By Alex Riley on Wed, 08 Feb 2017
In 2015, a team of scientists from China, Britain, and the U.S. found several populations of bacteria that had survived a death sentence. In samples taken from slaughterhouses and from samples of raw pork and chicken, they found that even one of the strongest antibiotics known, called colistin, couldn’t kill this strain of E. coli. Its resistance was linked to a single gene named mcr-1. This was the moment that modern medicine had feared for decades. Colistin is just a drug of last resort, a group of antibiotics often left unused because of their harmful or unknown side effects, and it had started to falter.
E. coli cells seen under a scanning electron microscope.
Over the next year, this strain of E. coli spread like a rash across the globe, reaching the U.S as early as May 2016 when it infected a 49-year-old woman from Pennsylvania. Other antibiotics are still effective against the strain, but the mcr-1 is just one of many genes that could usher in a post-antibiotic world in which even the most banal of ailments can be lethal.
The appearance of mcr-1 comes as no surprise. Ever since antibiotics were first discovered in the late 1920s, bacteria have been inventing new ways to survive and thrive in such chemicals, often to our own detriment—over 23,000 people die every year in the U.S. from antibiotic-resistant bacteria. In 2001, with the rising rate of bacterial resistance, the World Health Organization published its “Global Strategy for Containment of Antimicrobial Resistance.” In this report, they sought to guide and educate nations around the world in the correct use of antibiotics and “to encourage the research and development of new antimicrobial agents.”
In other words, they were running out of options.
That same year, in her new laboratory at Swinburne University in Melbourne, southeast Australia, Elena Ivanova was studying physical surfaces that could repel bacteria before they even had time to settle. In clinical settings, such as hospitals and dental practices, 80% of infections are caused by bacteria that cling to surfaces in such densities that no antibiotic can remove them. First, she tried making surfaces so smooth that bacteria would, theoretically, simply slide off. Although that was the case for some bacteria, many others—such as the common Staphococcus aureus, or staph—still managed to cling on and multiply.
“It was a partial success,” Ivanova admits. So she tried a different tack: Why not look to nature for solutions? After all, the first antibiotic discovered—penicillin—and many thereafter were originally extracted from fungi, bacteria, or other organisms that used them to defend themselves against their own foes. Ivanova didn’t have to look far. A couple in northeastern Australia had been amassing a vast collection of possible solutions in their kitchen freezer.
In 1997, Greg Watson, a PhD student at Griffith University in Brisbane, Australia, was just starting his work into atomic force microscopy, a technique that is more akin to how a record player works than your classic optical microscopy. Rather than magnifying an object visually, an atomic force microscope, also known as an AFM, amplifies by touch, detailing tiny topographies that are only a few atoms in height. After placing a sample—such as wool fibers or even viruses—into the microscope, a vibrating needle moves over its surface, up and down, while a highly sensitive laser tracks every movement. Each nanoscale nook and cranny is then translated into an image on a computer screen.
Without leaving the laboratory, Greg had become an explorer of untouched alien worlds. He loved his work, the thrill of seeing something to which everyone else was blind. But in his second year, he suddenly had less time on his favorite AFM—there was a new student, an undergraduate called Jolanta, who had asked specifically to work in the same lab. From the first day, they weren’t overly fond of each other. Jolanta thought he was arrogant and dismissive. And Greg, being quite arrogant and dismissive, thought she was just another student who was more likely to get in the way than to actually discover anything new.
Today, Greg and Jolanta Watson have been married for over 15 years and rarely spend any time apart. They live in the same house, work in their own laboratory at the University of the Sunshine Coast in Queensland, Australia, and even share the same birthday.
“As far as I’m aware, I have the world’s smallest marriage proposal.”
“We’re one of those couples that can be together…” Greg says. “…24/7,” Jolanta answers. (They interview on speakerphone together, too.)
The change in Greg and Jolanta’s relationship happened when they left the walls of their laboratory behind, discovering a mutual appreciation for the outdoors. Every lunch break away from their microscopes, they would walk through the native Australian bush that surrounded the University and wait for nature’s larger patterns to reveal themselves. From spring through into summer, for instance, they witnessed the boom and bust cycles of cicadas. Shortly after they emerged from the ground, these flying insects sang loudly from the branches and died a few weeks later. Their carcasses carpeted the floor and started to decompose. Or at least, their bodies did. Their wings were still in immaculate condition; the see-through membranes stretched taut over a fine lattice of veins would still be suitable for flight, if not for the absence of a body.
