Costas C. Papagiannitsis

An update of the evolving epidemic of blaKPC-2-carrying Klebsiella pneumoniae in Greece (2009–10)

OBJECTIVES To follow the epidemic of KPC-2-producing Klebsiella pneumoniae in Greece. METHODS 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). RESULTS 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. CONCLUSIONS 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).

Detection of carbapenemases in Enterobacteriaceae: a challenge for diagnostic microbiological laboratories

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.

An Ertapenem-Resistant Extended-Spectrum-β-Lactamase-Producing Klebsiella pneumoniae Clone Carries a Novel OmpK36 Porin Variant

ABSTRACT 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.

Emerging Klebsiella pneumoniae Isolates Coproducing KPC-2 and VIM-1 Carbapenemases

VIM-1-producing Klebsiella pneumoniae strains, increasingly isolated in Greece since 2002, constitute the majority of multiresistant clinical isolates of this species found in most Greek hospitals ([9][1]). Moreover, following reports on Klebsiella pneumoniae carbapenemase (KPC)-producing isolates

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Meropenem Hydrolysis Assay with NH4HCO3, a Reliable Tool for Direct Detection of Carbapenemase Activity

ABSTRACT 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 NH4HCO3 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.

Sequence of pNL194, a 79.3-Kilobase IncN plasmid carrying the blaVIM-1 metallo-β-lactamase gene in Klebsiella pneumoniae.

ABSTRACT The nucleotide sequence of pNL194, a VIM-1-encoding plasmid, is described in this study. pNL194 (79,307 bp) comprised an IncN-characteristic segment (38,940 bp) and a mosaic structure (40,367 bp) including blaVIM-1, aacA7, aadA1, aadA2, dfrA1, dfrA12, aphA1, strA, strB, and sul1. Tn1000 or Tn5501 insertion within fipA probably facilitated recruitment of additional mobile elements carrying resistance genes.

Characterization of Metallo-β-Lactamase VIM-27, an A57S Mutant of VIM-1 Associated with Klebsiella pneumoniae ST147

ABSTRACT VIM-27 metallo-β-lactamase, an Ala57 → Ser variant of VIM-1, was identified in three Klebsiella pneumoniae isolates belonging to sequence type 147. blaVIM-27 was part of a class 1 integron carried by non-self-transferable plasmids. Kinetic parameters and MIC determinations indicated that VIM-27 hydrolyzed most β-lactams, especially imipenem and cefoxitin, less effectively than VIM-1.

Rapid typing of extended-spectrum β-lactamase- and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolates by use of spectracell RA

ABSTRACT Enterobacteriaceae are important pathogens of both nosocomial and community-acquired infections. In particular, strains with broad-spectrum beta-lactamases increasingly cause problems in health care settings. Rapid and reliable typing systems are key tools to identify transmission, so that targeted infection control measures can be taken. In this study, we evaluated the performance of Raman spectroscopic analysis (RA) for the typing of multiresistant Escherichia coli and Klebsiella pneumoniae isolates using the SpectraCell RA bacterial strain analyzer (River Diagnostics). Analysis of 96 unrelated isolates revealed that RA generated highly reproducible spectra and exhibited a discriminatory power that is comparable to pulsed-field gel electrophoresis. Furthermore, adequate results were obtained for three collections of clinical isolates. RA was able to discriminate outbreak-related isolates from isolates that were not involved in an outbreak or transmission. Furthermore, it was found that the RA approach recognized clones, irrespective of the extended-spectrum β-lactamase type. It can be concluded that RA is a suitable typing technique for E. coli and K. pneumoniae isolates. Combining high reproducibility, speed, and ease-of-use, this technique may play an important role in monitoring the epidemiology of these important nosocomial species.

Relative Strengths of the Class 1 Integron Promoter Hybrid 2 and the Combinations of Strong and Hybrid 1 with an Active P2 Promoter

ABSTRACT The relative strengths of the uncommon promoters hybrid 2, hybrid 1 with an active P2 promoter (hybrid 1+P2), and strong+P2, which drive transcription of resistance genes in class 1 integrons, were evaluated using blaGES-1 as a reporter gene cassette. Hybrid 2 was stronger than hybrid 1. Coupling P2 with the strong promoter and with hybrid 1 caused a measurable increase in GES-1 expression.

Characterization of pKP1780, a novel IncR plasmid from the emerging Klebsiella pneumoniae ST147, encoding the VIM-1 metallo-β-lactamase

OBJECTIVES To determine the complete nucleotide sequence of the VIM-1-encoding plasmid pKP1780 from Klebsiella pneumoniae ST147 representing a distinct group of IncR replicons. METHODS The plasmid pKP1780 was from a K. pneumoniae clinical strain (KP-1780) isolated in Greece in 2009. Plasmid DNA was extracted from an Escherichia coli DH5α transformant and sequenced using the 454 Genome Sequencer GS FLX procedure on a standard fragment DNA library. Contig gaps were filled by sequencing of PCR-produced fragments. Annotation and comparative analysis were performed using software available on the Internet. RESULTS Plasmid pKP1780 (49 770 bp) consisted of an IncR-related sequence (12 083 bp) including replication and stability systems, and a multidrug resistance (MDR) mosaic region (37 687 bp). blaVIM-1 along with the aacA7, dfrA1 and aadA1 cassettes comprised the variable region of an integron similar to In-e541 from pNL194. The mosaic structure also included the strA, strB, aphA1 and mphA resistance genes as well as intact (n = 10) or defective (n = 3) insertion sequences and fragments of various transposons. CONCLUSIONS The mosaic structure of pKP1780 exhibited high similarity with the acquired region of the IncN plasmid pNL194, indicating the acquisition of the VIM-1-encoding MDR region from pNL194 by an IncR-type plasmid.