Exploring chronic disease
26 May 2008
As humans, our environment consistently exposes us to a variety of dangers. Tornadoes, lightning, flooding and hurricanes can all hamper our survival. Not to mention the fact that most of us can encounter swerving cars or ill-intentioned people at any given moment.
Thousands of years ago, humans realized that they could better survive a dangerous world if they formed into communities, particularly communities consisting of people with different talents. They realized that a community is far more likely to survive through division of labor– one person makes food, another gathers resources, still another protects the community against invaders. Working together in this manner requires communication and cooperation.
Inhabitants of a community live in close proximity and create various forms of shelter in order to protect themselves from external threats. We build houses that protect our families and larger buildings that protect the entire community. Grouping together inside places of shelter is a logical way to enhance survival.
With the above in mind, it should come as no surprise that the pathogens we harbor are seldom found as single entities. Although the pathogens that cause acute infection are generally free-floating bacteria – also referred to as planktonic bacteria – those chronic bacterial forms that stick around for decades long ago evolved ways to join together into communities. Why? Because by doing so, they are better able to combat the cells of our immune system bent upon destroying them.
It turns out that a vast number of the pathogens we harbor are grouped into communities called biofilms. In an article titled “Bacterial Biofilms: A Common Cause of Persistent Infections,” JW Costerton at the Center for Biofilm Engineering in Montana defines a bacterial biofilm as “a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface.” In layman’s terms, that means that bacteria can join together on essentially any surface and start to form a protective matrix around their group. The matrix is made of polymers – substances composed of molecules with repeating structural units that are connected by chemical bonds.
According to the Center for Biofilm Engineering at Montana State University, biofilms form when bacteria adhere to surfaces in aqueous environments and begin to excrete a slimy, glue-like substance that can anchor them to all kinds of material – such as metals, plastics, soil particles, medical implant materials and, most significantly, human or animal tissue. The first bacterial colonists to adhere to a surface initially do so by inducing weak, reversible bonds called van der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion molecules, proteins on their surfaces that bind other cells in a process called cell adhesion.
These bacterial pioneers facilitate the arrival of other pathogens by providing more diverse adhesion sites. They also begin to build the matrix that holds the biofilm together. If there are species that are unable to attach to a surface on their own, they are often able to anchor themselves to the matrix or directly to earlier colonists.
During colonization, things start to get interesting. Multiple studies have shown that during the time a biofilm is being created, the pathogens inside it can communicate with each other thanks to a phenomenon called quorum sensing. Although the mechanisms behind quorum sensing are not fully understood, the phenomenon allows a single-celled bacterium to perceive how many other bacteria are in close proximity. If a bacterium can sense that it is surrounded by a dense population of other pathogens, it is more inclined to join them and contribute to the formation of a biofilm.
Bacteria that engage in quorum sensing communicate their presence by emitting chemical messages that their fellow infectious agents are able to recognize. When the messages grow strong enough, the bacteria respond en masse, behaving as a group. Quorum sensing can occur within a single bacterial species as well as between diverse species, and can regulate a host of different processes, essentially serving as a simple communication network. A variety of different molecules can be used as signals.
“Disease-causing bacteria talk to each other with a chemical vocabulary,” says Doug Hibbins of Princeton University. A graduate student in the lab of Princeton University microbiologist Dr. Bonnie Bassler, Hibbins was part of a research effort which shed light on how the bacteria that cause cholera form biofilms and communicate via quorum sensing.
“Forming a biofilm is one of the crucial steps in cholera’s progression,” states Bassler. “They [bacteria] cover themselves in a sort of goop that’s a shield against antibiotics, allowing them to grow rapidly. When they sense there are enough of them, they try to leave the body.”
Although cholera bacteria use the intestines as a breeding ground, after enough biofilms have formed, planktonic bacteria inside the biofilm seek to leave the body in order to infect a new host. It didn’t take long for Bassler and team to realize that the bacteria inside cholera biofilms must signal each other in order to communicate that it’s time for the colony to stop reproducing and focus instead on leaving the body.
“We generically understood that bacteria talk to each other with quorum sensing, but we didn’t know the specific chemical words that cholera uses,” Bassler said.
Then Higgins isolated the CAI-1 – a chemical which occurs naturally in cholera. Another graduate student figured out how to make the molecule in the laboratory. By moderating the level of CAI-1 in contact with cholera bacteria, Higgins was successfully able to chemically control cholera’s behavior in lab tests. His team eventually confirmed that when CAI-1 is absent, cholera bacteria attach in biofilms to their current host. But when the bacteria detect enough of the chemical, they stop making biofilms and releasing toxins, perceiving that it is time to leave the body instead. Thus, CAI-1 may very well be the single molecule that allow the bacteria inside a cholera biofilm to communicate. Although it is likely that the bacteria in a cholera biofilm may communicate with other signals besides CAI-1, the study is a good example of the fact that signaling molecules serve a key role in determining the state of a biofilm.
Similarly, researchers at the University of Iowa (several of whom are now at the University of Washington) have spent the last decade identifying the molecules that allow the bacterial species P. aeruginosa to form biofilms in the lungs of patients with cystic fibrosis. Although the P. auruginosa isolated from the lungs of patients with cystic fibrosis looks like a biofilm and acts like a biofilm, up until recently, there were no objective tests available to confirm that the bacterial species did indeed form biofilms in the lungs of patients with the disease, nor was there a way to tell what proportion of P. aeruginosa in the lungs were actually in biofilm mode.
“We needed a way to show that the P. auruginosa in cystic fibrosis lungs was communicating like a biofilm. That could tell us about the P. auruginosalifestyle,” said Pradeep Singh, M.D., a lead author on the study who is now at the University of Washington.
Singh and his colleagues finally discovered that P. aeruginosa uses one of two particular quorum-sensing molecules to initiate the formation of biofilms. In November 1999, his research team screened the entire bacterial genome, identifying 39 genes that are strongly controlled by the quorum-sensing system.
In a 2000 study published in Nature, Singh and colleagues developed a sensitive test which shows P. auruginosa from cystic fibrosis lungs produces the telltale, quorum-sensing molecules that are the signals for biofilm formation.
