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.
Highlights
Development and spread of antibiotic resistance by pathogenic microorganisms is an increasing healthcare problem
Axc expression led to an increase of minimal inhibitory concentrations for carbapenems when introduced in a susceptible Escherichia coli strain and direct carbapenemase activity of Axc was demonstrated using in vitro imipenem conversion assays
An antibiogram of a clinical isolate of Achromobacter xylosoxidans from a patient treated for pneumonia revealed a multi-resistant character, but it was susceptible to meropenem (Supplemental Table 1)
Summary
Development and spread of antibiotic resistance by pathogenic microorganisms is an increasing healthcare problem. The emergence and spread of carbapenemases such as class A KPC4, a number of metallo beta-lactamases[5,6] (class B: IMP, VIM, NDM) and class D oxacillinases such as OXA-487, in combination with other resistance mechanisms[8], can jeopardize carbapenem efficacy, leaving little or no treatment options for patients. A comparative genomic exploration of two A. xylosoxidans isolates revealed many genes that could be involved in drug resistance, such as efflux pumps and β-lactamases Most of these genes were conserved between carbapenem susceptible and resistant strains, highlighting the difficulty in translating genomic data to the observed resistant phenotypes[22]. Since PCR analysis was negative for known carbapenemases, we performed a proteomic analysis which revealed the novel beta-lactamase Axc as highly abundant in the meropenem-resistant, but not in the susceptible isolate. The resistant and the susceptible clinical isolates are genetically almost identical, emphasizing the importance of mass spectrometry as a technique to investigate carbapenem resistance in A. xylosoxidans
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