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A team of Stanford
scientists
and engineers sets out to find where
cholera hides between outbreaks
--
and what triggers its emergence.
AUTUMN IS CHOLERA SEASON IN BANGLADESH.
AS THE MONSOON RAINS THAT HAVE DRENCHED THE COUNTRY FOR MONTHS BEGIN
TO WANE, THE CHOLERA BACTERIUM EMERGES FROM AN UNKNOWN HIDING PLACE
AND MISERY ENSUES. During the next few months,
thousands of Bangladeshis will swallow an infectious dose of the
infamous Vibrio cholerae, along with a meal or a drink of
water. Within as little time as 24 hours, the microbe provokes vomiting
and the hallmark of the disease -- torrential diarrhea that can
kill an adult within a day.
In this impoverished, largely rural country, many
patients have no recourse to medical care and must weather the symptoms
-- or die -- at home. Those who do make it to a hospital or clinic
may get a place on a "cholera cot" that has a hole in the middle
and a bucket underneath. They will be dosed with antibiotics and
fed large quantities of a low-tech version of Gatorade known as
oral rehydration solution -- sugar, salts and water in the proportions
necessary to restore the balance of fluids and solutes in the body.
Dehydration once killed between 50 and 70 percent of those who fell
ill with the most serious form of cholera, but with quick treatment,
the fatality rate today is a around one percent.
After sickening thousands, cholera mysteriously vanishes
again until just before the next monsoon, when a smaller, secondary
outbreak normally flares up. The best evidence suggests that cholera
originated in the area around the Bay of Bengal -- now Bangladesh
and the Bengal state of India. While much of the developing world
is vulnerable to occasional cholera epidemics, the disease is endemic
to Bangladesh and India, which endure outbreaks every year.
Though the number of cases fluctuates from year to
year, the pattern of semi-annual outbreaks bracketing the monsoon
has prevailed since epidemiologists began keeping tallies over 40
years ago. The pattern raises some obvious questions: Where does
the cholera bacterium take refuge between outbreaks? What coaxes
it out of hiding at such regular intervals? Can we stop its emergence,
or at least predict the time and location with enough precision
to warn the people at risk to take precautions?
Aiming to answer these questions, Stanford scientists
are launching an ambitious project to scrutinize the life of V.
cholerae in unprecedented detail. Already, they think they have
tracked this fugitive microbe to one of its lairs in the biofilm,
a durable, complex and organized slime layer that forms on almost
any moist surface (see sidebar). Using so-called gene chips,
which can simultaneously measure the activity of thousands of genes,
the researchers plan to interrogate the bacterium about the intimate
details of its metabolism and genetics. They hope to learn what
genes are turned on at different stages in the bacterium's life
cycle, pin down what those genes accomplish and then correlate these
patterns of gene expression with physical conditions in the organism's
natural environment in the Ganges River delta of Bangladesh.
Ultimately, the researchers plan to compile an inventory
of every biochemical reaction that V. cholerae can undertake
-- something scientists have accomplished for only two other organisms,
Escherichia coli and baker's yeast. With their knowledge
of the bacterium's ecology, they plan to build mathematical models
that link its participation in biofilms with environmental conditions
like water temperature and salinity. Their work could eventually
lead to an early warning system for cholera-prone areas like Bangladesh
or yield new ways to fight the disease by battling biofilms -- perhaps
by jamming the intercellular messages that keep the biofilm cohesive.
What makes this project unique, says professor of
medicine Gary Schoolnik, MD, is that it unites researchers with
different interests, talents and expertise who might not normally
have a reason to talk to each other. Schoolnik, who is also a professor
of microbiology and immunology, has an abiding interest in infectious
organisms. Another member of the group is his postdoctoral fellow
Fitnat Yildiz, PhD, a molecular biologist who will help identify
the genes expressed during biofilm growth and maturation. Emeritus
professor of mathematics Sam Karlin, PhD, is helping to pinpoint
the bacterium's genes by searching its complete genome sequence,
recently made available by the Institute for Genomic Research in
Rockville, Md. Two other key players who will be looking for connections
between the bacterium's behavior and its surroundings are Alfred
Spormann, PhD, a microbial physiologist who is an assistant professor
of civil and environmental engineering, and Craig Criddle,
PhD, an associate professor in the same department.
"This is essentially the new way to do business,"
Spormann says of the collaborative, interdisciplinary approach,
"because with the tools that each discipline brings, you can tackle
more complex questions that are intellectually and methodologically
hard for a single group to handle."
A meeting of minds might be just what's needed to
unravel the mysteries of cholera, a disease that poses particularly
knotty problems for epidemiologists. Unlike the measles virus, which
hops from person to person but does not establish itself in the
environment, V. cholerae lives quite comfortably and reproduces
outside the human body. In the lingo of epidemiology, the disease
has an environmental reservoir.
