A novel approach to understanding Biofilms

Chic, urbane, sophisticated and sometimes deadly-such are the inhabitants of the world's weirdest metropolis. Andy Coghlan reports from the underworld

THROUGH the murky gloom, you can just make them out- shadowy towers shaped like mushrooms. Circulating through and around them, apparently suspended in mid-air, there are ducts and channels of swishing liquid. Just for a moment, the murk clears. In the distance, you see skyscrapers of ghostly spheres piled one on top of the other.

The set of a futuristic movie on some distant planet? Not quite. This is slime city, a sprawling metropolis very much of this world. Inside, shielded from harm and replete with every creature comfort, live beings whose cosmopolitan lifestyle is only now coming to light.

Amazing cityscapes like this have existed on Earth for billions of years, built and populated by plain, humble bacteria such as Escherichia coli and salmonella. More properly known as biofilms or mucilages, slime cities thrive wherever there is water-in the kitchen, on contact lenses, in the gut linings of animals. When the urban sprawl is extensive, biofilms can be seen with the naked eye, coating the inside of water pipes or dangling slippery and green from plumbing.

Yet only in the past few years have scientists learnt how to observe the inner structures of biofilms using powerful microscopy techniques. What they are now discovering about those ghostly cityscapes is sending shock waves through microbiology.

Toxic food

For decades, microbiologists have based virtually all their ideas about bacteria-how fast they can grow, how they react to antibiotics, what they can eat, and so on-on the behaviour of colonies grown from single species on laboratory plates. But research now shows that in nature, bacteria seldom if ever grow in single-species colonies. Instead, different species live cheek-by-jowl in slime cities, helping each other to exploit food supplies and to resist antibiotics through neighbourly interactions. Toxic waste produced by one species might be hungrily devoured its neighbour. And by pooling their biochemical resources to build a communal slime city, several species of bacteria, each armed with different enzymes, can break down food supplies that no single species could digest alone.

The problem is that not all scientists have taken the new findings on board. "Serious errors have arisen from extrapolation of data from single-species laboratory cultures to explain bacterial behaviour in real ecosystems," warns Bill Costerton, a pioneer of slime research at the Montana State University in Bozeman. What's more, mistakes are still being made. Single-species cultures are still routinely used to assess the sensitivity of biofilm bacteria to antibiotics, says Costerton. And this despite the fact that biofilms can be 1500 times more resistant to antibiotics than a single colony, because they shelter their bacterial inhabitants so effectively.

If biofilms were rare, none of this might matter so much. But Costerton estimates that more than 99 per cent of all the planet's bacteria live in biofilm communities. Some do vital jobs. Sewage treatment plants, for instance, rely on biofilms to remove contaminants from water, while cows use them to digest grass in their forestomachs. But biofilms also wreak much havoc. Some are implicated in diseases as various as cystic fibrosis and the blood poisoning caused by infected catheters. Others secrete acids that nibble away at the toughest of metals and minerals, corroding anything from the legs on oil rigs to the teeth in your mouth.

Biologists have known about biofilms ever since bacteria were first studied. But in the past researchers were blinded by two flawed assumptions. The first was that, biochemically speaking, biofilm bacteria must behave much like solitary, free-roving microorganisms. The fact that many biofilm dwellers and free-rovers belong to the same bacterial species simply served to reinforce that view. It didn't seem likely that something as simple as an E. coli or salmonella bacterium could have two distinct modes of biochemical behaviour, one suited for the solitary life of scouring the waterways and bloodstreams, the other for structured group living.

But that is precisely what researchers are now discovering. While it's true that city dwellers have exactly the same genetic makeup as their free-roving cousins, their biochemistry is very different because they switch to using a different set of genes. It's a type of genetic flexibility that multicellular organisms simply don't have.

The second assumption was that biofilms contain disorderly clumps of bacteria located in no particular structure or pattern. Again, nothing could be further from the truth. Costerton, working with Zbigniev Lewandowski, a Bozeman colleague, and Douglas Caldwell's team at the University of Saskatchewan have dispelled that myth by using a high-powered microscopy technique to spy on biofilms as if viewing a city from a satellite.

The technique, confocal scanning laser microscopy, was first developed by cell biologists 15 years ago, but it was only taken up by biofilm researchers in the early 1990s. Its great advantage over other microscopic techniques is that it can magnify biofilms without destroying them. Previously, samples had to be dried out or treated with chemicals. What researchers didn't realise was that these methods were destroying the gel-like structures of biofilms.

They do now. When Costerton and his colleagues first used the confocal microscope in 1992, the complexity of what they saw astonished them and others alike. Since then, the researchers have examined a vast range of biofilms from sources as diverse as catheters, sewage pipes and the gut linings of animals.

