Kinkajou : What on earth is a biofilm?
Erasmus : A biofilm is a biological membrane composed of microorganisms typically adherent to a surface. Microorganisms may include bacteria, archaea, protozoa, fungi, algae and even viruses. An extracellular polymeric substance links cells together into a composite structure. Biofilms can be composed of a single species, however typically a number of species are present.
Cells within a biofilm are thought to act differently and to express a different range of genes to cells growing independently. The extracellular polymeric substance (EPS) includes polysaccharides, perhaps protein and even nucleic acids. The EPS can compose between 50 to 90% of the biofilm structure. It is thought that the extracellular polymeric substance (EPS) traps extracellular enzymes in close proximity to the microbial cells. This effectively allows an external digestion process to occur, keeping the degradation products of digestion or nutrients close to the bacteria in the biofilm, aiding absorption.
Some biofilms have even been found to contain water channels to assist in the distribution of nutrients and also to perhaps contain signalling molecules.
Kinkajou : So you mean that in effect bacteria are building cities?
Erasmus : There is a perception that bacteria are not social organisms. However this is not true. Bacteria do contain specific genetic coding for cooperation and intercellular signalling, much like the processes that occur within multicellular organisms. Biofilms must be regarded as a group or social form of planktonic type organisms.
Biofilms form spontaneously as bacteria inside the human body group together or replicate. The biofilm structure is most likely the normal growth habit of bacteria. Free planktonic bacteria are not likely to constitute a typical growth strategy.
Kinkajou :So what are the advantages of biofilm structures for bacteria?
Erasmus: The dense extracellular EPS matrix may allow biofilms to resist the action of detergents or antibiotics. The matrix structure improves the ability to withstand dehydration. Surfaces also enable bacteria to develop in media which contain nutrients that maybe too dilute for growth. When they are adherent to a surface, they can resist mechanical actions such as being washed away. Genetic interactions with exchange of DNA are facilitated by the presence of a stable matrix.
Because biofilms can be composed of many different organisms in many different interactive structures, they can colonise many different environments. They are complex structures. Even a simple dental plaque biofilm may contain up to 500 different bacterial species. Researchers have suggested that 60-80% of bacterial infections in the body are made up of bacteria in biofilm structures. Many bacteria prefer not function in a planktonic or free-floating format.
Biofilms can have different charge characteristics, namely being either anionic or cationic. Anionic polysaccharides confer the ability to allow cations such as calcium or magnesium to cross-link the polymer strands, increasing in the cohesion of the biofilm. Staphylococcal biofilms, such as may exist in contaminated medical devices are more typically catatonic.
Biofilms are not necessarily continuous monolayers or even multi-layers of microorganisms. Typically ,they have the structure of micro-colonies of bacterial cells typically encased in an EPS matrix and separated from other micro-colonies by interstitial voids or water channels.
Kinkajou : So we must really know a lot about biofilms.
Erasmus :Study of biofilms is made difficult by their complex structures. It is difficult to recreate biological situations such as infected catheters or infected hip prostheses in a microbiological culture dish situation, such as exists in the typical microbial laboratory. Pure cultures are almost non-existent outside of the laboratory.
Most research has therefore concentrated on external biofilms such as may exist on the interiors of pipes, on floors and counters, in water tanks, and storage areas. Biofilms are significant in many different environments. For example, the presence of microbes on a metal surface modifies deposition and dissolution of minerals within the fluid stored. This alters the electrochemical properties of metals and alloys, causing effects such as pitting corrosion of metal surfaces.
Most research in microbiology uses traditional methods developed at the turn of the century that focuses on the free planktonic forms of bacteria. Unfortunately, most bacteria in nature do not live like that. They actually cohabit with other bacteria in mixed surface dwelling communities. Even within the human body, there is a growing realisation that it is the bacterial community that needs to be studied or treated, not free planktonic bacteria.
Kinkajou : Are biofilms predominantly a bacterial phenomenon?
Erasmus :Most microorganisms are capable of adhering to surfaces and to each other, in effect creating biofilms. Biofilms exist in almost every possible niche on the planet. Some niches can be very specialised.
For example, the archaea are a group of organisms with the prokaryotic structure but with chemical pathways more commonly related to eukaryotes. They use a very large range of chemical energy sources including organic compounds, ammonia, metal irons and even hydrogen gas. They are capable of living in extreme environments such as hot springs or salt lakes. The ether linkages within the cell membranes are inherently more stable than the ester linkages found within the cell membranes of other bacteria or even eukaryotic cells. It is thought that this allows the archaea to function in these extreme environments. Another unique feature of the archaea is that they are the only known organism that can “metabolically” produce methane.
Kinkajou : If we are ever to terraform new planets, biological nano factories such as the archaea may well be the method we use.
Erasmus :And biofilms will be an important part of this.
Kinkajou : Tell us about the types of environments in which we may see biofilms.
Erasmus :As mentioned above, biofilms can be found even in hostile environments including Archaean organisms in hot Springs, salt pans, alkali flats and salt lakes, in glaciers and deep within the ground in Bore water such as Australia’s Great Artesian basin where iron reducing bacteria are responsible for blocking access pipes.
