Widespread emergence of OmpK36 loop 3 insertions among multidrug-resistant clones of Klebsiella pneumoniae

Abstract

Mutations in outer membrane porins act in synergy with carbapenemase enzymes to increase carbapenem resistance in the important nosocomial pathogen, Klebsiella pneumoniae (KP). A key example is a di-amino acid insertion, Glycine-Aspartate (GD), in the extracellular loop 3 (L3) region of OmpK36 which constricts the pore and restricts entry of carbapenems into the bacterial cell. Here we combined genomic and experimental approaches to characterise the diversity, spread and impact of different L3 insertion types in OmpK36. We identified L3 insertions in 3588 (24.1%) of 14,888 KP genomes with an intact ompK36 gene from a global collection. GD insertions were most common, with a high concentration in the ST258/512 clone that has spread widely in Europe and the Americas. Aspartate (D) and Threonine-Aspartate (TD) insertions were prevalent in genomes from Asia, due in part to acquisitions by KP sequence types ST16 and ST231 and subsequent clonal expansions. By solving the crystal structures of novel OmpK36 variants, we found that the TD insertion causes a pore constriction of 41%, significantly greater than that achieved by GD (10%) or D (8%), resulting in the highest levels of resistance to selected antibiotics. We show that in the absence of antibiotics KP mutants harbouring these L3 insertions exhibit both an in vitro and in vivo competitive disadvantage relative to the isogenic parental strain expressing wild type OmpK36. We propose that this explains the reversion of GD and TD insertions observed at low frequency among KP genomes. Finally, we demonstrate that strains expressing L3 insertions remain susceptible to drugs targeting carbapenemase-producing KP, including novel beta lactam-beta lactamase inhibitor combinations. This study provides a contemporary global view of OmpK36-mediated resistance mechanisms in KP, integrating surveillance and experimental data to guide treatment and drug development strategies.

Omic insights into various ceftazidime-avibactam-resistant Klebsiella pneumoniae isolates from two southern Italian regions

“…In our study, we confirmed that, in KPC producing- K. pneumoniae strains, mutations in the porine gene omp K36 were present and, as previously observed by other Authors ( Venditti et al., 2021 ) presumably correlate with high-level resistance to carbapenems ( Wong et al., 2019 ; David et al., 2022 ). In addition to the indel found in the omp K36 gene, our isolates harbored frameshift mutations in omp K35 and omp K37, leading to nonfunctional proteins, associated, also in this case, with lower carbapenemase MICs, as already described by other Authors ( Gaibani et al., 2020 ; Venditti et al., 2021 ).…”

The interaction of the F-like plasmid-encoded TraN isoforms with their cognate outer membrane receptors

“…We have recently shown that a GD insertion in L3 of OmpK36 in the recipient affected MPS formation with donors harbouring TraNβ 15 . More recently, while screening for other L3 insertions in a global genome collection of 16,086 K. pneumoniae isolates, we found Threonine-Aspartate (TD) and Aspartate (D) insertions in the important ST16 and ST231 lineages, respectively, which causes pore constriction of 41% and 8%, respectively 24 . Isogenic strains expressing these L3 insertions were recently generated from our wild type (WT) K. pneumoniae strain, ICC8001, itself derived from ATCC 43816 to study the impact of pore constriction on bacterial fitness and resistance to carbapenems 24 .…”

“…B . The crystal structure of OmpK36 WT+D adopts an open (shown in red) and closed (shown in blue) conformation (PDB ID: 7Q3T) 24 . Both conformations restrict the pore diameter by 8% relative to the WT protein.…”

“…In contrast, the naturally occurring D insertion in L3 had an intermediate conjugation efficiency phenotype, between the GD/TD insertion and the WT OmpK36. We have previously shown that the OmpK36 WT+D structure adopted two conformations, open or closed, with similar minimal pore diameters of 2.95 Å and 2.94 Å, respectively 24 . AlphaFold2 predicted the interaction of TraNβ with the L3 of OmpK36 WT+D in its open conformation (similar to the crystal structure), providing a plausible explanation on the propensity of L3 to adopt two conformations; when L3 adopts the open conformation, MPS can occur whereas the closed conformation causes steric clashes and prevents the insertion of TraNβ inside the pore.…”

“…More recently, while screening for other L3 insertions in a global genome collection of 16,086 K. pneumoniae isolates, we found Threonine-Aspartate (TD) and Aspartate (D) insertions in the important ST16 and ST231 lineages, respectively, which causes pore constriction of 41% and 8%, respectively 24 . Isogenic strains expressing these L3 insertions were recently generated from our wild type (WT) K. pneumoniae strain, ICC8001, itself derived from ATCC 43816 to study the impact of pore constriction on bacterial fitness and resistance to carbapenems 24 . The strains were engineered such that they do not express the alternative major porin OmpK35 as expression of this protein is frequently lost in clinical isolates.…”