Getting most scientists to accept this notion took
more than a decade of stubborn campaigning, mainly by Rita Colwell,
PhD, of the Maryland Institute of Biotechnology and her colleagues.
In the mid 1970s, Colwell, who is now director of the National Science
Foundation, announced she had discovered V. cholerae living
in the waters of the Chesapeake Bay, not far from Baltimore. Her
critics scoffed, disparaging her method of testing for the organism.
Yet when other scientists followed Colwell's lead, they found the
bacterium in unlikely and disconcerting places, far from its familiar
tropical haunts -- not just in the Chesapeake Bay but also in the
Gulf of Mexico and even in Morro Bay here in California. Schoolnik
notes that while nearly all cholera victims in the United States
are travelers infected abroad, every so often someone along the
Gulf Coast gets a "native" case after eating shellfish harboring
V. cholerae.
The bacterium's disembodied existence makes predicting
the time and location of outbreaks all the more difficult. Increasingly,
epidemiologists have turned to holistic studies that, in Spormann's
words, "cross scales," or strive to link environmental variables
such as temperature or rainfall with the organism's actions. In
short, the goal is to learn how changes in the bacterium's surroundings,
such as fluctuations in salinity or temperature, influence its behavior
and make it more likely to cross paths with people.
In Bangladesh, Schoolnik says, you have to start
with an understanding of the dominant influence on climate, the
monsoon. Every summer, monsoon rains deluge Bangladesh -- and precipitation
is measured in feet instead of inches. Two of Asia's largest rivers,
the Ganges and Brahmaputra, unite in Bangladesh, bringing yet more
water to the low-lying delta region of the country. The result is
widespread flooding that, in particularly soggy years like 1998,
swamps up to 70 percent of the country.
In the delta region, the floods temporarily transform
the environment, Criddle explains. Salinity plummets with the influx
of fresh water. Soaring nutrient levels nurture a bloom of phytoplankton
that, in turn, promotes a population explosion of tiny crustacean
grazers. Faster water flow means the bacteria will face stronger
shear forces.
Other scientists have worked out how some of these
changes influence V. cholerae. For instance, the bacterium
grows on and in the planktonic crustaceans, so a boom in their population
is likely to trigger an increase in the number of bacteria. So might
a population explosion of phytoplankton, which can provide food
for the bacteria through photosynthesis. However, no one has investigated
how the bacteria living in a biofilm respond to changes in the environment.
The crucial first step for the project is to show
that V. cholerae really does inhabit biofilms in the Ganges
delta. When growing in solution, cholera bacteria will spin a slime
layer on the wall of a flask and even at the boundary between air
and liquid. In fact, this penchant, rather than a deep curiosity
about biofilms, got Schoolnik interested in these slime cities in
the first place. "I would have been quite happy to ignore biofilms
for the rest of my life, except that we found that Vibrio cholerae,
when it grows in a certain colony type, produces an abundant amount
of polysaccharides on its surface."
To establish that the bacterium's behavior is the
same in the field, the group's Bangladeshi colleagues are deploying
acrylic discs about the size of a saucer. Attached to fishing line,
the disks hang at different depths in the water in a cholera-prone
site in the delta region of Bangladesh. Every two weeks, one of
the group's Bangladeshi colleagues will haul
in the discs and scrape off the accumulated biofilm. Half of the
slime will be tested on site for the presence of V. cholerae.
The rest will be express mailed to Stanford for more sensitive tests
that will search for bacterial RNA.
The first field tests, using an antibody specific
for the coat of V. cholerae, came up positive, although the
bacterium would not grow in culture. That's not unusual, because
bacteria in natural aquatic environments often refuse to proliferate
in culture medium.
To explore the bacterium's responses to environmental
change, Craig Criddle and his students have set up a small-scale
replica of the Ganges delta in a lab in the basement of the Terman
Engineering Building. Not a Hollywood-style miniature with plastic
trees and water buffalo, mind you, but a state-of-the-art biofilm
incubator that can replicate some of the conditions in the delta.
Outfitted with flow-through water circulation, the device, which
looks like a brawny coffee maker, is known as a biofilm annulator.
It is designed to grow biofilms and to measure their responses to
environmental variables like temperature, nutrient levels and water-flow
rates. The microbial action takes place within a clear plastic chamber
containing a fluid-filled inner cylinder with removable plastic
panels for bacterial attachment. Like a carousel, the inner cylinder
can spin at variable speeds, simulating different flow rates in
the river.
Criddle says one reason he became interested in the
project is that it represented a return to his discipline's roots
in public health. Environmental engineering got its start in the
1800s partly in response to cholera and other water-borne diseases.
