Massive Parallel Sequencing Provides New Perspectives on Bacterial Brain Abscesses

Abstract

m Rapid development within the field of massive parallel sequencing (MPS) is about to bring this technology within reach for diagnostic microbiology laboratories. We wanted to explore its potential for improving diagnosis and understanding of polymicrobial infections, using bacterial brain abscesses as an example. We conducted a prospective nationwide study on bacterial brain abscesses. Fifty-two surgical samples were included over a 2-year period. The samples were categorized as either spontaneous intracerebral, spontaneous subdural, or postoperative. Bacterial 16S rRNA genes were amplified directly from the specimens and sequenced using Ion Torrent technology, with an average of 500,000 reads per sample. The results were compared to those from culture-and Sanger sequencing-based diagnostics. Compared to culture, MPS allowed for triple the number of bacterial identifications. Aggregatibacter aphrophilus, Fusobacterium nucleatum, and Streptococcus intermedius or combinations of them were found in all spontaneous polymicrobial abscesses. F. nucleatum was systematically detected in samples with anaerobic flora. The increased detection rate for Actinomyces spp. and facultative Gram-negative rods further revealed several species associations. We suggest that A. aphrophilus, F. nucleatum, and S. intermedius are key pathogens for the establishment of spontaneous polymicrobial brain abscesses. In addition, F. nucleatum seems to be important for the development of anaerobic flora. MPS can accurately describe polymicrobial specimens when a sufficient number of reads is used to compensate for unequal species concentrations and principles are defined to discard contaminant bacterial DNA in the subsequent data analysis. This will contribute to our understanding of how different types of polymicrobial infections develop. O ur understanding of polymicrobial infections has been hindered by our limited possibilities for describing them. Recent investigations of bacterial brain abscesses using universal amplification of the bacterial 16S rRNA gene, followed by Sanger sequencing of cloned amplicons, have revealed that only a fraction of the bacteria present are identified by culture (1, 2). Nevertheless, this approach has limitations when it comes to detecting smaller subpopulations in a multispecies community, unless very high numbers of clones are sequenced (3). This is problematic, since the species structure of an abscess may change over time and pathogens important for establishing the infection potentially remain at only low concentrations in the more mature abscesses. Furthermore, the species that are important for maintaining and expanding the abscess might primarily exist close to the abscess wall and do not necessarily dominate in the pus obtained by aspiration. Rapid development within the field of massive parallel sequencing technologies (MPS) is about to provide the diagnostic laboratories with tools that can characterize even the most complex microbial communities. The aim of the present study was to use recent adva...

Actinomyces and Related Organisms in Human Infections

“…Based on data reported recently by Kommedal et al (15) (Table 2), 4 to 10 species were found in specimens from 12 spontaneous brain abscesses with an involvement of A. meyeri. In these specimens, Fusobacterium nucleatum, Parvimonas micra, and Streptococcus intermedius were the major concomitants, but also, Aggregatibacter aphrophilus, Campylobacter gracilis, Eikenella corrodens, and Eubacterium brachy were each detected in at least half of the specimens (15). All these organisms are inhabitants of the oral cavity.…”

“…In a recent comprehensive nationwide study conducted in Norway, the bacteriology of brain abscess specimens collected over a 2-year period was examined by culture and direct 16S rRNA gene sequencing, and positive specimens (n ϭ 52) were reanalyzed by using massive parallel sequencing (15). Of these specimens, 37 came from spontaneous brain abscesses, 3 were from spontaneous subdural infections, and 12 were from postoperative infections.…”

“…Although actinomycosis is relatively rare, at least in Western populations (8), recently reported observations implicating A. meyeri in brain abscesses (15) and Actinobaculum schaalii (currently Actinotignum schaalii) in urosepsis (16) and the introduction of advanced microbiological techniques, which can identify even very fastidious organisms, have resulted in an increased awareness of Actinomyces and other Gram-positive, non-sporeforming bacilli in clinical microbiology.…”

Characterization of the Pathogenicity of Streptococcus intermedius TYG1620 Isolated from a Human Brain Abscess Based on the Complete Genome Sequence with Transcriptome Analysis and Transposon Mutagenesis in a Murine Subcutaneous Abscess Model

“…Massively parallel sequencing is a powerful tool for the simultaneous identification of multiple bacteria. Kommedal et al suggested that Aggregatibacter aphrophilus, Fusobacterium nucleatum, and S. intermedius are key pathogens in the establishment of spontaneous polymicrobial brain abscesses; in particular, S. intermedius was frequently detected, with 24 S. intermedius-positive cases occurring among 52 patients (6).…”

Two Rapidly Growing Mycobacterial Species Isolated from a Brain Abscess: First Whole-Genome Sequences of Mycobacterium immunogenum and Mycobacterium llatzerense

“…Recent studies have also indicated that synergistic infectivity in polymicrobial populations may play an important role in the pathogenesis of brain abscesses, and it is possible that the mycobacterial species may have contributed to brain abscess formation in this patient (20,21). The increasing availability of molecular methods, such as whole-genome and metagenomic sequencing, to elucidate the microbial composition of brain abscesses is likely to drive the future development of optimal treatment strategies for these complex polymicrobial infections (22,23).…”