Legionella bacteria commonly form biofilms in the water cooling towers of air-conditioning systems. Other bacteria colonising air-conditioning equipment can compromise heat transfer functions. Bacteria can colonise galvanised iron water reticulation pipes, and are certainly present lining sewage pipes. Biofilms can form on kitchen floors and kitchen bench tops compromising sanitation in food preparation areas.
Streams and pools of water are commonly colonised by biofilms. Didymus algae forms a thick dense algal biofilm which chokes out many other plants and animals in Marine and freshwater environments. Australia’s biosecurity and quarantine laws attempt to limit the spread of this extremely harmful organism. Other biofilms can form food sources in water environments. Mosquito larvae for example feed on bacterial and algal microfilms. If you have slipped on a rock by the sea or in a stream, you have experienced a biofilm adhering to a surface.
Bacterial microfilms adhering to the holds of marine vessels can allow other Marine organisms such as barnacles to attach. They can also cause corrosion and limit the ability and speed of boats to travel.
Sewage treatment plants often use biofilms on filters to extract organic matter. Sand filters rely on biofilm development to assist in the process of purification of water for drinking. The water we drink could well be regarded as a waste product of the biofilm bacteria.
Bacterial biofilms are responsible for metabolising petroleum oil in spills, especially Marine systems. Bioremediation and biotransformation is increasingly used as a method of neutralising oil spills in the environment. Bioremediation is a process whereby microorganisms existing in the environment change their population structure to maximise their ability to use organic contaminants as a food source. Typical microbial cell densities adjacent to contaminated ground water sites show bacterial counts of the order of 100 cells per gram of soil.
Microorganisms are capable of utilising petroleum hydrocarbons, chlorinated organics and even nitro-aromatics as food sources. They also capable of locking up heavy metals and radionuclides in organic chemical structures.
Biofilms present in the mouth of animals are experienced by us as dental plaque. Bacteria (Streptococcus mutans especially) within this plaque are responsible for tooth decay and gum disease.
Biofilms are present in many plants. They can be useful or harmful. Yeast colonisation of the surface of grapes is important in the final structure and aromatic density of the wine produced. Nitrogen fixing Rhizobium bacteria exist symbiotically in the roots of plants and assist in the fixation of nitrogen facilitating growth.
Biofilm- based microbial fuel cells have been at the forefront of development in the production of bio-electricity.
Biofilms have been championed in the use of the production of bioethanol and other organic chemicals for use in industrial processes. They also important in biodegradation of waste matter, often with useful industrial by-products such as methane or hydrogen gas.
Biofilms are thought to form within the large intestine. Many modern probiotics include bacteria which assist the formation of biofilms and improve gut colonisation. It is suggested that the appendix maintains biofilms that may have a function in facilitating bacterial colonisation of the large intestine.
Pipelines can suffer from substantial corrosion from the action of microbial biofilms. While most corrosion is due to abiotic (non-living) factors, it is thought that up to 20% of corrosion is caused by microorganisms attached to the metal surface.
Microbial leaching is significant in mining processes. Microbes may oxidise minerals into metal sulphides. Typical minerals accessed include copper ores, uranium ores, silver ores, gold ore, cobalt and molybdenum ores. This microbial biofilm action occurs in the extremely hostile environment of low pH, high heavy metal concentrations and often unfavourable temperatures. This results in a bacterial micro-flora with highly specialised nutritional requirements and unusual metabolic processes. In microbial copper extraction acidified water of pH 1.5 to pH 3 containing acidophilic bacteria such as Thiobacillus ferrooxidans actively oxidise soluble ferrous iron and sulphide minerals, releasing soluble copper into the aqueous solution. This oxidation process is essentially similar to the same process involved in corrosion of metal surfaces.
Toxic algal blooms can create low oxygen environments harmful to the ecology of lakes, rivers and marine environments.
It has been suggested that biofilms form reservoirs for pathogenic organisms in diseases such as otitis media, bacterial endocarditis, lung infections in cystic fibrosis, and Legionella lung infections. It has been suggested that persistent middle ear infections may be due to the formation of organic biofilms within the middle ear cavity.
Bacterial Biofilms are likely to responsible for many hospital-acquired infections such as on the surfaces of catheters, implanted medical devices such as joint prostheses, osteomyelitic infections and on wounds. PCR analysis (Polymerase chain reaction amplification of DNA fragments) of wounds has revealed the presence of strictly anaerobic bacteria within biofilms on the exudative surface of chronic wounds.
Biofilms may also be present in bacterial vaginosis infections, urinary tract infections and, sinus infections. Research has shown evidence that sub-therapeutic levels of antibiotic such as the beta-lactams (notably), can induce biofilm formation in Staphylococcus aureus infection sites.
Research has shown evidence of many different bacterial signatures in the atherosclerotic lesions of patients with coronary heart disease. This suggests the presence of a functional biofilm within the atherosclerotic plaques.
Biofilm Infection Sites
Up to 20% of kidney stones involve biofilms interacting with mineral and organic substrates present in the urine.
It has been suggested that even leptospirosis organisms are capable of forming biofilms and that this may play a significant role in their ability to escape from immune system actions.
