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: Intracystic fluid was aseptically collected from 11 patients with postoperative maxillary cyst (POMC), and DNA was extracted from the POMC fluid. Bacterial species were identified by sequencing after cloning of approximately 580 bp of the 16S rRNA gene. Identification of pathogenic bacteria was also performed by culture methods. The phylogenetic identity was determined by sequencing 517-596 bp in each of the 1139 16S rRNA gene clones. A total of 1114 clones were classified while the remaining 25 clones were unclassified. A total of 103 bacterial species belonging to 42 genera were identified in POMC fluid samples by 16S rRNA gene analysis. Species of Prevotella (91%), Neisseria (73%), Fusobacterium (73%), Porphyromonas (73%), and Propionibacterium (73%) were found to be highly prevalent in all patients. Streptococcus mitis (64%), Fusobacterium nucleatum (55%), Propionibacterium acnes (55%), Staphylococcus capitis (55%), and Streptococcus salivarius (55%) were detected in more than 6 of the 11 patients. The results obtained by the culture method were different from those obtained by 16S rRNA gene analysis, but both approaches may be necessary for the identification of pathogens, especially of bacteria that are difficult to detect by culture methods, and the development of rational treatments for patients with POMC.ISRN dentistry. 01/2012; 2012:840483.
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ABSTRACT: PURPOSE: To determine the causative pathogens in eyes with bacterial conjunctivitis. DESIGN: Evaluation of diagnostic test or technology. PARTICIPANTS: Thirteen eyes diagnosed clinically with bacterial conjunctivitis and 12 eyes with normal conjunctival sac were studied. METHODS: The bacterial cell numbers were counted in the samples stained by ethidium bromide (EtBr). The microbiota was determined by the clone library method using polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA (rRNA) gene with universal primers. In addition, examinations of smears and cultures of samples were performed. MAIN OUTCOME MEASURES: Bacterial cell numbers determined by the EtBr staining method and microbiota analysis based on 16S rRNA gene of samples from eyes with bacterial conjunctivitis. RESULTS: The bacterial cell numbers in the eyes with bacterial conjunctivitis were significantly higher than those in the normal conjunctival sacs. Ten of 13 samples from the eyes with bacterial conjunctivitis had positive PCR results. The remaining 3 samples and all 12 samples from the normal conjunctiva had negative PCR results. In 5 of the 10 PCR-positive samples, the predominant species accounted for 84.5% or more of each clone library. In the remaining 5 samples, the predominant and the second dominant species accounted for 27.4% to 56.3% and 19.0% to 26.8%, respectively, of each clone library. The number of detected species in the clone libraries was between 8 and 20 (average ± standard deviation, 7.5±5.8). Bacteria were detected in 8 of the 10 bacterial conjunctivitis samples and in 5 of the 12 normal samples in the cultures. The number of species detected by cultures was 1 in the eyes with bacterial conjunctivitis and between 1 and 3 (mean ± standard deviation, 1.6±0.9) in the normal conjunctiva. CONCLUSIONS: These results showed that the bacterial cell number in a sample is a good method of determining bacterial conjunctivitis. The microbiota analysis detected a diverse group of microbiota in the eyes with bacterial conjunctivitis and showed that the causative pathogens could be determined based on percentages of bacterial species in the clone libraries. The combination of bacterial cell count and microbiota analysis is a good method for identifying the causative pathogens in cases of monomicrobial and polymicrobial conjunctivitis. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.Ophthalmology 12/2012; · 5.56 Impact Factor
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ABSTRACT: Saliva contains various microbes and host biological components that could be used for caries risk assessment. This review focuses on the research topics that connect dental caries with saliva, including both the microbial and host components within saliva.Journal of the California Dental Association 02/2013; 41(2):107-9, 112-8.