# An increase in environmental temperature within the growth range suppresses phage resistance in Escherichia coli

## TL;DR

This study shows that increasing temperature can suppress phage-resistant Escherichia coli, offering a potential non-antibiotic method for controlling these bacteria.

## Contribution

The study identifies a novel temperature-dependent vulnerability in phage-resistant E. coli that could be used for biocontrol without antibiotics.

## Key findings

- Phage-resistant E. coli strains exhibit increased membrane permeability and sensitivity to monocaprin.
- Elevated temperature (46°C) significantly reduces viability and regrowth of phage-resistant E. coli.
- Phage-resistant strains show increased Congo Red binding and autoaggregation at higher temperatures.

## Abstract

To develop countermeasures against phage-resistant bacteria without antibiotics, a detailed phenotypic characterization of phage-resistant Escherichia coli BW25113 was performed. Phage susceptibility testing of E. coli BW25113 deletion mutants involved in lipopolysaccharide (LPS) synthesis revealed that the first glucose residue of the LPS outer core was essential for infection by phage S127. From E. coli BW25113 cells that survived S127 exposure, four phage-resistant strains were isolated and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the phage-resistant strains had LPS with a smaller molecular mass compared with that of the E. coli BW25113 parental strain. Fluorescence microscopy after BacLight staining, along with comparisons of viable counts on non-selective versus selective media, indicated increased membrane permeability in the resistant strains, resulting in heightened sensitivity to monocaprin, a natural non-ionic surfactant. Furthermore, upon elevating the culture temperature from 30°C to 37°C, the resistant strains exhibited increased Congo Red binding and autoaggregation, which was not observed in the parental strain. Viability assays revealed that both the phage-resistant strains and deep rough mutants, such as ΔhldE and ΔwaaG, did not grow at 46°C. Notably, regrowth after phage S127 treatment at 37°C was significantly delayed in the E. coli BW25113 parental strain grown at 46°C than in that grown at 37°C. E. coli populations that became phage resistant owing to truncated LPS chains were highly sensitive to hydrophobic antibacterial substances and high temperatures, suggesting that these could be critical factors for controlling phage-resistant bacteria.

The application of phages in agriculture and food-producing environments often faces challenges in the control of phage-resistant bacteria. To effectively address this issue, a deeper understanding of the unique phenotypes associated with phage resistance is warranted. Few studies have suppressed the regrowth of phage-resistant populations without using antibiotics, based on detailed phenotypic characterization. Here, we report that the phage-resistant Escherichia coli population selected by lytic phage S127 was sensitive to elevated temperature and decreased viability at 46°C. Furthermore, Congo Red binding and autoaggregation, which have been reported to exhibit unique behaviors in E. coli deep rough mutants, were dependent on high culture temperature. Our findings highlight a novel, exploitable phenotype of phage resistance in host bacteria that could be applied to the biocontrol of phage resistance in foodborne pathogens without the use of antibiotics in practical settings.