Antifungal activity of some toothpastes and antiseptic solutions against yeasts isolated from the oral cavity of cancer radiated patients

This study aimed to investigate the antifungal efficacy of different types of toothpaste and some antiseptic solutions with varying concentrations against yeast isolated from the oral cavity of radiated patients suffering from head and neck cancer in radiotherapy unit in Suez Canal University teaching’s hospital, Egypt. Eight different species of yeast were isolated taxonomically, assigned to 1 phylum, two classes, one order, and three families and identified phenotypically. The antifungal activity of five kinds of toothpaste and three antiseptic solutions were investigated by agar well diffusion method against the eight isolated yeasts namely: Candida glabrata, C. albicans, C. tropicalis, C. kefyr, C. guilliermondii, C. parapsilosis, Dipodascus geotrichum and Geotrichum beigelii and expressed as the value of the clear zone diameter, however, Their activity was expressed by minimum inhibitory concentration (MIC). Results showed that toothpaste no 1, 3, 4 and 5 were effective against all tested taxa by variable inhibitory degrees. However, no. 2 showed efficacy only against C. glabrata, C. guilliermondii, and C. parapsilosis, Antiseptic solution no 1 showed the highest efficacy against all tested species except C. kefyr while no. 2 was effective against all the tested species except two taxa and finally no. 3 exhibited good antifungal activity against all the tested taxa. In conclusion, we recommended the use of toothpaste contained sodium monofluorophosphate, or sodium fluoride, as an active component followed by a mouthwash which contained Chlorohexidine or Cetylpridinium or miswak as active components to prevent colonization and development of oral candidiasis, especially in cancer patients. Key words– Candida, efficacy, Dipodascus geotrichum, Geotrichum beigelii, Suez Canal University. Introduction The oral cavity is a habitat for a large number of microbiota which coexists with another as a normal microbiome. However, Candida species are ovoid budding yeast-like fungi found as a normal commensal in the oral cavity, vagina, and skin and throughout the gastrointestinal tract of humans. Some factors leading to increased incidence of oral thrush and candidiasis as poor oral hygiene, nutritional deficiencies, high carbohydrate diet, diabetes mellitus, heavy cigarette smoking, dental prostheses, treatment by radiotherapy, immunosuppression, and HIV infection (Meghana et al. 2015). Thus the changes occur in mechanisms of host defense, and the buccal Microbial Biosystems 3(1): 25–33 (2018) ISSN 2357-0334 http://fungiofegypt.com/Journal/index.html Microbial Biosystems Copyright © 2018 Attia Online Edition


Introduction
The oral cavity is a habitat for a large number of microbiota which coexists with another as a normal microbiome.However, Candida species are ovoid budding yeast-like fungi found as a normal commensal in the oral cavity, vagina, and skin and throughout the gastrointestinal tract of humans.Some factors leading to increased incidence of oral thrush and candidiasis as poor oral hygiene, nutritional deficiencies, high carbohydrate diet, diabetes mellitus, heavy cigarette smoking, dental prostheses, treatment by radiotherapy, immunosuppression, and HIV infection (Meghana et al. 2015).Thus the changes occur in mechanisms of host defense, and the buccal environment may cause mucosal and periodontal opportunistic infections (Farah et al. 2010).Dental caries and related oral diseases like gingivitis and periodontitis are most common oral diseases throughout the world including both developed and developing countries and are continuously increasing with the change in eating custom between peoples of different age group and increased utilization of sugar (Saini et al. 2003).Yeasts are among opportunistic pathogenic fungi that able to infect immunocompetent patients and healthy individuals under certain conditions.Candida albicans is the most cause of oral thrush, endocarditis, vaginitis, and infection of nails, skin, and lungs and it is also the fungal species which most commonly isolated from infected root canals (Oztan et al. 2006).Beside C. albicans which is the major pathogen in oral candidosis infections other pathogenic taxa e.g. C. krusei, C. glabrata, and C. dubliniensis have been reported in both compromised and non-compromised hosts and has been isolated from oral plaque and saliva (He et al. 2006;Maijala et al. 2007;Zhu et al. 2008).
Toothpaste has a history that may be dated back to 4000 years ago (Tiwari et al. 2008) recently various studies have been carried out by several investigators on the antimicrobial efficacy of different dentifrices on oral microbes (Davies et al. 2004;Alsaimary 2008;Okpalugo and Ibrahim 2009;Manupati 2011;Andiara et al. 2014;Gibraiel et al. 2014;Nwakanma et al. 2014) and their effectiveness against the test microorganisms in vitro.Many types of toothpaste and mouthwash have been formulated over recent years to contain antibacterials compounds with the aim of preventing or reducing plaque, calculus, gingival inflammation or dental caries without specific antifungal compounds.Despite great improvements in the global oral health status, dental caries remains one of the most prevalent diseases (Abirami and Venugopal 2005;Marsh and Percival 2006;Hatti et al. 2007;Inetianbor et al. 2014;Glick et al. 2017).
This study aimed to evaluate the antifungal efficacy of different types of dentifrices toothpaste and some mouthwash solutions against recovered yeast from the oral cavity of cancer patients irradiated for head and neck cancer in Suez Canal University teaching's hospital, Egypt in vitro.

