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Background. The bacterial flora involved in brain abscess is often complex. In a previous study, using a metagenomic approach based on 16S ribosomal DNA (rDNA) amplification, we demonstrated that the diversity of the microbial flora involved in these infections was underestimated.Methods. We performed a 16S rDNA-based metagenomic analysis of cerebral abscesses from patients diagnosed from 2006 through 2010. All bacteria present in brain abscess specimens were identified, in view of the clinical and epidemiological characteristics of the patients.Results. Fifty-one patients were included in our study. By detecting polymicrobial infections in 19 patients, our strategy was significantly more discriminatory and enabled the identification of a greater number of bacterial taxa than did culture and conventional 16S rDNA polymerase chain reaction (PCR) and sequencing, respectively (P < 10−2). Data mining discriminated 2 distinct bacterial populations in brain abscess from dental and sinusal origin. In addition, of the 80 detected bacterial species, we identified 44 bacteria that had never been found in brain abscess specimens, including 22 uncultured bacteria. These uncultured agents mostly originated from the buccal or sinusal floras (P < 10−2) and were found in polymicrobial specimens (P < 10−2).Conclusions. Cloning and sequencing of PCR-amplified 16S rDNA is a highly valuable method to identify bacterial agents of brain abscesses.