Researchers from the University of California, Berkeley have developed a sensitive new imaging technique that reveals some details of the structure of biofilms, thus opening up attacks such as cholera, lung infections in patients with cystic fibrosis and even chronic sinusitis A large number of bacterial diseases that form biofilms and produce antibiotic resistance. Relevant papers were published in the journal Science on July 13.
Bacteria are not alone
Berk said that people generally think that bacteria are free-living organisms, and antibiotics are very easy to control them. However, scientists now realize that bacteria spend most of their lives in communities or biofilms, even in the human body. A single bacterium may be sensitive to antibiotics, and the biofilm is 1,000 times more resistant, most of which can only be removed by surgery,
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Transplants such as pacemakers, stents, and artificial joints are occasionally infected with bacteria that form biofilms. These biofilm sites periodically shed bacteria, and Berk calls them risk takers who can provoke acute infections and fever. Although antibiotics can remove these free-flowing bacteria and temporarily suppress the infection, the biofilm is still not damaged. The only solution is to remove the device covered by the cell membrane and replace it with a new sterile graft.
The long-lasting bacterial biofilm in the sinuses can provoke an immune response and lead to chronic sinus infections, manifested as fever and cold-like symptoms. By far, the most effective treatment is surgical removal of infected tissue.
Bacteria can also form persistent in the mucus-filled lungs of patients with cystic fibrosis, most of which are lifelong biofilms, which are important causes of chronic lung infections that lead to early death. Although long-term antibiotic treatment is helpful, they cannot completely eradicate the infection.
In order to study the biofilm formed by cholera bacteria, Berk constructed Zhu Diwen ’s former postdoctoral student, now a Harvard professor Xiaowei Zhuang (2007 Xiaowei Zhuang), in the basement of the University of California Berkeley Own super-resolution microscope. In order to reliably observe these cells during the formation of a "castle", Berk designed a new technique called continuous immunostaining, which enabled him to track four target molecules with four separate fluorescent dyes.
He found that within a period of about 6 hours, a single bacterium laid a protein glue to attach itself to the surface, and then they split to form daughter cells, while secreting proteins to make the daughter cells stick to each other. The daughter cells continue to divide until they form a colony, like a building made of bricks and stucco. At this time, the bacteria secrete a protein and sugar molecules to wrap the colony like a shell of a building.
Berk said these communities are separated by microchannels to ensure nutrients and waste enter and exit.
Berk said: "If we can find a drug to remove this glue protein, we can remove the building as a whole. Or if we can remove the binding protein, we can disintegrate everything and collapse the building. Antibiotics provide access. In the future, these may be targets for site-specific antibiotic drugs. "
Super-resolution microscope: painting with light
Berk is a biologist who has been trained in physics and optics, and is good at imaging protein structures: a group of researchers determined the atomic structure of the ribosome a few years ago.
He believes that the powerful new super-resolution optical microscope can reveal the unknown structure of the biofilm. Compared to standard optical microscopes, the resolution of super-resolution microscopes is 10 times higher at 20 nanometers. Thousands of images are compiled into a single snapshot by using a switchable photo fluorescent probe to highlight part of the image at once. This process is much like painting with light, shining the flashlight beam on the black screen and opening the camera's shutter. Each snapshot may take several minutes to compile, but for slow cell growth, this is fast enough to capture still images.
The problem is how to label cells with fluorescent dyes to continuously monitor their growth and division. Normally, biologists attach primary antibodies to cells and then submerge the cells to bind the primary antibodies with secondary antibodies attached to fluorescent dyes. Afterwards, the excess dye is washed away, the stained cells are irradiated and the fluorescence image is taken.
Berk believes that precision-balanced concentration of fluorescent staining (low enough to prevent background and high enough to effectively stain) can also play a role, so that there is no need to rinse excess dye because of fear of background light emission.
Berk said: "The classic method is to stain first, then decolorize, and take a single snapshot. We found a way to complete staining in solution and retain all fluorescent probes during imaging, so we monitored everything continuously , From a single cell all the way to a mature biofilm. Not just a snapshot, we are recording the entire image. "
He said: "This is a very simple and cool idea. Everyone thinks it is crazy. Yes, it is crazy, but it works."
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