MALDI-TOF MS is becoming the technique of choice for rapid bacterial identification at species level in routine diagnostics. However, some drawbacks concerning the identification of closely related species such as those belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex lead to high rates of misidentifications. In this work we successfully developed an approach that combines MALDI-TOF MS and chemometric tools to discriminate the six Acb complex species (A. baumannii, Acinetobacter nosocomialis, Acinetobacter pittii, A. calcoaceticus, genomic species "Close to 13TU" and genomic species "Between 1 and 3"). Mass spectra of 83 taxonomically well characterized clinical strains, reflecting the breadth of currently known phenetic diversity within the Acb complex, were achieved from intact cells and cell extracts and analyzed with hierarchical cluster analysis (HCA) and partial least squares discriminant analysis (PLSDA). This combined approach lead to 100% of correct species identification using mass spectra obtained from intact cells. Moreover, it was possible to discriminate two Acb complex species (genomic species "Close to 13TU" and genomic species "Between 1 and 3") not included in the MALDI Biotyper database.

Departamento de Tecnologia Farmacêutica Faculdade de Farmácia Universidade de Lisboa Av Prof Gama Pinto 1649 003 Lisboa Portugal

Laboratory of Bacterial Genetics National Institute of Public Health Prague Czech Republic

REQUIMTE Laboratório de Microbiologia Departamento de Ciências Biológicas Faculdade de Farmácia Universidade do Porto Porto Portugal

REQUIMTE Laboratório de Química Aplicada Departamento de Ciências Químicas Faculdade de Farmácia Universidade do Porto Porto Portugal

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