Prevalence of oral Candida carriage in Thai adolescents

Aim: Oral candidiasis is among the most common AIDS‐associated opportunistic infections. Adolescents remain at the highest risk of HIV infection and could suffer from oral candidiasis. However, information on oral Candida carriage in this population is limited. This study aims to evaluate the prevalence of oral Candida in Thai adolescents. Methods: Oral rinse samples from 80 healthy Thais (age: 15–17 years) were collected and analyzed for the prevalence of Candida species using culture‐based and polymerase chain reaction assays. Results: Twenty six adolescents (32.5%) carried Candida in the oral cavity. Candida albicans was detected in 28.75% (23/80). Non‐albicans Candida species were detected in 6.25% (5/80). The majority (92.3%, 24/26) of adolescents with Candida carried a single species. Two carried two species: one with Candida glabrata and Candida albicans, and the other with Candida parapsilosis and Candida albicans. Three adolescents harbored only non‐albicans species, with one carrying Candida tropicalis and two carrying Candida parapsilosis. Candida dubliniensis was not detected in this population. Most adolescents carried Candida at a low level (<500 c.f.u./mL). Conclusions: Oral Candida was present in approximately one‐third of adolescents. Candida albicans was the most prevalent (88.5%), and non‐albicans species were present in 19.2% of those with oral Candida.     

Colonization of Candida: prevalence among tongue‐pierced and non‐pierced immunocompetent adults

Oral Diseases (2010) 16 , 176–175 Objective: To evaluate the colonization of Candida at the tongue‐piercing site of immunocompetent individuals. Subjects and methods: Swabs samples were obtained from the anterior lingual mucosa of healthy young adults with tongue piercing (N = 115); 86 subjects with (non‐intra‐oral) facial piercing served as a comparison group. Candida colonization was examined by light microscopy after 5‐day incubation. Positive specimens were re‐cultured on Chromagar?Candida plates for species identifying. Results: Candida colonization was more prevalent among tongue‐pierced (20.0%) than facial‐pierced subjects (9.4%; P = 0.048). All colonies were of Candida albicans. No difference was found between current tongue ornament wearers (21.2%) and non‐wearers (19.5%; P = 0.803). In multivariate analysis, the only significantly positive influencing factors on colonization were tongue piercing (P = 0.034) and daily smoking of more than 10 cigarettes (P = 0.024). Conclusions: Piercing of the tongue was found to be a risk factor for colonization of Candida albicans, without an influence of whether or not an ornament is in place.     

The development and validation of a rapid genetic method for species identification and genotyping of medically important fungal pathogens using high‐resolution melting curve analysis

Accurate, rapid and economical fungal species identification has been a major aim in mycology. In this study, our goal was to examine the feasibility of a high‐resolution melting curve analysis (HRMA) of internal transcribed regions ITS1 and ITS2 in ribosomal DNA (rDNA) for a rapid, simple and inexpensive differentiation of eight clinically relevant Candida species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, Candida tropicalis, Candida guilliermondii, Candida dubliniensis and Candida lusitaniae). In addition, for the first time, we tested the applicability of HRMA to classify C. albicans strains into four previously described genotypes (A, B, C and D) using a primer set that spans the transposable intron region of 25S of rDNA. Type and unknown clinical oral isolates were used in this study and the melting curve analysis was compared with both amplicons' sequencing and agarose gel electrophoresis analysis. Real‐time PCR and subsequent HRMA of the two described rDNA regions generated distinct melting curve profiles that were in accord with sequencing and gel electrophoresis analysis, highly reproducible, and characteristic of each of the eight Candida species and C. albicans genotypes. Moreover, results were obtained in 4 h and without the need for any post‐amplification handling, so reducing time and cost. Owing to its simplicity and speed, this technique is a good fit for genotypic analysis of hundreds of clinical strains in large epidemiological settings.     

