Heini Torkko

Oral colonization by more than one clonal type of mutans streptococcus in children with nursing-bottle dental caries

By ribotyping the genetic diversity of mutans streptococci in six 1.5-3-yr-old children with nursing-bottle caries and in six caries-free, age-matched children and in their mothers was examined. The proportion of mutans streptococci in the dental plaque of the children and their levels in the saliva of the mothers were also examined. For ribotyping, chromosomal DNA of isolates obtained from the plaque of the children (3-12 isolates per child) and from the saliva of the mothers (4-13 isolates per mother) was digested with restriction endonuclease HindIII. The DNA fragments were hybridized to the plasmid pKK3535 which contains the rRNA operon of the Escherichia coli chromosome. The results showed that children with nursing-bottle caries exposed to frequent consumption of sucrose had a high proportion of mutans streptococci in plaque and four of them were colonized with more than one ribotype, whereas caries-free children had a low proportion of mutans streptococci in plaque and only one of them harboured more than one ribotype. Mothers of children with nursing bottle caries had similar levels and numbers of ribotypes of mutans streptococci in saliva as the mothers of the caries-free children. In both child groups, mothers were probably the main source of infection with mutans streptococci. Thus, children with nursing-bottle caries were not only heavily infected with mutans streptococci but also often colonized with more than one clonal type. In the childs acquisition of such clones, frequent sugar consumption may have an important role.

Salivary Levels of Suspected Periodontal Pathogens in Relation to Periodontal Status and Treatment

The primary ecological niche for suspected periodontal pathogens seems to be the subgingival area, even though periodontal pathogens are also frequently recovered from saliva. The interrelationship of different periodontal conditions and the salivary levels of suspected periodontal pathogens is not known. In the present study, salivary levels of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Campylobacter rectus, and Peptostreptococcus micros were determined by bacterial culture and related to clinical periodontal status in 40 subjects with either advanced, moderate, or initial/no periodontitis. Culture-positive subjects harbored the 5 bacterial species in mean numbers ranging from 2 x 105 to 6 x 107 colony-forming units (CFU)/mL saliva. A. actinomycetemcomitans was found in none and P. gingivalis in one of the subjects with initial periodontitis, whereas both species were found in 33% and 44%, respectively, of the subjects with moderate periodontitis and in 60% and 40%, respectively, of the subjects with advanced periodontitis. The mean numbers of CFU/mL of P. intermedia, C. rectus and P. micros were significantly higher in subjects with advanced periodontitis than in subjects with initial/no periodontitis. Ten patients with advanced periodontitis were treated mechanically and with adjunctive systemic metronidazole, and were re-examined 1 and 6 months after treatment. Periodontal treatment eradicated or significantly reduced the levels of salivary periodontal pathogens for half a year, whereas in untreated subjects, the levels and the detection frequencies generally remained fairly stable. In conclusion, the results showed that the salivary levels of periodontal pathogens reflect the periodontal status of the patient.

Simultaneous Detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by a Rapid PCR Method

The identification of periodontal pathogens by conventional methods is time-consuming and difficult. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.) and Porphyromonas gingivalis (P.g.) was developed for rapid and easy determination of these risk-indicator bacteria in human periodontal disease. The PCR primers were designed to hybridize to various regions of 16S rRNA genes, and a hot-start technique was used to obtain maximum sensitivity and specificity. This method can detect both of these bacteria in subgingival plaque samples at concentrations as low as 5 to 50 cells per sample. The sensitivity, however, was even 10 times better when the bacteria were analyzed in a water suspension. Since the only step between sample collection and the actual analysis is a brief centrifugation of the patient sample, the detection can be readily carried out in four hours. The performance of the method was studied with 36 patient samples. The results showed that the PCR method detected A.a. (44% vs. 25%, respectively) and P.g. (56% vs. 42%, respectively) more often than the conventional culture in plaque samples. Thus, our multiplex PCR method is rapid and more effective than conventional protocols in detecting these periodontal pathogens.

Identification of Bacteroides forsythus in Subgingival Dental Plaque with the Aid of a Rapid PCR Method

Bacteroides forsythus has been shown to be prevalent among patients with periodontitis. Conventional microbiological methods used to identify this bacterium, however, are laborious and time-consuming and are therefore not well-suited for screening purposes. We have developed a polymerase chain-reaction (PCR) method which is rapid, specific, and simple to perform and does not require other sample pre-treatment except a brief centrifugation. This method was applied to the detection of B. forsythus in subgingival plaque of 58 periodontitis patients. When compared with the results of conventional culturing, the PCR method always confirmed the culture-positive results, while none of the PCR negative samples was shown to be culture-positive. The PCR method appeared to give more than double the number of samples positive for B. forsythus than culturing (89.7% vs. 37.9%). The analysis requires less than 4 hrs to perform, and is specific only to B. forsythus and sensitive enough to detect fewer than 5 bacteria.

Effect of extraction of partly erupted third molars on subgingival microorganisms

This study was made to investigate the effect of extraction of third molars on subgingival microbes in 39 generally and gingivally healthy men with an average age of 20.2 years (SD 0.9). Microbial samples were taken from the pericoronal space of symptom-free partly erupted lower third molars and from the adjacent gingival pockets of the second molars. The samples were cultivated anaerobically. All partly erupted third molars were extracted from 20 subjects. A control group of 19 subjects was left untreated. Microbe sampling was repeated 2 and 5 months postoperatively with highly significant results. It was shown that at baseline the number of black-pigmented gram-negative bacteria and Fusobacterium species was more frequent in third molar than in second molar sites. The total bacterial count decreased significantly at the second molar sites after extraction of the third molars when compared with the control group. Before the extractions, black-pigmented gram-negative bacteria were detected in 45% of the test subjects and Actinobacillus actinomycetemcomitans in 20%. The respective postoperative figures were 30% for black-pigmented gram-negative bacteria and 10% for Actinobacillus actinomycetemcomitans. Capnocytophaga species were not affected by the extractions. The findings suggest that erupting third molars may harbor harmful bacteria that can be reduced by eradicating the foci.

Xylitol and the bactericidal effect of chlorhexidine and fluoride on Streptococcus mutans and Streptococcus sanguis

The present study was made to investigate the effect of xylitol on the bactericidal and bacteriostatic action of chlorohexidine diacetate (CHX) and sodium fluoride (F) in ATCC strains of Streptococcus mutans and S. sanguis. Standardized bacterial cell suspensions were used in tests for bactericidal effect and for inhibition of growth and sucrose fermentation. The results showed no interference of xylitol with the antibacterial effect of CHX and F combinations. Xylitol did not show any additive effect either but appeared inert in the combinations used.