Using a probe: A luminous spy molecule visualizes bacterial life

Russian scientists have synthesized an organic compound that can penetrate bacterial cells without destroying their natural habitat — biofilm. At the same time, this molecular "probe" has the ability to glow, which allows specialists to see the processes taking place in the "settlements" of microorganisms. According to experts, the technology will make it possible to understand how antibiotics work in natural conditions, and will help solve the problem of microbial resistance to drugs.

Luminous Molecular probe

SFU specialists have synthesized an organic compound that can stain bacteria and transmit information about their environment, changing optical properties depending on the conditions in which they find themselves. This will enable scientists to use a microscope to see the processes occurring in microorganisms in their natural state, which is important for the development of, for example, new antibiotics. Currently, biologists mainly study bacteria in laboratory dishes, but in living nature they form their own unique biocinoses — biofilms. Using a luminous molecular "probe" will help to study them without destroying them.

— Our work is devoted to the synthesis and research of a family of new fluorescent probes based on benzimidazolium. As a result of studying the spectral and fluorescent properties, it turned out that they demonstrate a high potential for using such compounds as pH-sensitive fluorescent probes. First of all, they are suitable for pH visualization in bacterial biofilms, which was demonstrated during pilot bio—imaging experiments using a fluorescence microscope," said Artem Pugachev, senior researcher at the Laboratory of Special Organic Synthesis at the Laboratory of Ecology and Molecular Biology of Microorganisms at the FOH Research Institute.

Now, when scientists take bacteria for analysis, they usually rip them out of the environment, we do seeding, and the microorganisms grow in a new environment, already in a laboratory dish, or we destroy them and do their chemical or genetic analyses, explained Mikhail, a researcher at the Institute for the Study of Aging at the Russian Gerontological Research and Clinical Center at Pirogov University. Bolkov.

— In their natural environment, they are inside biofilms and organize their own closed biocenosis, which protects them from immune factors or from some juices and all kinds of acids. They have their own infrastructure where they can interact with other microorganisms. And understanding how they will act in natural conditions will help us uncover possible targets for influencing them in a living body. This is very interesting, including for the creation of new antibiotics or antiseptics," the scientist said in an interview with Izvestia.

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In addition, there are not only harmful but also beneficial bacteria that need to be studied. For example, with the help of a probe, it is possible to understand how they function on the mucous membrane of the throat, the specialist added.

The selected substance is sensitive to the level of acidity and changes its optical and fluorescent properties depending on this parameter and is excited by the light of the visible spectrum. Once in the biofilm, for the first three hours it stains only the extracellular matrix of bacteria— the mucous layer in which they are immersed. But if the time is increased to 24 hours, the dye penetrates into the bacterial cells, which leads to the appearance of a second maximum glow. That is, they can be used to visualize the structure and physiology of bacterial biofilms, which makes them an excellent tool for microbiologists and ecologists, the developers said. They are also suitable for research in the field of physiology and neurophysiology.

A tool for new medicines

The main difficulty of the development was to create a molecular probe design that functions in the required pH range from 4.5 to 7.5, since most representatives of classical fluorescent compounds are not capable of this.

"In the near future, we plan to test other substituents in the molecular structure of the studied probes to improve their spectral characteristics, and only then we will try them on other models, for example, on cancer cells or on brain tissues," said Artem Pugachev.

The study of bacterial biofilms is one of the most urgent tasks of modern microbiology and medicine. In real conditions, bacteria are more likely to exist in this form, and in this state they are usually much more resistant to antibiotics, antiseptics, and environmental factors than free-floating cells. That is why biofilms underlie chronic infections, hospital-acquired infections, and resistance to therapy, Albert Rizvanov, head of the Personalized Medicine Center of Excellence at Kazan (Volga Region) Federal University, told Izvestia.

— Fluorescent dyes and probes for biofilm imaging already exist in the world, for example, markers of living and dead cells, extracellular matrix dyes or pH—sensitive probes. However, most of them either do not allow fine analysis of the microenvironment, or work in a narrow range of conditions. Therefore, the emergence of new pH-sensitive probes, specially adapted to a biologically significant range of acidity, is in high demand," said the specialist.

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Such dyes can be used not only to visualize the structure of biofilms, but also to study the effects of antibiotics, for example, to monitor how the pH and permeability of the matrix change during treatment. In addition, such tools are promising for screening and searching for new antibacterial compounds, allowing a quick assessment of whether a drug penetrates into a biofilm and whether it affects its physiology. In this sense, the development of Russian scientists fits into the global trend of creating "smart" molecular tools to combat antibiotic resistance, the scientist noted.

According to Valeria Gresis, an assistant at the agrobiotechnology department and head of the laboratory at the ATI RUDN University, so far we are talking about a niche method that requires further development and confirmation. At the same time, as a foundation for further research, the work looks reasonable.

The study was supported by the Priority 2030 program.