Research ArticleSYSTEMS BIOLOGY

Antithetic population response to antibiotics in a polybacterial community

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Science Advances  06 Mar 2020:
Vol. 6, no. 10, eaaz5108
DOI: 10.1126/sciadv.aaz5108
  • Fig. 1 Scheme illustrating the bacterial response of single-species cultures (left) and mixed-species cultures (right) to a specific antibiotic.

    Species 1 (magenta) has a tolerant response to an antibiotic (AB), while species 2 (blue) presents a sensitive response. Both sensitivities can be captured by measuring bacterial growth [optical density (OD)] over time and observing the resulting final outcomes. The mixed-species culture can have one of four different potential outcomes, described in the text.

  • Fig. 2 Single-species responses to ampicillin.

    (A and B) Growth curves of B. subtilis (A) and E. coli (B) under increasing concentrations of ampicillin. The maximum antibiotic concentration is Amax = 50 μg/ml in both cases. The horizontal double-headed arrow in (A) shows schematically the meaning of the time lag (Tlag) for the highest antibiotic concentration. (C and D) The corresponding median time lags for (A) and (B), respectively, as a function of the initial antibiotic concentration. Error bars represent SD over four replicates (see also fig. S1). The numbers in parentheses on top of some bars indicate the number of replicates in which growth was not observed within the time window of our experiment (70 hours). For the bar on the far right reaching the maximum of the y axis, none of the four replicates exhibited growth in 70 hours.

  • Fig. 3 Monoculture growth dynamics of the molecular titration model.

    (A) Sample trajectory (black line) in the phase space defined by the two variables P and A. The two nullclines of the system are shown in solid green and dashed magenta lines. Labels 1 to 3 correspond to the three portions of the trajectories indicated in fig. S6. The parameters are k1 = 0.15 (μM · s)−1, k2 = 0.0015 s−1, Pt = 2 μM, At = 65 μM, and γ = 5 × 10−4 s−1. (B) Free PBP as a function of instantaneous total antibiotic (AB) concentration, under the assumption that AB varies slowly [which happens in portions 2 and 3 of the trajectory shown in (A)]. The parameters for B. subtilis (black line) are the ones given above, while E. coli (red line) is described by two parameter changes: Pt = 28 μM and γ = 6 × 10−6 s−1. (C and D) Dynamics of the cell density resulting from the logistic equation (Eq. 8) for both species and different antibiotic concentrations. Parameters are those of the previous panels, plus kp = 2 μM, n = 2, μ = 6 × 10−4 s−1, and μ0 = − μ/10. The maximum antibiotic concentration is Amax = 140 μM, which approximately corresponds to the antibiotic concentration used in the experiments shown in Fig. 2 above. a.u., arbitrary units. (E and F) Corresponding temporal behavior of the free antibiotic. (G and H) Growth time lag (Tlag) as a function of the total antibiotic concentration.

  • Fig. 4 Test to compare the ampicillin inactivation rates of B. subtilis and E. coli.

    (A) Scheme of the experimental strategy. Cultures of B. subtilis, B. subtilis/E. coli, and E. coli are grown in the presence of ampicillin. After 20 hours, cells are removed from the media and the supernatant is used as media for independent new cultures of B. subtilis. Last, growth measurements are performed for the different cultures. (B) Bacterial growth (OD) as a function of time for B. subtilis in the supernatant from the B. subtilis monoculture (blue line), the mixed culture (green line), and the E. coli monoculture (orange line). The dashed line corresponds to a control, which contains B. subtilis in the absence of antibiotic. (C) Bar plot showing the quantification of the lag times of the B. subtilis monocultures in the presence of the three different supernatants. Error bars represent SE for three different replicas.

  • Fig. 5 Growth dynamics of the molecular titration model for B. subtilis (left column) and E. coli (right column) in the case of a mixed-species community.

