Calcium makes germs cluster: ion dilution leads cholera bacteria to disperse.

A chain of sugars on the surface of cholera-causing bacteria enables the pathogens to clump together in seawater and yet scatter in fresh water, new data suggest. Microbiologists propose that the dispersal facilitates seasonal outbreaks of cholera in coastal areas.

Vibrio cholerae bacteria naturally inhabit both fresh and salt water. Researchers have linked cholera outbreaks to high waters along the coast of Bangladesh, where sea level rises during the annual monsoon. Influxes of pathogen-bearing seawater, driven by monsoon winds, may alter conditions in coastal estuaries and trigger epidemics.

As many bacteria do, V. cholerae cluster on surfaces, such as the bodies of small aquatic animals. Certain genes, called vps genes, enable V. cholerae to stick together in bacterial communities, or biofilms, in both fresh and salt water.

But Katharine Kierek and Paula I. Watnick of the New England Medical Center in Boston recently discovered that V. cholerae can form biofilms in salt water even without the action of those genes. The team reports that finding in the September Applied and Environmental Microbiology.

To look for other factors that control the bacterial congregations, Kierek and Watnick tested a natural strain of V. cholerae and mutant strains unable to make one or both of two cell-surface structures. Both structures include a chain of sugars called the O-antigen polysaccharide, which triggers people's immune response to cholera.

The researchers attempted to grow biofilms of each strain in a solution containing calcium ions and other salts found in seawater. The natural strain and mutant strains with intact polysaccharide structures formed biofilms on submerged surfaces, but strains lacking the normal sugars didn't.

Then, to simulate a cholera biofilm entering fresh or estuary water, the investigators drained off the artificial seawater and submersed the biofilms in a calciumfree solution or added a compound that binds calcium ions. The biofilms disintegrated rapidly.

Removing other ions didn't have the same effect, Kierek and Watnick report in an upcoming Proceedings of the National Academy of Sciences.

"When you take calcium away, the biofilms fall apart, and the bacteria are essentially free-swimming," Watnick says. Presumably, she says, this happens when V. cholerae biofilms move into fresh water. Thus dispersed, the bacteria might readily find their way into a person's mouth, colonize the gut, and cause disease.

The novel findings imply that both calcium and V. cholerae's O antigen are vital to biofilm formation in seawater, says microbiologist Dianne K. Newman of the California Institute of Technology in Pasadena. Since the water in estuaries is relatively low in calcium, she says, the research also "provides a very simple environmental mechanism that could explain the mobilization of the pathogen."