J.F. Buijs

Similarity of Bacteriocin Activity Profiles of Mutans Streptococci within the Family When the Children Acquire the Strains After the age of 5

It has been shown that there is a window of infectivity for mutans streptococci between the ages of 19 and 31 months, when many children acquire mutans streptococci transmitted from their mothers. Part of the children that escape this window acquire mutans streptococci at a later age. In this group, maternal transmission is expected to be less prevalent. The present study compared the bacteriocin activity profiles of mutans streptococci isolated from mothers, fathers and children when the children acquired the mutans streptococci between the ages of 5 and 11. Twelve families were randomly selected from a group of 11–year–old children who were known to have acquired mutans streptococci during this age period. From the saliva of the mothers (n = 12), fathers (n = 8) and children (n = 12) approximately 30 mutans streptococci strains were isolated. All isolates were tested twice for bacteriocin activity against 21 indicator strains with a double–layer technique. Bacteriocin activity of strains was considered to be different when the number of strains against which bacteriocin was produced differed >1 or when the width of one or more inhibition zones differed ≥4 mm. In 7/12 mother–child pairs similar profiles were found. In the 8 father–child pairs similar profiles were only found on 2 occasions. In these 2 families, all 3 ( mother, father and child) harboured strains with a similar profile. In 4/8 father–mother pairs similar profiles were found. There was no correlation between the prevalence of mutans streptococci strains, the number of indicator strains against which the strains made bacteriocin, nor the mean size of the inhibition zones and the presence of similarity of bacteriocin activity profiles of mutans streptococci within the family members. The results show that even when a child acquires mutans streptococci after the age of 5, there may be similarity between mutans streptococci in mother, father and child, indicating that transmission between the family members occurs.

Prevalence of Streptococcus mutans and lactobacilli in 18-month-old children with cleft lip and/or palate

The prevalence of Streptococcus mutans and lactobacilli was determined in 62 18-month-old Dutch children with a cleft lip and/or palate. Plaque and saliva samples were collected, a dental examination was performed, and the parents were interviewed with a structured questionnaire regarding general health, dietary habits, fluoride exposure, and socioeconomic class. Appropriate dilutions of the plaque and saliva samples were cultured on selective media to count all viable bacteria, S. mutans and lactobacilli. S. mutans was detected in the saliva of 45% of the children, and lactobacilli was detected in 16%. Also, S. mutans was detected in 48% of the plaque samples and lactobacilli in 8%. Of all of the variables examined, consumption of more than three snacks and beverages between main meals was significantly associated with presence of S. mutans in saliva. Preoperative infant orthopedic treatment (i.e., wearing an acrylic plate from shortly after birth) was significantly associated with presence of lactobacilli in saliva. The presence of S. mutans in the plaque samples was also significantly associated with presence of lactobacilli in saliva. These results indicate that children with oral cleft are at an increased risk of being infected by S. mutans and lactobacilli at a very early age. Such early colonization indicates a high risk for caries in the primary dentition.

Protection of bovine enamel and dentine by chlorhexidine and fluoride varnishes in a bacterial demineralization model

In an in vitro demineralization model, the protective effect of two chlorhexidine varnishes, Cervitec (1% w/w chlorhexidine diacetate and 1% w/w thymol) and EC40 (40% w/w chlorhexidine diacetate), was compared with that of Fluor Protector, a varnish containing 0.1% w/w F. The demineralization model comprised an acidogenic Streptococcus mutans suspension in agarose placed on enamel or dentine specimens. The experiments extended over three serial 22-hour demineralization periods with fresh S. mutans suspensions for each period. To determine whether the varnishes released enough demineralization-inhibiting compounds, approximately 10 microliters of the varnishes was applied adjacent to the enamel and dentine specimens just before the first application of the S. mutans suspensions and left during the serial experiments (release study). In a separate series of experiments, the effect of the pretreatment of the enamal and dentine specimens with the various varnishes was tested (pretreatment study). In the release study the protective effect for enamel decreased in the order: EC40 = Fluor Protector >> Cervitec = no treatment. For dentine this order was: EC40 >> Fluor Protector = Cervitec > no treatment. In the pretreatment study, the enamel specimens were best protected by Fluor Protector (Fluor Protector >> Cervitec = EC40 > no treatment), while the dentine specimens were best protected by the chlorhexidine treatments (Cervitec = EC40 > Fluor Protector > no treatment). A 1:1 mixture of Cervitec and Fluor Protector was as effective as the most effective component alone. It is concluded that a varnish containing both fluoride and chlorhexidine may be useful, since it could give optimal protection to both enamel and dentine.

