Rebelling against resistance: the global threat of antibiotic-resistant bacteria

Like all living things, bacteria have evolved to deal with changes and pressures in their environments. Sometimes this has led to them evolving a super trait in their genes that gives them a competitive edge in survival – like the ability to resist antibiotics. Antimicrobial resistance poses a grave threat to public health, and scientists are looking for new ways to combat the rise of superbugs. Manuela Callari reports on the vanguard of current research, from the high-tech to the unexpectedly ancient.

Amy Cain gets a little paranoid whenever she prepares chicken for tea. She washes chopping boards and knives twice and makes sure there’s no cross-contamination happening. “Food preparation is the one thing I get funny about,” she says. 

Raw chicken can be contaminated with Campylobacter spp, Clostridium perfringens and Salmonella spp. The latter is the bug that causes typhoid, the infection Cain was fighting in 2007 while pondering what subject area she should take for her PhD. She chose food-borne infections. “I was like, I’m gonna get you,” she recalls.

Now an associate professor at Macquarie University, Sydney, Cain continues to peruse pesky bugs. Her current favourite is Acinetobacter baumannii, a superbug – a bacterium that survives currently available antibiotics. Declared one of the top three critical pathogens by the WHO, A. baumannii is so tough that it can live undisturbed on surfaces for months. Cain has isolated and desiccated it completely – rehydrated a year later, it was able to infect mice. It attaches to medical devices such as ventilator tubes and intravenous catheters and is responsible for up to 20% of infections in intensive care units.

AMR killed 1.27 million people in 2019 and played a role in another five ­million deaths – superbugs killed more people than AIDS and malaria together.

Cain and thousands of other scientists around the globe want to understand more about the world of superbugs to find better ways to protect us from one of our biggest unsolved health threats. Superbugs are everywhere. They’ve been found in playgrounds, farms, drinking water, sausages and seagulls, riding tiny plastic fibres in the ocean, in Finnish paper mills, 300 metres underground in an isolated cave in New Mexico and even in the Arctic.

Our immune system can usually keep them at bay. But in hospitals, drug-resistant bacteria become a serious issue. This is where the six ­scariest pathogens – Enterococcus faecium, Staphyl­­­ococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter spp, collectively known as ESKAPE – are found. Hospitals are also where sick, debilitated, wounded and immunocompromised people are – and they’re the perfect hosts.

“[Bacteria] are extremely opportunistic,” Cain says. “We have a rule in the lab: if you have a burn or even a small cut, you can’t come in at all, because they just find you.”

A study published in The Lancet in 2022 examined 494 million patient records and estimated that antimicrobial resistance (AMR) killed 1.27 million people in 2019 and played a role in another five ­million deaths – meaning superbugs killed more people that year than AIDS and malaria together. The researchers predicted that by 2050, 10 million people will die every year from AMR complications. 

In Australia, surveillance data are patchy, but a recent report found that AMR could kill more than 5000 people yearly within the next decade.

“It’s a much bigger issue than most people realise,” says Branwen Morgan, the Minimising Antimicrobial Resistance mission lead at CSIRO.

If antibiotics fail, even a routine surgery will become life-threatening; forget organ transplants and caesarean births. According to scientists, AMR is the biggest health crisis we’ll experience this century. “If nothing’s done, we’re going to see an ever-increasing rate of drug-resistant infections,” Morgan says.

How did we get into this mess?

Bacteria are clever beasts. Like all living things, they’ve evolved to deal with changes and pressures in their environments and sometimes evolve a super trait in their genes that gives them a competitive edge in survival – like the ability to resist antibiotics. These superbugs then pass on their super genes to their offspring. They also swap and share their super genes with other species. “They’re really talented at changing themselves and borrowing help from their friends,” says Cain.

The first antibiotic, penicillin, was discovered by Alexander Fleming in 1928. It wasn’t widely available until the 1940s, when it was used to treat infections caused by bacteria during World War II. By 1942 the first cases of of penicillin resistance had already been reported. In the 1950s and 1960s, antibiotics were heavily overprescribed, including for viral infections on which they had no effect.

The more often bacteria are exposed to anti­biotics, the greater the opportunity to develop ways to survive and grow in their presence. Compared to most European countries, Australia still has a higher antibiotic prescribing rate, with nearly 25% of prescriptions being given without evidence of benefit. 

Over the past three years, AMR has spiked globally; the COVID-19 pandemic has likely played a role. Studies found that 60–70% of hospitalised COVID patients received antibiotics, but less than 10% had primary or secondary bacterial infections, so most antibiotics used were unnecessary. 

In hospitals, the high use of antibiotics and disinfectants puts bacteria under so much pressure that those who survive, thrive.

“They’re so smart. They’ve been doing this for a long time – they will become resistant to anything you throw at them,” Cain says.

