Development and Use of Oral Microbiome Optimised Oxford Nanopore 16S Primers to Elucidate the Effect of Sialidase Inhibitors on a Plaque Community Biofilm Model

The oral microbiome is a diverse ecosystem that plays a critical role in health and disease and contains numerous bacterial species capable of metabolizing host-derived glycans, particularly sialic acids. Sialidase enzymes can be produced by both commensal and pathogenic bacteria, influencing biofilm formation and host-interactions. To investigate how sialidase activity might influence the oral microbiome we conducted a series of in vitro polymicrobial biofilm experiments and assessed community composition using 16S rRNA sequencing. As a first step, we optimized and validated Oxford Nanopore Technology (ONT) as a sequencing platform using an in-house sequencing workflow and compared it to the standard Illumina MiSeq technology. Through in silico and in vitro assessments, we identified primer bias in the standard ONT 16S primers and designed human oral microbiome (HOM) modified primers (HOM_27F-YM/1492R-D) to improve taxonomic resolution, achieving results comparable to the gold-standard Illumina 16S primers, particularly for key oral taxa. These HOM-optimized primers had an overall lower error rate and generated community profiles that closely matched to those produced by Illumina. We then used the same ONT workflow and modified 16S primers to evaluate the effect of sialidase inhibitors Oseltamivir and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) on Hydroxyapatite (HA)-coated minimum biofilm eradication concentration (MBEC) assay plate derived plaque biofilms from a whole plaque community model. Inhibitor-treated biofilms exhibited differences in relative abundance depending on the inhibitor combination used, with increased abundance of Streptococcus with Oseltamivir alone and Fusobacterium with both inhibitors combined. These findings demonstrate that ONT-based 16S sequencing is a viable, scalable method for oral microbiome research and suggest that sialidase activity can modulate microbial community structure in plaque biofilms.