Indigenous people in Australia dig pits next to a honeypot colony. Scientists think the honey can be used to make new antimicrobial drugs.
These hunts have become rarer, as many of the Tjupan people have moved away and, up until now, the exact antimicrobial properties of the ant honey remained unknown. But recently, scientists Andrew Dong and Kenya Fernandes from the University of Sydney, joined Ulrich, who runs the Honeypot Ants tours in Kalgoorlie, a city in Western Australia, on a honey-gathering expedition. Afterwards, they ran a series of experiments analyzing the honey’s antimicrobial activity—and confirmed that the Indigenous wisdom was true. The honey was effective against Staphylococcus aureus, a common pathogen responsible for sore throats, skin infections like boils and sores, and also sepsis, which can result in death. Moreover, the honey also worked against two species of fungi, Cryptococcus and Aspergillus, which can be pathogenic to humans, especially those with suppressed immune systems.
In the era of growing antibiotic resistance and the rising threat of pathogenic fungi, these findings may help scientists identify and make new antimicrobial compounds. “Natural products have been honed over thousands and millions of years by nature and evolution,” says Fernandes. “And some of them have complex and intricate properties that make them really important as potential new antibiotics. “
In an era of growing resistance to antibiotics and new threats of fungi infections, the latest findings about honeypot ants are helping scientists identify new antimicrobial drugs.
Danny Ulrich
Bee honey is also known for its antimicrobial properties, but bees produce it very differently than the ants. Bees collect nectar from flowers, which they regurgitate at the hive and pack into the hexagonal honeycombs they build for storage. As they do so, they also add into the mix an enzyme called glucose oxidase produced by their glands. The enzyme converts atmospheric oxygen into hydrogen peroxide, a reactive molecule that destroys bacteria and acts as a natural preservative. After the bees pack the honey into the honeycombs, they fan it with their wings to evaporate the water. Once a honeycomb is full, the bees put a beeswax cover on it, where it stays well-preserved thanks to the enzymatic action, until the bees need it.
Less is known about the chemistry of ants’ honey-making. Similarly to bees, they collect nectar. They also collect the sweet sap of the mulga tree. Additionally, they also “milk” the aphids—small sap-sucking insects that live on the tree. When ants tickle the aphids with their antennae, the latter release a sweet substance, which the former also transfer to their colonies. That’s where the honey management difference becomes really pronounced. The ants don’t build any kind of structures to store their honey. Instead, they store it in themselves.
The workers feed their harvest to their fellow ants called repletes, stuffing them up to the point that their swollen bellies outgrow the ants themselves, looking like amber-colored honeypots—hence the name. Because of their size, repletes don’t move, but hang down from the chamber’s ceiling, acting as living feedstocks. When food becomes scarce, they regurgitate their reserves to their colony’s brethren. It’s not clear whether the repletes die afterwards or can be restuffed again. “That's a good question,” Dong says. “After they've been stretched, they can't really return to exactly the same shape.”
These replete ants are the “treat” the Tjupan women dug for. Once they saw the round-belly ants inside the chambers, they would reach in carefully and get a few scoops of them. “You see a lot of honeypot ants just hanging on the roof of the little openings,” says Ulrich’s mother, Edie Ulrich. The women would share the ants with family members who would eat them one by one. “They're very delicate,” shares Edie Ulrich—you have to take them out carefully, so they don’t accidentally pop and become a wasted resource. “Because you’d lose all this precious honey.”
Dong stumbled upon the honeypot ants phenomenon because he was interested in Indigenous foods and went on Ulrich’s tour. He quickly became fascinated with the insects and their role in the Indigenous culture. “The honeypot ants are culturally revered by the Indigenous people,” he says. Eventually he decided to test out the honey’s medicinal qualities.
The researchers were surprised to see that even the smallest, eight percent concentration of honey was able to arrest the growth of S. aureus.
To do this, the two scientists first diluted the ant honey with water. “We used something called doubling dilutions, which means that we made 32 percent dilutions, and then we halve that to 16 percent and then we half that to eight percent,” explains Fernandes. The goal was to obtain as much results as possible with the meager honey they had. “We had very, very little of the honeypot ant honey so we wanted to maximize the spectrum of results we can get without wasting too much of the sample.”
