can develop resistance to polymyxin as a consequence of mutations in the PhoPQ regulatory system mediated by covalent lipid A modification. in polymyxin resistance. Surprisingly tandem deletion of or in the Δmutant or individual deletion of or failed to suppress 4-amino-l-arabinose addition to lipid A indicating that this Tg modification alone is not sufficient for PhoPQ-mediated polymyxin resistance in or in tandem or of individually complemented the Pm resistance phenotype in the Δmutant while episomal expression of individually did not. Highly polymyxin-resistant mutants of isolated from polymyxin-treated cystic fibrosis patients harbored mutant alleles of and background these mutant alleles enhanced polymyxin resistance. These results define ColRS and CprRS as two-component systems regulating polymyxin resistance in and mutations can contribute to high-level clinical resistance. INTRODUCTION The polymyxins (Pm) a family of cyclic oligopeptides with activity against and other Gram-negative pathogens are increasingly important in the treatment of invasive infections in critically ill patients and airway infections in those with cystic fibrosis (CF) (1 2 First-line treatment of these infections often involves intravenous or inhaled combinations of antipseudomonal beta-lactams aminoglycosides fluoroquinolones and other agents. Repeated use of these first-line agents imposes selection pressure leading to multidrug-resistant strains of (3-5). When this occurs the clinically available forms of Pm namely Pm B sulfate (PMB) and colistimethate the prodrug form of colistin (CST) (also known as Pm E) become key components of second-line regimens. Pm binds to lipopolysaccharide (LPS) the major constituent of the Gram-negative outer membrane promoting membrane permeabilization and diffusion of peptide through the periplasm to the inner membrane where Pm insertion disrupts cellular respiration and results in cell lysis (6). Unfortunately the prevalence of Pm-resistant (Pmr) clinical strains of and other Gram-negative pathogens is increasing (7-13); such strains are generally resistant to both PMB and CST. At a biochemical level Pm resistance of and other Gram-negative pathogens is strongly associated with covalent modification of LPS most specifically with the addition of 4-amino-l-arabinose (l-Ara4N) to the phosphate groups of its lipid A and core oligosaccharide components (14-16). Genes in the operon encode enzymes responsible for synthesis and transfer of l-Ara4N to LPS (17 18 This amino-sugar modification is thought to hinder charge interactions between phosphate groups within LPS and amino groups within the cyclic Pm oligopeptide. In contrast to their hierarchical regulation of Pm resistance in regulate Pm resistance convergently at least in part by activating transcription of the operon in response to antimicrobial peptide exposure or divalent cation depletion (17-20) or as a consequence of mutation (13 16 21 Recently the ParRS two-component system has also been found to play a role in Pm resistance in (26 27 We LY2484595 hypothesized that additional regulatory systems interact with these known two-component systems to modulate Pm resistance and that mutations in such systems might contribute to high-level clinical resistance. The primary objective of this study was to identify additional regulatory systems contributing to PhoPQ-mediated Pm resistance in highly resistant clinical strains; a secondary objective was to define loci encoding additional structural elements required for LY2484595 PhoPQ-mediated Pm resistance. MATERIALS AND METHODS Bacterial strains and growth conditions. Laboratory strains and clinical isolates of used in this study are listed in Table 1. Clinical isolates were from the sputum of patients followed in the CF clinic at Rigshospitalet Copenhagen Denmark; the Institutional Review Board of Massachusetts General LY2484595 Hospital reviewed and approved their use in this study. DH5α was used as a host for manipulation of recombinant plasmids. and were grown at 30°C or 37°C on lysogeny agar (LA) plates or in lysogeny broth (LB) with aeration. Antibiotics were used at the following concentrations for selection and maintenance of plasmids: 50 mg/liter kanamycin or 10 mg/liter gentamicin (GEN) for DH5α and 50 to 100 mg/liter GEN for PAK and its derivatives. Strains were stored at ?80°C in LB supplemented with 16% glycerol. Table 1 Strains LY2484595 of used in this work Molecular methods. Bacterial plasmids were isolated using the QIAprep spin kit (Qiagen Valencia CA) and.