Trying to outwit cholerae, the bacterium that causes cholera, is like playing against "Deep Blue," IBM's dedicated computer that defeated Kasparov, the world chess champion.

In a switch of metaphor, "The bacteria are really orchestrating the cholera infection," observed microbiologist Andrew Camilli, at Tufts University School of Medicine in Boston. He is senior author of a paper in today's Nature, dated June 6, 2002. Its title: "Host-induced epidemic spread of the cholera bacterium."

"The overall finding in our paper," Camilli told BioWorld Today, "is that cholera stool bacteria appear to have a greater ability to reinfect compared to laboratory-grown V.cholerae. And that could be an important factor in explosive cholera epidemics.

"Cholera outbreaks still occur," he pointed out, "though less frequently than in the past. For example, there have been some recent epidemics in Africa, in particular among Rwandan refugees a few years ago. Such situations are probably going to continue as populations increase in many poor areas of the world. There is a tremendous concentration of people sharing contaminated water," Camilli explained. "So someone infected with cholera will shed huge numbers of bacteria into the water supply. Then someone else downstream, a few minutes later, might drink some of that water.

"Fecal-oral spread," he continued, "is how cholera is transmitted during an epidemic. And that's why our work reported in Nature is important because we're saying that fecally contaminated water may carry Vibrios that are highly infectious - or to coin our phrase, hyperinfectious.'

"Cholera is still present in the developed countries of the world," Camilli went on. "Anywhere there's brackish or salty water in temperate regions you can find cholerae - even off the coast of the U.S., as in the Chesapeake Bay."

Camilli and his co-authors describe in Nature two main lines of experimentation - one in vivo with mice, the other in silico.

Mice, Gene Chips Yield Same Hyperinfectivity

"In the real world," he recounted, "cholerae infects only humans. People have found over the years that you can only infect na ve animal models. So we infected 5-day-old mice - not adults, who don't get cholera. We injected 100,000 bacteria into the stomach of these naive mice after anesthetizing them. We co-infected them with natural stool bacteria mixed 1-to-1 with laboratory-grown cholerae. Then the bacteria moved down to the small intestine - just as they do in humans - and multiplied there. That's the site that V. cholerae colonizes. It produced cholera toxin, and then the mice got diarrhea, becoming sick by 16 to 18 hours.

"At 20 hours we euthanized them, dissected out their small intestines, ground these up and plated them out to count how many bacteria were there now. And we usually saw tenfold to a 100-fold increase in the number of V. cholerae from what we had inoculated to the million that came out. We still don't know what's going on in that tremendous growth of the bacteria during the infection. It's all a black box. All we know is that the stool-grown bacteria outgrow the lab-grown."

Then the team turned to gene microarray analysis.

"First of all," Camilli narrated, "we were able to isolate large numbers of the bacteria from what we call the rice-water stools.' These human stool samples are just pure cholerae, containing virtually no other contaminating organisms. That was crucial because we could then isolate relatively pure RNA from the pathogens.

"We converted that material to cDNA, which we labeled with fluorescent dyes, and hybridized to a spotted DNA microarray. Most of the genes known from the V. cholerae genome, sequenced by [The Institute for Genome Research] a few years ago," Camilli recalled, "were synthesized and spotted on a glass slide. There were about 3,200 genes - 90 percent of the total genome - on the chip. Then we hybridized the complementary DNA to that, and were able to get relative expression levels of every one of those genes in the stool bacteria.

"We compared the gene expression to lab-grown V. cholerae because we wanted to know why stool bacteria are hyperinfectious relative to lab-grown bacteria. And we discerned about 200 genes that were different from most of the genes. Amongst those genes the most interesting things are what genes are not being expressed in the stool bacteria. All of the known virulence factors - the genes encoding cholera toxin - are not being expressed highly in the stool bacteria."

Bacterium Bides Time, Multiplies, Then Strikes

"Prior work in our lab told us that cholera toxin is not needed in the first few hours of infection. In fact, the genes are not turned on until three or four hours after the infective process has begun. That makes sense because cholera wants to adhere and multiply for awhile, to bring up the bacterial numbers before it starts making cholera toxin and producing diarrhea. Our microarray data on the stool bacteria are consistent with that. The incoming bacteria - the ones that are highly transmissible - have shut off the genes for cholera toxin and other virulence factors. Clearly, these genes must be turned on later in the infection to cause the diarrhea and consequent dehydration."

Camilli made the point, "What kills one in 100 people infected with cholera is dehydration, especially in small children with small body size. Oral rehydration therapy with electrolyte-spiked water is crucial," he added, "and that's what is used in the underdeveloped world to save peoples' lives.

"Another thing we proposed in the paper is that the hyperinfectious state may be due to turning on of genes for acid resistance, since the bacteria need to pass through the acidic stomach when they reinfect. But we didn't see any acid-resistance genes turned on."

As for clinical payoffs from his research, the Tufts microbiologist noted, "There's really no need for therapeutic drugs because at present antibiotics are sufficient. In the future, perhaps antibacterial resistance can become a problem, so we will then need new drug targets. At present, the major need is a designer vaccine to target the bacteria's surface antigens right as they're entering the human gut. The bacteria are few in number at that point," Camilli concluded, "and that's when you want to stop them."