Molecular Detection of Novel Anaerobic Species in

The ability to isolate in pure culture the bacteria that cause 1492R [2]. The amplified genes were singularized by cloning, reamplified with the same primers, and then together with amplidisease in humans has been a cornerstone of the advances made in medical microbiology over the past hundred years and an essential fied 16S rRNA genes from the cultivated bacteria were digested component of Koch’s postulates. The etiologic agents of the majorwith a range of restriction endonucleases. ity of the diseases suspected to be caused by bacteria have been The fragments were separated by electrophoresis to give restricdiscovered by this method. However, the advances made as a result tion-fragment-length polymorphism (RFLP) profiles, which were of the use of artificial culture media have perhaps been fortuitous used to group the cloned genes. Members of a group shared the because our knowledge of the microorganisms found on earth is same RFLP profiles for at least three restriction endonucleases. extremely limited; it has been estimated that õ1% of the bacteria The RFLP profiles from the cultivated isolates were then compared on earth have been cultured. It is also estimated that 50% of to those of the groups of cloned genes. Representative clones of the oral flora is unculturable, and the proportion of unculturable groups exhibiting profiles that did not match any of the cultivable organisms at other body sites remains unknown. organisms were sequenced. In recent years studies of the microbial composition of biomass Sequences were identified by means of Similarity-Rank software found at environmental sites have been facilitated by advances in [3]. The sequences were then aligned to related 16S rRNA molecular biology. More specifically, PCR has enabled the amplisequences, and phylogenetic analysis was performed with the fication of specific genes, and the development of automated DNA PHYLIP suite of programs [4]. sequencing methods has allowed the rapid characterization of both The bacteria isolated from two of the samples are shown in entire genomes and specific genes in large numbers of organisms. table 1. A similar range of organisms was recovered from both Genes encoding ribosomal RNA have been found to be particularly samples, and both the identity and numbers of the species isolated useful for the construction of phylogenetic trees reflecting the evowere typical. For each sample, there were three groups of cloned lution of bacteria [1]. This is because these genes have been con16S rRNA genes that did not match with the profiles of the cultured served throughout evolution because of the need to preserve funcorganisms. The results of the sequence analysis for these groups tion. are shown in table 2. Some regions are highly conserved, allowing the design of uniGroups 3.4 and 3.5 showed high similarity to Prevotella oris and versal PCR primers for the amplification of the gene, while other Porphyromonas endodontalis, respectively. P. oris was isolated in regions are more variable, with sufficient information present to the study, but the cultivated organism exhibited a different RFLP differentiate at the species level. The 16S rRNA gene has been profile to that of the cloned group. Group 3.6 showed only low found to be particularly useful because its size of Ç1,500 bases similarity to Prevotella oralis. Further phylogenetic analysis of is sufficient to generate meaningful information, while it is short this sequence indicated that this group may represent a new genus enough for easy sequencing. Thus, 16S rRNA genes have been related to Bacteroides and Prevotella. Group 9.2 was identified as amplified from a variety of sources, including the environment P. endodontalis, while 9.3 was found to show a similarity of 93.6% and human infections, and then sequenced to reveal the diversity to Prevotella buccae. This level of similarity is indicative of the of organisms present at the site. group representing a new species of Prevotella. Dentoalveolar abscesses are acute infections associated with the Group 9.4 showed 89.7% similarity to Eubacterium brachy. The teeth, arising from pulpal infection secondary to dental caries. oral asaccharolytic Eubacterium species are a heterogeneous group Cultural analyses have revealed they are associated with a reof organisms currently undergoing reclassification [5]; all of the stricted group of organisms, principally members of the genera currently recognized species are likely to be elevated to genus rank Fusobacterium, Peptostreptococcus, Prevotella, and Streptococcus. In the studies described here, we adapted methods for the detection of uncultivable bacteria in the environment to the analysis of the microflora associated with dentoalveolar abscesses. Table 1. Bacteria cultured from pus from dentoalveolar abscesses. Pus was aspirated from dentoalveolar abscesses by needle and