4.2 Identifying Gram-positive cocci

Your laboratory needs to be able to reliably confirm the identification of S. aureus and S. pneumoniae (see Figure 4).

Described image
Figure 4 How to identify S. aureus and S. pneumoniae.

Enterococci can show any of the three patterns of haemolysis. They are generally Lancefield Group D can be confirmed using biochemical tests such as aesculin hydrolysis or litmus milk reduction. They ferment a range of sugars including lactose.

For S. aureus, a presumptive identification is done by the characteristic appearance of colonies on agar and a positive catalase test. A minimum of two tests from the following must be used to confirm identification: tube coagulase, slide coagulase, or DNAse. Alternatively, MALDI-TOF can be used.

  • Why is more than one test needed to confirm S. aureus?

  • Bacteria, being living organisms, will occasionally not test the way the book says they should, so more than one test is needed to confirm. Some ‘coagulase negative’ Staphylococci will test positive on one of the coagulase tests. This is important for clinical reasons, to give the right treatment, and also for AMR surveillance. For example, methicillin-resistant, coagulase-negative Staphylococcus could be mis-reported as MRSA leading to inappropriate and unnecessary antibiotics and providing inaccurate surveillance data.

  • What system of controls for tests should be in place?

  • For any identification, not just for S. aureus, it is important to use positive and negative control organisms to make sure your reagents are working correctly. Ideally these organisms are reference strains obtained from the UK National Collection of Type Cultures (NCTC) or the American Type Culture Collection (ATCC). For these tests the controls are set up in parallel with the test organism.

S. pneumoniae is fastidious and hard to grow (Figure 5). For cultures grown in conditions that should support its growth, such as on blood agar under CO2, the organism is suspected by its typical appearance on solid media. This, plus a positive optochin sensitivity or bile solubility test, is usually sufficient to confirm species. Latex agglutination tests are also available. If using MALDI-TOF, optochin or bile solubility testing must also be performed, as MALDI-TOF is, at present, unable to distinguish between S. pneumoniae and viridans Streptococci.

Described image
Figure 5 Appearance of S. pneumoniae colonies on blood agar.

Activity 12: Testing for S. pneumoniae in your workplace

Timing: Allow 10 minutes

Think about your workplace activities and consider these questions.

  1. If you are able to culture and identify S. pneumoniae reliably in your laboratory, how are you differentiating it from other streptococcal species?
  2. If isolation of this organism is not reliable, or you do not usually work with this organism, what might be practical barriers to its identification?
  3. How might laboratory practice, for example the ease of isolating S. pneumoniae, affect AMR surveillance for this organism?
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Discussion

  1. Differentiation from other streptococcal species can be by appearance, haemolysis, specific confirmatory tests and by the fact that it only grows on blood-containing media under CO2.
  2. CO2 for incubation, and blood-containing media are both necessary for growth so these are limiting factors. Autolysis can occur in blood cultures. This is when the organism grows so fast in some media that it outstrips its nutrient supply, and no viable organism is detected on subculture. So you will need to have access to the right culture media and conditions to isolate this organism.
  3. Under-reporting from some laboratories who struggle to isolate the organism versus better equipped laboratories, could lead to bias if antibiotic resistance rates varied between the settings.

4 Identifying the key pathogens in global AMR surveillance

4.3 Identifying Gram-negative organisms