A sensitive and specific phenotypic assay for detection of metallo-β-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin.

Enterobacteriaceae producing carbapenemases, such as KPC or metallo-β-lactamases (MBLs), have emerged on several continents. Phenotypic tests are urgently needed for their rapid and accurate detection. A novel carbapenemase detection test, comprising a meropenem disk, and meropenem disks supplemented with 730 μg of EDTA, 1000 μg of dipicolinic acid (DPA), 600 μg of aminophenylboronic acid (APBA), or 750 μg of cloxacillin, was evaluated against Klebsiella pneumoniae isolates with KPC (n = 34), VIM (n = 21), IMP (n = 4) or OXA-48 (n = 9) carbapenemases, and carbapenem-resistant Enterobacteriaceae with porin loss in combination with an extended-spectrum β-lactamase (ESBL) (n = 9) or AmpC hyperproduction (n = 5). Commercially available diagnostics tablets from Rosco containing meropenem and the same inhibitors as described above (except EDTA) were also evaluated. An increased meropenem inhibition zone was sought in the presence of each added β-lactamase inhibitor. APBA had excellent sensitivity for detecting K. pneumoniae with KPC enzymes. Isolates with combined AmpC hyperproduction and porin loss were also positive in the APBA test but, unlike KPC producers, showed cloxacillin synergy. Both DPA and EDTA had excellent sensitivity for detection of MBL-producing K. pneumoniae. However, EDTA showed poor specificity, with positive results noted for 1/9 ESBL-producing isolates, for 4/34 KPC-producing isolates, and for 4/9 OXA-48-producing isolates, whereas all of these were negative when DPA was used. The in-house test distinguished accurately between several different mechanisms mediating reduced susceptibility to carbapenems in Enterobacteriaceae. The commercial combination tablets from Rosco performed similarly to the in-house test, with the exception of one false-positive MBL result and one false-positive KPC result among the OXA-48 producers.

Giske CG ，Gezelius L ，Samuelsen Ø ，Warner M ，Sundsfjord A ，Woodford N ... -  《-》

A simple test for the detection of KPC and metallo-β-lactamase carbapenemase-producing Pseudomonas aeruginosa isolates with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin.

We evaluated the ability of the combination disk test (CDT) and the Modified Hodge Test (MHT) to discriminate between various carbapenemase-producing Pseudomonas aeruginosa isolates (KPC, n = 36; metallo-β-lactamase (MBL), n = 38) and carbapenemase non-producers (n = 75). For the CDT, the optimal inhibitor concentrations and cut-off values were: 600 μg of 3-aminophenylboronic acid (APB) per disk (an increment of ≥4 mm), 1000 μg of dipicolinic acid (DPA) per disk (an increment of ≥5 mm) and 3000 μg of cloxacillin per disk (an increment of ≥3 mm). APB had excellent sensitivity (97%) and specificity (97%) for the detection of KPC enzymes. DPA detected MBL enzymes with a sensitivity and specificity of 97% and 81%, respectively. The MHT resulted in a low sensitivity (78%) and specificity (57%). The CDT could be very useful in daily practice to provide fast and reliable detection of KPC and MBL carbapenemases among P. aeruginosa isolates.

Pasteran F ，Veliz O ，Faccone D ，Guerriero L ，Rapoport M ，Mendez T ，Corso A ... -  《-》

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.

van Dijk K ，Voets GM ，Scharringa J ，Voskuil S ，Fluit AC ，Rottier WC ，Leverstein-Van Hall MA ，Cohen Stuart JW ... -  《-》

A simple phenotypic method for the differentiation of metallo-beta-lactamases and class A KPC carbapenemases in Enterobacteriaceae clinical isolates.

Tsakris A ，Poulou A ，Pournaras S ，Voulgari E ，Vrioni G ，Themeli-Digalaki K ，Petropoulou D ，Sofianou D ... -  《-》

Inhibitor-based methods for the detection of KPC carbapenemase-producing Enterobacteriaceae in clinical practice by using boronic acid compounds.

Pournaras S ，Poulou A ，Tsakris A 《-》