Pleiotropy complicates a trade-off between phage resistance and antibiotic resistance

Abstract

Bacteria frequently encounter selection by both antibiotics and lytic bacteriophages. However, the evolutionary interactions between antibiotics and phages remain unclear, in particular, whether and when phages can drive evolutionary trade-offs with antibiotic resistance. Here, we describe Escherichia coli phage U136B, showing it relies on two host factors involved in different antibiotic resistance mechanisms: 1) the efflux pump protein TolC and 2) the structural barrier molecule lipopolysaccharide (LPS). Since TolC and LPS contribute to antibiotic resistance, phage U136B should select for their loss or modification, thereby driving a trade-off between phage resistance and either of the antibiotic resistance mechanisms. To test this hypothesis, we used fluctuation experiments and experimental evolution to obtain phage-resistant mutants. Using these mutants, we compared the accessibility of specific mutations (revealed in the fluctuation experiments) to their actual success during ecological competition and coevolution (revealed in the evolution experiments). Both tolC and LPS-related mutants arise readily during fluctuation assays, with tolC mutations becoming more common during the evolution experiments. In support of the trade-off hypothesis, phage resistance via tolC mutations occurs with a corresponding reduction in antibiotic resistance in many cases. However, contrary to the hypothesis, some phage resistance mutations pleiotropically confer increased antibiotic resistance. We discuss the molecular mechanisms underlying this surprising pleiotropic result, consideration for applied phage biology, and the importance of ecology in evolution of phage resistance. We envision that phages may be useful for the reversal of antibiotic resistance, but such applications will need to account for unexpected pleiotropy and evolutionary context.

Keywords: Escherichia coli; bacteriophage; efflux pump; trade-off; virus.

Fig. 1.

Phage U136B relies on TolC. (A) An EOP receptor screen of phage U136B reveals TolC as the candidate OMP receptor. A phage that produces an equal number of plaques on a knockout as on wild-type bacteria has an EOP of 1.0 (dotted line). A “bd” at the lower, dashed line indicates that the EOP was below the limit of detection (∼10⁻⁷). (B) Genetic complementation with a plasmid containing tolC fully restores plaquing ability by phage U136B on a tolC knockout. Error bars = 95% CIs. (C) Bacterial growth curves show that phage U136B lyses wild-type bacteria in liquid culture (∼1.5 h) but has no effect on the tolC knockout in liquid culture. (D) Single-step growth curves confirm phage U136B cannot grow on a tolC knockout in liquid culture.

Fig. 2.

Phage U136B relies on LPS. (A) An EOP screen of phage U136B on LPS synthesis gene knockouts reveals genes important to phage replication: rfaC, rfaD, rfaE, and rfaP. A “bd” at the lower, dashed line indicates that the EOP was below the limit of detection (∼10⁻⁷). (B) Genetic complementation with plasmids containing respective rfa genes fully restores plaquing ability by phage U136B on the knockouts. Error bars = 95% CIs. (C) Schematic of genes involved in LPS synthesis, showing that the genes required by U136B affect the deep region of core polysaccharide (regions highlighted in red, blue, and yellow). Modified with permission from refs. and , with permission from American Society for Microbiology, with additional data from refs. and .

Fig. 3.

Trade-offs between phage resistance and antibiotic resistance. (A and B) MICs for phage-resistant isolates from the fluctuation experiment. (D and E) MICs for phage-resistant isolates evolved in + phage treatment communities. (G and H) MICs for phage-sensitive isolates evolved in control −phage populations. (C, F, and I) A phage-mediated trade-off between colistin resistance and tetracycline resistance is evident in the fluctuation experiment (in C), but this result is alleviated after evolution (in F) and doesn’t appear in the control treatment (in I). In C, F, and I, a jitter of ±7.5% has been added to the data points for visualization, but regression lines are based on the original, nonjittered data.

Fig. 4.

Evolution of antibiotic sensitivity in bacterial populations while under selection for phage resistance. (A) Total bacterial population densities and (B) tetracycline-resistant bacterial densities in the presence and absence of phage.