Method Direct Carbapenemase Detection
Ellen Smith Moland, Soo-Young Kim, Seong Geun Hong, Kenneth S. Thomson. Clinical
Microbiology Newsletter, Volume 30, Issue 11, 1 June 2008, Pages 79-85. Newer
-Lactamases: Clinical and Laboratory Implications, Part II.
The steps are:
1. Moisten Tris EDTA(TE) (catalog # 232211, BD Diagnostic Systems, Sparks,
MD) disk with 20 μl of sterile saline.
2. Inoculate moistened disk with a heavy inoculum of the test organism. We do this
in an empty Petri dish and use the wooden end of a sterile cotton swab to apply
the organism.
3. The disk should then be inverted before placing it on the agar. This ensures
optimal diffusion of the β-lactamase into the agar.
4. Add 20 μl of a 1:320 dilution of 2-mercaptopropionic Acid (2-MPA) (Thiolactic
acid or 2-Mercaptopropionic acid; catalogue # T31003-5G Sigma-Aldrich, St.
Louis, MO) to a second TE disk in a separate Petri dish. This disk does not have
to be flipped.
5. Inoculate susceptibility plate with lawn of 0.5 McFarland suspension of the test
isolate.
6. Place a carbapenem disk on susceptibility plate. I recommend imipenem as it
seems to be the most sensitive for this test.
7. Place the TE disk with test organism close to the imipenem disk but not touching
(1 mm separation is good).
8. Place the TE + 2MPA disk 10 mm away from the same carbapenem disk on the
opposite side.
9. Interpret test after overnight incubation. Examine zone margin for indentation
(Class A carbapenemase) or for elongation (metallo-beta-lactamase).
A possible modification is to simply rehydrate the TE disk with 20 μl of saline in lieu of
2-MPA, but this test may not be as sensitive.
Control Strains
KPC Positive Klebsiella pneumoniae ATCC BAA-1705 (D-05)
KPC negative Escherchia coli ATCC 25922
Chromosomal Metallo-β-lactamase Stenotrophomonas maltophilia ATCC 13636,
because a plasmid-mediated MBL is not commercially available at this time.