Development of a novel, simple and rapid chromogenic method to detect the presence of carbapenemase-producing Enterobacteriaceae

Background: Carbapenem resistance in Enterobacteriaceae poses a great public health concern, threatening human health worldwide. Production of carbapenemases is a crucial resistance mechanism to carbapenems. Development of reliable, simple and rapid methods for laboratory detection of carbapenemase is still required. The Nitro-Carba GKY test has been developed in the present study to detect carbapenemase in Enterobacteriaceae.

Methods: The Nitro-Carba test (NCT) is based upon the hydrolysis of nitrocefin (NTF) in the presence of carbapenem antibiotics, combined with a simple enzyme extraction method. A total of 87 molecularly-confirmed isolates including 31 carbapenemase producers (11 MBLs, 9 KPCs and 11 OXA-48s) and 56 non-carbapenemase producers (32 AmpC and 15 ESBL producers and 9 co-producers of ESBL and AmpC), were investigated with the NCT assay. The minimum inhibitory concentration (MIC) of imipenem (IPM), meropenem (MEM) and ertapenem (ERT) was also determined. To assess the performace of NCT assay, all isolates were lysed and added to wells containing carbapenems and water. The results can be observed visually following adding NTF by the change in colour from yellow to red in the presence of β-lactamases. The colour changes in wells containing cabapenems were denoted as presence of carbapenemase.

Results: The NCT assay showed very rapid detection of all 31 carbapenemase producers. The resulting times to detect the presence of carbapenemase ranged from 10 seconds to 12 min. The sensitivities of NCT for all antibiotics were 100%. No colour changes after 20 min in the wells with antibiotics were observed in non-carbarpenemase producers. ERT completely prevented hydrolysis of NTF by all AmpCs and ESBLs, while IPM and MEM did not prevent NTF hydrolysis by some strains. These findings indicated that ERT had the best specificity (100%) compared with IPM and MEM. The MIC results of IPM, MEM and ERT against all carbapenemase-producing strains ranged from 0.5-≥256, 0.25- ≥256 and 1-≥256 µg/ml, respectively.

Conclusion: The NCT assay proposed in this study is a very simple, reliable and cost-effective method, allowing visual observation of the results within 12 min. ERT shows the best sensitivity and specificity and it is suggested that this antibiotic should be adopted in future tests.