Transposon insertion sequencing of Pseudomonas aeruginosa identifies multiple intersecting pathways essential for extreme colistin resistance

Colistin is used to treat antibiotic resistant gram-negative infections, including those caused by Pseudomonas aeruginosa ( Pa ). Using a diverse collection of clinical isolates, we identified BWH047, a colistin-resistant isolate with an extremely high minimum inhibitory concentration (MIC, 1280 µg/mL). To characterize the genes conditionally essential for colistin resistance in BWH047, we employed transposon insertion sequencing and identified 20 gene candidates. In-frame deletion validated 75% of the candidates and identified genes in several novel pathways that contribute to colistin resistance, including algU and wapH . We also identified several candidate genes from previously reported colistin resistance pathways (e.g. arn , pmrAB ). We further investigated the impact of a colistin resistance-associated inner membrane DedA-family undecaprenyl phosphate flippase, which we named DpcA ( D edA of P seudomonas necessary for c olistin resistance A ). Deletion of dpcA in BWH047 restored sensitivity to colistin (MIC = 0.5 µg/mL) and resulted in several unique changes to the structure of lipopolysaccharide (LPS), including production of decreased amounts of the colistin resistance-conferring 4-amino-4-deoxy-L-arabinose (L-Ara4N) modification on lipid A. To date, this work represents the most complete analysis of colistin resistance in Pa and identifies novel intersecting pathways that contribute to extreme phenotypic resistance.