7.5.1 Isolation of E. faecium and E. faecalis

Primary inoculation of blood agar incubated aerobically or in 5–10% CO2 at 35–37°C for 24–48h will support the growth of many species of Enterococcus. Often, however, selective azide blood agar is used to inhibit Gram-negatives.

Activity 12: Isolating E. faecium and E. faecalis in your workplace

Timing: Allow about 10 minutes

Think about your workplace activities and/or reflect on what you have learned so far in the course and consider these questions.

  1. How could you isolate E. faecium and E. faecalis reliably from clinical specimens and differentiate these organisms from Streptococcus and from other Enterococcus species?
  2. What characteristic appearance would colonies of these organisms display on blood agar and what further tests might you use to confirm E. faecium and E. faecalis?
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Answer

  1. Selective solid media containing sodium azide for the suppression of the growth of Gram-negatives can be used as primary medium in addition to blood agar.
  2. Colonies are 1–3 mm in diameter, non-haemolytic or partially-haemolytic. A further catalase test would be negative.
  3. However, both these features are not specific, and further testing is necessary.

Due to the difficulty in distinguishing enterococci from each other and from streptococci, the identification of E. faecium and E. faecalis is not always straightforward. Most animal diagnostic laboratories therefore apply rudimentary identification routines for the enterococci, probably leading to many false positive and false negative results.

The following differential features enable the identification of the enterococci.

  • Enterococcus spp. express Lancefield group D surface antigen, enabling a differentiation from non-D streptococci.
  • Many enterococci hydrolyse aesculin in the presence of bile, and grow in the presence of 6.5% NaCl.
  • The application of a purposive combination of biochemical and antimicrobial susceptibility tests will enable a correct differentiation between group D streptococci and Enterococcus spp. in many cases. However, any stringent scheme should include PCR-amplification of Enterococcus genus-specific loci.
  • The use of commercial chromogenic agar media enable the differentiation between E. faecium and E. faecalis. Further analysis could include specific antimicrobial resistance tests and other phenotypic tests, or sequence analysis of the 16S rRNA gene.

Enterococci can be stored frozen, freeze dried or refrigerated, and may remain viable for several weeks on storage media slants in refrigeration, or even at room temperature.

7.5 Enterococcus faecium and E. faecalis

7.6 Campylobacter species