The social life of disease-causing bacteria
This essay was written by Michael Sargent and was first published in the 2004 Mill Hill Essays.
Infectious disease organisms that ravaged human communities before the twentieth century — cholera, diphtheria, typhoid, whooping cough and many others — typically spread as free-floating individual cells in air or water. Their effects on victims — the coughs, sneezes and diarrhoea that return the infection to the environment — are effective at spreading the disease because the organisms exist as single cells. Today scientists view the free-floating single microbial cell as an evolutionary strategy conducive to the widest spread of epidemics but it is not a lifestyle followed by most species of bacteria. Due to their capacity for social interaction, most microbes exist in close-knit communities, known to microbiologists as biofilms. Biofilms exist in nearly all natural habitats, sometimes playing a spectacularly useful service to human society but, as we shall see, also causing some troublesome diseases.
The slimy films that coat surfaces in any body of fresh water are familiar examples of biofilms that recently became newsworthy when they quickly and inevitably colonised the Princess Diana Fountain in London. A more thoughtprovoking instance of a biofilm can usually be found inside domestic drinking water taps! Huge permanent biofilms, which may weigh almost one hundred tonnes, play a vital role in secondary treatment of sewage effluents. Humans too — even the cleanest of us — are covered in an invisible biofilm of normally harmless bacteria that is not likely to be displaced by scrubbing with soap. Antonie van Leeuwenhoek of Delft, the first microscopist to observe microbes, was also the first to see the biofilms that coat teeth and the internal surfaces of the mouth with “very little living animalcules prettily a-moving”. As long as life-threatening infectious diseases were rife, the pioneer bacteriologists of a century ago ignored the apparently harmless bacteria adhering to the human body. Today, bacteria in biofilms cause about sixty-five percent of all reported bacterial infections, according to the Centers for Disease Control in Atlanta Georgia.
A number of mysterious and uncomfortable diseases, in which microbes were never convincingly implicated in the past, are now attributed to bacteria growing as a biofilm. Ironically, they are also most probably associated with some of the medical interventions that have contributed so much to improved 17 health care in the late twentieth century, such as artificial joints, mechanical heart valves and contact lenses. Perhaps one in a thousand of these devices will become infected. The problem is clearly vexing to doctors and patients but important scientific breakthroughs will be necessary before this risk is comprehensively eliminated.
So what is it about a biofilm that justifies another obscure word? In the early 1980s, photographic investigations of medical devices implanted in patients for long periods exposed the true extent of problems of infection in medical devices. Using scanning electron microscopy, a technique new at that time, to visualise surfaces at high magnification, investigators frequently discovered a seething mass of tightly packed bacteria attached to the surfaces of implants. These bacteria were unusual in several respects. In spite of the best efforts of bacteriologists, they could not be grown readily in the laboratory using traditional methods, nor were they eradicated by antibiotic drugs nor consumed by the white blood cells that normally attack infecting microbes. Nonetheless, analysis of DNA from these specimens indicated the presence of well-known bacteria to which most people have significant immunity and which responded to a range of antibiotics if they could be grown in the laboratory. Recent investigations have suggested that biofilms from a number of sources can also harbour hitherto completely unknown species of bacteria. As the ubiquity of microbial biofilms in nature became apparent, this lifestyle was recognised as a social behaviour, totally unlike that of free-floating microbes encountered in the laboratory.
The bacteria in biofilms form a tightly packed mass, enveloped in a sticky matrix that prevents individual cells escaping to start new colonies and which is permeated by channels through which water and nutrients circulate freely. Attachment of cells to a solid surface seems to be the usual cue for starting the assembly of a biofilm — free-floating colonies of bacteria encased in a matrix do not seem to exist. A set of genes are switched on that drive assembly of the matrix and strengthen the attachment to make it withstand almost any force. The matrix makes the biofilm physically strong and resistant to the scavenger cells which eat microbes that invade the mammalian body, and also resistant to antibiotics. This resistance owes something to the slow growth rate of the biofilm and the impermeability of the matrix to antibiotics rather than to the antibiotic resistance genes commonly transmitted between bacteria. However, the close-knit community at the heart of the biofilm is also a genetic melting pot in which genes transmit freely between different strains of bacteria, many hundred times more effectively than in free-living bacteria. Almost certainly biofilms are the key site of microbial evolution in nature and particularly of new disease-causing strains.
The capacity that biofilm bacteria show for social interaction is a co-operative behaviour known as “quorum-sensing”, and it accompanies the switch from the free-floating “loner” lifestyle to the socially integrated lifestyle of the biofilm. Co-operative social behaviour amongst certain microbes was known long ago but an understanding of its mechanism emerged only in the last decade. This began with the study of organisms that emit light — the bioluminescent bacteria. When grown in the laboratory these bacteria begin to emit light when the cell concentration reaches a critical density, because individual cells secrete a chemical “signal” that affects the entire community. With its evocative name, the quorum-sensing phenomenon reflects the custom of human committees to proceed with their deliberations only when a quorum is present, a feature of microbial life that is now well established. The quorum-sensing capabilities of medically significant biofilms probably contribute importantly to the development of many diseases, although this is hard to prove experimentally.
