Ørjan Samuelsen

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.

A sensitive and specific phenotypic assay for detection of metallo-β-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin

Enterobacteriaceae producing carbapenemases, such as KPC or metallo-β-lactamases (MBLs), have emerged on several continents. Phenotypic tests are urgently needed for their rapid and accurate detection. A novel carbapenemase detection test, comprising a meropenem disk, and meropenem disks supplemented with 730 μg of EDTA, 1000 μg of dipicolinic acid (DPA), 600 μg of aminophenylboronic acid (APBA), or 750 μg of cloxacillin, was evaluated against Klebsiella pneumoniae isolates with KPC (n = 34), VIM (n = 21), IMP (n = 4) or OXA-48 (n = 9) carbapenemases, and carbapenem-resistant Enterobacteriaceae with porin loss in combination with an extended-spectrum β-lactamase (ESBL) (n = 9) or AmpC hyperproduction (n = 5). Commercially available diagnostics tablets from Rosco containing meropenem and the same inhibitors as described above (except EDTA) were also evaluated. An increased meropenem inhibition zone was sought in the presence of each added β-lactamase inhibitor. APBA had excellent sensitivity for detecting K. pneumoniae with KPC enzymes. Isolates with combined AmpC hyperproduction and porin loss were also positive in the APBA test but, unlike KPC producers, showed cloxacillin synergy. Both DPA and EDTA had excellent sensitivity for detection of MBL-producing K. pneumoniae. However, EDTA showed poor specificity, with positive results noted for 1/9 ESBL-producing isolates, for 4/34 KPC-producing isolates, and for 4/9 OXA-48-producing isolates, whereas all of these were negative when DPA was used. The in-house test distinguished accurately between several different mechanisms mediating reduced susceptibility to carbapenems in Enterobacteriaceae. The commercial combination tablets from Rosco performed similarly to the in-house test, with the exception of one false-positive MBL result and one false-positive KPC result among the OXA-48 producers.

Emergence of clonally related Klebsiella pneumoniae isolates of sequence type 258 producing plasmid-mediated KPC carbapenemase in Norway and Sweden

BACKGROUND The class A carbapenemase KPC has disseminated rapidly worldwide, challenging the treatment of Gram-negative infections. This report describes the first KPC-producing Klebsiella pneumoniae isolates identified in Norway (n=6) and the second isolate from Sweden. METHODS Antimicrobial susceptibility profiles were determined using Etest. PCR and sequencing were used to determine the bla(KPC) variant, the surrounding genetic structure and the presence of AmpC and extended-spectrum beta-lactamase genes. PFGE and multilocus sequence typing (MLST) were used for epidemiological comparisons. Localization of bla(KPC) was investigated by S1 nuclease digestion, followed by PFGE and Southern blot hybridization. RESULTS All isolates expressed a multidrug-resistant phenotype with some variability in the carbapenem susceptibility profile. The Norwegian isolates carried bla(KPC-2), while the Swedish isolate carried bla(KPC-3). All isolates carried TEM-1, but were negative for bla(CTX-M) and bla(AmpC) genes. SHV-11 and SHV-12 were detected in the Norwegian isolates, while the Swedish isolate carried only SHV-11. Isolates from four patients were associated with import from Greece (n=3) and Israel. The other isolates were probably associated with local transmissions. PFGE and MLST showed that the isolates were clonally related, with three isolates displaying ST258, a single locus variant of ST11 previously associated with the clonal spread of CTX-M-15-producing K. pneumoniae in Hungary. In all isolates, bla(KPC) was located on plasmids as part of isoform a of Tn4401. CONCLUSIONS The emergence of KPC-producing isolates of K. pneumoniae in Norway and Sweden is associated with multiple import events and probable local transmission of a successful multiresistant ST258 clone, closely related to the CTX-M-15-producing ST11 clone previously described in Hungary.

Insights into the global molecular epidemiology of carbapenem non-susceptible clones of Acinetobacter baumannii

The global emergence of multidrug resistance (MDR) among Gram-negative bacteria has dramatically limited the therapeutic options. During the last two decades, Acinetobacter baumannii has become a pathogen of increased clinical importance due to its remarkable ability to cause outbreaks of infections and to acquire resistance to almost all currently used antibiotics, including the carbapenems. This review considers the literature on A. baumannii and data from multilocus sequence typing studies to explore the global population structure of A. baumannii and detect the occurrence of clonality, with the focus on the presence of specific resistance mechanisms such as the OXA-carbapenemases. The worldwide dissemination of MDR and carbapenem non-susceptible A. baumannii is associated with diverse genetic backgrounds, but predominated by a number of extensively distributed clones, such as CC92(B)/CC2(P) and CC109(B)/CC1(P), which have frequently been supplemented by acquired OXA-type carbapenemase genes.

