The Genetic Background and Culture Medium Only Marginally Affect the In Vitro Evolution of Pseudomonas aeruginosa Toward Colistin Resistance

Abstract

Background/Objectives: Colistin is a last-resort treatment for Pseudomonas aeruginosa multidrug-resistant infections, but resistance to it is emerging. While colistin resistance in P. aeruginosa is typically associated with chromosomal mutations inducing lipopolysaccharide (LPS) aminoarabinosylation, other mutations unrelated to LPS modifications have been proposed to influence the extent of colistin resistance. Here, we examined whether the genetic background and culture conditions affect the evolution of high-level colistin resistance in this bacterium. Methods: We performed in vitro evolution experiments in the presence or absence of increasing colistin concentrations with two phylogenetically distant reference strains in a standard laboratory medium and in two media mimicking P. aeruginosa growth during lung or systemic infections. Resistance-associated mutations were identified by comparative genomics, and the role of selected mutated genes was validated by allele replacement, deletion, or conditional mutagenesis. Results: Most colistin-resistant mutants carried mutations in genes belonging to four functional groups: two-component systems controlling LPS aminoarabinosylation (PmrAB, PhoPQ), LPS biosynthesis, the production of the polyamine norspermidine, and fatty acid metabolism. No mutation was exclusively and invariably associated with a specific strain or medium. We demonstrated that norspermidine is detrimental to the acquisition of colistin resistance upon PmrAB activation and that impaired fatty acid biosynthesis can promote colistin resistance, even if it increases susceptibility to other antibiotics. Conclusions: The evolution of colistin resistance in P. aeruginosa appeared to be only marginally affected by the genetic background and culture conditions. Notably, mutations in fatty acid biosynthetic genes represent a newly identified genetic determinant of P. aeruginosa colistin resistance, warranting further investigation in clinical isolates.

Keywords: PA14; PAO1; artificial sputum medium; colistin; fatty acids; human serum; in vitro evolution; lipid A; norspermidine; polymyxins.

Figure 1

Colistin MIC of the clones evolved in MH, ASM, or HS (as indicated in the figure) in the presence of colistin (filled symbols) and the control strains evolved for the same number of passages in the absence of colistin (empty symbols). MIC was determined after 20 h of growth in MH and represents the mode of at least three independent assays. The MIC for the parental strains PAO1 and PA14 was 0.25 μg/mL (not shown in the figure).

Figure 2

Effect of norspermidine on growth and colistin resistance. (A) Growth curves of PAO1, PAO1 PrpsA::arn, PAO1 pmrB^M292T, PAO1 ΔspeE2, PAO1 PrpsA::arn ΔspeE2, and PAO1 pmrB^M292T ΔspeE2 in MH supplemented with increasing colistin concentrations. (B) Growth curves of PA14, PA14 PrpsA::arn, PA14 pmrB^M292T, PA14 ΔspeE2, PA14 PrpsA::arn ΔspeE2, and PA14 pmrB^M292T ΔspeE2 in MH supplemented with increasing colistin concentrations. (C) Colistin MIC for the above-mentioned strains after 20 h of growth in MH. Growth curves are representative of at least three independent experiments giving similar results. MIC values correspond to the mode of at least three independent experiments.

Figure 3

Effect of suboptimal lpxA gene expression on colistin resistance. (A) Growth curves of PAO1, PAO1 PrpsA::arn, PAO1 pmrB^M292T, PAO1 araC-P_BAD::lpxA ΔlpxA, PAO1 PrpsA::arn araC-P_BAD::lpxA ΔlpxA, and PAO1 pmrB^M292T araC-P_BAD::lpxA ΔlpxA in MH supplemented with 0.125% arabinose and increasing colistin concentrations. (B) Growth curves of PA14, PA14 PrpsA::arn, PA14 pmrB^M292T, PA14 araC-P_BAD::lpxA ΔlpxA, PA14 PrpsA::arn araC-P_BAD::lpxA ΔlpxA, and PA14 pmrB^M292T araC-P_BAD::lpxA ΔlpxA cultured in MH supplemented with 0.125% arabinose and increasing colistin concentrations. (C) Colistin MIC for the above-mentioned strains after 20 h of growth in MH supplemented with 0.125% arabinose. Growth curves are representative of at least three independent experiments giving similar results. MIC values correspond to the mode of at least three independent experiments.

Figure 4

Effect of suboptimal accD gene expression on colistin resistance. (A) Growth curves of PAO1, PAO1 PrpsA::arn, PAO1 pmrB^M292T, PAO1 araC-P_BAD::accD ΔaccD, PAO1 PrpsA::arn araC-P_BAD::accD ΔaccD, and PAO1 pmrB^M292T araC-P_BAD::accD ΔaccD in MH supplemented with 0.5% arabinose and increasing colistin concentrations. (B) Growth curves of PA14, PA14 PrpsA::arn, PA14 pmrB^M292T, PA14 araC-P_BAD::accD ΔaccD, PA14 PrpsA::arn araC-P_BAD::accD ΔaccD, and PA14 pmrB^M292T araC-P_BAD::accD ΔaccD in MH supplemented with 0.5% arabinose and increasing colistin concentrations. (C) Colistin MIC for the above-mentioned strains after 20 h of growth in MH supplemented with 0.5% arabinose. Growth curves are representative of at least three independent experiments giving similar results. MIC values correspond to the mode of at least three independent experiments.

Figure 5

Kirby–Bauer disk diffusion assay for the speE2 deletion mutants and the lpxA and accD conditional mutants. (A) Antibiotic susceptibility profile of the speE2 deletion mutants and their parental strains on MH agar plates. (B) Antibiotic susceptibility profile of the lpxA conditional mutants (lpxA) and their parental strains on MH agar plates supplemented with 0.125% arabinose. (C) Antibiotic susceptibility profile of the accD conditional mutants (accD) and their parental strains on MH agar plates supplemented with 0.5% arabinose. Values represent the mean (±standard deviation) of three independent assays. Asterisks indicate statistically significant differences with respect to the wild-type strain PAO1 or PA14 according to the ANOVA test (* p < 0.05). Abbreviations: Rif, rifampicin; Gm, gentamicin; Cip, ciprofloxacin; Imp, imipenem; WT, wild type.