Not only are current antibiotics becoming increasingly ineffective, there are no new antibiotics in the pipeline. The development of new antibiotics is an expensive process, with low profit margins: antibiotics are typically used for short periods of time, and pharmaceutical companies are increasingly interested in developing drugs that treat chronic disease. The last time scientists discovered a novel class of antibiotics that would eventually make it to market was in 1984. Daptomycin was approved by the US FDA (Food and Drug Administration) in 2003, nearly two decades after its discovery.

Now scientists are racing to find alternatives.

Making allies from tiny viruses

Fernando Gordillo Altamirano spends lots of his time searching through crap. Literally. 

Every month, he receives a litre of fresh raw sewage from Melbourne Water from which he fishes bacteriophages. “It’s not the prettiest part of my job,” he admits – but it is a necessary one.

Bacteriophages – from the Greek “bacteria devourer”, also known as simply phages – are the most abundant organisms on Earth. They’re in sewage in big numbers. “Phages live where their prey live,” says Gordillo Altamirano, a medical doctor and microbiologist at Monash University. 

Phages are tiny viruses with an icosahedral head that contains their genome and a protein tail. They have been at odds with bacteria for as long as life has existed on Earth (see Bacteriophage assault, above).

Gordillo Altamirano uses superbugs as bait to fish for the right phage from his sewage samples. “It’s a process of luck,” he says. “But if it’s there, it’s going to bite.” The predator phage infects the bacteria and multiplies. 

These picky viruses infect neither mammalian cells nor the rest of the bacteria in the microbiome, avoiding the nasty side effects of antibiotics. But this specificity makes phage therapy extremely personalised and difficult to scale up. 

As “living” antibiotics, phages have several advantages and pitfalls. Because they can replicate quickly, initial dosages can be relatively low. But they won’t eradicate bacteria completely, because their survival depends on them. Unlike chemical antibiotics, they co-evolve with their prey, blunting a bacterium’s ability to develop resistance, at least in theory. In about half of patients, bacteria evolve to survive and the phage treatment becomes less effective, says Jean-Paul Pirnay, head of the Laboratory for Molecular and Cellular Technology (LabMCT) in Queen Astrid Military Hospital, Belgium. But evolutionary pressure comes at a cost: bacteria can lose virulence or antibiotic resistance in the process.

Phages were discovered over 100 years ago when Felix d’Herelle, a French-Canadian microbiologist at the Pasteur Institute in Paris, realised their potential to treat bacterial infections. In 1917, d’Herelle was studying a cholera outbreak when he noticed that some of the patients’ stool samples contained clear spots, which he called plaques.

D’Herelle hypothesised that the plaques were caused by a virus that was attacking the cholera bacteria. He added clear spots from the samples to cultures of cholera bacteria, which quickly died.

D’Herelle then went on to isolate and culture the bacteriophage, and he showed that it could be used to treat cholera infections in mice. In 1919, he was the first to use phages to cure a 12-year-old boy with severe dysentery. But within a few decades of their discovery, they were largely abandoned in favour of antibiotics, at least in the Western world. One of d’Herelle’s colleagues, a young Georgian scientist named George Eliava, returned home to found the institute that now bears his name.

Today, the Eliava Foundation’s Phage Therapy Centre treats thousands of Georgians every year, and hundreds of foreigners from 84 countries, including Australia. Most patients buy phage cocktails off the shelf of the centre’s pharmacy to treat non-resistant infections. Those with tougher bugs are treated with personalised therapies. 

Despite the encouraging anecdotal evidence from Georgia, in Australia, the EU and the US, phage therapy is only available on compassionate grounds. “It’s only a very, very small proportion of patients that would qualify,” says Gordillo Altamiro.

Phage Australia, a national network of phage researchers and clinician scientists, is running a phase I clinical trial to assess phages’ safety. Around the world, 11 other studies are currently recruiting. Biotech companies are pushing into this space, and the market for phage therapies is predicted to grow 17% by 2030 to approximately $84 million annually.  But phage therapy will not be widely available until large, randomised clinical trials are completed – a process likely to take years.

There are still plenty of questions left to answer. Scientists need to figure out the immune system’s response to phages. Some people seem to produce antibodies that quickly neutralise them; others, not at all. Whether to use phages before, together or after a standard course of antibiotics is still debated.

Pirnay envisions a future where bacterial DNA is extracted from a swab and sent to a secured server where complex AI-based algorithms predict the genome sequence of the phage most likely to kill the bacteria. The phage genome is then sent to a 3D bioprinter, which produces synthetic phages ready to use within an hour of the swab being taken.

But in the here and now, microbiologists are using next-gen technology to engineer phages to target superbugs and deliver a lethal payload.

Genomic scissors

Just like humans, bacteria can get sick from a viral infection. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR/associated protein 9) is a system bacteria use to protect themselves from viruses. The Cas9 enzyme is guided to the viral DNA by a short RNA molecule, and cuts the viral DNA in a targeted location. The bacteria can then destroy the virus.

