Long PCRs of transposons in the structural analysis of genes encoding acquired glycopeptide resistance in enterococci.

University of Tromsø, Norway.
BioTechniques (Impact Factor: 2.4). 03/1998; 24(3):432-7.
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: To investigate the epidemiologic characteristics of vancomycin-resistant enterococci (VRE) infection. An epidemiologic description by means of chromosomal DNA fingerprinting and transposon typing. A 2,200-bed tertiary care hospital in Korea. First VRE isolates were obtained from patients hospitalized from April 1997 to December 2001. The van genotypes of isolates were identified by means of multiplex polymerase chain reaction (PCR). The macrorestriction patterns of chromosomal DNA were determined by pulsed-field gel electrophoresis (PFGE). The transposon Tn1546 was typed by means of 2 sets of long PCR restriction fragment-length polymorphism analysis, which were ClaI restriction of a 10.4-kb region from orf1 to vanZ and DdeI restriction of a 4.4-kb region from vanR to vanX. VRE isolates were recovered from 215 patients. All were vanA genotype. PFGE analysis of the 215 isolates showed 172 types, including 21 clusters composed of 64 isolates and 151 types of as many isolates. Each type was composed of 2-10 isolates; the isolates within each PFGE cluster were detected within a 10-month period and mostly shared a transposon type. Transposon typing classified 169 strains into 15 types and 158 strains belonged to 4 major transposon clusters. Each of these 4 transposon clusters was isolated from patients treated in 5-22 different wards during a 31-52 month period and consisted of 9-80 PFGE types. Each of the other 11 types were found in only one strain. Our findings suggest that the horizontal transfer of Tn1546 has a major role in the nosocomial spread of vanA VRE. Clonal spread of VRE seemed to contribute to short-term dissemination in limited areas.
    Infection Control and Hospital Epidemiology 11/2006; 27(10):1081-7. · 4.02 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that "plasmid addiction systems" may contribute to the persistence of GREF in nonselective environments.
    Applied and Environmental Microbiology 02/2005; 71(1):159-68. · 3.95 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The purpose of this study was to study whether horisontal transfer of the satA gene could take place between isogenic strains of Enterococcus faecium in the gastrointestinal tract. Two separate groups of germ-free Sprague-Dawley rats were dosed orally with a single high dose of approximately 5×108 colony forming units (CFU) of the streptomycin resistant recipient E. faecium BM4105-Str. One week later, after the establishment of the recipient, a rifampicin, fusidin and virginiamycin resistant donor, E. faecium AHA15(,satA), containing a transferable satA gene was given orally in a single high dose of approximately 1×108 CFU to the gnotobiotic rats. The different strains of E. faecium were identified in faecal samples using the specific antibiotic resistance pattern for isolation on selective agar plates. In both groups of rats transconjugants E. faecium BM4105-Str (satA) were identified in faecal samples using specific phenotypic antibiotic resistance pattern on selective agar plates. High numbers of transconjugants were observed throughout the remaining experimental period of approximately 18 days, indicating horizontal transfer of the satA gene followed by persistence and proliferation of the transconjugants in vivo. The donor persisted in low numbers throughout the experimental period. The present study demonstrated that transfer of the satA gene encoding resistance to streptogramin A between isogenic E. faecium strains takes place under experimental conditions in the mammalian gastrointestinal tract. This indicates that a similar transfer may take place under natural conditions.
    07/2009; 11(4):241-247.