ABOVE: Carefully collected venom samples from black-necked spitting cobras (left) contain bacteria that grow on blood agar (middle) and MacConkey agar (right). STEVE TRIM AND STERGHIOS MOSCHOS

Update (May 31): The paper described in this article was published May 23 in Microbiology Spectrum.

For years, researchers have been looking to animal venoms for the next generation of antibiotics. That’s because these chemical cocktails possess potent antimicrobial activities in addition to their dangerous physiological ones—a fact that has also led to the widespread idea that, despite being connected to the outside world, venom glands are sterile environments.

That’s simply not true, posits a November 5 biorXiv preprint. As the title neatly summarizes, “Microbial adaptation to venom is common in snakes and spiders.”  

The research was led by Northumbria University RNA biologist Sterghios Moschos and venomologist Steve Trim, founder of the biotechnology company Venomtech, which aims to develop venom-based research tools and pharmaceuticals. It provides genetic and culture evidence that bacteria not only live in the venoms of several species of spiders and snakes, but are actively adapting to the venom gland environment—which could have implications for the evolution of venoms and the clinical management of bites.

A black-necked spitting cobra (Naja nigricollis)—one of the species studied—from Watamu, Kenya

“This paper is remarkable,” writes National University of Ireland Galway biologist John Dunbar in an email to The Scientist. Dunbar, who was not involved in the research, says that the evidence the study provides against the “traditional” view of venoms and venom glands as sterile environments is “strong”: “It totally throws a spanner in the works.”

University of Arizona medical toxinologist Leslie Boyer, who was also not involved in the work, agrees that the authors present convincing evidence for venom-dwelling microbes. But as a physician who has treated snakebite cases for decades, Boyer says she’s skeptical that the microbes described in the paper have immediate clinical relevance. “There’s a big difference between good basic science and meaningful applied clinical science. And this is the first step in a path that could conceivably lead to clinical implications, but it hasn’t got them yet,” she says.

Upending sterile dogma

The idea that animal venoms are germ-free likely arose sometime in the mid-20th century, when researchers began documenting the antimicrobial activities of venoms and their components. That laboratory science was then translated into clinical practice, says Boyer, in the sense that even to this day, snakebites are often not treated to prevent infection. Boyer describes the mentality as, “‘Oh heck, venom is so potent against everything biological, it would kill the germs anyway. It’s just going to kill everything in the wound, and nothing will live, and so we don’t have to worry about infection at all.’”

However, Sébastien Larréché, a clinical microbiologist at the Hôpital d'Instruction Des Armées Bégin in France, points out in an email to The Scientist that “new culture-free methods suggest that no anatomical area is really sterile in humans. By extrapolation, we can therefore assume that the venomous apparatus has its own microbiota.” Indeed, research has found microbes in the venom glands of squids, snails, and other animals, though investigations into venom gland microbiomes are currently few and far between, notes a 2019 Toxicon: X paper coauthored by Trim and Moschos. To remedy that, the paper announced the founding of a venom-microbiome research consortium called the Initiative for Venom Associated Microbes and Parasites, or iVAMP.

Steve Trim holding a vial of Naja nigricollis venom
Courtesty of STEVE TRIM

Seeing a gap in the literature, Moschos, Trim, and colleagues used both culture and culture-free methods in the new study to search for microbes in the venoms of five snake and two spider species. They also swabbed the animals’ mouths and fangs for comparison. They obtained bacterial 16S sequences from all of their samples, and culturable microbes from most of them, they report, although the cultured species represented only a fraction of what was revealed by DNA. The microbes detected in the venom samples differed markedly from the general oral flora for each species, with about 20 percent of the microbes detected in venom samples not found in the mouth. Additionally, the authors found that some of the culturable microorganisms—namely, strains of Enterococcus faecalis—flourish in the venom of black-necked spitting cobras (Naja nigricollis), likely thanks to mutations to genes involved with membrane integrity.

