Bacteriality

Exploring chronic disease

Category: biofilms

Excuse me, my what?!

Duh, Kineosphaeram, one of the over 600 bacterial species that may be living in your mouth or other areas of your body. If you don’t harbor Kineosphaeram, then perhaps your mouth is home to Bergeriella, Buttiauxella, Cedecea, Derxia, Faecalibacterium, Hallella, Mannheimia, Paludibacterm, Ruminococcus, Thermovirga, or Wolinella. The list goes on….

Looks like a few bacteria have visited this mouth.

If these bacterial species sound new to you, it’s because many of them are. Several of the species were just recently named after researchers led by Dr. Mark Stoneking of the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany conducted the first in-depth study of global diversity in the human mouth.[1] The team sequenced and analyzed variations in the bacterial gene encoding 16S rRNA, a component of the bacterial ribosome, in the salivary metagenome (bacterial population) of 120 healthy subjects from six geographic areas. The researchers proceeded to compare the sequences they found with a database of previously categorized 16S rRNA sequences to categorize the types of bacteria present.

These sequences could be assigned to 101 known bacterial genera, of which 39 were not previously reported from the human oral cavity; phylogenetic analysis suggests that an additional 64 unknown genera are present. The results suggest great diversity in the salivary microbiome within and between individuals that until this point had never been realized.

“The healthy human mouth is home to a tremendous variety of microbes including viruses, fungi, protozoa and bacteria,” said Professor William Wade from King’s College London Dental Institute. “The bacteria are the most numerous: there are 100 million in every millilitre of saliva and more than 600 different species in the mouth. Around half of these have yet to be named and we are trying to describe and name the new species.”

Are these bacteria helpful or up to no good? While some may not impact dental health, disease causing bacteria in the mouth are rampant – ranging from species that cause the dental plaque that leads to cavities to forms that weaken the gums or cause bad breath. For decades scientists have advised patients to brush their teeth (don’t forget to scrub for a full three minutes!), floss, and often use a variety of mouthwashes to rid the mouth of as many bacteria as possible. There is little worry that such procedures might kill commensal or “helpful” bacteria in the mouth, probably because most dental bacteria are seen as a menace to to salivary and dental health.

Three of the bacteria identified in the “healthy” subjects in Stoneking’s study are certainly not bacteria anyone wants to be carrying around – Neisseria. Treponema neisseria and Yersinia. Treponema and Neisseria can cause gonorrhea and syphilis respectively. Infection with Yersinia leads to a variety of symptoms including fever, abdominal pain, and diarrhea, which is often bloody. It has also been implicated in Reactive Arthritis.

At my last appointment, my dentist showed me an awesome video of biofilm (bacterial colonies) on the surface of normal teeth. The images were so cool that I asked him for permission to put the video up on this site, but, alas, it is copyrighted. My dentist proceeded to laud the virtues of regular flossing, a practice which I do regularly. (I swear!) In his opinion, flossing helps break up these biofilms and is critical to preventing tooth decay.

Interestingly, the Marshall Protocol does just that – although it uses pulsed, low-dose antibiotics – which have been shown to effectively destroy biofilms – and Benicar to get the job done with more vigor than a flossing addict could ever achieve. Take a certain MP patient (to protect her anonymity, I will call her “Mom”), who has been seeing the dentist for years due to tooth decay. I am told there is a ski home somewhere in Vail funded in large part by “Mom’s” regular dental work. She started the MP two years ago and now her dentist is more than a little surprised. At her last appointment, he said that he simply could not fathom the lack of plaque or tooth decay in any area of her mouth. Boy, does “Mom” wish she had started the MP earlier!

