Colistin resistance inPseudomonas aeruginosathat is not linked toarnB

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Abstract

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-CstR exhibited higher survival rates during conditions of 1% HCl and 42 °C 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.

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