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Stanford Report, April 21, 1999

Stanford researchers discover where cholera bacteria hides between outbreaks

BY MITCH LESLIE

Cholera outbreaks come and go, popping up suddenly and then abruptly disappearing again. Where the cholera bacterium hides between outbreaks has puzzled scientists for some time. But a new study by Stanford researchers may help resolve this mystery. The researchers have discovered evidence that the bacterium hunkers down within a durable slime layer, known as a biofilm, that is resistant to the chlorine used to disinfect municipal water systems.

Colonies of cholera bacteria come in two forms: rugose ­ named for its wrinkly texture ­ and smooth, said Fitnat Yildiz, PhD, lead author of the study in the March 30 issue of Proceedings of the National Academy of Sciences. Yildiz is a postdoctoral researcher in the lab of Gary Schoolnik, MD, professor of medicine and co-author of the paper.

Scientists have known for almost a decade that rugose colonies are resistant to chlorine. But the basis for this elusiveness remained a mystery, Yildiz said, until she and Schoolnik discovered that the rugose colonies extrude a protective slime composed of polysaccharides, or long chains of sugar molecules. Smooth colonies do not produce this material.

The researchers observed a marked difference in how chlorine affected smooth and rugose colonies. Smooth colonies were annihilated. But snug within their slime, rugose colonies withstood chlorine concentrations 10 to 20 times higher than those found in water treatment facilities. Confirming this result, Yildiz and Schoolnik found that adding slime to smooth colonies imparted chlorine resistance.

From the bacterium's perspective, this ooze is vital for another reason ­ it allows cells to band together and stick to a surface, forming a biofilm. More than a cluttered mass, a biofilm is a confederation of bacteria that may show an almost organismal degree of order, with tiny channels that transport food and waste running between the cells. Often containing multiple species, biofilms protect their residents and promote the absorption of nutrients.

Scientists have taken a greater interest in biofilms because of their recently recognized involvement in many diseases. Biofilms give rise to the dental plaque that coats our teeth and abrades our gums. Hardy biofilms may underlie some persistent infections of the urinary tract, middle ear and prostate. Tuberculosis and Legionnaire's disease may also involve biofilms. However, Yildiz and Schoolnik are the first to show that cholera bacteria join biofilms.

Using a technique that randomly disrupts bacterial DNA, Yildiz and Schoolnik identified 25 genes that participate in the production of bacterial slime. The genes are present in both kinds of colonies but apparently are only expressed in the rugose colonies, Yildiz said. The researchers are trying to find out what causes these genes to be expressed. They do know that starving smooth colonies can transform into rugose colonies in the lab, and this change may also happen in nature, she added.

The cholera bacterium's propensity to retreat into a biofilm may account for its persistence in chlorinated water supplies in the tropics. "That structure makes the bacteria resistant to chlorine, and chlorine is the first line of defense against aquatic pathogenic organisms," Yildiz said.

Yildiz and Schoolnik hope to confirm that cholera-containing biofilms form not only in the lab but also in nature. They are collaborating with scientists from Peru and Bangladesh ­ two places where cholera is endemic ­ to see if the rugose form of the bacteria is found there and if it is part of natural biofilms in aquatic sediments. They would also like to find the other species of bacteria that help cholera bacteria construct biofilms. SR