Anti-oral streptococci and anti-biofilm properties of Etlingera pavieana essential oil and its bioactive compounds proposed for an alternative herbal mouthwash.

Oral streptococci are the major group of bacteria in the oral cavity. Some of their species cause oral diseases that may lead to tooth loss and quality-of-life reduction, such as dental caries. One of prevention techniques to promote oral health is rinsing mouthwash after toothbrushing. This study aimed to determine the potential uses of local food, also remedy, plant in Thailand called Reaw-Horm or Etlingera pavieana for alternative herbal mouthwash. The essential oil from E. pavieana rhizome (Eo) is used for anti-streptococci including Streptococcus mutans and Streptococcus sobrinus and anti-biofilm activities. The main components of Eo are methyl chavicol (MC) and trans-anethole (TA). The disk diffusion method showed the inhibition zone of Eo in a dose-dependent manner. The minimum inhibitory concentration (MIC) of Eo and TA was >1.6 % v/v, and 0.4 % v/v of MC. Regarding anti-biofilm activities, MC showed nearly equal anti-biofilm formation of S. mutans and S. sobrinus, whereas Eo and TA acted toward S. sobrinus more than S. mutans biofilm. Sub-MIC killing effects on cells under biofilm were observed in Eo and MC. Therefore, MC was recommended as an active compound for anti-streptococci activities. Biocompatibility of Eo and MC were shown to be safe for epidermal cell lines. Herbal mouthwashes containing Eo were developed and had antioxidant and antimicrobial actions with established for 3 months. This study provides in vitro support on the use of herbal mouthwash with antioxidant and antimicrobial activities for dental caries prevention and well-being of individuals.

Evaluation of Streptococcus mutans Biofilm Formation and Acidogenicity of Infant Milk Formulas for Treating Cow Milk Allergy: An in vitro Study.

INTRODUCTION: When infants cannot consume breast milk, the most commonly available alternative milk formula is cow milk-based. Due to a rise in the prevalence of cow milk protein allergy (CMPA) among children, this study aimed to assess the biofilm formation and acidogenicity of cow milk-based formulas as well as milk formulas suggested for children with CMPA. METHODS: Cow milk-based formulas with 0%, 10%, or 18% sucrose added, partially hydrolyzed formula (pHF), extensively hydrolyzed formula (eHF), amino acid-based formula (AAF), and soy-based formulas with 0%, or 11% sucrose added were evaluated. Streptococcus mutans was used as a representative microorganism associated with caries. The acidogenicity after 24-h incubation was assessed by the pH of the formed biofilm and lactic acid formation. Biofilm formation was quantified using crystal violet staining. Additionally, the biofilm characteristics were determined using confocal laser scanning microscopy (CLSM). Comparisons were made among formulas without added sucrose to observe protein-based differences. Furthermore, formulas with different sucrose percentages were compared to explore the impact of sucrose content. RESULTS: When comparing the formulas without added sucrose, the biofilm formation in the cow milk-based formula and pHF were significantly greater than the soy-based formula, eHF, and AAF. In the presence of S. mutans, all formulas reduced the biofilm pH below the critical enamel pH. The cow milk-based formula and AAF showed a significantly lower biofilm pH than the pHF, soy-based, and eHF groups, while the lactic acid production was markedly higher in the cow milk-based formula, pHF and AAF, compared with the eHF and soy-based formula. Adding sucrose into the cow milk-based and soy-based formulas substantially increased biofilm mass. The biofilm pH of the cow milk-based formulas, with or without sucrose, was significantly lower than that of the soy-based formulas. The CLSM indicated distinct biofilm characteristics among the different protein-based formulas, with sucrose supplementation promoting S. mutans aggregation in cow milk-based formula biofilm and increased density and intact biofilm in the soy-based formula. CONCLUSION: All assessed milk formulas had caries-inducing factors, including those without supplemental sucrose. Among them, the eHF demonstrated the least caries-inducing factors, attributed to its minimal biofilm formation and the highest biofilm pH.

Oral Lactobacilli Related to Caries Status of Children with Primary Dentition.

Oral lactobacilli are members of a group of bacteria implicated in caries progression, although information regarding their transmission, colonization, and caries-associated species is not well established. This study isolated oral lactobacilli from a group of children with primary dentition for determination of Lactobacillus prevalence, detection of Streptococcus mutans, a major pathogen of caries initiation, and dental caries status of the children. Species of Lactobacillus isolates were determined from examination of 16S rDNA sequences. Subsequently, the most prevalent species was evaluated for involvement in caries status, and binding ability to type I collagen of all Lactobacillus isolates was determined in association with caries status. Multilocus sequence typing (MLST) of eleven loci was carried out to study strains of the predominant Lactobacillus sp. The detection of oral lactobacilli together with S. mutans was significantly associated with the highest dental caries indices, but there was no involvement of collagen-binding properties of Lactobacillus isolates in caries status. Lactobacillus fermentum was the most prevalent, and its presence was related to high scores of caries indices. MLST analysis of L. fermentum population could not specify a particular clone associated with caries status, but revealed sharing of identical L. fermentum strains among children in the same classrooms. Taken together, the data contributed useful information on the role of oral lactobacilli, in particular L. fermentum in dental caries.

