Direct detection of bacterial pathogens in brain abscesses by polymerase chain reaction amplification and sequencing of partial 16s ribosomal deoxyribonucleic acid fragments

Abstract

Objective: To evaluate the feasibility of detecting bacterial pathogens directly from the clinical brain abscess specimens by polymerase chain reaction (PCR) amplification and sequencing of bacterial 16S ribosomal deoxyribonucleic acid (rDNA).

Methods: A total of 14 specimens were tested by both culture and PCR amplification, targeting the full-length or a partial region of 16S rDNA. 16S rDNA is known to be conserved in bacteria. Sequencing of partial-length and full-length 16S rDNA was performed. The sequence data were compared with known sequences of 16S rDNA in the National Center for Biotechnology Information GenBank by using the Basic Local Alignment Search Tool (BLAST) algorithm. The species with the best match of similarity were regarded as the pathogenic species in the samples. We also developed a Streptococcus-specific multiplex PCR analysis for identifying members of the Streptococcus species, the most common pathogen of brain abscesses.

Results: The 10 culture-positive specimens were all PCR-positive for partial 16S rDNA, but only seven were positive for full-length 16S rDNA amplification. Bacterial DNA was not detected in the remaining four specimens with a negative culture. Species identification by phenotypes from culture was in agreement with that by sequencing results of partial-length (or full-length) 16S rDNA. The Streptococcus-specific PCR analysis could detect Streptococcus species correctly in one step.

Conclusion: Bacterial 16S rDNA sequences provide reliable clues to the identification of unknown pathogens. PCR analysis of 16S rDNA and sequencing may identify pathogens to the species level directly from brain abscesses. This approach is rapid and is useful especially in the identification of slow-growing and fastidious organisms.