Multidrug-resistant germs

Bacteria are masters of adaptation, which is why they can survive in even inhospitable places like hot springs, the deep sea and highly radioactive environments. Every time antibiotics are used, it encourages resistance: Sensitive bacteria are killed off, while others find a way to survive, reproduce and change their genetic makeup so there is no longer a target for the antibiotic. That is why drug-resistant pathogens are especially common in places where a lot of these medications are used, such as hospitals.

With darobactin, though, there is hope: “Our studies show that bacteria that have changed their target to elude darobactin are less dangerous,” Schäberle explains. He and his team are now working to optimize the substance known as the molecular lead: “Nature did not develop this substance for use in humans. We need to make it more active against the pathogens that affect us while also ensuring there are no toxic effects.” However, Schäberle also says it is clear that a single new drug will not be enough to overcome the current antibiotic crisis. “We need a whole toolbox – many different solutions that can and should be combined to fight life-threatening infections successfully.” Biodiversity offers many possibilities, he says. “We need to throw everything we can at it,” Schäberle emphasizes.

Medicinal toxins

One possibility that has been little explored in the field of antibiotic research to date is animal toxins. Schäberle’s colleague Dr. Tim Lüddecke, who also does research at Fraunhofer IME, where he leads the Animal Venomics working group, wants to change that. Lüddecke explains: “Animal toxins hold a wealth of potential for new drugs. A number of important medications are based on them, like captopril, a drug widely used to lower blood pressure that contains a slightly modified toxin derived from the terciopelo, a venomous South American snake.”

In August, he and his team discovered that individual components of the family of toxins found in house pseudoscorpions are highly effective against one of the most common – and dangerous – germs found in hospitals: methicillin-resistant Staphylococcus aureus, or MRSA. Twenty to 30 percent of people have this pathogen on their skin or mucous membranes without becoming ill from it. But if it enters the body, for instance through a surgical incision, it can cause infections that often end up being severe. MRSA lacks sensitivity to a broad range of antibiotics.

Lüddecke and his team are focusing their research on poisonous and venomous animals found in Germany, like the house pseudoscorpion (Chelifer cancroides), which averages just three millimeters in size and tends to prey on the booklice that cluster inside books. Worldwide, there are some 3,000 different species of pseudoscorpions, also known as book scorpions. “Compared to true scorpions, they do not have a poisonous stinger. Instead, they deliver their venom through pincer-like structures called pedipalps, which makes them really unusual,” Lüddecke explains. They use their venom to immobilize their prey, which also includes mites and fruit flies.

But how do people get the toxin out of these tiny creatures? “Oh, it’s a lot of work,” Lüddecke says with a laugh as he describes the process. The Fraunhofer IME team built an intricate apparatus to “milk” the toxin from the pseudoscorpions. “It took forever to collect enough venom for our chemical analysis. But luckily, we only had to go through the procedure once,” Lüddecke recalls. Once the toxin cocktail has been decoded, it can be synthesized chemically or made using biotechnology. “That’s not only true of pseudoscorpion venom. The same goes for all the animal poisons and venoms we work with,” says Lüddecke. These substances are typically complex compounds of many different toxins. “There are spiders whose venom consists of as many as 3,000 individual substances,” Lüddecke explains. The tiny pseudoscorpion is an arachnid, just like spiders. Its venom might not contain quite as many active ingredients, but it does have one that packs quite a wallop: checacin, the name Lüddecke and his team gave to one toxin that is highly effective in fighting MRSA. It also targets other common germs found in hospital settings, like E. coli and Pseudomonas aeruginosa, along with a number of pathogenic yeasts like Candida. “But the really key thing is its high efficacy against MRSA,” says Lüddecke.
 

He and his team are still in the early stages of their research, and the exact properties and mechanisms of action of checacin are not yet clear. “Before the toxin can be considered as a drug candidate, we need to study aspects like whether the molecules remain stable in blood serum or break down quickly.” If that is the case, it will prove unsuitable for use as an antibiotic. For now, it will be faster — because there are fewer obstacles and less time and cost are involved – to use it in antimicrobial coatings. MRSA is frequently transmitted via medical instruments and equipment. This could be an especially helpful way to lower the risk of immunosuppressed patients becoming infected with the dangerous bacteria while in the hospital.

Safely producing phage cocktails

Researchers at Fraunhofer ITEM are working on safe alternatives. In the Phage4Cure project, they are working with the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures GmbH and Charité – Universitätsmedizin Berlin to demonstrate the efficacy of phage therapy and bring the first-ever phage-based drug to market in the EU.
The cocktail of three different phages fights Pseudomonas aeruginosa bacteria, which frequently infect the lungs, causing severe inflammation. Most strains are resistant to a large number of antibiotics. Dr. Sarah Wienecke and Dr. Imke Korf, both experts on biotechnological production methods and phage biology, achieved a breakthrough at the Braunschweig location of Fraunhofer ITEM: They not only identified phages with therapeutic potential but also produced their phage cocktail under GMP conditions, a key prerequisite for drug approval. GMP stands for “good manufacturing practice,” a body of stringent requirements relating to quality assurance, for example in relation to drug purity and safety as well as accurate and detailed documentation of all process flows.

