Multilocus sequence typing and phenotypic properties of Streptococcus mutans from Thai children with different caries statuses.

BACKGROUND: Streptococcus mutans is studied for its acidogenic and aciduric characteristics, notably its biofilm formation in the presence of sucrose, toward its role in the caries process. Variations in both genotype and phenotype have been reported among clinical isolates of S. mutans. This study aimed to examine genotypic and phenotypic characteristics of S. mutans obtained from Thai children with varying caries statuses. METHODS: We determined the presence of S. mutans and caries status in 395 children aged 3-4 years. From 325 children carrying S. mutans, we selected 90 with different caries statuses-caries-free (CF; n = 30), low severity of caries (LC; n = 30), or high severity of caries (HC; n = 30). Three isolates of S. mutans were taken from each child, thus, a total of 270 isolates were obtained. Multilocus sequence typing (MLST) was used to genotype the isolates and assess their clonal relationships. The properties, including biofilm formation, collagen binding, and acid production and tolerance were also evaluated. RESULTS: Children with carious lesions showed a higher detection rate and number of S. mutans in saliva than those without caries. S. mutans from individuals with HC status showed the lowest biofilm formation ability, while this group had the highest detection rate of collagen-binding isolates. There was no difference in acid production or tolerance by caries status. Genotyping by MLST did not reveal any clone of S. mutans specific to CF status. This result remained even when we included MLST data from the open-access PubMLST database. MLST did identify clones containing only strains from caries-affected hosts, but tests of their phenotypic properties did not reveal any differences between S. mutans from these clones and clones that were from both caries-free and caries-affected children. CONCLUSIONS: The clonal relationships of S. mutans indicated by MLST were not associated with the status of dental caries in the host.

Effects of two novel denture cleansers on multispecies microbial biofilms, stain removal and the denture surface: an in vitro study.

BACKGROUND: The continuously increasing demand for removable denture appliances and the importance of adequate denture cleaning have led to the development of various denture cleansing products. The aim of this study was to evaluate the efficacy of two novel denture cleansing agents (GE and TM) and three commonly available cleansers (0.5% sodium hypochlorite; NaClO, 0.12% chlorhexidine gluconate; CHX, and Polident®; POL) on multispecies microbial biofilm formation, stain removal and physical properties of dentures. METHODS: The antimicrobial activities of denture cleansing agents were determined against major oral opportunistic pathogens including Streptococcus mutans, Staphylococcus aureus, Escherichia coli and Candida albicans, using time-kill assays. Multispecies microbial biofilms grown on acrylic resins for 72 h were generated to determine the antibiofilm effects of cleansing agents by confocal laser scanning microscopy (CLSM). Evaluations of the tea and coffee stain removal properties and the alterations in the physical properties of dentures were also performed. The toxicity of cleanser residues released from denture acrylics to fibroblast cells was investigated using MTT assay. RESULTS: All denture cleansing agents tested could effectively kill oral bacteria and Candida albicans. Furthermore, after immersion for more than 3 h, the cleansers Polident®, GE and TM could efficiently penetrate and inhibit multispecies denture biofilms with effects similar to 10 min of immersion in 0.5% NaClO. However, immersion in 0.12% CHX for 20 min showed less antibiofilm activity. The NaClO solution had the highest efficacy for removing stains from the artificial teeth. Conversely, the CHX solution enhanced tea and coffee staining, and the teeth immersed in this solution showed clinically unacceptable colour changes (ΔE > 5.5). However, the colour differences of teeth stained and immersed in POL, GE and TM cleansers were in the clinically acceptable range. There was no significant difference among the POL, GE and TM cleansers in terms of stain removal efficacy. The cleansers GE and TM did not alter the surface roughness and colour of the materials, moreover the residues of both cleansers did not exhibit cytotoxicity. CONCLUSION: Two novel denture cleansing agents containing natural products, GE and TM exhibited effective antimicrobial activity, antibiofilm and stain removal capabilities without toxicity or disturbance of the physical properties of acrylics.

Novel vinegar solution for denture-cleansing agent.

