Sequence heterogeneity of the PenA carbapenemase in clinical isolates of Burkholderia multivorans

Abstract

Multidrug-resistant gram-negative pathogens are a significant health threat. Burkholderia spp. encompass a complex subset of gram-negative bacteria with a wide range of biological functions that include human, animal, and plant pathogens. The treatment of infections caused by Burkholderia spp. is problematic due to their inherent resistance to multiple antibiotics. The major β-lactam resistance determinant expressed in Burkholderia spp. is a class A β-lactamase of the PenA family. In this study, significant amino acid sequence heterogeneity was discovered in PenA (37 novel variants) within a panel of 48 different strains of Burkholderia multivorans isolated from individuals with cystic fibrosis. Phylogenetic analysis distributed the 37 variants into 5 groups based on their primary amino acid sequences. Amino acid substitutions were present throughout the entire β-lactamase and did not congregate to specific regions of the protein. The PenA variants possessed 5 to 17 single amino acid changes. The N189S and S286I substitutions were most prevalent and found in all variants. Due to the sequence heterogeneity in PenA, a highly conserved peptide (18 amino acids) within PenA was chosen as the antigen for polyclonal antibody production in order to measure expression of PenA within the 48 clinical isolates of B. multivorans. Characterization of the anti-PenA peptide antibody, using immunoblotting approaches, exposed several unique features of this antibody (i.e., detected <500 pg of purified PenA, all 37 PenA variants in B. multivorans, and Pen-like β-lactamases from other species within the Burkholderia cepacia complex). The significant sequence heterogeneity found in PenA may have occurred due to selective pressure (e.g., exposure to antimicrobial therapy) within the host. The contribution of these changes warrants further investigation.

“Switching Partners”: Piperacillin-Avibactam Is a Highly Potent Combination against Multidrug-Resistant Burkholderia cepacia Complex and Burkholderia gladioli Cystic Fibrosis Isolates

“…One exception was B. multivorans AU15814, which possesses a band upon induction with piperacillin but has limited nitrocefinase activity. This lack of activity is likely due to the N132S amino acid substitution present in PenA, that we previously reported (17). The N132S substitution in PenA substantially diminishes the MIC (from 1,024 to 8 g/ml) toward cephalothin, which is also a first-generation cephalosporin.…”

“…as well as B. gladioli. A limitation of selecting PenA1 and AmpC1 is that these enzymes are highly heterogeneous within B. multivorans (16,17). In addition, between Burkholderia spp., the Pen-like ␤-lactamases vary in amino acid identity from 8% up to 37%.…”

“…Subsequently, both sets of cells and samples were prepared for immunoblotting as previously described (27). For detection of PenA, the polyclonal anti-PenA-peptide antibody was used as previously described (17).…”

“…In Bcc and B. gladioli, the expression of the genes encoding the Pen-like and AmpC ␤-lactamases, bla pen and bla ampC , respectively , is regulated by a LysR-type transcriptional regulator, PenR through a pathway similar to the AmpC/AmpR regulon found in Enterobacteriaceae (13,15). However, comprehensive characterization of these ␤-lactamases, as well as the PenR regulatory pathway, is otherwise lacking, with only PenA1, AmpC1, and PenR A from Burkholderia multivorans ATCC 17616 being well studied to date (13,(16)(17)(18). Adding to the complexity, the bla penA and bla ampC genes are highly heterogeneric among B. multivorans strains with PenA1 to PenA38 and AmpC1 to AmpC28 annotated thus far (whole-genome sequencing was conducted on 50 B. multivorans strains; of these 50 isolates, we identified 38 different PenAs and 28 different AmpCs by primary amino acid sequence comparisons) (16,17).…”

“…However, comprehensive characterization of these ␤-lactamases, as well as the PenR regulatory pathway, is otherwise lacking, with only PenA1, AmpC1, and PenR A from Burkholderia multivorans ATCC 17616 being well studied to date (13,(16)(17)(18). Adding to the complexity, the bla penA and bla ampC genes are highly heterogeneric among B. multivorans strains with PenA1 to PenA38 and AmpC1 to AmpC28 annotated thus far (whole-genome sequencing was conducted on 50 B. multivorans strains; of these 50 isolates, we identified 38 different PenAs and 28 different AmpCs by primary amino acid sequence comparisons) (16,17). The spectrum of activity of PenA2-38 and AmpC2-28 variants is under investigation.…”