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.

Genotyping of Candida albicans and Candida dubliniensis by 25S rDNA analysis shows association with virulence attributes in oral candidiasis.

OBJECTIVES: This study genotyped oral isolates of Candida albicans and C. dubliniensis by analyzing 25S rDNA transposable intron and evaluated their virulence attributes in oral candidiasis. DESIGN: C. albicans and C. dubliniensis were isolated from oral cavity of normal carriers (n = 100) and oral candidiasis patients (n = 100), genotyped by PCR, and virulence properties, namely, secreted phospholipase and proteinase activities (using an agar plate method) and binding to buccal epithelial cells, were determined. In addition, antifungal sensitivity was assayed for all Candida isolates. RESULTS: C. albicans genotypes A, B, C and D (C. dubliniensis) were identified. Genotype B was the most prevalent in both healthy and candidiasis groups and had highest buccal epithelial cell binding ability but lowest secreted phospholipase activity. Genotype C was the third most prevalent, with higher frequency in patients than normal carriers. Genotype A, the second most prevalent, was equally found in both groups. There were no significant differences in secreted proteinase activity among the three C. albicans genotypes. C. dubliniensis, the least prevalent, was more frequent in healthy carriers and demonstrated minimal levels of the virulence properties. When all Candida isolates were compared based on groups of subjects, only secreted phospholipase activity was significantly higher in isolates from candidiasis patients. All Candida isolates were susceptible to clotrimazole, fluconazole, miconazole and nystatin. CONCLUSIONS: Genotyping based on the 25S rDNA transposable intron region provided a simple method allowing studies of the pathogenicity of each genotype.

Cariogenic properties of Streptococcus mutans clinical isolates with sortase defects.

OBJECTIVE: In Streptococcus mutans, a Gram-positive pathogen of dental caries, several surface proteins are anchored by the activity of sortase enzyme. Although various reports have shown that constructed S. mutans mutants deficient of sortase as well as laboratory reference strains with a sortase gene mutation have low cariogenic potential, no known studies have investigated clinical isolates with sortase defects. Here, we examined the cariogenic properties of S. mutans clinical isolates with sortase defects as well as caries status in humans harboring such defective isolates. DESIGN: Sortase-defective clinical isolates were evaluated for biofilm formation, sucrose-dependent adhesion, stress-induced dextran-dependent aggregation, acid production, and acid tolerance. Additionally, caries indices of subjects possessing such defective isolates were determined. RESULTS: Our in vitro results indicated that biofilm with a lower quantity was formed by sortase-defective as compared to non-defective isolates. Moreover, impairments of sucrose-dependent adhesion and stress-induced dextran-dependent aggregation were found among the isolates with defects, whereas no alterations were seen in regard to acid production or tolerance. Furthermore, glucan-binding protein C, a surface protein anchored by sortase activity, was predominantly detected in culture supernatants of all sortase-defective S. mutans isolates. Although the sortase-defective isolates showed lower cariogenic potential because of a reduction in some cariogenic properties, deft/DMFT indices revealed that all subjects harboring those isolates had caries experience. CONCLUSIONS: Our findings suggest the impairment of cariogenic properties in S. mutans clinical isolates with sortase defects, though the detection of these defective isolates seemed not to imply low caries risk in the subjects harboring them.

Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates.

Mother-to-child transmission of mutans streptococci.

Mutans streptococci (MS) are the major group of pathogens implicated in dental caries. Like other infectious diseases, transmission of the causative microorganisms is the initial and essential step that should be understood relative to disease control and prevention. This review summarizes current knowledge regarding MS transmission, especially from mothers to their children. Included are methods used to study transmission, sources of MS, initial acquisition, factors concerning transmission and prevention of transmission. Information accumulated over many decades showed the involvement of MS transmission in the pathogenesis of caries, hence several preventive measurements have been proposed. Nevertheless, some essential aspects remain to be elucidated for more benefits of practical application.

Multilocus sequence typing of Streptococcus mutans strains with the cbm gene encoding a novel collagen-binding protein.

OBJECTIVE: Streptococcus mutans, an oral pathogen associated with infective endocarditis (IE), possesses two genes encoding collagen-binding proteins, namely cnm and cbm. In this study, we used multilocus sequence typing (MLST) of S. mutans with the cbm gene. DESIGN: Forty-five S. mutans strains including 15 strains with the cnm gene, 15 strains with the cbm gene, and 15 strains without these two genes were analysed by MLST. In addition, the collagen-binding properties as well as the abilities to adhere to and invade human umbilical vein endothelial cells (HUVEC) were also evaluated for all strains. RESULTS: In the groups of cnm-positive and cbm-positive strains, all properties, including collagen binding, adhesion, and invasion were significantly greater than those of the cnm-cbm-negative group. Moreover, MLST revealed three clonal complexes of S. mutans possessing the cbm gene. These three clones showed no close relatedness with clones of strains containing the cnm gene. Among three clones harbouring the cbm gene, two clones belong to serotype k, and appeared to be associated with the pathogenesis of IE due to their strong collagen binding and relatively enhanced abilities to adhere to and invade endothelial cells. However, such properties were relatively weak in the other non-serotype k clone possessing the cbm gene. CONCLUSIONS: MLST indicated a difference in evolution between S. mutans strains with the cbm gene and those with the cnm gene. In addition, this technique also suggested the importance of cbm-positive S. mutans clones relative to the pathogenesis of IE.

