Characterization of NWM-1, a novel subclass B3 metallo-β-lactamase found in a clinical isolate of Pseudomonas aeruginosa.

Abstract

Objectives: To investigate the carbapenem resistance mechanism of a carbapenem-resistant clinical Pseudomonas aeruginosa isolate.

Methods: A clinical isolate of P. aeruginosa was sent to the German National Reference Centre for multidrug-resistant Gram-negative bacteria for carbapenemase detection. Phenotypic tests for carbapenemase detection and an EDTA-combined disc test were positive, therefore PCR-screenings were done for the most prevalent metallo-β-lactamase (MBL) encoding genes. As no MBL gene could be found, whole-genome sequencing was performed. For characterization, heterologous expression in a E. coli strain with subsequent MIC testing and purification of the new MBL to determine enzyme kinetics with in vitro hydrolysis assays was performed.

Results: WGS revealed the putative gene for a B3 MBL located on the chromosome between several disrupted IS elements with 67% identity to EVM-1, which was named NWM-1. MIC studies and enzyme kinetics confirmed MBL activity. No activity against ceftazidime was observed.

Conclusions: The identification of NWM-1 shows the importance of WGS to identify yet unknown carbapenemases and underlines the diversity of subclass B3 β-lactamases. It also shows that although several carbapenemase variants have already been identified and characterized, there are always new variants to be found in clinical isolates.