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Olaug Elisabeth Torheim Bergum and her colleague Amanda Holstad Singleton have studied how E. coli bacteria react when exposed to the antibiotic Ciprofloxacin.

How E. coli defends itself against antibiotics

When the bacteria detect damage to their genetic material, they send out an SOS signal that alters the activity inside the cells.

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“The bacteria go into full emergency mode,” says Olaug Elisabeth Torheim Bergum.

She is a researcher at NTNU's Department of Clinical and Molecular Medicine.

Imagine that you have a very sore throat. You are sick, your throat hurts, and a visit to the doctor confirms that the pain is due to a bacterial infection. You get a prescription for antibiotics, which quickly sorts out your sore throat. You are pleased that the treatment has worked – but how did the bacteria experience the situation?

The bacteria sustain damage

“Antibiotic treatment causes damage to the bacteria. This damage can be many different things, but it often involves damage to the genetic material, to the DNA, and activates an SOS response in the bacteriua,” says Bergum.

She has studied how the pathogenic bacterium Escherichia coli reacts when exposed to small, non-fatal amounts of the antibiotic Ciprofloxacin.

Ciprofloxacin is currently one of the most widely used antibiotics in the world and works by attacking the DNA in bacterial cells.

“It binds to a protein that helps maintain the proper structure of the DNA by cutting and splicing the DNA strands. This is necessary because copying and reading DNA creates stress on the DNA molecule,” she explains.

When bacteria are damaged by antibiotics, they activate an SOS response. Instead of growing, the bacteria work to repair the damage and change so they can better tolerate antibiotics. As this happens, they change shape, becoming thin and elongated.

Initial repairs

In other words, the protein keeps the DNA strand in order while the bacterial cell carries on functioning. When the bacteria need to divide, it ensures that DNA replication occurs in a safe and orderly manner. However, when the antibiotic binds to the protein, this function is prevented and things become rather chaotic.

Bergum explains that this results in damage to the DNA, including the formation of single strands of incomplete DNA within the cell.

When these individual strands form, it is like lighting a match under a smoke detector. Other proteins detect the damaged DNA fragments, and the alarm goes off. This is a dramatic situation for the bacterial cells, which can also be seen with the naked eye.

“At this point, it's all hands on deck. Many common activities in the bacteria, such as replication, are put on hold. This is reflected in the bacteria changing shape. Usually, E. coli is rod-shaped, but when exposed to Ciprofloxacin, they become long filaments. The bacteria prioritise repairing the damage,” she says.

If they are unable to repair the damage flawlessly, they will move on to the next step.

“If the repairs are ineffective, the last resort is to alter the DNA. This is when they mutate. This response helps the bacteria to adapt and become resistant to antibiotics,” says Bergum.

The bacteria were grown in a bioreactor. In such a reactor, the researchers have complete control over the growth conditions. This allows them to obtain results that are easier to reproduce.

Developing resistance

Normally, a course of antibiotics will be given in such large doses that the damage to the DNA is too great to be repaired. However, at NTNU's Department of Clinical and Molecular Medicine, researchers are interested in finding out what happens when bacteria actually win the fight against the antibiotic through their SOS response.

Knowing exactly how the repair processes and mutations occur is important to counteract antimicrobial resistance. 

Bergum and her colleagues have therefore chosen to look at both how ‘SOS genes’ are activated and how the bacteria use proteins and small molecules to repair the damage caused by the antibiotic.

“When the alarm sounds, 60 different genes are activated inside the cell. It was previously thought that the genes are activated at different times. That is, the genes used to create the proteins needed in the initial phase of repair work are activated first, and then the next set of genes are activated," says Bergum.

However, the researchers at NTNU found that all the genes are activated at the same time.

“The regulation does not take place at the gene level but at the protein level,” says Bergum.

This is new knowledge, according to the researchers.

“Our results are different from those produced by other studies. This may be because we have grown the bacteria in a bioreactor, where we have complete control of the growth conditions. This gives us results that are easier to reproduce,” she says. 

Three levels of the SOS response

The researchers have used methods that allow them to measure gene activation, proteins, and small molecules.

“This study is the first to look at all three levels of the SOS response at the same time. We've also conducted frequent measurements, from one minute after the bacteria are exposed to antibiotics until two hours later. This provides a good understanding of the timeline,” says Bergum.

You might then think that preventing the development of resistance should not be a problem, as long as a large enough dose of the antibiotic is provided. But Bergum says that it is not that simple.

“When treating an infection, not all bacteria will be exposed to the antibiotic to the same extent. This might be due to different uptake between different tissues and that some bacteria are naturally more resistant. Therefore, some of the bacteria can develop resistance,” she says.

Contributing to new medicines

Bacteria can also develop resistance in nature.

“There's a lot of antibiotics in water and sewage, albeit in low doses. It's therefore important to reduce the use of antibiotics,” says Bergum.

The new insight into how the SOS response works will be useful in the development of new medicines.

“The world needs new antibiotics and more knowledge about the mechanisms of resistance. By learning more about how the SOS response works, substances can be developed that attack these mechanisms. These substances, called inhibitors, can then be administered together with antibiotics like Ciprofloxacin to prevent the development of resistance,” she says.

About the research

The study was part of the TAMIR project (Targeting antimicrobial resistance by inhibition of bacterial stress responses), led by Professor Marit Otterlei at NTNU. The work is funded by the Trond Mohn Foundation’s national research programme at AMR and NTNU.

Reference:

Bergum et al. SOS genes are rapidly induced while translesion synthesis polymerase activity is temporally regulated, Frontiers in Microbiology, vol. 15, 2024. DOI: 10.3389/fmicb.2024.1373344

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