Infections with Clostridioides difficile (for short: C. difficile) are often recurrent, because dormant stages of the bacterium persist in the intestine. Thus far, there was no drug that can effectively combat the dormant stages as well. A current One Health study led by the Helmholtz Centre for Infection Research (HZI) and the Friedrich-Loeffler-Institute (FLI) now shows that a natural product called chlorotonil A can do just that. The study was conducted in close cooperation with the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Greifswald University and the Leibniz Institute DSMZ. The research team hopes that chlorotonil A will be a candidate drug that might significantly reduce the risk of reinfection with C. difficile in the future. The study was published in the scientific journal Cell Host & Microbe.
“Infection with the bacterial pathogen C. difficile can lead to severe diarrhoeal diseases in humans and animals. Especially so, if the natural microbiome of the intestine is disturbed, for example by previous treatment with a broad-spectrum antibiotic,” says Prof Till Strowig, who leadsthe Department of Microbial Immune Regulation at the HZI. “This provides C. difficile with ideal conditions in the intestine such that it can proliferate with impunity - leading to severe diarrhoea and intestinal inflammation, which may even turn chronic.” The underlying reason is that the pathogen forms so-called dormant stages that remain in the intestine and can lead to a new infection at a later time - when conditions are more favourable. Since the C. difficile infection also has to be treated with antibiotics, which prevents the microbiome of the intestine from recovering, a veritable vicious circle develops. Up to 25 percent of the people with a first-time C. difficile infection experience recurrence at a later point in time. About ten percent of the newly afflicted patients die as a result of the consequences of reinfection. According to the Infection Protection Act, any infection with C. difficile must be reported to the public health department. The pathogen is transmitted via contact infections.
“Unfortunately, the antibiotics commonly used to treat a C. difficile infection today are not as effective and long-lasting as one would wish. Our study addresses this problem,” says Strowig, who directed the large-scale multidisciplinary collaborative study jointly with Prof Thilo Fuchs, who is the deputy head of the Institute of Molecular Pathogenesis at the FLI. “We wanted to find out whether or not, and to what extent, the natural substance chlorotonil A, which originates from a soil bacterium, is effective against C. difficile, and how it influences the microbiome of the gut,” says Fuchs.
Intensive research on chlorotonil A (ChA) and its chemical variants has been conducted at the HIPS for more than the past ten years in order to improve them as potential pharmaceutical agents and optimise them for possible future use in humans. Against the background of ever-increasing bacterial resistance, there is an urgent need to develop new antibiotic agents - and the researchers see the chlorotonil variants as promising candidate drugs. “ChA has a broad antibiotic spectrum against Gram-positive bacteria and effectively combats, e. g., Staphylococcus aureus, which is a hospital-acquired pathogen also known as MRSA. In addition, ChA is also effective against the malaria pathogen,” says Prof Rolf Müller, who is the Managing Director of the HIPS where he also leads the Department of Microbial Natural Products. “For our study, we put two candidate drugs into the running: ChA and chlorotonil B1-Epo2. We were interested to find out how their efficacy compares to that of the antibiotic vancomycin, which is often used to treat C. difficile infections.” The research team used a broad range of methods in the investigations and combined the expertise of the research institutions involved in the project. Microbiological methods, animal models as well as state-of-the-art methods of molecular biology (multi-omics) were applied in the research. "The multidisciplinary approach of our study was crucial in achieving such comprehensive and extremely satisfactory results," says Strowig. His colleague Fuchs adds: “A special feature of our study was the complementary integration of all data into a model that can ease the fight against zoonotic diseases.”
In microbiological studies, the researchers found the two chlorotonil variants to demonstrate similarly good efficacy against C. difficile as vancomycin. ChA proved to be effective in the mouse model as well. “In the series of tests on mice, the antibiotics were discontinued after some time - and then there was a surprise effect,“ says Arne Bublitz, a PhD student with the Department of Microbial Immune Regulation at the HZI and first author of the study. “The majority of the mice that had previously been treated with vancomycin became ill again. In contrast, not a single one of the mice that had been treated with ChA experienced a reinfection. We asked ourselves: ‘What exactly is going on here?’” Bublitz went on to investigate this question in further microbiological studies. He treated permanent stages of C. difficile with ChA and then tried to bring them back to life by providing for ideal conditions for the bacterium. That did not work though: No viable bacterial cells developed from the treated spores. And even in mice infected with ChA-treated permanent stages, the disease was significantly attenuated. “By washing with solvents, we were able to find out that the active ingredient ChA most likely lodges in the water-repellent envelope of the permanent stages and forms a kind of drug depot there,” explains Bublitz. “Once the cell begins to germinate, it comes into contact with the embedded ChA and dies. ChA is one of the first examples of an antibiotic that is effective in combating both bacterial cells and permanent stages in vivo at low active concentrations.”
Another research focus of the study addressed the effects of ChA on the microbiome. To this end, a research team from the FLI conducted studies on pigs - whose digestive system is very similar to that of humans. “Using a wide variety of multi-omics methods, we were able to show that ChA damages the pig microbiome significantly less than other antibiotics,” says Fuchs. “In addition, ChA had a clearly negative effect on the gene regulation of the intestinal pathogen,” Strowig adds: “And we also obtained encouraging results in the mouse model: After ChA administration, the intestinal microbiome recovered more quickly than was the case with the administration of other antibiotics. So ChA does indeed seem to act very specifically and to disrupt the protective function of the microbiome very little.” Both scientists emphasise that ChA, as an active substance, does not change the concentration of intestinal metabolites such as proline or short-chain fatty acids, which promote the growth of C. difficile - unlike conventional antibiotics.
Chlorotonil variants are promising candidate drugs for treatment of C. difficile infections - as is clearly shown by the results of the study. “Especially the effect of ChA on permanent stages, which cause new outbreaks of the disease, might be a milestone in the treatment of C. difficile infections,” says Strowig. “We hope that our study will contribute to the further advancement of natural product-based drug research in our search for new effective agents for the fight against infectious diseases. After all, they may be able to attack target structures of the pathogens that were previously inaccessible to known agents.”
ORIGINAL PUBLICATION:
Arne Bublitz, Madita Brauer, Stefanie Wagner, Walter Hofer, Mathias Müsken, Felix Deschner, Till R. Lesker, Meina Neumann-Schaal, Lena-Sophie Paul, Ulrich Nübel, Jürgen Bartel, Andreas M. Kany, Daniela Zühlke, Steffen Bernecker, Rolf Jansen, Susanne Sievers, Katharina Riedel, Jennifer Herrmann, Rolf Müller, Thilo M. Fuchs, Till Strowig: The natural product chlorotonil A preserves colonization resistance and prevents relapsing Clostridioides difficile infection. Cell Host & Microbe, 2023, DOI: 10.1016/j.chom.2023.04.003.
