As single cell organisms, bacteria are highly mobile. They swim using flagella, which are long and whip-like, and move across surfaces using hair-like pili. Scientists assumed that bacteria lost their mobility once they organized into aggregates known as biofilms, but a new study shows otherwise (1).  

Columbia University scientists and their colleagues at City University of New York and the University of Chicago, found that bacteria in biofilms use their pili to arrange themselves and optimize their access to nutrients and oxygen. The arrangement, once thought to be random, follows a highly ordered configuration that has implications for how substrates, including antibiotics, distribute throughout the biofilm. The findings, published in PLOS Biology, contribute to a deeper understanding of biofilm resilience and potential vulnerabilities that new antibiotics could exploit. 

“The paper successfully connects cellular organization with functional outcomes,” said microbiologist Jing Yan at Yale University who was not involved in the study. “This link is important because we can guess why it is advantageous for cells to organize in one way versus another, but we don’t have the appropriate tools to study that.” 

The scientists used scanning electron and fluorescence microscopy to image a Pseudomonas aeruginosa  biofilms formed by pipetting a few microliters of cell suspension onto agar-solidified media.  After several days, they noted two distinct zones: an ordered zone with cells aligned in vertical striations and a disordered zone with cells in various orientations.

Nutrients and oxygen need to move from outside the biofilm to the inside, and this movement creates different levels of substrates at different depths within the biofilm. The presence of oxygen gradients was critical for the formation of vertical striations. “It almost seemed like bacteria could somehow sense the oxygen gradient and then they aligned within that gradient,” said biologist and study coauthor Lars Dietrich. “Once you go into a region of the biofilm that has no oxygen left, the cells were randomly dispersed.” 

Nutrient availability also affected how the cells organized and behaved within the biofilm. The scientists experimented by growing biofilms with varying amounts of tryptone. While nutrients concentrated at the bottom of the biofilm where bacteria formed vertical striations, tryptone concentration decreased toward the top where bacteria were scattered at random.

Dietrich and his team hypothesized that the distinct cellular arrangements observed in mutant strains affected their susceptibility to antibiotics. Effective antibiotic penetration requires active transport into cells, which in turn requires metabolically active bacteria. The scientists grew biofilms and exposed them to the antibiotic tobramycin. They found that the regions where tobramycin was effective aligned with the areas where the balance between oxygen and tryptone was optimal, which were also the areas of highest metabolic activity. 

Lastly, they wondered about the mechanism behind these bacterial arrangements. They mutated several bacterial proteins involved in varied functions, from metabolism to pili extension. They found that mutants lacking functional pili and related regulatory proteins featured the striations observed in wild-type strains, but with wider bundles and greater spacing. This was likely due to defects in pilus extension, attachment, and retraction, all of which are crucial for maintaining the typical striated arrangement.

Moving forward, Dietrich’s group is interested in uncovering the exact mechanism by which pili participate in the vertical striation of bacteria within biofilms. His group also plans to study bacterial arrangements in a variety of biofilm systems to see if their observations apply across different settings. Dietrich pointed out that traditional antibiotic testing, typically conducted in liquid cultures where bacteria are isolated, fails to account for the complexities of biofilm architecture. "If you could somehow modulate how cells are arranged, and thereby also facilitate how substrates are being distributed, that could be a way of making existing antibiotics more efficient by helping them get into biofilms," he said.

Reference

1. Dayton, H. et al. Cellular arrangement impacts metabolic activity and antibiotic tolerance in Pseudomonas aeruginosa biofilms. PLOS Biol  22, e3002205 (2024).