The Impact of Insertion Sequences on O-Serotype Phenotype and Its O-Locus-Based Prediction inKlebsiella pneumoniaeO2 and O1

Klebsiella pneumoniaeis a nosocomial pathogen, pointed out by the World Helth Organisation (WHO) as "critical" regarding the highly limited options of treatment. Lipopolysaccharide (LPS, O-antigen) and capsular polysaccharide (K-antigen) are its virulence factors and surface antigens, determining O- and K-serotypes and encoded by O- or K-loci. They are promising targets for antibody-based therapies (vaccines and passive immunization) as an alternative to antibiotics. To make such immunotherapy effective, knowledge about O/K-antigen structures, drift, and distribution among clinical isolates is needed. At present, the structural analysis of O-antigens is efficiently supported by bioinformatics. O- and K-loci-based genotyping by polymerase chain reaction (PCR) or whole genome sequencing WGS has been proposed as a diagnostic tool, including the Kaptive tool available in the public domain. We analyzed discrepancies for O2 serotyping between Kaptive-based predictions (O2 variant 2 serotype) and the actual phenotype (O2 variant 1) for twoK. pneumoniaeclinical isolates. Identified length discrepancies from the reference O-locus resulted from insertion sequences (ISs) withinrfbregions of the O-loci. In silico analysis of 8130 O1 and O2 genomes available in public databases indicated a broader distribution of ISs inrfbs that may influence the actual O-antigen structure. Our results show that current high-throughput genotyping algorithms need to be further refined to consider the effects of ISs on the LPS O-serotype.