In most cases, says Costerton, the base of the biofilm is a bed of dense, opaque slime 5 to 10 micrometres thick. It is a sticky mix of polysaccharides, other polymeric substances and water, all produced by the bacteria. Soaring 100 to 200 micrometres upwards are colonies of bacteria shaped like mushrooms or cones. The more plentiful the food, the more crowded the bacterial skyscrapers.

Above street level comes more slime, this time of a more watery and variable consistency. Its chemical makeup varies, depending on the identity of the bacterial species making it. In most cities, says Costerton, you'll find a rich mix of species, each living in their own chemical ghettos or "microzones".

Similar cityscapes have been popping up on microscope computer screens elsewhere. At the Centre for Applied Microbiology and Research at Porton Down in Wiltshire, Bill Keevil and his colleagues have been using a microscope technique they invented in 1991 to study slime samples from North Sea oil rigs, dental plaques and other sources. Sparkling with reflections from the microscope's light beam, their skyscrapers resemble a city at night. "It looks like Manhattan when you fly over it," says Keevil.

Every city needs an infrastructure, and these little Manhattans are no exception. According to research in Costerton's laboratory, the biofilms are permeated at all levels by a network of channels through which water, bacterial garbage, nutrients, enzymes, metabolites and oxygen travel to and fro. Gradients of chemicals and ions between microzones provide the power to shunt the substances around the biofilm, as do currents from water flowing overhead. Individual bacterial species form microcolonies wherever their staple diet is in abundance. Often, their food is the waste product from a neighbouring microcolony and wafts in through one of the channels. The origin of these connecting channels is a mystery the researchers are itching to solve. Water alone might create the channels by pushing through weak spots in the slime structure. Or the bacteria might be involved in some way. "We don't know yet," says Costerton.

The researchers' microscope technique can also produce "movies". Some simply show the bacteria moving around the cities. More Big Brotherishly, Lewandowski and his colleagues have used fluorescent chemical markers to pry into which genes the bacteria are switching on and off and which proteins they're producing. "You can watch the critters at work," says Costerton. And what's clear in these movies is that bacteria are not the only inhabitants of slime city.

In many cases, fungi stake out their own turf, as do the algae that give some slimes their lurid green colour. And protozoans that consume bacteria can be seen scavenging for an easy meal. Video footage from Keevil's laboratory shows disc-shaped protozoa hunting for bacteria, and other kinds of protozoa spinning like Ferris wheels to suck in their prey.

Spying on slime cities is just the beginning. Biofilm researchers have their sights on a deeper issue-how organisms as simple as bacteria can produce such apparently complex worlds. The first requirement, plainly, is a surface. Biofilms, like cities, are born when individuals settle permanently in one place. Some bacteria settle because the surface, such as cellulose in grass, is edible. Others put down roots because the fluid washing over a surface, such as sewage in a pipe, is rich in nutrients the bacteria can digest.

Exactly how these "pioneer" bacteria anchor themselves to surfaces, nobody knows. Keevil thinks they rely on electrostatic forces. Costerton, on the other hand, thinks they glue themselves to surfaces with an unusually sticky form of slime called alginate. Using confocal scanning laser microscopy and a fluorescent chemical to track gene activation, Costerton's team has studied how Pseudomonas aeruginosa, a species of bacteria that clogs the lungs of people with cystic fibrosis, forms biofilms. The instant the bacteria dock to glass, they switch on certain genes involved in the synthesis of alginate, switching them off again once the bacteria are engulfed in alginate.

But sticking to a surface is not enough. The pioneer bacteria must also be persuaded to adapt their biochemistry to group living, giving up the selfish habits of the lone vagrant. Here, there's more agreement about the trigger. A bacterial chemical called homoserine lactone swings dramatically into action, signalling the pioneers to turn into stationary city dwellers (see "The secret language of bacteria", New Scientist, 16 September 1995, p 30).

The key role of this chemical first came to light in the early 1990s, thanks to teams led by microbiologists such as Gordon Stewart, of the University of Nottingham, and Pete Greenberg of the University of Iowa. Bacteria constantly discharge low levels of homoserine lactone, even when they are roving. They are equally capable of sniffing it out, by capturing the molecule on surface receptors. But the lactone's dramatic effects on bacteria only kick in when its concentration exceeds a certain threshold. Nothing happens until there's a critical mass of pioneer bacteria discharging the lactone all in one place. Only then will a biofilm form.

Towers of Babel

"The lactone is the communication signal that holds the city together," says Costerton. "The fascinating thing is that it works between species, so lots of different species can get together without it becoming the Tower of Babel."

