Colistin resistance in pseudomonas aeruginosa that is not linked to arnB

Purpose. It is known that the arnB (or pmrH) gene encoding uridine 5’-(beta-1-threo-pentapyranosyl-4-ulose diphosphate) aminotransferase plays a critical role in colistin resistance in Pseudomonas aeruginosa through the addition of 4-amino-4- deoxy-L-arabinose (L-Ara4N) to lipid A. In this study, we attempted to obtain a colistin-resistant mutant from an arnB-deleted mutant through exposure to colistin. Methodology. We constructed an arnB deletion mutant (P5ΔarnB :: nptIII) from a colistin-susceptible strain (P5) by allelic replacement mutagenesis, and colistin-resistant mutants were selected in vitro using P5 and P5ΔarnB :: nptIII. The growth rate, lipid A structure, biofilm-forming activity and cell viability in diverse stressful conditions (osmotic, oxidative, acidic and heat stress) were investigated. Expression of phoP, pmrA, parR, and cprR was evaluated by qRT-PCR. Results. An arnB deletion mutant was shown to develop colistin resistance through the addition of L-Ara4N to lipid A, despite a low survival rate (over 1000-fold lower than that of the wild-type strain) in the media with colistin. Two colistin-resistant mutants showed higher survival rates than colistin-susceptible strains against 5% NaCl. In the presence of acidic and heat stress, P5ΔarnB :: nptIII-Cst^R exhibited higher survival rates during conditions of 1% HCl and 42 ºC than the other strains. Both phoP and pmrA genes were overexpressed significantly in both colistin-resistant mutants, but parR and cprR genes were not. Conclusion. We revealed that colistin resistance could be developed despite arnB deletion in P. aeruginosa through the addition of L-Ara4N to lipid A, which was accompanied by diverse physiological changes.