Genetic and biochemical characterization of BIM-1, a novel acquired subgroup B1 MBL found in a Pseudomonas sp. strain from the Brazilian Amazon region

Abstract

Objectives: To characterize a novel acquired MBL, BIM-1, in a Pseudomonas #2 (subgroup P. guariconensis) strain isolated from the Aurá river located in the Brazilian Amazon hydrographic basin.

Methods: WGS using an Illumina® MiSeq System was used to characterize the genome of Pseudomonas sp. IEC33019 strain. Southern blotting/hybridization assays were performed to confirm the location of the MBL-encoding gene, blaBIM-1 (Belém Imipenemase). Antimicrobial susceptibility testing, cloning, and biochemical and phenotypic characterization were performed to determine BIM-1 kinetics.

Results: The IEC33019 strain showed high resistance rates to β-lactams, ciprofloxacin and aminoglycosides, being susceptible only to polymyxins and susceptible, increased exposure to aztreonam. WGS analysis revealed a novel acquired MBL-encoding gene, blaBIM-1, found as a gene cassette inserted into a class 1 integron (In1326) that also carried qnrVC1 and aadA11e. In1326 was located in a complex transposon, Tn7122, carried by a 52.7 kb conjugative plasmid (pIEC33019) with a toxin/antitoxin system (vapB/vapC). BIM-1 belongs to the molecular subgroup B1 and shares 70.2% and 64.9% similarity with SIM-1 and IMP-1, respectively. Kinetics analysis of BIM-1 showed hydrolytic activity against all β-lactams tested.

Conclusions: BIM-1 is a novel acquired MBL encoded by a gene carried by mobile genetic elements, which can be transferred to other Gram-negative bacilli (GNB). Because the IEC33019 strain was recovered from a river impacted by a populous metropolitan region with poor basic sanitation and served by limited potable freshwater, it would be important to establish the role of the BIM-1-producing GNB as nosocomial pathogens and/or as colonizers of the riverside population in this geographical region.

Figure 1.

Genetic structure of pIEC33019 conjugative plasmid carrying bla_BIM-1 in Pseudomonas #2 IEC33019 strain. (a) Circular map of pIEC33019. (b) Antimicrobial resistance domain containing In1326 (dashed box). (c)Truncated res site (ΔresI) with In1326 of module tnpAR-res. (d) Virulence-mediated domain. (e) Mobilization/conjugation domain. Grey arrows indicate genes encoding for hypothetical proteins.

Figure 2.

Amino acid sequence alignment of BIM-1 with other MBLs representative of subclasses B1 (IMP-1; ABK27309.1), B2 (CphA; CAA40386.1) and B3 (L1; CAA52968.1). Conserved residues in the first and second Zn²⁺ coordination sites are shown in red boxes, and dashes correspond to the sequence gaps. Numbering is according to the updated BBL scheme. The figure was obtained using MegAlign (Lasergene software package; DNAStar, Madison, WI, USA).

Figure 3.

Phylogenetic tree constructed based on the ClustalV alignment of BIM-1 and 16 acquired MBL groups described to date using MegAlign (Lasergene software package; DNAStar, Madison, WI, USA).