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Antimicrobial resistance (AMR) is one of the biggest public health concerns of our time [1]. Currently, resistance has been reported for almost all available antibiotics, from first-line to last-resort drugs [2]. AMR restricts treatment options, leads to increased morbidity and mortality of hospitalized patients and imposes a substantial financial burden on healthcare systems worldwide [3]. The limited pipeline of new antimicrobials and the continuous emergence of multidrug-resistant (MDR) organisms needs to be countered by novel antibacterial strategies coupled with rapid diagnostics to detect resistance, particularly in the case of broad-acting agents such as β-lactams and polymyxins. Traditionally, antimicrobial susceptibility testing is performed using culture-based methods, like minimum inhibitory concentration (MIC) determination assays, a lengthy process that is prone to misleading results, for example, in the case of clinical isolates that produce large amounts of capsule or biofilm. By contrast, polymerase-chain-reaction (PCR)-based tests like multiplex PCR allow the rapid identification of specific resistance factors but cannot predict susceptibility and are entirely insensitive to uncharacterized mechanisms of resistance. At the same time, despite the extensive progress of genotypic sequence-based approaches, such as long-read nanopore sequencing, these cannot yet be used as first-line diagnostics in part because genotype-to-phenotype predictions remain challenging [4,5]. With matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry being used routinely for bacterial identification in clinical settings, a range of MALDI-TOF-based diagnostics for the rapid detection of AMR are starting to be implemented [6,7]. More specifically, it is possible to discriminate some lineages of methicillin-resistant Staphylococcus aureus, identify certain strains of enterobacteria carrying plasmid-encoded βlactamases from the Klebsiella pneumoniae carbapenemase (KPC) family of enzymes, and detect Bacteroides fragilis strains that are resistant to β-lactams at the same time as performing bacterial identification. Alongside these identification-based approaches, efforts to identify both aminoglycoside and quinolone resistance using MALDI-TOF mass spectrometry have also been made. As resistance to these agents can arise through enzymatic modification of the antibiotic, this modification can be directly detected following incubation of the drug of interest with resistant bacteria [8,9]. Nonetheless, probably the most successful application of MALDI-TOF mass spectrometry for AMR detection to date has been functional screening for βlactamase activity. Bacteria are mixed with a β-lactam compound, and an increase in the PLOS PATHOGENS

The clue is in the lipid A: Rapid detection of colistin resistance