappin DF, Ramage G. Hydrolytic enzyme production is associated with Candida albicans biofilm formation from patients with type 1 diabetes. Mycopathologia. 2010 Oct;170(4):229-35.
• 19. Levine MJ. Development of artificial salivas. Crit Rev Oral Biol Med. 1993;4(3-4):279-86.
• 20. Park MS, Chung JW, Kim YK, Chung SC, Kho HS. Viscosity and wettability of animal mucin solutions and human saliva. Oral Dis. 2007 Mar;13(2):181-6.
• 21. Park MS, Chang JY, Kim YY, Kang JH, Kho HS. Physical and biological properties of yam as a saliva substitute. Arch Oral Biol. 2010 Feb;55(2):177-83.
• 22. Park MS, Chang JY, Kang JH, Park KP, Kho HS. Rheological properties of hyaluronic acid and its effects on salivary enzymes and Candida. Oral Dis. 2010 May;16(4):382-7.
• 23. Dodds MW, Johnson DA, Yeh CK. Health benefits of saliva: a review. J Dent. 2005 Mar;33(3):223-33.
• 24. Pesci-Bardon C, Fosse T, Serre D, Madinier I. In vitro antiseptic properties of an ammonium compound combined with denture base acrylic resin. Gerodontology. 2006 Jun;23(2):111-6.
• 25. Radford DR, Challacombe SJ, Walter JD. Denture plaque and adherence of Candida albicans to denture-base materials in vivo and in vitro. Crit Rev Oral Biol Med. 1999;10(1):99-116.
• 26. Hahnel S, Ettl T, Gosau M, Rosentritt M, Handel G, Bürgers R. Influence of saliva substitute films on the initial adhesion of Candida albicans to dental substrata prior to and after artificial ageing. Arch Oral Biol. 2010 May;55(5):391-6.
• 27. Tenovuo J, Lumikari M, Soukka T. Salivary lysozyme, lactoferrin and peroxidase: antibacterial effects on cariogenic bacteria and clinical applications in preventive dentistry. Proc Finn Dent Soc. 1991;87(2):197-208.
• 28. Güneri P, Alpöz E, Epstein JB, Çankaya H, Ates M. In vitro antimicrobial effects of commercially available mouth-wetting agents. Spec Care Dentist. 2011 Jul-Aug;31(4):123-8.

Publication Dates

• Publication in this collection
  21 May 2012
• Date of issue
  Feb 2012

History

• Received
  05 Oct 2011
• Accepted
  01 Dec 2011