Pseudomonas aeruginosa reveals high intrinsic resistance to penem antibiotics: penem resistance mechanisms and their interplay

Abstract

Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC beta-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC beta-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC beta-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8- and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC beta-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC beta-lactamase but not from the lower affinity of PBPs for penems.

FIG. 1

Chemical structures showing the charge(s) in the penems used in this study.

FIG. 2

Expression of OmpF in the outer membrane of P. aeruginosa and E. coli cells. Cells were grown at 37°C for 18 h on L agar containing various amounts of IPTG. The outer membrane proteins were prepared, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as described previously (10). Lanes: 1, PAO1 (IPTG, 0 mM); 2, PAO1/pKMF012 (IPTG, 1 mM); 3, PAO1/pKMF010 (IPTG, 0 mM); 4, PAO1/pKMF010 (IPTG, 0.02 mM); 5, PAO1/pKMF010 (IPTG, 0.06 mM); 6, PAO1/pKMF010 (IPTG, 0.25 mM); 7, PAO1/pKMF010 (IPTG, 1.0 mM); 8, E. coli K-12 (IPTG, 0 mM). An arrow shows the position corresponding to E. coli OmpF (37 kDa).

FIG. 3

Time course of the hydrolysis of various penems by the β-lactamase prepared from P. aeruginosa PAO1. The reaction mixture contained 0.5 μM β-lactamase and 10 μM penem in 50 mM sodium phosphate buffer (pH 7.0), and this was incubated for the indicated times. Each data point represents the level of residual antibacterial activity of the penem after incubation with β-lactamase for the indicated time.