Anaerobic bacteria dominate the cholesteatoma tissue of chronic suppurative otitis media patients.

The aim of this study was to investigate both the microbial composition and absolute abundance of clinically relevant bacteria in tissue specimens from patients with chronic suppurative otitis media with cholesteatoma (CSOM with cholesteatoma). Mastoid mucosa and cholesteatoma tissue from eleven subjects with CSOM with cholesteatoma, and mastoid mucosa from ten controls were examined using standard hospital culture swabs, Gram staining, bacterial 16S rRNA gene sequencing, Droplet Digital PCR (ddPCR), and multiplex PCR. Positive results from culture swabs were reported in half the CSOM with cholesteatoma samples and 1 control sample. In contrast, ddPCR detected bacterial genes copies in all 11 mucosa and cholesteatoma of CSOM subjects and 3 control samples. The average bacterial gene copies in tissue samples with CSOM with cholesteaotoma (1.6 ± 0.7 log10) was significantly higher compared to healthy controls (0.3 ± 1.6). These results were corroborated with Gram-staining that identified the large presence of Gram-positive cocci cells in the cholesteatoma tissue of CSOM subjects which were not seen in the mucosa of controls. The most abundant genus detected by sequencing in the mucosa and cholesteatoma of CSOM samples was Anaerococcus (93.5 % of all reads), and genus Meiothermus (0.9 %) in the control sample. The 3 samples with the highest sequencing reads (>300) were further analysed using multiplex PCR to identify the dominant Anaerococcus species. Anaerococcus hydrogenalis was the dominant species identified in these samples. In contract, commonly named ear pathogens, genera Staphylococcus and Pseudomonas, were detected in low numbers (<0.001 % of all sequencing reads) and low prevalence (2/16 samples) in the tissue samples of this study. The results show that culture severely underestimated the bacterial diversity in CSOM samples and investigating tissue rather than standard culture swabs might be advantageous to understanding the disease process. The high abundance of bacteria and the large presence of Gram-positive cells detected in the cholesteatoma tissue of CSOM compared to mucosa of CSOM or controls could be members from the genus Anaerococcus. Anaerococcus may well be a pathogen in CSOM with cholesteatoma, but their role in this condition requires further investigation.