Study population
A total number of 80 patients (40 females and 40 males) from the radiotherapy unit in Suez Canal University teaching's hospital were screened for oral yeast species load.Patients were classified as 40 elders (20 females and 20 males) aged between 50 to 78 years and 40 adults (20 females and 20 males) aged between 40 to 50 years old.All selected cancer patients were subjected to radiotherapy treatment of head and neck with a total dose of 44 grays.From each patient, successive samples were taken along the period of his/her treatment.The collected samples were routinely processed for the isolation of yeast.Ethical approval from patients was obtained for this study.Also, about 40 oral samples were collected from healthy volunteers (haven't any sign of oral disease) to compare between cancer patients and healthy peoples.

Sampling, isolation and identification of yeast species
The clinical specimens including saliva, palate mucosa, and tongue dorsum were collected with a sterile swab, immersed in 1 ml sterile saline tube 25 mM) and agitated for 30 seconds.Aliquot of 100 µl from each specimen was directly diluted into 10-fold and 100-fold in sterile saline.From each dilution, aliquots of 100 µl in triplicate were inoculated in the Sabouraud dextrose agar (SDA-Merc/Germany) supplemented with 0.1 mg/ml chloramphenicol and incubated at 37 ºC for 48 hours (Samaranayake et al. 2012).Isolates were phenotypically identified by standard-taxonomic yeasts criteria e.g. the production of germ-tube, typical microscopic appearances on cornmeal agar with Tween-80, chlamydospores production, colony morphology and pigment production on chromogenic medium.Yeast species were isolated and identified using appropriate keys according to Kurtzman et al. (2011).The identification of all recovered taxa was confirmed by the API 32 C AUX (bioMerieux, Marcy-l'Etoile, France) identification system for yeasts.Isolates were stored and maintained in Sabouraud dextrose broth accordingly at 4˚C to be used later.Recovered yeasts were examined by using bright field microscopy (Leitz Laborlux S, Germany).
The names of authors of fungal taxa are abbreviated according to Kirk and Ansell (1992).The systematic arrangement in the present list follows the latest system of classification appearing in the 10 th edition of Ainsworth and Bisby's Dictionary of the Fungi (Kirk et al. 2008) Name corrections, authorities, and taxonomic assignments of all taxa reported in this work were checked against the Index Fungorum database (www.indexfungorum.org).

Toothpaste and oral disinfectant solutions
Five different kinds of commercial toothpaste and three oral washes were selected as they the most used in our daily life then evaluates them as antifungal against recovered oral yeast, which was purchased from local pharmacies in Ismailia, Egypt.The different kinds of toothpaste ingredients and oral disinfectant solutions were listed in Table 1 and 2

Antifungal activity against isolated yeasts
A culture of each taxon (48 hr old) was suspended in 5 ml of sterile saline solution.A volume of 0.1ml of the microbe suspension was adjusted to 0.5 McFarland standards (approximately 1 X l0 6 to 5 X l0 6 CFU for yeast was seeded in the SDA plates.For estimation of toothpaste efficacy, five wells (6mm in diameter and 4 mm in depth) at equal distance in each seeded agar plate were created by sterile cork borer.Each well was filled with 1.0 g (0.8 ml) of toothpaste which withdrawn by a sterile syringe under sterile conditions, then plates were incubated at 37 ºC for 48 h.For assessing of oral disinfectants, three wells of each seeded agar plate were created by the same volume by cork borer.Mouthwash under investigation was added to each well (0.8 ml) and incubated for 48 h at 37 °C.The effect of toothpaste and disinfectant solution on yeast growth was determined by measuring the diameter of inhibition zone against 25 mg fluconazole discs® (Oxoid) as a positive control (Lai et al. 2001;Takahashi et al. 2006;and Laina et al 2016).

