Identification of enterococci and determination of their glycopeptide resistance in German and Austrian clinical microbiology laboratories

Abstract

OBJECTIVE: To determine exactly how German and Austrian routine laboratories perform tests for the identification of enterococci and determination of their glycopoptide resistance. METHODS: Six enterococcal test strains with different types of glycopeptide resistance (Enterococcus faecium, VanA; E. faecalis, VanA; E. faecium, VanB; E. faecalis, VanB; E. gallinarum, VanC1; E. casseliflavus, VanC2) were sent as anonymous isolates to 73 clinical microbiology laboratories (65 in Germany; eight in Austria). The participating laboratories had to identify the strains up to the species level and to determine their antibiotic susceptibilities to the glycopeptides vancomycin and teicoplanin by the test method(s) that are used daily in the corresponding laboratories. RESULTS: The analysis of the results received from 62 laboratories (56 from Germany, six from Austria) demonstrated that the most used routine method in susceptibility testing was the agar diffusion test, followed by the Etest, and the microbroth dilution procedure. The majority of participants had no difficulties in susceptibility testing with the VanA-type strains. However, the agar diffusion test was often not able to recognize clearly the VanB and VanC strains; some problems also arose with VanB isolates in the Etest. With the microbroth dilution method, the corresponding type of glycopeptide resistance was correctly determined in the majority of enterococcal test strains. Difficulties also arose in identification, especially with the VanC strains (E. gallinarum and E. casseliflavus), which were often falsely identified as E. faecium. The reasons for these errors are obviously based on the lack of important tests (such as motility and presence of a yellow pigment) in some commercially available identification test kits.

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources

“…In this study, both biochemical and PCR identification to the species level reliably distinguished E. faecalis from other Enterococcus species. The same was not true of E. faecium, as results of biochemical identification vs. PCR identification (Facklam and Collins 1989;Klare et al 1999), one of our goals in developing this protocol was to identify target enterococci that required the minimum of tests for confirmation. At least 19 species are currently assigned to the genus Enterococcus (Devriese et al 1993), and additional species have been proposed (Svec et al 2001;Teixeira et al 2001).…”

“…Furthermore, the majority (68·8%) of the isolates identified with low assurance by API 20 Strep were designated E. faecium , adding to the difficulties encountered in readily identifying the species. While motility testing is widely used in clinical studies to distinguish E. faecium (nonmotile) from the motile species E. casseliflavus and E. gallinarum (Facklam and Collins 1989; Klare et al. 1999), one of our goals in developing this protocol was to identify target enterococci that required the minimum of tests for confirmation.…”

Routine Molecular Identification of Enterococci by Gene-Specific PCR and 16S Ribosomal DNA Sequencing

“…The difficulty in phenotypic identification of unusual enterococcal strains is not unexpected, particularly by manual commercial kits (14,19,23,33), but incorrect identification at the genus level and the misidentification of strains that are frequent in clinical specimens, such as E. faecalis and E. faecium, point to the need for busy clinical laboratories not to rely only on commercial kits but also to do the fundamental additional tests which are necessary for genus and species identification of enterococci. In fact, only catalase and L-pyrollidonyl-␤-naphthylamide hydrolysis (PYR) tests have been used routinely by the three laboratories, and none of the laboratories used ad- on May 11, 2018 by guest http://jcm.asm.org/ ditional tests; moreover, it is also evident that in some cases the serological grouping was misleading or incorrectly done.…”

“…Commercially available kits are often used by clinical laboratories as an alternative to the numerous physiological tests needed to identify enterococcal species (6,9,10,11,37); nevertheless, all commercial kits vary in their performance and persistently show many drawbacks, especially in cases of atypical strains, and at best need supplementary manual tests, which somewhat impair their usefulness (14,17,23,33,34,36; P. A. d'Azevedo, C. G. Dias, A. L. S. Gonçalves, F. Rowe, and L. M. Teixeira, Abstr. 100th Gen. Meet.…”

Rapid Identification of Clinically Relevant Enterococcus Species by Fluorescence In Situ Hybridization

“…In turn, members of the VanC-type resistance group, such as Enterococcus gallinarum and Enterococcus casseliflavus, are intrinsically resistant to vancomycin but are mainly susceptible to penicillins. Species identification of enterococci by phenotypic methods is time-consuming (5-7, 14, 20, 21), however, and the misidentification of E. faecium as E. gallinarum or E. casseliflavus and vice versa is a frequent problem (4,7,12).…”