It turns out that P. aerugnosa secretes two signaling molecules, one that is long, and another that is short. Using the new test, the team was able to show that planktonic forms of P. aeruginosa produce more long signaling molecules. Alternately, when they tested the P. aeruginosa strains isolated from the lungs of patients with cystic fibrosis (which were in biofilm form), all of the strains produced the signaling molecules, but in the opposite ratio – more short than long.
Interestingly, when the biofilm strains of P. aeruginosa were separated in broth into individual bacterial forms, they reverted to producing more long signal molecules than short ones. Does this mean that a change in signaling molecular length can indicate whether bacteria remain as planktonic forms or develop into biofilms?
To find out, the team took the bacteria from the broth and made them grow as a biofilm again. Sure enough, those strains of bacteria in biofilm form produced more short signal molecules than long.
“The fact that the P. aeruginosa in [the lungs of cystic fibrosis patients] is making the signals in the ratios that we see tells us that there is a biofilm and that most of the P. aeruginosa in the lung is in the biofilm state,” states Greenberg, another member of the research team. He believes that the findings allow for a clear biochemical definition of whether bacteria are in a biofilm. Techniques similar to those used by his group will likely be used to determine the properties of other biofilm signaling molecules.
Once colonization has begun, the biofilm grows through a combination of cell division and recruitment. The final stage of biofilm formation is known as development and is the stage in which the biofilm is established and may only change in shape and size. This development of a biofilm allows for the cells inside to become more resistant to antibiotics administered in a standard fashion. In fact, depending on the organism and type of antimicrobial and experimental system, biofilm bacteria can be up to a thousand times more resistant to antimicrobial stress than free-swimming bacteria of the same species.
Biofilms grow slowly, in diverse locations, and biofilm infections are often slow to produce overt symptoms. However, biofilm bacteria can move in numerous ways that allow them to easily infect new tissues. Biofilms may move collectively, by rippling or rolling across the surface, or by detaching in clumps. Sometimes, in a dispersal strategy referred to as “swarming/seeding”, a biofilm colony differentiates to form an outer “wall” of stationary bacteria, while the inner region of the biofilm “liquefies”, allowing planktonic cells to “swim” out of the biofilm and leave behind a hollow mound.
Research on the molecular and genetic basis of biofilm development has made it clear that when cells switch from planktonic to community mode, they also undergo a shift in behavior that involves alterations in the activity of numerous genes. There is evidence that specific genes must be transcribed during the attachment phase of biofilm development. In many cases, the activation of these genes is required for synthesis of the extracellular matrix that protects the pathogens inside.
According to Costerton, the genes that allow a biofilm to develop are activated after enough cells attach to a solid surface. “Thus, it appears that attachment itself is what stimulates synthesis of the extracellular matrix in which the sessile bacteria are embedded,” states the molecular biologist. “This notion– that bacteria have a sense of touch that enables detection of a surface and the expression of specific genes– is in itself an exciting area of research…”
Certain characteristics may also facilitate the ability of some bacteria to form biofilms. Scientists at the Department of Microbiology and Molecular Genetics, Harvard Medical School, performed a study in which they created a “mutant” form of the bacterial species P. aeguinosa (PA). The mutants lacked genes that code for hair-like appendages called pili. Interestingly, the mutants were unable to form biofilms. Since the pili of PA are involved in a type of surface-associated motility called twitching, the team hypothesized this twitching might be required for the aggregation of cells into the microcolonies that subsequently form a stable biofilm.
Once a biofilm has officially formed, it often contains channels in which nutrients can circulate. Cells in different regions of a biofilm also exhibit different patterns of gene expression. Because biofilms often develop their own metabolism, they are sometimes compared to the tissues of higher organisms, in which closely packed cells work together and create a network in which minerals can flow.
“There is a perception that single-celled organisms are asocial, but that is misguided,” said Andre Levchenko, assistant professor of biomedical engineering in Johns Hopkins University’s Whiting School of Engineering and an affiliate of the University’s Institute for NanoBioTechnology. “When bacteria are under stress—which is the story of their lives—they team up and form this collective called a biofilm. If you look at naturally occurring biofilms, they have very complicated architecture. They are like cities with channels for nutrients to go in and waste to go out.”
Understanding how such cooperation among pathogens evolves and is maintained represents one of evolutionary biology’s thorniest problems. This stems from the reality that, in nature, freeloading cheats inevitably evolve to exploit any cooperative group that doesn’t defend itself, leading to the breakdown of cooperation. So what causes the bacteria in a biofilm to contribute to and share resources rather than steal them? Recently, Dr. Michael Brockhurst of the University of Liverpool and colleagues at the Université Montpellier and the University of Oxford conducted several studies in an effort to understand why the bacteria in a biofilm cooperate and share resources rather than horde them.
The team took a closer look at P. fluorescens biofilms, which are formed when individual cells overproduce a polymer that sticks the cells together, allowing the colonization of liquid surfaces. While production of the polymer is metabolically costly to individual cells, the biofilm group benefits from the increased access to oxygen that surface colonization provides. Yet, evolutionarily speaking, such a setup allows possible “cheaters” to enter the biofilm. Such cheats can take advantage of the protective matrix while failing to contribute energy to actually building the matrix. If too many “cheaters” enter a biofilm, it will weaken and eventually break apart.
After several years of study, Brockhurst and team realized that the short-term evolution of diversity within a biofilm is a major factor in how successfully its members cooperate. The team found that once inside a biofilm, P. fluorescensdifferentiates into various forms, each of which uses different nutrient resources. The fact that these “diverse cooperators” don’t all compete for the same chemicals and nutrients substantially reduces competition for resources within the biofilm.
When the team manipulated diversity within experimental biofilms, they found that diverse biofilms contained fewer “cheaters” and produced larger groups than non-diverse biofilms.
Similarly, this year, researchers from Johns Hopkins; Virginia Tech; the University of California, San Diego; and Lund University in Sweden recently released the results of a study which found that once bacteria cooperate and form a biofilm, packing tightly together further enhances their survival.
The team created a new device in order to observe the behavior of E. coli bacteria forced to grow in the cramped conditions. The device, which allows scientists to use extremely small volumes of cells in solution, contains a series of tiny chambers of various shapes and sizes that keep the bacteria uniformly suspended in a culture medium.
Not surprisingly, the cramped bacteria in the device began to form a biofilm. The team captured the development of the biofilm on video, and were able to observe the gradual self-organization and eventual construction of bacterial biofilms over a 24-hour period.
