Panagiota Giakkoupi

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).

IBC-1, a novel integron-associated class A beta-lactamase with extended-spectrum properties produced by an Enterobacter cloacae clinical strain.

ABSTRACT A transferable β-lactamase produced by a multidrug-resistant clinical isolate of Enterobacter cloacae was studied. Thebla gene was carried by a large (>80-kb) transmissible plasmid. Nucleotide sequence analysis of cloned fragments revealed that it was part of a gene cassette carried by a class 1 integron along with other resistance genes, includingaac(6′)-Ib. The encoded β-lactamase, designated IBC-1, was a novel class A enzyme that hydrolyzed ceftazidime and cefotaxime and was inhibited by tazobactam and, to a lesser extent, by clavulanate. Also, imipenem exhibited potent inhibitory activity against IBC-1. The enzyme consisted of 287 amino acid residues, including Ser-237, cysteines at positions 69 and 237a, and Arg-244, which may be implicated in its interaction with β-lactams. In amino acid sequence comparisons, IBC-1 displayed the highest similarity with the chromosomal penicillinase of Yersinia enterocolitica, a carbenicillinase from Proteus mirabilis GN79, the species-specific β-lactamases ofKlebsiella oxytoca, and the carbapenemase Sme-1. However, a phylogenetic association with established β-lactamase clusters could not be conclusively shown.

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

SPREAD OF INTEGRON-ASSOCIATED VIM-TYPE METALLO-BETA-LACTAMASE GENES AMONG IMIPENEM-NONSUSCEPTIBLE PSEUDOMONAS AERUGINOSA STRAINS IN GREEK HOSPITALS

ABSTRACT Fifty-eight imipenem-nonsusceptible (MIC ≥ 8 μg/ml) Pseudomonas aeruginosa strains isolated during May 2001 in 15 Greek hospitals were studied. Thirty-six isolates derived from nine hospitals carried VIM-type metallo-β-lactamase genes, as found by PCR. In 34 isolates, blaVIM was associated with class 1 integrons of various sizes. DNA sequencing indicated the presence of blaVIM-2 gene cassettes in a variety of integron structures. Random amplified polymorphic DNA typing suggested diversity of the blaVIM-positive strains. Synergy between 2-mercaptoacetic acid and imipenem indicated carbapenemase activity in 26 blaVIM-positive strains.

Discrepancies and Interpretation Problems in Susceptibility Testing of VIM-1-Producing Klebsiella pneumoniae Isolates

ABSTRACT Susceptibilities to β-lactam antibiotics of five VIM-1-producing Klebsiella pneumoniae isolates were determined by broth microdilution, Etest, disk diffusion, and the automated systems Vitek 2, Phoenix, and MicroScan. Significant discrepancies were observed in the determination of susceptibility to imipenem and meropenem. Interpretation problems by the automated systems were also noted.

Development and validation of a multiplex PCR assay for identification of the epidemic ST-258/512 KPC-producing Klebsiella pneumoniae clone

The Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-KP) sequence type (ST)-258/512 clone is the dominant clone by which KPC has disseminated worldwide. Standard typing methods are time-consuming and are therefore impractical for identification of this clone in the course of an outbreak. Through comparative genomic study, we have previously identified several presumably unique genes of this clone: 1) PILV-like protein (pilv-l), 2) transposase, IS66-family (is-66), and a 3) phage-related protein (prp). Our aims were to 1) test for the presence of these genes using a multiplex PCR in a large, multinational collection of KPC-KP isolates and to 2) validate this assay as a typing method for the identification of the ST-258/512 clone. KPC-KP isolates (n=160) that included both ST-258/512 (group A, n=114) and non-ST-258 (group B, n=46) strains were collected from the following countries: Greece, 20; Israel, 93; Italy, 19; USA, 25; and Colombia, 3. Group B included 30 different STs from various lineages. The pilv-l gene was present in 111/114 of ST-258 isolates, including all of the KPC-negative isolates resulting in a sensitivity of 97%. Using primers for a unique ST-258 pilv-l allele resulted in a specificity of 100%. The sensitivity values of is-66 and prp genes for detecting KPC-KP ST-258 were 83 and 89%, respectively, and the specificity values were 67 and 93%, respectively. PCR for the unique pilv-l ST-258 allele provides a reliable tool for rapid detection of the ST-258 clone. This method can be helpful both in the setting of an outbreak and in a large-scale survey of KPC-KP strains.

