People with lung diseases are treated long term with low doses of drugs called macrolides. They modulate the immune system but also have a bacteriostatic effect. Now researchers have found that these compounds – surprisingly – also create antibiotic resistance among people with cystic fibrosis with Pseudomonas aeruginosa infection. The research shows that antibiotics might work among people with cystic fibrosis if used correctly but requires testing for and investigating these types of resistance.
People with cystic fibrosis have a plethora of bacterial lung infections that often become chronic and can last for decades. They are therefore treated with a variety of varying antibiotic, immunomodulatory and supportive treatments. One of the important types of treatment is macrolides, which were originally developed as antibiotics, but for people with cystic fibrosis, they are typically prescribed as anti-inflammatory supportive treatment.
“People with cystic fibrosis typically become infected with Pseudomonas aeruginosa bacteria. According to international guidelines, these bacteria do not respond to macrolides, but our new study shows that this is not true” explains a main author of the study, Ruggero La Rosa, Senior Researcher, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby.
The researchers noticed several mutations in the known antibiotic-binding site for macrolides in our collection of clinical strains of Pseudomonas aeruginosa, indicating that the antibiotics develop resistance.
So, these macrolides help to fight the infections if used correctly, but if they are misused they could actually promote the development of chronic infections.
A little bit more space in a specific pocket
The researchers’ interest arose from their interest in the ribosome – one of the most important organelles inside the cell and the machinery within all cells that translates genetic templates into proteins. In their collection of bacterial strains, specifically in Pseudomonas aeruginosa, the researchers noticed an unusual number of mutations accumulating during infections of people with cystic fibrosis.
“The general idea is that the ribosomes are so important to the functioning of the cell so that, in principle, you should not be able to change them too much, because you would then somehow change their functioning and adversely affect the ribosome and the cell. So we were wondering why these mutations accumulate. As expected, many mutations might harm the cells, but some might actually benefit the bacteria, and we found an interesting mutation that was causing antibiotic resistance,” explains Ruggero La Rosa.
So apparently the benefits of antibiotic resistance were greater for the bacteria than any harm caused by changes to the otherwise stable and essential ribosome cell machinery. The findings made the researchers examine these mutations more closely.
"Whereas the antibiotics would normally block protein synthesis in the bacteria, the changes created a little bit more space in a specific pocket where the antibiotic binds, so that the translation of proteins could continue – even in presence of the antibiotics."
Pseudomonas aeruginosa is considered intrinsically resistant. And indeed, testing macrolide susceptibility, using standard methods used in every clinical laboratory, shows that the measure for resistance – the minimum inhibitory concentration – is extremely high, meaning that the strain looks like it is intrinsically resistant.
"But when one of our smart postdoctoral fellows started examining that, she got an idea,” says Ruggero La Rosa.
Postdoc Lise Goltermann realised that the standardised medium used for testing resistance was not at all similar to the specific environment of an infection.
“So, she started altering the medium composition and realised that, by changing the composition to match that of an airway infection, she could decrease the resistance measure for the wild-type standard laboratory strain. And then when she used the same media on our clinical strains with these specific mutations, the minimum inhibitory concentration readings dramatically changed,” explains Ruggero La Rosa.
Need to start testing for resistance
Pseudomonas aeruginosa is a major cause of airway infections and the predominant cause of illness and death among people with cystic fibrosis. Antibiotics to effectively treat Pseudomonas aeruginosa are therefore crucial. Equally important in treatment, however, are immunomodulatory compounds, such as the macrolides, since they reduce the production of proinflammatory cytokines and reduce chronic inflammation.
“Some of these people are treated very extensively with macrolides because of their effect on the immune system. But we have to be aware that, because macrolides are also antibiotics, we are generating antibiotic resistance without even knowing it and without even screening for antibiotic susceptibility or resistance,” says Ruggero La Rosa.
Ruggero La Rosa adds that this is worrying, because the mechanism of resistance is not completely known and because Pseudomonas aeruginosa is building up resistance genes.
“The good news is that Pseudomonas aeruginosa is sensitive to these macrolides and that they actually might work as antibiotics among people with cystic fibrosis. But we need to start testing for resistance and need to start considering macrolides as real antibiotics like other antibiotics. It is important to start looking at these types of resistance,” explains Ruggero La Rosa.
Susceptibility testing is the real challenge
The researchers have started to examine the types of resistance arising from the 529 isolates from people with cystic fibrosis. By analysing almost 6000 protein sequences, they have found many types of changes, and even though they have not finished the work yet, many of these changes worry them.
“This is still work in progress, but we have already found that, since the ribosomes are such central components of the cell, as soon as they mutate, you get a completely different phenotype of the bacteria. What we notice is that these mutations basically also change the expression of virulence factors,” says Ruggero La Rosa.
In acute infections, bacteria deploy several virulence factors to kill their host. But in chronic infections, the goal for bacteria is not to kill the host immediately. By changing their virulence factors, the host can survive and the bacteria can hide more easily when the immune system of the host is less active.
They want to be as silent as possible to survive for as long as possible. In this way, the infection becomes chronic, so changing their virulence factors can be a great advantage for bacteria.
Hoping to get closer
Thanks to a Novo Nordisk Foundation Challenge Programme grant obtained by Helle Krogh Johansen – co-principal investigator of the Infection Microbiology group of the Novo Nordisk Foundation Center for Biosustainability together with professor Søren Molin.
"We are building an infection model system to start asking such questions as how antibiotic resistance mutations affect the overall phenotype; how the infection changes if you get those mutations,” explains Ruggero La Rosa.
Until these studies are finished, Ruggero La Rosa says that the focus should be on how bacteria in clinical practice are tested for potential antibiotic resistance. Designing better susceptibility testing and making clinical decisions on treatment based on susceptibility testing is the real challenge. A clinical laboratory tests thousands of strains every day.
“So you need to have a reliable and standardised testing method, but our studies indicated that resistance is overlooked in the standardised medium used now. Maybe if we get closer to the media specific to the infection we can learn much more and carry out better diagnosis and eventually improve treatment. If we use them in effectively, macrolides are not just immune modulators but also effective antibiotics for treating people with Pseudomonas aeruginosa infections,” concludes Ruggero La Rosa.