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War in the intestines

New strategies can help fight the bacteria behind serious forms of diarrhea

LAURA DAVIÑAA Brazilian biologist who has been working for ten years in the United States has found a new option for controlling a variety of bacterium that causes bad diarrhea. This variety, enterohaemorrhagic Escherichia coli or EHEC, causes about 5,000 cases a year in Brazil of diarrhea, often bloody, followed by renal complications (about 600 thousand adults and children are hospitalized every year in the country as a result of acute diarrhea of a bacterial origin). However, it causes outbreaks with thousands of victims in other countries as well. In the United States, for instance, EHEC affects about 73 thousand people a year, of whom some 1,800 to 3,600 are admitted to hospital; 60 to 550 of them die as a result.

Vanessa Sperandio and her team from the University of Texas, which also includes Brazilian biologist Cristiano Moreira, tested 150 thousand synthetic molecules from this university and found one, identified by the abbreviation LED 209, which reduced the virulence of two varieties of bacteria that cause outbreaks of diarrhea in the United States, including EHEC. Presented in an article published in August in the journal Science, LED 209 bonds with a protein in the bacteria called histidine kinase and prevents them from producing the Shiga toxin, which aggravates the intestinal infection – the bacteria will remain in the intestine, but will be harmless.

“We don’t need to kill all the bacteria, but simply make sure that they stop producing toxins,” explains Vanessa, who has a degree in biology from the University of Campinas (Unicamp). “If we kill all the EHEC, those that survive will release more toxins, which will aggravate the clinical situation, and they could become resistant to medicines”. In the case of mice and rabbits, LED 209 produced satisfactory results against a diarrhea-causing type of bacteria that spreads throughout the organism and causes generalized infection, Salmonella typhimurium. However, this molecule is absorbed and disappears in the intestine, which is why it is not very effective against EHEC, which only lives in the bowel. “What we have to do now is to modify this molecule, so that it is not absorbed in the intestine,” explains Vanessa.

In March, based on this and other results, Vanessa received a grant of US$ 6.5 million from the United States National Institute of Health (NIH) – of which US$ 2.5 million have already been disbursed – to develop new medicines against bacteria that cause serious diarrhea, particularly EHEC, a public health problem that is not restricted to the United States. In 1996, 7,500 cases were registered in an outbreak in Sakai, in Japan. In Argentina, Chile and Uruguay, EHEC accounts for almost half of all cases of bloody diarrhea. Five to ten percent of those infected by EHEC, particularly children under the age of two, may also shortly afterwards present the so-called hemolytic uremic syndrome, characterized by anemia, by a drop in the platelet count (platelets are the blood cells responsible for coagulation), and by acute  kidney failure. The antibiotics used to stop other bacteria have the opposite effect on EHEC because they intensify production of the Shiga toxin and thus aggravate the destruction of the kidney cells.

Over the course of ten years, Vanessa has described in growing detail the EHEC mechanisms of survival and multiplication in the bowels. In 1999, in the journal PNAS, she showed that this bacteria lineage has surface proteins that work as environmental sensors, the so-called quorum sensing, which were previously identified in a type of bacteria that causes cholera. These sensors detect the quantity of two hormones – noradrenaline, produced in the intestines, and adrenaline, which is released by the adrenal glands – which trigger the body’s defenses against invading agents such as these bacteria. Vanessa showed that when there are a lot of these hormones nearby, the bacteria quietly wait for a more auspicious time; otherwise, they begin to multiply and settle in the intestine.

In another paper, published in 2003 (also in the journal PNAS), she explained the conflicting roles of noradrenaline and adrenaline. These two hormones help to protect the organism, but they can also benefit the bacteria, in two ways: first, by activating genes that result in the production of the Shiga toxin; and second, by enabling the flagella to work – the flagella are a type of tail that allows the bacteria to swim more easily. Another study that carried out by Vanessa’s team and published this year in Nature Reviews provides a detailed explanation of the chemical communications between the bacteria and the organism in which they settle: an adult’s intestines, which are about five meters long, houses 500 to 1,000 different species of bacteria that, if gathered together, would add up to a mass of 1.5 kg. Taken in conjunction with the contributions from other teams, this knowledge base can now help in the testing of and search for new medicines.

The most dangerous of the EHECs both in Brazil and other countries, the O157:H7, intensely studied by the team from Texas, has caused isolated cases of severe intestinal infections to date, sometimes accompanied by kidney problems, in the states of São Paulo, Rio de Janeiro, Bahia and Minas Gerais. The findings from São Paulo’s Federal University (Unifesp) and the Adolfo Lutz Institute up to now reveal at least ten cases a year in the state of São Paulo. In 2002, in the journal Emerging Infectious Diseases, researchers from these two institutions reported the first sporadic cases of diarrhea caused by this sub-type of the EHEC bacteria in residents of the city of São Paulo, one of whom also had the HIV virus, and from the city of Campinas. A month later the same team, coordinated by Beatriz Guth, from Unifesp, presented in the same journal the first case of hemolytic uremic syndrome associated with intestinal infection caused by another subtype of EHEC in an eight-month old baby who was a patient at Hospital São Paulo.

