OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo

Abstract

Carbapenem-resistance in Klebsiella pneumoniae (KP) sequence type ST258 is mediated by carbapenemases (e.g. KPC-2) and loss or modification of the major non-selective porins OmpK35 and OmpK36. However, the mechanism underpinning OmpK36-mediated resistance and consequences of these changes on pathogenicity remain unknown. By solving the crystal structure of a clinical ST258 OmpK36 variant we provide direct structural evidence of pore constriction, mediated by a di-amino acid (Gly115-Asp116) insertion into loop 3, restricting diffusion of both nutrients (e.g. lactose) and Carbapenems. In the presence of KPC-2 this results in a 16-fold increase in MIC to Meropenem. Additionally, the Gly-Asp insertion impairs bacterial growth in lactose-containing medium and confers a significant in vivo fitness cost in a murine model of ventilator-associated pneumonia. Our data suggests that the continuous selective pressure imposed by widespread Carbapenem utilisation in hospital settings drives the expansion of KP expressing Gly-Asp insertion mutants, despite an associated fitness cost.

Outbreak of KPC-producing Klebsiella pneumoniae ST15 strains in a Chinese tertiary hospital: resistance and virulence analyses

“…The strain that contained a nonsense mutation at amino acid position 248 (K248X) of OmpK36 was the highly carbapenem-resistant strain KP18069. However, a nonsense mutation of OmpK35 at amino acid position 63 (L63X) and the Gly115-Asp116 (GD) insertion of OmpK36 [19], which were identical to those of the highly carbapenem-resistant strain KP148 (CP040122) [18], were characterized in 100% (20/20) of the ST11 strains in this study.…”

“…In particular, the IPM MICs of 81.3% (26/32) of strains were 8 µg/mL or even less, which is considered to be the threshold of the high-dose continuous infusion of carbapenem (HDCI) strategy for anti-infection therapy [22]. The reason that KP18069 showed IPM and MEM MICs of both ≥ 64 µg/mL may be caused by the nonsense mutation (K248X) of OmpK36, which has been reported to be responsible for carbapenem resistance [19]. Thus, as the last choice to achieve complete killing of the KPC-producing K. pneumoniae ST15 strains, an HDCI strategy is suggested.…”

Genomic surveillance framework and global population structure forKlebsiella pneumoniae

“…In addition to the detection of carbapenemase genes, Kleborate also identified porin defects, which are known to contribute to the carbapenem-resistance phenotype 41,42 , in 36.5% of EuSCAPE genomes (including 60% of those with carbapenemase genes and 19.9% of those without carbapenemase genes). These defects included truncation/deletion of OmpK35 and/or OmpK36 (also considered in the original study) as well as GD or TD insertions in the OmpK36 β-strand loop 41 (not considered in original study, but here detected in 18.6% of genomes including 18 with no porin deletion).…”

“…It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted December 14, 2020. ; https://doi.org/10.1101/2020.12.14.422303 doi: bioRxiv preprint include fluoroquinolone resistance mutations in GyrA (codons 83 and 87) and ParC (codons 80 and 84), and colistin resistance from truncation or loss of MgrB and PmrB (defined as <90% amino acid sequence coverage). Mutations in the OmpK35 and OmpK36 osmoporins reportedly associated with reduced susceptibility to βlactamases 41,42 are also screened and reported for KpSC genomes, and include truncation or loss of these genes and OmpK36GD and OmpK36TD transmembrane βstrand loop insertions 41 . SHV β-lactamase, GyrA, ParC and OmpK mutations are identified by alignment of the translated amino acid sequences against a reference using BioPython, followed by interrogation of the alignment positions of interest (see Supplementary Text, Tables S12-S13 for a list of relevant positions).…”

Advances and Challenges in Studying Type III Secretion Effectors of Attaching and Effacing Pathogens

“…Indeed, native expression of a tagged effector is readily achievable by introducing a C-terminal tag onto the chromosome for effector visualization by immunofluorescence, or for use in tandem with co-immunoprecipitation and/or MS to probe for host protein interactors upon infection. Chromosomal manipulation by triparental conjugation works efficiently in A/E pathogens and other enteric pathogens (Mullineaux-Sanders et al, 2017 ; Watson et al, 2019 ; Wong et al, 2019 ). This conjugation protocol can similarly be used to generate scarless isogenic effector mutants in lieu of traditional gene disruption with antibiotic resistance cassettes or transposon elements (Cepeda-Molero et al, 2017 ).…”