Selection of AmpC β-Lactamase Variants and Metallo-β-Lactamases Leading to Ceftolozane/Tazobactam and Ceftazidime/Avibactam Resistance during Treatment of MDR/XDR Pseudomonas aeruginosa Infections

Abstract

Infections caused by ceftolozane-tazobactam and ceftazidime-avibactam-resistant P. aeruginosa infections are an emerging concern. We aimed to analyze the underlying ceftolozane-tazobactam and ceftazidime-avibactam resistance mechanisms in all multidrug-resistant or extensively drug-resistant (MDR/XDR) P. aeruginosa isolates recovered during 1 year (2020) from patients with a documented P. aeruginosa infection. Fifteen isolates showing ceftolozane-tazobactam and ceftazidime-avibactam resistance were evaluated. Clinical conditions, previous positive cultures, and β-lactams received in the previous month were reviewed for each patient. MICs were determined by broth microdilution. Multilocus sequence types (MLSTs) and resistance mechanisms were determined using short- and long-read whole-genome sequencing (WGS). The impact of Pseudomonas-derived cephalosporinases (PDCs) on β-lactam resistance was demonstrated by cloning into an ampC-deficient PAO1 derivative (PAOΔC) and construction of 3D models. Genetic support of acquired β-lactamases was determined in silico from high-quality hybrid assemblies. In most cases, the isolates were recovered after treatment with ceftolozane-tazobactam or ceftazidime-avibactam. Seven isolates from different sequence types (STs) owed their β-lactam resistance to chromosomal mutations and all displayed specific substitutions in PDC: Phe121Leu and Gly222Ser, Pro154Leu, Ala201Thr, Gly214Arg, ΔGly203-Glu219, and Glu219Lys. In the other eight isolates, the ST175 clone was overrepresented (6 isolates) and associated with IMP-28 and IMP-13, whereas two ST1284 isolates produced VIM-2. The cloned PDCs conferred enhanced cephalosporin resistance. The 3D PDC models revealed rearrangements affecting residues involved in cephalosporin hydrolysis. Carbapenemases were chromosomal (VIM-2) or plasmid-borne (IMP-28, IMP-13) and associated with class-1 integrons located in Tn402-like transposition modules. Our findings highlighted that cephalosporin/β-lactamase inhibitors are potential selectors of MDR/XDR P. aeruginosa strains producing PDC variants or metallo-β-lactamases. Judicious use of these agents is encouraged.

Keywords: Pseudomonas aeruginosa; antibiotic resistance; beta-lactamases; beta-lactams; cephalosporin; mechanisms of resistance.

FIG 1

(A) Main key sites and specific motifs of the PDC-1 enzyme observed in the crystal structure of PDC-1 from P. aeruginosa in the wild-type form (PDB 4GZB, 1.79 Å): Ω loop (red), YSN motif (pink), SVSK motif (green), KTG motif (blue) and H10 helix (orange). (B) Binding mode of ceftolozane (TOL, cyan) and ceftazidime (CAZ, pink) in the active site of PDC-1 for the acylation reaction obtained by MD simulation studies. Snapshots after 90 ns and 80 ns of simulation, respectively, are shown. The key sites and motifs are highlighted the same as in (A). Relevant hydrogen bonding interactions (dashed blue lines) and side-chain residues are shown and labeled. The SANC (structural alignment-based numbering of class C β-lactamases) is employed.

FIG 2

(A) Overall views of the PDC-1/TOL enzyme complex for the acylation reaction obtained by MD simulation studies and the homology model of PDC-223. The enzyme region (catalytic and R1 and R2 side-chain sites) that interacts with ceftolozane is highlighted in red. Note how PDC-223 would have a large extra cavity close to the R1 side chain. (B to F). Detailed view of the modified protein regions in the homology models of PDC-221 (B), PDC-477 (C), PDC-482 (D), PDC-478 (E), and PDC-483 (F). The modified residues are shown in yellow. The Ω-loop and YGN and SVSK motifs are highlighted in red, pink, and green, respectively. Potential polar interactions (attractive/repulsive) of the modified residues with others in the vicinity are shown as dashed lines.

FIG 3

(A) Schematic representation of the pJAS and pCHUAC plasmids harboring the bla_IMP-13 and bla_IMP-28 β-lactamases present in the ST175 isolates. (B) Detailed comparison of the immediate genetic environment surrounding the plasmid-borne bla_IMP-13 and bla_IMP-28 and the chromosomal bla_VIM-2 MBL-encoding genes. Predicted open reading frames are indicated by arrows displayed in the direction of transcription of each gene. Genes encoding bla_IMP-13, bla_IMP-28, and bla_VIM-2 β-lactamases are shown in red. Other genes involved in antimicrobial resistance are shown in orange. Genes associated with mobilization processes are indicated in dark blue. Mercury resistance genes and operons are shown in green. Yellow arrows represent genes involved in plasmid replication, transference, and maintenance. The ISPa17 element is indicated in purple. Tn402-like transposition modules are highlighted in light blue. Dark arrows indicate putative open reading frames of unknown function.