Now scientists are loading phages with CRISPR/Cas9 to turn that bacterium machinery against itself. “We can feed bacteria with short synthetic sequences programmed to cut a specific DNA sequence,” says David Sünderhauf, a microbiologist at the University of Exeter, UK.

Sünderhauf’s research targets antibiotic plasmids – circular strands of bacterial DNA where antibiotic resistance is encoded. CRISPR/Cas9 can break these plasmids apart, making the bacteria vulnerable to antibiotics again.

There are several advantages to targeting antibiotic plasmids. They are found in a broad range of drug-resistant bacteria but not bacteria in the microbiome or mammalian cells, which remain unaffected. If a superbug shares its antibiotic plasmids with another, the latter loses resistance – a highly valuable trait to treat antibiotic resistance in the environment, such as wastewater treatment plants, where these bacterial double agents could nullify a great number of superbugs in one sweep.

While gene editing shows promise in pre-clinical studies, many hurdles still exist. The greatest is resistance. “We know that anti-CRISPR proteins exist,” Sünderhauf says.

Bacteria can evolve CRISPR/Cas9 resistance by acquiring point mutations in the DNA sequence that are targeted by the guide RNA. These mutations can prevent the guide RNA from binding to the DNA, or stop the Cas9 enzyme from cutting the DNA at that location. For example, a bacteria could acquire a point mutation that changes one of the nucleotides in the DNA sequence that is targeted by the guide RNA. This would prevent the guide RNA from binding to the DNA, and the Cas9 enzyme would not be able to cut the DNA at that location.

Monoclonal antibodies are already used in immunotherapy to treat cancer and autoimmune diseases, but Freund wants to unleash them against bacteria.

One way to overcome this is by delivering two different editing tools targeting the same gene in two regions, because it is much less likely that two point mutations will occur simultaneously.

Another challenge is associated with microbial community complexity. Bacteria like to live in colonies that contain thousands of species, and nuances in resistance genes can be found even within single species. It is also difficult to predict how a community will respond to such changes. If a strain is removed from a population or its metabolism is affected, this may allow other, potentially more problematic species to outgrow it. 

Biological therapy

Some scientists are looking for allies within our own immune system.

We are born with trillions of B cells, a type of white blood cell responsible for producing antibodies. Antibodies are proteins that bind to specific antigens (molecules that are found on the surface of pathogens, such as bacteria and viruses). When an antibody binds to an antigen, it can help to neutralise the pathogen or mark it for destruction by other immune cells.

“We have more types of B cells than stars in the Milky Way galaxy,” says Natalia Freund, a microbiologist at Tel Aviv University, Israel. 

B cells are all “naive” until they encounter the targeted pathogen (see “B cell battlers”, opposite). Scientists have learned to identify such mono­clonal antibodies (an antibody that targets a ­specific antigen), isolate them and reproduce them in large quantities. Injected into a patient, monoclonal antibodies travel to their target, activate the immune system and attack that target just as antibodies produced by a person’s own B cells would. “We can give a patient an antibody that targets exactly what we want to stop,” says Freund.

Monoclonal antibodies are already used in immunotherapy to treat cancer and autoimmune diseases, but Freund wants to unleash them against bacteria. She has isolated monoclonal antibodies that hinder the growth of Mycobacterium tuberculosis (Mtb) in mice. Mtb is a highly infectious bacterium that travels through the air and is well adapted to humans. Tuberculosis (TB) is often latent and asymptomatic, so it can jump from person to person undetected. It affects 10 million people every year, killing 1.5 million. Recently, multi-drug-resistant forms have emerged, infecting half a million people yearly. “Once we run out of antibiotics, TB will be a major problem,” says Freund.

Mtb expresses almost 4000 proteins on its membrane, and identifying the one protein antibodies latch onto was laborious. Freund used Mtb as bait and went fishing in a bowl of B cells from patients who had recovered from TB to catch reactive B cells. She pinpointed a phosphate pump protein on the Mbt cell wall, which supplies energy to the bacterium and is highly specific and exists across all tuberculosis strains. She isolated and sequenced the monoclonal antibodies that blocked the action of the pump, then grew more of them. 

Tuberculosis-infected mice who received the treatment had a 50% reduction in Mtb growth compared to the control. There is a wide margin for improvement, but the study, published in Nature Communications, is an essential proof of concept.

Freund thinks that monoclonal antibodies could be used as adjunctive therapy with antibiotics to treat TB and other bacterial infections. “We are assessing [the treatment] in various preclinical models, [including] its ability to prevent reactivation [of the latent form] or infection altogether.”

