Early targeted antimicrobial therapy helps decrease costs and prevents the spread of antimicrobial resistance, including in Escherichia coli, the most frequent Gram-negative bacterium that causes sepsis. Therefore, rapid susceptibility testing represents the major prerequisite for knowledge-based successful antimicrobial treatment. To accelerate testing for antibiotic susceptibility, we have developed a new mass spectrometry-based assay for antibiotic susceptibility testing (MAAST). For proof of principle, we present an ampicillin susceptibility test for E. coli with a turnaround time of 90 min upon growth detection. Immediate empirically based broad-spectrum antibiotic therapy is a cornerstone in treatment of sepsis (8, 9). However, the early de-escalation of broad-spectrum antibiotic therapy based on knowledge of microbial identification and susceptibility not only reduces therapeutic costs (1, 2, 12) but also reduces the spread of antimicrobial resistance (3,5,10).Unfortunately, the turnaround time (TAT) of classical antibiotic susceptibility testing (AST) based on microbial growth inhibition is 7.5 to 14 h for direct AST from blood culture (BC) fluid or 31.5 to 36 h for indirect AST from subcultured bacteria (4). We have developed a mass spectrometry (MS)-based assay for antibiotic susceptibility testing (MAAST) that allows us to provide meaningful health care information on AST results in less than 90 min after primary microbial growth is detected in a BC that has turned positive.MAAST is based on the detection, identification, and quantification of antibiotics and their corresponding metabolization/ inactivation products as generated by ␤-lactamases. The feasibility of this test principle for the detection of ampicillin susceptibility in Escherichia coli is based on the hydrolysis of ampicillin by ␤-lactamases into ampicillin-penicilloic acid. The hydrolysis of ampicillin results in a mass shift of ampicillin from m/z 350 Da to m/z 368 Da, which allows easy detection of all compounds by MS (Fig. 1A). To increase the specificity of the assay, compounds were quantified after identification by collision-induced dissociation (CID) into compound-specific dissociation products.As known from drug monitoring, multiple compounds can be separated by high-pressure liquid chromatography (HPLC) and quantified by calculation of the peak area of the separated compounds. The combination of HPLC and mass spectrometry (LC-MS) allows simultaneous detection, identification, and quantification of ampicillin and metabolites (Fig. 1B). HPLC separation was performed using an Agilent series 1100 LC system (Agilent Technologies, Germany) with a Zorbax Eclipse XDB-C 18 column (Agilent Technologies) and a nonisocratic mobile phase consisted of a continuous gradient of 4 mM ammonium formiate and 90% methanol at 60°C. For MS detection, we used an HCT Ultra mass spectrometer (Bruker Daltonics) with an electrospray ionization (ESI) interface. Nitrogen served as a nebulizer and drying gas. Argon was used for CID. Mass spectrometric data w...

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