Oscillatory dynamics in a bacterial cross-protection mutualism

Abstract

Cooperation between microbes can enable microbial communities to survive in harsh environments. Enzymatic deactivation of antibiotics, a common mechanism of antibiotic resistance in bacteria, is a cooperative behavior that can allow resistant cells to protect sensitive cells from antibiotics. Understanding how bacterial populations survive antibiotic exposure is important both clinically and ecologically, yet the implications of cooperative antibiotic deactivation on the population and evolutionary dynamics remain poorly understood, particularly in the presence of more than one antibiotic. Here, we show that two Escherichia coli strains can form an effective cross-protection mutualism, protecting each other in the presence of two antibiotics (ampicillin and chloramphenicol) so that the coculture can survive in antibiotic concentrations that inhibit growth of either strain alone. Moreover, we find that daily dilutions of the coculture lead to large oscillations in the relative abundance of the two strains, with the ratio of abundances varying by nearly four orders of magnitude over the course of the 3-day period of the oscillation. At modest antibiotic concentrations, the mutualistic behavior enables long-term survival of the oscillating populations; however, at higher antibiotic concentrations, the oscillations destabilize the population, eventually leading to collapse. The two strains form a successful cross-protection mutualism without a period of coevolution, suggesting that similar mutualisms may arise during antibiotic treatment and in natural environments such as the soil.

Keywords: antibiotic resistance; cooperation; mutualism; oscillations; population dynamics.

Fig. 1.

Two strains of resistant bacteria can form a mutualism in a multidrug environment by protecting each other from antibiotics. (A) In an environment containing the antibiotics ampicillin and chloramphenicol, a mutualism forms between bacteria producing a β-lactamase enzyme (which protects against ampicillin) and bacteria producing a chloramphenicol acetyltransferase enzyme (which protects against chloramphenicol). (B) In our serial dilution experiments, we periodically diluted microbial cultures into fresh growth media, replenishing the supply of nutrients and antibiotics. We determined the size of each subpopulation by combining spectrophotometry measurements of the total culture density together with flow cytometry measurements of the relative abundances of each subpopulation. (C) A coculture of the two strains can survive above the concentrations at which the individual strains survive alone, indicating that the two populations form an obligatory mutualism. The black lines indicate the region beyond which only the coculture can survive. (D) An ampicillin-resistant monoculture can survive in high concentrations of ampicillin but cannot survive alone in chloramphenicol concentrations above 2.2 μg/mL. (E) Similarly, a chloramphenicol-resistant monoculture can survive in high concentrations of chloramphenicol but cannot survive alone in ampicillin concentrations above 2 μg/mL. The antibiotic concentrations in D and E are the same as in C. The populations shown in C–E were subject to five daily dilution cycles at 100×.

Fig. 2.

The subpopulation sizes of the two mutualists oscillate over a broad range of antibiotic concentrations, both inside and outside the region where the mutualism is obligatory. Many of the mutualisms settled into apparent period three oscillations, which had a period (3 days) longer than the period between successive exposures to antibiotics (1 day). These oscillations were substantial in magnitude with the relative abundances of the two mutualists changing by as much as 10⁴-fold. The red region of each subplot represents the size of the ampicillin-resistant subpopulation, and the blue region represents the size of the chloramphenicol-resistant subpopulation. The green line delineates the range of antibiotic concentrations above which neither strain can survive alone. In this experiment, the cocultures were diluted by 100× every 24 h into fresh media supplemented with antibiotics.

Fig. 3.

Oscillations in the relative abundances of the two mutualists are driven by the periodic dilution of the coculture into fresh media containing antibiotics. (A) Under periodic exposure to antibiotics, the relative abundances of the two mutualists in the coculture oscillate by as much as 10⁴-fold. (B) Under (pseudo)continuous exposure to antibiotics, the relative abundances of the two mutualists converge to an equilibrium ratio, and we do not see any sign of the large amplitude oscillations present in A. To transition from the periodic regime (A) to the (pseudo)continuous regime (B), we decreased the time between consecutive dilutions from ΔT = 24 h to ΔT = 1 h and the dilution strength from 100× to 1.2×. The death rate due to dilution, ln(dilution strength)/ΔT, is equivalent in both regimes. The detection limits on our flow cytometer were on the order of N_amp/N_chlor = 10^±3. In these experiments, the concentrations of ampicillin and chloramphenicol in the fresh media were 10 μg/mL and 5.1 μg/mL, respectively. See Fig. 2 and SI Appendix, Fig. S10 for the population sizes corresponding to these trajectories.

Fig. 4.

In the presence of daily 100× dilutions, the two mutualists undergo stable limit cycle oscillations. (A) We found that the relative abundance of the ampicillin-resistant subpopulation with respect to the chloramphenicol-resistant subpopulation settled into period three oscillations, even when test cultures started with different subpopulation abundances. The population size of these limit cycle trajectories remained close to the carrying capacity. (B) If the population size of each mutualist is sufficiently large, then the two mutualists settle into stable oscillations (colored trajectories); otherwise, the mutualism collapses (gray trajectory). (C) A discrete-time framework of an obligatory mutualism featuring a “healthy state” characterized by a limit cycle (white region) and a “collapsed state” (gray region). A boundary (called a separatrix) separates the sets of subpopulation compositions that converge to each state. (A and B) Experiments were carried out in an environment inside the region of obligatory mutualism (100× dilution strength, 24-h dilution cycle, 10 μg/mL ampicillin, and 5.1 μg/mL chloramphenicol). The blue trajectories present in A and B are the same time series. Open circles indicate the initial population composition for each trajectory.

Fig. 5.

The large oscillations in the relative abundance of the two strains (N_amp/N_chl) destabilize the mutualism in harsher environments, causing the population to collapse. At low chloramphenicol concentrations (7.6 μg/mL), cocultures can successfully recover from the extreme changes in the relative abundance caused by the oscillations. However, at higher chloramphenicol concentrations (17.1 and 38.4 μg/mL), the probability of collapse (portrayed by the gradient in the background of each subplot) increases rapidly when the relative abundance of the ampicillin-resistant population falls below 1:100, causing the oscillations to destabilize the mutualism. One extreme of the oscillation is more susceptible to collapse: When the population of ampicillin-resistant strain in the mutualism becomes too small, it can no longer protect the chloramphenicol-resistant strain from ampicillin, ultimately leading to the collapse of the coculture. Red circles denote the last time point when the overall size of the population remained above the detection limit (see SI Appendix, Fig. S21 for the time series of the population sizes of the shown trajectories). In these experiments, the media contained 10 μg/mL ampicillin and the specified amount of chloramphenicol.

Fig. 6.

A double-resistant strain can invade the mutualism and cause the oscillations to vanish, illustrating that the existence of oscillations depends on how resistance is allocated in the microbial population. (A) A double-resistant strain (carrying both resistance plasmids) does not require protection from the mutualistic strains to survive. (B) In the absence of the invader, the ampicillin- and chloramphenicol-resistant subpopulation oscillate. (C) After introducing the double-resistant strain at the beginning of the seventh cycle (dashed gray line) to a replicate culture of B, the double-resistant invader established in the microbial population, outcompeting the ampicillin-resistant subpopulation and removing the oscillations. (B and C) The concentrations of ampicillin and chloramphenicol were 10 μg/mL and 7.5 μg/mL respectively.