However, it is clear that the capacity of the bacterium Pseudomonas aeruginosa to establish a biofilm in mice with cystic fibrosis depends on an intact quorumsensing system. More than a hundred genes that are inactive in the bacterium in its free-living state are switched-on in biofilms in the lungs of cystic fibrosis patients. Some of these harm the victim or provide a means of evading the immune system. Individuals compromised by the genetic disease cystic fibrosis, fight a life-long battle against a biofilm. This lines their lungs and constantly threatens to overwhelm them with pneumonia. The lungs of most people are almost sterile because a biochemical pump that takes up sodium releases water and generates a steady flow of fluid that sweeps microbes towards the mouth. This pump is defective in victims of cystic fibrosis, rendering ineffective the usual defence to microbes that arrive in the lungs by chance. In such propitious circumstances, Pseudomonas aeruginosa readily establishes a dense biofilm in the lungs. This biofilm is notoriously resistant to antibiotics, even though the organism, in the laboratory, is sensitive to the antibiotic cloxacillin.
People suffering from cholera have become infected by exposure to freefloating single cells of Vibrio cholerae transmitted in water, but biofilms also play a role in the frightening biology of this disease. Once ingested, the microbe establishes a biofilm in the intestine, using the quorum-sensing facility to delay release of its notorious toxin until a substantial colony has developed. Consequently, the symptoms of the disease — the massive secretion of water by intestinal cells and the severe life-threatening diarrhoea — strike the victim suddenly, leaving no time to develop an immune response to the toxin. The chief reservoir of cholera in the environment is also a biofilm, one which is 18 associated with a marine phytoplankton bloom in the coastal waters of the Bay of Bengal. Free floating Vibrio cells are released from the biofilm only when it encounters fresh-water, typically when the sea penetrates the delta regions of the Ganges and Brahamaputra rivers, a typical trigger for cholera epidemics in Bangladesh.
In addition to these regular outbreaks, seven genetically distinct cholera pandemics have emerged from this extraordinary habitat in the last two centuries, to spread across the planet bringing misery and devastation to vulnerable places. The first to reach Europe, in 1832, went on to decimate the urban poor of most great cities of the world, including the Americas. Four more European pandemics followed before these terrifying visitations were curtailed by the introduction of closed sewers and uncontaminated piped water. The seventh pandemic, known as El Tor, appeared in the Far East in 1961 and spread to Peru in 1991 — the first appearance in the Americas in one hundred years. The source of these outbreaks, which continued throughout the 1990s, may have entered the coastal waters in the bilge of a Chinese freighter and then established as a biofilm in the prolific marine life of the Humboldt Current.
A threat from less virulent organisms than Vibrio cholerae is posed by organisms that form biofilms in innocuous-looking ecological niches. Listeriosis, caused by Listeria monocytogenes, is a serious disease in people whose immune system is compromised. It originates in biofilms found in what the food-making industry euphemistically describes as “sanitation-dead areas” in their facilities, from which they occasionally escape to contaminate unpasteurised food such as cheese. A pneumonia-like disease known as meliodosis arises from the luxuriant biofilms of Burkholderia pseudomallei that form in the rice fields of Southeast Asia. Field workers breathe in a fragment of the biofilm into their lungs and contract a low-level infection that may flare into a life-threatening infection during seasonal episodes of semistarvation.
A number of mysterious ill-defined infections probably originate in biofilms associated with unreliable water supplies or other places where aerosols are generated from bodies of stagnant water. As the concentration of disinfectants required to kill biofilm bacteria is typically at least a thousand-fold greater than for free-floating bacteria, they are likely to persist unless special efforts are made to remove them. An important issue facing the water industry is the need to ensure that disease-causing organisms do not take up residence in the biofilms that will inevitably colonise water-delivery pipes.
Biofilms formed in certain organs of the human body are the probable cause of 19 a number of rather intractable and neglected inflammatory conditions. These kinds of biofilm are found in sites with access to the environment, for instance even the gall bladder has a connection with the outside through the gut. Internal tissues such as bone become infected only if they have been exposed to the environment through an accident or a surgical intervention. Tooth decay and gum infections, too, are caused by organisms in biofilms. These vividly demonstrate the tenacity of biofilms by their ability to survive regular scouring with detergent- and disinfectant-laden toothpaste. Other conditions associated with biofilms include kidney stones, inflamed prostate gland, heart valve infection and middle ear infections. Typically, the bacteria are enveloped in a tough matrix that resists the efforts of physicians to release material that can be grown in the laboratory. If the bacteria causing these disorders can be grown successfully, they are usually sensitive to antibiotics although the infection itself may respond poorly and may never be entirely eradicated. Occasionally the only solutions are a grim last resort; surgery to remove an irredeemably infected limb or organ.