Multilocus sequence typing analysis of Streptococcus mutans strains with the cnm gene encoding collagen-binding adhesin.

Streptococcus mutans is one of the oral pathogens associated with infective endocarditis (IE). With respect to bacterial binding ability to the extracellular matrix, the Cnm protein, a cell surface collagen-binding adhesin of S. mutans, is known as one of the possible virulence factors with regard to IE. In this study, we aimed to determine the distribution of the cnm gene, which encodes Cnm, in a large number of clinical isolates of S. mutans from Thai subjects. Then, the cnm-positive strains were classified using a multilocus sequence typing (MLST) scheme, which we constructed previously. In addition, the data were analysed together with our previous MLST data of cnm-positive strains from Japan and Finland in order to evaluate the clonal relationship among S. mutans strains harbouring the cnm gene. The cnm gene was detected in 12.4 % of all 750 Thai isolates, and serotype f showed the highest rate of detection (54.5 %). According to the MLST data, two clonal complex groups were revealed as the important clones related to cnm-positive S. mutans from various origins of isolation. Moreover, the collagen-binding properties of S. mutans strains with the cnm gene were significantly greater than those of strains without the gene, although four cnm-negative strains classified into two sequence types (STs), ST110 and ST136, showed extremely high collagen-binding rates suggesting the presence of additional genes involved with collagen binding in these STs. Taken together, these results provided information on both epidemiological as well as evolutional aspects of S. mutans possessing the cnm gene.

Molecular and clinical analyses of the gene encoding the collagen-binding adhesin of Streptococcus mutans.

Streptococcus mutans is a known pathogen of dental caries and its major cell surface antigens have been widely investigated. Recently, an approximately 120 kDa Cnm protein with binding properties to type I collagen was identified, and its encoding gene (cnm) cloned and sequenced. In the present study, we sequenced cnm from 47 different clinical S. mutans strains and found that the nucleotide alignment of the collagen-binding domain was well conserved. We devised a PCR method for identifying the cnm gene, examined the prevalence of cnm-positive S. mutans strains in various mother-child groups, and assessed the significance of such strains for transmission and dental caries. The detection rate of cnm-positive strains was significantly lower in strains isolated from Japanese children in the 2000s (8.0 %) as compared to those isolated in the 1980s (15.8 %) (P<0.05). Furthermore, the presence of S. mutans possessing cnm in salivary specimens collected from 55 S. mutans-positive mother-child pairs was 40 and 32.7 % in the mothers and children, respectively. The frequency of cnm-positive children whose mothers were also positive was 72 %, which was significantly higher than that of cnm-positive children with negative mothers (P<0.0001, odds ratio 17.5). In addition, clinical parameters indicating dental caries were significantly increased in children with cnm-positive S. mutans in saliva (n=13), as compared to those with cnm-negative S. mutans (n=15) and S. mutans-negative children (n=20) (P<0.01). These results indicate that cnm-positive S. mutans strains are closely correlated with dental caries, while vertical transmission in cnm-positive mother-child pairs was also demonstrated.

Streptococcus mutans clonal variation revealed by multilocus sequence typing.

Streptococcus mutans is the major pathogen of dental caries, a biofilm-dependent infectious disease, and occasionally causes infective endocarditis. S. mutans strains have been classified into four serotypes (c, e, f, and k). However, little is known about the S. mutans population, including the clonal relationships among strains of S. mutans, in relation to the particular clones that cause systemic diseases. To address this issue, we have developed a multilocus sequence typing (MLST) scheme for S. mutans. Eight housekeeping gene fragments were sequenced from each of 102 S. mutans isolates collected from the four serotypes in Japan and Finland. Between 14 and 23 alleles per locus were identified, allowing us theoretically to distinguish more than 1.2 x 10(10) sequence types. We identified 92 sequence types in these 102 isolates, indicating that S. mutans contains a diverse population. Whereas serotype c strains were widely distributed in the dendrogram, serotype e, f, and k strains were differentiated into clonal complexes. Therefore, we conclude that the ancestral strain of S. mutans was serotype c. No geographic specificity was identified. However, the distribution of the collagen-binding protein gene (cnm) and direct evidence of mother-to-child transmission were clearly evident. In conclusion, the superior discriminatory capacity of this MLST scheme for S. mutans may have important practical implications.