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.

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    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.
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    Antimicrobial agents and chemotherapy 43, 1999, 483-491.
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    ABSTRACT: We report on a detailed study on the molecular diversity and evolutionary relationships of Tn1546-like elements in vancomycin-resistant enterococci (VRE) from humans and animals. Restriction fragment length polymorphism (RFLP) analysis of the VanA transposon of 97 VRE revealed seven different Tn1546 types. Subsequent sequencing of the complete VanA transposons of 13 VRE isolates representing the seven RFLP types followed by sequencing of the identified polymorphic regions in 84 other VanA transposons resulted in the identification of 22 different Tn1546 derivatives. Differences between the Tn1546 types included point mutations in orf1, vanS, vanA, vanX, and vanY. Moreover, insertions of an IS1216V-IS3-like element in orf1, of IS1251 in the vanS-vanH intergenic region, and of IS1216V in the vanX-vanY intergenic region were found. The presence of insertion sequence elements was often associated with deletions in Tn1546. Identical Tn1546 types were found among isolates from humans and farm animals in The Netherlands, suggesting the sharing of a common vancomycin resistance gene pool. Application of the genetic analysis of Tn1546 to VRE isolates causing infections in Hospitals in Oxford, United Kingdom, and Chicago, Ill., suggested the possibility of the horizontal transmission of the vancomycin resistance transposon. The genetic diversity in Tn1546 combined with epidemiological data suggest that the DNA polymorphism among Tn1546 variants can successfully be exploited for the tracing of the routes of transmission of vancomycin resistance genes.
    Antimicrobial Agents and Chemotherapy 04/1999; 43(3):483-91. · 4.57 Impact Factor