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.

Fig. 1

OmpK35_ST258 and OmpK36_ST258 porin variants do not impact on growth in vitro. a The OmpK35_ST258 frame-shift mutation results in a premature TGA stop codon. Sequence conservation with OmpK35_WT is lost within the first β-strand resulting in a truncated protein. b OmpK36 sequences share 95% identity, with two insertions (GD in loop 3 and LSP in loop 4) in OmpK36_ST258 (highlighted in green). The underlined PEFGGD is a conserved loop 3 motif in OmpK36 porins. c Outer membrane preparations demonstrate a loss of OmpK35 in ICC8003 and ICC8004. OmpK36_WT (ICC8001 and ICC8003) and OmpK36_ST258 (ICC8002 and ICC8004) are present in similar abundance in isogenic strains. d, e Growth, measured by OD₆₀₀, is not affected by the introduction of ST258 porins into ICC8001 in rich (Luria Bertani) media (d) or minimal (M9) media (e) with glucose as the sole carbon source (n = 3 repeats, error bars = s.d.)

Fig. 2

The impact of OmpK35_ST258 and OmpK36_ST258 substitution on resistance to antibiotics used in Gram-negative infections. a Meropenem, b Imipenem and c Ertapenem broth minimum inhibitory concentrations presented graphically in isogenic strains expressing the KPC-2 carbapenemase. Dotted lines represent sensitive and resistant EUCAST breakpoints. d Broth MIC of the isogenic KP strains expressing the KPC-2 or OXA-48 carbapenemase. Individual values are colour coded (green—sensitive, yellow—intermediate and red—resistant) according to their antibiotic resistance defined by EUCAST breakpoints. Antibiotic key: IPM Imipenem, MEM Meropenem, ETP Ertapenem. e Resistance to other antibiotics in different classes tested. Individual values are colour coded (green—sensitive, yellow—intermediate and red—resistant) according to their antibiotic resistance defined by EUCAST breakpoints. Antibiotic key: AMP Ampicillin, AMC Amoxicillin/Clavulanate (2:1), TZP Pipericillin/Tazobactam, CTX Cefotaxime, CZA Ceftazidime/Avibactam, CAZ Ceftazidime, FEP Cefipime, C_T Ceftolozone/Tazobactam, ATM Aztreonam, CIP Ciprofloxicin, TOB Tobramycin, AMK Amikacin, GEN Gentamicin, TGC Tigecycline

Fig. 3

Gly-Asp insertion in L3 of OmpK36 reduces pore diameter, restricts Meropenem diffusion and mediates Meropenem resistance. a Cartoon representation of the OmpK36_ST258 trimer. The OmpK36_ST258 mutations have been mapped onto the structure. All mutations are shown as orange sticks. The majority of the mutations are found in L3/4. Inset: close-up view of the pore constriction zone. In order to accommodate the GD insertion, L3 has undergone a conformational change, stabilised by a salt-bridge, with subsequent constriction of the pore relative to the OmpK36_WT (shown in grey cartoon). b Lateral view of OmpK36 monomer aligned to minimal pore diameter graph, calculated using the HOLE algorithm, demonstrating a reduction in minimal pore diameter in both OmpK36_ST258 and OmpK36_WT+GD compared with OmpK36_WT. c OmpK36 isoforms were reconstituted into proteoliposomes and Meropenem diffusion was assessed by liposomal swelling assay. Meropenem influx is reduced in OmpK36_ST258 compared with OmpK36_WT. This effect is abolished by GD deletion (OmpK36_ST258ΔGD) and reproduced by GD insertion (OmpK36_WT+GD) (n = 3 repeats, error bars = ± s.d). d Calculated Meropenem uptake rate over 20 seconds (ΔOD_400nm/Δt(s)) from (c). ****p < 0.0001, error bars ± s.e.m. e GD insertion in OmpK36 isoforms mediates Meropenem resistance assayed by agar dilution in KP (OmpK35_ST258 background with KPC-2). Substitution of OmpK36_WT with OmpK36_WT+GD increases the MIC to that of OmpK36_ST258, a phenotypic pattern reversed by GD deletion in the OmpK36_ST258ΔGD mutant

Fig. 4

The Gly-Asp insertion in Asp L3 causes a fitness disadvantage in an in vivo model of Ventilator-associated pneumonia. a Delivery of bioluminescent KP to the lung parenchyma by intubation recorded by 3D-diffuse light imaging immediately post inoculation. Images were reconstructed into 3D using 3D living image. b, c Mice were intubated with 500 CFU of ICC8001-ICC8004 or OmpK35_ST258/ΔOmpK36 (n = 10/strain, error bars ± s.d.). Enumeration of CFU in lungs and blood, collected at 36 h post infection, revealed no significant difference between ICC8001 and all strains tested except OmpK35_ST258/ΔOmpK36. **p < 0.001, ***p < 0.0002. d Competition between ICC8001-GFP and ICC8001-RFP was tested by intubating 250 CFU or each competing strain. Enumeration of CFU of each strain in lungs and blood, identified by their colony fluorescence, was performed at 36 h post infection. Top bar represents mean value, points below represent individual mice (n = 10 per competition, error bars ± 95% confidence interval). No significant difference between ICC8001-GFP and ICC8001-RFP was detected in either the lungs or blood (Fig. S9d). e Competition between ICC8001 and ICC8002 (OmpK35_ST258) and f ICC8001 and ICC8003 (OmpK36_ST258) result in non-significant fitness disadvantage in the lung, but significant attenuation in dissemination to the blood (Fig S9e, f) (n = 10 per competition, error bars ± 95% confidence interval). g, h ST258 configuration at both OmpK35 and OmpK36 loci (ICC8004) results in complete out-competition by WT configuration (ICC8001) at 36 h post inoculation in the lung and dissemination to the blood, with no recoverable ICC8004 at either site (n = 16, error bars ± 95% confidence interval). i Competition between OmpK36 isogenic pairs with or without Gly-Asp Loop 3 insertion demonstrates that in vivo disadvantage results from pore constriction. Top bar represents mean value (n = 20 mice, error bars ± 95% confidence interval), OmpK36_WT in competition with OmpK36_WT+GD (open circles, each point represents 1 mouse (n = 10)) or OmpK36_ST258 in competition with OmpK36_ST258ΔGD (closed circles, each point represents 1 mouse (n = 10))