Abstract
Interactions among beneficial mutations (that is, epistasis) are often strong enough to direct adaptation through alternative mutational paths. Although alternative solutions should display similar fitness under the primary selective conditions, their properties across secondary environments may differ widely. The extent to which these cryptic differences are to be expected is largely unknown, despite their importance—for example, in identifying exploitable collateral sensitivities among mutations conferring antibiotic resistance. Here we use directed evolution to characterize the diversity of mutational paths through which the prevalent carbapenemase Klebsiella pneumoniae carbapenemase-2 can evolve high activity against the clinically relevant antibiotic ceftazidime, an initially poor substrate. We identified 40 different substitutions—including many that are common in clinical settings—spread among 18 different mutational trajectories. Initial mutations determined four major groups into which the trajectories can be classified, a signature of strong epistasis. Despite similar final ceftazidime resistance, groups diverged markedly across multiple phenotypic dimensions, from molecular traits, such as in-cell stability and catalytic efficiency, to macroscopic traits, such as growth rate and activity against other β-lactam antibiotics. Our results indicate that cryptic yet consequential phenotypic differences can accumulate rapidly under strong selection, unpredictably shaping the long-term success of resistance enzymes in their journey across hosts and environments.
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All of the data needed to replicate the findings of this study are available in the manuscript and its Supplementary Information. Source data are provided with this paper.
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Acknowledgements
We thank L. J. González and A. J. Vila for valuable experimental suggestions and J. Barber and L. López-Merino for proofreading the manuscript. L.D. acknowledges support from the European Commission under the Horizon 2020 Framework Programme (Marie Skłodowska-Curie Individual Fellowship 101029953). A.C. acknowledges support from the Agencia Estatal de Investigación (Proyectos de Investigación, Desarrollo e Innovación (PID2019-110992GA-I00 and PID2022-142857NB-I00) and Centros de Excelencia Severo Ochoa (SEV-2016-0672 and CEX2020-000999-S)) and a Comunidad de Madrid Talento Fellowship (2019-T1/BIO-12882 and 2023-5A/BIO-28940).
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L.D. and A.C. conceived of the study idea. L.D. designed the methodology. L.D. performed the investigation with support from I.N. A.C. performed the formal analysis, supervised the study, wrote the original draft of the manuscript and acquired funding with support from L.D. A.C. and L.D. contributed equally to reviewing and editing the manuscript, with support from I.N.
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Extended data
Extended Data Fig. 1 Resistance trajectories plotted against the number of rounds for each lineage.
Colors indicate grouping based on positions associated with CAZ resistance: blue (D179Y), green (H274Y), yellow (L169P), and red (S171P). Panels show MIC values for CAZ, cefotaxime (CTX), cefoxitin (FOX), and imipenem (IMI), as labeled in the upper-right corner. Note that divergence in resistance profiles peaks around rounds 1–2, similarly to other traits (Fig. 3), underscoring how rapidly populations diverge after fixation of the first mutation.
Extended Data Fig. 2 Pairwise epistasis across the four-mutation landscape.
(a) Diagram of all mutational trajectories from the ancestral KPC-2 (bottom node). Conventions follow those in Fig. 2a, except that genotypes are numbered 1–16 (rather than labelled by the first letters of mutations) to facilitate the identification of genotypes shown in panel C. Genotype positions match those shown in Fig. 2a. (b) Distribution of epistasis values, calculated as the deviation of each double-mutant fitness from the additive expectation. (c) All possible pairwise interactions. At the top of each panel it is indicated which four genotypes are being considered. For example, the top-left panel compares the single effects of L169P and V240A on the ancestor (genotypes 1, 2 and 3) with their combined effect (genotype 6).
Extended Data Fig. 3 Biochemical characterization of selected enzymes.
(a) Progress curves of CAZ hydrolysis from a spectrophotometric assay. All reactions were performed with 500 nM enzyme and 50 μM ceftazidime. Hydrolysis of ceftazidime results in a decrease in absorbance at 260 nm. Curves correspond to the following variants: wild-type (black), D179P-L260M (blue), L169P-P94L (yellow), S171P-V240A-P94L-M49I (orange), H274Y (very light green), H274Y-V240A (light green), H274Y-V240A-V103L (green), and H274Y-V240A-V80E (dark green). (b) Steady-state kinetic parameters for CAZ hydrolysis for ancestral KPC-2 and the same variants as in A. Standard errors are provided in parentheses (n = 3).