After that, the researchers grew different microbes inside a nutrient rich broth. They added the broth to the different honey dilutions and incubated the mixes for a day or two at the temperature favorable to the germs’ growth. If the resulting solution turned turbid, it was a sign that the bugs proliferated. If it stayed clear, it meant that the honey destroyed them. The researchers were surprised to see that even the smallest, eight percent concentration of honey was able to arrest the growth of S. aureus. “It was really quite amazing,” Fernandes says. “Eight milliliters of honey in 92 milliliters of water is a really tiny amount of honey compared to the amount of water.”
Similar to bee honey, the ants’ honey exhibited some peroxide antimicrobial activity, researchers found, but given how little peroxide was in the solution, they think the honey also kills germs by a different mechanism. “When we measured, we found that [the solution] did have some hydrogen peroxide, but it didn't have as much of it as we would expect based on how active it was,” Fernandes says. “Whether this hydrogen peroxide also comes from glucose oxidase or whether it's produced by another source, we don't really know,” she adds. The research team does have some hypotheses about the identity of this other germ-killing agent. “We think it is most likely some kind of antimicrobial peptide that is actually coming from the ant itself.”
The honey also has a very strong activity against the two types of fungi, Cryptococcus and Aspergillus. Both fungi are associated with trees and decaying leaves, as well as in the soils where ants live, so the insects likely have evolved some natural defense compounds, which end up inside the honey.
It wouldn’t be the first time when modern medicines take their origin from the natural world or from the indigenous people’s knowledge. The bark of the cinchona tree native to South America contains quinine, a substance that treats malaria. The Indigenous people of the Andes used the bark to quell fever and chills for generations, and when Europeans began to fall ill with malaria in the Amazon rainforest, they learned to use that medicine from the Andean people.
The wonder drug aspirin similarly takes its origin from a bark of a tree—in this case a willow.
Even some anticancer compounds originated from nature. A chemotherapy drug called Paclitaxel, was originally extracted from the Pacific yew trees, Taxus brevifolia. The samples of the Pacific yew bark were first collected in 1962 by researchers from the United States Department of Agriculture who were looking for natural compounds that might have anti-tumor activity. In December 1992, the FDA approved Paclitaxel (brand name Taxol) for the treatment of ovarian cancer and two years later for breast cancer.
In the era when the world is struggling to find new medicines fast enough to subvert a fungal or bacterial pandemic, these discoveries can pave the way to new therapeutics. “I think it's really important to listen to indigenous cultures and to take their knowledge because they have been using these sources for a really, really long time,” Fernandes says. Now we know it works, so science can elucidate the molecular mechanisms behind it, she adds. “And maybe it can even provide a lead for us to develop some kind of new treatments in the future.”
Tardigrades can completely dehydrate and later rehydrate themselves, a survival trick that scientists are harnessing to preserve medicines in hot temperatures.
Formulating biologics to withstand drying and hot temperatures has been the holy grail for pharmaceutical researchers for decades. It’s a hard feat to manage. “Biologic pharmaceuticals are highly efficacious, but many are inherently unstable,” says Thomas Boothby, assistant professor of molecular biology at University of Wyoming. Therefore, during storage and shipping, they must be refrigerated at 2 to 8 degrees Celsius (35 to 46 degrees Fahrenheit). Some must be frozen, typically at -20 degrees Celsius, but sometimes as low -90 degrees Celsius as was the case with the Pfizer Covid vaccine.
For Covid, fewer than 73 percent of the global population received even one dose. The need for refrigerated or frozen handling was partially to blame.
The costly cold chain
The logistics network that ensures those temperature requirements are met from production to administration is called the cold chain. This cold chain network is often unreliable or entirely lacking in remote, rural areas in developing nations that have malfunctioning electrical grids. “Almost all routine vaccines require a cold chain,” says Christopher Fox, senior vice president of formulations at the Access to Advanced Health Institute. But when the power goes out, so does refrigeration, putting refrigerated or frozen medical products at risk. Consequently, the mRNA vaccines developed for Covid-19 and other conditions, as well as more traditional vaccines for cholera, tetanus and other diseases, often can’t be delivered to the most remote parts of the world.