What intrigues me about bacteria in the mouth is that scientists regard most of them as harmful to our health and have no problem with procedures that would seek to sterilize the mouth. But when one mentions other parts of the body – let’s say the gut – and points out that perhaps the majority of bacteria in that area are also causing inflammation and disease, the same researchers often strongly disagree. Currently bacteria in the gut are largely assumed to be “helpful”, although in many cases such thinking is based only on speculation. Perhaps some gut bacteria may help with metabolic breakdown, but it is quite possible that the environment in the gut more closely resembles that of the mouth – an environment that can easily be overtaken by pathogens. Under such circumstances, a treatment like the MP that kills bacteria in the gut is therapeutic against inflammatory diseases such as Crohn’s, colitis and myriad other bowel ailments. This is especially true since patients on the MP are reporting improvement and recovery from bowel diseases that have never previously been reversed.

Also interesting is that many bacteria in the mouth seem able to migrate down the esophagus and reach the interior organs of the body. For example,Porphyromonas gingivalis[2] and A. actinomycetemcomitans,[3] both of which cause decay in the mouth, have been repeatedly identified in artherosclerotic plaque. This strongly suggests that these bacteria may be wreaking havoc on the blood vessels and contributing to heart disease. In fact, biomedical research Trevor Marshall believes that arterial plaque is a result of chronic bacterial infection. Indeed, where arterial plaque was once thought to be made of cholesterol and lipids it is now known that it is largely composed of dead macrophages. Since bacteria can infect and kill macrophages the death of such cells and their accumulation in patients with heart conditions seems logically tied to bacterial infection. With the above in mind, it’s not surprising that patients on the MP have reported improvement and recovery from various cardiac conditions. Some have tests showing that after years on the MP the plaque in their arteries is greatly reduced.

So keep on brushing people, but I recommend doing the MP too. In a theoretical sense the MP “brushes” our insides – the places we can’t reach to kill pathogenic bacteria physically. For most MP patients doing so is proving to be quite rewarding.

REFERENCES

  1. Nasidze, I., Li, J., Quinque, D., Tang, K., & Stoneking, M. (2009). Global diversity in the human salivary microbiome. Genome Research. []
  2. Dorn, B. R., Dunn, W. A., & Progulske-Fox, A. (2001). Porphyromonas gingivalis traffics to autophagosomes in human coronary artery endothelial cells. Infection and Immunity, 69(9), 5698-708. []
  3. Kozarov, E. V., Dorn, B. R., Shelburne, C. E., Dunn, W. A., & Progulske-Fox, A. (2005). Human atherosclerotic plaque contains viable invasive Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Arteriosclerosis, Thrombosis, and Vascular Biology, 25(3), e17-8. []
  • Comments Off on Hey there, how’s your Kineosphaeram holding up?
  • Filed under: biofilms, microbiome, News Flash
  • Understanding Biofilms

    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.

    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

    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.”[1] 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.

    A biofilm in the gut.

    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.[2]

    “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.

    Sessile cells in a biofilm “talk” to each other via quorum sensing to build microcolonies and to keep water channels open.

    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.[3] 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.[3]

    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.

    Development

    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.[4]

    Biofilm bacteria can move in numerous ways: Collectively, by rippling or rolling across the surface, or by detaching in clumps. Individually, through a “swarming and seeding” dispersal.

    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…”[1]

    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).[5] 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.”[6]

    The biofilm life cycle in three steps: attachment, growth of colonies (development), and periodic detachment of planktonic cells.

    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.[7]

    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.

    Levchenko and team used this device to observe bacteria growing in cramped conditions.

    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.[6]

    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.

    Dr. Levchenko of Johns Hopkins and Hojung Cho, a biomedical engineering doctoral student

    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.

    Nodes of relapsing infection?

    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.[1]

    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.

    Planktonic bacteria are periodically released from a biofilm

    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.[8] 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.”

    The acceptance of biofilms as infectious entities

    Anton van Leeuwenhoek.

    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.[1]

    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.

    Biofilm in a swamp gas reactor.

    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.[9]

    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.

    Biofilm in acidic pools at Yellowstone National Park.

    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.”[10]

    Biofilms and disease

    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.

    Common sites of biofilm infection. One biofilm reach the bloodstream they can spread to any moist surface of the human body.

