Antibiotic Resistance

Cross-sectional infection model for skin infections: Histological section through a skin model grown in a test tube, which has been infected with Candida albicans – one of the commonest human fungal pathogens. Violet: Candida albicans, blue: skin model.

Dealing with Antibiotic Resistance

Antibiotic-resistant germs repeatedly spread panic among hospital staff and patients – and frequently cost human lives. The problem is that today’s antibiotics are powerless against these germs, and available treatments are often ineffective. Researchers are therefore looking for new ways of combating the mutant bacteria and fungi.

Do your tonsils hurt whenever you swallow? Are you constantly running to the bathroom, and feel a burning sensation when urinating? To treat these and other inflammatory infections, doctors prescribe a course of broad-spectrum antibiotics and the patient soon feels well again. But there are an increasing number of cases of infection caused by resistant bacteria or fungi that cannot be treated with the usual broad-spectrum antibiotics. This is a particular problem in hospitals. Despite the best efforts by hospital staff to comply with strict rules of hygiene, new outbreaks of infection keep on reoccurring.

One villain is the Acinetobacter bacillus, which upon entering the patient’s body can trigger an infection and cause blood poisoning – this is a potentially life-threatening condition. And then there’s the infamous “hospital germ”, MRSA or methicillin-resistant Staphylococcus aureus. What makes these bacteria so hard to combat is that their resistance is highly variable. Doctors often have to make an educated guess when choosing a suitable antibiotic. Each case requires a new investigation to determine which resistant strain is present, and decide what can be done to eliminate it. For an infected patient, every minute counts. The vital question is: which resistances does the bacterium carry, and which of the many available antibiotics will help?

Tracking down resistances

A number of clinics are working together with the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB in Stuttgart to track down resistant forms of pathogens. The number of resistances carried by bacteria is immense: some 60 to 80 different forms are known. Often, a single bacterium exhibits two or more resistances, hence the term “multi-resistant pathogens”. “By finding out which resistances a particular bacterium or fungus carries, we can enable doctors to target the treatment of infected patients by administering an antibiotic to which the pathogen is not resistant,” explains Prof. Dr. Steffen Rupp, department head and deputy director at the IGB.

Participating clinics send patient specimens containing resistant bacteria to the researchers at the IGB. The specimens are usually in the form of blood samples, but swabs and secretions are also possible. There are two causes of resistance. One is plasmids, i.e. DNA molecules that do not form part of the original genome and can replicate autonomously in bacterial or fungal cells. The other cause of drug resistance is mutations in the pathogen’s genome. The researchers therefore isolate the plasmids or bacterial DNA from the patient specimens, replicate this genetic material, and identify the sequence of individual building blocks. They then use a special software program to compare this sequence with a library of DNA sequences that code for known forms of drug resistance. In this way they can find out which resistance genes are present. It takes between one and four hours to complete the analysis. Then the doctor can be sure which antibiotic will help the patient.

Developing new antibiotics

An alternative route to combatting drug resistance is developing new antibiotics to which germs haven’t yet been able to build up a resistance. Researchers at the IGB are therefore designing screening systems to support this drug discovery process. They start by placing human tissue in contact with resistant bacteria or fungi, causing the tissue to become infected. Then they pit a whole library of active drug ingredients, containing tens of thousands of different components, against them. The tissue samples are deposited in microwell titer plates, arrays consisting of numerous tiny wells, to allow the researchers to keep track of which drug ingredients have been tested in combination with which samples.

A few days later, the researchers add a fluorescent dye to the samples, which is only absorbed by living human tissue cells. If light is emitted by the content of a well, this means that it contains live human cells. “In a sense, we’ve combined two screening processes in one,” says Rupp proudly. “If the cells are still alive, it proves that the active ingredient has destroyed the pathogen. Otherwise the bacterium would have invaded the human cells and killed them. Moreover, it provides us with a means of evaluating the toxicity of the drug ingredient, because if the cells are still alive it means that the drug has caused little, if any, cell damage.” In short, if a sample fluoresces, there is hope of finding a new active drug ingredient.

In this case, the researchers produce a larger quantity of the active ingredient and repeat the experiment using a series of different concentrations, for they need to know what dose is necessary to be sure of destroying the pathogen and at what level there is a risk of causing damage to human cells. However, this stage of the process demands a lot of patience, because numerous additional tests are required to guarantee the safety of the new active ingredient, and it may take five to ten years before it can be used to treat patients.