First, Andre Levchenko and Hojung Cho of Johns Hopkins recorded the behavior of single layers of E. coli cells using real-time microscopy. “We were surprised to find that cells growing in chambers of all sorts of shapes gradually organized themselves into highly regular structures,” Levchenko said.
Further observations using microscopy revealed that the longer the packed cell population resided in the chambers, the more ordered the biofilm structure became. As the cells in the biofilm became more ordered and tightly packed, the biofilm became harder and harder to penetrate.
Levchenko also noted that rod-shaped E. colithat were too short or too long typically did not organize well into the dense, circular main hub of the biofilm. Instead, the bacteria of odd shapes or highly disordered groups of cells were found on the edges of the biofilm, where they formed sharp corners.
Researchers often note that, once biofilms are established, planktonic bacteria may periodically leave the biofilm on their own. When they do, they can rapidly multiply and disperse.
According to Costerton, there is a natural pattern of programmed detachment of planktonic cells from biofilms. This means that biofilms can act as what Costerton refers to as “niduses” of acute infection. Because the bacteria in a biofilm are protected by a matrix, the host immune system is less likely to mount a response to their presence.
But if planktonic bacteria are periodically released from the biofilms, each time single bacterial forms enter the tissues, the immune system suddenly becomes aware of their presence. It may proceed to mount an inflammatory response that leads to heightened symptoms. Thus, the periodic release of planktonic bacteria from some biofilms may be what causes many chronic relapsing infections.
As Matthew R. Parsek of Northwestern University describes in a 2003 paper in the Annual Review of Microbiology, any pathogen that survives in a chronic form benefits by keeping the host alive. After all, if a chronic bacterial form simply kills its host, it will no longer have a place to live. So according to Parsek, chronic infection often results in a “disease stalemate” where bacteria of moderate virulence are somewhat contained by the defenses of the host. The infectious agents never actually kill the host, but the host is never able to fully kill the invading pathogens either.
Parsek believes that the optimal way for bacteria to survive under such circumstances is in a biofilm, stating that “Increasing evidence suggests that the biofilm mode of growth may play a key role in both of these adaptations. Biofilm growth increases the resistance of bacteria to killing and may make organisms less conspicuous to the immune system… ultimately this moderation of virulence may serve the bacteria’s interest by increasing the longevity of the host.”
Perhaps because many biofilms are sufficiently thick to be visible to the naked eye, the microbial communities were among the first to be studied by early microbiologists. Anton van Leeuwenhoek scraped the plaque biofilm from his teeth and observed what he described as the “animalculi” inside them under his primitive microscope. However, according to Costerton and team at the Center for Biofilm Research at Montana State University, it was not until the 1970s that scientists began to appreciate that bacteria in the biofilm mode of existence constitute such a major component of the bacterial biomass in most environments. Then, it was not until the 1980s and 1990s that scientists truly began to understand how elaborately organized a bacterial biofilm community can be.
As Robert Kolter, professor of microbiology and molecular genetics at Harvard Medical School, and one of the first scientists to study how biofilms developstates, “At first, however, studying biofilms was a radical departure from previous work.”
Like most microbial geneticists, Kolter had been trained in the tradition dating back to Robert Koch and Louis Pasteur, namely that bacteriology is best conducted by studying pure strains of planktonic bacteria. “While this was a tremendous advance for modern microbiology, it also distracted microbiologists from a more organismic view of bacteria, Kolter adds, “Certainly we felt that pure, planktonic cultures were the only way to work. Yet in nature bacteria don’t live like that,” he says. “In fact, most of them occur in mixed, surface-dwelling communities.”
Although research on biofilms has surged over the past few decades, the majority of biofilm research to date has focused on external biofilms, or those that form on various surfaces in our natural environment.
Over the past years, as scientists developed better tools to analyze external biofilms, they quickly discovered that biofilms can cause a wide range of problems in industrial environments. For example, biofilms can develop on the interiors of pipes, which can lead to clogging and corrosion. Biofilms on floors and counters can make sanitation difficult in food preparation areas.
Since biofilms have the ability to clog pipes, watersheds, storage areas, and contaminate food products, large companies with facilities that are negatively impacted by their presence have naturally taken an interest in supporting biofilm research, particularly research that specifies how biofilms can be eliminated.
This means that many recent advances in biofilm detection have resulted from collaborations between microbial ecologists, environmental engineers, and mathematicians. This research has generated new analytical tools that help scientists identify biofilms.
For example, the Canadian company FAS International Ltd. has just createdan endoluminal brush, which will be launched this spring. Physicians can use the brush to obtain samples from the interior of catheters. Samples taken from catheters can be sent to a lab, where researchers determine if biofilms are present in the sample. If biofilms are detected, the catheter is immediately replaced, since the insertion of catheters with biofilms can cause the patient to suffer from numerous infections, some of which are potentially life threatening.
Scientists now realize that biofilms are not just composed of bacteria. Nearly every species of microorganism – including viruses, fungi, and Archaea – have mechanisms by which they can adhere to surfaces and to each other. Furthermore, it is now understood that biofilms are extremely diverse. For example, upward of 300 different species of bacteria can inhabit the biofilms that form dental plaque.
Furthermore, biofilms have been found literally everywhere in nature, to the point where any mainstream microbiologist would acknowledge that their presence is ubiquitous. They can be found on rocks and pebbles at the bottom of most streams or rivers and often form on the surface of stagnant pools of water. In fact, biofilms are important components of food chains in rivers and streams and are grazed upon by the aquatic invertebrates upon which many fish feed. Biofilms even grow in the hot, acidic pools at Yellowstone National Park and on glaciers in Antarctica.
It is also now understood that the biofilm mode of existence has been around for millenia. For example, filamentous biofilms have been identified in the 3.2-billion-year-old deep-sea hydrothermal rocks of the Pilbara Craton, Australia. According to a 2004 article in Nature Reviews Microbiology, “Biofilm formation appears early in the fossil record (approximately 3.25 billion years ago) and is common throughout a diverse range of organisms in both the Archaea and Bacteria lineages. It is evident that biofilm formation is an ancient and integral component of the prokaryotic life cycle, and is a key factor for survival in diverse environments.”
