High-throughput cytological profiling uncovers genotype-phenotype associations in clinical isolates.

Abstract:

Understanding the functional impact of bacterial genetic diversity is crucial for linking pathogen variants to clinical outcomes. Here, we introduce a high-throughput cytological profiling pipeline optimized for (Mtb) clinical strains, integrating OD-calibrated feature analysis and high-content microscopy. Our system quantifies single-bacterium morphological and physiological traits related to DNA replication, redox state, carbon metabolism, and cell envelope dynamics. Applied to 64 Mtb clinical isolates from lineages 1, 2, and 4, the approach revealed that cytological phenotypes recapitulate genetic relationships and exhibit both lineage- and density-dependent dynamics. Notably, we identified a link between a convergent “small cell” phenotype and a convergent mutation that is associated with the presence of an antisense transcript, suggesting a potential non-canonical regulatory mechanism under selection. In summary, we present a resource-efficient approach for mapping Mtb’s phenotypic landscape, uncovering cellular traits that underlie its evolution and providing new insights into the functional consequences of bacterial genetic diversity.