LAURA DAVIÑA“Fortunately there have been no outbreaks, but the experts at research institutes and public health agencies are on alert, because this is an emerging disease,” Beatriz points out. In Brazil, cases of EHEC diarrhea are not only linked to poverty and lack of basic sanitation, but also to negligent hygiene. In a survey that she helped direct, Unifesp pediatrician Renato Lopes de Souza looked in intensive care units of the city of São Paulo hospitals for cases of children with hemolytic uremic syndrome who had previously shown symptoms of serious diarrhea. He found 13 such cases, which had been treated from January 2001 to August 2005, mainly at private hospitals (70% of the total). The researchers identified antibodies against bacteria of the EHEC group in seven children and the bacteria itself in other three, indicating an association. However, according to Beatriz, doctors may not always remember this potential link between serious kidney problems with prior bacterial infections. “The children with hemolytic uremic syndrome are already in a serious condition when they arrive at Intensive Care Units and the doctors rarely make the connection with diarrhea,” she says.

In search of a vaccine
In Brazil, another bacterium variety, enteropathogenic Escherichia coli (EPEC), is also more worrying. Less aggressive than EHEC, EPEC is likely to cause about 30 thousand cases a year of diarrhea in the more urban parts of Brazil and about 180 thousand cases in the less urban areas. In well-nourished children, EPEC causes intestinal infections that usually disappear within one week, demanding only oral rehydration. However, in the case of malnourished children, the bacteria can cause persistent diarrhea, which continues for more than two weeks, repetition of which can have a negative impact on growth as well as on mental development.

In experiments carried out on mice and on cells (in vitro) at the Butantan Institute, a team coordinated by Maria Leonor Oliveira managed to check the EPEC bacteria by stimulating the production of antibodies against intimin-beta, a protein,  by means of which these bacteria attach themselves to intestinal walls. According to the study published in October in the journal FEMS Immunology Medical and Microbiology, two intimin-beta fragments taken orally using Lactobacilli bacteria, such as the ones used to make yogurts and cheese, reduced by up to 80% the level of adhesion to human epithelial cells of the most common of the 12 varieties of EPEC.

“A vaccine against diarrhea is still needed because of the precarious state of basic sanitation, which makes it easier for these bacteria to spread, particularly among children in those areas that are far from the major cities,” explains Waldir Elias Jr., a researcher at Butantan who took part in this study. “Healthy adults,” added his colleague Roxane Piazza, “have natural immunity and have developed anti-intimin antibodies because of the continuous contact with EPEC.” The next stage will be to check whether this strategy can solve intestinal infections directly in mice and whether it works against other types of EPEC already found in Brazil. Until then, the best way to avoid diarrhea in new-born children will remain breast feeding. “Children who are breast fed don’t get EPEC diarrhea,” says Solange Barros Carbonare, a Butantan researcher who found antibodies against these bacteria in all the hundreds of samples of colostrum and mother’s milk that she has analyzed over the last few years.

The Butantan Institute experiment showed that the intimin blocking strategy could be used both for EPEC and for EHEC. Some types of EPEC and EHEC live in the intestines of oxen, cows, sheep, goats, monkeys, cats and dogs, and rarely cause these animals any problems. Humans get these bacteria from contact with animals, earth, water or polluted foods. “Any raw or non-pasteurized food can carry these bacteria,” points out Beatriz. “Nor can we rule out person to person transmission, from an adult carrier of the bacterium to a child, for instance, via dirty hands.”

The most recent outbreaks in the United States were caused by hamburger meat, spinach and tomatoes that had been contaminated by the bacteria, which can cause problems even in very low quantities. 100 EHEC is enough to unleash intestinal infection, whereas cholera, which is also characterized by diarrhea and strong dehydration, only starts after 100 million Vibrio cholerae bacteria have settled in the organism.

The projects
1. Fenotypical and molecular analysis of Escherichia coli samples of the O113 serogroup
2. Atypical enteropathogenic Escherichia coli
3. Evaluation of the activity of human antibodies against enteropathogenic E. coli and Shiga toxin producing E. coli in vitro and in vivo
1. Regular Research Awards
2. Thematic Project
3. Regular Research Awards
1. Beatriz Ernetina Cabilio Guth – Unifesp
2. Waldir Pereira Elias Junior – Instituto Butantan
3. Solange Barros Carbonare – Instituto Butantan
1. R$ 118,610.98 (FAPESP)
2. R$ 790,675.07 (FAPESP)
3. R$ 99,462.08 (FAPESP)