The fact that external biofilms are ubiquitous raises the question – if biofilms can form on essentially every surface in our external environments, can they do the same inside the human body? The answer seems to be yes, and over the past few years, research on internal biofilms has finally started to pick up pace. After all, it’s easy for biofilm researchers to see that the human body, with its wide range of moist surfaces and mucosal tissue, is an excellent place for biofilms to thrive. Not to mention the fact that those bacteria which join a biofilm have a significantly greater chance of evading the battery of immune system cells that more easily attack planktonic forms.
Many would argue that research on internal biofilms has been largely neglected, despite the fact that bacterial biofilms seem to have great potential for causing human disease.
Paul Stoodley of the Center for Biofilm Engineering at Montana State University, attributes much of the lag in studying biofilms to the difficulties of working with heterogeneous biofilms compared with homogeneous planktonic populations. In a 2004 paper in Nature Reviews, the molecular biologist describes many reasons why biofilms are extremely difficult to culture, such as the fact that the diffusion of liquid through a biofilm and the fluid forces acting on a biofilm must be carefully calculated if it is to be cultured correctly. According to Stoodley, the need to master such difficult laboratory techniques has deterred many scientists from attempting to work with biofilms. 
Also, since much of the technology needed to detect internal biofilms was created at the same time as the sequencing of the human genome, interest in biofilm bacteria, and the research grants that would accompany such interest, have been largely diverted to projects with a decidedly genetic focus. However, since genetic research has failed to uncover the cause of any of the common chronic diseases, biofilms are finally – just over the past few years – being studied more intensely, and being given the credit they deserve as serious infectious entities, capable of causing a wide array of chronic illnesses.
In just a short period of time, researchers studying internal biofilms have already pegged them as the cause of numerous chronic infections and diseases, and the list of illnesses attributed to these bacterial colonies continues to grow rapidly.
According to a recent public statement from the National Institutes of Health, more than 65% of all microbial infections are caused by biofilms. This number might seem high, but according to Kim Lewis of the Department of Chemical and Biological Engineering at Tufts University, “If one recalls that such common infections as urinary tract infections (caused by E. coli and other pathogens), catheter infections (caused by Staphylococcus aureus and other gram-positive pathogens), child middle-ear infections (caused by Haemophilus influenzae, for example), common dental plaque formation, and gingivitis, all of which are caused by biofilms, are hard to treat or frequently relapsing, this figure appears realistic.”
As Lewis mentions, perhaps the most well-studied biofilms are those that make up what is commonly referred to as dental plaque. “Plaque is a biofilm on the surfaces of the teeth,” states Parsek. “This accumulation of microorganisms subject the teeth and gingival tissues to high concentrations of bacterial metabolites which results in dental disease.”
It has also recently been shown that biofilms are present on the removed tissue of 80% of patients undergoing surgery for chronic sinusitis. According to Parsek, biofilms may also cause osteomyelitis, a disease in which the bones and bone marrow become infected. This is supported by the fact that microscopy studies have shown biofilm formation on infected bone surfaces from humans and experimental animal models. Parsek also implicates biofilms in chronic prostatitis since microscopy studies have also documented biofilms on the surface of the prostatic duct. Microbes that colonize vaginal tissue and tampon fibers can also form into biofilms, causing inflammation and disease such as Toxic Shock Syndrome.
Biofilms also cause the formation of kidney stones. The stones cause disease by obstructing urine flow and by producing inflammation and recurrent infection that can lead to kidney failure. Approximately 15%–20% of kidney stones occur in the setting of urinary tract infection. According to Parsek, these stones are produced by the interplay between infecting bacteria and mineral substrates derived from the urine. This interaction results in a complex biofilm composed of bacteria, bacterial exoproducts, and mineralized stone material.
Perhaps the first hint of the role of bacteria in these stones came in 1938 when Hellstrom examined stones passed by his patients and found bacteria embedded deep inside them. Microscopic analysis of stones removed from infected patients has revealed features that characterize biofilm growth. For one thing, bacteria on the surface and inside the stones are organized in microcolonies and surrounded by a matrix composed of crystallized (struvite) minerals.
Then there’s endocarditis, a disease that involves inflammation of the inner layers of the heart. The primary infectious lesion in endocarditis is a complex biofilm composed of both bacterial and host components that is located on a cardiac valve. This biofilm, known as a vegetation, causes disease by three basic mechanisms. First, the vegetation physically disrupts valve function, causing leakage when the valve is closed and inducing turbulence and diminished flow when the valve is open. Second, the vegetation provides a source for near-continuous infection of the bloodstream that persists even during antibiotic treatment. This causes recurrent fever, chronic systemic inflammation, and other infections. Third, pieces of the infected vegetation can break off and be carried to a terminal point in the circulation where they block the flow of blood (a process known as embolization). The brain, kidney, and extremities are particularly vulnerable to the effects of embolization.
A variety of pathogenic biofims are also commonly found on medical devices such as joint prostheses and heart valves. According to Parsek, electron microscopy of the surfaces of medical devices that have been foci of device-related infections shows the presence of large numbers of slime-encased bacteria. Tissues taken from non-device-related chronic infections also show the presence of biofilm bacteria surrounded by an exopolysaccharide matrix. These biofilm infections may be caused by a single species or by a mixture of species of bacteria or fungi.
According to Dr. Patel of the Mayo Clinic, individuals with prosthetic joints are often oblivious to the fact that their prosthetic joints harbor biofilm infections.
“When people think of infection, they may think of fever or pus coming out of a wound,” explains Dr. Patel. “However, this is not the case with prosthetic joint infection. Patients will often experience pain, but not other symptoms usually associated with infection. Often what happens is that the bacteria that cause infection on prosthetic joints are the same as bacteria that live harmlessly on our skin. However, on a prosthetic joint they can stick, grow and cause problems over the long term. Many of these bacteria would not infect the joint were it not for the prosthesis.”
Biofilms also cause Leptospirosis, a serious but neglected emerging disease that infects humans through contaminated water. New research published in the May issue of the journal Microbiology shows for the first time how bacteria that cause the disease survive in the environment.
Leptospirosis is a major public health problem in southeast Asia and South America, with over 500,000 severe cases every year. Between 5% and 20% of these cases are fatal. Rats and other mammals carry the disease-causing pathogen Leptospira interrogans in their kidneys. When they urinate, they contaminate surface water with the bacteria, which can survive in the environment for long periods.
