The persistent and chronic nature of infectious diseases can often be attributed to bacterial biofilms that are formed like matrices by growing colonies of bacteria. These biofilms protect the colonies from attack and prevent antibiotics from working effectively. Devising methods to penetrate biofilms to treat diseases such as chronic sinusitus, cholera, and lung infections has been difficult because of the limited knowledge of how they function.
Now researchers at UC Berkeley have combined super resolution microscopy with a labeling technique that allows tracking of growing and multiplying cells to study how biofilms form. Their newly discovered knowledge is already pointing at new genetic targets for future drugs to aim at to help disrupt cellular colonies.
From UC Berkeley:
The popular view of bacteria is that they are free-living organisms easily kept in check by antibiotics, Berk [Veysel Berk, postdoctoral fellow in the Department of Physics and the California Institute for Quantitative Biosciences] said. But scientists now realize that bacteria spend most of their lives in colonies or biofilms, even in the human body. While single bacteria may be susceptible to antibiotics, the films can be 1,000 times more resistant and most can only be removed surgically.
To study a biofilm formed by cholera bacteria (Vibrio cholerae), Berk built his own super-resolution microscope in the basement of UC Berkeley’s Stanley Hall based on a 2007 design by coauthor Xiaowei Zhuang, Chu’s former post-doctoral student who is now a professor at Harvard University. To actually see these cells as they divided to form “castles,” Berk devised a new technique called continuous immunostaining that allowed him to track four separate target molecules by means of four separate fluorescent dyes.
He discovered that, over a period of about six hours, a single bacterium laid down a glue to attach itself to a surface, then divided into daughter cells, making certain to cement each daughter to itself before splitting in two. The daughters continued to divide until they formed a cluster – like a brick and mortar building – at which point the bacteria secreted a protein that encased the cluster like the shell of a building.
The clusters are separated by microchannels that may allow nutrients in and waste out, Berk said.
“If we can find a drug to get rid of the glue protein, we can move the building as a whole. Or if we can get rid of the cement protein, we can dissolve everything and collapse the building, providing antibiotic access,” Berk said. “These can be targets for site-specific, antibiotic medicines in the future.”
Super-resolution microscopy obtains 10 times better resolution than standard light microscopy – 20 instead of 200 nanometers – by highlighting only part of the image at a time using photo-switchable probes and compiling thousands of images into a single snapshot. The process is much like painting with light – shining a flashlight beam on a dark scene while leaving the camera shutter open. Each snapshot may take a few minutes to compile, but for slow cellular growth, that’s quick enough to obtain a stop-action movie.
The problem was how to label the cells with fluorescent dyes to continuously monitor their growth and division. Normally, biologists attach primary antibodies to cells, then flood the cells with fluorescent dye attached to a secondary antibody that latches onto the primary. They then flush away the excess dye, shine light on the dyed cells and photograph the fluorescence.
Berk suspected that a critically balanced concentration of fluorescent stain – low enough to prevent background, but high enough to have efficient staining – would work just as well and eliminate the need to flush out excess dye for fear it would create a background glow.
“The classical approach is first staining, then destaining, then taking only a single snapshot,” Berk said. “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 a mature biofilm. Instead of one snapshot, we are recording a whole movie.”
Study abstract in Science: Molecular Architecture and Assembly Principles of Vibrio cholerae Biofilms