Kristin H. Dahl

Genetic methods for detection of antimicrobial resistance

Accurate and rapid diagnostic methods are needed to guide antimicrobial therapy and infection control interventions. Advances in real‐time PCR have provided a user‐friendly, rapid and reproducible testing platform catalysing an increased use of genetic assays as part of a wider strategy to minimize the development and spread of antimicrobial‐resistant bacteria. In this review we outline the principal features of genetic assays in the detection of antimicrobial resistance, their advantages and limitations, and discuss specific applications in the detection of methicillin‐resistant Staphylococcus aureus, glycopeptide‐resistant enterococci, aminoglycoside resistance in staphylococci and enterococci, broad‐spectrum resistance to β‐lactam antibiotics in gram‐negative bacteria, as well as genetic elements involved in the assembly and spread of antimicrobial resistance.

Effects of Phenotype and Genotype on Methods for Detection of Extended-Spectrum-β-Lactamase-Producing Clinical Isolates of Escherichia coli and Klebsiella pneumoniae in Norway

ABSTRACT Consecutive clinical isolates of Escherichia coli (n = 87) and Klebsiella pneumoniae (n = 25) with reduced susceptibilities to oxyimino-cephalosporins (MICs > 1 mg/liter) from 18 Norwegian laboratories during March through October 2003 were examined for blaTEM/SHV/CTX-M extended-spectrum-β-lactamase (ESBL) genes, oxyimino-cephalosporin MIC profiles, ESBL phenotypes (determined by the ESBL Etest and the combined disk and double-disk synergy [DDS] methods), and susceptibility to non-β-lactam antibiotics. Multidrug-resistant CTX-M-15-like (n = 23) and CTX-M-9-like (n = 15) ESBLs dominated among the 50 ESBL-positive E. coli isolates. SHV-5-like (n = 9) and SHV-2-like (n = 4) ESBLs were the most prevalent in 19 ESBL-positive K. pneumoniae isolates. Discrepant ESBL phenotype test results were observed for one major (CTX-M-9) and several minor (TEM-128 and SHV-2/-28) ESBL groups and in SHV-1/-11-hyperproducing isolates. Negative or borderline ESBL results were observed when low-MIC oxyimino-cephalosporin substrates were used to detect clavulanic acid (CLA) synergy. CLA synergy was detected by the ESBL Etest and the DDS method but not by the combined disk method in SHV-1/-11-hyperproducing strains. The DDS method revealed unexplained CLA synergy in combination with aztreonam and cefpirome in three E. coli strains. The relatively high proportion of ESBL-producing E. coli organisms with a low ceftazidime MIC in Norway emphasizes that cefpodoxime alone or both cefotaxime and ceftazidime should be used as substrates for ESBL detection.

Genetic linkage of the vanB2 gene cluster to Tn5382 in vancomycin-resistant enterococci and characterization of two novel insertion sequences

VanB-type vancomycin resistance is encoded by the vanB gene cluster, which disseminates by horizontal gene transfer and clonal spread of vancomycin-resistant enterococci (VRE). Genetic linkage of the vanB gene cluster to transposon Tn5382 and the insertion sequences IS16 and IS256-like has previously been shown. In this study linkage of defined vanB gene cluster subtypes to these elements was examined. All the vanB2 subtype strains studied (n=14) revealed co-hybridization of vanB and Tn5382, whereas the strains of vanB1 (n=8) and vanB3 (n=1) subtypes were Tn5382 negative. Conjugative cotransfer of the vanB2 gene cluster and Tn5382 was demonstrated for two strains. DNA sequencing of the vanX(B)-ORFC region in vanB2 strains confirmed that the vanB2 gene cluster is an integral part of Tn5382. No general pattern of linkage was observed with regard to IS16 and IS256-like. Two novel insertion sequences were identified in specific vanB2 subtype strains. (i) A 1611 bp element (ISEnfa110) was detected in the left flank of Tn5382. Its insertion site, lack of terminal inverted and direct repeats, and two conserved motifs in its putative transposase all conform to the conventions of the IS110 family. (ii) A 787 bp element (ISEnfa200) was detected in the vanS(B)-vanY(B) intergenic region. Its ORF encoded a putative protein with 60-70% identity to transposases of the IS200 family. No further copies of ISEnfa110 were found by colony hybridization of 181 enterococcal isolates, whereas ISEnfa200 was found in four additional vanB2 strains from the USA. The five strains had identical ISEnfa200 element insertion sites, and Tn5382 was located downstream from a pbp5 gene conferring high-level ampicillin resistance. These isolates showed related PFGE patterns, suggesting possible clonal spread of a VRE strain harbouring a Tn5382-vanB2-ISEnfa200 element linked to a pbp5 gene conferring ampicillin resistance.

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.

Nonconjugative Transposition of the vanB-Containing Tn5382-Like Element in Enterococcus faecium

ABSTRACT The vanB2 operon encoding glycopeptide resistance is an integral part of the putative conjugative transposon Tn5382. Characterization of clinical glycopeptide resistant derivatives from an epidemic ampicillin-resistant Enterococcus faecium strain showed precise chromosomal or plasmid insertions of a vanB2-containing Tn5382-like element. Conjugative transposition of the Tn5382-like element was not demonstrated in retransfer studies.

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.