Microbial gangs are organised killers

Who can forget Superman? Christopher Reeve with his dazzling white teeth, blue eyes and biceps of steel embodied the superhero – in films and in life. When he fell from his horse in 1995 while training for an equestrian event, Reeve was left quadriplegic but undefeated.

He continued his film career and his activism, campaigning for the spinally injured and the right to carry out human embryonic stem cell research.

Ultimately, this super man was beaten by a microbe. His pressure sores – the bane of the wheelchair-bound – had once again become infected. An allergic response to an antibiotic sent him into anaphylactic shock and heart failure. Reeve died on October 10, 2004.

It was probably not a superbug that brought down Superman. More likely it was a microbial monster of a different kind. A biofilm bears little resemblance to the individual rods or sphere-shaped cocci traditionally studied in biology class. It’s much more like a living animal composed of multiple cell types – bacteria, archaea, fungi, yeasts – communicating and working together. That makes biofilms almost bullet-proof when it comes to antibiotics. Like soldiers in an ancient Greek phalanx, the individuals cooperate to create a near invincible slime shield.

In the 1940s, antibiotics including penicillin and streptomycin brought our first great victories against infections such as typhus and TB or wounds that killed so many in the battlefields of World Wars I and II. But in the 21st century the evolution of superbugs may well push us back to where we were in the pre-antibiotic era. And it’s the way we use antibiotics that is driving this evolution.

The major infections that threaten populations these days are not fast and furious – they are slow and festering. They are the sores that threaten the wheelchair bound, diabetics or the elderly, persistent infections of the sinus, middle ear, airways and urinary tract, tooth decay and the infections that take hold around catheters, stents, synthetic heart valves and joints. Yet they are being treated much as Robert Koch prescribed more than 100 years ago – by taking a swab, seeing what grows in a broth and trying to kill that bug. The problem is the bug that grows in the broth does not represent everything in the infection. “What most clinicians fail to realise is that this is because most bacteria pursue a different infection strategy called biofilm,” says Randy Wolcott, director of the Southwest Regional Wound Care Centre in Lubbock, Texas.

The US National Institutes of Health estimate that more than 80% of problem infections are caused by biofilms. And 65% of hospital-acquired infections are attributed to biofilms introduced via catheters. “If you group them together, there’s no question it is bigger than any other medical problem we face,” says Wolcott who estimates biofilms claim hundreds of thousands of lives each year in the US alone.

With the new turn in the microbe wars, researchers are taking advice from Chinese general Sun Tzu’s The Art of War: “The opportunity of defeating the enemy will be provided by the enemy themselves.” Wolcott, for instance, uses smart gels that disrupt the command and control system of the biofilm, and identifies the genetic signature of the dozens of microbes present to deliver a lethal blow. His clinic reports vastly improved cure rates on patients whose only other option was amputation. This strategy owes much to a researcher who went back to the beginning to re-examine what he thought he knew about microbes.

“AN EMBARRASSING fall into the icy waters of Bugaboo Creek at the foot of the unclimbed south face route of Snowpatch Spire really focused my attention on bacterial biofilms,” wrote William Costerton in a 1978 article for Scientific American. The Canadian microbiologist was also an avid mountaineer and hoped to combine his passions to study the microbes of mountain streams. What he found surprised him. The fast flowing waters were almost sterile, yet the rock that caused his undoing was colonised with slime-forming bacteria. Though biofilms were well known from marine environments – they were known to cause the slime on the bottom of boats – the slimy granite cobblestones got Costerton thinking. If bacteria could gain such a tenacious foothold in a fast-flowing mountain stream, how might they behave in the human bloodstream?

Ever since Koch grew bacteria in broth cultures, microbiologists had pictured bacteria as floaters, existing in a “planktonic form”. But thinking about the slimy films, Costerton began to ask if these types of microbial colonies could explain some of the more persistent infections of the human body. He thought of the lungs of his son who suffered from cystic fibrosis – they also appeared to be covered in a slimy bacterial layer. In 1977, Costerton attended a meeting on lung infections in cystic fibrosis in Montreal. There he met Danish physician and cystic fibrosis specialist Niels Høiby, who had also been studying the clumps of slimy bacteria from the sputum of his cystic fibrosis patients. “I’d never seen anything like it,” he recalls. “The clumps resembled frogs’ eggs.” Høiby also found them in swabs from other persistent infections – in the urinary tract, ear and lung. He proposed the slime might be the reason immune cells could not clear the infections.

Høiby and Costerton formed an alliance to raise medical awareness of this microbial phenomenon. Costerton was a great communicator and played the lead role.

