λ phage Induction Under Infection-Associated Stressors in Escherichia coli: Implications for Antimicrobial Resistance

Abstract

Lysogenic phages, also known as temperate phages, play a crucial role in horizontal gene transfer and the dissemination of antimicrobial resistance (AMR). Upon induction, they may package bacterial DNA alongside or instead of their own, facilitating the spread of antibiotic-resistant genes. Phage induction can be triggered by the host bacterium's stress responses. While stress-induced prophage activation is well studied, most studies have investigated it under artificial or environmental conditions that do not accurately reflect the physiological environment during infection. This study aims to investigate how infection-relevant stressors (oxidative stress, elevated temperature, and nutrient availability), individually and in combination, affect prophage induction in Escherichia coli (E. coli) carrying prophage lambda (λ).

We hypothesized that each of these stressors influences induction differently, with potential synergistic effects when combined. To simulate infection-like conditions, E. coli cultures were exposed to physiologically relevant concentrations of hydrogen peroxide, fever-like temperatures, and limited nutrient availability, both individually and in combination. Prophage induction was assessed by measuring the ratio of phage to host DNA using droplet digital PCR (ddPCR). Additionally, a recA-GFP reporter system served as a proxy for phage induction and the activation of the SOS response.

Our results indicate that elevated temperature may have no effect on prophage induction, while nutrient limitation suppresses it. Moreover, nutrient deficiency appears to have the main effect on prophage induction when coupled with elevated temperatures. Lastly, higher concentrations of hydrogen peroxide cause bacterial stress, suggesting that they may increase prophage induction.

These findings improve our understanding of phage dynamics during infection, with implications for microbiota stability and the spread of AMR. This highlights the need to study other infection-relevant stressors (pH, antimicrobial peptides, etc.) and their combined effect and role in phage induction.

Popular Abstract

Bacteria Under Pressure: When Phages Jump Into Action

Phages are viruses that infect bacteria. They make bacteria their home and use their resources to replicate themselves. Once they’ve made enough copies, they pack themselves, burst the cell open, and leave to move into other houses (infect other bacteria), killing the original host in the process. Some phages can hide inside bacteria in a dormant state called a prophage, but certain stress signals can wake them up (induce them). Once awoke, they replicate, pack, burst the cell, and move on. Sometimes, during the packaging process, they accidentally take pieces of the bacteria’s DNA with them. When they infect the next bacterium, they can release this DNA, much like delivering genetic mail. If the mail contains instructions on how to resist antibiotics, bacteria become antibiotic-resistant. This mail-delivery system is called transduction.

Millions of deaths are caused by antibiotic-resistant bacteria, so understanding what wakes up the prophages and causes them to spread resistance is important. Thus, we ask: what stressors do bacteria face, and could one of those wake up the prophages, thereby helping the spread of antibiotic-resistant genes?

When bacteria invade our body, they are met by the immune system. Unfortunately, the immune system is not very welcoming. It hits them with heat (fever), hides nutrients from them, and traps them into immune cells, where it tries to kill them with toxic molecules such as hydrogen peroxide. To see if these stressors wake up the prophages, we tested them using Escherichia coli and phage lambda (λ) with two methods. One method, the GFP assay, measured fluorescence as an indicator of phage induction and SOS response. Where the higher fluorescence indicated increased phage induction. While the other method, digital droplet PCR (ddPCR) was more direct and measured the amount of bacterial and phage DNA. If there is more phage DNA than bacterial DNA, the prophage is awake (induced).

The results showed that when the bacterial cells are starved, prophages slip into an even deeper sleep, similar to hibernation. As long as the cells remain starved, they stay asleep (induction is reduced). Fever alone caused stress to the bacteria, but it did not seem to impact the prophages in any way. In addition, when starvation and fever were combined, starvation had the main effect on the prophages. Hydrogen peroxide showed signs that it might also wake up prophages. However, more research is needed to confirm this.

Overall, our findings highlight that it’s not always one stress signal that is enough to wake up a dormant prophage, but rather their synergistic effect. This shows the importance of studying these stress factors not in isolation, but in combination, as they would normally occur during an infection. Understanding how and when prophages wake up can help us better predict and understand the spread of resistance genes.

Master’s Degree Project in Molecular Biology 60 credits 2025
Department of Biology, Lund University
Advisor: Rolf Lood-Alayón
Department of Clinical Sciences, Lund, Division of Infection Medicine

• author: Jendris, Marija
• supervisor: Rolf Lood
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20242
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9214506
• date added to LUP: 2025-10-24 15:18:30
• date last changed: 2025-10-24 15:18:30