Development of a novel PCR method to comprehensively analyze salivary bacterial flora and its application to patients with odontogenic infections.
ABSTRACT The objective of this study was to develop a novel polymerase chain reaction (PCR) method to comprehensively analyze salivary bacterial flora.
The bacterial flora in the saliva of 10 healthy persons and 11 patients with odontogenic infections were examined using a DNA extraction method with a high level of cell destruction efficiency and a novel universal primer set to amplify approximately 580 bp of the 16S rDNA sequence.
Streptococcus (54.5%), Neisseria (14.7%), Actinomyces (8.4%), Gemella (4.1%), Granulicatella (3.8%), and Prevotella (1.4%) were dominant in a total of 1655 clones examined from the saliva of the healthy subjects. The dominant genera differed among the patients with odontogenic infections (a total of 823 clones) and were entirely different from those of the healthy subjects.
This novel comprehensive salivary bacterial flora analysis method may be a useful supportive method to identify causative agents of odontogenic infections.
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ABSTRACT: The aim of this study was to determine the influence of menstruation on the bacterial population of healthy Japanese women's vulvas, especially the labia minora. Labia minora swabs were obtained from 10 premenopausal, nonpregnant Japanese women at premenstruation and on day 2 of menstruation. Vaginal swabs were also obtained from 3 out of the 10 women. No significant difference was found in the average bacterial cell count between the menstruation and premenstruation samples. Molecular analysis using a 16S rRNA gene-based clone library method detected 22 genera from the labia minora swabs (total 20), with the genus Lactobacillus being predominant at both premenstruation and during menstruation in 7 out of the 10 women. Of the other 3 women, 2 showed various kinds of bacterial species, including oral and fecal bacteria, with Atopobium vaginae and Gardnerella vaginalis predominating in the remaining woman's vulva in both conditions. In total, 6 out of 10 cases (60%) showed significantly different microbiota of the labia minora between the two conditions. These results imply that menstruation may promote a distortion of the bacterial flora around the vulva, although it causes no significant increase of the bacterial count.01/2011; 64(1):76-80.
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ABSTRACT: Intestinal microbiotas of human subjects and effect of antibiotic treatment on them have been reported with cultivation independent methods. However, Japanese fecal microbiotas have not been studied enough. We have constructed a clone library method to obtain results within 3 d. In this study, intestinal microbiotas of 29 healthy Japanese adults, whose fecal samples were collected twice at 5 month intervals from each subject, were analyzed with our clone library method, and using those data as a benchmark effect of antibiotic treatment on intestinal microbiotas was evaluated. The fifty-eight fecal microbiotas were assessed based on percentages at genus level, and the variability was analyzed with a principal component analysis (PCA). PCA showed that the microbiotas divided into three groups depending on the large eigenvectors (genera Ruminococcus, Bacteroides, and Prevotella), and the dual samples from the twenty-two individuals have belonged to the same PCA group. It suggests that almost Japanese adults have own stable intestinal microbiota. The genera Ruminococcus and Bacteroides were present in almost subjects, while the genus Prevotella was found only in nine subjects (approximately 30%) which was preserved with 5 months intervals. Next, the microbiotas before and after antibiotic treatment were evaluated comparing with the 58 healthy adult microbiotas. The results showed that beta-lactams influenced profoundly on intestinal microbiotas and the effect of macrolides depended on the cases. It suggests that our clone library method could show overview of intestinal microbiota and would give us useful information about the effect of antibiotic treatment for daily clinical diagnosis.Biological & Pharmaceutical Bulletin 01/2011; 34(7):1011-20. DOI:10.1248/bpb.34.1011 · 1.78 Impact Factor
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ABSTRACT: The frequencies of etiologic bacterial agents of intrapleural infections reported until now have been widely varied, largely depending on the implemented detective methods. The aims of this study were to evaluate bacterial etiologies of bacterial pleurisy using a cultivation-independent method. Pleural fluids were collected from 42 febrile patients with hemipleural effusion. The bacterial flora was analyzed by a clone library method using amplified fragments of the 16S ribosomal RNA gene (rDNA) with universal primers in addition to conventional cultivation methods. Forty-two specimens were obtained from 26 patients with bacterial pleurisy, seven with mycobacterial pleurisy, and nine with other pleural effusions. In the 26 bacterial cases, 16 (61.5%) showed positive results for 16S rDNA sequencing analysis, of which 11 (42.3%) were also positive for cultivation method. In seven (43.8%) of the 16 polymerase chain reaction-positive cases, anaerobic phylotypes were predominantly detected. Anaerobic phylotypes (six of these seven cases) were not detected by cultivation method. In nine (34.6%) of the 26 bacterial pleural cases, the results from the clone library methods were not accordant with those of the cultivation method. In seven of these nine cases, the discrepancies between the two detection methods were due to the existence of anaerobes. The clone library analysis using the 16S rDNA of pleural fluid showed a higher incidence of anaerobic bacteria in infectious pleurisy than that previously expected and provided additional bacterial information for cultivation methods.Chest 03/2011; 139(3):600-8. DOI:10.1378/chest.10-0460 · 7.13 Impact Factor