Interactions between and within Streptococcus mutans and Streptococcus sobrinus isolated from humans harboring both species

Abstract— The prevalence of Streptococcus mutans and Streptococcus sobrinus was examined in plaque samples from small discrete areas of the buccal tooth surfaces of seven subjects. Strains of S. mutans and S. sobrinus were isolated and tested for bacteriocin-mediated interactions between and within the two species, using the stab inoculation technique. S. mutans and S. sobrinus did not colonize each tooth surface uniformly and, in plaque from small discrete sites, S. mutans and S. sobrinus were either undetected or present in different interspecies proportions. Within the same subject, there were no bacteriocin-mediated interactions between strains of the same mutans species and no difference in bacteriocin activity was found between the strains of S. mutans and S. sobrinus from different sites. When bacteriocin interactions were tested between isolated strains from all seven subjects a somewhat higher inhibition ability was found for producer strains isolated from plaque compared with those isolated from saliva. S. mutans appeared to be more bacteriocinogenic than S. sobrinus . Replacing the glucose in the medium with sucrose enhanced the bacteriocin activity of S. mutans towards other S. mutans strains but reduced the inhibitory interaction towards strains of S. sobrinus .     

Interactions between and within Streptococcus mutans and Streptococcus sobrinus isolated from humans harboring both species.

The prevalence of Streptococcus mutans and Streptococcus sobrinus was examined in plaque samples from small discrete areas of the buccal tooth surfaces of seven subjects. Strains of S. mutans and S. sobrinus were isolated and tested for bacteriocin-mediated interactions between and within the two species, using the stab inoculation technique. S. mutans and S. sobrinus did not colonize each tooth surface uniformly and, in plaque from small discrete sites, S. mutans and S. sobrinus were either undetected or present in different interspecies proportions. Within the same subject, there were no bacteriocin-mediated interactions between strains of the same mutans species and no difference in bacteriocin activity was found between the strains of S. mutans and S. sobrinus from different sites. When bacteriocin interactions were tested between isolated strains from all seven subjects a somewhat higher inhibition ability was found for producer strains isolated from plaque compared with those isolated from saliva. S. mutans appeared to be more bacteriocinogenic than S. sobrinus. Replacing the glucose in the medium with sucrose enhanced the bacteriocin activity of S. mutans towards other S. mutans strains but reduced the inhibitory interaction towards strains of S. sobrinus.     

Colonization of Streptococcus mutans and Streptococcus sobrinus genotypes and caries development in children to mothers harboring both species

The major route of early acquisition of mutans streptococci in humans is a vertical transmission from mother to child. The purpose of this longitudinal study was to examine the acquisition, distribution and persistence of Streptococcus mutans and Streptococcus sobrinus in children whose mothers harbored both species and to study the caries incidence in relation to colonization of these bacteria. Fifteen mother-child pairs were followed during the child's first 7 years. Stimulated salivary samples were taken from the mothers and the children. Plaque samples were also collected from the teeth and the tongue of the children. The samples were analyzed by cultivating techniques together with genomic fingerprinting and hybridizing. The caries experience was evaluated on the sampling occasions and retrospectively using the records of caries registrations from the community clinics. During the 7-year period 10 of the 15 children acquired mutans streptococci. Only 4 of them were colonized by both S. mutans and S. sobrinus despite the fact that their mothers harbored both species. In 2 of the children S. sobrinus was found later than S. mutans. A total of 26 genotypes were found in the children and 9 of them were identical to their mothers. New genotypes and a gain-loss pattern were noted especially in the children but also in their mothers. The groups of teeth first positive for the two species were the deciduous molars. The caries experience was low during the study period with 8 children showing no caries.     