Candida glabrata and Candida albicans co‐infection of an in vitro oral epithelium

J Oral Pathol Med (2010) 40 : 421–427 Background: Candida albicans is regarded as the leading of candidosis. However, Candida glabrata has emerged as an important pathogen of oral mucosa, occurring both singly or in mixed species infections, often with C. albicans. Compared with C. albicans, little is known about the role of C. glabrata in oral infection. The aim of this study was to examine single and mixed species infection of oral epithelium involving C. glabrata and establish its ability to invade and damage tissue. Methods: A reconstituted human oral epithelium (RHOE) was infected only with C. glabrata, or simultaneously with C. glabrata and C. albicans. The ability of both species to invade the tissue was examined using species specific peptide nucleic acid (PNA) probe hybridization and confocal laser scanning microscopy. Epithelial damage was assessed by measuring lactate dehydrogenase (LDH) activity. Results: Candida glabrata strains were able to colonize the RHOE, in a strain dependent manner. Candida glabrata single infection after 12 h, generally revealed no invasion of the RHOE, which contrasted with extensive tissue invasion demonstrated by C. albicans. Mixed infection showed that C. albicans enhanced the invasiveness of C. glabrata, and led to increased LDH release by the RHOE, which paralleled the observed histological damage. Conclusions: The results obtained demonstrating enhanced invasion and increased tissue damage caused by mixed C. glabrata and C. albicans infections have important clinical significance and highlight the need to identify Candida species involved in oral candidosis.     

Human oral keratinocyte E‐cadherin degradation by Candida albicans and Candida glabrata

J Oral Pathol Med (2010) 39 : 275–278 Background: E‐cadherin (E‐Cad) is a 120‐kDa adhesive protein found in adherens junctions of the digestive tract epithelium. We tested the ability of two Candida strains to degrade human E‐Cad in the Candida virulence factor perspective. Materials and methods: We set out to study oral mucosal E‐Cad degradation by clinical and reference strains of Candida albicans and Candida glabrata. We also included hyphal and secreted aspartic proteinase (Sap) mutants of C. albicans to test the effect of yeast/hyphal transition on the ability to degrade E‐Cad. The tests were performed at pH 4 and pH 6 to determine the effect of local tissue acidity on the activation of Saps. The C. albicans strains used were: CCUG 32723; clinical strain SC5314 which is known to be strongly invasive; hyphal mutants of SC5314: HLC52 (efg1/efg1), HLC54 (cph1/cph1 efg1/efg1) and JKC19 (cph1/cph1); clinical strain B1134; Sap 1–3 and Sap 4–6 mutants of SC5314. The C. glabrata strains used were ATCC 90030, and the clinical strains 5WT and G212. Results: The sonicated yeast cells of C. albicans JKC19 and SC5314, both in hyphal form, degraded E‐Cad at pH 4. The 10× concentrated growth media of the strains HLC‐52, HLC‐54, 32723 and B1134; all in yeast form, caused degradation at pH 4, HLC‐52 and HLC‐54 also at pH 6. The C. glabrata strains did not degrade E‐Cad. Conclusions: pH is a strain dependent triggering factor in activating yeast or hyphal form related Candida Saps in degrading epithelial cell associated E‐Cads.     

Correlation between enzyme production,germ tube formation and susceptibility to fluconazole in Candida species isolated from patients with denture‐related stomatitis and control individuals

Introduction: Phospholipase and proteinase secretion in yeasts of the genus Candida has been described as a relevant virulence factor. Also, germ tube formation by Candida albicans is associated with its invasive capacity and is considered an important pathogenic mechanism. Methods: To link the production of hydrolytic enzymes with the capacity to produce infection, 232 clinical isolates of yeasts from the oral cavity of 140 individuals wearing removable maxillary protheses were studied. The sample was composed of 70 patients with denture‐related stomatitis (DRS) and 70 individuals with normal palatal mucosa. For strains identified as C. albicans, the correlation between germ tube formation and their capacity to cause infection was studied and the presence of Candida dubliniensis was investigated. Susceptibility to fluconazole was evaluated. Results: Candida albicans was the only species producing phospholipase and germ tube. We observed a higher level of production of phospholipase in cases of infection compared with commensals. Significant differences between the two groups of C. albicans isolates were observed as to germ tube production. Only, Candida glabrata showed lower susceptibility to fluconazole. Conclusion: The results reinforced the idea that C. albicans is the most frequent and can be the most pathogenic yeast in oral candidosis. However, the strains isolated from DRS patients and healthy individuals showed the same virulence factors. It seems that several virulence attributes are involved in the infective process but no single factor contributes to Candida virulence. Candida dubliniensis was absent in the oral cavity of individuals with and without DRS.     

