Bettina Aasnæs

Comparative DNA Analysis of Two vanA Plasmids from Enterococcus faecium Strains Isolated from Poultry and a Poultry Farmer in Norway

ABSTRACT The DNA sequences of two plasmids carrying vanA, pVEF1 (39,626 bp) and pVEF2 (39,714 bp), were determined. Forty-three shared coding sequences were identified, and the only nucleotide difference was an 88-bp indel. A postsegregational killing system was identified. This system possibly explains the persistence of the vanA gene cluster in Norwegian poultry farms.

The AmpC phenotype in Norwegian clinical isolates of Escherichia coli is associated with an acquired ISEcp1-like ampC element or hyperproduction of the endogenous AmpC

OBJECTIVES The aim of the study was to examine resistance mechanisms associated with an AmpC phenotype in Norwegian clinical isolates of Escherichia coli. METHODS Clinical E. coli isolates (n = 106) with reduced susceptibility to third-generation cephalosporins without clavulanic acid synergy were collected from 12 Norwegian laboratories from 2003 to 2005. Twenty-two isolates with an AmpC phenotype were selected for further characterization by PFGE, isoelectric focusing, different PCR-based techniques, DNA sequencing, AmpC qRT-PCR, transfer studies and plasmid analyses. RESULTS The 22 isolates were not clonally related by the PFGE analysis. All isolates expressed a beta-lactamase with a pI of 9.0-9.2. Ten isolates contained a bla(CMY) gene, which was linked to an ISEcp1-like element in all cases. Twelve isolates had mutations or insertions in the promoter or the attenuator regions, leading to increased expression of the chromosomal ampC gene. One of these isolates had an ISEc10 element inserted upstream of the chromosomal ampC gene. CONCLUSIONS This is the first molecular study of Norwegian clinical E. coli isolates with an AmpC phenotype. Resistance was mediated either by expression of bla(CMY) from acquired ISEcp1-like-bla(CMY) elements, or by mutations or insertions in the chromosomal ampC gene control region leading to hyperproduction of the endogenous AmpC enzyme. There was no correlation between the level of ampC mRNA and the MICs of cephalosporins.

Evaluation of phenotypic tests for the detection of metallo-β-lactamase-producing Pseudomonas aeruginosa in a low prevalence country

OBJECTIVES To evaluate four phenotypic tests for the detection of metallo-beta-lactamase (MBL) production in Pseudomonas aeruginosa in a low MBL prevalence setting. METHODS Sixty clinical isolates of P. aeruginosa resistant to imipenem and/or meropenem and seven MBL-positive control strains were examined by: (i) MBL Etest; (ii) combined imipenem discs supplemented with EDTA (IPM-EDTA); (iii) beta-lactam discs on dipicolinic acid plates (DF-DIPI); and (iv) the Cica-beta test. Spectrophotometric analysis of crude cell extracts for imipenem hydrolysis along with consensus PCRs for bla(VIM) and bla(IMP) was used as reference methods. RESULTS Two clinical isolates (3%) were MBL-positive. The MBL Etest and IPM-EDTA test scored positive for all MBL-positive isolates, but showed specificities of 86% and 91%, and positive predictive values (PPVs) of only 20% and 29%, respectively. Adding resistance to ceftazidime (MIC >8 mg/L) as a criterion for MBL testing would reduce the number of isolates to be screened by 50% and increase the PPVs of the MBL Etest and IMP-EDTA test to 29% and 40%, respectively. The Cica-beta test correctly identified all MBL-negative isolates, but misidentified one MBL-positive clinical isolate as an extended-spectrum beta-lactamase (ESBL)-producer and one as inconclusive (producing multiple beta-lactamases). No reliable breakpoints could be defined for the DF-DIPI test due to overlapping inhibition zone diameters for MBL-positive and -negative isolates. CONCLUSIONS None of the phenotypic tests were optimal due to low sensitivity or specificity, resulting in low PPVs. Including ceftazidime resistance to the MBL-screening criteria would significantly improve the performance of the MBL Etest and IPM-EDTA disc test.

Cluster of linezolid-resistant Enterococcus faecium ST117 in Norwegian hospitals

Abstract A linezolid-resistant, vancomycin-susceptible Enterococcus faecium strain was isolated from 3 patients who had not received linezolid. The first patient was hospitalized in the same hospitals and wards as the 2 following patients. The E. faecium isolates were resistant to linezolid (minimum inhibitory concentration 8–32 mg/l), ampicillin, and high levels of gentamicin. Resistance to linezolid was associated with a G2576T mutation in 23S rDNA. The cfr linezolid resistance gene was not detected. The 3 isolates showed identical DNA fingerprints by pulsed-field gel electrophoresis, belonged to ST117, and harboured virulence genes esp, hyl, acm, efaAfm, srgA, ecbA, scm, pilA, pilB, and pstD typically associated with high-risk E. faecium genotypes. The linezolid-resistant E. faecium high-risk clone caused bacteraemia in the first 2 cancer patients and survived in the hospital environment for more than a year before appearing in the urethral catheter of the third patient.