“This led us to see if the bacteria build a protective casing around themselves for protection,” said Professor Mathieu Picardeau from the Institut Pasteur in Paris, France. 
Previously, scientists believed the bacteria were planktonic. But Professor Picardeau and his team have shown that L. interrogans can make biofilms, which could be one of the main factors controlling survival and disease transmission. “90% of the species of Leptospira we tested could form biofilms. It takes L. interrogans an average of 20 days to make a biofilm,” says Picardeau.
Biofilms have also been implicated in a wide array of veterinary diseases. For example, researchers at the Virginia-Maryland Regional College of Veterinary Medicine at Virginia Tech were just awarded a grant from the United States Department of Agriculture to study the role biofilms play in the development of Bovine Respiratory Disease Complex (BRDC). If biofilms play a role in bovine respiratory disease, it’s likely only a matter of time before they will be established as a cause of human respiratory diseases as well.
As mentioned previously, infection by the bacterium Pseudomonas aeruginosa (P. aeruginosa) is the main cause of death among patients with cystic fibrosis. Pseudomonas is able to set up permanent residence in the lungs of patients with cystic fibrosis where, if you ask most mainstream researchers, it is impossible to kill. Eventually, chronic inflammation produced by the immune system in response to Pseudomonas destroys the lung and causes respiratory failure. In the permanent infection phase, P. aeruginosa biofilms are thought to be present in the airway, although much about the infection pathogenesis remains unclear.
Cystic fibrosis is caused by mutations in the proteins of channels that regulates chloride. How abnormal chloride channel protein leads to biofilm infection remains hotly debated. It is clear, however, that cystic fibrosis patients manifest some kind of host-defense defect localized to the airway surface. Somehow this leads to a debilitating biofilm infection.
Because internal pathogenic biofilm research comprises such a new field of study, the infections described above almost certainly represent just the tip of the iceberg when it comes to the number of chronic diseases and infections currently caused by biofilms.
For example, it wasn’t until July of 2006 that researchers realized that the majority of ear infections are caused by biofilm bacteria. These infections, which can be either acute or chronic, are referred to collectively as otitis media (OM). They are the most common illness for which children visit a physician, receive antibiotics, or undergo surgery in the United States.
There are two subtypes of chronic OM. Recurrent OM (ROM) is diagnosed when children suffer repeated infections over a span of time and during which clinical evidence of the disease resolves between episodes. Chronic OM with effusion is diagnosed when children have persistent fluid in the ears that lasts for months in the absence of any other symptoms except conductive hearing loss.
It took over ten years for researchers to realize that otitis media is caused by biofilms. Finally, in 2002, Drs. Ehrlich and J. Christopher Post, an Allegheny General Hospital pediatric ear specialist and medical director of the Center for Genomic Sciences, published the first animal evidence of biofilms in the middle ear in the Journal of the American Medical Association, setting the stage for further clinical investigation.
In a subsequent study, Ehrlich and Post obtained middle ear mucosa – or membrane tissue – biopsies from children undergoing a procedure for otitis. The team gathered uninfected mucosal biopsies from children and adults undergoing cochlear implantation as a control.
Using advanced confocal laser scanning microscopy, Luanne Hall Stoodley, Ph.D. and her ASRI colleagues obtained three dimensional images of the biopsies and evaluated them for biofilm morphology using generic stains and species-specific probes for Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis. 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 46/50 children (92%) with both forms of otitis. Biofilms were not observed in eight control middle ear mucosa specimens obtained from cochlear implant patients.
In fact, all of the children in the study who suffered from chronic otitis media tested positive for biofilms in the middle ear, even those who were asymptomatic, causing Erlich to conclude that, “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.”
He 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.”
The emerging biofilm paradigm of chronic disease refers to a new movement in which researchers such as Ehrlich are calling for a tremendous shift in the way the medical community views bacterial biofilms. 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 Pathogenesis is an important part of this paradigm shift.
It was also just last year that researchers realized that biofilms cause most infections associated with contact lens use. In 2006, Bausch & Lomb withdrew its ReNu with MoistureLoc contact lens solution because a high proportion of corneal infections were associated with it. It wasn’t long before researchers at the University Hospitals Case Medical Center found that the infections were caused by biofilms. 
“Once they live in that type of state [a biofilm], the cells become resistant to lens solutions and immune to the body’s own defense system,” said Mahmoud A. Ghannoum, Ph.D, senior investigator of the study. “This study should alert contact lens wearers to the importance of proper care for contact lenses to protect against potentially virulent eye infections,” he said.
It turns out that the biofilms detected by Ghannoum and team were composed of fungi, particularly a species called Fusarium. His team also discovered that the strain of fungus (with the catchy name, ATCC 36031) used for testing the effectiveness of lens care solutions is a strain that does not produce biofilms as the clinical fungal strains do. ReNu contact solution, therefore, was effective in the laboratory, but failed when faced with strains in real-world situations.
Unfortunately, Ghannoum and team were not able to create a method to target and destroy the fungal biofilms that plague users of ReNu and some other contact lens solutions.
Then there’s Dr. Randall Wolcott who just recently discovered and confirmed that the sludge covering diabetic wounds is largely made up of biofilms. Whereas before Wolcott’s work such limbs generally had to be amputated, now that they have been correctly linked to biofilms, measures such as those described in thisinterview can be taken to stop the spread of infection and save the limb. Wolcott has finally been given a grant by the National Institutes of Health to further study chronic biofilms and wound development.
Dr. Garth James and the Medical Biofilm Laboratory team at Montana State University are also researching wounds and biofilms. Their latest article and an image showing wound biofilm was featured on the cover of the January-February 2008 issue of Wound Repair and Regeneration.
In just a few short years, the potential of biofilms to cause debilitating chronic infections has become so clear that there is little doubt that biofilms are part of the pathogenic mix or “pea soup” that cause most or all chronic “autoimmune” and inflammatory diseases.
In fact, thanks, in large part, to the research of biomedical researcher Dr. Trevor Marshall, it is now increasingly understood that chronic inflammatory diseases result from infection with a large microbiota of chronic biofilm and L-form bacteria (collectively called the Th1 pathogens). The microbiota is thought to be comprised of numerous bacterial species, some of which have yet to be discovered. However, most of the pathogens that cause inflammatory disease have one thing in common – they have all developed ways to evade the immune system and persist as chronic forms that the body is unable to eliminate naturally.