When he passed away in 2012 he was known as the “father of biofilms”. Høiby paid tribute in a 2014 scientific review: “His success was due to his entrepreneurship, convincing lectures and publications, combined with his charming and persuasive personality.”

CYNTHIA WHITCHURCH was starting her science degree at the University of Queensland in the late 1980s when the biofilm revolution hit. For more than 100 years, questions such as what sort of toxins bacteria produced were tackled by growing them in a broth. “When I was trained, we saw them as floating or swimming around oblivious to each other. The transformational idea was that they spend their time living on surfaces … It’s almost embarrassingly obvious in retrospect. But it took Bill Costerton to make it that way.”  Now based at the ithree institute at the University of Technology, Sydney, Whitchurch is pursuing the interest that captivated her since doing her PhD. How do bacteria move en masse across a surface?

It is the same problem posed by flocking birds or marching ants. How do the individuals coordinate their behaviours? One insight came in the 1990s with the discovery of how Vibrio fishceri, a luminescent bacteria that lights up the underside of the Hawaiian bobtail squid, coordinates its behaviour. Balmy Hawaiian nights may be brightly lit by moon or star light so the squid needs counter-illumination if it is to stealthily hunt the creatures swimming below. But the low densities of Vibrio found in the ocean do not glow. Much as a committee will not vote until it reaches a quorum, Vibrio first needs to reach a density of 10 billion cells per millilitre of water before it casts a light. Their breeding ground is a cavity on the underside of the squid called the light organ.  (It also has shades in case the night is cloudy.) Each evening the bacteria reach the necessary quorum and begin to luminesce. Each morning the squid turns downs the wattage by venting the Vibrio, perhaps to save nutrients. It’s an extraordinary story but what intrigued the microbiologists was how did free-living bacteria, supposedly oblivious to each other, sense each other’s presence? “To me the most fascinating understanding is that they can talk in order to co-ordinate their functions,” says Whitchurch. That mechanism, dubbed “quorum sensing”, is also key to the formation of biofilms.

When microbes hit a tooth or an artificial hip they attach within minutes. They send signals to recruit others, and once they sense a quorum, work to build a matrix. Within hours it encases them like a slimy tortoise shell that can account for 90% of the mass of the biofilm.  Quorum sensing also cues the bacteria to release toxins en masse – solo actors would do far less damage.

Studying biofilms that form at the interface between agar and plastic using time-lapse photography, Whitchurch has observed these films march forward like a phalanx.  But the microbes also act like organisms far higher up the evolutionary tree. Like sponges they work together to build complex structures. Within days, small microbial villages may become cities that are millimetres thick and replete with tower blocks and water-filled channels to supply nutrients and remove waste. Individual bacteria can take on specialised roles be it to produce building blocks for the shield or maintain surface attachment. “They talk, they build; they’re civil engineers. It’s an amazing time to be a microbiologist,” says Whitchurch. When the biofilm becomes overpopulated or nutrients become scarce, they also release multiple explorer microbes that are carried away by the current. Paul Stoodley shares the sense of awe. He heads a biofilm research lab at the College of Medicine, Ohio State University and spends a lot of time observing them under a microscope. “They are able to take on a variety of forms – it’s like the T-1000 robot in Terminator 2, who could melt then pool up to form another shape or structure. They can easily adapt as the environment changes around them.”

Whitchurch also stumbled upon another revelation about biofilms in 2002. The slime that encases the film is a complex goo. Costerton referred to it as the glyocalyx, reflecting the understanding that it was primarily made of sugars. Now it’s been dubbed the Extracellular Polymeric Substance or EPS to connote that it’s more of a dog’s breakfast – not only a variety of long sugary molecules but also bits of proteins, lipids and even snippets of DNA. This “extracellular” DNA (eDNA) was thought to be refuse, threads of DNA extruded as dying cells burst. But the vast quantity made Whitchurch question that, so she added an enzyme that dissolves DNA to the biofilm. To her amazement, the biofilms shrank. The simple experiment ended up as a ground-breaking publication in the journal Science. It turns out the structural properties of the double helix are exploited by biofilms, especially to reinforce their slime shield. A DNA-dissolving drug (Pulmozyme) has been used in cystic fibrosis patients to help disrupt the biofilm.

