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Disease and treatment

Major research initiative on chronic infections in cystic fibrosis

Cystic fibrosis is an important hereditary disease, but despite intense research, there are still many gaps in the knowledge of how this disease enables bacteria to produce deadly chronic infections in the lungs. Danish researchers will develop better methods to study chronic infections in the airways of people with cystic fibrosis.

Chronic infections threaten the health of many people with diseases such as diabetes, chronic obstructive pulmonary disease, gastric ulcer and cystic fibrosis.

Researchers have had great difficulty in studying chronic infections because they often act very differently in the laboratory from what they do in patients.

For example, some bacteria may appear to be susceptible to certain antibiotics when examined in the laboratory, but when patients are treated with the same antibiotics, the bacteria are refractory to antibiotics, or vice versa.

There is therefore an urgent need for new approaches to investigate chronic infections, which is exactly what the group of researchers behind a major new research project are developing.

The discoveries made in this new project may be clinically crucial for many patients, who today are victims of bacteria, which infect their body and cause severe disease.

“Together with antibiotics, the body can combat most acute infections itself. However, for many people, antibiotics do not kill the bacteria and the infections become chronic. For example, the lungs of patients with cystic fibrosis weaken slowly until they no longer function,” explains the leader of the research project, Helle Krogh Johansen, Clinical Professor and Chief Physician at Department of Clinical Microbiology, Rigshospitalet, Copenhagen and Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby.

The Novo Nordisk Foundation awarded a Challenge Programme grant of DKK 60 million to Helle Krogh Johansen and her collaborators to realize the project, which will last 6 years.

Chronic infections create problems for many people

Chronic infections occur predominantly among people with an underlying disease.

This may include cystic fibrosis, chronic obstructive pulmonary disease or primary ciliary dyskinesia, but it may also include chronic wounds among people who have obesity or diabetes. A third possibility is chronic peptic ulcer.

Cystic fibrosis is Helle Krogh Johansen’s area of expertise. This and other lung diseases can create a breeding ground for opportunistic bacteria that exploit the weakened lungs to colonize our bodies.

• In cystic fibrosis, a genetic disorder produces abnormally thick and dehydrated mucus in the lungs, comprising an ideal medium for bacteria to proliferate.

• In primary ciliary dyskinesia, a genetic disorder causes the microscopic hairs (cilia) in the lungs to not function well in removing small inhaled particles and mucus from the lungs. When mucus and particles accumulate, this creates an optimal medium for bacteria.

• In chronic obstructive pulmonary disease, mucus-producing cells replace much of the ciliated lung tissue and, like the other diseases, this provides bacteria with an excellent opportunity to proliferate.

Thus, although these three diseases have three different backgrounds, bacteria can colonize the dysfunctional lungs in all of them. In addition, the lungs in all three diseases are frequently colonized by the opportunistic pathogen Pseudomonas aeruginosa.

“Chronic infection of the lungs most often means being infected with P. aeruginosa. This is not usually an infectious pathogen that causes problems for people, but it is an opportunistic pathogen that strikes if it detects any signs of weakness. This may include the lungs of people with lung diseases or in the chronic wounds of people with diabetes or burn wounds. You can also get ear infections with P. aeruginosa if the water in the swimming pool is not sufficiently chlorinated,” explains Helle Krogh Johansen.

Patients with cystic fibrosis are treated lifelong with antibiotics

Although P. aeruginosa may be an opportunistic invader that exploits a pair of weak lungs to create an ecological niche for itself, this pathogen does not usually invade when a child with cystic fibrosis is born.

Infants with cystic fibrosis who crawl around the floor at home rarely get infected with P. aeruginosa initially.

Other bacteria such as Haemophilius influenzae, pneumococci and staphylococci are the first to colonize the lungs.

Once these undesirable pathogens initially arrive in the lungs, they begin to cause inflammation in the lung tissue, paving the way for P. aeruginosa to colonize the lungs later – most often in adolescence.

“We can treat these initial bacteria with antibiotics, but we cannot do much about P. aeruginosa other than keeping it at bay with antibiotics. Even large doses of antibiotics cannot eradicate it. Cystic fibrosis patients therefore require lifelong treatment with antibiotics to keep the infection in check and preserve as much of the lung tissue as possible,” says Helle Krogh Johansen.

Major biobank with bacteria from patients with cystic fibrosis

Helle Krogh Johansen has conducted research on P. aeruginosa for many years.

Her research has enabled her to discover the complexity of chronic infections with P. aeruginosa in the lungs of people with cystic fibrosis.

In 2004, Helle Krogh Johansen started systematically collecting Pseudomonas bacteria from young cystic fibrosis patients, and she has since then continuously stored thousands of bacteria from a large number of patients. Today this is one of the largest and most complete collections in the world, comprising P. aeruginosa isolates from many patients covering more than 16 years of infection, and it is therefore possible to examine how infections develop from the very first time P. aeruginosa is detected until the infection becomes chronic in the lungs of individual patients.

A study of genetic data from 34 people showed that P. aeruginosa changes over time and adapts well to the lungs of people with cystic fibrosis.

“We found that some people had almost identical strains of P. aeruginosa. They had apparently infected each other, perhaps in the hospital, and this led to a change of practice at Rigshospitalet so that patients can no longer infect each other with P. aeruginosa. We prefer that young people delay becoming infected with P. aeruginosa for as long as possible,” says Helle Krogh Johansen.

Bacteria become resistant to antibiotics

The study also showed that the bacteria often act differently in the laboratory and in patient lungs.

Some bacteria carry antimicrobial resistance genes but are only resistant in the lungs and not when tested in the laboratory.

