Influence of Transferable Genetic Determinants on the Outcome of Typing Methods Commonly Used for Enterococcus faecium

Abstract

A variety of methods is used for a molecular typing of Enterococcus spp. and related gram-positive bacteria including macrorestriction analysis using pulsed-field gel electrophoresis (PFGE), ribotyping, rapid amplification of polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP). To test the influence of transferable determinants on the outcome of different typing methods commonly used for enterococci, we established a homogenous strain collection of 24 transconjugants resulting from filter matings with antibiotic-resistant Enterococcus faecium. As expected, AFLP, RAPD, and PFGE all identified our model bacteria as strongly related. However, distinct differences in the resolving and discriminatory power of the tested methods could be clearly addressed. In PFGE, 22 of 24 transconjugants possessed less than a three-band difference to the recipient pattern and would be regarded as strongly related. Three different RAPD PCRs were tested; in two reactions, identical patterns for all transconjugants and the recipient were produced. One RAPD PCR produced an identical pattern for 18 transconjugants and the recipient and a clearly different pattern for the remaining 6 transconjugants due to a newly appearing fragment resulting from acquisition of the tetL gene. AFLP clusters all transconjugants into a group of major relatedness. Percent similarities were highly dependent on the method used for calculating the similarity coefficient (curve-based versus band-based similarity coefficient). Fragment patterns of digested plasmids showed the possession of nonidentical plasmids in most transconjugants. PFGE still could be recommended as the method of choice. Nevertheless, the more-modern AFLP approach produces patterns of comparable discriminatory power while possessing some advantages over PFGE (less-time-consuming internal standards). Plasmid fingerprints can be included to subdifferentiate enterococcal isolates possessing identical macrorestriction and PCR typing patterns.The "gold standard" for molecular typing of enterococci and related gram-positive bacteria, such as staphylococci and lactococci is still macrorestriction analysis via pulsed-field gel electrophoresis (PFGE) (13,14). In recent years, some alternative techniques have been successfully applied to the typing of enterococci below the species level. These include amplification-based methods, such as rapid amplification of polymorphic DNA (RAPD) (16, 26) and amplified fragment length polymorphisms (AFLP) (2, 19). These techniques are now applied more and more because they involve less time, comparably low costs, and only standard equipment. Fragments resulting from RAPD PCR typing are randomly amplified and resolved in common agarose gels. AFLP is used for a wide range of organisms with different applications and has been successfully applied to the study of the genetic relatedness of epidemiological unrelated strains of enterococci (33). AFLPresulting fragments are determined in a capillary or gel sequencer allowing detection of single base p...

Clonal Spread of a Vancomycin-Resistant Enterococcus faecium Strain among Bloodstream-Infecting Isolates in Italy

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Persistence of Animal and Human Glycopeptide-Resistant Enterococci on Two Norwegian Poultry Farms Formerly Exposed to Avoparcin Is Associated with a Widespread Plasmid-Mediated vanA Element within a Polyclonal Enterococcus faecium Population

“…Representative strains of all GREF PFGE types were examined for transferable vancomycin resistance by filter mating, as previously described (14). E. faecium UW 64/3 (Rif r Fus r ) was used as a recipient (53). Donor strains that failed to support transferable vancomycin resistance were retested once.…”

The Key Role of Pulsed-Field Gel Electrophoresis in Investigation of a Large Multiserotype and Multistate Food-Borne Outbreak of Salmonella Infections Centered in Pennsylvania

“…PFGE is a DNA-based fingerprinting method considered to be the "gold standard" for subtyping numerous bacterial pathogens (7,8,10,16,22,25,28,30). PFGE involves digesting intact bacterial chromosomes with restriction endonucleases that have specificities for sites in DNA that are relatively uncommon.…”