Molecular diagnosis of polymicrobial brain abscesses with 16S-rDNA-based next-generation sequencing

Area of Science:

• Microbiology
• Infectious Diseases
• Genomics

Background:

• Brain abscesses present a high mortality rate, challenging conventional antibiotic and surgical treatments.
• Accurate identification of causative bacteria is crucial for effective antibiotic therapy but is often hindered by prior antibiotic exposure and the complex polymicrobial nature of these infections.
• Traditional culture-based methods and Sanger sequencing of 16S PCR products have limitations in detecting the full spectrum of bacteria in brain abscesses.

Purpose of the Study:

• To evaluate the utility of 16S rRNA-based next-generation sequencing (NGS) for metagenomic analysis of intracranial abscesses (brain and epidural) and meningitis.
• To compare the diagnostic yield of NGS with conventional methods like Gram staining, culture, and Sanger sequencing.

Main Methods:

• Analysis of 79 samples from 54 patients with intracranial abscesses or meningitis using 16S rRNA gene PCR and Illumina MiSeq sequencing.
• Bioinformatic analysis of sequence reads using BLAST against the NCBI 16S bacterial database and MEGAN software.
• Establishment of a cut-off criterion using monobacterial meningitis samples to exclude contaminating sequences.

Main Results:

• The NGS workflow was successfully applied to 51 intracranial abscesses and 9 meningitis samples.
• NGS identified 86 bacterial taxa in brain abscesses, with Streptococcus intermedius and Fusobacterium nucleatum being the most prevalent species.
• Polymicrobial infections were detected in 31/51 intracranial abscesses, with NGS identifying up to 16 bacterial taxa per sample, revealing a more comprehensive microbial profile than traditional methods.

Conclusions:

• Next-generation sequencing (NGS) significantly expands the spectrum of bacteria detected in brain abscesses compared to conventional methods.
• The Illumina MiSeq platform is a suitable and powerful tool for metagenomic diagnostics of severe infections like brain abscesses.
• NGS provides valuable insights into the polymicrobial etiology of brain abscesses, aiding in more targeted therapeutic strategies.