Deciphering the Evolution of Cephalosporin Resistance to Ceftolozane-Tazobactam in Pseudomonas aeruginosa

• Published in: mBio Vol. 9; no. 6
• Main Authors: Barnes, Melissa D, Taracila, Magdalena A, Rutter, Joseph D, Bethel, Christopher R, Galdadas, Ioannis, Hujer, Andrea M, Caselli, Emilia, Prati, Fabio, Dekker, John P, Papp-Wallace, Krisztina M, Haider, Shozeb, Bonomo, Robert A
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 11-12-2018
• Subjects: AmpC; Anti-Bacterial Agents - pharmacology; antibiotic resistance; beta-lactam; beta-lactamase; beta-Lactamase Inhibitors - pharmacology; beta-Lactamases - chemistry; beta-Lactamases - genetics; beta-Lactamases - metabolism; ceftolozane; Cephalosporin Resistance; Cephalosporins - pharmacology; Escherichia coli - drug effects; Escherichia coli - genetics; Gene Expression; Kinetics; Mass Spectrometry; Microbial Sensitivity Tests; Molecular Dynamics Simulation; Mutant Proteins - genetics; Mutant Proteins - metabolism; Mutation, Missense; PDC-3; Protein Conformation; Protein Folding; Protein Stability; Pseudomonas aeruginosa - drug effects; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Tazobactam - pharmacology; Temperature; Therapeutics and Prevention; AmpC; PDC-3; antibiotic resistance; beta-lactamase; ceftolozane; beta-lactam; omega loop
• ISSN: 2161-2129; 2150-7511
• DOI: 10.1128/mBio.02085-18

produces a class C β-lactamase (e.g., PDC-3) that robustly hydrolyzes early generation cephalosporins often at the diffusion limit; therefore, bacteria possessing these β-lactamases are resistant to many β-lactam antibiotics. In response to this significant clinical threat, ceftolozane, a 3' aminopyrazolium cephalosporin, was developed. Combined with tazobactam, ceftolozane promised to be effective against multidrug-resistant
Alarmingly, Ω-loop variants of the PDC β-lactamase (V213A, G216R, E221K, E221G, and Y223H) were identified in ceftolozane/tazobactam-resistant
clinical isolates. Herein, we demonstrate that the
strain expressing the E221K variant of PDC-3 had the highest minimum inhibitory concentrations (MICs) against a panel of β-lactam antibiotics, including ceftolozane and ceftazidime, a cephalosporin that differs in structure largely in the R2 side chain. The
values of the E221K variant for both substrates were equivalent, whereas the
for ceftolozane (341 ± 64 µM) was higher than that for ceftazidime (174 ± 20 µM). Timed mass spectrometry, thermal stability, and equilibrium unfolding studies revealed key mechanistic insights. Enhanced sampling molecular dynamics simulations identified conformational changes in the E221K variant Ω-loop, where a hidden pocket adjacent to the catalytic site opens and stabilizes ceftolozane for efficient hydrolysis. Encouragingly, the diazabicyclooctane β-lactamase inhibitor avibactam restored susceptibility to ceftolozane and ceftazidime in cells producing the E221K variant. In addition, a boronic acid transition state inhibitor, LP-06, lowered the ceftolozane and ceftazidime MICs by 8-fold for the E221K-expressing strain. Understanding these structural changes in evolutionarily selected variants is critical toward designing effective β-lactam/β-lactamase inhibitor therapies for
infections.
The presence of β-lactamases (e.g., PDC-3) that have naturally evolved and acquired the ability to break down β-lactam antibiotics (e.g., ceftazidime and ceftolozane) leads to highly resistant and potentially lethal
infections. We show that wild-type PDC-3 β-lactamase forms an acyl enzyme complex with ceftazidime, but it cannot accommodate the structurally similar ceftolozane that has a longer R2 side chain with increased basicity. A single amino acid substitution from a glutamate to a lysine at position 221 in PDC-3 (E221K) causes the tyrosine residue at 223 to adopt a new position poised for efficient hydrolysis of both cephalosporins. The importance of the mechanism of action of the E221K variant, in particular, is underscored by its evolutionary recurrences in multiple bacterial species. Understanding the biochemical and molecular basis for resistance is key to designing effective therapies and developing new β-lactam/β-lactamase inhibitor combinations.