Reduced-Complexity Cultivation of Human Oral Anaerobic Bacteria Using a Microdroplet-Based Culturomics Platform

The human oral microbiota contains fastidious anaerobes that remain uncultivated due to resource competition and overgrowth by dominant species in conventional plating. Culturomics approaches leveraging droplet-based microfluidics offer an alternative by physically compartmentalizing microbial cells. We established a polydimethylsiloxane-based microdroplet platform to isolate anaerobic bacteria from human oral rinse samples. Droplet generation conditions were optimized using fluorescent beads, and bacterial loading at the collection-drop level was evaluated using Escherichia coli. Two downstream isolation strategies-Direct-plating (Method 1) and Enrichment (Method 2)-were compared against conventional serial dilution across various media compositions. Isolated strains were identified via 16S rRNA gene sequencing. At optimized flow rates (oil: 1.5 mL/h, core: 0.3 mL/h), the system produced uniform microdroplets with an average occupancy (λ) of 0.010-0.012 cells/droplet, corresponding to a high theoretical probability of single-cell occupancy among non-empty microdroplets. Each macro-collection drop delivered approximately 50-60 cells into recipient wells, serving as a reduced-complexity cultivation unit. Under the matched solid-medium condition, Method 1 achieved the highest taxonomic yield (14.3%), representing a 3.5-fold enhancement over conventional serial dilution (4.1%). Method 2 yielded 4.7%, indicating that liquid enrichment reintroduced growth bias toward fast-growing taxa. Among the tested media, unsupplemented Tryptic Soy Agar preserved the highest species diversity, whereas 5% sheep blood supplementation led to monoculture dominance by fast-growing taxa. Overall, the platform isolated diverse oral anaerobic species including fastidious taxa like Capnocytophaga, Cardiobacterium, and Eikenella. The microdroplet-based reduced-complexity platform may limit the dominance of rapidly growing taxa, providing a complementary culturomics tool for expanding human oral anaerobic bacterial resources.