9.1.2 Synergy testing

To confirm that an ESBL is the cause of the resistance the usual approach is to test for synergy with clavulanic acid for ceftazidime, cefotaxime and cefepime, by one of several methods including:

• commercially available combination disk test (CDT) (Figure 14),
• double disk synergy test (DDST) (Figure 15)
• automated systems such as VITEK II^TM or Phoenix^TM .

Figure 14 Combination disk testing using commercial Rosco disks: A) Rosco disk inoculator; B) Rosco test plate.

Key:

• CTX+C = cefotaxime + clavulanic acid
• CTXCX = cefotaxime + cloxacillin
• CTXCC = cefotaxime + clavulanic acid + cloxacillin
• CAZ+C = ceftazidime + clavulanic acid
• CAZCX = ceftazidime + cloxacillin
• CAZCC = ceftazidime + clavulanic acid + cloxacillin.

Activity 10 Interpreting a DDST

Timing: Allow 5 minutes

How would you interpret the results of the double disk synergy test in Figure 15? Does the test organism have an ESBL?

Figure 15 Double disk synergy test set up to compare the zone for cefotaxime alone with the zone for cefotaxime adjacent to the disk with the beta lactamase inhibitor (clavulanic acid).

Answer

The test organism has an ESBL.

Discussion

The cefotaxime is inactivated by the ESBL (no zone) on its own but works ‒ preventing growth and leaving a clear zone – in the part of the agar where the clavulanic acid has diffused out from its disk. The clavulanic acid is protecting the cefotaxime from the ESBL. (Note the clavulanic acid disk contains co-amoxiclav (AMC) so there is also amoxicillin present. This combination has given a small zone around the disk, but not enough for this antibiotic to be useful in clinical practice.)