The recent outbreak of E. coli infections that began in Germany sickened more than 4,000 people in 16 countries and caused an unusually high number of cases of hemolytic-uremic syndrome, a life-threatening complication that destroys red blood cells and damages the kidney. An international team of researchers seeking to understand the genetic makeup of the bacteria responsible for the outbreak has compared its genome to the genomes of 11 related strains of E. coli from around the world. Their analyses show that the deadly strain likely emerged when a less pathogenic E. coli picked up a critical set of virulence genes that made it more lethal in humans.

This sharing of genes among bacteria, known as horizontal gene transfer, is common among bacteria, and the new study illustrates how it contributes to changes in virulence and the rapid evolution of infectious pathogens. The team, led by Howard Hughes Medical Institute investigator Matthew Waldor, published its findings in an online article in the New England Journal of Medicine on July 27, 2011.

“It rapidly became clear that this was an unusual -- perhaps new -- pathogen that was giving rise to this unprecedented epidemic of hemolytic-uremic syndrome.”
Matthew K. Waldor

Waldor says it was clear from the early stages of the outbreak that the bacteria causing the illness were not typical. The bacteria produced a toxin called Shiga toxin; Shiga-toxin producing E. coli have been associated with previous outbreaks of hemolytic-uremic syndrome (HUS). Most Shiga-toxin producing E. coli are classified as enterohemorrhagic, but laboratory studies showed that the outbreak was not caused by this type of E. coli. Instead, initial tests suggested that the strain appeared similar to a separate type of E. coli known as enteroaggregative E. coli. Although enteroaggregative E. coli are widespread, Waldor says they are not generally thought to be highly virulent.

The process of conjugation, shown in this animation, is one method bacteria use to share genes.
Video: HHMI Biointeractive

“It rapidly became clear that this was an unusual -- perhaps new -- pathogen that was giving rise to this unprecedented epidemic of hemolytic-uremic syndrome,” says Waldor, who is at Brigham and Women’s Hospital and Harvard Medical School. “Nearly 25 percent of people who had symptoms developed HUS. This is by far the largest epidemic of HUS in history.”

Waldor, an expert in pathogenic bacteria that infect the gut, says his interest was piqued immediately by the unusual nature of the German outbreak. Other research groups took notice as well, Waldor said. The outbreak began in May, and by the beginning of June, a team at the Beijing Genomics Institute had released a preliminary draft of the genome sequence of the responsible bacteria and scientists around the world set to work analyzing the data.

Waldor decided that with the right technology and the right team, he could contribute to the scientific community’s understanding of the virulent pathogen, which was continuing to cause disease and disrupting commerce in Europe. Scientists including James Nataro of the University of Virginia, David Rasko of the University of Maryland, and Karen Krogfelt and Flemming Scheutz of the Statens Serum Institute in Copenhagen, assembled a collection of E. coli samples for analysis. These included E. coli from a patient infected during the current outbreak, plus samples of enteroaggregative E. coli from patients in Peru, Chile, Mexico, Africa, and Denmark. The complete DNA sequences of the genomes of these 12 isolates were determined using sequencing technology from Pacific Biosystems, which Waldor says is particularly well suited to producing complete bacterial genomes because it generates very long “reads” -- uninterrupted sequences of DNA. Eric Schadt led the sequencing and analysis work at Pacific Biosciences.

When the team compared the sequences of these strains – supplementing their analysis with 40 other previously sequenced E. coli isolates -- it was clear that bacteria responsible for the outbreak shared all the hallmarks of enteroaggregative E. coli, but unlike most bacteria of this type, included a small segment of DNA encoding Shiga toxin. That DNA segment corresponded to a prophage -- an integrated virus. This finding meant that the enteroaggregative E. coli had acquired the genes for Shiga toxin because it was infected by a virus, also called a phage. Phage infection is a major mechanism of gene transfer between bacteria. They also identified several other virulence and antibiotic-resistance genes that the strain also appeared to have acquired via horizontal gene transfer as it evolved.

“Horizontal gene flow is very common in bacteria,” Waldor says – noting that in E. coli, transfer of genes between individuals is thought to account for about 20 percent of the genome. “Ultimately, during the evolution of contemporary strains, only 80 percent came from mom, and the rest of it came from the next-door neighbors,” he explains. The new findings underscore an idea that already has a great deal of evidence behind it: horizontal gene transfer has been particularly important in the evolution of pathogens.

Waldor cautions that it is too soon to consider the strain that caused the outbreak “hyper-virulent.” Other epidemiological factors, such as the degree of contamination of the suspected food source (sprouts), could have influenced the severity of the epidemic. “We think it could be the bug, but really we don’t know that with certainty at this point,” he says. However, the particular combination of virulence factors in the outbreak strain together with the presence of Shiga toxin likely contribute to the pathogen’s virulence. Taken together, Waldor says, his team’s findings highlight ways in which bacterial genomes can undergo rapid evolution, leading to the emergence of new pathogens.