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

William M McGee; Arvind Verma; Marjaana Viirtola; Scott R Kronewitter; Jason R Neil; James L Stephenson Jr

doi:10.1016/j.jmsacl.2021.05.001

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

J Mass Spectrom Adv Clin Lab. 2021 May 29:20:25-34. doi: 10.1016/j.jmsacl.2021.05.001. eCollection 2021 Apr.

Authors

William M McGee¹, Arvind Verma², Marjaana Viirtola², Scott R Kronewitter¹, Jason R Neil¹, James L Stephenson Jr¹

Affiliations

¹ Thermo Fisher Scientific, Cambridge, MA, USA.
² Thermo Fisher Scientific, Vantaa, Finland.

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.