Some L-form bacteria are able to evade the immune system because, long ago, they evolved the ability to reside inside macrophages, the very white bloods cells of the immune system that are supposed to kill invading pathogens. Upon formation, L-form bacteria also lose their cell walls, which makes them impervious to components of the immune response that detect invading pathogens by identifying the proteins on their cell walls. The fact that L-form bacteria lack cell walls also means that the beta-lactam antibiotics, which work by targeting the bacterial cell wall, are completely ineffective at killing them.
Clearly, transforming into the L-form offers any pathogen a survival advantage. But among those pathogens not in an L-form state, joining a biofilm is just as likely to enhance their ability to evade the immune system. Once enough chronic pathogens have grouped together and formed a stable community with a strong protective matrix, they are likely able to reside in any area of the body, causing the host to suffer from chronic symptoms that are both mental and physical in nature.
Biofilm researchers will also tell you that, not surprisingly, biofilms form with greater ease in an immunocompromised host. Marshall’s research has made it clear that many of the Th1 pathogens are capable of creating substances that bind and inactivate the Vitamin D Receptor – a fundamental receptor of the body that controls the activity of the innate immune system, or the body’s first line of defense against intracellular infection.
Thus, as patients accumulate a greater number of the Th1 pathogens, more and more of the chronic bacterial forms create substances capable of disabling the VDR. This causes a snowball effect, in which the patient becomes increasingly immunocompromised as they acquire a larger bacterial load.
For one thing, it’s possible that many of the bacteria that survive inside biofilms are capable of creating VDR blocking substances. Thus, the formation of biofilms may contribute to immune dysfunction. Conversely, as patients acquire L-form bacteria and other persistent bacterial forms capable of creating VDR-blocking substances, it becomes exceptionally easy for biofilms to form on any tissue surface of the human body.
Thus, patients who begin to acquire L-form bacteria almost always fall victim to biofilm infections as well, since it is all too easy for pathogens to group together into a biofilm when the immune system isn’t working up to par.
To date, there is also no strict criteria that separate L-form bacteria from biofilm bacteria or any other chronic pathogenic forms. This means that L-form bacteria may also form into biofilms, and by doing so enter a mode of survival that makes them truly impervious to the immune system. Some L-form bacteria may not form complete biofilms, yet may still possess the ability to surround themselves in a protective matrix. Under these circumstances one might say they are in a “biofilm-like” state.
Marshall often refers to the pathogens that cause inflammatory disease as an intraphgocytic, metagenomic microbiota of bacteria, terms which suggest that most chronic bacterial forms possess properties of both L-form and biofilm bacteria. Intraphagocytic refers to the fact that the pathogens can be found inside the cells of the immune system. The term metagenomic indicates that there are a tremendous number of different species of these chronic bacterial forms. Finally, microbiota refers to the fact that biofilm communities sustain their pathogenic activity.
For example, when observed under a darkfield microscope, L-form bacteria are often encased in protective biofilm sheaths. If the blood containing the pathogens are aged overnight, the bacterial colonies reach a point where they expand and burst out of the cell, causing the cell to burst as well. Then they extend as huge, long biofilm tubules, which are presumably helping the pathogens spread to other cells. The tubules also help spread bacterial DNA to neighboring cells.
Clearly, there is a great need for more research on how different chronic bacterial forms interact. To date, L-form researchers have essentially focused soley on the L-form, while failing to investigate how frequently the wall-less pathogens form into biofilms or become parts of biofilm communities together with bacteria with cell walls. Conversely, most biofilm researchers are intently studying the biofilm mode of growth without considering the presence of L-form bacteria. So, it will likely take several years before we will be better able to understand probable overlaps between the lifestyles of L-form and biofilm bacteria.
Anyone who is skeptical about the fact that biofilms likely form a large percentage of the microbiota that cause inflammatory disease should consider many of the recent studies that have linked established biofilm infections to a higher risk for multiple forms of chronic inflammatory disease. Take, for example, studies that have found a link between periodontal disease and several major inflammatory conditions. A 1989 article published in British Medical Journal showed a correlation between dental disease and systemic disease (stroke, heart disease, diabetes). After correcting for age, exercise, diet, smoking, weight, blood cholesterol level, alcohol use and health care, people who had periodontal disease had a significantly higher incidence of heart disease, stroke and premature death. More recently, these results were confirmed in studies in the United States, Canada, Great Britain, Sweden, and Germany. The effects are striking. For example, researchers from the Canadian Health Bureau found that people with periodontal disease had a two times higher risk of dying from cardiovascular disease.
Since we know that periodontal disease is caused by biofilm bacteria, the most logical explanation for the fact that people with dental problems are much more likely to suffer from heart disease and stroke is that the biofilms in their mouths have gradually spread to the moist surfaces of their circulatory systems. Or perhaps if the bacteria in periodontal biofilms create VDR binding substances, their ability to slow innate immune function allows new biofilms (and L-form bacteria as well) to more easily form and infect the heart and blood vessels. Conversely, systemic infection with VDR blocking biofilm bacteria is also likely to weaken immune defenses in the gums and facilitate periodontal disease.
In fact, it appears that biofilm bacteria in the mouth also facilitate the formation of biofilm and L-form bacteria in the brain. Just last year, researchers at Vasant Hirani at University College London released the results of a study which found that elderly people who have lost their teeth are at more than three-fold greater risk of memory problems and dementia.
At the moment, Autoimmunity Research Foundation does not have the resources to culture biofilms from patients on the treatment and, even if they did, current methods for culturing internal biofilms remain unreliable. According to Stoodley, “The lack of standard methods for growing, quantifying and testing biofilms in continuous culture results in incalculable variability between laboratory systems. Biofilm microbiology is complex and not well represented by flask cultures. Although homogeneity allows statistical enumeration, the extent to which it reflects the real, less orderly world is questionable.”
As discussed thus far, biofilms form spontaneously as bacteria inside the human body group together. Yet people can also ingest biofilms by eating contaminated food.
According to researchers at the University of Guelph in Ontario Canada, it is increasingly suspected that biofilms play an important role in contamination of meat during processing and packaging. The group warns that greater action must be taken to reduce the presence of food-borne pathogens like Escherichia coli and Listeria monocytogenes and spoilage microorganisms such as thePseudomonas species (all of which form biofilms) throughout the food processing chain to ensure the safety and shelf-life of the product. Most of these microorganisms are ubiquitous in the environment or brought into processing facilities through healthy animal carriers.