Inhibitory effect of alpha-mangostin on <Emphasis Type="Italic">Candida</Emphasis> biofilms

The objective of this study was to determine the inhibitory effect of alpha-mangostin on Candida biofilms. Candida species including Candida albicans, Candida krusei, Candida tropicalis, and Candida glabrata were tested. Candida biofilms were formed in flat-bottomed 96-well microtiter plates. The metabolic activity of cells within biofilms was quantified using the XTT assay. The results demonstrated that alpha-mangostin showed a significant anti-biofilm effect on both developing biofilms and preformed biofilms of Candida species. It may be concluded that alpha-mangostin could be an anti-biofilm agent against Candida species. Further in vivo investigations are needed to uncover the therapeutic values of this medicinal plant.     

Correction to: Synergistic effect of thymoquinone and nystatin in the treatment of oral candidiasis; an in vitro study

The effectiveness of antifungal agents may be insufficient against resistant strains in some cases of oral candidiasis. The aim of this study was to evaluate the antifungal effect of thymoquinone against Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei strains and the synergistic antifungal activity of these strains in combination with nystatin. To evaluate in vitro antifungal activity and interactions between thymoquinone and nystatin, substances were tested against Candida albicans ATCC 10,231, C. tropicalis ATCC 750, C.krusei ATCC 6258 and C. glabrata ATCC 2001 standard strains both individually and combinationally via microdilution method. MIC and ΣFIC index value were analysed. The Kruskal Wallis test and Bonferroni test were used for statistical evaluations. Statistical significance was set at p < 0.05. A statistically significant difference was observed between the mean ranks of all Candida species and doses of thymoquinone, nystatin, and the combination thymoquinone-nystatin (p < 0.05). MIC values for thymoquinone were determined as 15 μg/mL for C. albicans, C. tropicalis and C. krusei while it was 30 μg/mL for C. glabrata. Moreover, MIC for nystatin was found as 1.875 μg/mL for C. albicans, C. tropicalis and C. krusei, whereas it was 7.5 μg/mL in C. glabrata. Interaction assays and ΣFIC index value revealed that, TQ and nystatin have a synergistic effect against to all strains. Thymoquinone was found to have antifungal activity on Candida species and synergistic effect when combined with nystatin.

     

Prevalence and antifungal susceptibility profiles of Candida glabrata,Candida parapsilosis and their close-related species in oral candidiasis

ObjectiveTo evaluate the importance of Candida glabrata, Candida parapsilosis and their close-related species, Candida bracarensis, Candida nivariensis, Candida metapsilosis and Candida orthopsilosis in patients with oral candidiasis and, to determine the in vitro activities of antifungal drugs currently used for the treatment.MethodsOne hundred fourteen isolates of C. glabrata and 97 of C. parapsilosis, previously identified by conventional mycological methods, were analysed by molecular techniques. In vitro antifungal susceptibility to fluconazole, itraconazole, miconazole, and nystatin was evaluated by CLSI M44-A2 disk diffusion test, and by CLSI M27-A3 microdilution for fluconazole.ResultsAll C. glabrata isolates were identified as C. glabrata sensu stricto, 93 out of 97 C. parapsilosis isolates as C. parapsilosis sensu stricto, three as C. orthopsilosis and one as C. metapsilosis. Candida glabrata was mainly isolated in mixed cultures but C. parapsilosis complex was more frequent in pure culture. Candida metapsilosis and C. orthopsilosis were isolated as pure culture and both species were susceptible to all antifungal agents tested. Most C. glabrata isolates were susceptible to miconazole and nystatin, but resistant to fluconazole and itraconazole. Azole cross resistance was also observed. Candida parapsilosis isolates were susceptible to fluconazole although azole cross resistance to miconazole and itraconazole was observed.ConclusionThis study highlights the importance of accurate identification and antifungal susceptibility testing of oral Candida isolates in order to have an in-depth understanding of the role of C. glabrata and C. parapsilosis in oral candidiasis.     

Isolation of Candida dubliniensis in denture stomatitis

Objective

To describe the isolation of Candida dubliniensis from a patient with denture stomatitis and to compare with the presence of yeasts in the oral cavities of denture wearers.

Design

One hundred and fifty-two Candida isolates were recovered through oral swabs from denture as well as the underlying mucosa from 100 patients wearing denture. For detection and identification of fungal isolates, standard phenotypic and genotypic methods were used.