Glycan-binding specificities of Streptococcus mutans and Streptococcus sobrinus lectin-like adhesins

Since the adhesion of bacteria to the tooth surface is a prerequisite for dental plaque and subsequent caries development, a promising caries preventive strategy could be to block the lectin?Cglycan-mediated adherence of cariogenic bacteria. The aim of the study was to evaluate potential differences in glycan-binding specificities of two Streptococcus mutans strains (DSM 20523 and DSM 6178) and Streptococcus sobrinus (DSM 20381). A competitive enzyme-linked lectin-binding assay was used to identify the binding specificities of isolated bacterial surface lectins. Blotting of the microbial proteins on neoglycoprotein-coated PVP membranes enabled a qualitative protein analysis of all specific bacterial lectins. Different glycan-binding sites could be identified for the S. mutans strains in comparison to S. sobrinus. An earlier reported glycan-binding specificity for terminal galactose residues could be confirmed for the S. mutans strains. For the S. sobrinus strain, more than one glycan-binding specificity could be found (oligomannose and terminal sialyl residues). Each of the tested strains showed more than one surface lectin responsible for the specific lectin-binding with varying molecular weight (S. mutans, 90/155?kDa and S. sobrinus, 35/45?kDa). The established experimental setup could be used as future standard procedure for the identification of bacterial lectin-derived binding specificities. The findings from this study might serve as basis for the design of an individual ??glycan cocktail?? for the competitive inhibition of lectin-mediated adhesion of mutans streptococci to oral surfaces.     

变形链球菌Ⅰ型和Ⅲ型合成葡聚糖的研究

目的 通过对Ⅰ型ＪＢＰ（ｃ型）和Ⅲ型６７１５（ｇ型）变链菌在不同状态葡糖基转移酶（ＧＴＦ）所合成的水不溶性和葡聚糖（ＩＧ）和水溶性葡聚糖（ＳＧ）的测定来研究两种变链菌的ＧＴＦ－Ⅰ和ＧＴＦ－Ｓ是否存在差异。方法 制备ＪＢＰ和６７１５的含酶上清液（ｃｅｌｌ－ｆｒｅｅ ｅｎｚｙｍｅ）及含酶提取液（ｃｅｌｌ－ｂｏｕｎｄ ｅｎｚｙｍｅ）,以蔗糖为底物,恒温培养后分别收集ＩＧ和ＳＧ。     

Differences in cariogenicity between fresh isolates of Streptococcus sobrinus and Streptococcus mutans

Streptococcus sobrinus is known to possess cariogenic properties in vitro. It can produce acid in large amounts and it has the capacity to adhere to enamel and other surfaces. However, most studies on cariogenicity have been performed with laboratory strains that have been subcultured over long periods of time. Therefore, the cariogenicity and acidogenicity of 9 fresh isolates of both S. sobrinus and Streptococcus mutans from human dental plaque were compared. The bacteria were inoculated into the oral cavity of rats. The rats were fed diet SSP 20/5, containing 20% sucrose and 5% glucose. After the experimental period of 42 days, the amount of caries was assessed and bacterial counts were determined using monoclonal antibodies. Four out of 9 S. sobrinus strains and 3 out of 9 S. mutans strains did not colonize the rats. Colonizing strains constituted 39-78% of the total anaerobic cultivable microflora. The numbers of advanced dentinal lesions in the fissures of the rats colonized with S. mutans were significantly lower than those colonized with S. sobrinus (p less than 0.05). S. sobrinus produced acid more rapidly than S. mutans in a pH-stat system at pH values between 6.5 and 5.0 (p less than 0.01). The results indicate that fresh isolates of S. sobrinus are more cariogenic in rats than fresh isolates of S. mutans. This is possibly due to differences in glycolytic properties of these two species.     