How insects can serve medicine

© Photo Andreas Vilcinskas/Fraunhofer IME

So far, so good. But how do the researchers choose the active ingredients for their screening tests? Where do they get their inspiration for new medicines? The answer is found in nature, according to researchers in Giessen at the LOEWE Center for Insect Biotechnology and Bioresources – a cooperative venture between the Fraunhofer Institute for Molecular Biology and Applied Ecology IME, the Justus-Liebig University (JLU) and TH Mitttelhessen University of Applied Sciences. The insects that fill most people with fear -- the carrion beetle, the rat-tail maggot, the common green bottle fly, and many other creepy-crawly creatures -- can indeed be useful to science.

What is so special about the genetic makeup of these creatures that they are able to kill off multi-resistant, alien bacteria? This is the question that the scientists aim to answer together with colleagues from French pharmaceuticals group Sanofi. A joint research team has been set up at Sanofi’s research laboratory in Frankfurt to work on this project. “Instead of fumbling around in the dark, we want to approach the question from a scientific point of view. Nature has provided solutions for almost every conceivable problem. By learning from nature, we hope to be able to create new pharmaceutical products,” says Professor Rainer Fischer, director of Fraunhofer IME. To do so, the research team led by Professor Vilcinskas in Giessen extracts genetic material from insects, replicates it in a culture medium, and performs tests to determine whether the active agents it contains are capable of killing pathogenic organisms.

Biofilms – crawling with bacteria

Makroalgen in Nahaufnahme
© Photo Fraunhofer EMB

Macroalgae (Chondrus crispus)

When looking for ways of combating drug-resistant germs, the role of biofilms should not be forgotten. These films can form on almost any damp surface, from rocks and the walls of buildings to door handles in hospitals, and as deposits on human skin and teeth. All of these places are crawling with bacteria and fungal spores, many of which can be resistant to antibiotics. They present no danger to people in full health, but if they come into contact with a weakened immune system, these resistant germs can multiply explosively, in which case treatment with antibiotics is ineffective.

Researchers at the Fraunhofer Research Institution for Marine Biotechnology EMB have found a possible way of preventing the formation of biofilms. “In collaboration with colleagues at the University of Lübeck and the Lübeck University of Applied Sciences, we have been able to isolate family of substances from macro algae and determine their effect on multi-resistant germs,” reports EMB team leader Dr. Ronny Marquardt. To do this, the researchers isolated various classes of compounds from algae, each containing up to a hundred different substances. They found that when these isolated classes of compounds are placed on a surface, no biofilm is created. In other words, substances derived from algae could help to prevent the formation of biofilms. The scientists are now conducting further experiments to determine which individual substances or groups of substances are responsible for this effect.

Another unanswered question is what mechanisms the substances use to achieve this effect. And: is there a mechanism that enables the multi-resistant germs to be killed while allowing the “good” bacteria to continue growing unharmed? As far as the door handle in the hospital is concerned, it wouldn’t matter if all bacteria were destroyed. But if used in an antiseptic hand rinse for staff and patients, it would be more useful if only the resistant germs were prevented from reproducing, because if all germs were sterilized it would adversely affect the skin flora. In around three years’ time, substances derived from algae could be available to protect surfaces such as door handles and floors, and as an additive to cleaning products. For skin applications, it is likely to take longer – say five or ten years.

Imitating insects: How can biofilms be destroyed?

The researchers at the LOEWE Center in Giessen are among those trying to eradicate biofilms. To do so, they are taking a closer look at insects whose larvae grow in a protective biofilm. The brood produces a special cocktail of enzymes that break down the biofilm. “These enzymes offer numerous possibilities for medical applications,” says Fischer. “Active drug ingredients can only penetrate the outer layers of a biofilm, whereas if the biofilm is weakened by an enzyme, we have a better chance of reaching the bacteria in the deeper layers and destroying pathogenic germs.”

The molecular biologists at the LOEWE Center have already managed to isolate these enzymes, and prove that their method works in principle. But there is still a long road ahead, and it will probably be another ten years before the enzymes can be used in a clinical environment. Possible applications for the enzymes are not restricted to fighting multi-resistant germs. The researchers also have one of the most widespread biofilms in their crosshairs: dental plaque. This biofilm consists of around 500,000 microbial species, not all of which are dangerous to health. Only those that reduce the acidity level contribute to the development of caries. “If we could specifically inhibit these germs, dental plaque would no longer be a cause of caries,” explains Fischer.