The potential use of chlorhexidine (CHX) and hexetidine-containing mouth rinse in maintaining toothbrush sterility

The study was carried out with the aim of demonstrating quantitatively the presence of microorganisms adhered to toothbrush bristles and to determine the potential of using antimicrobial agent (such as chlorhexidine gluconate (CHX) and hexetidine (HX)) in commercialized mouth rinses to reduce microbial contamination. The study was carried out by enumerating the total colony counts of bristles-adhered microbes after three weeks of normal oral hygiene followed by rinsing the toothbrushes with CHX, HX, tap water and deionized water independently following a strict planned schedule. Rinsing toothbrush with tap water was included in the study as a control due to the normal way of cleaning toothbrush after use in every home. Whereas, sterilized deionized water do not contain any ions, minerals and is microbes-free. The total colony counts of microbes obtained from the toothbrush rinsed with tap water, deionized water, CHX and HX were 62.6×106 CFU mL-1, 74.4×106 CFU mL-1, 2.4×106 CFU mL-1 and 7.6×106 CFU mL-1, respectively. Staphylococcus aureus, Actinomyces naeslundii and Clostridium sp. were isolated from toothbrush rinsed with tap water. Staphylococcus aureus and Peptostreptococcus sp. were obtained from toothbrush rinsed with deionized water. Actinomyces sp. and Clostridium sp. were recovered from toothbrush rinsed with CHX and only Staphylococcus aureus was obtained from toothbrush rinsed with HX. Although toothbrush rinsed with mouth rinses containing antimicrobial agent such as CHX and HX still harbour microorganisms, but the microbial load has been very much lowered compared to the control toothbrush. Thus, this indicates that toothbrush rinsing with mouth rinse after the normal oral hygiene is very convenient and cost effective to reduce toothbrush contamination

Effect of phenotypic switching on the biological properties and susceptibility to chlorhexidine in Candida krusei ATCC 14243

Phenotypic switching is characterized as a virulence factor of Candida spp. This study was carried out to evaluate the phenotypic switching ability of C. krusei ATCC 14243 and to determine its effect on the biological properties, adherence capacity and susceptibility towards chlorhexidine digluconate (CHX). To induce switched generations C. krusei was cultured under nitrogen-depleted growth conditions by adding phloxine B. These phenotypically switched colo- nies were designated as the 1st generation. Subsequent sub-culturing was per- formed to produce the 2nd, 3rd and 4th switched generations. The recovery of the 3rd generation was the highest at 85.7% while that of the 4th generation was lower at 70.8%, and the recovery of the 1st and 2nd generations gradually reduced to 46.6% and 36.4%, respectively. All generations of C. krusei were susceptible towards CHX. The unswitched C. krusei was the most susceptible but the least adherent to coated hard surfaces. The 2nd generation was the least susceptible, but with the highest adherent ability. The minimum inhibition concentration and minimal fungicidal concentration of C. krusei of all genera- tions were determined at 0.4 mg mL 1. These observations suggest that the switching activity of C. krusei induces changes to its biological properties and susceptibility towards CHX

Gaining more insight into the determinants of candida species pathogenicity in the oral cavity

Candida infection (candidiasis) is potentially life threatening and can occur in almost all anatomical sites, including the mouth. Candida species are in fact the most common fungal pathogens isolated from the oral cavity and frequently cause superficial infections such as oral candidiasis and denture-associated erythematous stomatitis. Whilst systemic dissemination of Candida from intraoral foci is rare and largely due to severe deficits of the host immune defenses, the development of localized oral candidiasis is most commonly related to a variety of non-immune determinants such as Candida virulence factors and permissive oral microenvironment. In particular, phenotypic switching and dental biofilm have emerged as major determinants for the pathogenicity of Candida and are currently the subject ofintense research. An understanding of the molecular aspects underlying the biological behavior of Candida will be the key to the development of effective preventive as well as therapeutic measures for invasive and oral candidiasis