Greg and Jolanta suspected that the wings had some antibacterial properties that somehow stopped the normal process of decomposition. Back in the lab, they both used the AFM to take a much closer look. As they scanned the surface, the microscopic became mountainous on screen, with huge pillars erupting from the wing’s surface. A year earlier, another group of scientists had noted the presence of similar “nipples” covering the wings and eyes of hawkmoths. They had proposed that they were used as antireflective coatings, scattering any light that hit their surface and camouflaging their presence from predators.
This made sense for cicadas, too. Greg and Jolanta certainly had a hard time spotting them in the trees, even on the brightest days of the Australian summer, though they suspected that couldn’t be the only explanation. But before they could investigate further, other studies and life got in the way. In 1999, they moved into their first flat together. A few years later, Greg proposed to Jolanta by etching the words “Let’s get married” into a piece of silicon and then asking her to scan the sample using the AFM. “As far as I’m aware, I have the world’s smallest marriage proposal, being only eight to ten atoms high,” Jolanta says. They married a month later, on their birthday.
A Quick Death
In 2004, Greg received an email from Ivanova, the researcher from Swinburne University. At the time, she was writing a book on antibacterial surfaces—both natural and synthetic—and asked Greg if he had any ideas from his own research. After her super-smooth surfaces, she had been testing whether lotus leaves—a classic example of an anti-wetting surface—were antibacterial and found them wanting. Covered in a thick meadow of tiny hairs, these leaves don’t allow water to spread out. Known as the lotus effect, the hairs and the air between them hold water droplets aloft, which are then removed by wind or by gravity, taking any contaminants with them. “We thought that bacteria would behave as a water drop on these super-hydrophobic surfaces,” Ivanova says. “They would simply slide off.” But many bacteria, such as staph, were still able to attach and grow, she found.
Greg suggested that she look at insect wings and sent her some of his cicada samples. In Ivanova’s lab, Pseudomonas aeruginosa, a common human pathogen, was able to settle on the surface of the wings just fine. But after just three minutes, they were dead. Under the microscope, the nanoscale pillars of the cicada wing seem to have punctured the cell walls of the bacteria, causing their innards to spill out and leading to a quick death. To ensure their demise, Ivanova and her colleagues added a couple of fluorescent dyes to the wing’s surface that bind to the DNA inside bacteria, glowing red in those that have punctured membranes and green in those that are healthy. Under their bench-top microscope, the surface looked like a bloody battle had just taken place. As Ivanova wrote at the time, “This is the first reported example of a naturally existing surface with a physical structure that exhibits such effective bactericidal properties.”
Nanoscale pillars on a cicada wing have pierced this bacterium, causing it to spill its contents and die.
But it wasn’t effective enough. The cicada wings only killed one of two main types of bacteria. Like the rod-shaped E. coli, the outer cell walls of P. aeruginosa are very thin and easily ruptured by the cicada wings. But the cell walls of other bacteria, such as staph and the species that causes pneumonia and other respiratory infections, Streptococcus pneumoniae, are filled with a strong matrix of sugars that can be up to five times thicker than that of E. coli. With this tough exterior, the cicada wing was less a bed of nails and more a surface to spread upon without competition from their more fragile kin. “It was a partial success,” Ivanova says. “We had to look for something else.”
By 2010, Greg and Jolanta had moved on from insect wings. Rather than being focused on a single topic, their research projects are guided by a combination of short attention spans and child-like curiosity. Ever since they first scratched the surface of cicada wings in the late 1990s, they had matured from students into scientists, but they refused to grow up. “As soon as you start playing in science, when you’ve got that curiosity, something always happens,” Greg says. One day he’ll be working on crab burrows, for example. The next he’ll be thermal imaging water dragons in urban environments. And another day Jolanta would find herself sitting at her lab bench carefully skinning a dead gecko.
A speck of dust sits atop the skin of a box-patterned gecko, seen here under a scanning electron microscope.