Calcification of a Cariogenic Streptococcus and of Corynebacterium (Bacterionema) matruchotii

The main aim of this investigation was to challenge the idea that cariogenic streptococci do not calcify. Calcium uptake or calcification of Streptococcus mutans C180-2, proven to be an acidogenic and cariogenic strain, was compared with calcium uptake and calcification of Corynebacterium (Bacterionema) matruchotii, known as a ready calcifier. Bacteria were grown on Brain Heart Infusion Agar (BHIA) and on well-buffered semi-synthetic E-agar, both containing 1.4 mmol/L calcium, 2 g/L glucose, initial pH 7.4. Calcium uptake from BHIA by C. matruchotii (25 mmol Ca/kg wet bacterial cell mass), but not by S. mutans, was found. Grown as a plaque-like lawn on E-agar, the S. mutans cell mass concentrated calcium to 63 ± 11 mmol/ kg compared with 145 ± 61 mmol/kg in C. matruchotii. X-ray diffraction confirmed the presence of crystalline apatite in the bacterial cell masses. Electron microscopy revealed crystals and mineralized deposits in both organisms. Heavy calcifications in some cells of S. mutans were seen. Calcification was partly inhibited by magnesium ion and by methanehydroxybisphosphonate. S. sobrinus 6715, as well as freshly isolated S. mutans and S. sobrinus from patients, concentrated very large quantities of calcium, up to 500-fold from the medium, when maintained for several weeks on E-agar of initial pH 7.6. Our observations widen the view on acidogenic bacteria as mineralization agents and support the notion that members of the mutans group of streptococci may be involved in events that trigger heavy intracellular calcifications and, possibly, dental calculus formation.

In vitro Demineralization of Enamel by F-sensitive and F-resistant Mutans Streptococci in the Presence of 0, 0.05, or 0.5 mmol/L NaF

Lactate production and accompanying enamel demineralization by fluoride-sensitive and fluoride-resistant mutans streptococci were studied in an in vitro demineralization model in the presence of 0, 0.05, or 0.5 mmol/L NaF. The fluoride-resistant strains were derived from laboratory strains or were recently isolated strains from xerostomic patients on high-dose fluoride therapy. The demineralization model was composed of a cell suspension in a glucose-agarose gel overlying a bovine enamel block. Lactate and calcium content of the agarose were determined after 22-hour incubations at 37°C. Fluoride-resistant variants of Streptococcus sobrinus 6715-15 produced less lactate and caused less demineralization than did the parent strain even in the presence of fluoride. On the other hand, fluoride-resistant variants of Streptococcus mutans C180-2 and of S. mutans GS-5 produced more acid and caused greater demineralization than did their respective parent strains, both in the absence and presence of fluoride. Two recently isolated fluoride-resistant S. mutans strains produced more lactate and demineralized enamel more than did two recently isolated S. mutans strains from normal human subjects, both in the presence of 0 and 0.05 mmol/L NaF. It is concluded that adaptation to fluoride resistance does not invariably reduce the cariogenicity of mutans streptococci nor the effectiveness of fluoride in preventing demineralization.

Plaque composition, fluoride tolerance and acid production of mutans streptococci before and after the suspension of the use of fluoride toothpastes.