Results

Forty-five of 100 denture wearers suffered from denture stomatitis. Seventy-three Candida isolates were recovered from 38 denture wearers without denture stomatitis. In this group, Candida albicans was the predominant species (58.9%), followed by Candida tropicalis (15.1%), Candida guilliermondii (13.7%), Candida glabrata (9.6%), and Candida parapsilosis (2.7%).Seventy-nine isolates were yielded from 40 patients suffering from denture stomatitis. C. albicans was also the most frequently isolated species (58 isolates, 73.4%), followed by C. glabrata and C. tropicalis (7 isolates each, 8.9%), and Saccharomyces cerevisiae (2 isolates, 2.5%). One isolate was yielded of the following species: Candida famata, Candida krusei, C. parapsilosis and C. guilliermondii. Moreover 1 isolate was phenotypic and genotypic identified as C. dubliniensis genotype 1.

Conclusions

C. albicans is the predominant fungal species isolated from denture wearers. C. dubliniensis could be isolated from adults with denture stomatitis.     

Diversity,frequency and antifungal resistance of Candida species in patients with type 2 diabetes mellitus

Objective: To determine number, species of Candida and Candida resistance to antifungal therapy according to the metabolic control state and the associated salivary changes in patients with type 2 diabetes mellitus (DM2).

Materials and methods: Samples of non-stimulated saliva were collected from 52 patients with DM2. Salivary pH was measured and cultured on Sabouraud glucose agar and the values of CFU/ml were calculated. The species were presumptively identified using CHROMagar Candida^® plates, and identification was confirmed by polymerase chain reaction (PCR). C. albicans isolates were cultured on SGA tetracycline agar with nystatin and fluconazole diffusion disks to measure susceptibility.

Results: Sixty six percent of the yeasts isolated were Candida albicans, followed by C. glabrata (20.7%). In patients with decompensated DM2, there was an inverse association between HbA1c value and salivary pH. At higher levels of salivary acidification, a greater diversity and quantity of yeasts of the genus Candida were observed. With nystatin, higher inhibition was observed at lower pH.

Conclusions: The antifungal therapies could be more effective if it consider, qualitative salivary characteristics as pH, that could determine the susceptibility of species of Candida to at least to nystatin, which is the most used antifungal for treatment to oral candidiasis in patients with DM2.     

The isolation,identification and molecular analysis of Candida spp. isolated from the oral cavities of patients with diabetes mellitus

Previous studies have shown a high incidence (77%) of isolation of Candida spp. from the oral cavities of patients with type 1 diabetes mellitus. The aim of the present study was to assess the prevalence of yeast in the oral cavities of patients suffering from type 1 and type 2 diabetes mellitus. The patients were classified according to the level of diabetic control (HbA1_c), and further stratified on the presence or absence of dental prosthesis. Oral rinse samples were assessed for the growth of yeast and the degree of colonization. Oral isolates were defined to the species level by both phenotypic and novel molecular methods. The overall proportion (60%) of diabetic patients who had Candida spp. isolated from the oral cavity was similar to that previously reported. Local oral factors, such as the presence of dentures, seemed to have a greater influence than diabetic status on the amount and species of Candida isolated from the oral cavities of diabetic patients. Diabetic patients with dentures had more non‐albicans Candida isolated from their mouths than dentate diabetic patients. Candida dubliniensis was isolated from diabetic patients and may have a predilection for dentate patients.     

Oral epithelium–Candida glabrata interactions in vitro

Background: Oropharyngeal candidiasis is a common opportunistic infection and Candida glabrata is the second or third most frequently isolated species from oropharyngeal candidiasis lesions, after Candida albicans. The aim of this study was to study the cytokine‐inducing and cell‐damaging potential of C. glabrata in oral epithelial cells and compare this to C. albicans. Methods: Oral epithelial cell lines and primary gingival epithelial cells were cocultured with C. glabrata strains GDH2269 and 94‐11 or C. albicans strains SC5314 and ATCC28366. Supernatants were analysed for the presence of interleukin‐1α (IL‐1α), IL‐8 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) by enzyme‐linked immunosorbent assay. The cytotoxity of different strains was determined using the CytoTox‐96 assay. Results: Compared to C. albicans, C. glabrata induced different proinflammatory cytokine responses in oral epithelial cells; a high level of GM‐CSF induction was only detected in C. glabrata‐infected cells and not in C. albicans‐infected cells, regardless of the origin of these cells (cell lines or primary cells) or the strain used. Like C. albicans, C. glabrata induced an IL‐1α response by oral epithelial cells, but this response was both strain‐dependent and epithelial cell origin‐dependent. Unlike C. albicans, C. glabrata failed to induce a strong IL‐8 response in any of the cell systems studied. Finally, in these studies C. glabrata showed lower cytotoxicity than C. albicans. Conclusions: C. glabrata is less cytotoxic than C. albicans and induces different proinflammatory cytokine responses in oral epithelial cells.     

