Development of phenotypic and genotypic methods for the identification of NmcA β-lactamases in carbapenem-resistant Enterobacter spp. and Klebsiella aerogenes

Enterobacterales resistant to carbapenems owing to their acquisition of carbapenemase genes have been reported worldwide. Enterobacter cloacae complex produces chromosomally encoded inducible Ambler class C β-lactamase but is usually susceptible to carbapenems. However, some studies have reported the incorporation of the NmcA carbapenemase gene, the only inducible carbapenemase classified as Ambler class A, into the chromosomes of members of this complex. Although only a few cases showing NmcA production have been reported, detection methods are urgently needed. Therefore, we developed a double-disk synergy test and loop-mediated isothermal amplification method to detect NmcA producers. The double-disk synergy test was performed by placing a disk of carbapenem (meropenem) 20 mm (center to center) from the disk of a third-generation cephalosporin (cefpodoxime). The loop-mediated isothermal amplification primers were designed to specifically recognize bla NmcA . NmcA-producing Enterobacter ludwigii isolate NR1491 was used as a positive control. The double-disk synergy test results revealed a D-shaped inhibition zone around the cefpodoxime disk, whereas the inhibition zone adjacent to the meropenem disk was blunted. No other D-shaped inhibition zones were observed for other types of carbapenemase producers. The loop-mediated isothermal amplification assay successfully detected NmcA and IMI producers with high homology to NmcA. The detection limit for this assay was 10 −1 CFU per tube, which is 100-fold more sensitive than the PCR assay for bla NmcA detection. The accuracy, sensitivity, and specificity of these assays are high, and these tests are expected to be used for the detection of NmcA producers in clinical laboratories. IMPORTANCE NmcA, which is associated with carbapenem resistance, has occasionally been detected in the Enterobacter cloacae complex. NmcA is the only inducible carbapenemase whose expression is regulated by transcriptional regulator genes. We developed phenotypic and genotypic methods for the identification of NmcA β-lactamases and evaluated their usefulness in clinical laboratories. The multiplex double-disk synergy test method was developed as a phenotypic method to identify NmcA producers by confirming the phenomenon (induction mechanism) exclusive to NmcA producers. We also developed a loop-mediated isothermal amplification assay as a genotyping method to detect NmcA producers by specifically recognizing bla NmcA . These methods demonstrated high accuracy, sensitivity, and specificity and are expected to be applied for the detection of NmcA producers in clinical settings.