Determination of minimum inhibitory concentration (MIC)
The MIC of the different toothpaste and the mouthwash against the tested organisms were determined.The minimum inhibitory concentration (MIC) was the lowest concentration of the toothpaste and the Mouthwash that inhibit the growth of yeast cells, as indicated by the absence of growth (Houshmand et al. 2013).

Statistical analysis:
Statistical analysis was performed using Analytical Software SPSS® 13.0 (2005).The values were compared by one way ANOVA followed by Duncan's test and the result considered significant when p<0.05.All the experiments were held in triplicates.

Results and Discussion
A total of 668, 616 yeast colony-forming units (CFU) from elder patients female and male and 514 (CFU) and 490 (CFU) from adult patients female and male respectively were recovered during the entire study.Only a total number of 100 CFU were recovered from the healthy volunteers.All identified taxa were deposited in Suez Canal University Fungarium (SCUF) at Arab Society for Fungal Conservation.From the cancer patients eight species namely: Candida glabrata, C. albicans, C. tropicalis, Candida kefyr, C. guilliermondii, C. parapsilosis, Dipodascus geotrichum and Geotrichum beigelii were recovered while from the healthy volunteers only Candida tropicalis and Saccharomyces cerevisiae were obtained (Fig. 1, Table 3).Taxonomically, isolated species were assigned to 1 phylum, two classes, one order, and three families.Order Saccharomycetales accommodates the most significant range of species (7 species) followed by Trichosporonales (1 species).Taxa with uncertain position were distributed among subclasses, orders and families.
On the higher taxanomic level, Ascomycota was represented only by seven species (87.5 % of the total species number) and Basidiomycota (1 species, 12.5 %).Isolated species belonged to three genera.The dominant geneus was Candida (6 species; 75% of the total isolates), and the remaining genera were represented only by one species each.The species genus ratio (S/G) per family, however, shows that family with uncertain position (Incertae sedis) in Saccharomycetales was the most diverse taxonomical rank by recording a ratio of (6) followed by Dipodascaceae (2).Head and neck cancer patients treated with radiotherapy were suffering from oral mucositis, fungal infection, salivary gland dysfunction (xerostomia), dental caries and taste dysfunction, however, these due to dysfunction in the salivary gland which leads to a decrease in saliva secretion (defense protein) which protect the mouth from microbes (Khan et al. 2010).
Most studies on the antimicrobial activity of toothpaste and antimicrobial compounds have focused on inhibition of bacterial growth, but are less focused on the antifungal activity (Adwan et al. 2012).There is an increasing incidence of neck cancer in adult as in children, which by far leads to increase the oral fungal infection.These types of patients need toothpaste and mouthwash with antifungal activity following brushing.So, this study was designed to evaluate the antifungal efficacy of different dentifrices toothpaste and some antiseptic solutions against yeast isolated from the oral cavity of cancer patients.
Our data revealed that all the five different toothpaste brands showed varying inhibitory activity degree against the oral tested isolates without dilution as shown in Table ( 4).Among the five toothpastes, brand-1 showed the highest zone of inhibition on C. albicans (2.43 ±0.03).However, brand-3 showed the highest zone of inhibition on C. parapsilosis (3.06 ±0.07); brand-4 and brand-5 showed the highest zone of inhibition on Dipodascus geotrichum (2.60 ±0.0, 3.20 ±0.10) respectively.Brand 2 showed the least effect on yeast species.However, it had no effects on C. albicans; C. tropicalis, C. kefyr, D. geotrichum and G. beigelii.-A,b,c,.. Means values in the same column marked with unlike letters are significantly different (p<0.05).
Toothpaste 1 contained the active component sodium monofluorophosphate which give an antifungal effect against all the tested species.However, it gives a high zone of inhibition on Candida albicans (2.43 ±0.030), this result was confirmed previously by Flisfisch et al. (2008) who reported that sodium monofluorophosphate has un activity against C. albicans.
Toothpaste 2 contained the active component sodium fluoride, which had no effects on C. albicans; C. tropicalis, Candida kefyr, Dipodascus geotrichum and Geotrichum beigelii.These data were in contrast with Satari (1990) who reported that fluoride demonstrates some antibacterial and antifungal effects.Fluoride inhibited glycolysis and prevented the transfer of glucose into cells (Ellepol and Amaranayake 2001).