Streptococcus mutans strains harboring collagen-binding adhesin

A previously unidentified 120-kDa protein was detected in Streptococcus mutans strain Z1 and was involved in the cold-agglutination of the strain. We have identified the gene, designated cnm, as being involved in the agglutination of strain Z1 following random mutagenesis. The amino acid sequence of the deduced Cnm protein exhibited high similarity to those of collagen-binding adhesins from staphylococci and other organisms. To confirm whether the protein is involved in collagen-binding, we cloned a cnm gene fragment, overexpressed it in E.coli, and prepared crude extracts. The extracts containing recombinant protein exhibited binding to immobilized collagen and laminin but not to fibronectin. Compared with the parental strain Z1, the cold-agglutination-negative mutant 05A02 exhibited reduced binding to collagen and laminin but retained that to fibronectin. This gene was detected in some strains of S. mutans. Therefore, the cnm gene encoded a new strain-specific member of the collagen-binding adhesin family.     

Caries prevalence associated with Streptococcus mutans and Streptococcus sobrinus in Japanese schoolchildren

International Journal of Paediatric Dentistry 2012; 22: 342–348 Background. Streptococcus mutans and Streptococcus sobrinus are known to be associated with dental caries in humans. Aim. We used a polymerase chain reaction method to detect S. mutans and S. sobrinus in 128 Japanese schoolchildren and then compared their presence with the dental caries experience. Design. Plaque samples were collected from all erupted tooth sites with a sterile toothbrush, and dental examinations were performed to determine the numbers of decayed and filled teeth in primary (dft) and permanent (DFT) dentition using the WHO caries diagnostic criteria. Fisher’s PLSD test was employed to compare caries scores between combinations of the detected bacteria. Results. Streptococcus mutans and S. sobrinus were present in 38.3% and 68.0%, respectively, whereas 14.8% were positive for S. mutans alone, 44.5% for S. sobrinus alone, and 23.5% for both S. mutans and S. sobrinus, with 17.2% negative for both. The DFT, dft, and total (DFT + dft) scores for subjects positive for both S. mutans and S. sobrinus were significantly higher than those positive for S. mutans alone (P < 0.05, in triplicate). Conclusion. These results suggest that schoolchildren harbouring both S. mutans and S. sobrinus have a significant higher dental caries experience in both permanent and primary teeth as compared to those with S. mutans alone.     

变形链球菌和远缘链球菌耐氟菌株超微结构观察

目的：探讨变形链球菌和远缘链球菌耐氟突变后,其超微结构变化及氟化物对细菌结构的影响。方法：将变形链球菌和远缘链球菌分为3组：A为正常对照组;B为加氟孵育组;C为耐氟菌株组。透射电镜观察3组细菌的超微结构。结果：与正常亲代菌株相比,加氟孵育后的菌株及耐氟菌株菌体出现不同程度超微结构改变,包括胞浆电子密度减低,细胞肿胀,内容物外溢,远缘链球菌链状结构消失等。结论：变形链球菌和远缘链球菌与氟共同孵育及耐氟突变后,其超微结构发生了改变,表现为自溶活动及解链活动增强。     

变形链球菌遗传Ⅰ型JBP和Ⅲ型6715表面形态的电镜观察

对变形链球菌遗传Ⅰ型和Ⅲ型菌力表面形态的电镜观察显示，仅遗传Ⅲ型菌体表面存在毛状外膜。但此差异形成的机理以及是否影响变形链球菌的粘附尚需进一步探讨。     

Homology of glucosyltransferase gene and protein sequences from Streptococcus sobrinus and Streptococcus mutans

The sequences of glucosyltransferase genes from Streptococcus sobrinus (gtfI) and Streptococcus mutans (gftB) were compared and show a high degree of homology. There is a 57.7% homology of nucleotides in the genes and a 56.7% homology of amino acids in the deduced protein sequences. The G + C content for the protein-coding region is 43.6% for S. sobrinus and 41.2% for S. mutans. Internal repeating sequences present in both proteins exhibit some difference in sequence pattern.     

