Detection and biochemical characterization of BioF, a novel B2 metallo-β-lactamase from Pseudomonas sp.

MAURICIO JAVIER LOZANO; PABLO EMILIANO TOMATIS; JUAN HILARIO CAFIERO; MARIA CARLA MARTINI; MARIA FLORENCIA DEL PAPA; CAROLINA VACCA; ANTONIO LAGARES

Evento virtual

Encuentro; ISME Virtual Microbial Ecology Summit; 2020

International Society for Microbial Ecology

The worldwide rise in antibiotic resistant (AR) bacteria poses a serious threat for global health. The environment is considered a source of antibiotic resistant organisms and the resistance genes they carry. We have previously identified a novel metallo-β-lactamase (MBL) gene in a high-throughput sequencing (HTS) of high molecular weight plasmids present in a bacterial collection isolated from a biopurification system (BPS), used on-farm to treat pesticide-polluted water. This gene was designated blaBioF and sequence comparison indicates that it belongs to the subclass B2 of MBLs. The isolate carrying the blaBioF gene was identified as a Pseudomonas sp. strain BF61 according to 16S DNA sequencing. We confirmed the resistance of BF61 to carbapenems and that metal chelating agents EDTA and DPA inhibited the carbapenemase activity, giving support that this strain encodes an active MBL. Next, we performed a HTS of total DNA of strain BF61. Analyses based on average nucleotide identity indicated that isolate BF61 fits in the P. fluorescens complex and most likely belongs to a new species within the genus Pseudomonas. In silico analysis performed to define the genomic location of blaBioF showed inconclusive results, with the MBL containing contig predicted as plasmidic or chromosomal, depending on the algorithm used. Interestingly, IslandViewer4 predicted that this gene is coded in a genomic island, suggesting a horizontal acquisition. We next performed steady-state kinetic measurements to assess the capacity of a recombinant mature BioF protein to hydrolyze β-lactams in vitro. BioF showed mainly carbapenemase activity (Kcat/KM values of 1 to 3 × 105 (M-1 s-1)), with a poor efficiency against some cephalosporins. The results presented here emphasize the importance of the exploration of AR in the environmental microbiota, as a first step towards the prediction of novel resistance mechanisms before they emerge clinically.