tRNA intergenic spacer PCR (tDNA-PCR) was evaluated for its usefulness in the differentiation of enterococcal species of human and animal origin. This technique was carried out for 124 strains belonging to 17 enterococcal species and generated DNA fragments, which were separated by capillary electrophoresis. tDNA-PCR enabled us to discriminate for all species tested. Enterococcus faeciumshowed minor but reproducible differences with Enterococcus durans, while Enterococcus hirae was easily distinguishable. Enterococcus avium, Enterococcus malodoratus, and Enterococcus raffinosus generated highly similar though distinctive patterns.

The discriminatory power, speed, and interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) combined with capillary electrophoresis was evaluated for the identification of streptococci. This method was carried out in three different laboratories under highly standardized conditions for 54 strains belonging to 18 different species. It was concluded that interlaboratory reproducibility of tDNA fingerprints produced by means of capillary electrophoresis was sufficiently high to permit the exchange between different laboratories and the construction of common libraries which can be consulted for comparison with fingerprints obtained independently in separate laboratories. In a second step, 17 other species were included in the study and examined in one of the participating laboratories. All Streptococcus species studied, except S. mitis, S. oralis, S. parasanguinis, S. pneumoniae, S. thermophilus, and S. vestibularis, showed distinguishable tDNA fingerprints. A database of well-characterized strains was constructed to enable computeraided identification of unknown streptococcal isolates.Traditionally the clinically most important Streptococcus species have been identified by Lancefield carbohydrate antigen detection and the application of a few biochemical or physiological tests. Difficulties arise when less-prevalent species are to be dealt with. Lancefield groups are not speciesspecific (5, 10), and certain species groups (2) are notoriously difficult to differentiate phenotypically (8).A number of genotypic methods have been evaluated for the identification of streptococci: amplified ribosomal DNA restriction analysis (7, 9), amplification of ddl genes (6), and sequencing of the MnSOD gene (13). tRNA intergenic length polymorphism analysis (tDNA-PCR) (15) has been used not only for the differentiation of streptococcal species (3, 12) but also for Acinetobacter (4, 16), staphylococci (11), Listeria (14), and enterococci (1). Thus far the interlaboratory reproducibility of this kind of genotypic identification technique has been ill studied although it is crucial with regard to the ability to compare fingerprints generated in different laboratories and with regard to the construction of publicly accessible DNA fingerprint data banks. Here we evaluated the interlaboratory reproducibility of tDNA-PCR in combination with capillary fluorescent electrophoresis and its suitability for identification in routine diagnostics. MATERIALS AND METHODSBacterial strains. Fifty-four BCCM-LMG culture collection strains (University of Ghent, K. L. Ledeganckstraat 35, B-9000 Ghent) belonging to 18 streptococcal species were used to standardize the method of tDNA-PCR and to evaluate its interlaboratory reproducibility (Table 1). The collection was extended with 47 strains of the BCCM-LMG culture collection belonging to 17 other streptococcal species (Table 2). Ten collection strains were subjected to blind testing in all three laboratories. DNA preparation. Bacterial cells were grown overnight on Co...

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