Bacterial cell culture media E. faecium

Bacterial cell culture media E. faecium
CHROMID® VRE from Biomerieux

Protocol tips

Protocol tips
-Incubation : 48 hours

Publication protocol

Deidentified stool specimens from patients who provided samples for Clostridium difficile testing were used for this evaluation. One microliter of remnant stool was inoculated and aseptically streaked for isolation onto each of the five chromogenic media: the InTray Colorex VRE (BioMed Diagnostics, White City, OR), chromID VRE (bioMérieux, Marcy l'Étoile, France), VRESelect (Bio-Rad, Marnes-la-Coquette, France), HardyCHROM VRE (Hardy Diagnostics, Santa Maria, CA), and Spectra VRE (Remel, Lenexa, KS), along with BEAV agar (6 μg/ml vancomycin) (Enterococcosel; BD Diagnostics, Sparks, MD) and BEAV broth (6 μg/ml vancomycin) (BEAV broth; Hardy Diagnostics). At the time of this study, BEAV agar was routinely used for VRE surveillance at the Johns Hopkins Hospital. For the purposes of this study, BEAV broth was added in order to enhance VRE detection performance. The media and BEAV broths and all chromogenic media were incubated in the dark, in non-CO2, at 35 to 37°C. The identification of the enterococci from the chromogenic agars was based on the observation of appropriately colored colonies and supplemental testing (Gram stain, catalase, and l-pyrrolidonyl-β-naphthylamide enzyme [PYR]) if required, as per the manufacturers' instructions. Vancomycin susceptibility tests were performed using Etest (AB bioMérieux, Durham, NC) for the VRE isolates recovered from each medium.

At 24 h, BEAV broths with black color development were subcultured to 5% sheep blood agar and incubated for an additional 18 to 24 h. Catalase-negative PYR-positive colonies were sent to the BD Phoenix automated microbiology system (BD Diagnostics, Sparks, MD) for identification to the species level and susceptibility testing. The vancomycin susceptibility results were reported using current Clinical and Laboratory Standards Institute interpretive guidelines (vancomycin resistant, MIC > 32 μg/ml). A positive result for a chromogenic agar that did not match the results of the BEAV broth was assessed by subculturing the organisms from each chromogenic medium and BEAV broth to 5% sheep blood agar again for reidentification and repeat susceptibility, as described above, using BD Phoenix and Etest. If the identification of the colored colony did not match the Phoenix identification, it was considered an incorrect identification. Appropriately colored colonies growing directly on chromogenic medium identified as VRE and the results of the subculture from positive BEAV plates and broth served as a combined true-positive gold standard for this study after the resolution of discrepancies by repeat testing.

Of the 400 specimens tested, four were excluded from the analysis because they grew only VRE Enterococcus raffinosus, an organism for which none of the chromogenic agars have FDA approval. Of the 396 remaining specimens, 297 (75%) were negative for VRE. The performance characteristics of each medium, BEAV agar, and BEAV broth were compared using combined data. Overall, the sensitivities of all of the chromogenic media (89.9 to 94.9%) were higher than that of the BEAV agar (84.8%). chromID had the highest sensitivity (94.9%). All of the chromogenic media recovered significantly more VRE than did the BEAV agar. There was no statistical difference in VRE detection performance among the five chromogenic media

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