Biofilms study reveals how multiple bacteria species manage to coexist

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

A new study by Dartmouth scientists in Current Biology uses experiments and modeling to delve into how three species of biofilm bacteria coexist—and when they move out on their own.

One species, Pseudomonas aeruginosa, a versatile pathogen known to be antibiotic resistant, dominated over the other two bacteria. But the species migrated in search of greener pastures when the surface became too crowded rather than staying behind to compete with its cohabitants. By striking out on its own, Pseudomonas allowed the whole colony of bacteria to thrive.

“Pseudomonas’ dispersal behavior allows for the three species to coexist where otherwise they would not,” says corresponding author Carey Nadell, an assistant professor of biological sciences at Dartmouth. “This is the first case of showing explicitly that dispersal has very important ecological consequences when you’re thinking about 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, so understanding how they interact could enhance understanding of these infections.

“We wanted to know how biofilms can support a diversity of species or strains because we know that bacteria are really good 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 motivation—if they’re so good at these antagonistic behaviors, how do they coexist in these tightly 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” an equal number of each bacterium in each environment then used fluorescence microscopy to examine how the relative abundance of the different species changed over time.

In the liquid culture, P. aeruginosa boomed and completely outcompeted the other two species after about three days. However, in the biofilm environment, the researchers saw a very different dynamic play out.

At first, the E. faecalis and E. coli populations grew more rapidly than P. aeruginosa, but after a few days, the P. aeruginosa population rapidly increased and began to displace the other two species. However, the P. aeruginosa population shrank once the biofilm became densely crowded, which allowed the other two species to bounce back. Shortly thereafter, P. aeruginosa began to take over once again—and the cycle repeated.

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

To test this hypothesis, the researchers repeated the experiment with a genetically engineered 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 seen in the liquid culture and supporting their model’s findings.

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

“It’s important to push for more ecological realism. The inferences you gain from well-mixed liquid bacterial cultures often may not apply to biofilm environments, which are much more common in the real world,” he says. “It points to the importance of studying these communities in a context with a little bit of added 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 bacteria often grow in their marine environment.