OmpK35 and OmpK36 porin variants associated with specific sequence types of Klebsiella pneumoniae.

OmpK35 and OmpK36 porin variants associated with specific sequence types of Klebsiella pneumoniae Constantinos C. Papagiannitsis, Panagiota Giakkoupi, Stathis D. Kotsakis, Eva Tzelepi, Leonidas S. Tzouvelekis, Alkiviadis C. Vatopoulos, Vivi Miriagou Department of Microbiology, National School of Public Health, Athens, Greece, Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens, Greece, Department of Microbiology, Medical School, University of Athens, Greece, Central Public Health Laboratory, Hellenic Centre of Disease Control and Prevention, Vari, Greece

Characterisation of IncA/C2 plasmids carrying an In416-like integron with the blaVIM-19 gene from Klebsiella pneumoniae ST383 of Greek origin

The complete nucleotide sequences of three multidrug resistance (MDR) IncA/C-like plasmids from Enterobacteriaceae isolates carrying the VIM-type carbapenemase-encoding integrons In4863 (blaVIM-19-aacA7-dfrA1-ΔaadA1-smr2) or In4873 (blaVIM-1-aacA7-dfrA1-ΔaadA1-smr2) were determined, which are the first In416-like elements identified in Greece. Plasmids pKP-Gr642 and pKP-Gr8143 were from Klebsiella pneumoniae ST383 isolates, whereas plasmid pEcl-Gr4873 was from an Enterobacter cloacae ST88 isolate. Sequencing showed that pKP-Gr642 (162787bp) and pKP-Gr8143 (154395bp) consisted of the type 1 IncA/C2 conserved backbone, the blaCMY-2-like gene-containing region, and the ARI-B (with the sul2 gene) and ARI-A (with a class 1 integron) resistance islands, like the plasmid pUMNK88_161 from the USA. The third plasmid, pEcl-Gr4873 (153958bp), exhibited extensive similarity with the type 2 IncA/C2 plasmid pR55 from France. pEcl-Gr4873 carried only one resistance island of a hybrid transposon structure inserted in a different location to ARI-A in type 1 A/C2 plasmids. In all three plasmids, the In416-like integrons In4863 or In4873 were identified within non-identical class II transposon structures. All three In416-like-carrying regions presented significant similarities with the MDR region of the IncA/C2 plasmid pCC416 from Italy, carrying the prototype In416 integron (blaVIM-4-aacA7-dfrA1-ΔaadA1-smr2). These findings provided the basis for speculations regarding the evolution of IncA/C2 plasmids with In416-like integrons, and confirmed the rapid evolution of some IncA/C2 plasmid lineages. Considering the broad host range of IncA/C2 molecules, it seems that pKP-Gr642, pKP-Gr8143 and pEcl-Gr4873 plasmids might support the diffusion of In416-like integrons among Enterobacteriaceae.

Characterization of pKP1433, a Novel KPC-2-Encoding Plasmid from Klebsiella pneumoniae Sequence Type 340

ABSTRACT The nucleotide sequence of pKP1433 (55,417 bp), a blaKPC-2-carrying plasmid from Klebsiella pneumoniae sequence type 340, was determined. pKP1433 displayed extensive sequence and structural similarities with the IncN plasmids possessing the KPC-2-encoding Tn4401b isoform. However, the replication, partitioning, and stability of pKP1433 were determined by sequences related to diverse non-IncN plasmids.

Supplementation of growth media with Zn2+ facilitates detection of VIM-2-producing Pseudomonas aeruginosa.

