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Antimicrobial resistance is a major public-health concern. We evaluate chlorhexidine role in selection of resistant Pseudomonas aeruginosa mutants and their antibiotic cross-resistance. Mutation frequency and mutation rate after short-term exposure to sub-inhibitory concentrations of chlorhexidine were compared to those after spontaneous chlorhexidine-exposure, in P. aeruginosa PAO1 strain. Chlorhexidine-resistant mutants were generated, either by serial passage in increasing chlorhexidine concentrations or by single exposure to lethal