Bug that bears the mark of death

While researching a strain of typhoid wreaking havoc in Vietnam, British scientists made a chilling discovery. At the heart of the typhoid bug they glimpsed the Black Death. Steve Farrar reports.

It was not supposed to be there. When you pit yourself against the uncanny ingenuity of bacteria you expect surprises. But the last thing Gordon Dougan imagined he would see when he peered into the genetic heart of the bug that causes typhoid was a glimpse of bubonic plague.

Yet the latest sequencing data to emerge from the Sanger Centre in Cambridge shows that a whole chapter of the genetic guidebook for Salmonella typhi, the nasty little bug that kills 600,000 people a year, is identical to that of Yersinia pestis, the even nastier one responsible for the Black Death.

Dougan, a professor of biochemistry at Imperial College, London, and an adviser to the World Health Organisation, was recently appointed director of the Wellcome Trust-funded Centre for Molecular Microbiology. But for all his experience, he admits this discovery caught him unawares. "It was a complete bolt from the blue," he says.

The data emerged from the pathogen sequencing unit of the Sanger Centre, whose hit list of bugs has revealed the genetic secrets of several killer microbes, including those responsible for tuberculosis and meningitis.

Alarmed by the emergence of a virulent and drug-resistant form of typhoid in Vietnam, Dougan and colleagues pushed their pathogen, Salmonella typhi, to the front of the unit's queue in the hope of finding new ways to tackle the disease. With the genome all but complete, the analysis has now begun. It makes for chilling reading.

The biggest surprise came when a newly decoded sequence of plague DNA also being deciphered at the Sanger Centre was unexpectedly found - by Mike Prentice at St Bartholomew's Hospital in London - to match with typhoid. Dougan was immediately alerted. Inside the nucleus of Salmonella typhi Dougan's team had already found three separate pieces of DNA. The first was a massive coil some 4.5 million bases long that essentially defined the bacteria, telling it how to grow, function and survive. Alongside it were two plasmids, smaller loops of free-swimming genetic data.

One of the plasmids contained an astonishing array of offensive and defensive genes, which probably explain the potency of the strain of typhoid wreaking havoc in Vietnam. The nature of the other plasmid was a mystery, until Dougan realised that this was where the plague DNA must rest.

It was not simply a matter of two bacteria swapping the odd gene, something witnessed many times before. This plasmid contained some 50 to 60 genes that help make plague so virulent. It was genetic shoplifting on an unprecedented scale. That two of the most unpleasant pathogens to afflict humanity were involved made it doubly disturbing.

The Vietnamese bug appears to be an enhanced microbe, fortified with the genes of other pathogens. Those who succumbed to it experienced a particularly nasty attack of typhoid, but no one has recorded any plague-like symptoms. Neither do the scientists expect any. Just what that plasmid actually does is not yet known, but the chances are that the genes it carries give this strain of typhoid some advantages. That is bad news for humanity.

Typhoid may have been virtuallyeradicated in the United Kingdom, but in Vietnam it is all too real. Those who catch it - 1 per cent of the Vietnamese population and at least 16.6 million worldwide - suffer fever, haemorrhaging, diarrhoea and often painful bowel perforations. Some die, mostly children. Its spread is aided by poor sanitation and lack of clean water. Treating it is a serious burden on poorer nations.

In 1993, doctors working in the Mekong Delta began seeing people afflicted with a particularly nasty type of typhoid that was impervious to many medicines. Its spread was also witnessed by a newly assembled team of scientists. The Oxford University-Wellcome Trust Clinical Research Unit was established within the Centre for Tropical Diseases in the Cho Quan Hospital, Ho Chi Minh City, in 1991, precisely to tackle such problems.

The unit, under the direction of Oxford clinician Jeremy Farrar, matched British experts with a Vietnamese team led by T. T. Hien. "It is impossible for developing countries to find enough money to make use of the massive increase in scientific knowledge or for London, Oxford or New York to address the problem of typhoid from the comfort of the developed world," Farrar says. The Wellcome initiative bridges that gap.

John Wain, one of the unit's experts, who is now back with Dougan in London, treated many typhoid sufferers during his time at Cho Quan. Generally, treatment was successful. Most of the patients were discharged after a short programme of antibiotics.

Wain's records note that in 1994, they included a nine-year-old boy from the Mekong Delta. He knows the youngster returned home healthy but cannot recall his face. The genetic code of the bug he was carrying, however, is far more familiar.

Dougan needed Salmonella typhi DNA to sequence, and a sample taken from the child was chosen for the task. Back in the UK, those genes are starting to surrender their secrets, and Dougan's team, with Bart Barrell and Julian Parkhill at the Sanger Centre, has discovered what makes this particular typhoid strain so vicious.

That first plasmid, the one not carrying plague DNA, turned out to contain genes that give the microbe a host of defensive capabilities as well as pepping up its virulence. There are genes to fend off ten common antibiotics - one pumps tetracycline out of the bacteria before it can do any harm, another cloaks the chemical target that trimethoprim homes in on. One gene protects the microbe from manmade pollutants such as mercury, while another stretch of genes extends the bug's life in victims' blood.

However, despite this valuable genetic survival pack, acquired about a decade ago, the microbe was still vulnerable to a new generation of antibiotics called fluoroquinolones. These drugs are cheap and easy to administer and doctors across Vietnam, prompted by the Cho Quan experts, have been trained in their use. But the bug has begun to devise its own defence. It needs four mutations to its genome to gain full resistance to fluoroquinolones. In the space of a few years it has managed two. "We are watching evolution in progress, and all we can do is delay it," Dougan says.

Already the medicines are having a declining effect and it is only a matter of time before they become useless. Sadly, the cost of alternatives is prohibitive for Vietnam.

From the viewpoint of disease-causing bacteria, mankind has been a wonderful challenge. Those bugs that target humans have grown up alongside us. The speed at which bacteria can evolve - a new generation every 20 minutes in Salmonella typhi - allows them to exploit fresh opportunities from changes in our lifestyle. There is growing evidence that this is how many new diseases evolved in past millennia. Bubonic plague might have emerged as little as 1,500 years ago when a microbe that previously caused only a mild stomach upset mutated into Yersinia pestis, possibly in response to people moving into crowded communities and near constant contact with disease-carrying fleas and rats.

Similarly, tuberculosis might have evolved from a bovine bacterium some 15,000 years ago in response to the domestication of cattle. The emergence of Aids and the spread of hepatitis B and C remind us that if anything, this process is getting worse, spurred on by the unprecedented social upheavals of our time.

There are suggestions that the new strain of typhoid originated in central Asia, possibly within Afghan refugee camps. Plans are afoot to investigate this possibility. The genetic-screening techniques that Dougan's team has developed to study the spread of Salmonella typhi will ultimately be used to track local outbreaks back to their source. They will also enable scientists to spot the emergence of different strains and find the hotspots where their evolution is at a frenzy.

"These places are like an open sore on the body, a reservoir for the evolution of pathogenic micro-organisms," Dougan says. "Infectious diseases are still emerging. We need to be on our toes."

While sequencing will help identify genetic targets for new antibiotics and screening will pick out those most vulnerable to infection, there is no doubt we are up against an ingenious and determined foe. All the tricks Dougan's team has seen with Salmonella typhi will be employed by many different bacteria to create new diseases or revitalise old ones. The bugs have been playing this genetic card game for aeons. The latest technological breakthroughs have allowed us to peek at some of our opponents' cards -but we still barely know the rules.

The longer it takes for us to learn, the more vulnerable we will be to a new epidemic. Its genesis may already have begun.

More info at: www.sanger.ac.uk/Project/S_typhi/