Identification of Candida species in the clinical laboratory: a review of conventional,commercial, and molecular techniques

In healthy individuals, Candida species are considered commensal yeasts of the oral cavity. However, these microorganisms can also act as opportunist pathogens, particularly the so‐called non‐albicans Candida species that are increasingly recognized as important agents of human infection. Several surveys have documented increased rates of C. glabrata, C. tropicalis, C. guilliermondii, C. dubliniensis, C. parapsilosis, and C. krusei in local and systemic fungal infections. Some of these species are resistant to antifungal agents. Consequently, rapid and correct identification of species can play an important role in the management of candidiasis. Conventional methods for identification of Candida species are based on morphological and physiological attributes. However, accurate identification of all isolates from clinical samples is often complex and time‐consuming. Hence, several manual and automated rapid commercial systems for identifying these organisms have been developed, some of which may have significant sensitivity issues. To overcome these limitations, newer molecular typing techniques have been developed that allow accurate and rapid identification of Candida species. This study reviewed the current state of identification methods for yeasts, particularly Candida species.     

Efficacy of denture cleansers on denture liners contaminated with Candida species

As poor denture hygiene is related to Candida colonisation, disinfectant solutions have been proposed as an effective method of preventing denture stomatitis. This study assessed the efficacy of denture cleansers on Candida albicans and Candida glabrata adherence on denture liners. Another aim was to correlate materials’ surface roughness (Ra) to Candida adherence. Specimens of three denture liners (soft and hard polymethyl methacrylate (PMMA)-based and soft silicone-based) were prepared and had their Ra measured. Specimens were randomly divided to adherence assays with C. albicans or C. glabrata. After contamination with the fungi, specimens were treated with an enzymatic cleanser solution, a cleanser solution or a 0.5% NaOCl solution by soaking for 3, 15 or 10 min, respectively. Control group specimens were soaked in distilled water for 15 min. Number of remaining Candida cells after treatment was determined by light microscopy (×400). Analysis of variance (α = 0.05) showed that Ra of the silicone-based liner was lower than that of the PMMA-based liners (p < 0.05). The overall results showed high C. glabrata adherence (p < 0.001), while the lowest levels of remaining Candida cells were found for the treatment with 0.5% NaOCl (p = 0.0019). No difference among denture cleansers and control was found (p = 0.19). There was no correlation between Ra and C. albicans or C. glabrata adherence in all materials tested. The only treatment able to reduce both Candida species adherence on all materials tested was 0.5% NaOCl solution.     

Identification of Candida dubliniensis among oral yeast isolates from an Italian population of human immunodeficiency virus‐infected (HIV+) subjects

Candida dubliniensis, an emerging oral pathogen, phenotypically resembles Candida albicans so closely that it is easily misidentified as such. The aim of the present study was to evaluate the usefulness of two phenotypic methods, growth at 45°C and 2,3,5‐triphenyltetrazolium chloride (TTC) reduction, for confirming presumptive identification of C. dubliniensis and C. albicans by colony color on CHROMagar Candida (CAC) medium. A combination of these methods was used to establish the prevalence of oral C. dubliniensis in an Italian population of 45 human immunodeficiency virus (HIV)‐infected subjects. Twenty‐two samples (48.9%) were positive for yeasts on CAC medium producing a total of 37 fungal isolates. The colony color and 45°C growth ability test correctly identified all C. dubliniensis and C. albicans isolates (5/37, 13.5%, and 16/37, 43.2%, respectively), while assessment of TTC reduction misidentified one C. albicans isolate. The isolation rate of C. dubliniensis was 11.1% (5/45 patients). All of the C. dubliniensis isolates were highly susceptible to fluconazole (MIC = 0.5 µg/ml). The combination of CAC medium screening with growth at 45°C and TTC reduction tests may represent a simple, reliable and inexpensive identification protocol for C. dubliniensis.     

