Research ArticleMICROBIOLOGY

Rapid and robust evolution of collateral sensitivity in Pseudomonas aeruginosa antibiotic-resistant mutants

See allHide authors and affiliations

Science Advances  05 Aug 2020:
Vol. 6, no. 32, eaba5493
DOI: 10.1126/sciadv.aba5493
  • Fig. 1 Analysis of early steps in the evolution of P. aeruginosa wild-type strain and antibiotic-resistant mutants in the presence of CAZ.

    (A) Scheme of the resultant phenotype after the evolution of PA14, single (nfxB177, parR87, mexZ43, orfN50, and nuoD184) and multiple (MDR6 and MDR12) mutants, in the presence of CAZ [left part of (A)] or in the absence of antibiotic [LB; right part of (A)] for 3 days (see Materials and Methods). Pyomelanin hyperproduction was observed in 27 of 32 populations evolved in the presence of CAZ (red-colored cells). (B) Diagram showing convergence toward hypersusceptibility to TOB in the different genetic backgrounds and replicates, analyzed after short-term evolution on CAZ. In all cases, acquisition of a pyomelanogenic phenotype is associated with collateral sensitivity to TOB, irrespective of the genetic background of the evolving strain. MIC values of TOB and CAZ are included in table S2. (C) Isolation of pyomelanogenic clones from each 27 pyomelanogenic population [left part of (A); red-colored cells] obtained in the presence of CAZ. As shown in the figure, the early steps of evolution in presence of CAZ of P. aeruginosa, which lead to collateral sensitivity to TOB and pyomelanin production, are conserved among the different antibiotic-resistant mutants analyzed. (D) Pyomelanogenic phenotype of mexZ43 mutant after 1-day evolution in the presence of CAZ or in the absence of antibiotic (LB). Pyomelanin hyperproduction was observed in 28 of 32 populations evolved on CAZ. Photo credits for (C) and (D): Inés Poveda, Centro Nacional de Biotecnología. Permission for using these images is not required.

  • Fig. 2 General model illustrating evolution of heterogeneous pyomelanogenic populations of P. aeruginosa subjected to TOB/CAZ sequential evolution.

    Evolution of a heterogeneous population containing a pyomelanogenic (CAZ resistant) subpopulation starts when TOB is added at time zero (t0). In the presence of TOB, there is an extinction of TOB-hypersusceptible pyomelanogenic mutants (red-colored cells) and TOB becomes ineffective (t1). Then, treatment is switched to CAZ, and TOB-resistant cells (contoured gray-colored cells) become TOB-hypersusceptible (t2). Treatment would be switched back to TOB, resulting in the elimination of TOB-hypersusceptible cells (t3). This strategy would also be potentially applicable to initial populations not resistant to CAZ (t1), being reduced to a first step on CAZ (leading to t2), followed by a second step on TOB (resulting in t3).

  • Fig. 3 Analysis of TOB/CAZ sequential evolution of heterogeneous pyomelanogenic populations of P. aeruginosa.

    (A) Diagram showing the evolution of heterogeneous populations (dubbed +1, +2, +3, and +4) containing each parental strain: PA14, nfxB177, parR87, mexZ43, orfN50, MDR6, or MDR12 and four individual pyomelanogenic clones belonging to the same genetic background, during first step of sequential evolution in the presence of TOB (left) and second step in the presence of CAZ (right), for 6 days (see Materials and Methods). In the case of parR87, only three pyomelanogenic clones from independent CAZ-evolved populations (see Fig. 1, A and C) could be isolated. Hypersusceptibility to TOB (contoured and noncontoured red-colored cells) is observed in 23 of 27 populations (see table S4), and pyomelanin production (noncontoured red-colored cells) is observed in 17 of 27 populations. (B) Analysis of extinction of the pyomelanogenic part of the heterogeneous pyomelanogenic populations after a first step of sequential evolution in the presence of TOB [(A) section, left]. The phenotype (color) of 20 clones isolated from each heterogeneous population, after 3 days of TOB evolution, was observed in liquid medium and compared with the color (brown) of each pyomelanogenic parental strain (upper left corner of each plate). In agreement with data shown in table S4, which points to the extinction of pyomelanogenic populations by comparison of CAZ MIC value of each heterogeneous population with the ones of their parental strains and pyomelanogenic clones, the color of the 540 clones analyzed indicated that pyomelanogenic clones were extinct after first step of sequential evolution on TOB. Photo credits for (B): Fernando Sanz-García, Centro Nacional de Biotecnología.

  • Fig. 4 MICs (μg/ml) of TOB for heterogeneous pyomelanogenic populations of P. aeruginosa after TOB/CAZ sequential evolution.

    Evolution of TOB MICs (μg/ml) of heterogeneous pyomelanogenic populations after sequential evolution on TOB/CAZ (see Fig. 3). Each plot shows the TOB MIC values for a parental strain (PA14, parR87, orfN50, nfxB177, mexZ43, MDR6, or MDR12), indicated as t0 in the x axis, and for four heterogeneous pyomelanogenic populations (represented as black circles) after first evolution on TOB (indicated as TOB in the x axis), followed by second evolution on CAZ (indicated as CAZ in the x axis). Only three heterogeneous pyomelanogenic populations of parR87 were analyzed. TOB MICs decreased after switching from TOB to CAZ by up to 128-fold in PA14, 48-fold in nfxB177, 21-fold in parR87, 96-fold in orfN50, 43-fold in mexZ43, 4-fold in MDR6, and 11-fold in MDR12. MIC values are shown in table S4.

  • Fig. 5 Diagram showing the degree of convergence of cross-resistance and collateral sensitivity in heterogeneous pyomelanogenic populations of P. aeruginosa obtained after TOB/CAZ sequential evolution.

    Collateral sensitivity and cross-resistance to antibiotics from different structural families were analyzed in the 27 populations obtained after sequential evolution. A population is classified as “susceptible” or “resistant” when there was an MIC change with respect to the parental strain value. Triangles indicate antibiotics where a predominant change toward resistance (red) or susceptibility (green) with respect to the parental strain was observed. Thickness of the triangle depends on the percentage of conservation of said phenotype. MIC values (μg/ml) are included in table S5. AMK, amikacin; ATM, aztreonam; FOF, fosfomycin; ERY, erythromycin; CHL, chloramphenicol; LEV, levofloxacin; TET, tetracycline.

  • Table 1 MICs (μg/ml) of different antibiotics for the single and multiple P. aeruginosa PA14 mutants used in this work.

    MICs ≥2-fold of the MICs for the wild-type PA14 strain are highlighted in bold. TOB, tobramycin; TGC, tigecycline; CAZ, ceftazidime; CIP, ciprofloxacin; IPM, imipenem.

    TOBTGCCAZCIPIPM
    PA141610.0940.75
    nfxB1771321.531
    parR871.5810.1252
    orfN5033230.192
    nuoD1842410.0470.75
    mexZ431.5810.381.5
    MDR6*2481.50.191.5
    MDR12326410.51.5

    *MDR6 mutant presents mutations in nfxB, phoQ, frr, and pmrB.

    †MDR12 mutant presents mutations in fusA, orfN, pmrB, mexZ, gabP, ptsP, and nuoD.

    Supplementary Materials

    • Supplementary Materials

      Rapid and robust evolution of collateral sensitivity in Pseudomonas aeruginosa antibiotic-resistant mutants

      Sara Hernando-Amado, Fernando Sanz-García, José Luis Martínez

      Download Supplement

      This PDF file includes:

      • Fig. S1
      • Tables S1 to S6

      Files in this Data Supplement:

    Stay Connected to Science Advances

    Navigate This Article