Back in the year 1832, cholera took 7,000 lives in London alone - it was the first of seven global cholera pandemics. Twenty years later, on Sept. 7, 1852, a prominent medical figure, John Snow by name, persuaded the London city fathers to remove the handle from the water pump serving the municipality's Broad Street - and deaths quickly dropped off. Snow's show and tell proved that cholera was caused by fecally contaminated water from the city's faulty sewage system.

That was then - 151 years ago. During that interval, six more cholera pandemics swept the world - one in Russia and New York, one in Egypt and Arabia, and also ones in Italy, France, Indonesia and finally Peru. In Peru, pandemic No. 7 rages to this day, with 150,000 diagnosed cases recorded to date. Elsewhere in Latin America, cholera recently struck down 400,000 men, women and children, killing 4,000.

Today, clinical and research infectious disease specialist Paula Watnick follows John Snow's historic hunch. Her teaching hospital is at Tufts New England Medical Center, affiliated with Tufts University in Boston. Besides attending to infectious-disease patients, Watnick's research laboratory focuses on bacterial infection, particularly cholera.

Watnick said, quoting World Health Organization data, that "between 1999 and 2000, there have been 222,000 [cholera] cases diagnosed worldwide, of whom 8,400 died."

"Whenever there's a breakdown in the socioeconomic infrastructure and hygienic facilities become unavailable," she noted, "cholera epidemics arise - this has been the case in present-day Iraq."

Watnick is senior author, along with first author Dr. Katharine Kierek, of an article in the Proceedings of the National Academy of Sciences (PNAS), first released online Nov. 3, 2003. Her paper is titled "The Vibrio cholerae 0139 0-antigen polysaccharide is essential for Ca2+ -dependent biofilm development in seawater."

"Our PNAS paper's overall finding," Watnick observed, "is that when bacteria are carried from marine environments into fresh water environments, some, such as V. cholerae, leave multicellular surface-attached biofilm structures to enter the single-cell state. In this state, V. cholerae may be more easily ingested by human hosts.

"Dr. Rita Colwell and colleagues have shown that when seawater is forced into rivers by environmental factors such as high winds, the incidence of cholera increases. Many factors may account for this. V. cholerae may be brought to waters that are commonly used by people in daily activities, these bacteria may multiply more rapidly in the lower salinity environments, and they may be more infectious. An additional possibility that we have suggested," Watnick added, "is when the bacteria move from seawater into fresh water, the large decrease in calcium concentration causes V. cholerae to become dissociated from surfaces, thus increasing the concentration of unattached bacteria in the water. These bacteria may be more likely to be ingested."

One Lone Vibrio Bug Is Easier To Digest

"We're just beginning this research," Watnick went on. "We are beginning to define the differences between biofilms that form in fresh water and seawater. It seems that biofilms formed in fresh water require exopolysaccharide synthesis, while those formed in seawater do not. Exopolysaccharides," she explained, "are sugars that form the outermost covering of the cell when they are present. So when the bacterium is in a nutrient-rich environment, it makes these sugars. This renders them sticky, and they attach to surfaces.

"But in seawater," Watnick recounted, "the concentration of calcium is 10 times higher than that of fresh water. The calcium helps to bind the bacteria to surfaces and each other in the absence of exopolysaccharide synthesis. We call this the calcium-dependent biofilm. In order to identify bacterial factors that were essential for forming a biofilm in seawater, we used a product known as Instant Ocean,' which creates an artificial sea. This product is made for seawater fish tanks." Watnick recounted. "Using a well-established technique for isolating biofilm-defective bacteria, we made a library of transposon-insertion mutants. We inoculated a different mutant in each well and grew it overnight. Then we washed all the bacteria out, leaving behind the ones that were stuck to the sides of the wells, and, therefore, biofilm-associated. These bacteria were stained with crystal violet; the dye that makes Gram-positive organisms purple. Mutants in wells without purple stain remaining were considered unable to form a biofilm.

"In the absence of exopolysaccharide synthesis, the outermost covering of the bacterium is the capsule," Watnick explained. "This is a covering made out of chains of sugars very closely associated with the bacterium. This capsule is formed of so-called O-antigen subunits. We found that the O-antigen and O-antigen-based capsule are required for forming a biofilm in seawater. Thus, while nutrients and exopolysaccharide synthesis are required for forming biofilms in fresh water, we have now found that calcium and the O-antigen polysaccharide is required for forming biofilms in seawater.

"Because the signals and environmental requirements for making a biofilm in seawater and fresh water are completely different, our results suggest that the bacterium occupies a different ecological niche in each environment," she said.

"What we want to do now," Watnick continued, "is to monitor the salinity and calcium content of environments where cholera is prevalent and to correlate this data with numbers of V. cholerae in the environment and cholera incidence. These environmental studies are really the challenge. You can't rush them; it takes several years to determine whether your hypothesis is validated.

"In collaboration with Dr. G. B. Nair at the International Centre for Diarrheal Diseases in Dhaka, Bangladesh," Watnick said, "we're planning a study to test these findings in the environment in Bangladesh. He's part of the Center for Health and Population Research in that country."

An Ounce Of Prevention Beats Cholera Infection

"As for the relevance of our work to human patient," Watnick suggested, "it's relevant to prevention rather than treatment. For instance, if at certain times of the year when the salinity of rivers increase, suggesting mixing with sea water, one might need to use smaller-size pore filters or non-filter-based methods to sterilize water," she concluded.

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