New Method Tracks How Bacteria Behave Inside Human Cells

Assessing how dangerous a bacterium is has traditionally come down to one question: does it grow when exposed to a treatment, or does the treatment stop it? But infection involves more than survival. Whether bacteria enter human cells, how they spread or cluster, and whether they trigger DNA damage linked to inflammation or disease can matter just as much.

To capture this, researchers at the HUN-REN Biological Research Centre and the Hungarian Centre of Excellence for Molecular Medicine developed MALVINA (Machine Learning-Based Virulence Interaction Analysis), which detects fluorescently labeled bacteria inside human cells and applies machine learning to microscopy images. The method measures bacterial entry efficiency, intracellular accumulation, and induced DNA damage within the same experimental system. “One of the strengths of the method is that we can see the details of infection cell by cell. We can distinguish whether a few bacteria enter many cells, or whether large numbers accumulate in only a few cells, while also measuring the damage they cause,” said Bence Bognár, co-first author of the study published in Nature Communications.

By linking these processes at the single-cell level, and to bacterial genotype where known, MALVINA can separate infection patterns that look identical when averaged across a population. “What was particularly exciting in the experiments was that even with the same bacterial strain, individual host cells could behave very differently. Our method allowed us not only to observe these cell-to-cell differences, but also to quantify them,” added co-first author Terézia Kovács.

Tested on four E. coli strains, the method revealed distinct virulence profiles, including a colorectal tumor-derived strain that invaded cells efficiently, accumulated inside them, and caused DNA damage. “The special feature of this method is that we can see, within the same individual host cell, how many bacteria have entered and what damage the cell has suffered. This gives a much more precise picture of the consequences of infection,” said corresponding author Szilvia Juhász. 

The researchers also found that bacterial strains can alter each other’s behavior: an invasive strain linked to inflammatory bowel disease promoted entry of an otherwise harmless strain, while a genotoxic strain suppressed invasion by non-genotoxic rivals, partly through the genotoxin colibactin. Drug testing showed some treatments reduced viable bacteria while increasing invasion, indicating that drugs can reshape virulence as well as kill pathogens.

“MALVINA is powerful because it does not look at bacteria in isolation. It shows how they behave inside human cells and in the presence of other microbes. Infection is therefore not just a matter of bacterial numbers, but a process shaped by host cells, neighboring microbes, and drugs together,” said Viktória Lázár, another corresponding author. A patent application has been filed for the approach, which the team suggests could help identify invasive strains and inform drugs designed to disarm pathogens rather than simply kill them.