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January 29, 2018

Botulinum Toxin Jumps to Ubiquitous Bacterial Family

Source: Francois Lebreton, Daria Van Tyne and Ann Tisdale, Massachusetts Eye and Ear

  • When not used to stiffen the muscles and smooth out the wrinkles on your face, botulinum toxin is regarded as one of the deadlier substances in the world—so much so that it has been (and most likely still is) used as a biological weapon by many nation states. Yet, until now, scientists only needed to worry about the deadly neurotoxin being produced by a single bacterial species, Clostridium botulinum, most often associated with foodborne illness. Now, a collaborative team of investigators led by scientists at Harvard Medical School have discovered botulinum toxin in a strain of Enterococcus—hardy microbes that thrive in the gastrointestinal tracts of nearly all land animals, including our own—isolated from cow feces sampled at a South Carolina farm.

    The findings from the new study were published recently in Cell Host Microbe through an article entitled “Identification of a Botulinum Neurotoxin-like Toxin in a Commensal Strain of Enterococcus faecium.”

  • Click Image To Enlarge +
    Botulinum toxin was able to jump into a new type of bacteria, Enterococcus, through plasmids, mobile structures that contain DNA independently of the chromosomes and can be swapped from one bacterium to another. [Elena Hartley]

    "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," explained senior study investigator Min Dong, Ph.D., a scientist in Boston Children's Hospital's Department of Urology and Harvard Medical School 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, Dr. Dong and his colleagues reported the eighth, BoNT/X, made by C. botulinum and the first new botulinum toxin to be found in close to 50 years.

    "The enterococcal isolate carrying the toxin luckily remains susceptible to key antibiotics," noted lead study investigator Sicai Zhang, Ph.D., a postdoctoral fellow in Dr. Dong's laboratory. "It was found only once from a single animal, and no signs of botulism disease were observed." 

    When the research team 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. The researchers are currently testing BoNT/En on cultured neurons to determine if it is toxic to humans.

    Additionally, the researchers were curious to determine exactly how the botulinum toxin jumped from one bacterial species to another. As they suspected, they found that BoNT/En botulinum toxin genes were carried by a plasmid. Plasmids are mobile structures that contain DNA independently of the 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

  • Click Image To Enlarge +
    Zhang et al. report that a commensal strain of Enterococcus faecium carries a conjugative plasmid encoding a BoNTlike toxin gene. Thus, a commensal organism can acquire and possibly disseminate BoNT genes. [Zhang et al., 2018, Cell Host & Microbe 23, 1–8]

    This ability to swap genes is what worries many researchers. Could a potent toxin from C. botulinum end up in a multidrug-resistant, human E. faecium strain? Many investigators now seem to think that it is at least theoretically possible.

    "Enterococcus is a central hub for gene transfer within the gut, and that makes it potentially scary," Dr. Dong remarked.

    With current technology being what it is today, the research team was able to rapidly sequence 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," stated co-lead investigator Francois Lebreton, Ph.D., an instructor at Harvard Medical School who specializes in examining the genome sequences of these microbes. "There was no reason to suspect its existence."

    Dr. Lebreton has been investigating the evolution of enterococci from its commensal Paleozoic origins its 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," Dr. Lebreton 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."

    When bioinformaticians started mining the genome data from the newly sequenced E. faecium, to discover new toxins and virulence genes, the 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," said study co-author Andrew Doxey, Ph.D., assistant professor at the University of Waterloo. "It represents scientific collaboration and data sharing at its best."

    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," Dr. Lebreton stated. "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. They research team 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," Dr. Lebreton concluded "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."

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