Effect of Piper betle

The study aimed to identify the HWP1 gene in non-Candida albicans Candida species and the differential expression of HWP1 following treatment with Piper betle and Brucea javanica aqueous extracts. All candidal suspensions were standardized to 1×106 cells/mL. The suspension was incubated overnight at 37 °C (C. parapsilosis, 35°C). Candidal cells were treated with each respective extract at 1, 3, and 6 mg/mL for 24 h. The total RNA was extracted and reverse transcription-polymerase chain reaction was carried out with a specific primer of HWP1. HWP1 mRNAs were only detected in C. albicans, C. parapsilosis, and C. tropicalis. Exposing the cells to the aqueous extracts has affected the expression of HWP1 transcripts. C. albicans, C. parapsilosis, and C. tropicalis have demonstrated different intensity of mRNA. Compared to P. betle, B. javanica demonstrated a higher suppression on the transcript levels of HWP1 in all samples. HWP1 was not detected in C. albicans following the treatment of B. javanica at 1 mg/mL. In contrast, C. parapsilosis and C. tropicalis were shown to have HWP1 regulation. However, the expression levels were reduced upon the addition of higher concentration of B. javanica extract. P. betle and B. javanica have potential to be developed as oral health product

Assessment of Antifungal Activity of Bakuchiol on Oral-Associated Candida

Bakuchiol is an active component of Psoralea glandulosa and Psoralea corylifolia, used in traditional Chinese medicine. The study aimed at investigating the antifungal activity of bakuchiol on planktonic and biofilm forms of orally associated Candida species. The antifungal susceptibility testing was determined by the broth micro dilution technique. Growth kinetics and cell surface hydrophobicity (CSH) of Candida were measured to assess the inhibitory effect of bakuchiol on Candida planktonic cells. Biofilm biomass and cellular metabolic activity were quantitatively estimated by the crystal violet (CV) and the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assays. All Candida strains have been shown to be susceptible to bakuchiol with the MIC ranges from 12.5 to 100 μg/mL. Significant decrease in specific growth rates and viable counts demonstrates the inhibitory effect of bakuchiol on Candida planktonic cells. A brief exposure to bakuchiol also reduced CSH of Candida (P<0.05), indicating altered surface properties of yeast cells towards hydrophobic interfaces. Biofilm biomass and cell metabolic activity were mostly decreased, except for C. glabrata (P=0.29). The antifungal properties of bakuchiol on Candida species in this in vitro study may give insights into the application in therapeutic strategy against Candida infections

Assessment of Antifungal Activity of Bakuchiol on Oral-Associated Candida spp

Bakuchiol is an active component of Psoralea glandulosa and Psoralea corylifolia, used in traditional Chinese medicine. The study aimed at investigating the antifungal activity of bakuchiol on planktonic and biofilm forms of orally associated Candida species. The antifungal susceptibility testing was determined by the broth micro dilution technique. Growth kinetics and cell surface hydrophobicity (CSH) of Candida were measured to assess the inhibitory effect of bakuchiol on Candida planktonic cells. Biofilm biomass and cellular metabolic activity were quantitatively estimated by the crystal violet (CV) and the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assays. All Candida strains have been shown to be susceptible to bakuchiol with the MIC ranges from 12.5 to 100 g/mL. Significant decrease in specific growth rates and viable counts demonstrates the inhibitory effect of bakuchiol on Candida planktonic cells. A brief exposure to bakuchiol also reduced CSH of Candida ( &lt; 0.05), indicating altered surface properties of yeast cells towards hydrophobic interfaces. Biofilm biomass and cell metabolic activity were mostly decreased, except for C. glabrata ( = 0.29). The antifungal properties of bakuchiol on Candida species in this in vitro study may give insights into the application in therapeutic strategy against Candida infections

The role of Candida albicans candidalysin ECE1 gene in oral carcinogenesis

Oral squamous cell carcinoma is associated with many known risk factors including tobacco smoking, chronic alcoholism, poor oral hygiene, unhealthy dietary habits and microbial infection. Previous studies have highlighted Candida albicans host tissue infection as a risk factor in the initiation and progression of oral cancer. C. albicans invasion induces several cancerous hallmarks, such as activation of proto-oncogenes, induction of DNA damage and overexpression of inflammatory signalling pathways. However, the molecular mechanisms behind these responses remain unclear. A recently discovered fungal toxin peptide, candidalysin, has been reported as an essential molecule in epithelial damage and host recognition of C albicans infection. Candidalysin has a clear role in inflammasome activation and induction of cell damage. Several inflammatory molecules such as IL-6, IL-17, NLRP3 and GM-CSF have been linked to carcinogenesis. Candidalysin is encoded by the ECE1 gene, which has been linked to virulence factors of C albicans such as adhesion, biofilm formation and filamentation properties. This review discusses the recent epidemiological burden of oral cancer and highlights the significance of the ECE1 gene and the ECE1 protein breakdown product, candidalysin in oral malignancy. The immunological and molecular mechanisms behind oral malignancy induced by inflammation and the role of the toxic fungal peptide candidalysin in oral carcinogenesis are explored. With increasing evidence associating C albicans with oral carcinoma, identifying the possible fungal pathogenicity factors including the role of candidalysin can assist in efforts to understand the link between C albicans infection and carcinogenesis, and pave the way for research into therapeutic potentials

