A disc diffusion assay for detection of class A, B and OXA-48 carbapenemases in Enterobacteriaceae using phenyl boronic acid, dipicolinic acid and temocillin.
Class A and B carbapenemases in Enterobacteriaceae may be detected using carbapenemase inhibition tests with boronic acid derivatives (BA) and dipicolinic acid (DPA)/EDTA, respectively. However, for OXA-48 (like) carbapenemases, no specific inhibitor is available. Because OXA-48 confers high-level temocillin resistance, a disc diffusion assay using temocillin as well as BA and DPA inhibition tests was evaluated for detection of class A, B and OXA-48 carbapenemases. The test collection included 128 well-characterized non-repeat Enterobacteriaceae isolates suspected of carbapenemase production; that is, with meropenem MICs ≥ 0.5 mg/L, including 99 carbapenemase producers (36 KPC, one GES, 31 MBL, four KPC plus VIM, 25 OXA-48, two OXA-162), and 29 ESBL and/or AmpC-producing isolates. PCR and sequencing of beta-lactamase genes was used as a reference test. Phenotypic carbapenemase detection was performed with discs (Rosco) containing meropenem (10 μg), temocillin (30 μg), meropenem + phenyl boronic acid (PBA), meropenem + DPA, meropenem + BA + DPA, and meropenem + cloxacillin (CL). Absence of synergy between meropenem and BA and/or DPA and a temocillin zone ≤10 mm was used to identify OXA-48. The sensitivity for identification of class A, B and OXA-48 carbapenemases was 95%, 90% and 100%, with 96-100% specificity. In non-Proteus species, the sensitivity for class B carbapenemase detection was 97%. All isolates without PBA or DPA synergy and a temocillin disc zone ≤10 mm were OXA-48 (like) positive. In conclusion, carbapenemase inhibition tests with PBA and DPA combined with a temocillin disc provide a reliable phenotypic confirmation method for class A, B and OXA-48 carbapenemases in Enterobacteriaceae.
Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands; Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands.
K
K
van Dijk
G M
GM
Voets
J
J
Scharringa
S
S
Voskuil
A C
AC
Fluit
W C
WC
Rottier
M A
MA
Leverstein-Van Hall
J W T
JW
Cohen Stuart
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis*; D001431:Bacteriological Techniques / Q000379:methods*; D001897:Boronic Acids / Q000378:metabolism; D004755:Enterobacteriaceae / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D010406:Penicillins / Q000378:metabolism*; D010848:Picolinic Acids / Q000378:metabolism; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000032:analysis*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001897:Boronic Acids; D004791:Enzyme Inhibitors; D010406:Penicillins; D010848:Picolinic Acids; C031367:temocillin; D001618:beta-Lactamases; C063912:carbapenemase; C004860:dipicolinic acid
Boronic acid; Enterobacteriaceae; KPC; MBL; OXA-48; carbapenemase; dipicolinic acid; temocillin