Biofilm study reveals how multiple species of bacteria manage to coexist

Biofilms, slimy communities of bacteria, grow on all kinds of surfaces: from glaciers and hot springs to plant roots, your bathtub and refrigerator, wounds and medical devices such as catheters . Most biofilms are composed of multiple bacterial species, but how these species manage to live together remains unclear.

A new study by Dartmouth scientists in Current biology uses experiments and modeling to understand how three species of biofilm bacteria coexist and when they move on their own.

One species, Pseudomonas aeruginosa, a versatile pathogen known to be resistant to antibiotics, dominated the other two bacteria. But the species migrated in search of greener pastures when the surface became too populated rather than staying put to compete with its co-inhabitants. By attacking alone, Pseudomonas allowed the entire colony of bacteria to thrive.

“The dispersal behavior of Pseudomonas allows all three species to coexist where they otherwise would not,” says corresponding author Carey Nadell, assistant professor of biological sciences at Dartmouth. “This is the first case explicitly showing that dispersal has very important ecological consequences when considering biofilms as a community.”

The researchers examined a community of three bacterial species: P. aeruginosa, Escherichia coli and Enterococcus faecalis. All behave as opportunistic pathogens and are frequently isolated from catheter-associated urinary tract infections. Understanding how they interact could therefore improve the understanding of these infections.

“We wanted to know how biofilms can support a diversity of species or strains, because we know that bacteria are very efficient at killing each other,” says first author Jacob Holt, a graduate student in Nadell’s research group. who led the study. “So that was a big motivator: If they are so good at these antagonistic behaviors, how can they coexist in these closely associated communities?”

To investigate, the researchers grew the three species on a glass surface conducive to biofilm development and in a well-mixed liquid culture. They “seeded” equal numbers of each bacteria in each environment, then used fluorescence microscopy to examine how the relative abundance of different species changed over time.

In the liquid culture, P. aeruginosa exploded and completely outcompeted the other two species after about three days. However, in the biofilm environment, the researchers observed very different dynamics.

Initially, populations of E. faecalis and E. coli grew faster than those of P. aeruginosa, but after a few days, the P. aeruginosa population grew rapidly and began to displace the other two species. However, the P. aeruginosa population declined once the biofilm became densely populated, allowing the other two species to rebound. Soon after, P. aeruginosa began to take over again and the cycle repeated itself.

When the team tested different theoretical models to explain these cycles, the best model was surprisingly simple. “The fundamental mechanism is very simple,” says Holt. “When a dominant species reaches a very high abundance, it selectively removes itself from the system, allowing other species to remain.”

To test this hypothesis, the researchers repeated the experiment with a genetically modified mutant strain of P. aeruginosa that lacks the ability to disperse. In this case, the biofilm became completely dominated by P. aeruginosa, mirroring the results observed in the liquid culture and supporting the conclusions of their model.

These findings highlight the importance of conducting research in realistic settings, says Nadell.

“It is important to promote more ecological realism. The conclusions that can be drawn from well-mixed liquid bacterial cultures often do not apply to biofilm environments, which are much more common in the real world,” he says . “This highlights the importance of studying these communities in context with a little more realism.”

The team plans to build on this realism component in future research. For their next project, Holt and Nadell are working to grow Vibrio cholera, the bacteria that causes cholera, on shrimp shells, the substrate on which the bacteria often grow in their marine environment.