The Role of nmcR, ampR, and ampD in the Regulation of the Class A Carbapenemase NmcA in Enterobacter ludwigii.

Various carbapenemases have been identified in the Enterobacteriaceae. However, the induction and corresponding regulator genes of carbapenemase NmcA has rarely been detected in the Enterobacter cloacae complex (ECC). The NmcA-positive isolate ECC NR1491 was first detected in Japan in 2013. It was characterized and its induction system elucidated by evaluating its associated regulator genes nmcR, ampD, and ampR. The isolate was highly resistant to all ß-lactams except for third generation cephalosporins (3GC). Whole-genome analysis revealed that bla NmcA was located on a novel 29-kb putatively mobile element called EludIMEX-1 inserted into the chromosome. The inducibility of ß-lactamase activity by various agents was evaluated. Cefoxitin was confirmed as a strong concentration-independent ß-lactamase inducer. In contrast, carbapenems induced ß-lactamase in a concentration-dependent manner. All selected 3GC-mutants harboring substitutions on ampD (as ampR and nmcR were unchanged) were highly resistant to 3GC. The ampD mutant strain NR3901 presented with a 700 × increase in ß-lactamase activity with or without induction. Similar upregulation was also observed for ampC and nmcA. NR1491 (pKU412) was obtained by transforming the ampR mutant (135Asn) clone plasmid whose expression increased by ∼100×. Like NR3901, it was highly resistant to 3GC. Overexpression of ampC, rather than nmcA, may have accounted for the higher MIC in NR1491. The ampR mutant repressed nmcA despite induction and it remains unclear how it stimulates nmcA transcription via induction. Future experiments should analyze the roles of nmcR mutant strains.

Rapid species identification and epidemiological analysis of carbapenem-resistant Acinetobacter spp. by a PCR-based open reading frame typing method.

The spread of carbapenem-resistant Acinetobacter spp. has become a global problem. In this study, 18 carbapenem-resistant Acinetobacter calcoaceticus-baumannii (ACB) complexes, identified using a conventional biochemical method at our hospital during 2004-2013, were studied for species identification and epidemiological analyses. Species identification was performed using matrix-assisted laser desorption ionization-time-of-flight MS, a partial sequence analysis of rpoB and a PCR-based ORF typing (POT) method. The POT method can not only identify the species of ACB complexes but also simultaneously determine the international epidemic clones and the genetic identities of Acinetobacterbaumannii in several hours. Carbapenem resistance gene detection by PCR, molecular epidemiological analysis by PFGE and Pasteur Institute multilocus sequence typing (MLST) analysis were performed. All three methods identified 18 isolates as A. baumannii (n=10), Acinetobacterpittii (n=4) and Acinetobacternosocomialis (n=4). A metallo-ß-lactamase gene in all strains of A. pittii and A. nosocomialis and an ISAba1 gene in the upstream of the blaOXA-51-like gene in eight strains of A. baumannii were detected, respectively, as carbapenemase-related genes. Results from PFGE demonstrated that nine strains of A. baumannii were closely related genetically. Results of MLST analysis showed that A. baumannii are classifiable to sequence type 2. These results were consistent with those obtained using the POT method. This POT method can easily and rapidly identify the international epidemic clones and the identities of A. baumannii. It can be a useful tool for infection control.