“Producing phages is challenging, since no two are alike,” Wienecke explains. The bacteria-eating viruses have their individual preferences for factors such as temperature, culture medium and oxygenation. As a result, the three phages used to fight Pseudomonas aeruginosa are not produced together but rather in separate processes. They are then combined at a later stage. The scientists have developed a production platform that can also be used for other phages when individual adjustments are made. “The infrastructure for phage production is in place. We now know where to make adjustments to create ideal conditions for each phage. And the more phages we produce, the more experience we gain, which also translates to greater process acceleration,” Korf explains.

But before doctors could administer the phage cocktail to the first test subject at Charité, preclinical studies were needed to test for aspects such as undesirable side effects and toxicity and to determine the dose-response relationship. Dr. Dorothee Winterberg, head of the Preclinical Toxicology department at Fraunhofer ITEM in Hannover, comments: “The preparation is inhaled. We were able to show that this is safe because in the animal model, the phages did not go anywhere but where they were supposed to: the lungs. They did not migrate to the blood or other organs.” And that means there is no concern about side effects here, either. No negative effects were observed from high concentrations or daily inhalation of the substance for up to 14 consecutive days.
On the whole, the study results were so good that the German Federal Institute for Drugs and Medical Devices (BfArM) greenlit the start of clinical trials at Charité, which have been under way since the fall of 2023. The trials started with healthy volunteers and are now being conducted with patients suffering from chronic Pseudomonas aeruginosa infection of the lungs. Initial results are scheduled for publication in the fall of 2025.

But phages are not simply an improved version of antibiotics; bacteria can develop resistance to them, too. This means combining the two may be the ideal route. “We treated infected lung sections with our phage preparation and antibiotics and saw that it works much better than using just one or the other,” says Dr. Franziska Dahlmann, Group Leader Infection and Immunology. In the future, she hopes it will be possible to mix and administer phage cocktails on a targeted basis to kill off bacteria that have mutated to elude antibiotic therapy.
“Compounding” at pharmacies would be an ideal way to connect people with these substances, her colleagues Wienecke and Korf say: The cocktails could be customized and prepared individually there for each patient. This would eliminate the need for costly, time-consuming clinical trials while allowing for fast responses to emerging resistance and flexibility in adjusting the composition of the phage cocktails.

In the PhagoFlow project, they teamed up with the pharmacy, microbiology and surgery departments at the Bundeswehr Hospital in Berlin and with the Leibniz Institute DSMZ to test this approach – with good results. The pilot project was financed by the German Federal Joint Committee. “What I can tell you at this point is that the phage preparations were very helpful for individual patients at the Bundeswehr Hospital. We’re happy about every single case. When you spend this long researching something, it’s really nice to see that you’ve been able to make an impact.” But before this solution can enter widespread use, there needs to be a way to provide pharmacies with much larger volumes of phages for the different species of bacteria. After all, just like a bartender needs a variety of ingredients to do their job, a compounding pharmacist will also need access to a wide range of phage preparations to make customized cocktails for their patients. The phage production platform from Fraunhofer ITEM has created the conditions for large-scale mass production, but GMP requirements are too stringent to be able to speed things up much more at this point. “It would be important to agree on minimum requirements going forward so the production process can be streamlined and sped up to the degree needed,” Korf says.

Wastewater as treasure trove

Dr. Belinda Loh from the Fraunhofer Institute for Cell Therapy and Immunology IZI in Leipzig agrees that we cannot afford to continue to ignore phages in the fight against multidrug-resistant germs. With that in mind, she and her research group have teamed up with hospitals in central Germany to work on developing phage-based treatments as well.

To identify bacteriophages that are effective against common problem germs, Loh and her team make regular visits to wastewater treatment plants. “The dirtier the water, the better,” she says. After all, to find phages, you have to go where their hosts – bacteria – are clustered. “It’s a process of coevolution. You can’t find one without the other,” Loh explains. “It’s not exactly one of my favorite jobs. The water is so dirty that in some cases it isn’t even really liquid anymore, and the smell is truly awful,” she admits. But the effort is worthwhile, since the wastewater has proven to be a true treasure trove. By now, she has a collection of about 200 phages, abou half of them effective against Klebsiella pneumoniae, a much-feared germ found in hospitals that can cause severe pneumonia or even sepsis (blood poisoning) in vulnerable patients. It is increasingl resistant to commonly used antibiotics and can also contribute to further infections. Loh has also discovered phages that are effective at fighting other problematic pathogens such as Pseudomonas aeruginosa and multidrug-resistant enterococci.

