ORIGINAL ARTICLE Direct antimicrobial susceptibility testing from the blood culture pellet obtained for MALDI-TOF identification of Enterobacterales and Pseudomonas aeruginosa J. M. López-Pintor 1 & C. Navarro-San Francisco 1,2 & J. Sánchez-López 1 & A. García-Caballero 1 & E. Loza Fernández de Bobadilla 1,2 & M. I. Morosini 1,2 & R. Cantón 1,2 Received: 5 December 2018 /Accepted: 27 January 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract To standardize the methodology for conducting direct antimicrobial susceptibility testing (AST) of Enterobacterales and Pseudomonas aeruginosa causing bacteremia from positive blood culture pellets. Two methods for processing positive blood cultures with Enterobacterales and P. aeruginosa were compared: a conventional method for identification and AST versus a direct method obtaining a pellet for both matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) identification and direct AST. A total of 157 (145 Enterobacterales, 12 P. aeruginosa) positive blood cultures were included. Microorganism identification showed 100% concordance between both methods at species and genus level. Definitive AST results were obtained 24 h earlier with the rapid method than the conventional one (p < 0.001). Of the 2814 MICs generated, there were discrepancies with respect to the conventional method in 47 (1.7%), 0.3% being very major (VME) and 1.3% major (ME) errors. Better results for AST were obtained when colony counts with the pellet were ≥ 10 5 cfu/ml. The essential agreement (EA) for antibiotics tested in Enterobacterales was at least 97%, except for ampicillin (95%). Regardless of colony count, the greatest discrepancies were observed for first/s-generation cephalosporins and aminoglycosides. In P. aeruginosa, EA was at least 92%, except for piperacillin-tazobactam (84%) and cefepime (76%). No VME occurred except for ceftazidime (8%). ME occurred in piperacillin/tazobactam (16%), ticarcillin, ceftazidime, tobramycin, amikacin, and colistin (8% each). Direct use of the blood culture pellet permits fast AST in bacteremia of Enterobacterales, enabling the clinicians to perform an early treatment adjust- ment. However, for Pseudomonas aeruginosa, the data needs expanding to improve the reliability of this technique. Keywords Bacteraemia . Gram-negative bacilli . Blood culture pellet . Direct antimicrobial susceptibility testing . MALDI-TOF Introduction A key function of Clinical Microbiology laboratories is to obtain accurate and timely antimicrobial susceptibility testing (AST) data, especially in sepsis or critically ill patients. Bloodstream infections (BSI) are the main cause of morbidity and mortality in hospitalized patients and timely start of ap- propriate antibiotics has an important impact on patient out- come. A reduction of this timeline to less than one working day is very useful, reducing morbidity and mortality as well as the overall costs for healthcare systems [1]. Moreover, accel- erating the AST has a great impact on patient therapy and is also critical for the overall success of health care institutions’ antimicrobial stewardship programs, particularly in critically ill patients [2–4]. In the hospital setting, microorganisms are subjected to a high antibiotic pressure, resulting in multidrug- resistant pathogens which imply great difficulties for their correct treatment [5]. Different methods to obtain earlier AST results than with current standard methods are under development. Both EUCAST and CLSI are developing new clinical breakpoints Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10096-019-03498-y) contains supplementary material, which is available to authorized users. * C. Navarro-San Francisco carolinansf@yahoo.es 1 Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Carretera Colmenar Viejo Km 9, 1, 28034 Madrid, Spain 2 Red Española de Investigación en Patología Infecciosa (REIPI), Instituto de Salud Carlos III, Madrid, Spain European Journal of Clinical Microbiology & Infectious Diseases https://doi.org/10.1007/s10096-019-03498-y