Klebsiella pneumoniae outer membrane porins OmpK35 and OmpK36 play roles in both antimicrobial resistance and virulence

Abstract

OmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. In this study, a virulent clinical isolate was selected to study the role of these two porins in antimicrobial resistance and virulence. The single deletion of ompK36 (ΔompK36) resulted in MIC shifts of cefazolin, cephalothin, and cefoxitin from susceptible to resistant, while the single deletion of ompK35 (ΔompK35) had no significant effect. A double deletion of ompK35 and ompK36 (ΔompK35/36) further increased these MICs to high-level resistance and led to 8- and 16-fold increases in the MICs of meropenem and cefepime, respectively. In contrast to the routine testing medium, which is of high osmolarity, susceptibility tests using low-osmolarity medium showed that the ΔompK35 mutation resulted in a significant (≥ 4-fold) increase in the MICs of cefazolin and ceftazidime, whereas a ΔompK36 deletion conferred a significantly (4-fold) lower increase in the MIC of cefazolin. In the virulence assays, a significant (P < 0.05) defect in the growth rate was found only in the ΔompK35/36 mutant, indicating the effect on metabolic fitness. A significant (P < 0.05) increase in susceptibility to neutrophil phagocytosis was observed in both ΔompK36 and ΔompK35/36 mutants. In a mouse peritonitis model, the ΔompK35 mutant showed no change in virulence, and the ΔompK36 mutant exhibited significantly (P < 0.01) lower virulence, whereas the ΔompK35/36 mutant presented the highest 50% lethal dose of these strains. In conclusion, porin deficiency in K. pneumoniae could increase antimicrobial resistance but decrease virulence at the same time.

FIG. 1.

Creation of the porin deletion and complemented strains. (A) Physical map of the ompK35 and ompK36 genes. The positions of the putative promoters (arrows) and terminators (stem-loop structures) identified are highlighted. DNA fragments KO (dotted lines) are the region to be deleted from porin genes. DNA fragments 1 and 2 (dark lines) are the flanking regions of fragment KO. (B and C) Amplify the DNA fragments from K. pneumoniae NVT2001S using PCR. The generated DNA fragments were ligated into the suicide vector pKAS46, resulting in plasmid pKO35, pKO36, pCOM35, and pCOM36. The porin deletion (B) and complemented (C) constructs were transformed into K. pneumoniae to generate the porin deletion and complemented strains, respectively. (D) A single recombination event through DNA fragment 1 or 2 gave rise to one of two possible single-crossover strains. (E) The second recombination event can result in either restoration of the origin gene or a mutant double-crossover strain.

FIG. 2.

OMP profiles (A) and phenotype comparison (B) of evaluated strains. (A) OMP profiles from strains grown in high-osmolarity MHB (lane 1) and low-osmolarity NB (lanes 2 to 8). Lanes 1 and 2, NVT2001S; lane 3, ΔompK35 strain; lane 4, ΔompK36 strain; lane 5, ΔompK35/36 strain; lane 6, ΔompK35/36::35 strain; lane 7, ΔompK35/36::36 strain; lane 8, ΔompK35/36::35/36 strain. (B) The K. pneumoniae stains on blood agar incubated at 37°C overnight. When the concentrated regions of colonies are compared on blood agar, the phenotype of these OmpK36-loss strains formed more glistening colonies than that of strains without OmpK36 loss.

FIG. 3.

Effect of OmpK35 and OmpK36 on bacterial growth (A1 and A2), susceptibility to normal human serum (B1 and B2), and phagocytosis (C1 and C2). (A1, A2, B1, and B2) The data illustrated are representative of three independent experiments, all of which showed the similar results. (A1 and A2) Generation time (derived from the three growth curve data between 1 and 3 h) for OmpK35/36-loss strain was about 30 ± 0.5 min, while that for other strains was all about 27 ± 0.5 min. (B1 and B2) Generation times (derived from the three growth curve data between 2 and 6 h) for OmpK35/36-loss strain was about 76 ± 4 min, while that for other strains was all about 57 ± 3 min. (C1 and C2) Each point on the curves represents the means of three independent experiments ± the standard deviation. In time points of 15 and 30 min, the phagocytic resistances of both OmpK36-loss and OmpK35/36-loss strains were significantly (P < 0.05) lower than those of OmpK35-loss and OmpK35/36-non-loss strains.

FIG. 4.

Effect of OmpK35 and/or OmpK36 loss on mice lethality. The survivals after intraperitoneal injection with 3 × 10² CFU of K. pneumoniae were counted over 14 days. The data points represent the percentage of mice surviving in each experimental group over time (n = 12 mice per strain). In experiment A or B, each of the strains was inoculated at different groups of 6 mice at the same day, while the data illustrated are pooled from two independent experiments. (A) Survival curves for K. pneumoniae NVT2001S and its porin deletion strains. (B) Survival curves for K. pneumoniae ΔompK35/36 and its porin-complemented strains. In both experiments, the survival curves for both OmpK36-loss and OmpK35/36-loss strains were significantly (log-rank test, P < 0.01) different from that for the OmpK35/36-non-loss strain, while no significantly different was found between the curves for the OmpK35-loss strain and OmpK35/36-non-loss strain.