The next generation of weapons against antibiotic-resistant superbugs

For the past 70 years, antibiotics have given us the upper hand against microbial invaders. Now the bugs are fighting back. Dyani Lewis takes a look at the next generation of ‘evolution-proof’ weapons being developed. 

It’s not an alien spaceship. It’s a bacteriophage. Scientists are turning to these ‘bacteria eaters’ to tackle superbugs.
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At his North Adelaide practice, Peter-John Wormald has the unenviable job of unblocking the noses of people with chronic sinusitis. Many of his patients have spent years on antibiotics that have failed to budge their infection, providing the perfect breeding ground for resistant superbugs.

For four out of five of them, surgery is the answer. Wormald carves away inflamed tissue, widens sinus openings and flushes out accumulated pus. For the stubborn one in five, however, Wormald is conducting one of the world’s first clinical trials into phage therapy, a century-old idea that predates modern antibiotics.

Such trials are desperately needed. We are on the threshold of returning to the dark ages that preceded antibiotics. In the late 19th century, tuberculosis was humanity’s greatest scourge. Robert Koch, who identified the causative Mycobacterium tuberculosis, estimated that “one-seventh of all humans die of tuberculosis”. The disease is now on its way to becoming untreatable once more. Every year, nearlyhalf a million new cases of multidrug-resistant tuberculosis (MDR-TB) occur world-wide. Extensively drug-resistant tuberculosis (XDR-TB) strains, impervious to the most potent antibiotics in our arsenal, have already reared their head.

Even normally benign bacteria like Staphylococcus aeureus – golden staph – which harmlessly colonises our skin and nostrils, can cause lethal infections if they hitch a ride into the bloodstream on a catheter or through a surgical incision.

In the post-antibiotic era, drug-resistant strains could render elective surgery, like knee or hip replacements, too risky. According to a 2016 review from Britain led by economist Jim O’Neill, by 2050 drug-resistant infections could kill 10 million people each year – higher than the toll from cancer.

Our profligate use of antibiotics is responsible. Antibiotics unleash massive die-offs within bacterial ecosystems. In a textbook case of evolution, that antibiotic-laden environment selects for the rare bacterium that happens to have acquired a resistance gene. By mechanisms that range from shredding or pumping out the antibiotic to shielding vulnerable targets, resistance genes enable bacteria to dodge whatever weapons we throw at them.

Thus armed, a single bacterial cell can quickly blossom into a resistant army of trillions. The resistance genes, which are carried on rings of DNA called plasmids, can be shared with unrelated bacteria like the latest piece of malware. Since the first use of penicillin more than 70 years ago, this story has played out for every antibiotic deployed. In most cases, resistance emerges within a few short years.

In 2015 a new shock emerged. Researchers in China reported finding E. Coli bacteria in pigs, chicken and humans that had acquired a plasmid that made them resistant to colistin, an antibiotic of last resort. It’s not often used in humans because it causes kidney damage, which is why it had remained effective. But in China, and other places, this cheap antibiotic has been a mainstay of animal farming. Indeed 70% of our global use of antibiotics is for the purpose of accelerating growth in farm animals, reducing the time needed to get them ready for market. The 2015 report in Lancet Infectious Diseases made it clear that resistant bugs in farm animals could spread their malware to bacteria that infect humans – till then a hypothetical risk. China banned the use of colistin in farm animals a year later.

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Until recently, we’ve managed to outmanoeuvre disease-causing bacteria. When one antibiotic succumbed to resistance, there was another popping out of the pipeline. Over the past few decades, however, the steady flow has dried to a trickle.

Pharmaceutical makers see little profit in the costly development of new antibiotics. That’s because, at least in the near future, the majority of infections will still be treatable by available low-cost antibiotics. That leaves a relatively small market for the new drug. “For a pharmaceutical company, the economics just don’t make any sense,” says chemist Mark Blaskovich from the University of Queensland. “Why would you invest in something where it’s a single two-week course and you’re cured, as opposed to an ongoing therapy [like new treatments for cancer] costing thousands, or hundreds of thousands, of dollars a year.”

According to a recent report from the PewCharitable Trust, just 40 antibiotics are chugging along the drug development pipeline, compared to several hundred drugs for cancer. To make matters worse, bacteria are increasingly arming themselves with multiple resistance mechanisms. When it comes to treating gonorrhoea and tuberculosis, for example, doctors now have few drugs that still work.

