Brilliance vre agar

Samples were directly inoculated on Brilliance VRE agar (Oxoid, United Kingdom), as well as into bile aesculin azide broth (Liofilchem, Italy) supplemented with 6 μg/ml vancomycin (BEAV broth). The inoculated culture media were incubated aerobically at 37 °C and were examined for growth at 24 h and 48 h. The identification of the enterococci growing on Brilliance VRE agar was based on the observation of appropriately colored colonies – indigo to purple for E. faecium and light blue for E. faecalis. Growing colonies were transferred to a 5% Blood agar plate (BAP) and re-incubated for 24 h.
The positive BEAV broths that developed black color were subcultured to 5% BAP and chromID CPS Elite (bioMérieux, France) and incubated for an additional 24 h. All suspected VRE, isolated from Brilliance VRE agar and BEAV broth were identified using Vitek 2 Compact (bioMérieux, France). In cases of low-level discrimination between E. gallinarum and E. casseliflavus, motility and pigment production tests were also done.

A collection of previously characterized isolates of E. faecium (n = 13), Enterococcus faecalis (n = 11), Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus raffinosus and Enterococcus hirae was selected for testing. The isolates originated from NCTC collections (n = 12), a retrospective collection of bacteremia isolates from Cambridge University Hospitals (CUH) NHS Foundation Trust (2006–2012, n = 15), and from a meat sample (n = 1) (Table 1). They included isolates with vanA and vanB acquired resistance, and in the case of E. faecalis and E. faecium bacteremia isolates, a range of diverse hospital-adapted STs Raven et al., 2016 (link), Raven et al., 2016 ). The organisms were grown from glycerol bead vials stored at −80°C onto Columbia blood agar (CBA, Oxoid) and 200 μl of a 0.5 McFarland suspension was streaked using the four-quadrant technique onto chromID VRE agar (bioMérieux) and Brilliance VRE agar (Oxoid). Growth was assessed after 24 and 48 hours aerobic incubation at 37 °C. Vancomycin minimum inhibitory concentration was measured using Etest (bioMérieux).

Cherry-pit-sized volumes from the stool samples provided were used for broth enrichment in thioglycolate broth (Heipha, Eppelheim, Germany) for 16–24 hours at 37°C. Subsequently, 10 μl preincubated broth was cultured on Brilliance ESBL selective agar (Oxoid, Basingstoke, UK) for the selection of third-generation cephalosporin-resistant bacteria. This agar is made for selective growth of ESBL-positive Enterobacteriaceae. An additional 10 μl for each was incubated on Brilliance VRE agar (Oxoid) for the selection of vancomycin-resistant enterococci (VRE) and on CHROMagar MRSA (CHROMagar, Paris, France) for the selection of methicillin-resistant Staphylococcus aureus (MRSA).
Agar plates were incubated at 37°C for 40–48 hours. All colonies that looked suspicious for Enterobacteriaceae on Brilliance ESBL selective agar (blue, green, brown colonies) were isolated, while suspected Gram-negative nonfermentative rod-shaped bacteria (i.e., yellow or yellowish-brown or greenish-brown colonies) were discarded. Similarly, only colonies that appeared suspicious for MRSA and VRE were selected for further analysis. Suspected enterococci were of blue or violet color; suspected MRSA were of mauve color.
All suspicious isolates were frozen at −80°C in Microbank tubes (Pro-Lab Diagnostics, Bromborough, UK) until further assessment.