Purified PCR products were sequenced with an ABI Prism cycle sequencing kit with a BigDye Terminator cycle sequencing reaction kit according to the manufacturer's instructions (PE Applied Biosystems, Foster City, CA). The primers used were 9F (see above) and 533R (TTACCGCGGCTGCTG) (14
). Sequencing reactions were run on an ABI model 3100 DNA sequencer. Approximately 500 bases were obtained first to determine identity or approximate phylogenetic position. Full sequences (about 1,500 bases with five or six additional sequencing primers were obtained for novel species (11
). The sequences were compared to the known sequences of >10,000 microorganisms in our database and 100,000 sequences in the Ribosomal Database Project (http://wdcm.nig.ac.jp/RDP/html/index.html
) and GenBank databases by BLAST (http://www.ncbi.nlm.nih.gov
). Phylogenetic trees were constructed by the neighbor-joining method of Saitou and Nei (15
). TREECON, a software package for the Microsoft Windows environment, was used for the construction and drawing of evolutionary trees (16
Our data demonstrate that the combination of BACTEC media for bacterial growth and 16S rRNA sequencing for bacterial identification allows the recovery of diverse and novel oral bacterial species from the blood following both tooth brushing and tooth extraction. With the BACTEC media for bacterial growth, we recovered 98 different species from the three groups (Fig. ). As expected, the predominant species recovered were Streptococcus spp. and other low-G+C Firmicutes. In addition, we recovered representatives from four other bacterial phyla, namely, Fusobacteria, Actinobacteria, Bacteroidetes (e.g., Prevotella spp.), and Proteobacteria (e.g., including species of Eikenella, Campylobacter, and Haemophilus). The most predominant species recovered were Streptococcus spp., followed by Peptostreptococcus micros, Veillonella dispar or V. parvula and Dialister pneumosintes. Staphylococcus spp. were detected in only 9 (0.5%) of the ~1,800 blood samples processed in this study, and they were most likely from skin contamination during blood drawing. Using the LYTIC 10 Anaerobic/F BACTEC media also allowed the detection of a high percentage of anaerobes, e.g., 9% genus Prevotella, 5% genus Actinomyces, and 5% genus Fusobacterium. We also identified 19 species or phylotypes which had not been cultivated prior to this study (highlighted in red and blue in Fig. ), of which 3 were first recovered in this study (highlighted in red in Fig. ).
FIG. 1. Phylogenetic tree of bacterial species and phylotypes identified in the blood following tooth brushing and tooth extraction with or without antibiotic prophylaxis. Strains in red are putative new species from this study. Species in blue are cultivable (more ...)
The advantage of BACTEC media for bacterial isolation from blood over the traditionally used lysis filtration method has also been documented in prior studies. For example, Lucas et al. reported obtaining a greater range of bacterial species by using the BACTEC system compared to the cell lysis filtration technique (8
). In another study, Heimdahl et al. used lysis filtration for bacterial recovery from blood and found that bacteremia did not extend much beyond 10 min after a dental procedures (5
). However, in our results with BACTEC media for 151 bacteremic subjects, 20% were still bacteremic at 20 min, 9% at 40 min, and 6% at 60 min. This suggests that the BACTEC method is superior to the lysis filtration method for detecting low levels of bacteremia.
Antibiotic prophylaxis reduced the incidence of bacteremia from dental extraction (Table ). It also resulted in bacteremia with fewer bacterial species, which were cleared from the blood in a shorter time (i.e., mostly within 20 min) (Fig. ). Although antibiotic prophylaxis reduced the bacteremia of several species of streptococci, as expected, it does not seem to affect species of proteobacteria (e.g., Eikenella corrodens) and Prevotella.
Bacterial species found (i.e., ≥2%) in blood samples following tooth brushing and dental extraction