    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. [10]

    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.”[11]

    Hundreds of microbial biofilm colonize the human mouth, causing tooth decay and gum disease.

    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.”[12]

    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.

    Microbes that colonize vaginal tissue and tampon fibers can become pathogenic, causing inflammation and disease such as Toxic Shock Syndrome.

    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.[13]

    Cells of Staphylococcus epidermidis causing devastating disease as they grow on the cuff at a mechanical heart valve.

    “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. [14]

    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.

    When the immune response is compromised, Pseudomonas aeruginosabiofilms are able to colonize the alveoli, and to form biofilms.

    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.[15]

    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.

    Biofilms have the potential to cause a tremendous array of infections and diseases

    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.[16]

    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.

    Otitis media, or inflammation of the inner ear, is caused by biofilm.

    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. [17]

    “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.

    Fungal biofilm can form in contact lens solution leading to potentially virulent eye infections

    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.[18]

    Biofilm bacteria and chronic inflammatory disease

    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).[19][20] 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.[21]

    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.[22]

    Diagram of the Vitamin D Receptor and capnine.

    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.[23]

    Dental plaque as seen under a scanning electron microcroscope.

    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.[24]

    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.”[10]

    How else do we acquire biofilm bacteria?

    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.

    A biofilm on a piece of lettuce

    “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.[25] 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.

    Effectively targeting biofilm infections

    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.[19][20]

    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.[11]

    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.[26]

    After antibiotics are applied to a biofilm, a number of cells called “persisters” are left behind.

    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.

    Model of biofilm resistance based on persister survival. An initial treatment of high-dose constant antibiotic kills planktonic cells and the majority of biofilm cells. But persisters remain alive and resurrect the biofilm, causing the infection to relapse

    “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.[27] 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.”[28]

    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.[29] 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.

    After antibiotics are applied to a biofilm, a number of cells called “persisters” are left behind

    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.

    REFERENCES

    1. Costerton, J. W., Stewart, P. S., & Greenberg, E. P. (1999). Bacterial biofilms: a common cause of persistent infections. Science (New York, N.Y.), 284(5418), 1318-22. [] [] [] []
    2. Higgins, D. A., Pomianek, M. E., Kraml, C. M., Taylor, R. K., Semmelhack, M. F., & Bassler, B. L. (2007). The major Vibrio cholerae autoinducer and its role in virulence factor production.Nature, 450(7171), 883-6. []
    3. Singh, P. K., Schaefer, A. L., Parsek, M. R., Moninger, T. O., Welsh, M. J., & Greenberg, E. P. (2000). Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature, 407(6805), 762-4. [] []
    4. Stoodley, P., Purevdorj-Gage, B., & Costerton, J. W. (2005). Clinical significance of seeding dispersal in biofilms: a response. Microbiology, 151(11), 3453. []
    5. O’toole, G. A., & Kolter, R. (1998). Flagellar and Twitching Motility Are Necessary for Pseudomonas Aeruginosa Biofilm Development. Molecular Microbiology, 30(2), 295-304. []
    6. Cho, H., Jönsson, H., Campbell, K., Melke, P., Williams, J. W., Jedynak, B., et al. (2007). Self-Organization in High-Density Bacterial Colonies: Efficient Crowd Control. PLoS Biology, 5(11), e302 EP -. [] []
    7. Brockhurst, M. A., Hochberg, M. E., Bell, T., & Buckling, A. (2006). Character displacement promotes cooperation in bacterial biofilms. Current biology: CB, 16(20), 2030-4. []
    8. Parsek, M. R., & Singh, P. K. (2003). Bacterial biofilms: an emerging link to disease pathogenesis. Annual review of microbiology, 57, 677-701. []
    9. Kraigsley, A., Ronney, P., & Finkel, S. Hydrodynamic effects on biofilm formation. Retrieved May 28, 2008. []
    10. Hall-Stoodley, L., Costerton, J. W., & