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Uncovering the Evolutionary Pathways Toward Triple-Resistant Bacterial Populations

Benjamin Adkins, Quentin Berry, Ashley Jennings, Shubhangini Shah, and Lindsey McGee

Earlham College | Richmond, IN

Abstract

Phenotypic Results

Phenotype Results

Methods

Conclusions

Antibiotic resistance is a prominent challenge to the treatment of bacterial infections. Phage therapy, a possible treatment alternative, has become a developing topic in the medical world. To gain a better understanding of the evolutionary pathways of E. coli, bacteria (EC-WT and EC-R-WA11) were evolved over ten days in varying conditions: growth, ampicillin, gallium nitrate, and ampicillin/gallium. After ten days, each of the evolved bacteria was tested with a spot assay to determine phage resistance, and the growth fitness of each bacterial population at varying concentrations of ampicillin and gallium nitrate was tested by assay in a 96-well plate. We observed that the bacteria that were selected for in ampicillin conditions gained resistance to ampicillin but not to gallium while the bacteria that was selected in gallium conditions gained resistance to both gallium and ampicillin. All populations that began with the ancestor EC-R-WA11 kept the resistance to phage even though phage resistance was not selected for during the course of this experiment. The sequencing results showed an outline of the mutations that the bacteria gained to have these adaptations that allowed them to survive.

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Spot Assays Testing for Phage-Resistance

Figure 2. The spot assay protocol was performed to determine phage resistance in evolved populations. Each population (ECWT, ECWA11) and condition (growth, ampicillin, gallium nitrate, and ampicillin + gallium nitrate) were assayed with a control (LB broth) and phage (WA11) to test phage resistance of evolved bacterial strains. Each assay consisted of 150µL of bacteria and 3µL of each control. Both the control and the WA11 performed in triplicate. Plates were then incubated at 37°C for 24 hours and infections were identified by presence of plaques on the petri plate.

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Experimental Evolution Design

Figure 1. An experimental evolution generated adapted bacterial populations to ampicillin conditions, gallium nitrate conditions, conditions alternating daily between ampicillin and gallium nitrate, and growth conditions. Transfers were conducted with E. coli strain C wild-type or E. coli strain C resistant to the bacteriophage WA11. Populations were transferred to fresh flasks every 24 hours for 10 days with their designated selective conditions. Populations were assayed in 96-well plates to determine growth fitness under ampicillin or gallium nitrate exposure.

Growth Fitness of Evolved Populations Treated with Ampicillin and Gallium Nitrate

Figure 3. Growth fitness data over 24 hours at increasing concentrations of ampicillin or gallium nitrate.

Red = adapted to ampicillin, Blue = adapted to gallium nitrate, Purple = adapted to alternating ampicillin and gallium nitrate, and Green = adapted to favorable growth conditions (absence of ampicillin and gallium nitrate.

Ampicillin

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�Gallium

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Alternating

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Growth

Sequencing Results

Increasing Concentrations of

Ampicillin or Gallium Nitrate

• We hypothesized that combinatorial selective pressures with phage, antibiotics, and heavy metals would deter multi-resistant phenotypes from arising.
• We assessed how ecological complexity influences the underlying genetic architecture.
• We found many populations, even those selected for growth only, overcame any constraints of adaptation and resulted in the ability of our populations to evolve multi-treatment resistance.

Boyd, S. M., Rhinehardt, K. L., Ewunkem, A. J., Harrison, S. H., Thomas, M. D., & Graves Jr, J. L. (2022). Experimental Evolution of Copper Resistance in Escherichia coli Produces Evolutionary Trade-Offs in the Antibiotics Chloramphenicol, Bacitracin, and Sulfonamide. Antibiotics, 11(6), 711.

McGee, L. W., Barhoush, Y., Shima, R., & Hennessy, M. (2023). Phage‐resistant mutations impact bacteria susceptibility to future phage infections and antibiotic response. Ecology and Evolution, 13(1), e9712.

Graves Jr, J. L., Ewunkem, A. J., Ward, J., Staley, C., Thomas, M. D., Rhinehardt, K. L., ... & Harrison, S. H. (2019). Experimental evolution of gallium resistance in Escherichia coli. Evolution, Medicine, and Public Health, 2019(1), 169-180.

Jeje, O., Ewunkem, A. J., Jeffers-Francis, L. K., & Graves Jr, J. L. (2023). Serving Two Masters: Effect of Escherichia coli Dual Resistance on Antibiotic Susceptibility. Antibiotics, 12(3), 603.

Funding Sources: National Science Foundation; Stephen and Sylvia Tregidga Burges Endowed Fund for Student Research; Faculty Collaborative Research Matthews Fund; Student Faculty Research in Physics and Biological Science Fund

Maintenance of Phage-Resistance for EC-WA11 Populations

Figure 5. Spot Assay. Clear zones indicate bacteriophage infection for EC-WT. EC-R-WA11 is resistant to WA11 infection. All evolved EC-R-WA11 populations maintained phage resistance throughout the course of the experiments. A) ancestors EC-WT and EC-R-WA11, and B) representative image for evolved populations.

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Figure 4. Growth fitness data at 40 mgL ampicillin or 1500 mg/L gallium nitrate. Populations were compared to the ancestors at a single treatment concentration. Surprisingly, populations selected for growth only fared equally well or better than other selective conditions when exposed to ampicillin or gallium nitrate.

An ANOVA was used to determine the overall effects of bacterial population, treatment concentration, and the interaction on the response variable, optical density (OD600).

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Pairwise contrasts were conducted to compare the OD600 of E. coli ancestors to each evolved population of E. coli for each treatment type. Dunnett test corrected for multiple comparisons.

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Error bars represent SEM

* represents p<0.05

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