Genetic and biochemical characterization of an acquired subgroup B3 metallo-β-lactamase gene, blaAIM-1, and its unique genetic context in Pseudomonas aeruginosa from Australia

Abstract

Three clinical Pseudomonas aeruginosa isolates (WCH2677, WCH2813, and WCH2837) isolated from the Women's and Children's Hospital, Adelaide, Australia, produced a metallo-β-lactamase (MBL)-positive Etest result. All isolates were PCR negative for known MBL genes. A gene bank was created, and an MBL gene, designated bla(AIM-1), was cloned and fully characterized. The encoded enzyme, AIM-1, is a group B3 MBL that has the highest level of identity to THIN-B and L1. It is chromosomal and flanked by two copies (one intact and one truncated) of an ISCR element, ISCR15. Southern hybridization studies indicated the movement of both ISCR15 and bla(AIM-1) within the three different clinical isolates. AIM-1 hydrolyzes most β-lactams, with the exception of aztreonam and, to a lesser extent, ceftazidime; however, it possesses significantly higher k(cat) values for cefepime and carbapenems than most other MBLs. AIM-1 was the first mobile group B3 enzyme detected and signals further problems for already beleaguered antimicrobial regimes to treat serious P. aeruginosa and other Gram-negative infections.

Fig 1

Evidence of movement of ISCR15 and bla_AIM-1, and relatedness of AIM-1-positive strains. All panels a marker (lane M), WCH2677 (lane 1), WCH2813 (lane 2), and WCH2837 (lane 3). (A) SpeI digestion. (B) I-CeuI digestion. (C) S1 digestion and probing with bla_AIM-1. (D) SpeI digestion and probing with ISCR15.

Fig 2

Alignment of the amino acid sequence of AIM-1 with those of L1 and THIN-B. Differences in the amino acid sequences are noted by a single letter representing the amino acid change within a particular sequence. Conserved residues and residues involved in the coordination of the zinc ions are denoted with asterisks. Numbering is according to the updated BBL scheme (11).

Fig 3

Schematic representation of WCH2677 carrying bla_AIM-1 (the arrows in the gene boxes indicate the direction of transcription). The sequence upstream of bla_AIM-1 and the oriIS for ISCR1, ISCR3, ISCR5, and IS1294 is highlighted. The putative ribosomal binding site is underlined, and the start codon of bla_AIM-1 is indicated in boldface type. GenBank accession numbers are in parentheses.