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Rapid identification and characterisation of drug-resistant bacterial pathogens have an important role in diagnostic and antimicrobial stewardship. Response time in the diagnosis of not only the etiological agent but also in antimicrobial susceptibility results is of utmost importance in patient treatment. In this study, matrix-assisted laser desorption ionisation–time of flight (MALDI-TOF) mass spectrometry (MS) was used to screen for biomarkers of ESCAPE (vancomycin-resistant   E   nterococcus faecium , methicillin-resistant   S   taphylococcus aureus , hypervirulent NAP1/ribotype 027  Clostridioides  [ Clostridium ]  difficile , multidrug resistant   A   cinetobacter baumannii , multidrug resistant   P   seudomonas aeruginosa , and carbapenem-resistant   E   nterobacteriaceae ) pathogens to predict antimicrobial resistance or hypervirulence. Several biomarkers of drug-resistant genotypes in  S. aureus, A. baumannii, P. aeruginosa , and  K. pneumoniae , as well as hypervirulence in  C. difficile , were detected. The fastest possible susceptibility testing with MALDI-TOF MS is simultaneous detection of a characteristic drug-resistant peak and species identification in the same spectra generated in routine processing. According to our approach, resistance or virulence biomarker peaks can be identified while performing routine microbiology analysis, and no additional assays nor prolonged incubation time is needed. Outstanding biomarker peaks detected in our study should be further analysed by additional methods to identify the specific proteins involved.

Screening of biomarkers of drug resistance or virulence in ESCAPE pathogens by MALDI-TOF mass spectrometry