Covered in a patchwork of tan and beige, they had found the deceased box-patterned gecko on one of their lunchtime walks and placed a sample of its skin under the microscope as soon as they could. They knew there was a wealth of research into the gecko’s sticky feet, but there had been little interest in the rest of the body. This time using an electron microscope—a method that compiles a high-resolution image of the surface by firing charged particles through the sample and detecting how and when they are blocked en route—they once again found the surface to be covered in a carpet of tiny pillars, each one around a millionth of a meter wide, or one-fifth of the diameter of a single red blood cell. Pointed at the tip and tapering toward a slightly thicker base, these hair-like projections were much larger than those of the cicada wings and spaced much more widely apart. In these gaps, however, there were nanoscale pillars similar to the cicada wing’s.
To Greg and Jolanta, this hierarchy of hair-like pillars was reminiscent of the surface of a lotus leaf. In 2015, along with their colleague and friend David Green, then at the University of Queensland, they demonstrated that this similarity in structure bestowed a similarity in function. Gecko skin was anti-wetting and self-cleaning, with pearl-like droplets of water rolling off the surface much more easily than off a duck’s back.
But where the lotus leaf had failed to stymie bacteria, the lizard triumphed. The surface was lethal. After cleaning and cutting the gecko skin into small squares, Green had added a sample of the small, rod-shaped bacterium that causes gingivitis, Porphyromonas gingivalis, to the surface. In total, he added around 10 million microbes every day for a week. What’s more, this mass of microbes was given everything that they needed for a good life: a constant temperature of 98.6˚ F, an atmosphere without oxygen, and a daily ration of food. Regardless, after the week, nearly all were punctured and torn, their cellular carcasses strewn over the gecko skin. “Bacteria are trying to move and settle on the surface,” Green says. “And they’re just getting spiked and skewered by these long hairs.”
A bacterium, shown here in false color, has been pierced by the nanoscale features of gecko skin.
Even those thicker-skinned bacteria—such as Streptococcus mutans, a small but sturdy species that causes tooth decay—aren’t impervious. Their deaths were unexpected and much harder to explain. Rather than sitting atop the hairs, these smaller bacteria slipped in-between them. In their study, the researchers suggest two theories: one, that these bacteria are squashed or stretched to death rather than being skewered, their cell walls rupturing under the pressure; or two, perhaps the tiny pillars at the base of the hair-like projections puncture the bacteria instead.
“There are three or maybe four possible killing mechanisms,” Green says. However, in such a nascent field of research, the details of how each of these occurs is still more speculation than science, Ivanova says. Working on dragonfly wings (another gift from Greg), she is now comparing how different species are tuned to kill different types of bacteria, revealing their weak spots in the process. “What are forces that are really important to be reproduced on the surface, to achieve the maximum of bactericidal efficiency?” she asks. “That is now my priority. This work is evolving.”
“Nature is a wonderful problem solving mechanism,” Green says. “And it can be translated directly into technologies.” In late 2016, he made his first synthetic gecko skin. By pressing a stamp of real gecko skin into soft polyvinyl plastic, he was able to create a mold into which molten acrylic was poured and left to harden. After 30 minutes, the mold was peeled away and the replica skin was placed underneath an AFM. Like a rover mapping a new planet, the needle and laser scanned the surface and found this simulacrum to be nearly as good as the real thing. The hairs were a little shorter and more bulbous, but it was close enough. And, being 88% effective against soft-shelled microbes and 66% against hard-shelled species, the replica was just as efficient a bactericide as the real skin. More importantly, this “proof of concept” surface was lethal to the most resistant of bacteria: a biofilm.
When bacteria divide, grow, and clump together they can be almost impossible to separate. “Once the biofilm is already formed, it’s basically hopeless,” Ivanova says. “You can’t get rid of it. Antibiotics and chemicals don’t really affect bacteria which are hidden inside the biofilm.” By producing a thin slime of sugary molecules, the bacteria collectively shield themselves from any threat that might be lethal to a loner.
On these surfaces, there is no antibacterial chemistry at work, just physics.
