A New Perspective on Bacterial Biofilm Defenses

Caltech researchers have reintroduced a classic technique to image the formation and growth of individual cells that make up biofilms, sticky masses of millions of cells that are often responsible for antibiotic-tolerant infections. The method will help answer longstanding questions about how biofilms behave, offering insights that have the potential to help combat them in the context of chronic infections.

The research was conducted in the laboratory of Dianne Newman, the Gordon M. Binder/Amgen Professor of Biology and Geobiology and Merkin Institute Professor, and was led by postdoctoral scholar Georgia Squyres. A paper describing the work appeared in the journal Proceedings of the National Academy of Sciences on October 6.

Biofilms as a whole have properties that individual cells do not possess. This includes group behaviors that protect them from antibiotics, like a defensive matrix. Squyres and Newman wanted to understand how cells in biofilm work together to build these defenses.

To do this, researchers first need to be able to image and track the behavior of all biofilm cells at once. Biological imaging is often done with fluorescent proteins, but these require oxygen to function. Crucially, the interior cores of biofilms are often devoid of oxygen, meaning that the usual methods to label cells fluorescently do not work.

In the new study, Squyres reintroduced a fluorescent technique to label the medium that cells grow in, rather than the cells themselves, using a cheap, nontoxic dye; the dye made the cells dark against a bright background. This method works even in the biofilm core and increases the amount of time that biofilms can be imaged at high resolution. By combining this method with an algorithm to detect the behavior of individual cells, Squyres was able to track biofilm growth and cell dynamics over many days.

The team used Pseudomonas aeruginosa, a commonly studied pathogen that is often responsible for infections, but the technique could be applied to any bacterial species that forms biofilms.

"What Georgia has done was technically challenging on multiple levels and is a big accomplishment," Newman says. "But even more than the technique, what impresses me is her vision for what to do with it. Leveraging the unique features of bacterial biofilms, she is asking questions that are opening new frontiers in developmental and cell biology."

With the new technique, Squyres answered an open question about how biofilm develops. Individual cells in a biofilm are surrounded by a gooey substance called the extracellular matrix, which includes DNA. This extracellular DNA, or eDNA, is released into the matrix when cells die and explode in a process called lysis. This eDNA is a major component of biofilm, helping cells stick together, giving it structural stability, and also retaining certain metabolites needed for biofilm development. Some antibiotics also get stuck in eDNA, which prevents them from reaching their intended cellular targets. Given that eDNA is released when cells die, certain cells in the biofilm effectively sacrifice themselves in order to keep up a continuous supply of eDNA in the matrix.

To understand how these sacrificial cells are chosen in the biofilm, Squyres wanted to visualize which cells were undergoing lysis in the biofilm and observe if they were located at any particular positions within the larger structure. She discovered that around one in every 10,000 cells lyses per hour, and that these cells are located at specific positions within the biofilm that are patterned by gradients of nutrients like carbon and oxygen. By mapping these lysis events, she was able to understand how the biofilm's eDNA matrix gets its shape.

"Antibiotic tolerance in biofilms is coordinated by individual cells, and my hope is that this work gives a new framework for how to study their behavior," Squyres says.

Next, Squyres plans to continue to develop these imaging techniques and use them to examine other components of the extracellular matrix and how they interact to give the biofilm its properties.

The paper is titled "Single-cell lysis patterns morphogenesis of eDNA in the matrix of Pseudomonas aeruginosa biofilms." Funding was provided by the Resnick Sustainability Institute at Caltech, the Damon Runyon Cancer Research Foundation, and the National Institutes of Health.