The evolution of colistin resistance in CF patients Colistin is a cyclic cationic antimicrobial peptide that is highly active against many Gram-negative bacteria, including Pseudomonas aeruginosa (P. aeruginosa). They exert their bactericidal activity through disruption of the cell membrane leading to leakage of cell contents and cell death. This is achieved at least in part by binding to lipopolysaccharide (LPS), a major component of the Gram negative cell surface, through interactions with phosphates and fatty acids of LPS core and lipid A moieties. The arnB operon is directly controlled by pmrAB, and the gene products are responsible for the synthesis of N4-aminoarabinose that binds to lipid A and reduces binding of colistin to LPS, whe-reby resistance arises. Colistin resistant P. aeruginosa have rarely been reported in cystic fibrosis (CF) patients. This is surprising because colistin is commonly used for inhalation and intravenous therapy in CF patients worldwide. A CF patient has chronically carried colistin sensitive mucoid P. aeruginosa for a long time. Sud-denly, 17 in November 2007 a colistin resistant non mucoid clone appeared alongside the primary mucoid clone in the patient’s sputum samples. A number of consecutive P. aeruginosa isolates has been obtained from this patient before and after the occurrence of the colostin resistance clone these events give us the opportunity to study the evolution of colistin resistance in the clinical set-ting. Aim of the project To characterize and quantify the evolution of colistin resistance in P.aeruginosa isolated from a CF patient in respect to growth physiology and response to different classes of antibiotics and eluci-date potential resistance mechanisms. Methodology Isolate independent clones of P.aeruginosa from consecutive patient samples from a chronically infected CF patient by classic microbiology techniques. Characterize the isolated clones with respect to growth physiology and antibiotic sensitivity to structurally different classes of antibiotics such as b-lactams, fluoroquinolones and aminoglyco-sides in addition to colistin. This will be performed by standard microbial growth experiments and MIC determination. Analyse whole genome sequencing data to identify the molecular mechanisms underlying the dif-ferent phenotypes. Characterize “recreated mutants” with the identified mutations with respect to growth physiology and antibiotic sensitivity with strains. Molecular characterization of known targets of antibiotic resistance in the isolated clones using cloning and sequencing. Contact lector Anders Folkesson, afol@vet.dtu.dk, DTU Vet, Bülowsvej 27, 1870 Frederiksberg C
Projects 14
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