Streptococcus mutans and Streptococcus sobrinus biofilm formation and metabolic activity on dental materials

Abstract

Objectives. To examine potential correlations between streptococcal biofilm formation and lactate production in streptococcal biofilms formed on the surface of dental materials with different surface characteristics. Materials and methods. Samples of a glass-ionomer cement (Ketac Molar) and a ceramic (Empress 2) were incubated with whole saliva and suspensions of Streptococcus mutans ATCC 25175 or Streptococcus sobrinus ATCC 33478 for initiating single-species biofilm formation for either 4 or 24 h. The relative amount of adherent, viable cells was determined using a Resazurin and a MTT assay. Metabolic activity was assessed by quantifying lactate production with a modification of the commercial Clinpro Cario L-Pop kit. Results. Both assays identified similar S. sobrinus biofilm formation on the two substrata; for S. mutans, the MTT test showed significantly fewer streptococci on the glass-ionomer cement than on the ceramic. Concerning metabolic activity, for S. sobrinus, significantly higher lactate production was observed for biofilms formed on the glass-ionomer cement in comparison to the ceramic, whereas similar values were identified for S. mutans. Conclusions. Within the limitations of the study, the results suggest that the pure amount of adherent streptococci does not a priori indicate the metabolic activity of the cariogenic bacteria organized in the respective biofilm. Thus, comparisons between the relative amount of adherent streptococci and their metabolic activity may allow for an improved understanding of the effect of dental material surfaces on the formation and metabolic activity of streptococcal biofilms.     

A simple biochemical scheme for the differentiation of Streptococcus mutans and Streptococcus sobrinus.

A collection of wild-type and reference strains of Streptococcus mutans and Streptococcus sobrinus was compared using a set of fermentation and enzymic tests in order to establish a simple scheme for the differentiation of these two species isolated from selective media. Tests for the production of acid from N-acetylglucosamine, arbutin and melibiose, and the presence of alpha-galactosidase and alpha-glucosidase activities were useful in differentiating these species. Strains of Streptococcus mutans unable to ferment melibiose were also frequently unable to ferment other sugars; a consideration of the genetic control and organization of the genes involved in melibiose metabolism indicates that certain of the conventional characterization tests are not measurements of independent biochemical capabilities.     

PCR detection of Streptococcus mutans and Streptococcus sobrinus in plaque samples from Mongolian mother-child pairs

Early childhood caries results in a considerable burden of pain and suffering as well as poorer general health. Streptococcus mutans (serotypes c, e and f ) and Streptococcus sobrinus (serotype d and g) are the species closely associated with dental caries. The exact age at which their colonization occurs in children is controversial. The objective of this study was to detect S. mutans and S. sobrinus in plaque samples of Mongolian mother-child pairs by PCR and to compare their presence with the caries status. Dental examination and caries risk assessment using the Cariostat^® carried out on 320 children aged 6–30 months and their mothers. The presence of S. mutans and S. sobrinus was checked by PCR. The caries prevalence and DEFT scores of mothers enrolled in the present study were 98% and 11.5±0.7, respectively. In children, the prevalence and deft scores of the 6–18-month-olds were 29% and 1.3±0.2 while those of the 19–30-month-olds were 59% and 3.4±0.4 correspondingly. Twenty nine percent of the 6–18-months old children of high-risk mothers and 53.1% of the 19–30-months old children of high-risk mothers had high caries risk (P<0.001). There was a statistically significant correlation between caries risk of 19–30 month-old children and their mothers (P<0.001). In mothers, the prevalence of S. mutans and S. sobrinus was 79% and 33%, respectively; 54% harbored S. mutans alone, 8% harbored S. sobrinus alone, 25% harbored both strains. In children, 45% were positive for S. mutans alone, 9% were positive for S. sobrinus alone, 18% were positive for both strains. Either or both strains were detected in 67.3% of 6–18-months old children and 76.5% of 19–30-months old children. In conclusion, our results showed that S. mutans and/or S. sobrinus first colonized infants’ teeth from 6–18 months, and the colonization increased with increasing age, so that by 30 months of age, 76.5% of children harbored the bacteria.     

