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. 2016 Sep 26;1:16175.
doi: 10.1038/nmicrobiol.2016.175.

A Competitive Trade-Off Limits the Selective Advantage of Increased Antibiotic Production

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A Competitive Trade-Off Limits the Selective Advantage of Increased Antibiotic Production

Ylaine Gerardin et al. Nat Microbiol. .
Free PMC article

Abstract

In structured environments, antibiotic-producing microorganisms can gain a selective advantage by inhibiting nearby competing species1. However, despite their genetic potential2,3, natural isolates often make only small amounts of antibiotics, and laboratory evolution can lead to loss rather than enhancement of antibiotic production4. Here, we show that, due to competition with antibiotic-resistant cheater cells, increased levels of antibiotic production can actually decrease the selective advantage to producers. Competing fluorescently labelled Escherichia coli colicin producers with non-producing resistant and sensitive strains on solid media, we found that although producer colonies can greatly benefit from the inhibition of nearby sensitive colonies, this benefit is shared with resistant colonies growing in their vicinity. A simple model, which accounts for such local competitive and inhibitory interactions, suggests that the advantage of producers varies non-monotonically with the amount of production. Indeed, experimentally varying the amount of production shows a peak in selection for producers, reflecting a trade-off between benefit gained by inhibiting sensitive competitors and loss due to an increased contribution to resistant cheater colonies. These results help explain the low level of antibiotic production observed for natural species and can help direct laboratory evolution experiments selecting for increased or novel production of antibiotics.

Conflict of interest statement

statement The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1. Colicin producers inhibit sensitive competitors in their vicinity, promoting their own growth as well as that of nearby resistant, non-producing cheaters
a, The colicin E2 operon contains toxin, immunity, and lysis genes under an SOS promoter induced by DNA damage. b, Producer strain releases colicin (red hexagons) which kills a sensitive strain, but is ineffective against a resistant strain. Strains are differentially labeled with fluoresceist reporters. c, Two-strain co-culture on solid media (containing 16 ng/mL mitomycin C) shows representative producer colonies (red) inhibiting growth of nearby sensitive (blue), but not resistant (green), colonies (scale bars = 1 mm). Sensitive colonies do not grow within the inhibition radius ri. d, Co-culture of all three strains together, showing the resistant strain can act as a production cheater. Left, growth across entire surface of representative plate; right, zoomed image of 1 cm2 box region (scale bar = 1 mm). Resistant colonies are small when competing with the sensitive strain (dash arrow) but they gain in size when growing in the vicinity of producer colonies (solid arrow).
Figure 2
Figure 2. Producers have an advantage over resistant cheaters only at high densities of sensitive competitors and low densities of producers
a, Selection for production relative to the non-producing resistant cheater in three-way competitions on agar for varying seeding densities of sensitive and producer cells (producer density [P] = cheater density [C], mean of 4 replicates). b, Mean growth of producer colonies (arbitrary units) in pairwise co-culture with sensitive competitors (blue) versus resistant (green, line showing reciprocal fit, slope=−1). Gain from killing, measured as the difference between the two lines (arrows), appears and further increases as the seeding density of competitors increases beyond a critical density equal to 1/πri2 (dotted line). Error bars show s.d. of 3 replicate plates, [P]=0.7 CFU/cm2. c, The growth of individual cheater colonies (arbitrary units, green dots) decreases with their distance to producer colonies (d is distance to the nearest producer colony; sensitive density [S]=20,000 CFU/cm2, [P]=[C]=3 CFU/cm2; pooled data from 4 replicates; line shows smoothened average by local linear regression. Inset: example of two representative cheater colonies indicated by × and (scale bar = 1 mm). d, Mean cheater growth increases linearly with producers at high sensitive density (solid gray line shows fit, slope = 0.98 ± 0.09 at 95% c.i.), but is not helped by producers at low sensitive density (dashed gray line, slope = 0.13 ± 0.05 at 95% c.i.). Each series is normalized to the mean growth at the lowest producer density (n=4, error bars show s.d.).
Figure 3
Figure 3. A simple model of competition and inhibition predicts that selection for antibiotic production is maximized at intermediate production levels
a, Steps in simulating model: (1) Random seeding of producers (red), sensitive (blue) and cheater (green) colonies at given densities (scale bar = 2 mm); (2) killing sensitive colonies inside inhibition zones of radius ri around producer colonies; (3) final growth of each colony is determined by the amount of resource available to it in a grazing zone of radius rg around it. Resources in overlapping grazing zones of two or more colonies are equally shared among them. b, Selection for production increases monotonically with the density of sensitive species and decreases with the density of producers (mean of 20 simulations per parameter set). c, Selection for production η is maximized at an intermediate level of production ri* (n=50 simulations per parameter set, error bars show s.e.m.). Panels show sample simulations at indicated points (scale bars = 2 cm). Parameter values are given in Supplementary Table 1; see Supplementary Fig. S9 for effects of adding cost to the model and Supplementary Fig. S10 for the effect of varying rg.
Figure 4
Figure 4. The advantage of producers over cheaters is maximized at an intermediate level of antibiotic production
a, Selection for production in three-way competitions as a function of varying levels of colicin induction via mitomycin C ([S]=2,000 CFU/cm2; high density: [P]=[C]=20 CFU/cm2, low density: [P]=[C]=2 CFU/cm2; mean of 4 replicate plates for each point, error bars show s.d.). Left-most points represent no-killing (NK) controls, where the sensitive competitor was replaced with resistant. Differences between these experiments and the simulation results (Fig. 3c) may be attributable to model parameters such as production cost (Supplementary Fig. S9), grazing zone radius (Supplementary Fig. S10), cooperative toxicity (Supplementary Fig. S2), or antibiotic diffusivity (Supplementary Fig. S11). b, Mean growth (arbitrary units) of producer colonies increases monotonically with production level (n=4, error bars show s.d.). Insets: representative colonies from highlighted data points. c, Growth (arbitrary units) of individual cheater colonies close to producer colonies increased with colicin induction (each series is pooled data from all replicates in the high [P] condition; solid lines are smoothened averages calculated by local linear regression). See Supplementary Fig. S12 for low [P] data. d, Mean growth (arbitrary units) of resistant colonies at low and high producer density for varying production levels (n=4, error bars show s.d.).

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