Unexpected challenges in treating multidrug-resistant Gram-negative bacteria: resistance to ceftazidime-avibactam in archived isolates of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a notoriously difficult-to-treat pathogen that is a common cause of severe nosocomial infections. Investigating a collection of β-lactam-resistant P. aeruginosa clinical isolates from a decade ago, we uncovered resistance to ceftazidime-avibactam, a novel β-lactam/β-lactamase inhibitor combination. The isolates were systematically analyzed through a variety of genetic, biochemical, genomic, and microbiological methods to understand how resistance manifests to a unique drug combination that is not yet clinically released. We discovered that avibactam was able to inactivate different AmpC β-lactamase enzymes and that blaPDC regulatory elements and penicillin-binding protein differences did not contribute in a major way to resistance. By using carefully selected combinations of antimicrobial agents, we deduced that the greatest barrier to ceftazidime-avibactam is membrane permeability and drug efflux. To overcome the constellation of resistance determinants, we show that a combination of antimicrobial agents (ceftazidime/avibactam/fosfomycin) targeting multiple cell wall synthetic pathways can restore susceptibility. In P. aeruginosa, efflux, as a general mechanism of resistance, may pose the greatest challenge to future antibiotic development. Our unexpected findings create concern that even the development of antimicrobial agents targeted for the treatment of multidrug-resistant bacteria may encounter clinically important resistance. Antibiotic therapy in the future must consider these factors.

FIG 1

(A) BLIs. (B) Scheme of interaction of avibactam (I) with a β-lactamase (E) showing formation of the Michaelis-Menten complex (E:I), the formation of the acyl-enzyme (E-I), and recyclization of avibactam to regenerate active compound and allow repetition of the reaction. (C) Known resistance mechanisms in P. aeruginosa, including a mucoid layer, outer membrane porins, efflux pumps, PBP alterations, and β-lactamase upregulation. P. aeruginosa has a mucoid layer outside the outer membrane; increased thickness of this layer can lead to antibiotic resistance. Antibiotics enter the cell through porins in the outer membrane. Loss of these porins can lead to antibiotic resistance. P. aeruginosa also can carry efflux pumps in the outer membrane (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM); when these structures are present, antibiotics can be pumped out of the cell, leading to resistance. β-Lactamases can be present in the periplasmic space of the bacteria, which are able to break down β-lactam antibiotics and/or BLIs, leading to resistance to these compounds. The regulation of the chromosomal AmpC in P. aeruginosa is illustrated, which involves a complex relationship between peptidoglycan breakdown, β-lactam exposure, and gene regulation leading to overexpression of the AmpC enzyme. Lastly, the PBPs in the peptidoglycan layer can be altered to prevent interaction of β-lactam antibiotics with their targets.

Fig 2

Diversilab rep-PCR dendrogram analysis of the 54 clinical P. aeruginosa strains used in these experiments with the wild-type P. aeruginosa 18SH (which has a stably derepressed AmpC) included for comparison.

FIG 3

Western blot of the various P. aeruginosa clinical isolates tested with an antibody raised to the 18SH AmpC protein. Isolate names in boldface are strains resistant to CAZ-AVI combination treatment. “PDC-3 prot” is the purified PDC-3 protein as a size comparison for the AmpCs.