Now, researchers are close to understanding precisely how it works. When the lactone concentration reaches a threshold level it signals the bacteria to produce proteins called "sigma factors". Vital for changing vagrants into city dwellers, these powerful proteins are subunits of the enzyme RNA polymerase, which helps to transcribe the information present in genes into a form that cells can use. Acting inside bacteria, each sigma factor will switch on a job lot of up to 40 genes simultaneously. Entire biochemical pathways, dormant in free-roving bacteria, spring to life, enabling bacteria to produce the sugary polymers, like alginate, on which slime cities are founded.

At the same time, sigma factors shut down many genes required by free-rovers. New arrivals in the city no longer need such tough cell walls, so they close down genes involved in making cell-wall proteins

In all, as many as 30 to 40 per cent of the proteins present in bacterial cell walls differ between city dwellers and free-rovers, says Costerton. "Some of the targets for antibiotics are not there any more either, so the bugs become desperately difficult to kill," he says. In many cases, they become resistant to penicillin and even powerful chlorine-based disinfectants.

This newfound genetic hardiness, combined with the physical shelter provided by slime cities, explains why biofilms that form on the surfaces of prosthetic devices, contact lenses and catheters are such a problem. It also explains why biofilms in sewage works, water pipes and cooling towers provide such safe havens for bugs like Legionella pneumophila, the cause of Legionnaire's disease, and Cryptosporidium parvum, a protozoan parasite that causes severe stomach upsets.

If researchers can discover a "reverse" sigma factor, or a chemical that neutralises homoserine lactone, it might be possible to dissolve biofilms by sending in the equivalent of an evacuation signal. At present, the only chemical that reliably lays the cities to waste is plain old bleach, which turns the slime into carbon dioxide and water and kills the bacteria. But milder alternatives are in prospect.

Costerton has found that methyl cellulose, a chemical discharged by bacteria when they return to vagrancy, can evacuate an entire city if the concentration is high enough. A further option is alginate lyase, a bacterial enzyme that dissolves slime. Researchers have also dislodged the cities using pulses of sound. Marianne Walch of the US Naval Surface Warfare Center in Silver Spring, Maryland, will reveal details of this latest technique at an international meeting on biofilms next month at Snowbird in Utah.

Order or anarchy?

However, the key question for most biologists is not how to destroy slime cities. It's about who-or rather what-governs the form and apparent orderliness of their structures. Maybe their street plans and infrastructure are the result of specific genetic programmes that have evolved in bacteria to make them cooperate in specific ways when the conditions are right-programmes, perhaps, that are activated at the same time as sigma factors. Or maybe the orderliness is an illusion, the fortuitous outcome of opportunist organisms randomly exploiting whatever niches are available. Opinions remain divided.

The bacteria simply go where the food is, says Julian Wimpenny of the University of Wales in Cardiff. "Don't say the cells are responsible for this design-it's a haphazard result of concentrations of nutrients."

But Costerton, for one, thinks there's more to it than that. He sees biofilms less like colonies of self-serving automatons and more like the cells of tissues of multicellular organisms, where selfish instincts must be subordinated to the wider interests of the group. "All of a sudden, instead of individual organisms, you have communication, cell cooperation, cell specialisation and a basic circulatory system, as in plants or animals," says Costerton. "It's a big intellectual break."

To back up his argument, Costerton points to the inhospitable terrain of the cow's rumen, home to one of the first biofilms he and his colleagues investigated. At least five different species of bacteria are needed to digest cellulose in the rumen. Only by collaborating in slime cities can these bacteria succeed in the complex task of breaking down cellulose.

On one side of the biofilm-the cellulose "coalface", as Costerton puts it-Fibrobacter succinogenes bacteria break down the cellulose into glucose. On the other side are mushroom-shaped colonies of butyrovibrio bacteria that specialise in breaking down this glucose into butyrate. In turn, the butyrate is degraded into acetate by a clump of neighbouring bacteria that are different again. And in the final step, the acetate is converted into methane by a nearby ball-shaped colony of specialist methanogen bacteria. "What's waste for one bacterial colony is the nutrient for the next," says Costerton.

But the collaboration seems to run even deeper than that. Since oxygen normally poisons methanogens, these bacteria need help to survive in the oxygen-rich environment of the biofilm. That help comes from a fifth species of bacteria, which forms a protective seal around each methanogen colony.

It's hard to imagine all these interactions arising from random opportunistic mechanisms, argues Costerton.

Whatever the truth, there's no denying that slime cities have a distinctly social ring to them. After all, they seem to be founded on the familiar trade-off between individual and collective needs that governs all good cities and societies. Citizenship carries a price. The bacteria must use some of their resources and energy to produce sticky slime and infrastructure that will in the end be used by many different species. But the dividends of group living can-when the conditions are right-make this investment worthwhile even for individuals that are purely selfish.

A few years ago, all this would have seemed fanciful. But now the secrets of the city are leaking out, and microbiology is slowly turning a new, slimy corner.