Synergism effect of tunicamycin and amphotericin B causes suppression to the MP65 and ERG3 gene in oral associated-Candida albicans and C. dubliniensis

Combination therapy can be used for the treatment of fungal infections, especially for those caused by antifungal-resistant fungi. In the present study, in vitro interactions and mechanisms between tunicamycin (TM) and amphotericin B (AmB) against Candida spp. were evaluated. The nature of the interactions determined by spectrophotometric method in a checkerboard assay was interpreted using nonparametric models of fractional inhibitory concentration index (FICI). We also evaluated the potential activity of TM in association with AmB on selected virulence gene (MP65 and ERG3) in C. albicans and C. dubliniensis. It was found that TM can work synergistically with AmB against C. dubliniensis; the minimum inhibitory concentration of TM and AmB decreased about fourfold, with an FICI of 0.5. The effect of combination has also significantly decreased the expression level of MP65 and ERG3 in which these genes have long been recognized as virulence traits in the pathogenicity of Candida (P ˂ 0.05). Our results suggest that the combination has the ability to reduce the growth of Candida in vitro

Anti-hyphal properties of potential bioactive compounds for oral rinse in suppression of Candida growth

Most oral rinses contain active ingredients with limited antifungal activity, particularly against hyphae development. This has encouraged us to search for bioactive compounds effective against preventing morphology transition of the dimorphic Candida. The antifungal activity of the selected bioactive compounds against seven species of oral-associated Candida was screened before the evaluation of their potency to inhibit hyphal formation. The identified potent inhibitor was then further investigated on its effectiveness through a brief treatment test on germination, adhesion, cellular morphology and gene expression. Bakuchiol, hydroxychavciol and pseudolaric acid B showed antifungal activity while luteolin and sakuranetin were determined to be inactive against the oral-associated Candida species at a concentration below 1 mg/mL. Hydroxychavicol was determined as a potent inhibitor against hyphal growth of Candida albicans. Hydroxychavicol delayed the germination process of C. albicans by affecting the expression of RAS1, NRG1 and HWP1 genes up to 1 h after the treatment. In addition, the treatment caused minimal changes to the cellular morphological structure. But, hydroxychavicol showed poor anti-adherence activity against the germinated cells. In summary, hydroxychavicol is one of the identified bioactive compounds that possessed anti-candida properties against oral-associated Candida species and appeared potent in inhibition of hyphae of C. albicans by affecting its ultrastructural morphology and gene regulation. This finding could suggest development of hydroxychavicol as a bioactive compound that can potentiate the activity of oral rinse in preventing the colonization of dimorphic Candida in the oral cavity

Polymicrobial interactions between Streptococcus mitis, Streptococcus sanguinis and oral associated Candida albicans on an in vitro salivary biofilm and differential expression of ALS1, ALS2 and ALS3 genes

Interactions between oral microorganisms contribute to formation of polymicrobial communities on surfaces and prosthetics. Since streptococci are early colonizers, the ability of Candida albicans to adhere to streptococci paves the way for an additional surface for candidal colonization and propagation. This study aimed to investigate the molecular response of C. albicans to two species of streptococci, i.e. Streptococcus mitis and Streptococcus sanguinis in in vitro salivary biofilms. Single, dual and mixed species salivary biofilms were formed for 24 h. Biofilm biomass and cellular metabolic activity of microbial species were assessed by crystal violet (CV) staining and XTT reduction assay, respectively, followed by scanning electron microscopy. ALS1, ALS2 and ALS3 genes were screened and quantified using qPCR. CV and XTT analysis showed a significant increase in biofilm biomass and cellular metabolic activity of two dual species and mixed species in comparison to single species biofilm. SEM analysis revealed formation of candidal hyphae under influence of streptococci. ALS1 and ALS3 were significantly overexpressed in a mixed species biofilm, in comparison to dual species and single species biofilms. Presence of S. mitis and S. sanguinis assisted in the proliferation of C. albicans which may augment tissue invasion. This study proposes substantial contribution of bacteria in propagation of C. albicans biofilm. Hence, in oral fungal conditions, promotion of the infection may be contingent upon the bacterial constituent, a prospect which is frequently neglected and requires more research