While health authorities like the World Health Organisation exhort the community to limit their use of antibiotics to slow the spread of resistant bacteria, it won’t eliminate the hordes of resistant microbes already in our midst. In 2014, at the time of announcing the O’Neill-led review, then British prime minister David Cameron warned: “If we fail to act, we are looking at an almost unthinkable scenario where antibiotics no longer work and we are cast back into the dark ages of medicine.”. Now we are “beyond a tipping point”, according to Michael Gillings, a microbiologist at Macquarie University, Sydney. “It might have been possible to stop this 40 to 50 years ago,” he says, “now we have the problem forever.”

As antibiotic after antibiotic invariably fails, scientists are searching for alternative weapons. One of those weapons was discovered amid the fog of World War I. In the summer of 1915, an outbreak of severe haemorrhagic dysentery raged through the ranks of French soldiers stationed at Maisons-Laffitte on the outskirts of Paris. Several of the soldiers were hospitalised, and French-Canadian microbiologist Félix d’Hérelle, working at the Institut Pasteur in Paris, was sent to investigate. He discovered that stool samples from the soldiers not only contained the dysentery-causing bacterium Shigella but also the antidote – “un microbe invisible” which, when added to Shigella in a dish, killed it.

Across the channel, British microbiologist Frederick Twort had also stumbled across bacteria-killing agents in his culture dishes. Both turned out to be bacteriophages, or ‘phages’ for short. These ‘bacteria eaters’ are viruses that specifically infect and kill bacteria, much as the flu virus infects our own cells.

Sage of phage: Felix d’Herelle first had the idea of harnessing phage to treat infection in 1915.
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It was d’Hérelle that first had the idea of harnessing phages as a clinical treatment. In 1919, he unleashed his poo-derived concoction on a small group of Parisian children with dysentery. The trial was a success, and phage therapy quickly gained the attention of the medical community. By the 1940s, however, it had all but been abandoned in the West in favour of the newfangled – and less finicky – antibiotics.

In the former Eastern bloc, phage therapy is alive and kicking. Specialist institutes in Georgia and Poland have carved out a niche supplying personalised phage remedies for stubborn bacterial infections. Now, in the face of failing antibiotics, researchers in Western countries are following their lead.

Nevertheless, rigorous clinical trial data remains sparse. Researchers like Wormald, who also leads otolaryngology head and neck surgery at Adelaide and Flinders universities, are now playing catch-up to test whether phage therapy is as effective as its proponents have long suggested.

Twice a day for two weeks, nine of his patients flush out their sinuses with a briny solution containing phages that kill golden staph, the most common culprit in chronic sinusitis. This usually harmless microbe lives on most people’s skin and nostrils, but can overrun nasal cavities, wounds or surgical incisions when a person’s immune system is at a low ebb or the microbial ecosystem has been thrown out of whack with antibiotics. At the end of the two weeks, Wormald sees modest improvement. Three months on, the results are dramatic. “They are absolutely 100%,” he says. The likely explanation is that phages stay on the job: as long as there are bacteria, they hang around and attack.

Wormald uses a cocktail of four different phages that together kill about 95% of golden staph strains circulating in the human population. Other cocktails – such as one used to treat leg ulcers – also combine phages to target several different bacterial species at once. Cocktails guard not only against treatment failure but against resistance, says Wormald: “While [the bacteria] might try to become resistant to one strain of the phage, they’re likely to be hammered by the others.”

In another recent trial conducted at wound centres in the US state of Washington, phages were dabbed onto the gangrenous toes of six people with diabetes. It was a last-ditch attempt to spare their toes. In all cases the phage preparation – obtained from the George Eliava Institute in Tbilisi, Georgia – did the trick.

Even more promising, researchers in the US announced in April 2017 the successful deploymentof phages to treat a man on the brink of death due to a multidrug-resistant Acinetobacter baumannii infection.

The phages were introduced via catheters into the man’s abdominal cavity, and injected intravenously. It is the first report of phage therapy thwarting a systemic infection with no apparent side-effects; the caveat is it was a single case and is yet to be published in the peer-reviewed literature.

Like guided missiles, phages target enemy bacteria with great precision. In principle that makes them safe – both for us and for the legions of beneficial microbes that inhabit our bodies. “They have a very low potential to do harm,” says phage researcher Steve Abedon of Ohio State University.

As phage therapy becomes more widespread, will phage-resistant bacteria emerge? Yes, but unlike the arms race between bacteria and antibiotics, which requires humans to keep upgrading their weaponry to stay in the game, phages do it by themselves. As bacteria evolve resistance to the phages that prey on them, phages evolve new ways to target them.