Isolation rates of Pseudomonas aeruginosa producing zinc-dependent class B metallo-β-lactamases (MBLs) mainly of the VIM and IMP types are increasing worldwide (7). These enzymes exhibit wide hydrolysis spectra, including carbapenems, and are strongly inhibited by chelating agents such as EDTA. Based on the latter property, various MBL-detecting assays have been developed (1). EDTA-imipenem synergy tests are widely utilized in hospitals in Greece, where the incidence of VIM-positive P. aeruginosa is considered among the highest in Europe (www.rivm.nl/earss). However, observations in various hospitals in Athens and the reference laboratory of the National School of Public Health (NSPH) indicate suboptimal sensitivity of the EDTA-based methods. In this study, we attempted to evaluate the effect of zinc supplementation of the test medium on the performance of these methods. Forty-three P. aeruginosa isolates submitted for testing in the NSPH from 13 hospitals during 2006 due to difficulties in interpreting the results of the EDTA-imipenem synergy methods were included in the study. MICs of β-lactams were determined by the Etest (AB Biodisk, Solna, Sweden). PCR assays for detection of MBL genes were performed as described previously (5, 6). The identities of MBL genes were confirmed by sequencing of the respective amplicons. Phenotypic detection of MBLs was performed in Mueller-Hinton agar (MHA) as well as in the same medium in which 70 mg/liter ZnSO4·7H2O had been incorporated (Zn2+ at a final concentration of 250 μM) as suggested by Lee et al. (4). Imipenem-EDTA synergy was assessed with the MBL-Etest (with a ≥8-fold decrease in the MIC of imipenem in the presence of EDTA considered a positive result) as well as two in-house techniques: the double-disk synergy test (DDST), using imipenem (10 μg) and EDTA disks (930 μg) in a 20-mm center-to-center distance, and the combination disk test (CDT), using an imipenem (10 μg) disk alone and containing 930 μg EDTA (with a ≥7-mm increase in inhibition zone considered a positive result) (3). The effects of the Zn2+ supplementation on the Etest MICs of imipenem and ceftazidime were also determined. Twenty-seven (63%) of the 43 isolates carried blaVIM-2 (group A). The remaining 16 isolates (37%) were negative for MBL genes (group B). Imipenem MICs for group A isolates ranged from 1 to >32 μg/ml. The respective range for group B isolates was 4 to 32 μg/ml. Imipenem MICs were in good agreement with those reported by the hospital laboratories. Sensitivity problems (false negatives) were noticed with all three EDTA-based methods employed. The higher sensitivity score was observed with DDST followed by MBL-Etest and CDT. Also four of the group B isolates appeared false positive (Table ​(Table1),1), producing slight although reproducible synergy images. Imipenem MICs of the false-positive isolates ranged from 8 to 32 μg/ml. TABLE 1. Properties of 43 P. aeruginosa isolates Incorporation of Zn2+ in the growth medium resulted in a significant increase in the sensitivity of all three MBL detection methods without compromising specificity. More specifically, in Zn2+-supplemented MHA, the MBL-Etest and CDT correctly identified 27 (sensitivity 100%) and 26 (sensitivity 96%) group A isolates, respectively, while performance of the conventional testing techniques was poor. Likewise, zinc supplementation increased the number of group A isolates characterized as MBL positive by the DDST from 12 to 18, thus improving sensitivity from 44 to 67% (Table ​(Table1).1). A plausible explanation for the positive effect of Zn2+ on MBL detection in P. aeruginosa is that Zn2+ may facilitate formation of functional MBL molecules in the periplasmic space. Also, the relatively high Zn2+ concentrations during growth reduce expression of P. aeruginosa porins and consequently carbapenem diffusion rates (2), further enhancing the effects of carbapenemase activity. This explanation is compatible with the increase in the apparent resistance levels to imipenem and ceftazidime that was more pronounced among VIM-2 producers (Table ​(Table11). Twenty-seven of the 43 submitted P. aeruginosa isolates (15 group A and 12 group B) were readily and correctly characterized in the NSPH by at least one conventional EDTA-based phenotypic method, likely suggesting technical problems in the hospital laboratories. Nevertheless, in a number of isolates, MBL production was not apparent. Despite the limitations of this preliminary study (a relatively small number of VIM-producing isolates), our findings suggest that Zn2+ supplementation may be a useful adjunct for MBL detection in P. aeruginosa and warrants further investigation.