Affinity of the new cephalosporin CXA-101 to penicillin-binding proteins of Pseudomonas aeruginosa

Abstract

CXA-101, previously designated FR264205, is a new antipseudomonal cephalosporin. The objective of this study was to determine the penicillin-binding protein (PBP) inhibition profile of CXA-101 compared to that of ceftazidime (PBP3 inhibitor) and imipenem (PBP2 inhibitor). Killing kinetics, the induction of AmpC expression, and associated changes on cell morphology were also investigated. The MICs for CXA-101, ceftazidime, and imipenem were 0.5, 1, and 1 microg/ml, respectively. Killing curves revealed that CXA-101 shows a concentration-independent bactericidal activity, with concentrations of 1x the MIC (0.5 microg/ml) producing a > 3-log reduction in bacterial load after 8 h of incubation. Live-dead staining showed that concentrations of CXA-101 as low as 0.5x the MIC stopped bacterial septation and induced an intense filamentation, which is consistent with the documented high affinity of PBP3. CXA-101 was found to be a potent PBP3 inhibitor and showed affinities > or = 2-fold higher than those of ceftazidime for all of the essential PBPs (1b, 1c, 2, and 3). Compared to imipenem, in addition to the obvious inverse PBP2/PBP3 affinities, CXA-101 showed a significantly higher affinity for PBP1b but a lower affinity for PBP1c. Furthermore, CXA-101, like ceftazidime and in contrast to imipenem, was found to be a very weak inducer of AmpC expression, consistent with the low PBP4 affinity documented.

FIG. 1.

Results for killing kinetics experiments. (A) Results of the killing curves measured in terms of reduction of viable CFU/ml over time. (B) Results of the killing curves in terms of the OD₆₀₀ change. The concentrations tested were 0×, 0.5×, 1×, 2×, and 4× the MICs (0.5 μg/ml for CXA-101 and 1 μg/ml for ceftazidime [CAZ] and imipenem [IMP]). Mean values for three experiments ± the standard deviation are shown.

FIG. 2.

Results of Live/Dead staining. (A) Images obtained at 2 h of incubation with 0.5× MIC concentrations of CXA-101 (CXA), ceftazidime (CAZ), or imipenem (IMP). (B) Images obtained at 8 h of incubation with 4× MIC concentrations of each antibiotic. Live cells are green stained, and dead cells are red stained.

FIG. 3.

Representative example of the SDS-polyacrylamide gels labeled with Bocillin FL and incubated in the presence of CXA-101, ceftazidime (CAZ), or imipenem (IMP). Range of concentrations tested: 0.0156 to 2 μg/ml.