When the immune response is compromised, Pseudomonas aeruginosa biofilms are able to colonize the alveoli, and to form biofilms.
Lung Alveoli Biofilms
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Kinkajou : Wow! It’s obvious that Biofilms have the potential to cause a tremendous array of infections and diseases. Tell us a few more specifics about
biofilms in medicine.
Erasmus :Using laser scanning microscopy, researchers have obtained three dimensional images of the biopsies and evaluated them for biofilm morphology using generic stains and species-specific probes for Haemophilus influenzae, Streptococcus pneumonia and Moraxella catarrhalis. (Typical bacteria found in middle ear infections). Effusions, when present, were also evaluated for evidence of pathogen specific nucleic acid sequences (indicating presence of live bacteria). The study found mucosal biofilms in the middle ears of over 90% of children with middle ear infections.
Otitis media, or inflammation of the inner ear, is caused by biofilm bacteria Biofilms may also be present in asymptomatic patients. This raises the possibility of the risk they pose for disease exacerbations over time.
The researchers concluded from their studies of the symptomatic “It appears that in many cases, recurrent disease stems not from re-infection as was previously thought and which forms the basis for conventional treatment, but from a persistent biofilm.”
Middle Ear Infection
The researchers went on to state that the discovery of biofilms in the setting of chronic otitis media represented “a landmark evolution in the medical community’s understanding about a disease that afflicts millions of children world-wide each year and further endorses the emerging biofilm paradigm of chronic infectious disease.”
Kinkajou : So who cares about a few minor ear infections in children?
They are easy to treat and generally go away.
Erasmus :There was a big long term health study of families in New Zealand. The most important factor in determining whether a child will be sitting in a prison cell as a young adult was not their socio-economic status or whether their parents were divorced or separated. The key determinant was whether the child could hear. Most of these children had glue ears, which are full of mucous like fluid but are generally not regarded as infected by doctors. The fluid is hard to see, so the condition is rarely diagnosed. Children who have hearing problems fall behind at school. By the time the problem of “glue ear” typically resolves as the child gets older, the child is so far behind their classmates that they will never catch up. Problem kids who are labelled as stupid but who really have low grade hearing problems, in effect become problem adults.
Biofilms are at the centre of this insidious process.
Glue ears are treated with antibiotic therapy with mixed success. Complex biofilms existing in the middle ear cavity even in apparently uninfected ears may well lie at the heart of this problem. Currently, the treatment rationale is that antibiotics target associated throat swelling around Eustachian tube openings. Reducing bacteria here with antibiotic therapy reduces swelling, allowing mucous drainage from the glue affected middle ear. Rationale is everything when therapy is being used as it changes the combination of antibiotics and other treatments used.
Those scientists who support an emerging biofilm paradigm of chronic disease feel that biofilm research is of utmost importance because of the fact that the infectious entities have the potential to cause so many forms of chronic disease.
The Marshall Hypothesis is an important part of this paradigm shift.
Kinkajou : Do biofilms colonise obvious wet places in the human body such as eyes that most doctors usually regard as “clean”.
Erasmus :Researchers have indeed proposed that biofilms cause most infections associated with contact lens use. Once bacterial cells form a biofilm on contact lens surfaces, the cells become resistant to lens solutions and immune to the body’s own defence systems. Fungal biofilms can form in contact lens solutions leading to potentially virulent eye infections.
Eye Drop Bottles
Kinkajou : Any other information on biofilms in the human body?
Kinkajou : Some researchers investigated biofilms containing K. pneumonia and P. aeruginosa in lung tissue. It was discovered that both bacteria can exist in a mixed culture biofilm such as in the bronchial Airways even though typical growth rates for pseudomonas aeruginosa are much slower than those for Klebsiella pneumoniae.
Klebsiella grew localised micro-colonies covering proxy 10% of the affected area. Pseudomonas in contrast rapidly colonised the entire surface. (Undoubtedly due to the ability of Pseumonads to be motile).
Klebsiella succeeds in surviving in this environment due to its ability to attach to the pseudomonas aeruginosa biofilm. In the long term, Klebsiella grows more rapidly and out- competes the Pseudomonas aeruginosa bacteria in the surface layers of the bronchial biofilm, dominating the surface of the biofilm. Biofilms can change their nature over time, with early coloniser and late coloniser bacteria, much akin to the situation of de novo new forest growth.
Do Biofilm bacteria have a role in chronic inflammatory diseases in humans?
Erasmus :Recently,, the potential of biofilms to cause debilitating chronic infections has become so clear that it is being proposed that biofilms are part of the pathogenic mix or “pea soup” that cause most or all chronic “autoimmune” and inflammatory diseases.
The Marshall hypothesis suggests that chronic inflammatory diseases result from infection with a large microbiota of chronic biofilm and L-form bacteria (collectively called Th1 pathogens. Th1 describes their ability to activate the cellular immune system as opposed to the antibody based immune system). It has been suggested that the bacteria in chronic inflammatory disease states have all developed ways to evade the immune system and to persist as chronic forms that the body is unable to eliminate naturally.