<i>In vivo</i> competition experiments in <i>K</i>. <i>pneumoniae</i> ATCC 13883 and porin mutants.

The relative fitness of porin mutants in comparison with parental strain (ATCC 13883) or between porin mutants was determined by competition experiments in co-cultures. The values for each mouse are represented individually. Violet diamond, ATCC 13883 wild type strains. Orange square, ΔOmpK35 mutant. Red square, ΔOmpK36 mutant. Green square, OmpK36GD mutant. Blue circle, ΔOmpK35ΔOmpK36 mutant. Pink circle, ΔOmpK35OmpK36GD mutant.</p

Lung infection experiments in <i>K</i>. <i>pneumoniae</i> ATCC 13883 and 10.85 as well as their isogenic porin mutants ΔOpmK35/OmpK36GD.

Survival curves after intranasal infection are represented in panels A1 (for ATCC 13883) and A2 (for 10.85). Organ burden after 24, 48 or 72h is represented in panels B1 to D1 for ATCC 13883 and B2 to D2 for 10.85. Violet diamond, ATCC 13883 or 10.85 wild type strains. Pink circle, ΔOmpK35OmpK36GD mutant. For survival studies the results were analysed using Long-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test. To compare bacterial load in organs during lung infection, results were compared using Mann-Whitney unpaired t tests. The analyses were performed using Prism7 (GraphPad Software). The differences between the wild type and the porin mutants were not statistically significant (P < 0.05) in any case.</p

<i>In vitro</i> competition experiments in <i>K</i>. <i>pneumoniae</i> ATCC 13883 and 10.85 porin mutants.

The relative fitness of porin mutants in comparison with parental strain (ATCC 13883 or 10.85) or between porin mutants was determined by competition experiments in co-cultures and expressed as a percentage of the mutant or wild type cells versus total population at each time point. In vitro growth conditions, MH broth with continuous shaking at 37°C. Violet diamond, ATCC 13883 or 10.85 wild type strains. Orange square, ΔOmpK35. Red square, ΔOmpK36 mutant. Green square, OmpK36GD mutant. Blue circle, ΔOmpK35ΔOmpK36 mutant. Pink circle, ΔOmpK35OmpK36GD mutant.</p

Carbapenem MICs against ATCC 13883 and porin mutants with <i>bla</i>_CTX-M-15, <i>bla</i>_IMP-4 or <i>bla</i>_KPC.

Carbapenem MICs against ATCC 13883 and porin mutants with blaCTX-M-15, blaIMP-4 or blaKPC.</p

Bacterial strains used in this study.

Bacterial strains used in this study.</p

Minimum spanning tree of 1,557 <i>K</i>. <i>pneumoniae</i> strains based on their MLST profile.

Each circle corresponds to a distinct ST, with its size being proportional to the number of strains of that particular ST (for scale, ST258 contains 552 isolates). Within an ST, the proportion of strains harbouring either a GD or TD insertion in the loop L3 of ompK36 is shown as a sector coloured in red and pink, respectively. STs carrying these mutations are also circled in grey.</p

Real-time RT-PCR in <i>K</i>. <i>pneumoniae</i> ATCC 13883 and 10.85 porin mutants.

The expression of rpoD was used to normalize results. The levels of expression of each mutant are shown relative to the wild type strain ATCC 13883 or 10.85.</p

Antibiotic MICs <i>K</i>. <i>pneumoniae</i> ATCC 13883 and porin mutants.

Antibiotic MICs K. pneumoniae ATCC 13883 and porin mutants.</p

Channel restriction of OmpK36 variants.

Comparison of the reference OmpK36 structure under PDB accession 5nupA (A) against predicted structural models of OmpK36 (B) and OmpK36GD mutant (C) from ATCC 13883, showing progressive restriction of the porin channel. The conformation visualised in panel B, in particular the loop 6 in yellow which can be seen partially obstructing the channel, is not associated with a carbapenem resistance phenotype, contrary to the GD mutant shown in panel C. Panel D consists of the multiple alignment of the 3 corresponding sequences, along with a representation of the predicted secondary structures designated as follows; B for barrel, T for turn, and L for loop. Signal peptide is not shown in 5nupA sequence (Panel D).</p

Maximum likelihood tree of 1,557 <i>K</i>. <i>pneumoniae</i> strains.

A phylogenetic tree was built using a 2,253,033 bp long core alignment. Contextual information relevant to the collection was visualized using Phandango and includes ST (of which the major ones are indicated on the tree); GD or TD insertion in the loop L3 of ompK36, in black and red, respectively; presence or absence of ompK36, in orange and purple, respectively; presence or absence of ompK35, in orange and purple, respectively. Additional metadata include year(date) of isolation, in a gradient from purple to yellow; source and geographical region of isolation in a rainbow gradient; and presence of major beta-lactamases (bla) alleles identified, in dark blue.</p