A disc diffusion assay for detection of class A, B and OXA-48 carbapenemases in Enterobacteriaceae using phenyl boronic acid, dipicolinic acid and temocillin

Abstract

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.

Comparison of the Modified Hodge Test and the Carba NP Test for Detection of Carbapenemases in Enterobacteriaceae Isolates

“…Van Dijk ve arkadaşları 23 da, çalışmamızla uyumlu olarak, MHT'nin duyarlılığının %99 gibi yüksek, özgüllüğünün %59 gibi düşük olduğunu bildirmişlerdir. Yapılan çeşitli çalışmalarda, bu yöntemin duyarlılığı %58-99 ve özgüllüğü %38.9-100 arasında büyük değişkenlik göstermekte; çalışılan izolatların taşı-dığı karbapenemaz tipi duyarlılığı, karbapenemazlar dışında mekanizmaların varlığı ise özgüllüğü belirgin ölçüde etkilemektedir 1,17,20,[22][23][24] (3) 3 (100) 3 (100) K.oxytoca (2) 2 (100) 2 (100) E.cloacae (2) 2 (100) 2 (100) Toplam (9) 8 (88.9) 8 (88.9) tipi enzimlerin daha iyi saptandığı, metallo-beta-laktamazların saptanmasının zayıf olduğu bildirilmektedir 1,4,20,22 . Ancak çalışmamızda, MHT ile dört OXA-48 (K.pneumoniae), bir NDM-1 (K.pneumoniae) ve bir IMP (E.coli) saptanamamıştır.…”

“…Çalışmamızda testin özgüllüğü %60.3 gibi düşük bir oranda bulunmuştur. GSBL (özellikle CTX-M tipi) veya AmpC betalaktamazların azalmış geçirgenlik ile birlikteliğinin, MHT'de yalancı pozitifl iklere neden olabildiği bildirilmiştir 3,5,[20][21][22][23]25 . Ülkemizde GSBL enzimleri yaygın olup, en sık CTX-M tipi enzimler saptanmaktadır 26 .…”

Evaluation of Carbapenemase Screening and Confirmation Tests with Enterobacteriaceae and Development of a Practical Diagnostic Algorithm

“…Critical diameters and MICs of ertapenem (ETP), meropenem (MEM), and imipenem (IPM) and automated microdilution expert systems have been evaluated as screening methods (14,(18)(19)(20). For carbapenemase confirmation, the modified Hodge test is recommended by CLSI and various commercial and in-house combined-disk tests (CDTs) using boronic acid derivatives and EDTA/dipicolinic acid as specific inhibitors have been described (13,(19)(20)(21)(22)(23)(24)(25). In 2014, EUCAST published new guidelines for the detection of resistance mechanisms, including carbapenemases, in which a CDT is recommended for carbapenemase confirmation (14,22,25).…”

“…For carbapenemase confirmation, the modified Hodge test is recommended by CLSI and various commercial and in-house combined-disk tests (CDTs) using boronic acid derivatives and EDTA/dipicolinic acid as specific inhibitors have been described (13,(19)(20)(21)(22)(23)(24)(25). In 2014, EUCAST published new guidelines for the detection of resistance mechanisms, including carbapenemases, in which a CDT is recommended for carbapenemase confirmation (14,22,25). Recently, Nordmann et al described a new inhibitor-based biochemical assay for carbapenemase detection, the Carba NP test, which has been published in two versions: the Carba NP-I assay provides a positive or negative result (carbapenemase detected/not detected), whereas the Carba NP-II test has been designed to also discriminate among carbapenemase classes A, B, and D (26)(27)(28)(29).…”

Evaluation of Two Phenotypic Screening Tests for Carbapenemase-Producing Enterobacteriaceae

“…As previously reported, the sensitivity of synergy tests for Proteus spp. is low because of swarming (18); in our study, the only unidentified CPE is indeed an NDM-producing P. mirabilis isolate for which the synergy with dipicolinic acid was present but not sufficient. The specificity of OXA-48 producers found in our study was slightly less powerful than the study of van Dijk et al (98.1% versus 100%, respectively) (18).…”