Hans Blaschek of the University of Illinois has discovered that biofilms form on much of the other food products we consume as well.
“If you could see a piece of celery that’s been magnified 10,000 times, you’d know what the scientists fighting foodborne pathogens are up against,” says Blaschek.
“It’s like looking at a moonscape, full of craters and crevices. And many of the pathogens that cause foodborne illness, such as Shigella, E. coli,and Listeria, make sticky, sugary biofilms that get down in these crevices, stick like glue, and hang on like crazy.”
According to Blaschek, the problem faced by produce suppliers can be a triple whammy. “If you’re unlucky enough to be dealing with a pathogen–and the pathogen has the additional attribute of being able to form biofilm—and you’re dealing with a food product that’s minimally processed, well, you’re triply unlucky,” the scientist said. “You may be able to scrub the organism off the surface, but the cells in these biofilms are very good at aligning themselves in the subsurface areas of produce.”
Scott Martin, a University of Illinois food science and human nutrition professor agrees, stating,”Once the pathogenic organism gets on the product, no amount of washing will remove it. The microbes attach to the surface of produce in a sticky biofilm, and washing just isn’t very effective.”
Biofilms can even be found in processed water. Just this month, a study was released in which researchers at the Department of Biological Sciences, at Virginia Polytechnic Institute isolated M. avium biofilm from the shower head of a woman with M. avium pulmonary disease. A molecular technique called DNA fingerprinting demonstrated that M. avium isolates from the water were the same forms that were causing the woman’s respiratory illness.
Although the mainstream medical community is rapidly acknowledging the large number of diseases and infections caused by biofilms, most researchers are convinced that biofilms are difficult or impossible to destroy, particularly those cells that form the deeper layers of a thick biofilm. Most papers on biofilms state that they are resistant to antibiotics administered in a standard manner. For example, despite the fact that Ehrlich and team discovered that biofilm bacteria cause otitis media, they are unable to offer an effective solution that would actually allow for the destruction of biofilms in the ear canal. Other teams have also come up short in creating methods to break up the biofilms they implicate as the cause of numerous infections.
This means patients with biofilm infections are generally told by mainstream doctors that they have an untreatable infection. In some cases, a disease-causing biofilm can be cut out of a patient’s tissues, or efforts are made to drain components of the biofilm out of the body. For example, doctors treating otitis media often treats patients with myringotomy, a surgical procedure in which small tubes are placed in the eardrum to continuously drain infectious fluid.
When it comes to administering antibiotics in an effort to target biofilms, one thing is certain. Mainstream researchers have repeatedly tried to kill biofilms by giving patients high, constant doses of antibiotics. Unfortunately, when administered in high doses, the antibiotic may temporarily weaken the biofilm but is incapable of destroying it, as certain cells inevitably persist and allow the biofilm to regenerate.
“You can put a patient on [a high dose] antibiotics, and it may seem that the infection has disappeared,” says Levchenko. “But in a few months, it reappears, and it is usually in an antibiotic-resistant form.”
What the vast majority of researchers working with biofilms fail to realize is that antibiotics are capable of destroying biofilms. The catch is that antibiotics are only effective against biofilms if administered in a very specific manner. Furthermore, only certain antibiotics appear to effectively target biofilms. After decades of research, much of which was derived from molecular modeling data, Marshall was the first to create an antibiotic regimen that appears to effectively target and destroy biofilms. Central to the treatment, which is called the Marshall Protocol, is the fact that biofilms and other Th1 pathogens succumb to specific bacteriostatic antibiotics taken in very low, pulsed doses. It is only when antibiotics are administered in this manner that they appear capable of fully eradicating biofilms.
In a paper entitled “The Riddle of Biofilm Resistance,” Dr. Kim Lewis of Tulane University discusses the mechanisms by which pulsed, low dose antibiotics are able to break up biofilms, while antibiotics administered in a standard manner (high, constant doses) cannot. According to Lewis, the use of pulsed, low-dose antibiotics to target biofilm bacteria is supported by observations she and her colleagues have made in the laboratory.
Some researchers claim that antibiotics cannot penetrate the matrix that surrounds a biofilm. But research by Lewis and other scientists has confirmed that the inability of antibiotics to penetrate the biofilm matrix is much more of an exception than a rule. According to Lewis, “In most cases involving small antimicrobial molecules, the barrier of the polysaccharide matrix should only postpone the death of cells rather than afford useful protection.”
For example, a recent study that used low concentrations of an antibiotic to killP. aeruginosa biofilm bacteria found that the majority of biofilm cells were effectively eliminated by antibiotics in a manner that did not differ much from what is observed when the same antibiotic concentrations are administered to single planktonic cells.
Thus, since antibiotics can generally penetrate biofilms, some other factor is responsible for the fact that they cannot be killed by standard high dose antibiotic therapy. It turns out that after antibiotics are applied to a biofilm, a number of cells called “persisters” are left behind. Persisters are simply cells that are able to survive the first onslaught of antibiotics, and if left unchecked, gradually allow the biofilm to form again. According to Lewis, persister cells form with particular ease in immunocompromised patients because the immune system is unable to help the antibiotic “mop up” all the biofilm cells it has targeted.
“This simple observation suggests a new paradigm for explaining, at least in principle, the phenomenon of biofilm resistance to killing by a wide range of antimicrobials,” states Lewis. “The majority of cells in a biofilm are not necessarily more resistant to killing than planktonic cells and die rapidly when treated with [an antibiotic] that can kill slowly growing cells.”
Thus, a dose of antibiotics – particularly in immunocompromised patients – eradicates most of the biofilm population but leaves a small fraction of surviving persisters behind. Unfortunately, in the same sense that the beta-lactam antibiotics promote the formation of L-form bacteria, persister cells are actually preserved by the presence of an antibiotic that inhibits their growth. Thus, paradoxically, dosing an antibiotic in a constant, high-dose manner (in which the antibiotic is always present) helps persisters persevere.
