Where No Botulinum Toxin Has Gone Before

Toxin found for first time in Enterococci bacteria

Where No Botulinum Toxin Has Gone Before
Enterococci bacteria. Image: James Archer/CDC

Enterococci are hardy microbes that thrive in the gastrointestinal tracts of nearly all land animals, including our own, and generally cause no harm. But their ruggedness has lately made them leading causes of multi-drug-resistant infections, especially in settings like hospitals where antibiotic use disrupts the natural balance of intestinal microbes.

So the discovery of a new toxin in a strain of Enterococcus is raising scientific eyebrows. Isolated from cow feces sampled at a South Carolina farm, the bug was unexpectedly found to carry a toxin resembling the toxin that causes botulism. The finding was reported in Cell Host and Microbe.

Get more HMS news here

“This is the first time a botulinum neurotoxin has been found outside of Clostridium botulinum—and not just the toxin, but an entire unit containing the toxin and associated proteins that prevent the toxin from being degraded in the GI tract,” said Min Dong, Harvard Medical School assistant professor of surgery at Boston Children’s Hospital and one of the world’s experts on botulinum toxins.

The toxin, dubbed BoNT/En, is the ninth botulinum toxin to be described. Last August, Dong and colleagues reported the eighth, BoNT/X, made by C. botulinum—the first new botulinum toxin to be found in close to 50 years.

Should we be scared?

No. At least, not yet, said Sicai Zhang, a postdoctoral fellow in Dong’s lab and one of three co-first authors on the new report.

“The enterococcal isolate carrying the toxin luckily remains susceptible to key antibiotics,” said Zhang. “It was found only once from a single animal, and no signs of botulism disease were observed.”

When Sicai and his colleague Jie Zhang tested the toxin in rodents in the lab, it had little or no effect. Only when they manipulated the toxin to better target mouse and rat neurons did it become potent, shutting down nerve function and causing paralysis.

Dong’s lab is now testing BoNT/En in cultured human neurons to find out whether it’s toxic to humans.

Making the leap

How could this botulinum toxin jump from one bacterial species to another? Teams led by Dong’s collaborators, Michael Gilmore, the HMS Sir William Osler Professor of Ophthalmology at Massachusetts Eye and Ear, and Andrew Doxey, a bioinformatician at the University of Waterloo, found that the BoNT/En botulinum toxin genes were carried by a plasmid.

Plasmids are mobile structures that contain DNA independently of chromosomes and can be swapped from one bacterium to another. Plasmids are quite common in enterococci. In fact, they have been associated with the acquisition of resistance to vancomycin, a last-resort antibiotic, and transfer of resistance to the fearsome Staphylococcus aureus.

Down on the farm

Gilmore’s lab sequenced the toxin producing E. faecium strain as part of a much wider search for the origins of enterococcal antibiotic resistance and disease-causing ability.

“We were not looking for a neurotoxin in E. faecium,” said Francois Lebreton, HMS instructor of ophthalmology at Mass. Eye and Ear and another co-first author on the paper, who specializes in examining the genome sequences of these microbes. “There was no reason to suspect its existence.”

“This is a unique discovery of a botulinum neurotoxin in a bacterium that is both ubiquitous in animals and a serious problem in human health.” —Francois Lebreton

Lebreton has been investigating the evolution of enterococci from their commensal Paleozoic origins to their rise as a hospital threat.

“In intensive agriculture, antibiotics are administered to farm animals to promote weight gain in often crowded facilities. We believe that this creates an environment in the animal gut that allows antibiotic-resistant enterococci to thrive and come into contact with humans,” he explained. “We know that the highly antibiotic-resistant E. faecium strain we fight in the hospital is very closely related to strains found in the GI tracts of farm-raised animals.”

From farm to computer

Doxey’s lab specializes in mining genome data to discover new toxins and virulence genes. Analyzing the newly sequenced E. faecium genome, the lab’s computer programs quickly spotted the genetic sequence for the novel botulinum toxin.

“The way that we discovered this toxin using computational methods is different from how toxins used to be identified in the past, and may become a standard approach in biomonitoring,” Doxey said. “It represents scientific collaboration and data sharing at its best.”

A perfect storm?

The newly discovered toxin does raise some concern that botulinum toxin could turn up in antibiotic-resistant enterococci, perhaps stemming from gene transfer in the gut of an animal harboring both C. botulinum and Enterococcus.

“This is a unique discovery of a botulinum neurotoxin in a bacterium that is both ubiquitous in animals and a serious problem in human health,” said Lebreton. “E. faecium is in the gut of nearly every human; it is extremely tough and survives a lot of stresses, often including efforts to disinfect hospital surfaces. A hospital-adapted, antibiotic-resistant, hard-to-kill bug carrying a neurotoxin would be a worst-case scenario.”

Exactly what animal this ninth botulinum toxin is meant to target remains unknown. Gilmore’s lab continues to expand and study its collection of enterococcal isolates.

“Most of what we know about Enterococcus comes from the few strains circulating in the hospital,” said Lebreton. “It’s possible that BoNT/En, or even other novel toxins, will turn up in other enterococci isolated from the wild. We just never looked for those before.”

The fine print

This study was funded by grants from the National Institutes of Health (AI132387, NS080833, AI083214, AI072360, and AI108710), Swedish Research Council (2014-5667), Wenner-Gren Foundation and Swedish Cancer Society. Additional support came from a grant (FunGCAT) from IARPA (The Intelligence Advanced Research Projects Activity), National Institute of General Medical Sciences (F32GM121005), Natural Sciences and Engineering Research Council of Canada (NSERC Discovery Grant), an Ontario Early Researcher Award, the NIH-funded Harvard Digestive Disease Center (P30DK034854), Boston Children’s Hospital Intellectual and Developmental Disabilities Research Center (P30HD18655) and the Burroughs Wellcome Fund.

Sicai Zhang (Dong Lab), Francois Lebreton (Gilmore lab) and Michael Mansfield (Doxey Lab) were co-first authors on the study. Coauthors were Shin-Ichiro Miyashita, Jie Zhang and Liang Tao of the Dong Lab; Julia Schwartzman of the Gilmore Lab; Geoffrey Masuyer, Markel Martínez Carranza and Pål Stenmark of Stockholm University; and Michael Gilmore, who is also founder and Principal Investigator of the Harvard-wide Program on Antibiotic Resistance. Andrew Doxey and Min Dong are both corresponding authors.

Adapted from a post on Vector, the Boston Children’s clinical and research innovation blog.