Y. CHARRETIER¹, S. M. DIENE¹, D. BAUD¹, S. CHATELLIER², E. SANTIAGO-ALLEXANT³, A. VAN BELKUM⁴, G. GUIGON³, J. SCHRENZEL<sup>1

1</sup>Genomic Research Laboratory, Service of Infectious Diseases, Geneva University Hospitals, Geneva Switzerland, ²Innovation Unit, bioMérieux SA, La Balme Les Grottes, France, ³Innovation Unit, bioMérieux SA, Marcy l’Etoile, France, ⁴R&D Microbiology Unit, bioMérieux SA, La Balme Les Grottes, France

Multidrug-resistant Acinetobacter baumannii infection has recently emerged as a worldwide clinical problem and colistin is increasingly being used as a last-resort therapy. Despite its favorable bacterial killing property, resistance and heteroresistance to colistin has been described. The purpose of this study was to identify resistance mechanisms selected during short antimicrobial exposure and to evaluate the in vitro fate of colistin resistance after withdrawal of antibiotic pressure using global epidemic strains. Three clinical isolates representing global clones were selected and exposed to colistin at the MIC or above. Four resistant mutants were isolated in a one-step liquid-based culture. A second screening of the mutants with or without colistin in the culture medium helped to select phenotypic revertants. Targeted sequencing of the pmrCAB operon was performed to correlate phenotypic and genotypic resistance markers. Whole genome sequencing, population analysis profiling and quantitative RT-PCR were performed on a subset of strains. Use of high and low colistin concentrations allowed the selection of mutants resistant or heteroresistant to colistin. Each mutant showed at least one genetic change in either pmrA or pmrB. Population analysis profiling revealed heteroresistance. Whole genome sequencing demonstrated that mutations were exclusively found in pmrCAB. Quantitative RT-PCR demonstrated the diversity in pmrCAB regulation. Reversion by compensatory mutations was observed only in a single mutant through ISAba1 mobilization. Colistin resistance is easily and rapidly acquired in vitro and its fate appeared to be at least strain-dependent and probably clone-specific. This report demonstrates the involvement of mutations in pmrAB resulting in colistin (hetero)resistance.