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.

To follow the epidemic of KPC-2-producing Klebsiella pneumoniae in Greece. KPC-2-producing isolates (n = 378) were collected during January 2009–April 2010 in 40 Greek hospitals. bla_KPC and bla_VIM were detected by PCR. Carbapenemase production was confirmed by spectrophotometry. Sequences flanking bla_KPC-2 and their plasmid carriers were studied. Isolates were typed by PFGE and multilocus sequence typing (MLST). All 378 isolates were bla_KPC-2 positive; 18 also carried bla_VIM-1/VIM-4. Higher isolation frequencies were observed in Athens and Crete. Isolates were classified into 13 PFGE types and 11 sequence types (STs). ST258 was predominant (n = 322), followed by ST147 (n = 20), ST383 (n = 9), ST133 (n = 6), ST274 (n = 4) and ST323 (n = 3). Of the remaining isolates, seven were distributed into five STs (11, 17, 340 and the novel 494 and 495) and seven were not typed. bla_KPC-2 could not be transferred from ST258 isolates, in contrast to isolates of ST17, ST133, ST147, ST274, ST494 and ST495. All bla_KPC-2-encoding plasmids were of similar size (∼100 kb) and showed indistinguishable restriction fragment length polymorphism (RFLP) patterns except those from the ST340 isolates. Sequences flanking bla_KPC-2 revealed that the Tn4401a isoform was present in plasmids from all STs except ST340 containing Tn4401b. Co-production of VIM enzymes was observed in isolates of ST147, ST323 and ST383. 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.

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 β-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.

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.

Colistin-resistant/carbapenem-resistant Acinetobacter baumannii is a significant challenge for antibiotic treatment and infection control policies. Since 2012, in Central Greece an increase of colistin/pan- resistant A. baumannii has occurred, indicating the need for further analysis. A total of 86 colistin-resistant/carbapenem-resistant out of 1228 A. baumannii clinical isolates, consecutively collected between 2012 and 2014 in a tertiary Greek hospital of Central Greece, as well as one environmental isolate from surveillance cultures were studied. Molecular typing and mechanisms of resistance to colistin and to carbapenems were assessed, whereas, epidemiological and clinical data of the patients were reviewed. During the study period, the rate of colistin resistance gradually increased and reached 21.1 % in 2014. All colistin-resistant/carbapenem-resistant A. baumannii belonged to 3LST ST101 clone that corresponds to the international clonal lineage II. Carbapenem resistance was associated with the presence of bla oxa-23-like, while resistance to colistin probably correlated with G54E and R109H amino acid substitutions in PmrA and PmrC, respectively. Epidemiological data of the patients indicated that the first detection of colistin-resistant/carbapenem-resistant ST101 clone in the University Hospital of Larissa (UHL) was associated with a patient who previously had received colistin, while, the movement of the infected patients into the hospital probably resulted to its spread.