Molecular and Biochemical Characterization of Novel PAM-like MBL Variants, PAM-2 and PAM-3, from Clinical Isolates of Pseudomonas Tohonis.

Background There is no comprehensive study on PAM-like MBLs. Objectives Our aim was to characterize novel B3 MBL variants, PAM-2 and PAM-3, from Pseudomonas tohonis clinical isolates. Methods We evaluated the antimicrobial susceptibility and the MBL gene composition of three novel P. tohonis clinical isolates identified at a Japanese hospital, using the broth microdilution method and WGS, respectively. We characterized the PAM-2 and PAM-3 proteins using recombinant protein expression and biochemical evaluations. Results Low carbapenem MICs (meropenem MIC = 0.125-1 mg/L) were observed for all three P. tohonis isolates; however, the isolates produced MBLs. We identified bla(PAM-2) and bla(PAM-3) as potential genes, belonging to a novel subclass of B3 MBLs. Their genomic sequence was similar to that of bla(PAM-1) from Pseudomonas alcaligenes. PAM-2 and PAM-3 comprised 287 amino acids and exhibited 90% amino acid identity with PAM-1, 73% identity with POM-1 from Pseudomonas otitidis and 61% identity with L1 from Stenotrophomonas maltophilia. Biochemical evaluations of recombinant PAM-2 and PAM-3 revealed similar k(cat)/K-m ratios and demonstrated catalytic activity against all the tested beta-lactams, except for aztreonam. In addition, the k(cat)/K-m ratio for imipenem was 40-fold lower than that for meropenem. Conclusions P. tohonis harbours a species-specific PAM-family MBL gene. This enzyme has higher hydrolytic activity against meropenem compared with that against imipenem.