But in the case of low, pulsed dosing, where an antibiotic is administered, withdrawn, then administered again, the first application of antibiotic will eradicate the bulk of biofilm cells, leaving persister cells behind. Withdrawl of the antibiotic allows the persister population to start growing. Since administration of the antibiotic is temporarily stopped, the survival of persisters is not enhanced. This causes the persister cells to lose their phenotype (their shape and biochemical properties), meaning that they are unable to switch back into biofilm mode. A second application of the antibiotic should then completely eliminate the persister cells, which are still in planktonic mode.
Lewis has found that the feasibility of a pulsed, or cyclical biofilm eradication approach depends on the rate at which persisters lose resistance to killing and regenerate new persisters. It also depends on the ability to manipulate the antibiotic concentration – something that is done quite effectively by patients on the Marshall Protocol who carefully dose their antibiotics at different levels, allowing constant variation in antibiotic concentration. Although Lewis speculates that allowing the concentration of an antibiotic to drop could potentially lead to resistance towards the antibiotic, she is quick to add that if two or more antibiotics are used to target a biofilm at one time, such resistance would not occur. Again, since the Marshall Protocol uses a total of five bacteriostatic antibiotics, usually taken two or three at a time, concerns of resistance are essentially negligible.
“It is entirely possible that successful cases of antimicrobial therapy of biofilm infections result from a fortuitous optimal cycling [pulsed dosing] of an antibiotic concentration that eliminated first the bulk of the biofilm and then the progeny of the persisters that began to divide,” states Lewis.
Lewis’ work has been supported by other research teams. Recently, researchers at the University of Iowa found that subinhibitory (extremely low dose) concentrations of the bacteriostatic antibiotic azithromycin significantly decreased biomass and maximal thickness in both forming and established biofilms. These extremely low concentrations of azithromycin inhibited biofilms in all but the most highly resistant isolates. In contrast, subinhibitory concentrations of gentamicin, which is not a bacteriostatic antibiotic, had no effect on biofilm formation. In fact, biofilms actually became resistant to gentamicin at concentrations far above the minimum inhibitory concentration.
Researchers at Tulane University recently confirmed yet again that low, pulsed dosing is a superior way of targeting treatment-resistant biofilm bacteria. According to the team, who mathematically modeled the action of antibiotics on bacterial biofilms, “Exposing a biofilm to low concentration doses of an antimicrobial agent for longer time is more effective than short time dosing with high antimicrobial agent concentration.”
Similarly, a bioengineer led team at the University of Washington recently created an antibiotic- containing polymer that releases antibiotic slowly onto the surface of hospital devices, such as catheters and prostheses, to reduce the risk of biofilm-related infections.
“Rather than massively dosing the patient with high levels of released antibiotic, this strategy allows the release of extremely low levels of this very potent antibiotic over long periods of time,” explained Buddy Ratner, PhD, Professor and Director of the Engineered Biomaterials Program at the University of Washington, Seattle. “We calculated the amount released at the surface that would kill 100% of the bacteria entering the surface zone.”
When challenged by Dr. Leonard A. Mermel from Brown University School of Medicine on the issue that long-term use of pulsed, low-dose antibiotics might allow for increased resistance on the part of the bacteria being treated, Ratner responded, “Dr. Mermel’s concerns are, in fact, why we developed this system for [antibiotic] release. Bacteria that live through antibiotic dosing can go on to produce resistant strains. If 100% of the bacteria approaching the surface are killed, they can’t produce resistant offspring. The classical physician approach, dosing the patient systemically and heavily to rid the patient of persistent bacteria, can lead to those resistant strains. Our approach releases miniscule doses compared to what a physician would use, but releases the antibiotic where it will be optimally effective and least likely to leave antibiotic-resistant survivors.”
Although taken orally, the MP antibiotics are taken in the same manner as those administered by Ratner and team. Because they too are dosed at optimal times in extremely small doses, the chance that long-term antibiotic use might foster resistant bacteria is again, essentially negligible, especially when multiple antibiotics are typically used.
Key to the ability of the Marshall Protocol to effectively target biofilm bacteria is the fact that the specific pulsed, low-dose bacteriostatic antibiotics used by the treatment are taken in conjunction with a medication called Benicar. Benicar binds and activates the Vitamin D Receptor, displacing bacterial substances and 25-D from the receptor, so that it can once again activate the innate immune system. Benicar is so effective at strengthening the innate immune response that the patient’s own immune system ultimately helps destroy the biofilm weakened by pulsed, low-dose antibiotics.
Thus, it is not enough for patients on the Marshall Protocol to simply take specific pulsed, low-dose antibiotics. The activity of their innate immune system must also be restored so that the cells of the immune system can actively combat biofilm bacteria, the matrix that surrounds them, and persister cells.
How do we know that the Marshall Protocol effectively kills biofilm bacteria? Namely because those patients to reach the later stages of the treatment do not report symptoms associated with established biofilm diseases. Patients on the MP who once suffered from chronic ear infections (OM), chronic sinus infections, or periodontal disease find that such infections resolve over the course of treatment. Furthermore, since we now understand that biofilms almost certainly form a large part of the chronic microbiota of pathogens that cause chronic inflammatory and autoimmune diseases, the fact that patients can use the Marshall Protocol to recover from such illnesses again suggests that the treatment must be effectively allowing them to target and destroy biofilms.
Because all evidence points to the fact that the MP does indeed effectively target biofilm bacteria, it is of utmost importance that people who suffer from any sort of biofilm infection start the treatment. Knowledge of the Marshall Protocol has yet to reach the cystic fibrosis community, but there is great hope that if people with the disease were to start the MP, they could destroy the P. aeruginosabiofilms that cause their untimely deaths. In the same vein, people with a wide range of infections, such as those infected with biofilm during surgery, can likely restore their health with the MP.
It is to be hoped that the clinical data emerging from the Marshall Protocol study site, which shows patients recovering from biofilm-related diseases, will inspire future researchers to invest a great deal of energy into further research aimed at identifying and studying the biofilm bacteria – bacteria that almost certainly form part of the microbiota of pathogens that cause inflammatory disease. In the coming years, as the technology to detect biofilms becomes even more sophisticated, it is almost certain that a great number of biofilms will be officially detected and documented in patients with a vast array of chronic diseases.
Amy Proal graduated from Georgetown University in 2005 with a degree in biology. While at Georgetown, she wrote her senior thesis on Chronic Fatigue Syndrome and the Marshall Protocol.