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.

FIG. 1.

(a) Macrorestriction patterns of SmaI-digested genomic DNA of 24 transconjugants and recipient R (negated gel image). Lanes: M, SmaI-digested genomic DNA of S. aureus NCTC 8325; 1 to 24, transconjugants 1 to 24; R, recipient isolate. (b) Cluster analysis according to the method of Claus et al. (8) by using a band-based similarity coefficient-generated matrix (Dice).

FIG. 2.

PCR RAPD typing for 24 transconjugants (lanes 1 to 24) and recipient R (negated gel image). (a) RAPD1 with primer 1; (b) RAPD2 with primer 2; (c) RAPD3 with primer 3. Lane M, molecular size standard. In panels a and b, SPP1 DNA was cleaved with EcoRI; panel c is a mix of pBR322 cleaved with Bgl2 and HinfI.

FIG. 3.

AFLP typing for 24 transconjugants (1 to 24) and recipient (25). (a) Pearson coefficient evaluation; (b) Dice coefficient evaluation.

FIG. 4.

(a) Patterns of HindIII-digested plasmids from transconjugants 1 to 24 and recipient R (negated gel image). M, Molecular size marker (lambda DNA cleaved with EcoRI and HindIII). (b) Cluster analysis according to the method of Claus et al. (8) by using a band-based similarity coefficient-generated matrix (Dice).