In addition to the use of the phages themselves, Loh and her team are also researching therapeutic uses for individual phage proteins. She explains: “A phage is made up of DNA inside a protein envelope. It also produces further proteins that can kill off bacteria. To destroy the bacteria, we use certain phage proteins that poke holes in the cell wall and cell membrane.” Loh likens an indi­vidual bacterium to a water balloon full of water, which is then pierced with ultra-thin needles: “At some point, it bursts.” The advantage of this method is that antibacterial proteins are not limited to highly specific strains of bac­teria, instead having broader effects. “But simplifying the drug approval process is much more important,” Loh says. This is because unlike viruses, the therapeutic pro­teins can be produced as what are known as biologics. They could be used like chemical antibiotics. The issue, Loh says, is finding suitable proteins: “So, I look closely at every single phage and identify all the genes that could be of interest. Then I pro­duce the proteins separately in bacteria and test them.”

But although phage research is currently gaining momentum, it will still likely be some time before the first preparations hit the market. Until then, it is even more important to curb the spread of dangerous pathogens and minimize the number of infections, especially in hospitals and nurs­ing homes. To that end, a team of researchers at the Fraunhofer Institute for Manufacturing Engineering and Automation IPA in Stuttgart had a simple yet highly effective idea that has already been tested successfully at two hospitals: antimicrobial wall paint that reliably kills bacteria and viruses. Dr. Christina Bauder, head of the Applied Coating Technology team, explains: “We added photocatalytically active pigments to the paint. They are activated by natural light or artificial indoor light, at which point they form radicals that react with the surfaces of the germs and destroy them.” The patho­gens do not even need to come into direct contact with the wall surface, either; it is sufficient if the air circula­tion brings them into the immediate vicinity. “The nice thing about our method is that the chemical reaction continues indefinitely. The photocatalyst isn’t used up,” Bauder points out. That is because it uses oxygen and water from the air inside the room to produce radicals that are harmless to humans. And that means the paint remains effective permanently, unlike other antimicro­bial coatings that gradually release substances to kill germs. “Once those substances are used up, the protec­tion wanes. That can’t happen with our method. Even in darkness, our photocatalyst has residual activity lasting at least 24 hours,” Bauder says. The results of the field tests conducted at the Oberschwabenklinik medi­cal center in Ravensburg and at Kantonsspital Graubün­den hospital in the Swiss city of Chur are impressive: The paint was highly effective, and swabs taken by researchers showed that the walls were nearly germ-free.

Even with all these approaches and potential solu­tions, the antibiotic crisis is still very dangerous, as there is a particularly acute issue: Antibiotics are not lucrative. Prices are low, development costs high. “We should actually be able to do more about this creeping pandemic, as Lothar Wieler, former president of the Robert Koch Institute, once called it,” says Dr. Dorothee Winterberg from Fraunhofer ITEM. There are many research initia­tives, she says, but unfortunately not enough money. Winterberg sighs: “It’s enough to drive you crazy at times.” Prof. Till Schäberle from Fraunhofer IME agrees. “The market isn’t going to solve this on its own. The state needs to step in,” he adds. He has high hopes for large global funding initiatives like CARB-X, whose support­ers include the Bill & Melinda Gates Foundation, the U.S. and Canadian governments and the German Federal Ministry of Education and Research (BMBF). Schäberle is firm in his prediction: “It is necessary, so we will see action on this front.”

Whether phages or antibiotics are used, successful treat­ment depends on rapid diagnostic systems, which ideally identify the pathogen causing an infection, along with any resistance, right there at the hospital. A team at the Fraunhofer Institute for Cell Therapy and Immunology IZI, Bioanalytics and Bioprocesses institute branchin Potsdam is working on this. Emily Mattig, a technical employee in the Point-of-Care Technologies working group, explains: “The traditional method of detection through a blood culture takes anywhere from five to seven days. Our goal is to do it in four hours.” Cutting the time involved can save lives in cases of severe infection, especially sepsis, also known as blood poisoning. The trick: Mattig and her team detect the bacteria based on their DNA instead of incubating the blood sample in spe­cial culture media and waiting for potential microorgan­isms to slowly grow so they can be identified. To achieve this, the researchers first isolate the bacterial DNA, reproduce it and then deposit it onto a microarray – a chip pre-loaded with numerous DNA counterparts from various bacteria.

“If the DNA binds to one of these counterparts, which we call probes, we’ve found the pathogen,” Mattig explains. She and her colleagues have succeeded in developing modified probes to cover resistance as well: “In the case of resistance, it’s not enough to simply detect the gene. We need to find specific point mutations in the gene. But we can identify them, too.” The microarray is tiny, holding 5,000 probes in an area of only about three square centimeters. The entire diagnostic system would fit into a hospital exam room with no problem. And that means in the future, there might be no need to involve external labs, a time-consuming process.