Size-based sorting of dynamic bacterial clusters

Group A Streptococcus (GAS) forms highly deformable aggregates with broad variations in size and morphology, complicating controlled separation and biological analysis. Reliable methods to isolate fractions of GAS clusters with defined properties are essential for studying host-pathogen interactions that depend on cluster size. Here, we present a simple deterministic lateral displacement (DLD) microfluidic device to separate complex suspensions of bacterial aggregates into two size-enriched fractions. We use a DLD with a small displacement angle to accommodate the large range of particle sizes above the critical size. We introduce an intermediate outlet, in addition to the conventional zigzag and displacement outlets, to collect the aggregates which exhibit a large dispersion due to their broad variety in shape and sizes close to the device critical diameter. In this way, we can demonstrate fractionation of GAS clusters with >90% purity based on effective size while causing minimal fragmentation or additional aggregation, as demonstrated by image analysis and dual-colour experiments. Finally, we show biological relevance through a live immune-cell assay, where human immune cells migrate more rapidly in the presence of larger GAS clusters than in smaller clusters or single bacteria. These results demonstrate that DLD-based separation provides biologically meaningful fractions of bacterial aggregates and enables new analyses of how cluster size influences immune responses.