A Simple and Cost-Effective Protocol for Enriching 'Ca. Saccharimonadia' from Human Saliva to Enable Efficient Genome Sequencing

Background"Candidatus Saccharimonadia", a lineage within the Candidate Phyla Radiation (CPR), is characterized by small cell size, reduced genomes, and an epibiotic lifestyle dependent on bacterial hosts. Despite their presence in diverse environments, including the human oral microbiota, their role in human health remains largely unexplored. Observational studies have linked increased "Ca. Saccharimonadia" abundance in human saliva with inflammatory conditions such as periodontitis and inflammatory bowel disease, while recent in vivo evidence suggests potential anti-inflammatory properties. Investigating "Ca. Saccharimonadia" is challenging due to their fastidious nature, making standard culturing methods impractical. Current genomic studies rely on either co-cultivation with bacterial hosts or shotgun metagenomics, both requiring advanced technical expertise and costly resources. To address this limitation, we developed an affordable and efficient protocol for enriching "Ca. Saccharimonadia" from human saliva samples, facilitating genome sequencing. Our protocol, termed the "Ca. Saccharimonadia" Enrichment (SE) protocol, consists of two mandatory phases (detachment from host bacteria and weight/size-based separation) along with an optional DNA degradation phase. We validated this method on two human saliva samples. ResultsAfter SE protocol, the two saliva showed a substantial increase in "Ca. Saccharimonadia" DNA concentration. The enriched samples enabled high-quality metagenome-assembled genomes (MAGs) to be obtained via shotgun metagenomics. ConclusionsThis study presents a cost-effective and scalable approach to studying "Ca. Saccharimonadia", overcoming prior limitations and expanding opportunities for future research on their role in human health and disease.