Terror of further devastating outbreaks inspired the improvements
in sanitation that we take for granted today: filtration and chlorination
of drinking water, sewers and sewage treatment. He says the current
research also harks back to the pioneering work of Dr. John Snow,
Queen Victoria's physician, who inaugurated the field of epidemiology
with his scrupulous studies of cholera outbreaks in London in 1848-49
and 1853-54.
In the next few months, Criddle and his students
will be growing biofilms in the annulator and then tweaking the
conditions to represent what happens during the monsoon: lowering
the sodium concentration to mimic the infusion of fresh water, raising
the input of sugars and nitrogen to simulate a bloom of algae and
speeding up the spinning cylinder to replicate faster water flow.
What they expect is that under the right combination of conditions,
the biofilm will dissolve and the bacteria will swarm into the water
-- presumably what happens during outbreaks in Bangladesh.
When that happens, samples of the bacteria will be
whisked off to Schoolnik's lab for genetic profiling. Schoolnik
and Yildiz will use the DNA microarray, or gene chip, technology
invented by Stanford associate professor of biochemistry Patrick
Brown, MD, PhD, to find out which genes are turned on and off in
the bacteria. "This gives us a chance to look into the heart and
soul of this microbe," says Criddle.
"Biofilm growth is very different from growth in
the planktonic, free-living state," says Yildiz. The scientists
expect that many genes active during the biofilm stage will be turned
off during the planktonic stage, and vice versa.
Once the scientists know what genes are active during
different phases of the bacterium's life, they hope to figure out
what each of those 3,545 genes accomplishes. That might sound like
a lifetime's work, but a few shortcuts are available. For instance,
if history is any guide, about half of V. cholerae's genes
will have the same function as those of well-studied bacteria like
E. coli. Moreover, based on a gene's nucleotide sequence,
it's possible to make an educated guess about the function of the
protein that gene encodes. From there, Spormann and his students
plan to test their hypotheses by growing the bacteria under different
sets of conditions in a chemostat, a microbial incubator, and then
subjecting the microbes to genetic profiling. For instance, if they
hypothesize that a particular gene is involved in breaking down
sugars, they can add sugar to the chemostat and then see if the
gene is activated.
In the end, this step-by-step approach will yield
a complete diagram of the bacteria's capabilities -- not a single
biochemical pathway but a biochemical network, showing all the interacting
pathways.
Once the different sub-projects are complete, the
scientists will know V. cholerae in greater depth than almost
any other organism. The knowledge should pay
off with practical benefits. For example, using a combination of
field and lab data, it may be possible to build mathematical models
that could forecast when and where an outbreak is likely, Criddle
says. That V. cholerae takes refuge in a biofilm suggests
a new goal for preventive programs in the field: disrupting biofilms,
either chemically or physically.
For those of us living in the developed world, it's
easy to get complacent about cholera. Although cholera is not the
great killer it was in the 19th century, when a single epidemic
in 1849 killed one-tenth of the population of St. Louis, Mo., the
disease still casts a pall over much of the world. Epidemics are
commonplace in Africa and parts of Asia, and the disease has recently
returned to South America after a century of absence. It haunts
human calamities, popping up in Rwandan refugee camps in Zaire,
for example, and most recently in flooded Mozambique. "It's
an infectious agent still capable of producing pandemics that involve
millions and millions of people," Schoolnik says to explain his
continuing interest in V. cholerae.
In Bangladesh, fear of cholera led indirectly to
another public health disaster. Beginning in the 1960s, international
aid organizations like UNICEF started digging millions of wells
to provide what they hoped would be safer water, free from pathogens.
Millions of Bangladeshis switched to this new water source. But,
as scientists discovered in 1993, about half of the wells are contaminated
with high levels of arsenic, and perhaps 10 million people in Bangladesh
and millions more in India have been exposed to this cancer-causing
chemical, which can also produce suppurating skin lesions and vision
problems.
Some scientists worry that cholera's range could
expand as global warming heats subtropical seas to temperatures
that V. cholerae likes. Whether that happens or not, Schoolnik
points out that environmental changes already under way in south
Asia will worsen cholera outbreaks. The monsoon floods that spread
the bacterium are growing more severe because of deforestation in
the Himalayas, which means greater runoff into the Ganges and Brahmaputra.
And farmers in the Ganges delta are using more and more fertilizer
to boost productivity. As nitrates and phosphates from the fertilizer
enter the water, they cause bigger blooms of algae, which should
mean more cholera cases.
Even with our best efforts, Schoolnik cautions, we
won't ever eliminate V. cholerae as we did the smallpox virus.
"It has an extra-intestinal lifestyle that is very robust," he says.
"Our hope is that we can prevent incursions into humans." SM
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