Long PCRs of transposons in the structural analysis of genes encoding acquired glycopeptide resistance in enterococci
University of Tromsø, Norway. BioTechniques
(Impact Factor: 2.95).
Glycopeptide-resistant enterococci (GRE) associated with multiple antibiotic resistance present a major challenge to clinical practice and infection control due to limited or nonexistent antimicrobial treatment options. The genes encoding VanA- and VanB-type glycopeptide resistance have been shown to reside on transposons Tn1546 and Tn1547, respectively. These transferable genetic elements may carry the resistance determinants between and within different ecological niches. Molecular epidemiological studies of nosocomial outbreaks of VanA- and VanB-type GRE indicate horizontal transfer of glycopeptide resistance genes as an important mechanism for the spread of GRE. To target infection control and better understand the epidemiology of GRE, outbreak investigations and molecular epidemiological studies should therefore apply at least two different approaches, i.e., molecular-typing methods to analyze bacterial genomic heterogeneity and structural analysis of mobile resistance determinants. Here we describe the development and use of long PCRs in the structural analysis of vanA and vanB gene clusters in GRE.
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- "For PCR, bacterial DNA was prepared using a Dynabeads DNA DIRECT kit (Dynal, Oslo, Norway) as described (Haaheim et al. 1998) and for southern hybridization, bacterial genomic DNAs were prepared as described (Pitcher et al. 1989). "
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ABSTRACT: The characterization of a novel insertion sequence (IS) in vanB2-containing Enterococcus faecium was conducted.
Direct PCR amplification of ORFC region of Tn5382 from DNA extracted from vanB2-containing E. faecium, and sequence analysis were performed. A novel IS was identified. It is 1418 bp in length and contains one putative open reading frame that is similar to transposase. There exists inverted terminal repeats of 12 bp, but direct repeats are not present. According to high similarity to putative transposases of IS3 members, such as, IS150, IS861, IS1077 and IS911, we designated it ISEnfa3.
Since ISEnfa3 was detected in all vanB2-containing strains examined so far, it could be used as a tool for epidemiological study.
Letters in Applied Microbiology 02/2003; 36(3):186-90. DOI:10.1046/j.1472-765X.2003.01292.x · 1.66 Impact Factor
Available from: Rob J L Willems
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ABSTRACT: The genetical relatedness between epidemiologically linked fecal VRE strains from poultry farmers (n = 5) and their broilers (n = 7) at five avoparcin-exposed Norwegian farms was examined. Pulsed-field gel electrophoresis (PFGE) of bacterial chromosomal digests and structural analysis of vanA resistance elements was performed. Animal and human Enterococcus faecium strains at one farm were genetically closely related with indistinguishable vanA elements and a single band position difference in PFGE analysis. Examination of the vanA elements in genetically unrelated strains by restriction enzyme digestion of Tn1546 long-PCR amplicons and ORF2-vanR intergenic sequencing revealed a pool of at least two distinct vanA gene cluster groups in the two reservoirs. The results indicate that transmission of VanA glycopeptide resistance in enterococci between human and animal at avoparcin-exposed farms can occur by direct transfer of VRE strains as well as horizontal spread of resistance genes between strains.
Microbial Drug Resistance 02/1998; 4(4):313-8. DOI:10.1089/mdr.1998.4.313 · 2.49 Impact Factor
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