To understand the scope of the challenge, consider this: In the U.S., more than 984 million doses of Covid-19 vaccine have been distributed so far. Each one needed refrigeration that, even in the U.S., proved challenging. Now extrapolate to all vaccines and the entire world. For Covid, fewer than 73 percent of the global population received even one dose. The need for refrigerated or frozen handling was partially to blame.
Globally, the cold chain packaging market is valued at over $15 billion and is expected to exceed $60 billion by 2033.
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Freeze-drying, also called lyophilization, which is common for many vaccines, isn’t always an option. Many freeze-dried vaccines still need refrigeration, and even medicines approved for storage at ambient temperatures break down in the heat of sub-Saharan Africa. “Even in a freeze-dried state, biologics often will undergo partial rehydration and dehydration, which can be extremely damaging,” Boothby explains.
The cold chain is also very expensive to maintain. The global pharmaceutical cold chain packaging market is valued at more than $15 billion, and is expected to exceed $60 billion by 2033, according to a report by Future Market Insights. This cost is only expected to grow. According to the consulting company Accenture, the number of medicines that require the cold chain are expected to grow by 48 percent, compared to only 21 percent for non-cold-chain therapies.
Tardigrades to the rescue
Tardigrades are only about a millimeter long – with four legs and claws, and they lumber around like bears, thus their nickname – but could provide a big solution. “Tardigrades are unique in the animal kingdom, in that they’re able to survive a vast array of environmental insults,” says Boothby, the Wyoming professor. “They can be dried out, frozen, heated past the boiling point of water and irradiated at levels that are thousands of times more than you or I could survive.” So, his team is gradually unlocking tardigrades’ survival secrets and applying them to biologic pharmaceuticals to make them withstand both extreme heat and desiccation without losing efficacy.
Boothby’s team is focusing on blood clotting factor VIII, which, as the name implies, causes blood to clot. Currently, Boothby is concentrating on the so-called cytoplasmic abundant heat soluble (CAHS) protein family, which is found only in tardigrades, protecting them when they dry out. “We showed we can desiccate a biologic (blood clotting factor VIII, a key clotting component) in the presence of tardigrade proteins,” he says—without losing any of its effectiveness.
The researchers mixed the tardigrade protein with the blood clotting factor and then dried and rehydrated that substance six times without damaging the latter. This suggests that biologics protected with tardigrade proteins can withstand real-world fluctuations in humidity.
Furthermore, Boothby’s team found that when the blood clotting factor was dried and stabilized with tardigrade proteins, it retained its efficacy at temperatures as high as 95 degrees Celsius. That’s over 200 degrees Fahrenheit, much hotter than the 58 degrees Celsius that the World Meteorological Organization lists as the hottest recorded air temperature on earth. In contrast, without the protein, the blood clotting factor degraded significantly. The team published their findings in the journal Nature in March.
Although tardigrades rarely live more than 2.5 years, they have survived in a desiccated state for up to two decades, according to Animal Diversity Web. This suggests that tardigrades’ CAHS protein can protect biologic pharmaceuticals nearly indefinitely without refrigeration or freezing, which makes it significantly easier to deliver them in locations where refrigeration is unreliable or doesn’t exist.
The tricks of the tardigrades
Besides the CAHS proteins, tardigrades rely on a type of sugar called trehalose and some other protectants. So, rather than drying up, their cells solidify into rigid, glass-like structures. As that happens, viscosity between cells increases, thereby slowing their biological functions so much that they all but stop.
Now Boothby is combining CAHS D, one of the proteins in the CAHS family, with trehalose. He found that CAHS D and trehalose each protected proteins through repeated drying and rehydrating cycles. They also work synergistically, which means that together they might stabilize biologics under a variety of dry storage conditions.
“We’re finding the protective effect is not just additive but actually is synergistic,” he says. “We’re keen to see if something like that also holds true with different protein combinations.” If so, combinations could possibly protect against a variety of conditions.
Commercialization outlook
Before any stabilization technology for biologics can be commercialized, it first must be approved by the appropriate regulators. In the U.S., that’s the U.S. Food and Drug Administration. Developing a new formulation would require clinical testing and vast numbers of participants. So existing vaccines and biologics likely won’t be re-formulated for dry storage. “Many were developed decades ago,” says Fox. “They‘re not going to be reformulated into thermo-stable vaccines overnight,” if ever, he predicts.
