Carbapenemase-producing bacteria have now spread all over the world. Infections caused by those bacteria are difficult to treat. Therefore, there is an urgent need for accurate and fast detection of carbapenemases in diagnostic laboratories. In this review, we summarize screening methods for suspected isolates, direct assays for confirmation of carbapenemase activity (e.g. the Carba NP test and matrix-assisted laser desorption ionization time-of-flight mass spectrometry carbapenem hydrolysis assay), inhibitor-based methods for carbapenemase classification, and molecular-genetic techniques for precise identification of carbapenemase genes. We also propose a workflow for carbapenemase identification in diagnostic laboratories.

Apart from the epidemic of KPC-2-producing K. pneumoniae belonging to ST258 in Greece, diffusion of bla(KPC-2) to at least 10 additional STs has taken place. Notably, strains from three of the latter STs (147, 323 and 383) were found to carry both bla(KPC-2) and bla(VIM).

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Carbapenem-resistant Klebsiella pneumoniae caused an outbreak in a hospital in Rome, Italy. The clinical isolates were tested by antimicrobial susceptibility testing, pulsed-field gel electrophoresis, multilocus sequence typing, plasmid typing, and ␤-lactamase identification. The OmpK35 and OmpK36 porins were analyzed by SDS-PAGE, and their genes were amplified and sequenced. Complementation experiments were performed using a recombinant unrelated ompK36 gene. An ertapenem-resistant and imipenem-and meropenem-susceptible clone was identified and assigned to the sequence type 37 lineage by MLST; it carried SHV-12 and CTX-M-15 ESBLs, did not produce the OmpK35 due to a nonsense mutation, and expressed a novel OmpK36 variant (OmpK36V). This variant showed two additional amino acids located within the L3 internal loop, one of the highly conserved domains of the protein. Two isolates of the same clone also exhibited resistance to imipenem and meropenem, due to the loss of OmpK36 expression by a nonsense mutation occurring in the ompK36V variant gene. These were the first carbapenem-resistant K. pneumoniae isolates identified within the hospital. Screening for the ompK36V gene of unrelated K. pneumoniae isolates derived from patients from 2006 to 2009 demonstrated the high frequency of this gene variant as well as its association with ertapenem resistance, reduced susceptibility to meropenem, and susceptibility to imipenem.The susceptibility to carbapenems in the Enterobacteriaceae family is no longer guaranteed since the increasing occurrence of carbapenem-resistant isolates (26, 38). Carbapenem resistance can arise by acquisition of resistance genes encoding metallo-␤-lactamases (MBLs) and non-metallo-carbapenemases (of the KPC, GES, or OXA type) but has also been associated with the AmpC cephalosporinase or extended-spectrum ␤-lactamase (ESBL) production and alterations of the expression of the major nonspecific porins. The latter mechanism has been reported worldwide in clinical isolates of Klebsiella pneumoniae, Escherichia coli, Enterobacter spp., and others (11, 12, 14-18, 23, 24, 31, 35). The alteration of porin expression may be caused by several different events, including the disruption of the gene by insertion sequences, the termination of translation by nonsense mutations, and the downregulation of transcription by mutations occurring within the promoter (12,18,29). In particular, the effect of the alteration of OmpK35 and OmpK36 expression in ESBL-producing K. pneumoniae isolates has been demonstrated to confer a high level of ertapenem resistance, with the MICs of imipenem and meropenem being raised (11,12,14,16,17,24,39).Sequence analysis of the ompK36 genes of clinical isolates of Enterobacter aerogenes, E. coli, and K. pneumoniae demonstrated the importance of transmembrane ␤-strand loop 3 (L3) in the specific uptake of ␤-lactams (3,8,36). The L3 loop constitutes the channel eyelet of enterobacterial porins, extends inside the barrel, and constricts the pore (1). The mutations occurring in L3 drastic...

A comparison of a matrix-assisted laser desorption ionization-time of flight mass spectrometric (MALDI-TOF MS) meropenem hydrolysis assay with the Carba NP test showed that both methods exhibited low sensitivity (approximately 76%), mainly due to the false-negative results obtained with OXA-48-type producers. The addition of NH 4 HCO 3 to the reaction buffer for the MALDI-TOF MS assay dramatically improved its sensitivity (98%). Automatic interpretation of the MALDI-TOF MS assay, using the MBT STAR-BL software, generally agreed with the results obtained after manual analysis. For the Carba NP test, spectrophotometric analysis found six additional carbapenemase producers. Nosocomial infections caused by carbapenem-resistant Enterobacteriaceae and Pseudomonas spp. are now emerging worldwide and are difficult to treat (1). Previous data have shown that strict epidemiological intervention based on the rapid detection of carbapenemase production can prevent the spread of those bacteria (2). Therefore, the introduction of rapid and sensitive methodologies for the detection of carbapenemase-producing bacteria is of utmost importance. Recently, two new highly sensitive and rapid methods for the direct detection of carbapenemase activity were developed. In 2011, two research groups demonstrated that matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) can detect carbapenemase activity, based on mass spectral profiles obtained from carbapenem molecules (3, 4). The second method, the Carba NP test, is a biochemical method used in the detection of carbapenemase activity in Enterobacteriaceae, Pseudomonas spp., and Acinetobacter species (5-7). This test is based on a decrease in pH resulting from the hydrolysis of the ␤-lactam ring of carbapenem molecules, which is detected using phenol red as a pH indicator.The aim of this study was to validate the efficiency of a MALDI-TOF MS meropenem hydrolysis assay and the Carba NP test for the detection of carbapenemase producers. The performances of both methods were compared with that of a recently published modification of the MALDI-TOF MS assay (MALDI-TOF BIC) in an effort to increase the reliability of detecting OXA-48 producers (8). Furthermore, the automatic interpretation of all methods was evaluated.The methods were tested against a group of 124 Enterobacteriaceae and 37 Pseudomonas aeruginosa isolates from collections of the Faculty of Medicine and University Hospital in Plzen (Czech Republic), the National Medicines Institute in Warsaw (Poland), the Robert Koch Institute in Wernigerode (Germany), and the University Hospital in Larissa (Greece). The isolates were previously characterized, as described below. All isolates were tested for extended-spectrum ␤-lactamase (ESBL) and AmpC expression by the ESBL double-disk synergy test (DDST) with cefotaxime, ceftazidime, aztreonam, and amoxicillin-clavulanate disks in the absence and presence of cloxacillin (250 g/ml) (9). Susceptibility to carbapenems was determined by using imipenem, merope...