Honey: an overlooked remedy 

In the fight against AMR, scientists are rediscovering the antibacterial power of one ancient remedy. Honey has been used as a wound dressing since long before bacteria were discovered. All kinds of honey have some antimicrobial activity: its high sugar content and low pH create an unwelcome environment for microbes. 

While that’s not enough to treat an infection, some kinds of honey can be powerful antimicrobials. Some bees add an enzyme called glucose ­oxidase to their honey, which – when it comes in contact with moisture, for example from wound fluid – converts sugar into hydrogen peroxide, a weak bleach that kills bacteria. Many types of Australian honey are high in glucose oxidase. 

But then, some honey has extra power. The ­nectar of the Mānuka flower (Leptospermum scoparium) contains the compound dihydroxyacetone (DHA). Over time, DHA converts into another chemical with potent antimicrobial traits, methyl­glyoxal (MGO). While the most famous Mānuka honey comes from New Zealand, over 80 Leptospermum species grow in Australia, including L. scoparium.

Nural Cokcetin, a microbiologist at the University of Technology Sydney, tested 45 of them and found that a third had high activity – some even higher than Mānuka. In the lab, MGO-rich honey kills antibiotic-resistant and susceptible bugs alike and doesn’t seem to trigger resistance.

“We are unlikely to see resistance anytime soon. Honey has already been used for thousands of years, and there’s no resistance,” says Cokcetin.

Why isn’t exactly clear. Besides sugars, glucose oxidase and MGO, honey contains a whole bunch of phenolic acids and antimicrobial peptides, from either the bees or the flowers they feed on. Over 200 components orchestrate attacks on the bacteria; the bug struggles to fight them all off at once. 

Honey-based ointment, alginate dressing, gel pads, adhesive dressings, nasal and throat sprays are widely available at pharmacies. But GPs often overlook them in favour of modern antibiotics. “People look at honey as an alternative medicine. But if we use it before [the infection] becomes a huge problem, we can save those antibiotics for when we really, really need them,” says Cokcetin.

Preventing infection altogether 

Treating antibiotic-resistant bacterial infections poses challenges for scientists and doctors, so perhaps the solution lies much earlier in the process.

Bacterial vaccines have a long history, dating back to 1896, when Almroth Wright developed the typhoid vaccine: a live, attenuated strain of the Salmonella typhi bacteria. Like viral vaccines, bacterial vaccines target specific antigens on the microbial cell wall, initiating an immune response that helps the body recognise and neutralise these pathogens. “Selecting good candidate vaccine antigens can be difficult,” says Kate Seib, a professor of microbiology at Griffith University, in Queensland.

Advances in genomics have made the task easier. Scientists can analyse a bacterium’s genome and examine potential antigens to identify the ones that are exposed on its membrane and accessible to the immune system. 

Since bacteria can mutate easily, scientists must identify antigens that remain constant over time and across strains. Many bacterial vaccines are multi-component: they target several antigens and can provide broader coverage against different strains, reducing the likelihood of bacterial escape through simultaneous mutation of all antigens.

There are several bacterial vaccines in the pipeline. Seib is running a phase I clinical trial of a vaccine against Neisseria gonorrhea, a bacterium transmitted through unprotected sex that has developed resistance to all but one class of antibiotics. It is well-adapted to humans and excellent at evading our immune system. 

Gonorrhea cases are most prevalent in low- and middle-income countries, where access to antibiotics can be challenging. “In some parts of Africa, there’s not a clinic on the street corner,” says Seib. “Vaccines can prevent some of these issues.”

Vaccines against four of the WHO’s priority pathogens – Salmonella, Streptococcus pneumoniae, Haemophilus influenzae type b, and Mtb – exist, but are not equally distributed worldwide. The S. pneumoniae vaccine, for example, has dramatically reduced mortality in the US and Europe but is not widely available in sub-Saharan Africa, where most cases are detected. “[Equal distribution of vaccines] is not something that needs to happen in the future. It’s something that needs to happen now,” says Pilar Garcia-Vello, an AMR scientist and former AMR global coordinator at WHO.

She says vaccine uptake is essential in all parts of the world and across society. “If there is herd immunity, there is a decrease in the circulation of pathogens. That will make a huge impact.”

To each their own 

None of the people working on AMR thinks one single therapy will replace the use of antibiotics. Instead, each will be essential in the AMR toolbox.

The amount of research in the AMR field is somewhat reassuring, yet many questions remain unanswered. One common problem for many novel therapies is the regulatory framework around which they should be produced and ­marketed. Licensing viruses, gene-editing tools or animal products as medicines can be challenging. 

Cain, the superbugs expert, says everyone has their part to play. “A collaborative, multisectoral and transdisciplinary approach is 100% the way to go,” she says. “When half of the world’s population doesn’t have access to flushing toilets or proper sanitation, it doesn’t matter how well one country is doing. If resistance arises anywhere in the world, it will spread anywhere else.”