Direct detection of OXA-48-like carbapenemase variants with and without co-expression of an extended-spectrum β-lactamase from bacterial cell lysates using mass spectrometry

Abstract

Introduction: Antibiotic-resistant Gram-negative bacteria are of a growing concern globally, especially those producing enzymes conferring resistance. OXA-48-like carbapenemases hydrolyze most β-lactam antibiotics, with typically low-level hydrolysis of carbapenems, but have limited effect on broad-spectrum cephalosporins. These are frequently co-expressed with extended spectrum β-lactamases, especially CTX-M-15, which typically shows high level resistance to broad-spectrum cephalosporins, yet is carbapenem susceptible. The combined resistance profile makes the need for successful detection of these specific resistance determinants imperative for effective antibiotic therapy.

Objectives: The objective of this study is to detect and identify OXA-48-like and CTX-M-15 enzymes using mass spectrometry, and to subsequently develop a method for detection of both enzyme types in combination with liquid chromatography.

Methods: Cells grown in either broth or on agar were harvested, lysed, and, in some cases buffer-exchanged. Lysates produced from bacterial cells were separated and analyzed via liquid chromatography with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS).

Results: The intact proteins of OXA-48, OXA-181, and OXA-232 (collectively OXA-48-like herein) and CTX-M-15 were characterized and detected. Acceptance criteria based on sequence-informative fragments from each protein group were established as confirmatory markers for the presence of the protein(s). A total of 25 isolates were successfully tested for OXA-48 like (2), CTX-M-15 (3), or expression of both (7) enzymes. Thirteen isolates served as negative controls.

Conclusions: Here we present a method for the direct and independent detection of both OXA-48-like carbapenemases and CTX-M-15 β-lactamases using LC-MS/MS. The added sensitivity of MS/MS allows for simultaneous detection of at least two co-eluting, co-isolated and co-fragmented proteins from a single mass spectrum.

Keywords: ATCC, American Type Culture Collection; Antimicrobial-resistant organisms; CDC, Centers for Disease Control and Prevention; CPO, carbapenemase-producing organism; CRE, carbapenem-resistant Enterobacterales; CSD, charge state distribution; CTX-M-15; Carbapenem-resistant Enterobacterales; Carbapenemase; Carbapenemase-producing organisms; ESBL, extended-spectrum β-lactamase; ESI, electrospray ionization; LC, liquid chromatography; Liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MW, molecular weight; Mass Spectrometry; OXA-48; OXA-48-like; PCR, polymerase chain reaction; TOF, time-of-flight (mass spectrometry); Tandem mass spectrometry; m/z, mass-to-charge ratio; β-Lactamase.

Fig. 1

Mass spectra of intact OXA-48-like proteins in m/z and MW space. Spectra of intact protein elutions for m/z 650–1300 correspond to a) OXA-48, c) OXA-181, and e) OXA-232. Molecular weights of b) OXA-48, d) OXA-181, and f) OXA-232 as well as additional co-eluting proteins were calculated through deconvolution of the corresponding mass spectra. The data is from Klebsiella ozaenae AR Bank #0051 (OXA-181 and CTX-M-15), K. pneumoniae AR Bank #0066 (OXA-232), and Enterobacter aerogenes AR Bank #0074 (OXA-48) of the Gram-Negative Carbapenemase Detection Panel (CDC and FDA AR Isolate Bank).

Fig. 2

Fragmentation of a) +32, b) +33, c) +34, d) +35, and e) +36 charge states of OXA-48, with selected shared fragments highlighted in blue. Data from E. aerogenes AR Bank #0074.

Fig. 3

Sequence information provided by fragment ions for each charge state with set acceleration voltages for charges +32 through +36. Each charge state has a unique symbols, with blue representing b-ions and red representing y-ions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Comparison of fragmentation spectra of the +33 charge state between a) OXA-48 (E. aerogenes AR Bank #0074), b) OXA-181 (K. ozaenae AR Bank #0051) and c) OXA-232 (K. pneumoniae AR Bank #0066), with selected fragment ions highlighted in blue.

Fig. 5

Mass spectra of CTX-M-15, with a) the charge state distribution of the intact protein, with inset showing the deconvoluted MW of all proteins present in the spectrum from 10,000 to 40,000 Da, and b) the fragmentation spectrum of the +33 charge state, with selected fragment ions highlighted in red. It should be noted that the intensity scale for b) has been set to 0.5 from 1.0, wherein the y₆₃⁷⁺ and y₆₃⁺⁶ ion intensities correspond to 1.0 (100%) and 0.92 (92%), respectively. Data from K. ozaenae AR Bank #0051. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Isotopic distribution and overlap of the +33 charge states of OXA-48-like variants with CTX-M-15, from separate bacterial isolates. Data from K. ozaenae AR Bank #0051 (OXA-181 and CTX-M-15), K. pneumoniae AR Bank #0066 (OXA-232), and E. aerogenes AR Bank #0074 (OXA-48).

Fig. 7

Detection of both OXA-181 and CTX-M-15 using LC-MS/MS. a) Extracted ion chromatogram (EIC) of OXA-181, with b) MS/MS spectrum of OXA-181; c) EIC of CTX-M-15 with d) MS/MS spectrum. Selected OXA-181 fragments are highlighted in blue, while CTX-M-15 fragments are highlighted in red. Data from K. ozaenae AR Bank #0051.

Fig. 8

Fragmentation spectrum of both OXA-181 and CTX-M-15 following co-isolation and co-fragmentation. Fragments corresponding to OXA-181 are highlighted in blue, while those corresponding to CTX-M-15 are highlighted in red. Isotopic distributions of individual selected fragments are shown in the lower panel. Data from K. pneumoniae AR Bank #0140. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)