Achieving high levels of Carbapenem resistance disadvantages Klebsiella pneumoniae during host infection.

Elucidation of the molecular mechanism that restricts Carbapenem diffusion in a resistant OmpK36 isoform and confers a fitness disadvantage in murine pneumonia.

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Behind the paper of ‘OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo’ by Joshua L.C. Wong, Maria Romano, Louise E. Kerry, Hok-Sau Kwong, Wen-Wen Low, Stephen J. Brett, Abigail Clements, Konstantinos Beis and Gad Frankel published in Nature Communications.

Klebsiella pneumoniae typifies a successful modern pathogen. It thrives in the nosocomial niche, colonising and causing invasive disease in hospitalised patients. Antibiotics are the cornerstone of therapy in K. pneumoniae sepsis and we rely heavily on a class called Carbapenems. Previous waves of antimicrobial resistance have rendered other antibiotics ineffective (e.g. 3rd generation cephalosporins and extended-spectrum beta-lactamases (ESBLs)) leaving Carbapenems as fundamentally important last-line agents. Carbapenems have been a victim of their own success; they exhibit a broad-spectrum of antimicrobial activity, they remain effective in the face of common Gram-negative bacterial resistance mechanisms (e.g. ESBL and AmpC expression) and have a good side effect profile. However, as has been seen since the inception of targeted antibiotic chemotherapy in humans, resistance has emerged and acquired Carbapenem resistance in K. pneumoniae is rapidly increasing in prevalence1.

Figure 1: Left panel. The architecture of the wild type OmpK36 monomer is composed of 16-stranded b-barrel that traverses the outer-membrane forming a central pore. Loop 3 (red) arises from the extracellular face, folding back into the barrel, forming a constriction point approximately halfway down the barrel. This restriction determines pore diameter and contributes to permeability, limiting diffusion by solute size and charge. Right panel. Carbapenem resistance is achieved in ST258 strains by two synergistic mechanisms. There is a diffusion defect, from OmpK35 truncation and absence in the outer membrane combined with OmpK36 pore constriction, limiting the access of Carbapenems to the periplasm. Hydrolysis by carbapenemase enzymes, like KPC-2 which associates with ST258 strains, inactivates the small amount of drug that is able to enter the periplasm.

Resistance is mediated by two synergistic mechanisms that are genetically distinct (Figure 1). First, carbapenemases abrogate drug activity by Carbapenem hydrolysis in the periplasm. These enzymes are most commonly encoded on large self-transferable plasmids2. In tandem, modification (usually loss) of chromosomally encoded outer-membrane porins has occurred. These porins function as non-specific channels and are embedded in the otherwise impermeable outer membrane and ensure the influx and efflux of nutrients and waste metabolites respectively3. Hydrophilic drugs, like Carbapenems enter via this route and therefore modification of these proteins can alter their diffusion. Small amounts of drug that can enter the periplasm are efficiently hydrolysed and inactivated by carbapenemases.

The two major porins in K. pneumoniae are OmpK35 and OmpK364. In our paper, we study a common combination of mutations in these proteins from a globally successful Carbapenem-resistant sequence type, ST2585. We set out to answer two overarching questions. Firstly, what effect do these proteins have on the level of Carbapenem resistance and how do these mutations achieve this? Secondly, do these mutations benefit Klebsiella during infection, or are they neutral with respect to virulence as has been suggested for mutations in other successful antibiotic resistant bacteria?6

Figure 2
Figure 2: In this work we solved the crystal structure of the resistant porin, OmpK36ST258. Here we show the trimeric form of the porin, which is the structure that forms in the outer membrane of K. pneumoniae. Residues highlighted in orange represent those found only in the resistant isoform. These changes were not found to influence resistance. We demonstrated that a Gly-Asp insertion in loop 3 resulted in an extended conformation in this region allowing a salt-bridge to form between a aspartate (red) with an arginine (blue). Overall, the changes in this region restricted the pore diameter, reduced Carbapenem diffusion and increased resistance to these important drugs.

To answer these questions, we generated a series of K. pneumoniae strains where the OmpK35 and OmpK36 sequences from a lab strain were sequentially replaced with those from an ST258 strain. This revealed that the ST258 porins increased resistance to Carbapenems. The OmpK35 mutation had a straightforward explanation- the protein is truncated and therefore the pore cannot not be formed. The resistance conferred by OmpK36ST258, posed a more interesting question as the sensitive isoform is very similar to the resistant form. The crystal structure provided the answer, a two amino acid (Glycine-Aspartic acid) insertion at the constriction point in the pore (extracellular Loop 3), reduced the pore diameter, retarding the diffusion of antibiotics (Figure 2). Removing the two amino acids from the resistant isoform reversed Carbapenem resistance and inserting them into the sensitive isoform generated a resistant phenotype.

We answered the second question by delivering these strains into the lungs of mice. Strains expressing the resistant isoforms were able to cause infection indistinguishable from sensitive K. pneumoniae. However, when we competed strains expressing sensitive and resistant porins against each other the sensitive isoforms always out-competed the resistant ones. This suggested a significant disadvantage resulting from changes to the porins that increase Carbapenem resistance. This is contrary to the existing theory that traits conferring resistance to successful antibiotic resistant clones have no fitness cost. These findings are important as they not only shed light on a poorly elucidated aspect of K. pneumoniae infection biology, but also support continuing strategies that aim to both limit the excessive use of antibiotics and employ narrow spectrum agents in clinical practice when possible7.   


1.       Cassini, A. et al. Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: a population-level modelling analysis. Lancet Infect. Dis. (2019). doi:10.1016/S1473-3099(18)30605-4

2.       Mathers, A. J., Peirano, G. & Pitout, J. D. D. The Role of Epidemic Resistance Plasmids and International High-Risk Clones in the Spread of Multidrug-Resistant Enterobacteriaceae. Clin. Microbiol. Rev. 28, 565–591 (2015).

3.       Nikaido, H. Porins and specific diffusion channels in bacterial outer membranes. J. Biol. Chem. 269, 3905–8 (1994).

4.       Tsai, Y.-K. et al. Klebsiella pneumoniae Outer Membrane Porins OmpK35 and OmpK36 Play Roles in both Antimicrobial Resistance and Virulence †. Antimicrob. Agents Chemother. 55, 1485–1493 (2011).

5.       David, S. et al. Epidemic of carbapenem-resistant Klebsiella pneumoniae in Europe is driven by nosocomial spread. Nat. Microbiol. 1–11 (2019). doi:10.1038/s41564-019-0492-8

6.       Klemm, E. J., Wong, V. K. & Dougan, G. Emergence of dominant multidrug-resistant bacterial clades: Lessons from history and whole-genome sequencing. Proc. Natl. Acad. Sci. U. S. A. 115, 12872–12877 (2018).

7.       Public Health England. Start Smart-Then Focus Antimicrobial Stewardship Toolkit for English Hospitals. (2015).

Joshua Wong

Clinical Research Fellow, Imperial College London