The purported venom microbes that the team was able to grow were remarkably resilient to the animals’ toxins. “Normally, you put fifteen [milligrams per milliliter] of any antimicrobial substance [in their growth medium], and not only is the bacteria of interest not going to grow, nothing will grow,” explains Moschos. “In this instance, our bacteria grew quite happily [in that and higher concentrations of venom]. They were not inhibited in growth at all.” The paper notes that one strain showed no ill effects from venom whatsoever, even at a venom protein concentration of 50 mg/ml, while a vancomycin-resistant clinical isolate of the same species was wiped out by roughly one-fifth that amount.

Larréché, who was not involved in the study, calls the microbiological methods used by the team “robust” while noting that “there remains a risk of contamination by the bacteria present on the fangs when the venom is ejected.” He concedes that risk is difficult to remove, as “it is difficult (and above all dangerous for the operator and for the snake) to carefully disinfect the fangs before sampling.”

University of Queensland venom scientist Samantha Nixon, who also did not participate in the research, says the sampling of the fangs and mouth in addition to the venoms to try to rule out contamination was “quite clever” and “really a strength of this study compared to previous ones.” Still, she also wonders if the venom samples were truly free of fang microbes. “When I collect spider venoms myself, I often get a bit of debris off the spider fangs” simply because the electrostimulation used to expel venom makes the spiders twitch a bit, which can dislodge and scatter dirt from their bodies. She adds, however, that the authors’ protocols were sound: “I have been thinking about it. I’m not sure if there’s a much better way to go about it.”   

Regardless, she says, the study “is adding to a building body of evidence that there really are microbes in the venom glands, and that they probably do play quite an important role.”

Arms race or function?

Moschos and Trim say that these bacteria have likely been overlooked for so long despite extensive research on snake and spider venoms because of the way these venoms are usually prepared. Shortly after collection, most venoms undergo lyophilization—a kind of freeze-drying—to prolong their shelf life. But the process destroys any living bacteria, so culture methods for microbe detection would be off the table. That leaves DNA-based methods, which also prove tricky due to the enzymes and other chemicals in the venom that can interfere with PCR.

In fact, the team says that one of the big challenges of this research was figuring out how to extract DNA from venom. “I have never seen anything like it,” describes Moschos, whose research predominately focuses on obtaining genetic material from tricky samples such as exhaled breath. “We had to play around a little bit,” he says, to get the extraction protocols to work.

Moschos and Trim say they hope other researchers will take their methods and try them on other venomous species. Their hunch is that there’s nothing particularly special about the species they used; microbes are simply able to evolve to live anywhere, even in glands producing highly antimicrobial venoms. Now they and their colleagues want to know: Where do the bacteria come from and what are they doing there?

Poecilotheria regalis, one of the spiders examined in the study

The bacteria are likely opportunistic, Moschos and Trim suggest. As Dunbar phrases it, venom is “a perfectly nutritious broth” thanks to all the proteins and other biomolecules in it. All a microbe has to do to tap into this resource is survive it. Trim says that the chance of bacterial residence may be the reason those antimicrobials are in venom in the first place—to ensure that its biochemical makeup remains sufficiently potent for hunting or protection against predation for the days, weeks, or even months that it is stored in the glands before use. “If the bacteria are metabolizing your arsenal, you can see the potential for the venoms to be deactivated,” he says. There may well be an arms race between microbes and venomous animals, Trim explains, with animals evolving to produce greater amounts and more powerful antimicrobials so that their toxic cocktail lasts long enough for use, while the microbes evolve ways of subverting these compounds.

In support of this idea, Moschos says that the team found “statistically significant enrichment for mutations that suggest that the bacteria are actively evolving, constantly trying to get into the venom.” This included mutations in genes associated with antimicrobial resistance and with membrane integrity, which could be essential to withstand the venoms’ lipid-chopping toxins.

However, the relationship between venom gland microbes and their hosts may not be entirely antagonistic. Venomous animals such as blue-ringed octopuses of the genus Hapalochlaena use bacterially produced toxins in their venoms, Nixon notes; if venom gland microbiomes are present in most or all venomous animals, it’s possible that such mutualistic partnerships with bacteria are more widespread than currently thought.

