Rapid MRSA detection via tandem mass spectrometry of the intact 80\xa0kDa PBP2a resistance protein

Christopher Mullen,Johan Finell,William M Mcgee,Joni Rannisto,Jason R Neil,Arvind Verma,Marjaana Viirtola,Annika Kotovuori,Scott R Kronewitter,James L Stephenson,John E P Syka,Herdis Friedrich

doi:10.1038/s41598-021-97844-w

Abstract

Treatment of antibiotic-resistant infections is dependent on the detection of specific bacterial genes or proteins in clinical assays. Identification of methicillin-resistant Staphylococcus aureus (MRSA) is often accomplished through the detection of penicillin-binding protein 2a (PBP2a). With greater dependence on mass spectrometry (MS)-based bacterial identification, complementary efforts to detect resistance have been hindered by the complexity of those proteins responsible. Initial characterization of PBP2a indicates the presence of glycan modifications. To simplify detection, we demonstrate a proof-of-concept tandem MS approach involving the generation of N-terminal PBP2a peptide-like fragments and detection of unique product ions during top-down proteomic sample analyses. This approach was implemented for two PBP2a variants, PBP2amecA and PBP2amecC, and was accurate across a representative panel of MRSA strains with different genetic backgrounds. Additionally, PBP2amecA was successfully detected from clinical isolates using a five-minute liquid chromatographic separation and implementation of this MS detection strategy. Our results highlight the capability of direct MS-based resistance marker detection and potential advantages for implementing these approaches in clinical diagnostics.

Highlights

Treatment of antibiotic-resistant infections is dependent on the detection of specific bacterial genes or proteins in clinical assays
In an expansion of our effort to characterize and develop mass spectrometry (MS)-based resistance marker detection approaches, we propose a targeted method for direct detection of penicillin-binding protein 2a (PBP2a) through the implementation of intact protein liquid chromatographic (LC) separation and high-resolution MS for methicillin-resistant Staphylococcus aureus (MRSA) identification
Confirmation of the wild-type target was observed in LC separated MRSA samples subject to gas phase ion-ion proton-transfer charge reduction (PTCR) and MS detec

Summary

Introduction

Treatment of antibiotic-resistant infections is dependent on the detection of specific bacterial genes or proteins in clinical assays. In an expansion of our effort to characterize and develop MS-based resistance marker detection approaches, we propose a targeted method for direct detection of PBP2a through the implementation of intact protein liquid chromatographic (LC) separation and high-resolution MS for MRSA identification. The approach to PBP2amecA detection, in the absence of induction, is scaled down to a five-minute LC–MS/MS method to detect smaller fragments of the intact protein and validated with clinical patient and quality control (QC) MRSA and MSSA strains These results highlight the advantage of antibiotic resistance identification through direct detection of those proteins responsible, has the potential to improve and automate clinical workflows, providing a level of MS-based discrimination not previously available in this area of disease management

Methods

Results

Conclusion