Oral Candida carriage,quantification, and species characterization in oral submucous fibrosis patients and healthy individuals

Aim: To determine the prevalence of oral Candida carriage, candidal quantification, and various subtypes of Candida species in oral submucous fibrosis patients and healthy individuals. Methods: The study comprised 30 clinically‐diagnosed and ‐staged oral submucous fibrosis patients aged 20–40 years, and 20 age‐ and sex‐matched controls. Buccal mucosa was sampled by sterile swab technique. Each sample was inoculated on Sabouraud’s dextrose agar and CHROMagar culture media. Candida species identification was done using the KB006 Candida identification kit. Results: Eleven (36.67%) cases in the study group, and two (10%) cases in the control group, yielded Candida on culture. The value of CFU/mL increased with an increased duration of betel quid chewing habit. All Candida‐positive oral submucous fibrosis patients complained of a burning sensation. Candida albicans and Candida tropicalis were the most common species in the oral submucous fibrosis cases. Candida dubliniensis was isolated in both the study and control groups. Conclusions: Our observations in this study affirm that oral submucous fibrosis favors the colonization of Candida. Mucosal alterations due to the underlying disease process or betel quid chewing, coupled with other factors, might lead to candidal colonization, even in the absence of clinically‐related mycotic manifestations.     

In vitro activity of a monoclonal killer anti‐idiotypic antibody and a synthetic killer peptide against oral isolates of Candida spp. differently susceptible to conventional antifungals

Background/aims: A monoclonal killer anti‐idiotypic antibody (mAbK10) and a synthetic killer peptide, acting as internal images of a microbicidal, wide‐spectrum yeast killer toxin (KT) have been recently shown to express candidacidal in vitro and an in vivo therapeutic activity against experimental mucosal and systemic candidosis models caused by a reference strain of Candida albicans (10S). Material and methods: The in vitro candidacidal activity of mAbK10 and synthetic killer peptide was compared using a colony forming unit assay against a large number of isolates of different Candida spp., obtained from oral saliva of adult diabetic (type 1 and 2) and nondiabetic subjects from Parma (Italy) and London (UK). Results: Both the KT‐mimics exerted a strong dose‐dependent candidacidal activity, probably mediated by the interaction with β‐glucan KT receptors on target yeast cells, against all the tested strains, regardless of their species and pattern of resistance to conventional antifungal agents. Conclusions: These observations open new perspectives in the design and production of candidacidal compounds whose mechanism reflects that exerted in nature by killer yeasts.     

Extracellular proteinases of Candida species pathogenic yeasts

The increased incidence of severe disseminated infections caused by the opportunistic yeast‐like fungi Candida spp. highlights the urgent need for research into the major virulence factors of these pathogens—extracellular aspartic proteinases of the candidapepsin and yapsin families. Classically, these enzymes were considered to be generally destructive factors that damage host tissues and provide nutrients for pathogen propagation. However, in recent decades, novel and more specific functions have been suggested for extracellular candidal proteinases. These include contributions to cell wall maintenance and remodeling, the formation of polymicrobial biofilms, adhesion to external protective barriers of the host, the deregulation of host proteolytic cascades (such as the complement system, blood coagulation and the kallikrein–kinin system), a dysregulated host proteinase–inhibitor balance, the inactivation of host antimicrobial peptides, evasion of immune responses and the induction of inflammatory mediator release from host cells. Only a few of these activities recognized in Candida albicans candidapepsins have been also confirmed in other Candida species, and characterization of Candida glabrata yapsins remains limited.     

Oral environmental factors affecting number of microbes in saliva of complete denture wearers

Summary The purpose of this study was to clarify which oral environmental factors affected number of microbes in saliva in an edentulous environment. We enrolled 68 edentulous subjects in the study. Numbers of total anaerobic bacteria and Candida species in saliva were determined. Age, sex, un‐stimulated salivary flow rate, pH and viscosity of saliva, histatin level in saliva, tongue coating status, tongue pressure, denture plaque status, material of denture base, duration of edentulism, frequency of self oral health care and number of cigarettes per day were also investigated as oral environmental factors. Correlation between number of total anaerobic bacteria or Candida species and each oral environmental factor was determined with the Spearman rank correlation coefficient. Stepwise logistic regression analysis was used to identify which factors were significantly associated with level of total anaerobic bacteria and Candida species. Correlation and stepwise logistic regression analyses revealed associations between un‐stimulated salivary flow rate, tongue coating status, denture plaque status or frequency of self oral health care and number of total anaerobic bacteria. The correlation analysis showed a significant correlation between age and number of total anaerobic bacteria. Stepwise logistic analysis revealed associations between pH of saliva or viscosity of saliva and level of anaerobic bacteria; it also revealed associations between histatin level in saliva or un‐stimulated salivary flow rate and level of Candida species. We conclude that salivary flow rate, in particular, affects number of salivary microbes in an edentulous environment.