Researchers from the Technical University of Denmark have demonstrated a new mechanism behind how bacteria develop antimicrobial resistance and thus a basis for chronic lungs infection among people with cystic fibrosis. The discovery can be developed into a new diagnostic tool that can guide treatment.
People with cystic fibrosis face a universal truth: bacteria in their lungs eventually become more and more resistant to antibiotics.
Slow growth is one way the bacteria adapt to and become tolerant of therapeutic antibiotics, even if the bacteria are not actually resistant to them.
When the bacteria grow slowly, their uptake of antibiotics changes, but so far researchers have not understood very well why and how the bacteria transition from growing rapidly to growing slowly and from being antibiotic sensitive to being antibiotic tolerant.
Researchers from the Technical University of Denmark have revealed this mechanism in a new study in which they reversed evolution.
“The problem has been that determining why the bacteria have different phenotypes is very difficult when they are in equilibrium, since they are in the lungs of people with chronic cystic fibrosis. We therefore had to alter this equilibrium to reveal the secret of the bacteria,” explains a researcher behind the new study, Ruggero La Rosa, Senior Researcher, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby.
The research has been published in Nature Communications.
Tracking how bacteria evolve over 1,000 generations
The background for the new results is the many years of research by Søren Molin and Helle Krogh Johansen at Rigshospitalet in Copenhagen.
The doctors and researchers at Rigshospitalet have painstakingly collected samples from people with cystic fibrosis, with some bacteria having lived for more than 50 years in their lungs.
These bacteria are as well adapted to this environment as they can possibly be. They grow very slowly, are in balance with the host’s immune system and are very unresponsive to antibiotics.
The researchers removed the bacteria from their state of equilibrium by placing them in an environment with plenty of nutrients, no immune system and no antibiotics. They then mapped the genetic modifications the bacteria made over the next 1,000 generations as they acclimatised to their new environment.
“The bacteria need to find a new point of equilibrium. They have the space and nutrients to grow, and they gradually revert to their origin before growing in the lungs of a person with cystic fibrosis. By mapping the genetic changes from slow to rapid growth and from antibiotic tolerance to antibiotic susceptibility, we can also understand the reverse process, which is the key to infections in the lungs of patients with cystic fibrosis,” says Ruggero La Rosa.
Bacteria revert to their genetic origins
The researchers used various molecular biological and genetic techniques to reveal how the genetic expression changed in the bacteria.
For example, they found that the bacteria in the lungs of a person with cystic fibrosis had cleaved off a piece of a gene that is important for metabolising nitrogen.
This means that the bacteria are poor at utilising nitrogen to grow, and they thus grow more slowly and become more tolerant of antibiotics.
However, as the bacteria evolved through the 1,000 generations, they repaired the functionality of this gene so that it was turned on again and the bacteria could grow more rapidly again. This also made them more susceptible to antibiotics.
Another example is the components bacteria use to produce the biofilm in which they grow in the lungs. The biofilm is a major part of how bacteria protect themselves against antibiotics but also against other bacteria.
In the lungs of a person with cystic fibrosis, the bacteria overexpress genes for producing biofilm, but these genes were turned off as the bacteria developed over time in the laboratory after the adaptation of the 1,000 generations.
“When we bring the bacteria back to very rapid growth, we can follow the genetic adaptations and thereby understand how the bacteria conversely adapted to the life in the lungs of people with cystic fibrosis. This adaptive laboratory evolution gives us completely new insights,” explains Ruggero La Rosa.
Slow growth can be a surprising evolutionary advantage
Søren Molin explains that the new study has some major perspectives.
The research provides new insights into how bacteria can adapt to new environments.
A mantra in evolutionary microbiology has always been that rapid growth is the key to success. For bacteria in the lungs, however, the situation is different, because they apparently benefit from growing slowly.
“It came as a surprise that the bacteria the lungs compete by growing not rapidly, but slowly. Our new data has convinced us that the increased tolerance to antibiotics is a major reason for this, and on the whole, it is very likely that stressful environments generally select for reducing the growth rate of bacteria. This is an interesting conclusion,” says Søren Molin.
Søren Molin also says that researchers noticed for several years that bacteria in the lungs of people with cystic fibrosis rarely develop traditional antibiotic resistance. The researchers also therefore wondered how these bacteria can survive continuous antibiotic treatment.
“We now know several reasons for this, but based on this study, we think that a significant reason is the slow growth, which makes sensitive bacteria tolerant. This helps to emphasise that traditional antibiotic resistance is not the only major problem. Increased tolerance can be an equally major problem,” adds Søren Molin.
May make diagnosis more precise
The other interesting perspective in the research result is clinical.
Søren Molin says that the results indicate that slow growth becomes a relevant factor for the bacteria after 2–3 years in the lungs of a person with cystic fibrosis.
The growth of the bacteria can therefore be used to indicate the degree of antibiotic resistance and therefore an increased risk of chronic infection.
If doctors regularly take samples from the lungs of people with cystic fibrosis, they can constantly monitor when the bacteria slow down their growth rate. This is a sign that they are now becoming more tolerant and that treatment may need to change.
“We have research projects in which we are investigating whether we can use bacterial growth to indicate antibiotic tolerance and thus chronic infection. If this is true, this can have great clinical significance. Currently identifying the point at which the bacteria begin to develop a chronic infection is difficult, but if we can determine this point, we can also target treatments more aggressively when it happens. This will mean that we can keep chronic infection at bay for longer than we can today,” says Søren Molin.
The researchers also state that, although the discovery was made for bacteria from the lungs of people with cystic fibrosis, the principle may be the same for other chronic infections, for which this insight may also have clinical significance.
These include lung infections among people with chronic obstructive pulmonary disease, chronic wounds among people with diabetes and people with stubborn urinary tract infections or chronic intestinal infections.