    (A and B) Dynamics of the cell density for the two species in mixed cultures (gray solid lines) in comparison with their behavior in isolation (dashed lines) for a total antibiotic concentration of 280 μM. (C and D) Corresponding temporal behavior of the free antibiotic (the single-culture case has been scaled up twofold for comparison). (E and F) Predicted time lag difference between the monoculture and the coculture conditions for the two bacterial species. Parameters are those of Fig. 3, plus ka = 0.2 s−1. The maximum antibiotic concentration is Amax = 280 μM.

  • Fig. 6 Experimental observations of mixed-species cultures of B. subtilis and E. coli.

    (A and B) Fluorescence marker signal reporting on the growth of the two species: YFP for B. subtilis and CFP for E. coli, respectively. (C and D) Median time lags for (A) and (B), respectively, as a function of the initial antibiotic concentration. Error bars represent SD over four replicates. The numbers in parentheses on top of some bars indicate the number of replicates in which growth was not observed within the time window of our experiment (70 hours). (E and F) Time lag difference between the mixed-species and single-species conditions as a function of the total antibiotic concentration (Amax = 100 μg/ml). (G) Composite phase and fluorescence microscopy images after 48 hours in coculture. B. subtilis cells express YFP (represented here in magenta), and E. coli cells express CFP (represented in blue). The low antibiotic concentration condition (left column) contains media with ampicillin (0.39 μg/ml), and the high antibiotic condition (middle column) contains 25 μg/ml.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaz5108/DC1

    Supplementary Text

    Fig. S1. Growth curves of monocultures of B. subtilis and E. coli in the presence of increasing concentrations of ampicillin.

    Fig. S2. B. subtilis tolerance to ampicillin is not due to gain of resistance.

    Fig. S3. Growth curves of monocultures of B. subtilis and E. coli in the presence of low concentrations of another β-lactam antibiotic.

    Fig. S4. Growth curves of monocultures of B. subtilis PY79 and NCBI 3610 in the presence of increasing concentrations of different antibiotics.

    Fig. S5. Ampicillin is not degraded passively in the medium.

    Fig. S6. Model dynamics and sensitivity analysis.

    Fig. S7. Growth curves and fluorescent signal of mixed culture in the presence of increasing concentrations of ampicillin.

    Table S1. Strains used in this study.

    Notebook S1. Code to reproduce the modeling results of Fig. 3.

    Notebook S2. Code to reproduce the modeling results of Fig. 5.

    Notebook S3. Code to reproduce the modeling results of fig. S6 (A and B).

    Notebook S4. Code to reproduce the modeling results of fig. S6C.

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Text
    • Fig. S1. Growth curves of monocultures of B. subtilis and E. coli in the presence of increasing concentrations of ampicillin.
    • Fig. S2. B. subtilis tolerance to ampicillin is not due to gain of resistance.
    • Fig. S3. Growth curves of monocultures of B. subtilis and E. coli in the presence of low concentrations of another β-lactam antibiotic.
    • Fig. S4. Growth curves of monocultures of B. subtilis PY79 and NCBI 3610 in the presence of increasing concentrations of different antibiotics.
    • Fig. S5. Ampicillin is not degraded passively in the medium.
    • Fig. S6. Model dynamics and sensitivity analysis.
    • Fig. S7. Growth curves and fluorescent signal of mixed culture in the presence of increasing concentrations of ampicillin.
    • Table S1. Strains used in this study.
    • Legends for notebooks S1 to S4

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    Other Supplementary Material for this manuscript includes the following:

    • Notebook S1 (.ipynb format). Code to reproduce the modeling results of Fig. 3.
    • Notebook S2 (.ipynb format). Code to reproduce the modeling results of Fig. 5.
    • Notebook S3 (.ipynb format). Code to reproduce the modeling results of fig. S6 (A and B).
    • Notebook S4 (.ipynb format). Code to reproduce the modeling results of fig. S6C.

    Files in this Data Supplement:

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