套式PCR检测唾液中变形链球菌及远缘链球菌

目的 探索一种快速、简便地从人类唾液中同时检测变形链球菌和远缘链球菌的方法。方法 分别以变形链球菌gtfI和远缘链球菌gtfB基因设计两组成套引物，首先用套式PCR（二次PCR）检测变形链球菌和远缘链球菌标准株和临床株，然后用套式PCR直接从唾液中检测这两种细菌。结果 变链菌（血清型c,e,f)的标准株及临床株第1次PCR扩增产物为517bp，第2次扩增产物为468bp；远缘链球菌（血清型d,g)及道勒链球菌（血清型h)的标准株及临床株第1次PCR扩增产物为712bp，第2次扩增产物为663bp;其他异种菌均不能扩增出产物，因此该PCR检测具有高度的特异性。细菌纯培养物及唾液PCR检测的敏感性分别是：第1次PCR为10^5CFU，第2次PCR为10^3CFU。结论 套式PCR能快速在人类唾液中同时检测变形链球菌和远缘链球菌。该检测方法有望运用于临床检测，对揭示两种细菌与龋病发生关系的研究具有一定价值。     

Susceptibility of Streptococcus mutans and Streptococcus sobrinus to cell wall inhibitors and development of a novel selective medium for S. sobrinus

Representative strains of Streptococcus mutans and Streptococcus sobrinus showed differences in susceptibility to members of the monobactam group of beta-lactam antibiotics: S. sobrinus was less sensitive than S. mutans. The minimum inhibitory concentrations of aztreonam (AZT) and carumonam, both of which belong to this group, were 2,000 microg/ml for S. sobrinus and 125 microg/ml for S. mutans. Further addition of fosfomycin, bacitracin and sodium chloride to Mitis Salivarius agar (MS) supplemented with AZT resulted in growth inhibition of S. mutans and oral streptococci other than S. sobrinus, and was therefore used as a selective medium for S. sobrinus (MS-SOB medium). The average growth recovery of laboratory and clinically isolated strains of S. sobrinus on MS-SOB medium was 74.1% compared to that on MS medium. Seventy-eight percent of clinical samples in which S. sobrinus was detected yielded pure growth of S. sobrinus on MS-SOB medium.     

Rapid detection of the cariogenic pathogens Streptococcus mutans and Streptococcus sobrinus using loop-mediated isothermal amplification

INTRODUCTION: Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. In this study, we developed a rapid, sensitive method for detecting these major cariogenic pathogens using loop-mediated isothermal amplification (LAMP). The assay procedure is quite simple: the amplification is carried out in a single tube under isothermal conditions at 63 degrees C, and the result can be obtained in less than 1 h. METHODS: Initially, a set of six primers was designed by targeting S. mutans-specific and S. sobrinus-specific regions, identified using the genomic subtractive hybridization technique. We evaluated the specificities and sensitivities of these assays. Furthermore, we detected and quantified these bacteria in saliva and carious dentin from eight children. RESULTS: The sensitivities of the S. mutans-specific and S. sobrinus-specific LAMP methods, examined using agarose gel electrophoresis, were each one cell for a 30-min reaction. The detection limits using real-time turbidimetry analysis were 1 to 10(7) cells (3.28 x 10(1) to 3.28 x 10(8) fg S. mutans template DNA) per reaction tube and 1 to 10(5) cells (2.72 x 10(3) to 2.72 x 10(8) fg S. sobrinus template DNA) per reaction tube. Using these assays, we detected and quantified these cariogenic bacteria for evaluation of the LAMP assay for clinical diagnosis. CONCLUSIONS: Our results suggest that the LAMP-based assay in combination with subtractive hybridization is valuable for preparing species-specific primers for closely related species. Furthermore, the LAMP-based assay will be a useful tool for the rapid and sensitive prediction of dental caries.