Multiphase flow-enabled microfluidic sorting reveals cellular chain length-dependent pathogenic heterogeneity

Delineating morphology-related pathogenic heterogeneity of micro- and nano-sized pathogens remains challenging due to the absence of precise and gentle sorting methods. Here, we report a multiphase flow-enabled microfluidic approach for label-free, high-resolution sorting of Streptococcus mutans (S. mutans) based on cellular morphology. By co-flowing viscoelastic fluid (S. mutans spiked with polyethylene oxide, PEO) and Newtonian fluid (PBS) in the microchannel, the synergistic effect of elastic and inertial lift forces leads to chain length-dependent migration and sorting of S. mutans (0.5 μm in diameter) into short- (0.6–2 μm), medium- (2–4.5 μm), and long-chain (4.5–15 μm) subpopulations, achieving separation efficiencies > 77.8 %, purities > 85 %, and viabilities > 98.5 %. Notably, we reveal that the cellular chain length is a critical factor for pathogenicity of S. mutans. The long-chain subpopulation exhibits enhanced biofilm formation, stronger resistance to phagocytosis, and higher MIC₅₀ of antibacterials compared to medium- and short-chain subpopulations. Our microfluidic sorting method offers a new tool for studying pathogenic heterogeneity.