The kit is intended for detection of:
• Klebsiella pneumoniae carbapenemase (KPC) and Metallo-beta-lactamase (MBL) in Pseudomonas aeruginosa
• Metallo-beta-lactamase (MBL) and Oxacillinase in Acinetobacter spp.
Principle
This kit contains five cartridges of tablets containing Imipenem 10μg and Imipenem in combination with inhibitors of different β-lactamases. Inhibitors are added to differentiate isolates with resistance mechanisms from those without resistance mechanisms (see explanation below).
Reduced susceptibility to carbapenems is observed when:
1. The organism produces a metallo-beta-lactamase (MBL) that hydrolyses carbapenems efficiently. MBLs are inhibited by dipicolinic acid (DPA) and EDTA. Synergy (ghost zone) between imipenem and DPA or/and EDTA indicates the presence of a MBL. If here is no zone around Imipenem 10μg, synergy should be checked at a closer distance between Imipenem 10μg and Imipenem + DPA.
2. The organism produces a Klebsiella pneumoniae carbapenemase (KPC). KPC enzymes are inhibited by Phenylboronic Acid. However, Phenylboronic Acid inhibits also the AmpC (class C cephalosporinases). In order to raise Kit’s specificity, Cloxacillin High (AmpC inhibitor) is included to distinguish between these two. Thus, different inhibition zone using Imipenem + Phenylboronic Acid and Imipenem + Cloxacillin High indicates the presence of a KPC enzyme.
Interpretation
For Pseudomonas aeruginosa:
1. Measure the inhibition zone around Imipenem 10μg (IMI10) and compare it with the zones around Imipenem + Phenylboronic Acid (IMPBO) and Imipenem + Cloxacillin High (IPCX4). If the zone difference around:
• IMPBO and IMI10 is ≥4mm
• IPCX4 and IMI10 is <3mm
the organism demonstrates KPC activity.
Note: If IPCX4 zone is ≥5mm than IMI10, the strains do not produce carbapenemase. (Do not test with the kit).
2. Measure the inhibition zones around Imipenem 10μg (IMI10) and compare it with Imipenem + DPA (IM+DP) and Imipenem + EDTA (IM10E). If the zone difference around
• IM+DP and IMI10 is ≥5mm
and/or
• IM10E and IMI10 is ≥8mm
the isolate produces a Metallo-beta-lactamase.
If there is no IMI10 inhibition zone (9mm) and
• IM+DP inhibition zone is ≥12mm
• IM10E inhibition zone is ≥13mm
Synergism is indicated, thus a positive result.
Note: Test only Ceftazidime resistant isolates. Otherwise false MBL-positive isolates may be obtained (if Ceftazidime sensitive).
Heinrichs et al showed that Imipenem + DPA (but not Meropenem + DPA) could be used for detecting MBLs in Pseudomonas aeruginosa, with a sensitivity of 99 % and a specificity of 95 %. Meropenem + DPA should be used for detecting MBLs in Enterobacteriaceae (kit 98015).
For Acinetobacter spp.
1. Same method as applies for the detection of MBLs in Ps. Aeruginosa.
2. Oxacillinases are influenced by EDTA resulting in a weak synergism between Imipenem + EDTA, while DPA has no effect. This can be used for oxacillinase detection from Acinetobacter spp.
