Phenotypic and genetic analysis of Klebsiella pneumoniae strains isolated from community-acquired pneumonia patients in Rostov-on-Don in 2021–2023

Introduction. Here, we present a study of 33 Klebsiella pneumoniae strains isolated in Rostov-on-Don in 2021–2023 from patients with community-acquired pneumonia. The bacterial strains were analyzed according to the traits known to be linked to hypervirulence. The aim of the study was to compare of the strains by analyzing phenotypic (siderophore activity, hypermucoviscosity, bacteriophage sensitivity) and genotypic (presence of plasmids, siderophore genes, and rmpA and rmpA2 genes) properties. Materials and methods. Assessment of bacteriophage sensitivity, hypermucoviscosity using “string test”, siderophore activity on chrome azurol S containing plates, high-molecular weight plasmids content, and whole-genome sequencing. Results. Sequencing of 11 strains differing in mucoviscosity allowed to establish that all hypermucoviscous strains contained the rmpA gene, whereas the rmpA2 gene was either absent or contained single nucleotide insertions or deletions, leading to a reading frame shift. The same mutations in rmpA2 were observed in non-mucoviscous strains, all of which lacked the rmpA gene. The strains differed by the presence of four siderophore clusters, the number of which did not correlate with the siderophore activity. The lack of rmpA and salmochelin biosynthesis genes but presence of its receptor gene in the non-mucoviscous strains suggest that they have deletions, leading to the loss of the hypermucoviscous phenotype. A study of 33 strains showed that they were able to dissociate, forming dark and light colonies, which were observed in both hypermucoviscous and non-hypermucoviscous strains. In dark but not light clones obtained from hypermucoviscous strains, this property was preserved. In contrast, both clone variants of non-hypermucoviscous strains retained this property. An analysis of different clones of 17 strains showed that dark vs light clones had reduced siderophore activity and bacteriophage sensitivity. The clone genomes did not differ in the siderophore clusters, but rmpA was revealed only in the dark clones of hypermucoviscous strains. In non-mucoviscous strains, this gene was not found in both clones, whereas differences in siderophore activity and bacteriophage sensitivity were preserved. Conclusion. K. pneumoniae hypermucoviscosity phenotype is associated with the presence of at least the rmpA, while intact rmpA2 is not required. The differences in the colony morphology, clone siderophore activity, and bacteriophage sensitivity are not related to rmpA and rmpA2, but rather result from an unknown yet mechanism.