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 {micro}g/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 (DedA of Pseudomonas necessary for colistin resistance A). Deletion of dpcA in BWH047 restored sensitivity to colistin (MIC = 0.5 {micro}g/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. Author summaryPseudomonas aeruginosa is a bacterium that causes a wide variety of infections. It is especially problematic given its propensity to become resistant to antibiotics. One antibiotic used to treat multidrug-resistant P. aeruginosa infections is colistin. In this study, we investigated colistin resistance mechanisms in a patient-derived, extremely phenotypically resistant P. aeruginosa isolate, BWH047, using transposon insertion sequencing and mass spectrometry. We identified 13 genes conditionally essential for colistin resistance and investigated the role of one of these genes, dpcA, on the composition of the bacterial outer membrane, the target of colistin. Additionally, our study identified novel colistin resistance genes residing in several intersecting pathways that could be targeted to prevent the development of antimicrobial resistance.