Conversely, other bacteria do not appear to have antimicrobial resistance genes but turn out to be resistant to antibiotics in the clinic.

“We also found, to our great surprise, that bacteria often develop the resistance genes in patient lungs several years before they become resistant to antibiotics in the laboratory. And when they become resistant in the laboratory, they suddenly become multidrug resistant in the lungs,” explains Helle Krogh Johansen.

Bacteria act differently in patient lungs than in the laboratory

Another study by Helle Krogh Johansen’s group shows that the bacteria also act differently in the lungs and in a petri dish in the laboratory.

In these experiments, the researchers analysed how all bacterial genes are expressed (the transcriptome) in patient lungs from sputum samples taken directly from cystic fibrosis lungs. They compared this with the transcriptome from the same bacteria in laboratory media.

This study also showed that bacteria act very differently in the lungs and in the laboratory.

“Again, we found clear evidence that laboratory tests usually differ greatly from what happens in real life. We also realized that it is very difficult to determine what takes place in the lungs of people with cystic fibrosis when they develop chronic infections with multidrug-resistant P. aeruginosa by merely examining secretions in petri dishes in the laboratory. We need a completely different way of doing such investigations for them to become clinically relevant,” says Helle Krogh Johansen.

Tiny tissue cultures to study lung tissue

The many acknowledgements of the shortcomings of the researchers’ opportunities to investigate infections with P. aeruginosa in a realistic research regimen led to the current research project.

Overall, this project aims to improve model systems for examining chronic infections with P. aeruginosa in the lungs of people with cystic fibrosis, chronic obstructive pulmonary disease and primary ciliary dyskinesia but also chronic wounds or peptic ulcers.

A team of international researchers led by Helle Krogh Johansen is collaborating on developing an organoid infection model.

Organoids are tiny three-dimensional tissue cultures or miniature organs that researchers can cultivate in the laboratory.

The researchers isolate stem cells from, for example, a biopsy, bronchoalveolar lavage or a nasal scraping.

They can then cultivate the cells in the laboratory and make them form tiny three-dimensional ball-like structures, in which the surface of the lung tissue faces inward into the structure.

However, this is not practical for examining tissue surfaces, so the researchers have developed a method to make the tiny structures turn inside out, so that the entire surface of the resulting structure will represent the surface of the lung tissue to which the bacteria adhere.

Nose and lung tissue almost identical

Helle Krogh Johansen explains that the researchers can also use cell scrape biopsies from the nose to make the organoids.

The tissues of the nose and lungs are almost identical physiologically, which is why researchers often call the nose and lungs the united airways.

Together with otolaryngologists from Rigshospitalet, Helle Krogh Johansen has taken the initiative to develop a new standard for treating people with cystic fibrosis by simply opening and cleaning the sinuses surgically so that antibiotics can penetrate and eradicate P. aeruginosa before they migrate from the nose and sinuses and colonize the lungs.

“This may help to delay P. aeruginosa infecting the lungs of people with cystic fibrosis,” explains Helle Krogh Johansen.

Researchers already have a checklist

Helle Krogh Johansen explains that the ingenuity of their newly developed model system is that researchers can go back to the 16-year-old bacterial isolates that they have stored in the freezer and try to recreate what happened in the early stages of infection in the patients.

They can determine the genome composition of the bacteria, they know about the trends in antimicrobial resistance and they know which treatment the patients have received throughout their infection.

“We have the checklist, and then we just have to try to recreate this in the model system. We can then also start manipulating the set-up and determine how the infection would have developed if we had done things differently,” explains Helle Krogh Johansen.

Discovering markers for developing chronic infections

Helle Krogh Johansen thinks that this research project can mark a new era in research on cystic fibrosis and chronic infections related to other diseases.

She hopes that the research project will, for example, find markers that can be used to predict when an infection with P. aeruginosa is becoming resistant to antibiotics.

This may relate to the expression of the bacteria or changes in a person’s immune cells and may provide clues about the future direction of the disease and the best way to treat it.

“The dream is to find markers that can quickly reveal when antimicrobial resistance is developing. We could also find other bacteria that contribute to P. aeruginosa developing antimicrobial resistance, and these are the ones we need to treat. Our model system enables us to discover these things,” says Helle Krogh Johansen.

Ensuring clinical relevance

The research project also aims to ensure that the results become quickly translated into benefits for patients. Otherwise, says Helle Krogh Johansen, she would never have begun the project.

She expects the research to make a difference, because everything so far indicates that the experimental set-up is robust and much better than previous experimental set-ups involving, for example, mice and rats.

“The greatest advantage is that we use people’s own airway tissue. This is like making a miniature model of the person, and we can therefore also use the model to determine something about the individual and deliver personalized medicine to people with persistent infections. The disease develops differently for each person, and we can reproduce it precisely for each person and study what could have been done or could be done to improve their condition,” says Helle Krogh Johansen.

In 2019, the Novo Nordisk Foundation awarded a Challenge Programme grant to Helle Krogh Johansen for the project Persistent Bacterial Infections (PERFECTION).

Helle Krogh Johansen
Dr. med., Chief Physician
The research area is (shared research between RH and DTU) bacterial airway infections in patients with cystic fibrosis (CF). Most CF patients have bacteria in their lungs from early childhood until they die prematurely. The bacterial lung infections in CF patients, is an excellent model to study infectious disease for which antibiotic treatment is challenged by frequent lack of success. Equally important is that modern human life-style, as well as increases in the average population life span, will create problems with long-term bacterial infections that are difficult or impossible to treat. Moreover, the rising global problem of antibiotic resistance threatens to become the biggest health risk within the next 20-30 years. Our research is directly addressing the problem of antibiotic resistance.