The images reveal for the first time that E. coli, which are responsible for 80 to 90 percent of all UTIs, pass through at least four distinct developmental stages during the course of an infection. Researchers hope to further define these stages and use them as guides in the search for new drugs.
The study, to be published this week in the early online edition of the Proceedings of the National Academy of Sciences, also reveals that bacteria will sometimes shift into an inactive state, creating reservoirs of infection within the bladder that might be responsible for some of the recurrent UTIs that plague many women.
To image the bacteria in action through a videomicroscope, researchers made the bacteria glow by adding a phosphorescent green protein. The movies show the bacteria rapidly changing themselves and their interactions with each other to collectively hijack bladder cells and use them as safe havens for replication.
"It just boggles the mind what these bacteria can do, in terms of sensing and responding to their environment and each other," says Scott J. Hultgren, Ph.D., the Helen Lehbrink Stoever Professor of Molecular Microbiology and lead investigator of the study. "This has never been seen before in live host tissue, and parts of this process are probably present in a multitude of different kinds of pathogens."
UTIs, which mainly occur in women, are the second most common type of bacterial infection. Linked to poor hygiene, sexual behavior and migration of intestinal flora, they are believed to cause around $1.6 billion in medical expenses every year in the United States. Scientists estimate half of all women will experience a UTI at some point in their lives, and additional recurrent UTIs will affect 20 to 40 percent of these patients.
Clinicians had assumed that E. coli and other bacteria that cause UTIs were not invading cells of the urinary tract, but in June 2003, Hultgren's lab produced images of E. coli forming biofilms inside bladder cells. Biofilms are networks of single-celled pathogens that cooperate with each other to form structures that are resistant to attack.
"Once these bacteria begin to replicate inside their target cell, they almost behave more like a multicellular organism," Hultgren explains. "Some kind of switch occurs, probably due to processing of environmental cues, and instead of acting like individual bacteria, they behave more in a multicellular manner, working together to defeat the cell's defenses."
Hultgren and his coauthors divided E. coli's infectious process into four stages. In the first, bacteria enter bladder cells and begin replicating rapidly. In the second stage, they decrease their size and replication rate and begin to form the intracellular bacterial community (IBC), a podlike structure that Hultgren compared to a marble in a balloon.
In the third stage, bacteria begin to break out of the IBC and swim away.
"It's like peeling an onion," says Sheryl S. Justice, Ph.D., a postdoctoral fellow in Hultgren's lab and one of three lead authors of the paper. "They come off the outside of the IBC in successive layers."
During the fourth stage, some of the bacteria from the dispersed IBC become filaments, taking on long, thin, needle-like shapes. The new shapes may help them evade the immune system and seek chances to start new infections both in the urinary tract of their hosts and in new hosts.
The movies also reveal that groups of E. coli will sometimes shift into an inactive or quiescent state.
"We don't actually know when this happens, but at some point, that's all you see -- small numbers of bacteria inside the cells no longer replicating," Hultgren says. "They've entered this quiescent state and are presumably no longer causing any symptoms, and the question is whether that quiescent reservoir can now provide seeds for recurrent infections."
Hultgren's lab is working to better understand the distinctions between the various stages of development, including the signals that trigger the changes from one stage to the next.
"There's such complex genetic circuitry involved here that we're going to have to start thinking about this like electrical engineers," Hultgren says. "But the more we can understand this network, the better our chances of figuring out ways to interrupt it."
Justice SS, Hung C, Theirot JA, Fletcher DA, Anderson GG, Footer MJ, Hultgren SJ. Differentiation and developmental pathways of uropathogenic Escheria coli in urinary tract pathogenesis. Proceedings of the National Academy of Sciences, early online edition, week of Jan. 19-23, 2004.
Funding from the National Institutes of Health, the Food and Drug Administration, the David and Lucille Packard Foundation and the Stanford Bio-X Program.