Furthermore, Nixon points out that there may well be dynamic changes in venoms and their microbiomes. “Many venomous animals will modulate their venom in response to environmental stimuli and life changes,” she says, “so I think the question of whether the venom gland responds to changes in the microbiome is really interesting,” and potentially one that could be investigated using venom-producing organoids.  

Clinical questions remain

Whatever role they play in venomous animals, if venom-dwelling bacteria can infect skin and other tissues, they could cause direct harm to the creatures bitten, including humans. Moschos suspects the venom-dwelling bacteria are especially pathogenic. “The moment the venom hits that tissue, and there’s massive lysis, massive cell death for the host—that creates a soup of nutrients that the bacteria are quite happy they’ve been injected into. In the snakebite wound, they can sit there and just eat, eat, eat, reproduce, grow.”

“The oppression from the venom is suddenly removed, and they’re free to go into full exponential [growth],” adds Trim.

Indeed, Moschos, Trim, and colleagues’ discovery of Enterococcus faecalis in spitting cobra venom is particularly intriguing because this species is known to be responsible for many snakebite wound infections. However, the research stopped short of directly evaluating the clinical relevance of it or any other potentially venom-vectored microbe.

There’s a big difference between good basic science and meaningful applied clinical science. And this is the first step in a path that could conceivably lead to clinical implications, but it hasn’t got them yet.

–Leslie Boyer, University of Arizona

Dunbar says the prevailing assumption that all infections of venomous bites are from opportunistic microbes living on the skin or in the environment never really made sense to him, particularly when it comes to spider bites. “In my line of work, I’ve been cut and scratched walking through South American swamps, Asian jungles, and African deserts and have yet to experience a bacterial infection,” he notes, making him skeptical that a bite from “a tiny spider with fangs so small you can’t even see the puncture wound” would become infected by skin microbes. “It seems much more plausible that pathogenic and antibiotic resistant bacteria in the venom or on the fangs that has penetrated the skin barriers is much more likely to be responsible for establishing an infection.”

Dunbar and his colleagues have investigated the idea for false widow (Steatoda nobilis) bites, finding that the bacteria on the spiders’ fangs are distinct from the bacteria on their bodies and include antibiotic-resistant human pathogens. However, the study didn’t examine the spiders’ venom or venom glands—that research is in the works, says Dunbar. He adds that the team did find that the venom “had no inhibitory effect [on] bacteria, suggesting that bacteria potentially transferred during a bite would not be inhibited directly from the venom activity.”

Boyer doesn’t quite buy this line of reasoning for snakebites. “I want to see what happens when those germs go into patients,” she says. “I just don’t believe that the germs that are being propelled in there by snake fangs are having a statistically important impact on the health of my patients in the US,” she says, expressing doubt that microbes adapted to living peaceably in their cold-blooded hosts would thrive as pathogens in human tissues.

Boyer adds that in her experience, infections of snakebite wounds are rare to begin with—though that observation is clouded by antibiotic use by primary care physicians that patients often see separately. She also concedes that in other parts of the world, including sub-Saharan Africa where black-necked spitting cobras are found, among many other venomous snakes, infections are more common. But that may be due to myriad factors, she says, including the lengthier duration of time that usually passes before cases reach a hospital and the kind of first aid given. Traditional healers sometimes make incisions into bite wounds or rub them with botanical mixtures, for instance, which may increase the potential for infection, she says.

Larréché expresses similar reservations and says further research is needed on a diversity of snakes, as “the authors only used ophidian species whose bites are known to be regular sources of infections.” He adds: “In my opinion, it would have been appropriate to add species such as [members of the viper genera] Vipera or Echis to this panel, the bite of which, on the contrary, is almost never complicated by infection.”

For now, he says the study hasn’t changed his mind—he still maintains that opportunistic skin or environmental microbes, not venom-derived ones, are to blame for bite-associated infections.

Moschos, on the other hand, says his work has convinced him, arguing that it’s unlikely a coincidence that the most common microbe in their cultures is also the most common culprit in snakebite infections. “If it looks like a duck and walks like a duck, then it’s probably not a chicken,” he says.