Toothpaste 3 and 4 contained the same active component sodium fluoride, but they had a potent fungal inhibition effect on the all tested oral yeast species.They had a high zone of inhibition on Candida parapsilosis (3.06 ± 0.66) and Dipodascus geotrichum (2.60 ±.0.0) respectively.Although the toothpaste 2 contained the same active components sodium fluoride, toothpastes 3 and 4 were more active against the most isolated oral yeast species because they contained other several components that may make a synergistic effect with the active components to become more active.Toothpaste 3 contained sorbitol, water, hydrated silica, sodium lauryl sulfate, flavour, cellulose gum, sodium saccharin, eugenol, while the toothpaste 4 contained aqua, hydrated silica, sodium lauryl sulfate, cellulose gum, aroma, sodium saccharin, carbomer, C177891, trisodium phosphate, limonene, C174160 as shown in table 1.the different toothpastes activity was expressed by minimum inhibitory concentration (MIC), The MIC of tested toothpastes ranged between 0.50 to 1.2 % (w/v).It could be due to the different type and concentration of the active ingredients incorporated in each type as shown in table 4.
The three tested antifungal oral wash solutions showed varying inhibitory effect against the tested isolates without dilution.The three antifungal types of mouthwash 1, 2 and 3 showed the highest zone of inhibition against C. glabrata as 4.60±0.05,4.40± 0.05 and 3.33±.33respectively.However, mouthwash (1) showed zero effects on C.kefyr, mouthwash (2) showed no effect on C. albicans and C. guilliermondii (Table 5).The different antifungal compounds activity was expressed by minimum inhibitory concentration (MIC), which ranged between 0.56 to 8.8 % (w/v).These could be due to the different type and concentration of the active ingredients incorporated in each type as shown in table 5.
The use of antifungal mouthwash after brushing is necessary for complete removal of fungi from the oral cavity of cancer patients which treated with radiotherapy, however, in our work, the antiseptic wash no 1 contains chlorohexidine as an active antifungal component which may be the main factor for the inhibition against all the isolated oral yeast species except C. kefyr.
These results in agreement with previous work carried out by Shrestha et al. (2011) that chlorhexidine molecule is a highly cationic chlorophenyl bisbiguanide and keenly binds to negatively charged surfaces including epithelial cells.Besides, it was shown to adsorb onto enamel and salivary proteins.Also, about 30% of the total chlorhexidine dose may be retained in the mouth for 24 hours after a 1-minute rinse, although most of the agent is removed from the oral cavity within the first hour.Besides all of these, it can be used as antifungal therapy for candida infection, as it induces coagulation of nucleoproteins, inhibits budding, and makes changes in the cell wall that lead to cytoplasm component escape and yeast death.
Nystatin based antiseptic wash no 2 inhibited most of the isolates but showed no effect on both C. albicans and C. guilliermondii.These results proved previously by Ebelle et al. (2017) which showed that the potency of nystatin is both fungistatic and fungicidal in vitro against a great number of yeast fungi.Nystatin acts by binding to sterols in the cell membrane of susceptible Candida species with a resultant change in membrane permeability allowing leakage of intracellular components.Nystatin exhibits no significant activity against bacteria, virus or protozoa.
Antiseptic wash no 3 contained acetyl pyridinium chloride as an active component, it inhibited all the tested oral isolates.McDonnell and Russell (1999) reported that the cetylpyridinium chloride (CPC) is a cationic quaternary ammonium compound that is usually used in mouthwashes to avoid or treats candidiasis and bacterial infections.CPC alters the surface tension of the cell wall structure which may lead to cell wall leakage.

Conclusions
Oral cavity fungi always make oral complications in immunocompromised patients, especially in cancer radiated patients.So it is urgently required a specific toothpaste and antiseptic oral wash that have a potent antifungal activity to decrease the oral candidiosis.From this study, we recommended the use of toothpaste contained sodium monofluorophosphate, or sodium fluoride, as an active components and mouthwash which contained Chlorohexidine,

Table 1
. Ingredient details of toothpaste formulations tested for antifungal potential.

Table 2
Ingredient details of three antiseptic solutions tested for antimicrobial potential.

Table 3
Presence/absence of recovered yeast species during the study from cancer patients.

Table 4
Inhibition zone diameter in (mm) of the 5 tested toothpastes against isolated yeast species.

Table 5
Inhibition zone diameter in (mm) of the 3 antifungal compounds against isolated yeast species.Data represented in means of triplicates ± standard deviation.-A,b,c,..Means values in the same column marked with unlike letters are significantly different (p<0.05).