Extending stability outside the cold chain, even for a few days, can have profound health, environmental and economic benefits.
Instead, this technology is most likely to be used for the new products and formulations that are just being created. New and improved vaccines will be the first to benefit. Good candidates include the plethora of mRNA vaccines, as well as biologic pharmaceuticals for neglected diseases that affect parts of the world where reliable cold chain is difficult to maintain, Boothby says. Some examples include new, more effective vaccines for malaria and for pathogenic Escherichia coli, which causes diarrhea.
Tallying up the benefits
Extending stability outside the cold chain, even for a few days, can have profound health, environmental and economic benefits. For instance, MenAfriVac, a meningitis vaccine (without tardigrade proteins) developed for sub-Saharan Africa, can be stored at up to 40 degrees Celsius for four days before administration. “If you have a few days where you don’t need to maintain the cold chain, it’s easier to transport vaccines to remote areas,” Fox says, where refrigeration does not exist or is not reliable.
Better health is an obvious benefit. MenAfriVac reduced suspected meningitis cases by 57 percent in the overall population and more than 99 percent among vaccinated individuals.
Lower healthcare costs are another benefit. One study done in Togo found that the cold chain-related costs increased the per dose vaccine price up to 11-fold. The ability to ship the vaccines using the usual cold chain, but transporting them at ambient temperatures for the final few days cut the cost in half.
There are environmental benefits, too, such as reducing fuel consumption and greenhouse gas emissions. Cold chain transports consume 20 percent more fuel than non-cold chain shipping, due to refrigeration equipment, according to the International Trade Administration.
A study by researchers at Johns Hopkins University compared the greenhouse gas emissions of the new, oral Vaxart COVID-19 vaccine (which doesn’t require refrigeration) with four intramuscular vaccines (which require refrigeration or freezing). While the Vaxart vaccine is still in clinical trials, the study found that “up to 82.25 million kilograms of CO2 could be averted by using oral vaccines in the U.S. alone.” That is akin to taking 17,700 vehicles out of service for one year.
Although tardigrades’ protective proteins won’t be a component of biologic pharmaceutics for several years, scientists are proving that this approach is viable. They are hopeful that a day will come when vaccines and biologics can be delivered anywhere in the world without needing refrigerators or freezers en route.
Jamie Rettinger with his now fiance Amie Purnel-Davis, who helped him through the clinical trial.
Melanoma is the deadliest form of skin cancer. About 85,000 people are diagnosed with it each year in the U.S. and more than 8,000 die of the cancer when it spreads to other parts of the body, according to the Centers for Disease Control and Prevention (CDC).
There are two peaks in diagnosis of melanoma; one is in younger women ages 30-40 and often is tied to past use of tanning beds; the second is older men 60+ and is related to outdoor activity from farming to sports. Light-skinned people have a twenty-times greater risk of melanoma than do people with dark skin.
"When I graduated from medical school, in 2005, melanoma was a death sentence" --Diwakar Davar.
Jamie had a follow up PET scan about six months after his surgery. A suspicious spot on his lung led to a biopsy that came back positive for melanoma. The cancer had spread. Treatment with a monoclonal antibody (nivolumab/Opdivo®) didn't prove effective and he was referred to the UPMC Hillman Cancer Center in Pittsburgh, a four-hour drive from his home in western Ohio.
An alternative monoclonal antibody treatment brought on such bad side effects, diarrhea as often as 15 times a day, that it took more than a week of hospitalization to stabilize his condition. The only options left were experimental approaches in clinical trials.
Early research
"When I graduated from medical school, in 2005, melanoma was a death sentence" with a cure rate in the single digits, says Diwakar Davar, 39, an oncologist at UPMC Hillman Cancer Center who specializes in skin cancer. That began to change in 2010 with introduction of the first immunotherapies, monoclonal antibodies, to treat cancer. The antibodies attach to PD-1, a receptor on the surface of T cells of the immune system and on cancer cells. Antibody treatment boosted the melanoma cure rate to about 30 percent. The search was on to understand why some people responded to these drugs and others did not.