Molecular and epidemiological characterization of carbapenemase-producing Enterobacteriaceae in Norway, 2007 to 2014

The prevalence of carbapenemase-producing Enterobacteriaceae (CPE) is increasing worldwide. Here we present associated patient data and molecular, epidemiological and phenotypic characteristics of all CPE isolates in Norway from 2007 to 2014 confirmed at the Norwegian National Advisory Unit on Detection of Antimicrobial Resistance. All confirmed CPE isolates were characterized pheno- and genotypically, including by whole genome sequencing (WGS). Patient data were reviewed retrospectively. In total 59 CPE isolates were identified from 53 patients. Urine was the dominant clinical sample source (37%) and only 15% of the isolates were obtained from faecal screening. The majority of cases (62%) were directly associated with travel or hospitalization abroad, but both intra-hospital transmission and one inter-hospital outbreak were observed. The number of CPE cases/year was low (2–14 cases/year), but an increasing trend was observed. Klebsiella spp. (n = 38) and E. coli (n = 14) were the dominant species and blaKPC (n = 20), blaNDM (n = 19), blaOXA-48-like (n = 12) and blaVIM (n = 7) were the dominant carbapenemase gene families. The CPE isolates were genetically diverse except for K. pneumoniae where clonal group 258 associated with blaKPC dominated. All isolates were multidrug-resistant and a significant proportion (21%) were resistant to colistin. Interestingly, all blaOXA-48-like, and a large proportion of blaNDM-positive Klebsiella spp. (89%) and E. coli (83%) isolates were susceptible in vitro to mecillinam. Thus, mecillinam could have a role in the treatment of uncomplicated urinary tract infections caused by OXA-48- or NDM-producing E. coli or K. pneumoniae. In conclusion, the impact of CPE in Norway is still limited and mainly associated with travel abroad, reflected in the diversity of clones and carbapenemase genes.

A nationwide study of mechanisms conferring reduced susceptibility to extended-spectrum cephalosporins in clinical Escherichia coli and Klebsiella spp. isolates

Abstract Background: Enterobacteriaceae exerting a high level of extended-spectrum cephalosporin (ESC) resistance have increased significantly in Norway in the last decade. Various mechanisms acting alone or in concert mediate variable levels of ESC resistance and pose great challenges in the implementation of screening strategies and treatment. This study was undertaken to document the prevalence of underlying mechanisms conferring resistance to ESCs in a nationwide collection of clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca, before the increase in extended-spectrum β-lactamase (ESBL)-producing strains. Methods: Consecutive E. coli (n = 2213), K. pneumoniae (n = 303), and K. oxytoca (n = 66) isolates from 23 Norwegian diagnostic laboratories were collected and examined for reduced susceptibility to ESCs. Isolates displaying minimum inhibitory concentrations (MICs) of > 2 mg/l by Etest to cefpodoxime and/or MICs > 1 mg/l to any other ESCs were included (n = 54; 35 E. coli, 11 K. pneumoniae, and 8 K. oxytoca). Isoelectric focusing for the detection of β-lactamases, and polymerase chain reactions (PCRs) with subsequent sequencing for detection of ESBLs CTX-M, TEM, and SHV, plasmid-mediated AmpC, OXA subtypes, and alterations of porin genes ompC and ompF, and quantitative reverse transcriptase (RT)-PCR for investigation of enhanced expression of chromosomal ampC were performed. Results: Eight E. coli isolates (0.4%) were ESBL producers and 20 (1.0%) were hyperproducers of the chromosomal ampC. Three K. pneumoniae isolates (1.1%) were ESBL producers, and all K. oxytoca isolates (n = 8; 13.6%) were OXY-hyperproducers. No definite mechanisms for reduced susceptibility to ESCs could be inferred for 7 E. coli (0.4%) and 8 K. pneumoniae (3.0%) isolates. Conclusions: This study identified chromosomal AmpC-hyperproducing E. coli and OXY-hyperproducing K. oxytoca in addition to ESBLs in Enterobacteriaceae as major mechanisms of resistance to ESC, and documented their rates of prevalence for the first time in Norway.

Dissemination and characteristics of a novel plasmid-encoded carbapenem-hydrolyzing class D β-lactamase, OXA-436, found in isolates from four patients at six different hospitals in Denmark

ABSTRACT The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D β-lactamase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates, including Enterobacter asburiae (n = 3), Citrobacter freundii (n = 2), and Klebsiella pneumoniae (n = 1), were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4 to 92.8% identity to the amino acid sequences of other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to that of OXA-48 and OXA-181, with activity against penicillins, including temocillin; limited or no activity against extended-spectrum cephalosporins; and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ∼314-kb IncHI2/IncHI2A plasmid belonging to plasmid multilocus sequence typing sequence type 1 in a region surrounded by chromosomal genes previously identified to be adjacent to blaOXA genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with functional properties similar to those of OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and the plasmid location of blaOXA-436 illustrate its potential for further dissemination.

