Long PCRs of transposons in the structural analysis of genes encoding acquired glycopeptide resistance in enterococci.
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.
SourceAvailable from: Lars B Jensen
Article: Horizontal Transfer of the satA Gene Encoding Streptogramin A Resistance Between Isogenic Enterococcus faecium Strains in the Gastrointestinal Tract of Gnotobiotic RatsPart of this study has been presented at the 2nd World Congress on Anaerobic Bacteria and Infections, Nice, France, October 1998[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.Microbial Ecology in Health and Disease 07/2009; 11(4):241-247. DOI:10.1080/08910609908540834
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ABSTRACT: Molecular subtyping of the VanA-type resistance element Tn1546 in an international collection of 81 genomically diverse vancomycin-resistant enterococci (VRE) from human, animal, and environmental reservoirs was evaluated by restriction analysis of long-range PCR amplicons (PCR-RFLP), single gene PCRs, Southern blot analysis of genomic digests, and partial DNA sequencing, A dominant Tn1546-RFLP in accordance with Enterococcus faecium BM4147 was detected in 43 of the 49 long-range PCR positive strains from ecologically diverse sources in several European countries and the US. Tn1546-like elements from the 32 (40%) long-range PCR negative strains were typed into 17 different groups by single-gene PCRs and Southern blot analysis of the ORF1, ORF2, vanS-vanH, vanX-vanY, and vanZ regions, All these isolates showed deletions in the ORF1 and/or vanZ primer binding regions explaining the failure of long-range PCR amplification, Enlarged vanS-vanH or vanX-vanY fragments were detected in 7 (22%) and 16 (50%) of the long-range PCR negative strains, respectively, The enlarged vanS-vanH regions of five clinical isolates from the US (n = 2), Ireland (n = 2), and Norway (n = 1) contained identical IS1251-like insertions indicating intercontinental spread of the vanA gene cluster, Intergenic vanS-vanH IS1251 insertions have so far not been reported in European studies, Structural rearrangements of Tn1546-like elements may represent single recombination events that can serve as fingerprints in the molecular examination of vanA gene cluster evolution and transmission, The optimal strategy for such analysis has yet to be determined. Two alternative long-range PCRs with subsequent RFLP analysis were successfully used to type the majority of vanA gene clusters in an ecologically and geographically heterogeneous VRE strain collection, but failed to detect and type a group of variant Tn1546-like elements truncated in the left-end ORF1/ORF2 region, Further subtyping of such variants should specifically target the polymorphic vanS-vanH and vanX-vanY regions.Microbial drug resistance (Larchmont, N.Y.) 03/2000; 6(1):49-57. DOI:10.1089/mdr.2000.6.49 · 1.99 Impact Factor