Although fluoride toothpastes are widely used for caries prevention, little is known about the impact of fluoride dentifrices on plaque composition. Also the issue of adaptation of mutans streptococci to grow in vivo in a fluoride environment has received little attention. Such an adaptation may be of interest as it has been suggested that adapted mutans streptococci may show reduced glycolytic activity thereby being less cariogenic. In the present experiments the impact of the suspension of the use of fluoride toothpastes on plaque composition, fluoride tolerance and acid production of mutans streptococci was studied. Pooled plaque samples from the lingual surfaces of the lower incisors were collected from individuals (n = 13) just before and 7 weeks after they had replaced their fluoride toothpastes (0.1-0.15% F) with a non-fluoride one. The samples were analysed for fluoride and the numbers and proportions of streptococci, Streptococcus mutans, Streptococcus sobrinus, Actinomyces species, and lactobacilli, respectively. The fluoride tolerance of the mutans streptococci was estimated by culture of the plaque samples on TYCSB agar supplemented with of 0, 1, 2, 3, 4, and 5 mmol/l fluoride (NaF) at pH 7.2. From each plaque sample six S. mutans strains were isolated for the measurement of the rate of acid production (Vap) at pH 7 in the presence of 0, 5, and 10 mmol/l F. The overnight final pH was measured in cultures of the S. mutans strains with excess of glucose and 0, 5, and 10 mmol/l F. The results showed that the removal of the fluoride pressure from plaque did not affect the numbers or proportions of the various species and genera of bacteria. The fluoride tolerance of the mutans streptococci, and the Vap or the overnight final pH of the isolated strains had not changed. These results suggest that the use of fluoride toothpaste had not affected plaque composition, nor fluoride tolerance or acidogenicity of mutans streptococci. Probably the amount of fluoride delivered by fluoride dentifrices to dental plaque is too low to induce such adaptations.

Protective Effect of Topically Applied Fluoride in Relation to Fluoride Sensitivity of Mutans Streptococci

The aim of the present in vitro experiments was to determine whether the protection of enamel by topically applied fluoride against demineralization by bacterial acids would depend on the fluoride sensitivity of the bacteria. Glucose-agarose gel suspensions of fluoride-sensitive and fluoride-resistant mutans streptococci were placed on bovine enamel specimens with different amounts of fluoride. One group of specimens was untreated, a second group had been pretreated with a F-lacquer, and a third group had been pretreated with the F-lacquer and rinsed subsequently with a KOH-solution, to remove deposited CaF2. After 22-hour incubations at 37°C, the amounts of calcium and lactate and the pH of the agarose gels were determined. This procedure was repeated on three consecutive days. Two parent S. mutans strains, one parent S. sobrinus strain, and five fluoride-resistant derivatives were tested. Both pretreatments gave a significant protection to the enamel specimens. For the S. mutans strains, the degree of protection did not depend on the fluoride sensitivity of the strains. For the S. sobrinus strains, the results suggested a reduced protection against demineralization by the fluoride-resistant derivatives. Only from the second group of enamel specimens was enough fluoride released for inhibition of bacterial metabolism. Presumably, it was released by the dissolution of CaF2. It is concluded that a possible adaptation of mutans streptococci in dental plaque to frequent exposures to fluoride will not necessarily decrease the caries-preventive effects caused by topically applied fluoride agents.

Protective Effect of Hexetidine against in vitro Bacterial Demineralisation of Bovine Enamel and Dentin in the Presence of Fluoride

Bovine enamel and dentin specimens were overlaid with acidogenic Streptococcus mutans suspensions in agarose. In this model, the minimal demineralisation-inhibiting concentrations (MDIC) of hexetidine was determined in the presence of fluoride. A commercially available mouthwash containing 0.1% (2.9 mmol/l) hexetidine was diluted serially and added to the bacterial suspensions together with 0, 5.3, or 26.3 mumol/l fluoride (NaF). After 22 h of incubation at 37 degrees C the bacterial suspensions were removed and assessed for calcium and lactate. The results showed significant inhibitory effects of hexetidine on the demineralisation of the enamel specimens with a MDIC between 15 and 31 mumol/l hexetidine. In the presence of fluoride, approximately fourfold higher concentrations of hexetidine were needed for a significant additional protection of the enamel. No synergistic effect between hexetidine and fluoride was observed. For the demineralisation of the dentin specimens, the MDIC of hexetidine had a value between 31 and 61 mumol/l. At both these concentrations the dentin specimens were relatively less protected in the presence than in the absence of fluoride, and some synergistic effect between hexeditine and fluoride was observed.