Molecular Epidemiology of Metallo-β-Lactamase-Producing Pseudomonas aeruginosa Isolates from Norway and Sweden Shows Import of International Clones and Local Clonal Expansion

ABSTRACT Scandinavia is considered a region with a low prevalence of antimicrobial resistance. However, the number of multidrug-resistant (MDR) Gram-negative bacteria is increasing, including metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa. In this study MBL-producing P. aeruginosa isolates identified in Norway (n = 4) and Sweden (n = 9) from 1999 to 2007 were characterized. Two international clonal complexes (CC), CC111 (n = 8) and CC235 (n = 2), previously associated with MBL-producing isolates, were dominant. CC111 isolates (ST111/229; serotype O12; blaVIM-2) included clonally related isolates identified in Skåne County, Sweden (n = 6), and two isolates associated with importation from Greece and Denmark. In all CC111 isolates, blaVIM-2 was located in integron In59.2 or In59 variants. The two CC235 isolates (ST235/ST230; serotype O11; blaVIM-4) were imported from Greece and Cyprus, were possibly clonally related, and carried blaVIM-4 in two different integron structures. Three isolates imported from Ghana (ST233; serotype O6; blaVIM-2), Tunisia (ST654; serotype O11; blaVIM-2), and Thailand (ST260; serotype O6; blaIMP-14) were clonally unrelated. ST233 was part of a new CC (CC233) that included other MBL-producing isolates, while ST654 could also be part of a new CC associated with MBL producers. In the isolates imported from Ghana and Tunisia, blaVIM-2 was part of unusual integron structures lacking the 3′ conserved segment and associated with transposons. The blaVIM gene was found to be located on the chromosome in all isolates. Known risk factors for acquisition of MBL were reported for all patients except one. The findings suggest that both import of successful international clones and local clonal expansion contribute to the emergence of MBL-producing P. aeruginosa in Scandinavia.

Occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in the European survey of carbapenemase-producing Enterobacteriaceae (EuSCAPE): a prospective, multinational study

BACKGROUND Gaps in the diagnostic capacity and heterogeneity of national surveillance and reporting standards in Europe make it difficult to contain carbapenemase-producing Enterobacteriaceae. We report the development of a consistent sampling framework and the results of the first structured survey on the occurrence of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in European hospitals. METHODS National expert laboratories recruited hospitals with diagnostic capacities, who collected the first ten carbapenem non-susceptible clinical isolates of K pneumoniae or E coli and ten susceptible same-species comparator isolates and pertinent patient and hospital information. Isolates and data were relayed back to national expert laboratories, which made laboratory-substantiated information available for central analysis. FINDINGS Between Nov 1, 2013, and April 30, 2014, 455 sentinel hospitals in 36 countries submitted 2703 clinical isolates (2301 [85%] K pneumoniae and 402 (15%) E coli). 850 (37%) of 2301 K pneumoniae samples and 77 (19%) of 402 E coli samples were carbapenemase (KPC, NDM, OXA-48-like, or VIM) producers. The ratio of K pneumoniae to E coli was 11:1. 1·3 patients per 10 000 hospital admissions had positive clinical specimens. Prevalence differed greatly, with the highest rates in Mediterranean and Balkan countries. Carbapenemase-producing K pneumoniae isolates showed high resistance to last-line antibiotics. INTERPRETATION This initiative shows an encouraging commitment by all participants, and suggests that challenges in the establishment of a continent-wide enhanced sentinel surveillance for carbapenemase-producing Enterobacteriaeceae can be overcome. Strengthening infection control efforts in hospitals is crucial for controlling spread through local and national health care networks. FUNDING European Centre for Disease Prevention and Control.

Identification of NDM-1-producing Enterobacteriaceae in Norway

Reference Centre for Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, N-9038 Tromso, Norway; Department of Medical Microbiology, Drammen Hospital, Drammen, Norway; Department of Medicine, Drammen Hospital, Drammen, Norway; Laboratory for Medical Microbiology, Levanger Hospital, Levanger, Norway; Research Group for Host-Microbe Interactions, Department of Medical Biology, Faculty of Health Sciences, University of Tromso, Tromso, Norway

Species identification and molecular characterization of Acinetobacter spp. blood culture isolates from Norway