Also, when it comes to new phage varieties, nature has been prodigious. Phages are the most abundant life form on the planet and they are everywhere. A drop of seawater, a smear of poo, a clump of dirt – all teem with a multitude of phage types. This makes for an essentially limitless pool from which to fish for new weaponry. In theory, for every bothersome bacterium there should be a phage to prey on it.

That said, it hasn’t all been smooth sailing for the phage field. The largest randomised clinical trial of phage therapy to date – carried out in 2016 in Bangladesh with 100 children suffering diarrhea – was a failure, with the phage cocktail providing no benefit. There were, however, mitigating factors. The phages only targeted Escherichia coli (E. coli) and it turned out that more than a third of the children weren’t infected with this bacterium. For the rest of the children, the phage dose was too low to be effective. Whatever the reason for the trial’s failure, the results played to the perception of phages as a finicky therapy that won’t easily replace antibiotic pills.

Phage therapy faces other hurdles. One is finding commercial backers. “They’re the most ubiquitous biological entity on Earth,” says Abedon, “so they’re just free for the taking.” This makes them unattractive to pharmaceutical companies. Without investment to cover the cost of conducting expensive clinical trials, phage therapy will struggle to become anything more than a niche treatment.

Vincent Fischetti, a phage researcher at Rockefeller University, New York, also predicts “an uphill battle” in gaining regulatory approval. Regulators favour welldefined single-molecule drugs. A complex cocktail of viruses could prove challenging, he says. Once approved, the effectiveness of phage formulations would also need to be carefully monitored and modified in step with the march of bacterial evolution. This is similar to the annual revamp that the flu vaccine gets – and why you need a fresh jab each year – but it’s another mark against phages, potentially making them less attractive to investors.

“A flea has smaller fleas that upon him prey,” wrote Jonathan Swift. So too is the bacterium preyed on by bacteriophages.
Ami Images / Getty Images

If phages themselves are too messy, Fischetti has another solution: commandeer their weaponry.

Once phages home in on their target, they act like hypodermic syringes, injecting their genetic blueprint into their bacterial host. Within minutes that blueprint is directing the assembly of dozens of new phage progeny. To break out, they drill holes in the tough outer wall of the bacterial cell by deploying deadly enzymes called lysins. These lysins themselves are being developed as single-molecule patentable drugs, closer to what pharmaceutical companies and regulatory agencies are accustomed to.

Lysins also have the advantage over phages – and traditional antibiotics – of being extremely efficient killers. In 2001, Fischetti’s team showed that, within seconds, a purified phage lysin obliterated 15 different strains of the pneumonia-causing bacteria Streptococcus pneumoniae in test tubes but were less deadly towards harmless throat bacteria.

Their ability to specifically target bacteria is not as finely honed as that of phages, but is superior to antibiotics. For instance, Fishcetti says it is possible to direct specific lysins to wipe out all streptococci, or all staphylococci, or all pneumococci. Nor do lysins just work in test tubes; they have rescued mice from death by multidrug-resistant golden staph infections.

In 2016, Fischetti and colleagues unexpectedly found that lysins could even chase down and kill Streptococcus pyogenes, the bacterium responsible for “strep throat”. The pyogenes hide away inside throat cells, where antibiotics are unable to reach them, making strep throat highly resistant to treatment. Remarkably the lysin did not harm the throat cells.

“Lysins are right up there as probably the best chance of killing organisms at least as well as current antibiotics,” Fischetti says.

Lysins are not only potent antimicrobials; they also have a reputation for being evolution-proof. That’s because lysins target peptidoglycan, a fundamental building block of the bacterial cell wall. Any tweaking of these building blocks to evade lysins would likely be deadly to the cell, too. “Lysins have evolved to target substrates that the bacteria can’t change very easily,” Fischetti explains. Of course, he and others aren’t taking any chances. They look all the time, he says, and haven’t found one organism that’s resistant to lysin.

Despite the impressive results coming out of preclinical trials, progress towards human trials of lysins has been sluggish. That’s about to change. Fischetti has been working with the company ContraFect on a lysin treatment for golden staph blood infections. A phase II clinical trial will kick off mid-2017, following on from a successful phase I safety trial that ended in 2016. Fischetti expresses confidence that the trial, which will take about two years to complete, will be a success: “From what I see in animal studies and [other] experiments, it works every time.”

One of the reasons that superbugs emerge is because antibiotics are so deadly. In the wake of the microbial holocaust, resistant organisms thrive. Roy Robins-Browne at Melbourne’s Doherty Institute thinks there’s a better way to treat the troublesome bacteria that cause infections. Instead of trying to kill them, just defang them.