In hospitals and other clinical settings, biofilms lead to 80% of all infections, many emerging after bacteria coat an implant with their colonies, outcompeting the person’s own cells in the so-called “race for the surface.” Currently, implants such as titanium hips and teeth are coated in a variety of antibacterial veneers that vary in their efficacy. Silver nanoparticles, for example, are commonly used but can build-up to toxic levels and are only effective over short time scales. And the longer lasting methods such as quaternary ammonium compounds—the main antibacterial used in hand solutions and cosmetics—are ineffective against an ever-increasing legion of resistant bacteria.
But with new materials, the implant itself could be the antibacterial agent. On the acrylic gecko skin, less than 4% of the surface had any evidence of bacteria. And 95% of cells within these regions were dead. In other words, the surface had stopped biofilms before they had even chance to form.
On these surfaces, there is no antibacterial chemistry at work, just physics and hundreds of millions of years of evolutionary trial and error copied in contemporary materials. By coating implants with a gecko-like surface, Green thinks that bacteria can be kept at bay while promoting the growth of our larger cells that use the hairs as attachment points. “The gecko skin surface has similar feature designs to the patterns found in human tissues,” he says. Recently, Green has been using gecko skins to grow and study stem cells in the lab, as well as creating reparative tissue linings for damaged organs. “There seems to be a match between these structures that we gather from nature and the structures in our tissues.”
Today, working closely with Kenneth Lee from the Chinese University of Hong Kong, Green is scaling up his gecko skins, printing large sheets of the antibacterial surface in a variety of synthetic materials that could be used for a range of products and settings. “We don’t need gecko skin anymore,” Lee says. “We can mimic that surface into plastics.”
Since the first outbreak of SARS in China and Hong Kong in 2002, many citizens have become increasingly wary of quotidian behaviors such as pressing the button to an elevator or grabbing a doorknob, Lee says. “They are really paranoid about touching them in case they get infected by all sorts of germs.” Those people’s fears were his initial inspiration for a real-world application of replica gecko skins, but along with Green, he is also looking into surfaces for hospitals, from wallpapers to surgical gowns, soap dispensers, and even bed linens. For the latter, “it would be a very thin coating you could apply directly on the textile material.” Green says. Early tests suggest that the pillars would be resistant to everyday wear and tear. “They don’t break because they’re such small scale,” Green says. Plus, he adds, the surface is so fine and the structures so microscopic that patients wouldn’t feel anything.
But any bacteria certainly would. In soon-to-be published research, Green and Lee have showed that MRSA, a versatile pathogen that is impervious to many antibiotics and causes a wide variety of ailments in clinical settings around the world, had no resistance to the hairs on a gecko’s skin.
‘That Fascinating First Step’
Although other scientists have taken the lead, it may not have happened without Greg and Jolanta’s walks outdoors. They have provided the raw materials to test their own and other researchers’ theories. In teaming up with Ivanova, whose work on antibacterial surfaces was well underway, the couple may have stumbled upon several solutions that could reduce our dependency on chemicals.
In the future, bacteria may evolve resistance to physical antibacterial surfaces, Greg says. “Organisms are clever and evolution is a pretty wonderful strategy for circumventing anything that you’re trying to do,” he says, including his own work. “But at least it gives you another way.”
Like the process of evolution itself, Greg and Jolanta had no goal and no end point for any of their projects. But they forged ahead, driven by an unquenchable urge to know more about the places on Earth that are hidden by their diminutive scale. Such basic research is an approach to science that is often criticized for wasting money.
But because our understanding of how the world works is so piecemeal, so full of haze and darkness, taking a wide-angle approach is sometimes vital for scientific progression. After all, penicillin was a chance discovery in 1928 that no one could have predicted or foreseen its importance in the future. “Science is becoming so money driven and outcome driven, it’s losing that beauty,” Jolanta says. “But Greg and I are very much determined, whether to our detriment or not, to just explore.”
“Most people are very structured, like they’ve already solved the problem before they’ve even started their research,” Greg says. “They miss that fascinating first step, that curiosity. Just go out there into the world and just look. You don’t have to look far.” On their way home from a recent fishing trip to Canberra, Greg and Jolanta picked up a two and half-meter long monitor lizard and put it in the backseat of their car. To most people it was road kill to be left for scavengers. To Greg and Jolanta it was a chance to discover something new. “We have an extra freezer now,” Jolanta says.