At the same time, there was a growing understanding of the role that bacteria in the gut, the gut microbiome, plays in helping to train and maintain the function of the body's various immune cells. Perhaps the bacteria also plays a role in shaping the immune response to cancer therapy.
One clue came from genetically identical mice. Animals ordered from different suppliers sometimes responded differently to the experiments being performed. That difference was traced to different compositions of their gut microbiome; transferring the microbiome from one animal to another in a process known as fecal transplant (FMT) could change their responses to disease or treatment.
When researchers looked at humans, they found that the patients who responded well to immunotherapies had a gut microbiome that looked like healthy normal folks, but patients who didn't respond had missing or reduced strains of bacteria.
Davar and his team knew that FMT had a very successful cure rate in treating the gut dysbiosis of Clostridioides difficile, a persistant intestinal infection, and they wondered if a fecal transplant from a patient who had responded well to cancer immunotherapy treatment might improve the cure rate of patients who did not originally respond to immunotherapies for melanoma.
The ABCDE of melanoma detection
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Clinical trial
"It was pretty weird, I was totally blasted away. Who had thought of this?" Jamie first thought when the hypothesis was explained to him. But Davar's explanation that the procedure might restore some of the beneficial bacterial his gut was lacking, convinced him to try. He quickly signed on in October 2018 to be the first person in the clinical trial.
Fecal donations go through the same safety procedures of screening for and inactivating diseases that are used in processing blood donations to make them safe for transfusion. The procedure itself uses a standard hollow colonoscope designed to screen for colon cancer and remove polyps. The transplant is inserted through the center of the flexible tube.
Most patients are sedated for procedures that use a colonoscope but Jamie doesn't respond to those drugs: "You can't knock me out. I was watching them on the TV going up my own butt. It was kind of unreal at that point," he says. "There were about twelve people in there watching because no one had seen this done before."
A test two weeks after the procedure showed that the FMT had engrafted and the once-missing bacteria were thriving in his gut. More importantly, his body was responding to another monoclonal antibody (pembrolizumab/Keytruda®) and signs of melanoma began to shrink. Every three months he made the four-hour drive from home to Pittsburgh for six rounds of treatment with the antibody drug.
"We were very, very lucky that the first patient had a great response," says Davar. "It allowed us to believe that even though we failed with the next six, we were on the right track. We just needed to tweak the [fecal] cocktail a little better" and enroll patients in the study who had less aggressive tumor growth and were likely to live long enough to complete the extensive rounds of therapy. Six of 15 patients responded positively in the pilot clinical trial that was published in the journal Science.
Davar believes they are beginning to understand the biological mechanisms of why some patients initially do not respond to immunotherapy but later can with a FMT. It is tied to the background level of inflammation produced by the interaction between the microbiome and the immune system. That paper is not yet published.
Surviving cancer
It has been almost a year since the last in his series of cancer treatments and Jamie has no measurable disease. He is cautiously optimistic that his cancer is not simply in remission but is gone for good. "I'm still scared every time I get my scans, because you don't know whether it is going to come back or not. And to realize that it is something that is totally out of my control."
"It was hard for me to regain trust" after being misdiagnosed and mistreated by several doctors he says. But his experience at Hillman helped to restore that trust "because they were interested in me, not just fixing the problem."
He is grateful for the support provided by family and friends over the last eight years. After a pause and a sigh, the ruggedly built 47-year-old says, "If everyone else was dead in my family, I probably wouldn't have been able to do it."
"I never hesitated to ask a question and I never hesitated to get a second opinion." But Jamie acknowledges the experience has made him more aware of the need for regular preventive medical care and a primary care physician. That person might have caught his melanoma at an earlier stage when it was easier to treat.
Davar continues to work on clinical studies to optimize this treatment approach. Perhaps down the road, screening the microbiome will be standard for melanoma and other cancers prior to using immunotherapies, and the FMT will be as simple as swallowing a handful of freeze-dried capsules off the shelf rather than through a colonoscopy. Earlier this year, the Food and Drug Administration approved the first oral fecal microbiota product for C. difficile, hopefully paving the way for more.
An older version of this hit article was first published on May 18, 2021