Improved in silico and in vitro methods for Escherichia coli LPS outer core typing

The Gram-negative bacterial envelope comprises the outer membrane, periplasmic space with the peptidoglycan layer and inner (cytoplasmic) membrane. A lipopolysaccharide (LPS) layer linked to the outer membrane is essential for survival in most species of bacteria, primarily by providing structural stability and regulating selective chemical permeability. These functions make the LPS layer a key pathogenicity determinant, protecting bacteria from host defences. At the same time, LPS serves as a common receptor for multiple bacteriophage orders, making it a crucial point of bacterial vulnerability. LPS outer core typing is traditionally performed using immunoblotting and PCR. With the increasing availability of sequenced genomes, PCR has emerged as the primary method for typing. This study presents a set of nine oligonucleotides designed for typing the five LPS outer core structures of <i>Escherichia coli</i>, R1, R2, R3, R4 and K-12, both <i>in vitro</i> and <i>in silico</i>. Our method was able to successfully type 99% of strains within a comprehensive dataset of 4,549 complete genomes. Using these new methods, we identify previously unreported hybrid LPS structures involving R2 and K-12 outer core types and establish associations between LPS outer core types and <i>E. coli</i> phylogenetic groups, pathogenicity, O-antigen polysaccharides, sequence types and capsule types. We also introduce LPSTyper, a Python-based command-line tool that enables rapid and precise LPS outer core typing in <i>E. coli</i> genome sequences. Together, the expanded dataset of LPS outer core types and enhanced laboratory and computational tools for their detection provide new insights into LPS structural evolution and its role in <i>E. coli</i> ecology and pathogenicity. We anticipate that these tools will improve the efficiency of future research and diagnostics of this important bacterial envelope structure.