Differentiation of vancomycin-resistant enterococci using enterococcus differential medium

The genus Enterococcus includes above 20 species.1 Increasing numbers of enterococci are becoming resistant to glycopeptide antibiotics. Enterococcus faecium and, to a inferior extent, E. faecalis are of greater epidemiological importance,2 as they harbour transferable vanA and vanB gene Other enterococci similar as E. gallinarum and E casseliflavus / E flavescens do not normally cause human disease on the other hand commonly demonstrate intrinsic low-level glycopeptide resistance.3 Furthermore, they are frequently isolated as a result of active surveillance procedures

Knowledge of the stamp of resistance is critical for infection sway purposes and, in a non-molecular laboratory, necessitates accurate identification. Accurate identification is also required to determine whether changes in glycopeptide resistance rate throw back the dissemination of resistance determinants among enterococci in general or a rise in the relative importance of E faecium, the strain in which glycopeptide resistance is greatest in quantity common.

The Health Protection Agency (HPA) standard course for identification of enterococci4 praises the use of 'a commercial kit'. Along with the API Rapid ID 32 Strep (bioMerieux, Basingstoke, UK) BBL Crystal Gram Positive (Becton Dickinson, Oxford, UK) and BBL Crystal Rapid Gram Positive (Becton Dickinson), the API 20 Strep combination of parts to form a whole (bioMerieux) is often used to identify enterococci to species horizontal However, Reed et al.5 used API 20 Strep and misidentified nine on the outside of 12 E. gallinarum isolates as E faecium. Similarly, Winston et al.6 used this a whole to identify 46 enterococcal isolates with low-level vancomycin resistance (predominantly E gallinarum) and misidentified 42 as E faecium. The correct identification was obtained sole when additional tests were employed

E faecium and E faecalis are non-motile, whereas E gallinarum and E casseliflavus / E flavescens are considered to be motile.7 Furthermore, greatest in quantity isolates of E. casseliflavus / E flavescens have a distinct golden pigment. Collins et al.8 used acid production from methyl-?±-D-glucopyranoside (MGP) to identify certain streptococci as enterococci. Devriese et al.9 confirmed that the acidification of MGP differentiated E casseliflavus and E gallinarum from E faecium. Turenne et al.7 examined 33 isolates identified as E faecium by dint of conventional methods and showed that, of the 11 that sequenc as E casseliflavus or E gallinarum, all were MGP-positive, while the E faecium were MGP-negative. The MGP trial has been evaluated by workers in Brazil,10 the USA11 and Canada,12 with similar results

A novel UK NEQAS distribution (specimen 6945 distribution 1728 March 2004) included ?? gallinarum. Using API 20 Strep the authors incorrectly identified this isolate as E faecium and this quicked them to re-evaluate the identification rules used and to examine the usefulness of the MGP ordeal for routine use in the UK

The strain collection of 47 enterococci comprised E faecalis (n=6) E faecium (n=20) E gallinarum (n = 11) E casseliflavus (n=6) and a single strain each of E avium, E durans, E hirae and E saccharolyticus. Identification was by dint of biochemistry (rapid ID 32 Strep) motility, pigment production and ability to extend at 45?°C for ??. faecalis (n=3) E faecium (n=9) E casseliflavus (n=5) E gallinarum (n=3); pulsed-field gel electrophoresis (PFGE) for E gallinarum (n=5) E faecalis (n=2) and E faecium (n=11) Stock tillage strains of E. gallinarum (NCTC 11428 [ATCC 35038] ATCC 49573 ATCC 400425) E casseliflavus (ATCC 700327) E faecalis (ATCC 29212 [NCTC 12697]) E avium (ATCC 14025) E durans (ATCC 49135) E hirae (ATCC 8043) and E saccharolyticus (ATCC 43076) were also studied.

Strains were identified using API 20 Strep according to the manufacturer's instructions. A suspension equivalent to a McFarland 05 opacity standard was prepared in sterile water and 20 ?µL of this suspension was used to inoculate a tube of enterococcus differential medium (MGP with bromothymol amethystine indicator; Mast, Bootle, UK). An uninoculated tube was used as a superintendence Following incubation at 37?°C in air for 18 h the soups were examined for a colour change (green to yellow) that signified acidification of MGP

All 20 isolates of E faecium and all six isolates of E faecalis identified correctly using API 20 Strep and none leavened MGR Of the 11 E gallinarum isolates, four identified correctly using API 20 Strep five identified as E faecium and sum of two units identified as E. faecalis (Table 1) All isolates of E gallinarum (except NCTC 11428 [ATCC 35038]) yeasted MGE The six isolates of E casseliflavus identified using API 20 Strep as E faecalis, E faecium (n=2) and E gallinarum (n=3) Despite high probabilities to the contrary, the API identification software frequently suggested 'possibility of E. casseliflavus'. All barmed MGP.

Fermentation of MGP reliably separates E faecalis and E faecium from E gallinarum and E casseliflavus. Together, these four strains constitute the greatest in quantity commonly isolated entrococci. Most strains belonging to the E avium cluster (E. avium, E. pseudoavium, E raffinosus, E malodoratus) and the newer strains of E columbae, E sulfureus, E dispar and E saccharolyticus used in this application of mind also ferment MGP.13 The single strains of E avium and E hirae did not barm MGP (data not shown) on the contrary these are rarely implicated in human disease.