RESEARCH ARTICLE The cost of resistance to colistin in Acinetobacter baumannii: a proteomic perspective María Fernández-Reyes 1 * , Manuel Rodríguez-Falcón 2 * , Cristina Chiva 2 , Jerónimo Pachón 3 , David Andreu 2 and Luis Rivas 1 ** 1 Center for Biological Research (CIB-CSIC), Madrid, Spain 2 Proteomics Unit, Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Barcelona, Spain 3 Services of Infectious Diseases, Virgen del Rocío University Hospitals, Sevilla, Spain Colistin resistance in Acinetobacter baumannii, a pathogen of clinical concern, was induced in the susceptible strain ATCC 19606 by growth under increasing pressure of the antibiotic, the only drug universally active against multi-resistant clinical strains. In 2-D difference gel electropho- resis (DIGE) experiments, 35 proteins with differences in expression between both phenotypes were identified, most of them appearing as down regulated in the colistin-resistant strain. These include outer membrane (OM) proteins, chaperones, protein biosynthesis factors, and metabolic enzymes, all suggesting substantial loss of biological fitness in the resistant phenotype, as sub- stantiated by complementary experiments in the absence of colistin. Results shed light on the scarcity of widespread clinical outbreaks for resistant phenotypes. Received: May 17, 2008 Revised: September 28, 2008 Accepted: October 13, 2008 Keywords: Acinetobacter baumannii / Antibiotic resistance / Biological fitness / Colistin / Poly- myxin 1632 Proteomics 2009, 9, 1632–1645 1 Introduction The successful thriving of bacteria in the antibiotic era has set off an alarm situation in the therapeutic community, as the antimicrobial armamentarium against many species of clinical importance is nearing exhaustion. These disquieting prospects, however, must be offset against the fact that induction of resistance entails an additional metabolic bur- den to the resistant organism, frequently resulting in loss of fitness and thus limited dissemination in the absence of antibiotic [1]. Successful microbes, on the other hand, over- come this problem through compensating and/or free-cost mutations, together with allelic exchange between suscep- tible and resistant strains [2]. This scenario is particularly true for antibiotics with a well-defined single target, but the situation becomes more complicated for those with not fully defined and possibly multiple targets, such as colistin (poly- myxin E). Polymyxins (PMXs), a family of cyclic decapeptides pro- duced by Bacillus polymyxa, have an N-terminal fatty acid chain and over several (.50%) 2,4-diaminobutyric acid resi- dues conferring them, respectively, the amphipathicity and cationic character that underlie their antibiotic properties [3, 4]. PMXs show strong bactericidal activity on Gram nega- tives, together with anti-endotoxic activity [5, 6]. Since cyto- toxicity precluded systemic administration of PMXs, they were reserved for topical applications while other antibiotics with better toxicity profiles were promoted. However, as the efficacy of the latter was again eroded by the appearance of Correspondence: Professor David Andreu, Department of Experi- mental and Health Sciences, Pompeu Fabra University, Barce- lona Biomedical Research Park, Dr. Aiguader 88, 08003 Barce- lona, Spain E-mail: david.andreu@upf.edu Abbreviations: OM, outer membrane; PMXs, polymyxins; SRP, signal recognition particle * These authors contributed equally to this work. ** Additional corresponding author: Dr. Luis Rivas; E-mail: luis.rivas@cib.csic.es DOI 10.1002/pmic.200800434  2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com