OBJECTIVES The study investigated the species distribution, antibiotic susceptibility patterns and genotypic resistance characteristics of 113 consecutive blood culture isolates of Acinetobacter species collected between 2005 and 2007 throughout Norway. METHODS Species identification was performed by partial rpoB sequence analysis, and verified by 16S rDNA and recA sequence analyses. Susceptibility testing was performed by agar disc diffusion and Etest. Distribution of OXA carbapenemase genes and epidemic clonality of Acinetobacter baumannii isolates were detected by PCR assays. Analyses of blaOXA-51-like variants and quinolone resistance-determining regions (QRDRs) were done by sequencing. RESULTS The most prevalent species in the collection were Acinetobacter genomic species (gen. sp.) 13TU (46.9%) and Acinetobacter gen. sp. 3 (19.5%), followed by A. baumannii (8.8%) and Acinetobacter lwoffii/Acinetobacter gen. sp. 9 (7.1%). Carbapenem resistance was observed in one blaOXA-23-like-positive A. baumannii isolate. Quinolone resistance was detected in five isolates from the Acinetobacter calcoaceticus-A. baumannii complex, of which two had point mutations in the QRDRs, including one novel ParC mutation. None of the A. baumannii isolates belonged to European/international clones I, II or III. Six blaOXA-51-like variants, including two novel variants, were identified. CONCLUSIONS Acinetobacter gen. sp. 13TU and Acinetobacter gen. sp. 3 were predominant in Norwegian blood cultures, in contrast to in other countries where A. baumannii has dominated. The study demonstrated the importance of genotypic identification to determine the exact epidemiology of non-baumannii Acinetobacter species.

The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria : report from the EUCAST Subcommittee

Whole genome sequencing (WGS) offers the potential to predict antimicrobial susceptibility from a single assay. The European Committee on Antimicrobial Susceptibility Testing established a subcommittee to review the current development status of WGS for bacterial antimicrobial susceptibility testing (AST). The published evidence for using WGS as a tool to infer antimicrobial susceptibility accurately is currently either poor or non-existent and the evidence / knowledge base requires significant expansion. The primary comparators for assessing genotypic-phenotypic concordance from WGS data should be changed to epidemiological cut-off values in order to improve differentiation of wild-type from non-wild-type isolates (harbouring an acquired resistance). Clinical breakpoints should be a secondary comparator. This assessment will reveal whether genetic predictions could also be used to guide clinical decision making. Internationally agreed principles and quality control (QC) metrics will facilitate early harmonization of analytical approaches and interpretive criteria for WGS-based predictive AST. Only data sets that pass agreed QC metrics should be used in AST predictions. Minimum performance standards should exist and comparative accuracies across different WGS laboratories and processes should be measured. To facilitate comparisons, a single public database of all known resistance loci should be established, regularly updated and strictly curated using minimum standards for the inclusion of resistance loci. For most bacterial species the major limitations to widespread adoption for WGS-based AST in clinical laboratories remain the current high-cost and limited speed of inferring antimicrobial susceptibility from WGS data as well as the dependency on previous culture because analysis directly on specimens remains challenging. For most bacterial species there is currently insufficient evidence to support the use of WGS-inferred AST to guide clinical decision making. WGS-AST should be a funding priority if it is to become a rival to phenotypic AST. This report will be updated as the available evidence increases.

A long-term low-frequency hospital outbreak of KPC-producing Klebsiella pneumoniae involving Intergenus plasmid diffusion and a persisting environmental reservoir.

Background To study the molecular characteristics of a long-term, low frequency outbreak of bla KPC-2 in a low prevalence setting involving the hospital environment. Methodology/Principal Findings KPC-producing bacteria were screened by selective chromogenic agar and Real-Time PCR. The presence of antibiotic resistance genes was ascribed by PCRs and subsequent sequencing, and the KPC-producing isolates were phylogenetically typed using PFGE and multi-locus sequence typing. Bla KPC-2-plasmids were identified and analysed by S1-nuclease-PFGE hybridization and PCR based replicon typing. A ∼97 kb IncFII plasmid was seen to carry bla KPC-2 in all of the clinical isolates, in one of the isolates recovered from screened patients (1/136), and in the Klebsiella pneumoniae and Enterobacter asburiae isolates recovered from the environment (sinks) in one intensive care unit. The K. pneumoniae strain ST258 was identified in 6 out of 7 patients. An intergenus spread to E. asburiae and an interspecies spread to two different K. pneumoniae clones (ST27 and ST461) of the bla KPC-2 plasmid was discovered. K. pneumoniae ST258 and genetically related E. asburiae strains were found in isolates of both human and environmental origins. Conclusions/Significance We document a clonal transmission of the K. pneumoniae ST258 strain, and an intergenus plasmid diffusion of the IncFII plasmid carrying bla KPC-2 in this outbreak. A major reservoir in the patient population could not be unveiled. However, the identification of a persisting environmental reservoir of strains with molecular determinants linked to human isolates, suggests a possible role of the environment in the maintenance of this long-term outbreak.