Emergence of mutation‐based linezolid‐resistant invasive Enterococcus faecalis in a haemodialysis patient in Norway

To the Editor Linezolid, an oxazolidinone antibiotic, has been available as a therapeutic alternative against antibiotic-resistant Gram-positive cocci since 2000. Linezolid inhibits bacterial protein synthesis by binding to the A site pocket at the ribosomal peptidyltransferase centre in domain V of the 23S rRNA genes. Point mutations in this region, in particular a G2576U mutation, have been associated with linezolid resistance in both enterococci and staphylococci (1, 2). Recently, the first Enterococcus human isolate with plasmid mediated transferable linezolid resistance encoded by the cfr (chloramphenicolflorfenicol resistance) gene was recovered from a patient in Thailand (3). Although resistance to linezolid in clinical isolates was reported prior to the release of the drug (4), surveillance programmes in both the US and Europe confirm that resistance rates remain very low (1, 2). Here, we report the first case of linezolidresistant Enterococcus identified in Norway. An elderly male with chronic renal failure due to hypertensive and diabetic nephropathy commenced haemodialysis therapy in October 2011. An episode of dialysis catheter-related sepsis with Serratia marcescens was treated with cefotaxime combined with ciprofloxacin in early January 2012. In mid-January, an acute myocardial infarction, cardiac failure and cerebrovascular insult resulted in a prolonged admission to the intensive care unit (ICU). He was isolated in a single room under contact precautions for the duration of his ICU stay due to concurrent Clostridium difficile enterocolitis. In mid-February, linezolid therapy was initiated due to a central line-related bacteraemia with coagulase-negative staphylococci, susceptible only to linezolid and vancomycin. Intravenous linezolid 600 mg 9 2 was administered for a total of 19 days. The patient was thereafter transferred to a single room in the cardiology ward and attended the renal dialysis unit regularly for haemodialysis. Ten days after discontinuing linezolid treatment, he again developed clinical sepsis. Multiple blood cultures yielded Enterococcus faecalis, identified utilizing the Vitek 2 GP card (bioM erieux, Marcy l’Etoile, France) and confirmed by ddl-specific PCR (5). The isolate was resistant to linezolid (MIC 16 mg/L), high-level resistant to gentamicin and susceptible to ampicillin, vancomycin and teicoplanin, using Etest (bioM erieux) and EUCAST clinical breakpoints. The E. faecalis strain was also isolated from the dialysis catheter tip and insertion site. Contact precautions were reinstated. The patient responded well to ampicillin treatment and was finally discharged to a rehabilitation centre by the end of April. All 32 patients attending the renal dialysis unit were screened for faecal carriage of the E. faecalis strain. Rectal swabs were incubated in BBL Enterococcosel Broth (BD, Franklin Lakes, NJ, USA) and subcultured to BBLColumbia CNA Agar with a 10 lg Linezolid disc. Only the index patient screened positive for linezolid-resistant E. faecalis. Interestingly, the patient screened positive again 4 months after cessation of linezolid therapy. Analysis for the mechanism of linezolid resistance included PCR analysis (6) for the cfr gene that was negative. The cfr-primers utilized showed 100% match to the previously reported E. faecalis cfr sequences. Amplification of 23S rDNA encoding domain V and subsequent NheI digestion (7) revealed heterozygosis for the G2576T mutation. The development of mutation-based linezolid resistance in staphylococci and enterococci was initially considered unlikely due to the presence of multiple copies of the 23S rRNA gene. The rate-limiting step in the development of linezolid resistance in enterococci appears to be the initial mutation occurring under antimicrobial selective pressure, as subsequent replacement of wild-type genes with mutant copies occurs rapidly by homologous recombination (8). The level of linezolid resistance expressed correlates with the number of mutated 23S rRNA genes. At least two mutated copies are required to detect a resistant phenotype. The linezolid MIC value of 16 mg/L of our isolate corresponds well with the finding of two or three mutated genes. Risk factors for the development of mutation-based linezolid resistance in enterococci

Urinary tract isolates of Enterobacteriaceae from hospitalized patients in the Arkhangelsk region, Russia: Antimicrobial susceptibility and characterization of extended-spectrum beta-lactamases strains

From the 1 Department of Medical Biology, Faculty of Health Science, University of Troms ø , 2 Reference Centre for Detection of Antimicrobial Resistance (K-res), Department of Microbiology and Infection Control, University Hospital of North Norway, Troms ø , Norway, 3 Department of Microbiology, Virology and Immunology, Northern State Medical University, 4 Regional Clinical Hospital, Arkhangelsk, Russia, and 5 Division of Infectious Disease Control, Norwegian Institute of Public Health, Oslo, Norway Scandinavian Journal of Infectious Diseases, 2010; 42: 797–800

P867 Carbapenem resistance mechanisms in Norwegian clinical isolates of Pseudomonas aeruginosa

1Reference Centre for Detection of Antimicrobial Resistance, University Hospital of North Norway, Department of Microbiology and Infection Control, Tromso, Norway, 2University of Tromso, Department of Microbiology and Virology, Tromso, Norway, 3Karolinska Institutet-MTC, Karolinska University Hospital, Solna, Department of Clinical Microbiology, Stockholm, Sweden, 4University Hospital of North Norway, Department of Microbiology and Infection Control, Tromso, Norway and 5Rikshospitalet, Institute of Medical Microbiology, Oslo, Norway.