The DNA reinforced slime barrier is only part of the explanation for how biofilms can be up to 10,000 times more resistant to antibiotics than bacteria in the planktonic form. For one thing, most antibiotics throw a spanner in the works of dividing or metabolising cells but it turns out mature biofilms harbour dormant spore-like cells in their interior. So any antibiotics that do penetrate the biofilm will not be able to eradicate the so-called “persisters”. Administering low levels of antibiotics can also backfire. The survivors activate stress-response genes that see them building bigger and more robust biofilms. The members of a biofilm are also immersed in each other’s DNA, allowing for a never-ending swap meet – a challenge when some of the participants carry antibiotic-resistant genes. Microbiologists have long been aware of the so-called horizontal gene transfer. The new understanding of biofilms explains why this is so prevalent.

Biofilms also foil the immune system. And not only by means of  their impenetrable slime. In 2007, Høiby’s team observed a contingent of immune cells zero in on bacteria – then they saw an ambush. Poking out of the biofilm was a war machine built of sugars and lipids. Called a “rhamnolipid canon” it blasted holes in the immune cells, a strategy coordinated by quorum sensing.

Unable to clear nasty biofilm infections, immune cells congregate on the outskirts and continue to mount their blunted attack. The toxins, like free radicals, fired at the biofilm end up damaging the surrounding healthy tissue. So, explains Høiby, biofilm infections in cystic fibrosis patients end up destroying lung tissue; in artificial knee joints they eat away at the bone. And biofilm infections in the stomach, colon, liver and prostate are associated with the development of cancer.

Yet biofilms aren’t all bad. Most of our resident bacteria, our microbiome, resides in biofilms – they are essential to our health. Problems occur when a particular biofilm ecosystem becomes imbalanced, perhaps when a catheter introduces skin-based microbes deep into the body. Most biofilms also manage not to rile the immune system. How these well-behaved biofilms achieve that harmony remains to be explained.

Meanwhile, the war against bad biofilms needs to be won. Following Sun Tzu’s advice, researchers have been like espionage agents, stealing biofilms’ battle plans and cracking their communication codes. And Randy Wolcott’s patients have been benefitting.

Wolcott exudes cheery Texan gentility and dedication to his patients. For 20 years he has borne witness to the pressure sores of the bedridden and the ulcers of diabetics. In 2005 he met Tommy Haney, a diabetic and guitarist to country music star Loretta Lynn. At 58, Haney was at his lowest ebb. His son had been killed in a grizzly bear attack in Alaska, and Haney was losing the fight against infections in his extremities. He had already lost some toes but amputation had not stopped the infection. Nor had antibiotics. His swollen red leg was now at risk.

Wolcott got in touch with the Centre for Biofilm Engineering in Montana where Costerton was the director. And what he learnt, he put into practice on Haney. A cultured swab of Haney’s wound yielded methicillin-resistant Staphylococcus aureus, also known as “golden staph”. But when he sent off a sample for DNA testing, it revealed the signatures of 65 other organisms. He made up a gel containing a combination of antibiotics more than 200 times greater than would be used in a drip or tablet form.

It also contained compounds to disperse the biofilms. One was xylitol, the artificial sweetener you’d find in Diet Coke. Like us, bacteria like its sweet taste but when they incorporate it into the structure of the biofilm it makes for a flimsy slime shield. Another was lactoferrin, which seems to scoop away the iron the biofilm needs. Before applying the gel, Haney’s wounds were aggressively scraped to disrupt the biofilm and rouse any persister cells into action to make them vulnerable to the antibiotics. The treatment saved Haney’s leg. Since then similar treatments at the clinic have raised cure rates from around 50% to 90%, Wolcott and his colleagues reported in the Journal of Wound Care in 2011. The more recent gels include hamamelitannin, an extract of witch-hazel bark that inhibits quorum sensing.

Other biofilm-specific treatments that block the communication codes are waiting in the wings. While the biofilm is a mixture of different species, it is no tower of Babel. A chemical called cyclic-di-GMP is  a universal signal for bacteria to aggregate. Last year Høiby’s group showed they could disperse a Pseudomonas aeruginosa biofilm growing on silicone implants in mice by decreasing the levels of cyclic-di-GMP. Very low concentrations of the gas nitric oxide also act as a dispersal signal for many bacterial species, as Staffan Kjelleberg, director of the Singapore Centre on Environmental Life Sciences Engineering, discovered. Kjelleberg says they have built a “double warhead” where a nitric oxide donor is attached to the antibiotic cephalosporin.

The hope is that biofilm-based tactics will disseminate fast. Not only could hundreds of thousands of lives be saved, but we might stop our headlong descent into the “end of antibiotics era”. As Wolcott put it “the ‘one and done’ strategy of treating infections as if they are planktonic is not and cannot work for biofilm”. But he adds “the good news is we can accumulate new knowledge faster than the bacteria evolve”.