Infectious diseases caused by multidrug-resistant (MDR) Enterobacteriaceae have exponentially increased in the past decade, and are a major concern in hospitals. In the first part of the work, we compared the proteome profile of MDR and susceptible clinical isolates of Escherichia coli and Klebsiella pneumoniae in order to identify possible biological processes associated with drug resistance and susceptible phenotypes, using a label-free approach. In the second part, we used an immunoproteomics approach to identify immunoreactive proteins in the same isolates. A total of 388 and 377 proteins were identified in MDR and susceptible E. coli, respectively, evidencing that biological processes related to translation are upregulated in E. coli MDR, while there is an upregulation of processes related to catalytic activity in K. pneumoniae MDR. Both MDR strains show downregulation of processes related to amino acid activation and tRNA amino-acylation. Our data also suggest that MDR strains have higher immunoreactivity than the susceptible strains. The application of high-throughput mass spectrometry (MS) and bioinformatics to the study of modulation of biological processes might shed light on the characterization of multidrug resistance in bacteria. (C) 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.