Klebsiella pneumoniae expresses two major porins: OmpK35 and OmpK36. These well-conserved proteins are located in the bacteria outer membrane permitting the entrance of nutrients and other hydrophilic molecules, such as beta-lactam antibiotics. They contain a well-conserved domain in the internal loop 3 (L3). Mutations in this domain may change the size or properties of the channel impairing the entrance of large electro-negative molecules, such as antibiotics, but not nutrients, conferring resistance to the bacteria. We have previously published that for some carbapenems, the resistance profile of K. pneumoniae was related to the presence of non-carbapenemase genes and OmpK36 porin defects: 1) absence of OmpK36, ΔOmpK36; or 2) presence of GlyAsp duplication in L3, OmpK36GDdup.
In order to understand the role of porins in antibiotic resistance and bacterial adaptation, we constructed a set of porin mutants in K. pneumoniae ATCC13883. Antibiotic resistance by micro-dilution method, growth curves and in vitro and in vivo competition experiments were performed to evaluate the consequences of porin loss in antimicrobial susceptibility and bacterial fitness. Porin expression, measured by real time RT-PCR, show that OmpK35 normally compensates OmpK36 absence but it is not up-regulated in an OmpK36GDdup background. Our data reveal that even in the absence of a carbapenemase gene, ΔOmpK36 or OmpK36GDdup increased K. pneumoniae carbapenem resistance to clinically relevant levels. Competition experiments show that OmpK36GDdup is fitter than ΔOmpK36 mutant. Finally, the analysis of GenBank K. pneumoniae porin sequences shows convergent evolution in OmpK36_L3 variants in unrelated strains.
Between the two more common OmpK36 mutations found in K. pneumoniae clinical isolates, ΔOmpK36 or OmpK36GDdup, the latter is more beneficial in terms of fitness, suggesting that OmpK36GDdup is better adapted. This mutation seems an efficient balance between resistance and fitness, what may explain the increasing proportion of OmpK36GDdup among K. pneumoniae clinical isolates.