Extended Data Fig. 4 Western blot of KPC-2 and its variants in periplasmic extracts of E. coli TOP10.
(a–d) Periplasmic abundance of representative lineages from groups H274 (a, green), L169 (b, yellow), D179 (c, blue), and S171 (d, red) after 1 and 5 h of growth. (e) Immunoblot of selected KPC variants in the periplasm of E. coli TOP10 after 10 h of incubation. Isolates from each lineage are identified by the top label and color-coded as in previous panels. The bottom label indicates the evolutionary round of each isolate. Note that for groups D1 & D2 and S1 & S2, the earliest isolate shown (R2 and R1, respectively) is ancestral to both descendant lineages. Repeated round labels (R3 and R4, respectively) represent isolates sampled after lineage splitting and are arranged in natural alphanumeric order (for example, round 3 D1, round 3 D2).
Extended Data Fig. 5 Periplasmic stability of selected variants.
Bar plots display the log intensity ratio of initial (1 h) and final (5 h) bands for each variant, quantified from the Western blots in Extended Data Fig. 4. Panels and panel labels follow the same arrangement as in Extended Data Fig. 4. For convenience, lineage labels are included in the upper left of each panel. Colors correspond to those in Extended Data Fig. 2. Error bar represents the control’s 95% confidence interval across gels (mean ± 2 SEM; n = 8, Extended Data Fig. 4a–d).
Extended Data Fig. 6 Marked trade-offs between catalytic efficiency and periplasmic stability.
(a) Catalytic efficiency shows a weak, positive, nonsignificant correlation with MIC increases (Pearson’s r = 0.37, P = 0.47). (b) Periplasmic stability exhibits a strong negative correlation with MIC increases (Pearson’s r = –0.93, P = 0.007). (c) Combining catalytic efficiency and periplasmic stability (as in ref. 83) still strongly negatively correlates with MIC increases (Pearson’s r = –0.91, P = 0.0109). Although higher stability should intuitively increase MIC by providing greater enzyme availability, this expectation only holds true in the absence of the activity-stability trade-offs observed here. Note these factors do not offset each other effectively because losses in stability are orders of magnitude greater than gains in catalytic efficiency.
Extended Data Fig. 7 Growth rates of consecutive mutants within plateaus.
(a) Optical density changes over 12 h of growth. The black line represents the strain with wild-type KPC-2, gray lines represent earlier plateau strains, and colored lines represent subsequent strains. (b) Boxplot summarizing maximum growth rates from the growth curves above. X-axis labels, excluding the control strain, consist of a bottom label indicating the evolutionary line (as in Table S2) and a top label indicating which round the isolate was taken from (round 2: R2; round 3: R3; round 4: R4). Following R’s default convention, boxes span the first (Q1) to third (Q3) quartiles, central lines mark the median, and whiskers reach the most extreme points within 1.5 times the interquartile range.
Extended Data Fig. 8 Growth rates along evolutionary trajectories in the absence of antibiotic.
(a) Optical density changes over 12 h of growth. The black line represents the strain with wild-type KPC-2, thick colored lines represent the first round strains, and the thiner colored lines represent plateau strains. (b) Boxplot summarizing maximum growth rates from the growth curves above. X-axis labels, excluding the control strain, indicate the evolutionary line (as in Table S2) and the round from which each isolate was taken (that is, rounds 1–4, labeled R1–R4). Following R’s default convention, boxes span the first (Q1) to third (Q3) quartiles, central lines mark the median, and whiskers reach the most extreme points within 1.5 times the interquartile range. (c) Growth rates in the absence of antibiotic show a moderate negative correlation with those measured in the presence of subinhibitory concentrations of CAZ, albeit non-significant (R = –0.65, P = 0.11). Solid line indicates the ideal one-to-one correspondence.
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Dabos, L., Nedjari, I. & Couce, A. Cryptic phenotypic variation emerges rapidly during the adaptive evolution of a carbapenemase. Nat Ecol Evol (2025). https://doi.org/10.1038/s41559-025-02804-6
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DOI: https://doi.org/10.1038/s41559-025-02804-6
