Vivi Miriagou

Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe

Plasmid-acquired carbapenemases in Enterobacteriaceae, which were first discovered in Europe in the 1990s, are now increasingly being identified at an alarming rate. Although their hydrolysis spectrum may vary, they hydrolyse most β-lactams, including carbapenems. They are mostly of the KPC, VIM, NDM and OXA-48 types. Their prevalence in Europe as reported in 2011 varies significantly from high (Greece and Italy) to low (Nordic countries). The types of carbapenemase vary among countries, partially depending on the cultural/population exchange relationship between the European countries and the possible reservoirs of each carbapenemase. Carbapenemase producers are mainly identified among Klebsiella pneumoniae and Escherichia coli, and still mostly in hospital settings and rarely in the community. Although important nosocomial outbreaks with carbapenemase-producing Enterobacteriaceae have been extensively reported, many new cases are still related to importation from a foreign country. Rapid identification of colonized or infected patients and screening of carriers is possible, and will probably be effective for prevention of a scenario of endemicity, as now reported for extended-spectrum β-lactamase (mainly CTX-M) producers in all European countries.

Carbapenemase-Producing Klebsiella pneumoniae Bloodstream Infections: Lowering Mortality by Antibiotic Combination Schemes and the Role of Carbapenems

ABSTRACT Carbapenemase-producing Klebsiella pneumoniae strains (CP-Kps) are currently among the most important nosocomial pathogens. An observational study was conducted during 2009 to 2010 in two hospitals located in a high-prevalence area (Athens, Greece). The aims were (i) to evaluate the clinical outcome of patients with CP-Kp bloodstream infections (BSIs), (ii) to identify predictors of mortality, and (iii) to evaluate the various antibiotic schemes employed. A total of 205 patients with CP-Kp BSIs were identified: 163 (79.5%) were infected with KPC or KPC and VIM, and 42 were infected with VIM producers. For definitive treatment, 103 patients received combination therapy (two or more active drugs), 72 received monotherapy (one active drug), and 12 received therapy with no active drug. The remaining 18 patients died within 48 h after the onset of bacteremia. The all-cause 28-day mortality was 40%. A significantly higher mortality rate was observed in patients treated with monotherapy than in those treated with combination therapy (44.4% versus 27.2%; P = 0.018). The lowest mortality rate (19.3%) was observed in patients treated with carbapenem-containing combinations. In the Cox proportion hazards model, ultimately fatal disease (hazards ratio [HR], 3.25; 95% confidence interval [CI], 1.51 to 7.03; P = 0.003), the presence of rapidly fatal underlying diseases (HR, 4.20; 95% CI, 2.19 to 8.08; P < 0.001), and septic shock (HR, 2.15; 95% CI, 1.16 to 3.96; P = 0.015) were independent predictors of death. Combination therapy was strongly associated with survival (HR of death for monotherapy versus combination, 2.08; 95% CI, 1.23 to 3.51; P = 0.006), mostly due to the effectiveness of the carbapenem-containing regimens.

Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues

Acquired carbapenemases are emerging resistance determinants in Gram-negative pathogens, including Enterobacteriaceae, Pseudomonas aeruginosa and other Gram-negative non-fermenters. A consistent number of acquired carbapenemases have been identified during the past few years, belonging to either molecular class B (metallo-beta-lactamases) or molecular classes A and D (serine carbapenemases), and genes encoding these enzymes are associated with mobile genetic elements that allow their rapid dissemination in the clinical setting. Therefore, detection and surveillance of carbapenemase-producing organisms have become matters of major importance for the selection of appropriate therapeutic schemes and the implementation of infection control measures. As carbapenemase production cannot be simply inferred from the resistance profile, criteria must be established for which isolates should be suspected and screened for carbapenemase production, and for which tests (phenotypic and/or genotypic) should be adopted for confirmation of the resistance mechanism. Moreover, strategies should be devised for surveillance of carbapenemase producers in order to enable the implementation of effective surveillance programmes. The above issues are addressed in this article, as a follow-up to an expert meeting on acquired carbapenemases that was recently organized by the ESCMID Study Group for Antibiotic Resistance Surveillance.

Identification and screening of carbapenemase-producing Enterobacteriaceae

Carbapenem-hydrolysing β-lactamases are the most powerful β-lactamases, being able to hydrolyse almost all β-lactams. They are mostly of the KPC, VIM, IMP, NDM and OXA-48 types. Their current extensive spread worldwide in Enterobacteriaceae is an important source of concern, as these carbapenemase producers are multidrug-resistant. Detection of infected patients and of carriers are the two main approaches for prevention of their spread. Phenotypic and molecular-based techniques are able to identify these carbapenemase producers, although with variable efficiencies. The detection of carriers still relies mostly on the use of screening culture media.

Imipenem Resistance in a Salmonella Clinical Strain Due to Plasmid-Mediated Class A Carbapenemase KPC-2

ABSTRACT A Salmonella enterica serotype Cubana isolate exhibiting resistance to most β-lactam antibiotics, including oxyimino-cephalosporins and imipenem, was isolated from a 4-year-old boy with gastroenteritis in Maryland. β-Lactam resistance was mediated by a conjugative plasmid that encoded KPC-2, a class A carbapenemase previously found in a Klebsiella pneumoniae isolate from the Maryland area as well. Sequence analysis of the flanking regions indicated a potential association of blaKPC-2 with mobile structures.

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).

Replicon Typing of Plasmids Encoding Resistance to Newer β-Lactams

Polymerase chain reaction–based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems.

Escherichia coli with a Self-Transferable, Multiresistant Plasmid Coding for Metallo-β-Lactamase VIM-1

ABSTRACT An Escherichia coli strain exhibiting decreased susceptibility to carbapenems was isolated from a hospitalized patient in Greece. The strain carried a self-transferable plasmid coding for metallo-β-lactamase VIM-1. blaVIM-1, along with aacA7, dhfrI, and aadA, was included as a gene cassette in a novel class 1 integron. A Citrobacter freundii ampC-derived gene, not associated with the integron, was also located in the same plasmid.

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.

IS26-Associated In4-Type Integrons Forming Multiresistance Loci in Enterobacterial Plasmids

ABSTRACT Three distinct multiresistant loci from enterobacterial plasmids each comprised an integron and an IS26-associated sequence. Sequence comparison suggested a common ancestral structure that derived from an IS26 insertion into the 5′ conserved segment of an In4-type integron and evolved through acquisition of gene cassettes and IS26-mediated recruitment of additional resistance genes of diverse origin.