PURPOSE: To study the antimicrobial effects of a novel vinegar-based denture cleansing agent on oral Streptococci and Candida species and the inhibitory effects on pre-formed bacterial and Candida biofilms on denture base. METHODS: The microorganisms used in this study were Streptococcus mutans (S. mutans), Streptococcus sobrinus (S. sobrinus), Streptococcus sanguinis (S. sanguinis), Candida albicans (C. albicans), and Candida glabrata (C. glabrata). The antimicrobial activity of novel vinegar solution was evaluated by time kill assay and biofilm grown on denture base. RESULTS: Time kill assay showed that vinegar exhibited the highest antibacterial effect on S. sobrinus, S. sanguinis, and S. mutans after 15 min of treatment. A 99.9% reduction in C. glabrata and C. albicans required more than 4 and 6 h of treatment, respectively. Vinegar significantly inhibited streptococcal biofilm, with an approximately 6 log-reduction at 30 min of treatment. The results demonstrated that viable Candida cells in biofilm reduced in excess of 6-log CFU/mL after 3 h treatment with vinegar. Moreover, the vinegar-based denture cleanser inhibited bacterial and Candida biofilm formation compared to the control group without treatment with statistical significance. CONCLUSION: A novel vinegar-based denture cleansing agent showed moderate antibacterial properties, but required a slightly longer immersion time to attain anticandidal effects compared to Polident and 0.2% CHX.

Geraniol and thymoquinone inhibit Candida spp. biofilm formation on acrylic denture resin without affecting surface roughness or color.

PURPOSE: This study was designed to investigate the in vitro effects of geraniol (GE) and thymoquinone (TQ) on Candida biofilms on denture acrylic and any accompanying changes in acrylic surface roughness or color. METHODS: The susceptibility of Candida species to GE and TQ was determined using the broth microdilution method and time-kill assay. A minimum biofilm eradication concentration (MBEC) assay was performed using 7-day Candida biofilms grown on denture acrylic. RESULTS: The minimum inhibitory concentration (MIC) of GE and TQ for Candida spp. was 256 and 32 µg/mL, respectively. The Candida strain complete kill rates for GE and TQ at 5-fold MIC were determined after 1 h of incubation. At 5-fold MIC, GE and TQ inhibited the preformed biofilm activity (MBEC80) of all Candida strains on denture acrylic by more than 80% after treatment for 3 h. At sub-MIC levels, GE and TQ prevented the development of C. albicans and C. tropicalis hyphae. SEM images demonstrated that GE and TQ damaged the fungal cell membrane and induced cell lysis. On the other hand, GE and TQ at 10-fold MIC did not alter the surface roughness or color of the denture acrylic. CONCLUSION: GE and TQ are interesting natural substances that could be developed as promising disinfectants for removable dentures.

Missense mutation in CgPDR1 regulator associated with azole-resistant Candida glabrata recovered from Thai oral candidiasis patients.

OBJECTIVES: Non-albicans Candida (NAC) species are increasingly identified as pathogens causing oral candidiasis. Incidence rates for azole resistance among NAC species have been continuously reported. This study aimed to evaluate the azole susceptibility profiles and to characterise the azole resistance mechanisms of oral clinical NAC isolates. METHODS: In vitro susceptibility patterns of 85 NAC species isolates were determined by the broth microdilution method. Azole resistance-related genes (ERG3, ERG11 and PDR1) of Candida glabrata isolates were sequenced to determine the presence of nucleotide substitutions. Expression levels of various resistance-related genes were also evaluated by RT-qPCR in azole-susceptible, susceptible dose-dependent (SDD) and resistant Candida isolates. RESULTS: Two C. glabrata isolates (2.4% of all NAC isolates) were resistant to all three azoles tested (fluconazole, itraconazole and ketoconazole). All clinical isolates of Candida tropicalis and Candida kefyr were susceptible to azoles. Silent mutations were found in the CgERG11 and CgERG3 genes of clinical C. glabrata isolates. Interestingly, two missense mutations in CgPDR1 (N768D and E818K) were identified only in resistant C. glabrata isolates. The presence of a CgPDR1 missense mutation in resistant isolates is associated with overexpression of its own product as well as multidrug transporters including ABC and MFS transporters. CONCLUSION: A gain-of-function (GOF) mutation in CgPDR1 is associated with upregulation of various drug transporters, which appears to serve as a primary mechanism for azole resistance in the detected C. glabrata isolates. Therefore, analysis of GOF mutations in the PDR1 regulator provides a better understanding of the development of antifungal resistance.