Major porin channels, OmpK35 and OmpK36, were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot; porin genes were amplified and sequenced, and their expression was assessed by reverse transcriptase-PCR. The C-NS isolates belonged to three pulsotypes and to the clone ST11, produced the SHV-5 ESBL and/or DHA-1 AmpC-type cephalosporinase, did not express OmpK36, and had a reduced expression of OmpK35. The C-S isolates differed from their C-NS counterparts only by porin expression profiles. Resistance to carbapenems in Enterobacteriaceae is mediated either by the production of various carbapenemases or by the high-level extended-spectrum β-lactamase (ESBL) or AmpC cephalosporinase

expression combined with alterations of major porin channels. In the case of the porin deficiency, carbapenems Compound Library chemical structure reach low concentrations in the periplasmic space and their activity may then be compromised by large amounts of enzymes with weak carbapenemase activity (Livermore R428 in vivo & Woodford, 2006; Martínez-Martínez, 2008). In the Czech Republic, Gram-negative bacteria with acquired carbapenemases are sporadic, with the first isolates of that kind (Pseudomonas aeruginosa and Serratia marcescens) identified in 2008 (Hrabák et al., 2009b; J. Hrabák, unpublished

data). Recently, Klebsiella pneumoniae isolates nonsusceptible to carbapenems (C-NS) were recovered in some hospitals and collected by the National Reference Laboratory for Antibiotics in Prague. None of

these Bumetanide had carbapenemase activity, but they expressed either an ESBL or an AmpC-like β-lactamase (P. Urbášková & J. Hrabák, unpublished data). The aim of this study was to characterize all nonrepetitive K. pneumoniae C-NS isolates in one of the largest Czech hospitals, and to compare these with C-S K. pneumoniae isolates from the same patients. The University Hospital in Plzeň is a teaching center with 1800 beds. Between January 2007 and June 2008, all nonrepetitive K. pneumoniae C-NS isolates according to the EUCAST guidelines [minimal inhibitory concentrations (MICs) of imipenem or meropenem, >2 μg mL−1] (http://www.eucast.org) were collected. Only if available, carbapenem-susceptible (C-S) K. pneumoniae isolates recovered from the same patients were included as well (in the case of stool samples, these were identified as the only Enterobacteriaceae other than Escherichia coli). Species identification was performed by enterotest 12 (Pliva Lachema Diagnostika, Brno, Czech Republic). MICs of antimicrobials were determined by broth dilution as proposed by European Committee for Antimicrobial Susceptibility Testing (EUCAST) (2003) and interpreted using its guidelines (http://www.eucast.org). Carbapenem MICs were confirmed by E-test (AB Biodisk, Solna, Sweden). Imipenem hydrolysis activity of the crude extracts was assayed by spectrophotometry (Woodford et al., 2004).