In Vitro Activity of Penem-1 in Combination with β-Lactams against blaKPC-Possessing Klebsiella pneumoniae Isolates

The widespread dissemination of blaKPC-possessing Klebsiella pneumoniae isolates represents a serious threat for hospitalized patients (4, 8). blaKPC-possessing K. pneumoniae isolates are resistant to all β-lactams, commercially available β-lactam-β-lactamase inhibitor combinations, quinolones, and frequently to aminoglycosides (5, 8). Furthermore, blaKPC-possessing K. pneumoniae isolates can manifest a phenotype of nonsusceptibility to tigecycline and colistin (6, 7). As a result, new therapeutic strategies against infections due to blaKPC-possessing K. pneumoniae isolates should be quickly developed. Penem-1 (Wyeth Research) is a novel serine-reactive β-lactamase inhibitor with potent activity against class A, C, and D enzymes (1, 10). We recently demonstrated that this methylidene penem is a potent inhibitor of the KPC-2 carbapenemase (with a Km or Ki of 60 nM and a kcat/kinact of 250, where kinact is the rate constant of enzyme inactivation) (9). Data regarding the in vitro activity of penem-1 in combination with various commercially available β-lactams against blaKPC-possessing K. pneumoniae clinical isolates are not yet available. In this work, we tested the in vitro activity of penem-1 in combination with four β-lactams against a collection of 42 previously characterized blaKPC-possessing K. pneumoniae clinical isolates that produce multiple β-lactamases (5). MICs of β-lactams and β-lactams combined with penem-1 (at a constant concentration of 4 μg/ml) were determined by the agar dilution method according to Clinical and Laboratory Standards Institute (CLSI) criteria (2) using cation-adjusted Mueller-Hinton agar (BBL; Becton Dickinson) and a Steers replicator that delivers 104 CFU/10-μl spot. Piperacillin, cefotaxime, aztreonam (Sigma Chemical Co.), and cefepime (Apotex Corp.) were combined with penem-1. The following ATCC control strains were used: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and K. pneumoniae ATCC 700603. MIC results, including those for the combinations with penem-1, were interpreted according to the CLSI criteria established for the β-lactams when tested alone (3). As shown in Fig. ​Fig.1,1, piperacillin and aztreonam exhibited very high MICs (MIC50s, ≥512 μg/ml) when tested against blaKPC-possessing K. pneumoniae isolates whereas cefotaxime and cefepime exhibited lower MICs (MIC50s, 64 and 32 μg/ml, respectively). When penem-1 was tested alone, this inhibitor did not show activity against blaKPC-possessing K. pneumoniae strains (MICs for all strains, >128 μg/ml) (data not shown). When penem-1 was combined with piperacillin, aztreonam, or cefotaxime, the MICs remained mostly in the resistant range. In contrast, cefepime in combination with penem-1 showed significantly better results, with an overall proportion of susceptible strains of 88.1% (Fig. ​(Fig.11). FIG. 1. Distributions of MICs of piperacillin, aztreonam, cefotaxime, and cefepime and their combinations with penem-1 (Pen-1) obtained using the agar diffusion method. Results were interpreted according to CLSI criteria (3). Dashed line, susceptibility (S) cutoff. ... In conclusion, we show here that penem-1 possesses good ability to lower the MICs of β-lactams (usually by at least three to four 2-fold dilutions from those of the β-lactam alone) when tested against blaKPC-possessing K. pneumoniae clinical isolates. This new β-lactamase inhibitor undergoes novel reaction chemistry when acylated in the active site and is superior to clavulanate and tazobactam (5, 9). The combination with cefepime represents a possible therapeutic option against infections due to blaKPC-possessing K. pneumoniae isolates. Further in vitro studies should be performed to establish the possible spectrum of application of β-lactam-penem-1 combinations against different types of multidrug-resistant Gram-negative organisms.