Proteomic identification of Axc, a novel beta-lactamase with carbapenemase activity in a meropenem-resistant clinical isolate of Achromobacter xylosoxidans

Abstract

The development of antibiotic resistance during treatment is a threat to patients and their environment. Insight in the mechanisms of resistance development is important for appropriate therapy and infection control. Here, we describe how through the application of mass spectrometry-based proteomics, a novel beta-lactamase Axc was identified as an indicator of acquired carbapenem resistance in a clinical isolate of Achromobacter xylosoxidans. Comparative proteomic analysis of consecutively collected susceptible and resistant isolates from the same patient revealed that high Axc protein levels were only observed in the resistant isolate. Heterologous expression of Axc in Escherichia coli significantly increased the resistance towards carbapenems. Importantly, direct Axc mediated hydrolysis of imipenem was demonstrated using pH shift assays and ¹H-NMR, confirming Axc as a legitimate carbapenemase. Whole genome sequencing revealed that the susceptible and resistant isolates were remarkably similar. Together these findings provide a molecular context for the fast development of meropenem resistance in A. xylosoxidans during treatment and demonstrate the use of mass spectrometric techniques in identifying novel resistance determinants.

Figure 1

Comparative proteomic analysis of meropenem resistant and susceptible Achromobacter xylosoxidans clinical isolates. (A) Tryptic digests of protein extracts of the meropenem resistant (AchroR, Table 1) and susceptible (AchroS, Table 1) isolate were analysed by LC-MS/MS. Spectra were assigned to peptides based on database searching. Identified spectra were then assigned to the corresponding proteins and the total number of spectra per protein were counted. Each circle represents one protein with the number of spectra observed in the resistant and the susceptible isolate. Hence, proteins on the diagonal were observed in similar counts in both isolates. Axc (arrow), a classA PenP-family beta-lactamase, is the most prominent outlier. See also Table S2 and Supplemental Fig. S2. (B) The full amino acid sequence of Axc, with the peptides identified by LC-MS/MS analysis underlined. Conserved residues from serine beta-lactamases, Ser-X-X-Lys, Ser-Asp-Asn and the active site Glu, are in bold. (C) Extracted ion chromatograms of m/z values corresponding to tryptic peptides of Axc in the meropenem resistant isolate (AchroR, upper panel) and susceptible isolate (AchroS, lower panel). The corresponding tryptic peptides are indicated above the corresponding peaks.

Figure 2

Genomic context of axc in Achromobacter xylosoxidans strains and comparison of Axc with other class A beta-lactamases (A) Axc (a putative PenP class A beta-lactamase, 1.17 e-54) and the gene encoding its putative transcriptional repressor (axcR), were found in both clinical isolates (AchroS and AchroR, Table 1). Three other fully sequenced genomes of Achromobacter xylosoxidans were examined for the presence of axc; NH44784_1996 (ENA assembly GCA_000967095.1), C54 (ENA assembly GCA_000758265.1) and NBRC_15126/ATCC27061 (ENA assembly GCA_000508285.1). Only within the strain NH44784_1996 (used as a reference to search our proteomics data), axc and the putative regulator are also present. (B) Unrooted cladogram obtained for 176 class A beta-lactamases including Axc. The class A β-lactamase protein sequences of Gram negative bacteria were obtained by querying the refseq_protein database using Blastp (<1e-10, http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi) and a consensus β-lactamase-alignment. Duplicate sequences and sequences causing a strong overrepresentation of branches produced in the tree were removed. Names of known carbapenames (orange) and names of identifier of branches are indicated. Axc is indicated with a red dot. The closest homologue to Axc, a β-lactamase from R. saidenbachensis (WP_029709665), is also indicated.

Figure 3

Axc has carbapenemase activity. (A) Axc was expressed in E. coli and cell extracts were tested for the ability to hydrolyse imipenem. Bars show the A431/A560 ratio, which is a measure for the shift in pH due to imipenem hydrolysis. (B) NMR-based identification of imipenem hydrolysis by Axc. The structure of imipenem is shown, with the proton numbering used in the spectrum. The line color indicates the incubation time (red, 10 min at room temperature or black, 10 hrs at 6 °C). Imipenem hydrolysis is accompanied by the loss of the H-6 multiplet at 3.4 ppm, and a shift in the H-9 doublet, resulting in a decrease of the doublet at 1.3 ppm. Strains used (see also Table 1): KPC (JC113): Carbapenem resistant Klebsiella pneumoniae. E. coli Axc (JC107): E. coli strain C43(DE3), containing plasmid pET21-Axc; E. coli_control (JC108): E. coli strain C43, containing plasmid pET21-PPEP-1 (Pro-Pro endopeptidase 1). IPTG: isopropyl β-D-1-thiogalactopyranoside.