Recognition of the singular biology of biofilms has created an important new dimension to understanding infections that arise when medical devices are implanted in the human body. They are sterile when implanted and the installation is made under the most stringent conditions of sterility that can be managed, but the risk of infection is always present. Bacteria that normally lurk on human skin seem to have little difficulty in migrating through a skin lesion to colonise an implanted medical device; they even reach implants through the blood stream from urinary infections or dental abscesses. Generally, physicians and surgeons reckon that aggressive antibiotic therapy from the start should eradicate bacteria before a biofilm establishes but there is always a significant chance that a few bacteria will survive even the most strenuous treatment. After periods that may extend to many weeks, the surviving bacteria may establish a biofilm that is well adapted to withstand the attacks of the immune system. Once formed, biofilms are a serious threat that is eliminated only by removing the device and allowing normal healing processes to eliminate the infection. If this fails the infection can lead to systemic blood poisoning that may be difficult to arrest and may be fatal.
Biofilms that cause inflammation may contain members of the genera Proteus, Streptococcus, Haemophilus, the yeast Candida (endocarditis) and other fungi sometimes in mixed infections. Staphylococcus epidermidis and the somewhat less common Staphylococcus aureus are particularly important causes of infection of medical devices through their highly developed capacity to form biofilms. Staphylococcus aureus is particularly dangerous because of its armoury of toxins: once established in the blood stream little can be done to 20 check its advance. Families of victims of this kind of septicaemia are usually astonished at the speed and intensity of the onslaught.
The potential harm caused by biofilm-forming Staphylococci has another serious dimension which originates in genetic elements that confer resistance to antibiotics. The most notorious of these is MRSA (methicillin resistant Staphylococcus aureus) — first identified in Britain around 1960 — which evolved early in the history of antibiotic therapy to carry resistance to five important drugs. If these strains establish inside the body, the patient may be in mortal danger from blood poisoning as most antibiotics are unlikely to arrest the disease. Until recently, the antibiotic vancomycin was the one remaining weapon against MRSA. The medical profession holds this in reserve solely for MRSA infections so that a vancomycin resistance gene does not emerge in other organisms through indiscriminate use. Unfortunately, this last resort may already be compromised as rare vancomycin resistant strains of MRSA already exist.
The increase in cases of septicaemia caused by MRSA in the last decade has been matched by increased numbers of people infected, but without showing symptoms. The organism is usually present on the skin or in more protected niches such as nostrils or tonsils in health-care workers and in members of the public and can be passed on inadvertently to compromised patients. This is a steadily growing threat in the developed world but is notably less common in the Netherlands and Scandinavia. The reason for this probably lies in a longterm commitment to reducing the chance of cross-infection in hospital. In these countries, some hospitals are able to screen staff and patients for MRSA carriers and they attempt to eradicate the infection by a rigorous disinfection of the skin and other suspected sites of infection such as tonsils. The scale of MRSA infection elsewhere is so great that any radical infection-control strategy more extensive than rigorous hand washing and hospital cleanliness seems overwhelmingly difficult. The shrill voices in the media that have so much to say about MRSA are unaware that this organism is part of the much bigger scientific problem of how to control microbial biofilms.
Can anything else be done to reduce the health hazards associated with microbial biofilms? Materials that would prevent the growth of microbial biofilms when incorporated in medical devices to be implanted in human tissues have been at the top of the wish lists of health-equipment inventors for many years. Materials for implants with antibiotics or disinfectants immobilised on their surfaces that might kill bacteria have been explored. Indeed, the literature is full of “promising leads” but robust and successful applications are notably elusive. When science reaches this kind of impasse, imagination and lateral thinking is required to make further progress. Not for the first time, scientists have looked for inspiration in the interactions of individual microbes with other species; this time for clues to how biofilms are suppressed in nature.
Almost ten years ago, some Australian biologists noticed the absence of a biofilm on fronds of a red alga growing in Sydney harbour; an occurrence they considered strange which they traced to secreted chemicals that prevented bacterial colonisation. These chemicals are members of a family of compounds known as furanones that block the receptors for a class of molecules that affect quorum-sensing. They seem to be non-toxic in the mammalian body and are relatively stable; moreover, they seem to prevent the formation of Pseudomonas aeruginosa biofilms in the lungs of mice with cystic fibrosis. Promising news is also emerging from studies of a family of small peptides that have a role in the proliferation of biofilms of MRSA; certain derivatives can prevent biofilms forming. A third type of molecule, the only known organic compound of the element boron that exists in the living world, furanosyl borate diester, is a recently discovered quorum-sensing chemical. This substance, unlike other quorum-sensing chemicals, affects many bacterial species and suggests that it may be possible to find an agent that blocks biofilm formation. With three important leads, the pharmaceutical industry now has a great opportunity to find agents that combine the facility of disrupting biofilms with an antibiotic. Many hurdles must be cleared before troublesome microbial biofilms in the human body cease to be a problem, but it looks as though progress is possible.3