β-lactam Resistance in Pseudomonas aeruginosa: Current Status, Future Prospects

Abstract

Pseudomonas aeruginosa is a major opportunistic pathogen, causing a wide range of acute and chronic infections. β-lactam antibiotics including penicillins, carbapenems, monobactams, and cephalosporins play a key role in the treatment of P. aeruginosa infections. However, a significant number of isolates of these bacteria are resistant to β-lactams, complicating treatment of infections and leading to worse outcomes for patients. In this review, we summarize studies demonstrating the health and economic impacts associated with β-lactam-resistant P. aeruginosa. We then describe how β-lactams bind to and inhibit P. aeruginosa penicillin-binding proteins that are required for synthesis and remodelling of peptidoglycan. Resistance to β-lactams is multifactorial and can involve changes to a key target protein, penicillin-binding protein 3, that is essential for cell division; reduced uptake or increased efflux of β-lactams; degradation of β-lactam antibiotics by increased expression or altered substrate specificity of an AmpC β-lactamase, or by the acquisition of β-lactamases through horizontal gene transfer; and changes to biofilm formation and metabolism. The current understanding of these mechanisms is discussed. Lastly, important knowledge gaps are identified, and possible strategies for enhancing the effectiveness of β-lactam antibiotics in treating P. aeruginosa infections are considered.

Keywords: AmpC; PBP3; antibiotic efflux; antibiotic resistance; carbapenem; carbapenemase; cephalosporin; cystic fibrosis; nosocomial infection; β-lactamase.

Figure 1

Roles of penicillin-binding proteins (PBPs) in peptidoglycan synthesis. (A) NAG-NAM chains are not cross-linked before processing by PBPs. (B) LMM PBPs 4, 5 and 7 (DD-carboxypeptidases) cleave terminal D-alanyl residues from some pentapeptides, regulating levels of cross-linking. (C) HMM PBP 1a, 1b, 2, 3, 3a (DD-transpeptidases) cross-link pentapeptide-containing side chains to penta-, tetra-, or tri-peptides of adjacent NAG-NAM chains while simultaneously removing terminal D-alanyl residues. (D) Mature peptidoglycan contains a mixture of cross-linked and unlinked peptides. NAG, N-acetyl glucosamine; NAM, N-acetyl muramic acid.

Figure 2

Core structures of β-lactam subclasses used in P. aeruginosa treatment, and the terminal D-alanine-D-alanyl residues of peptidoglycan pentapeptide. (A) Penicillins. (B) Cephalosporins. (C) Carbapenems. (D) Monobactams. (E) D-alanine-D-alanyl residues. The β-lactam ring is indicated in red and mimics the terminal D-alanine-D-alanyl of the peptidoglycan pentapeptide precursor. Figure adapted from [101,107].

Figure 3

Mechanisms of β-lactam resistance in P. aeruginosa.

Figure 4

Structure of PBP3 in complex with meropenem. The transpeptidase domain is shown in red. The domain shown in blue is thought to play a role in protein-protein interactions. The membrane-spanning helix and small cytoplasmic part of PBP3 are not included in the structure. Meropenem shown in yellow is bound to the catalytic serine S294 (in black). Amino acid residues that are commonly substituted in clinical isolates are coloured green with side chains displayed. The image is based on protein structure PDB 3PBR_1 [102].

Figure 5

Regulation of ampC expression. (A) Under normal cellular conditions expression of ampC is repressed. LMM PBPs such as PBP4 hydrolyse uncross-linked peptidoglycan pentapeptides to tetrapeptides. During recycling of peptidoglycan, peptidoglycan fragments (the majority being NAG-NAM tetrapeptide cleaved from the NAG-NAM chains by lytic transglycosylases) are imported into the cytoplasm. NAG is removed by NagZ, after which NAM is cleaved from the peptide side chain by AmpD. NAG, NAM and the peptide side chains are used in synthesis of new peptidoglycan. Excess peptidoglycan precursor UDP-NAM pentapeptide formed through recycling as well as de novo synthesis binds to the AmpR regulator protein, which acts as a repressor inhibiting ampC expression. (B) β-lactams cause upregulation of ampC. Increased peptidoglycan recycling occurs because of the presence of β-lactams, which also inhibit conversion of tetrapeptides to pentapeptides by LMMs PBPs. The resulting peptidoglycan fragments (primarily NAG-NAM pentapeptide but also NAG-NAM tripeptide [not shown]) are imported into the cytoplasm. In the recycling pathway, AmpD becomes saturated because of increased amounts of peptidoglycan fragments, increasing the intracellular concentrations of the AmpR activator molecules NAG-NAM pentapeptide, NAM-pentapeptide and NAG-NAM tripeptide. Increased export of UDP-NAM pentapeptide for peptidoglycan synthesis also occurs. The activator molecules outcompete UDP-NAM pentapeptide for binding to AmpR and the AmpR-activator complexes trigger increased expression of ampC. UDP, uridine diphosphate; NAG, N-acetyl glucosamine; NAM, N-acetyl muramic acid; pentapeptide, L-alanine-γ-D-Glutamate-meso-DAP-D-Ala-D-Ala.

Figure 6

Locations of amino acid variants in AmpC that contribute to β-lactam resistance.