New Imaging Technique Reveals Structure of Bacterial Biofilms

August 9, 2012

A new imaging technique developed at the University of California, Berkeley, for the first time reveals the structure of the bacterial biofilms that are responsible for many infectious diseases.

A new imaging technique developed at the University of California, Berkeley, for the first time reveals the structure of the bacterial biofilms that are responsible for many infectious diseases. The method uses fluorescent labeling and super-resolution light microscopy, and was developed by a team led by Veysel Berk, a post-doctoral fellow in the Department of Physics and the California Institute for Quantitative Biosciences at UC Berkeley, in the laboratory of Nobel laureate Steven Chu.

Bacterial biofilms, or “sticky plaques,” are the cause of many persistent bacterial infections, such as cholera, chronic sinusitis, and lung infections in cystic fibrosis patients. When bacteria form these colonies, or biofilms, they become highly resistant to antibiotics. Most biofilms can only be removed surgically.

The new imaging technique reveals the structures of these biofilms and how bacteria grow to form them, and identifies several potential targets for potential drugs that could break up the bacterial community and expose the bugs to antibiotics.

Berk and his team applied single-molecule methods based on optical microscopy, to continuously image live biofilms, on length scales of tens of nanometers to tens of micrometers. “Before this work, these bacterial communities were largely studied in terms of their average composition, appearance, and bulk biochemistry,” said Chu, in a statement. “By introducing a new in vivo fluorescence tagging structure, the molecular and architectural roles of three specific matrix proteins and the extracellular polysaccharides of a growing Vibrio cholera biofilm were visualized as a series of three-dimensional images.”

The standard tagging process involves staining and then flushing away excess dyes before taking single snapshot images with light miscroscopy. Berk, however, used a critically balanced concentration of fluorescent stain that was high enough to enable efficient staining but low enough to prevent background glow, and thus eliminate the need to flush out excess dye. He called the technique “continuous immunostaining.”

“We found a way to do staining and keep all the fluorescent probes inside the solution while we do the imaging, so we can continuously monitor everything, starting from a single cell all the way to the mature biofilm, ” Berk said in a published statement.

“Instead of one snapshot, we are recording a whole movie.”

Berk and his team discovered that V. cholera biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Over a period of six hours, a single bacterium laid down “glue” to attach itself to a surface, then divided into daughter cells, also bound to each other. The daughter cells continued to divide until they formed a cluster, at which point the bacteria secreted a protein that encased the cluster. The imaging revealed complementary architectural roles of four essential matrix constituents: RbmA, which provided cell-cell adhesion; Bap1, which allowed the developing biofilm to adhere to surfaces; and mixtures of Vibrio polysaccharide, RbmC, and Bap1, which formed envelopes that encased the cell clusters.

Targeting these matrix constituents may make it possible to dissolve the structures and providing antibiotic access, Berk said. Berk and his coauthors reported their findings in the July 13 issue of the journal Science.