Development of a novel chromogenic method, Penta-well test, for rapid prediction of β-lactamase classes produced in clinical Enterobacteriaceae isolates

We developed a novel chromogenic method, Penta-well test, which enables the rapid detection and classification of β-lactamases in clinical Enterobacteriaceae isolates. This test is based on a combination of nitrocefin and 3 β-lactamase inhibitors specific to classes A, B, and/or C, with nitrocefin hydrolysis by β-lactamases being assessed by optical density measurements at 490. nm. When the cutoff value for each β-lactamase class was determined (0.09, 0.4, and 0.55 for class A, class B, and class C β-lactamase producers, respectively), the sensitivity and specificity of classification were 93.5% and 68.8% for class A, 93.8% and 100% for class B, and 86.7% and 100% for class C, respectively. Moreover, this method allowed accurate β-lactamase classification in 20 of 23 (87.0%) isolates producing plural class of β-lactamases. Thus, the Penta-well test can provide information that would be useful in the accurate detection and classification of β-lactamases produced by causative bacteria.