Yothin Teethaisong1, Ismini Nakouti2, Katie Evans2, Griangsak Eumkeb2, Glyn Hobbs3. 1. School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University,Byrom Street, Liverpool L3 3AF, UK; School of Preclinic, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. 2. School of Preclinic, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand. 3. School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University,Byrom Street, Liverpool L3 3AF, UK. Electronic address: G.Hobbs@ljmu.ac.uk.
Abstract
OBJECTIVES: In this study, a rapid and simple chromogenic method for screening of carbapenemase-producing Enterobacteriaceae (CPE), namely the Nitro-Carba test (NCT), was developed. METHODS: The NCT was validated using a total of 31 carbapenemase-producing isolates [9 Klebsiella pneumoniae carbapenemase (KPC), 11 metallo-β-lactamase (MBL) and 11 OXA-48] and 56 non-carbapenemase-producing isolates. The assay relies on the hydrolysis of nitrocefin by carbapenemases in the presence of carbapenem antibiotics. Carbapenemases were extracted with lysis buffer prior to addition to wells with and without imipenem (IPM), meropenem (MEM) and ertapenem (ETP). Following addition of nitrocefin, a change in colour from yellow to red, indicating carbapenemase production, was observed within 20min. The susceptibility profiles of each bacterial isolate were also investigated. RESULTS: The NCT detected all 31 CPE within a timeframe of only 10s to 12min. All carbapenemase-producers hydrolysed nitrocefin in all wells. No colour change in wells with carbapenems was observed in non-carbapenemase-producers. The sensitivity for all three carbapenems was 100%, whilst the specificity of IPM, MEM and ETP was 64.3%, 91.1% and 100%, respectively. IPM, MEM and ETP had minimum inhibitory concentrations (MICs) against all carbapenemase-producing strains ranging from 0.5μg/mL to ≥256μg/mL, 0.25μg/mL to ≥256μg/mL and 1μg/mL to ≥256μg/mL, respectively. OXA-48-producing isolates showed lower MICs compared with MBL- and KPC-producing isolates. CONCLUSION: This assay is a promising method for detecting CPE rapidly. The NCT is a simple and reliable method capable of detecting CPE even in carbapenem-susceptible strains.
OBJECTIVES: In this study, a rapid and simple chromogenic method for screening of carbapenemase-producing Enterobacteriaceae (CPE), namely the Nitro-Carba test (NCT), was developed. METHODS: The NCT was validated using a total of 31 carbapenemase-producing isolates [9 Klebsiella pneumoniae carbapenemase (KPC), 11 metallo-β-lactamase (MBL) and 11 OXA-48] and 56 non-carbapenemase-producing isolates. The assay relies on the hydrolysis of nitrocefin by carbapenemases in the presence of carbapenem antibiotics. Carbapenemases were extracted with lysis buffer prior to addition to wells with and without imipenem (IPM), meropenem (MEM) and ertapenem (ETP). Following addition of nitrocefin, a change in colour from yellow to red, indicating carbapenemase production, was observed within 20min. The susceptibility profiles of each bacterial isolate were also investigated. RESULTS: The NCT detected all 31 CPE within a timeframe of only 10s to 12min. All carbapenemase-producers hydrolysed nitrocefin in all wells. No colour change in wells with carbapenems was observed in non-carbapenemase-producers. The sensitivity for all three carbapenems was 100%, whilst the specificity of IPM, MEM and ETP was 64.3%, 91.1% and 100%, respectively. IPM, MEM and ETP had minimum inhibitory concentrations (MICs) against all carbapenemase-producing strains ranging from 0.5μg/mL to ≥256μg/mL, 0.25μg/mL to ≥256μg/mL and 1μg/mL to ≥256μg/mL, respectively. OXA-48-producing isolates showed lower MICs compared with MBL- and KPC-producing isolates. CONCLUSION: This assay is a promising method for detecting CPE rapidly. The NCT is a simple and reliable method capable of detecting CPE even in carbapenem-susceptible strains.