Mutational background influences P. aeruginosa ciprofloxacin resistance evolution but preserves collateral sensitivity robustness

Abstract

Collateral sensitivity is an evolutionary trade-off whereby acquisition of the adaptive phenotype of resistance to an antibiotic leads to the nonadaptive increased susceptibility to another. The feasibility of harnessing such a trade-off to design evolutionary-based approaches for treating bacterial infections has been studied using model strains. However, clinical application of collateral sensitivity requires its conservation among strains presenting different mutational backgrounds. Particularly relevant is studying collateral sensitivity robustness of already-antibiotic-resistant mutants when challenged with a new antimicrobial, a common situation in clinics that has hardly been addressed. We submitted a set of diverse Pseudomonas aeruginosa antibiotic-resistant mutants to short-term evolution in the presence of different antimicrobials. Ciprofloxacin selects different clinically relevant resistance mutations in the preexisting resistant mutants, which gave rise to the same, robust, collateral sensitivity to aztreonam and tobramycin. We then experimentally determined that alternation of ciprofloxacin with aztreonam is more efficient than ciprofloxacin–tobramycin alternation in driving the extinction of the analyzed antibiotic-resistant mutants. Also, we show that the combinations ciprofloxacin–aztreonam or ciprofloxacin–tobramycin are the most effective strategies for eliminating the tested P. aeruginosa antibiotic-resistant mutants. These findings support that the identification of conserved collateral sensitivity patterns may guide the design of evolution-based strategies to treat bacterial infections, including those due to antibiotic-resistant mutants. Besides, this is an example of phenotypic convergence in the absence of parallel evolution that, beyond the antibiotic-resistance field, could facilitate the understanding of evolution processes, where the selective forces giving rise to new, not clearly adaptive phenotypes remain unclear.

Keywords: Pseudomonas aeruginosa; antibiotic resistance; collateral sensitivity; convergent evolution; phenotypic convergence.

Fig. 1.

Diagram showing robustness of CS to tobramycin and aztreonam in PA14 and in different mutational backgrounds submitted to short-term ALE on ciprofloxacin. Cross-resistance and CS to antibiotics from different structural families were analyzed in PA14 and the mutational backgrounds parR87, orfN50, nfxB177, mexZ43, MDR6, and MDR12 (four replicate populations for each) submitted to ALE in the presence of tobramycin, aztreonam, or ciprofloxacin for 3 d. Intensity of the color is proportional to the log-transformed FC regarding the MIC of the respective parental strain. Since control populations evolved in the absence of antibiotics may present, on rare occasions, subtle changes (below or above 2- or 0.5-fold, respectively) in their susceptibility to antibiotics with respect to the MIC of parental strains, changes in MICs above or below 2- or 0.5-fold, respectively, were considered physiologically relevant to classify a population as “resistant” (purple) or “susceptible” (orange). MIC values (µg/mL) of populations evolved in the presence of tobramycin, aztreonam, or ciprofloxacin are included in SI Appendix, Tables S1–S3, respectively. MIC values (µg/mL) of control populations evolved in the absence of drugs are included in SI Appendix, Table S4. ATM, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; FOF, fosfomycin; IPM, imipenem; TOB, tobramycin.

Fig. 2.

General model illustrating evolution of antibiotic-resistant mutants of P. aeruginosa submitted to the alternation of ciprofloxacin with tobramycin or aztreonam or the combination of ciprofloxacin with tobramycin or aztreonam. (A) Evolution starts when different antibiotic-resistant mutants are treated with ciprofloxacin at time 0 (t₀). Then, there is evolution toward ciprofloxacin resistance and CS to tobramycin and aztreonam (purple cells), rendering ciprofloxacin ineffective (t₁). Subsequently, treatment is switched to tobramycin (TOB) or aztreonam (AZT) that may result in the elimination of cells susceptible to tobramycin and aztreonam (t₂). (B) Evolution starts when different antibiotic-resistant mutants are treated with a ciprofloxacin–tobramycin or a ciprofloxacin–aztreonam combination at time 0 (t₀). Since ciprofloxacin-resistance acquisition leads to CS to tobramycin and aztreonam, it may be expected that drug combinations result in a reduced rate of adaptation or the elimination of cells (t₁).

Fig. 3.

Diagram showing the efficacy of the alternation of ciprofloxacin with tobramycin or aztreonam and the combination of ciprofloxacin with tobramycin or aztreonam for driving extinction of P. aeruginosa PA14 and different antibiotic-resistant mutants. (A) Short-term evolution of PA14 and six mutational backgrounds (nfxB177, parR87, mexZ43, orfN50, MDR6, or MDR12), four replicate populations of each parental strain, was performed during 6 d: 3 d in the presence of ciprofloxacin (CIP) or the absence of antibiotic (control populations), leading to ciprofloxacin-resistant populations (purple cells), and 3 d in the presence of tobramycin (TOB) or aztreonam (AZT). CS to tobramycin and aztreonam was observed in 21 and 19 out of 28 populations after a first step on ciprofloxacin (SI Appendix, Table S3). Populations that were extinct at the end of the experiment are represented in black, while surviving populations are colored in gray. Most of the populations (24 out of 28) submitted to short-term ALE in the presence of tobramycin grew after 3 d. However, short-term ALE in the presence of aztreonam led to extinction of 11 out of 28 populations. This evolutionary strategy was efficient in driving extinction of ciprofloxacin-resistant mutants belonging to nfxB177, parR87, mexZ43, orfN50, and MDR6, but ineffective in driving extinction of PA14 and MDR12. (B) Short-term evolution of PA14 and six mutational backgrounds (nfxB177, parR87, mexZ43, orfN50, MDR6, or MDR12), four replicate populations of each parental strain, was performed during 3 d in the presence of the ciprofloxacin–tobramycin (CIP-TOB) or the ciprofloxacin–aztreonam (CIP-ATM) combination. Growth of the 84 control populations was confirmed in the three drugs independently used at the concentrations present in the drugs combinations. A total of 25 out of 28 populations submitted to short-term ALE in the presence of the ciprofloxacin–aztreonam combination were extinct, while it occurred in 23 out of 28 populations submitted to short-term ALE in the presence of the ciprofloxacin–tobramycin combination. These results indicate that CS may not only improve treatment when drugs are applied sequentially, but it may also serve to optimize combinatory therapy.

Fig. 4.

Diagram showing genetic causes of ciprofloxacin-resistance acquisition and mutational background dependence. Classical mutations regularly found in clinical isolates from patients treated with ciprofloxacin, within gyrAB, nfxB, and mexS, were acquired (black boxes) during ALE in the presence of ciprofloxacin for 3 d in 28 populations belonging to PA14 and 6 different mutational backgrounds (parR87, orfN50, nfxB177, mexZ43, MDR6, and MDR12). We identified 15, 5, 3, and 1 different variants of mexS, nfxB, gyrA, and gyrB, respectively. As observed, early steps of ciprofloxacin-resistance evolution are dependent on the mutational background (Table 2).