In 2013, Robins-Browne and his team unveiled regacin, a molecule that effectively defangs the deadly diarrhoea-causing enteropathogenic E. coli (EPEC). Regacin, turns off the “virulence” genes that make this bacteria such a bad ass, preventing them from setting up camp in the intestine and pumping out poisonous toxins. In mice, regacin works against an EPEC-like mouse pathogen. So far, Robins-Browne hasn’t found any regacin-resistant mutants. In humans, he says, using a drug like regacin could turn the deadly EPEC back into a harmless strain of E. coli, without fear of resistance emerging. But Robins-Browne is yet to find backing from the pharmaceutical industry to develop the drug further. “You can argue till you’re blue in the face about how this doesn’t select for resistance,” he says, “but they don’t believe you.”

While Robins-Browne’s strategy is to disarm bacteria, there is another way of curbing their aggression: make them think they don’t have the numbers to mount a successful attack.

Some bacteria have a clever way of sensing the size of their ranks. Known as “quorum sensing”, researchers first stumbled on this phenomenon in the 1960s in an unlikely place: the bobtail squid. It needs camouflage from predators below that can see it silhouetted against the light. The squid solves that problem by virtue of a light organ on its underbelly that houses luminescent bacteria. These microbial light bulbs glow only once they reach a threshold population. The bacteria ‘ping’ each other with a protein that lets the others know they are there. The more ‘pings’, the greater their number. When there are enough ‘pings’, the whole group turns on its bioluminescence.

Researchers subsequently discovered quorum sensing can be used for more aggressive purposes. Disease-causing bacteria use it to coordinate a successful invasion. As soon as there’s a ‘quorum’, they pump out toxins and tissue-digesting enzymes. In about two-thirds of infections, the bacteria also hunker down into resilient, slime-shrouded biofilms that protect them against the immune system and antibiotics.

It wasn’t long before scientists were looking for ways to silence this microbial chatter. Drugs that do this are called quorum quenchers. “Quorum sensing inhibition jams the communication lines,” explains microbiologist Tom Coenye from the University of Gent in Belgium.

This tricks the bacteria into acting as if they don’t have the numbers to mount an offensive or build a biofilm. That makes it easier for the immune system – or antibiotics – to come in and counter the would-be invaders.

Dozens of quorum quenchers have been identified. In petri dishes, Coenye has found, they prevent bacteria from building biofilm fortresses. In mice, quorum quenchers have reduced the severity of Pseudomonas aeroginosa lung infections.

But quorum quenchers have their limitations. Most people seek treatment when their infections are well and truly established – too late for quorum quenchers to work. Their role, therefore, will be in preventing the spread of infections in the first place.

One notorious source of drug-resistant infection in hospitals is via medical devices such as catheters or replacement joints. That’s why Helen Blackwell, a chemist at the University of Wisconsin, is developing ways of impregnating catheters and replacement hips with chemicals that act as quorum quenchers. Preventing post-operative infections could help eliminate the need for antibiotics and reduce the chance for resistance to evolve and spread.

Meanwhile, Coenye is hoping quorum quenchers will make current antibiotic therapies more effective. His work on hamamelitannin – a naturally occurring quorum quencher found in the bark of witch hazel – makes golden staph biofilms more susceptible to antibiotics.

The upshot would be that antibiotics could be used at lower doses and for shorter durations, reducing the chance of antibiotic resistance and extending the useful life of the antibiotics we currently rely on.

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With deaths from drug-resistant infections now estimated at 700,000 a year globally, there’s no doubt our seven-decade dream run with antibiotics is drawing to a close.

Recognising the dangers, governments and pharmaceutical companies are beginning to respond. In 2016, nearly 100 pharmaceutical companies pledged to work with governments to reinvigorate the antimicrobial development pipeline.

Overall scientists are thinking broadly. In addition to phages, lysins, quorum quenchers and virulence disruptors, the war chest includes probiotic capsules packed with beneficial bacteria to edge out troublemakers, as well as vaccines and drugs such as host defence peptides that ramp up the body’s own natural defences.

For now this is an arms race that we are losing. To buy time, our best bet is to rob resistant bacteria of their evolutionary advantage by minimising the use of antibiotics.

With sufficient investment in research and development, the next generation of smart weapons will hopefully be ready before antibiotics are rendered useless. If the researchers are right, our new weapons should protect us a lot longer than 70 years.

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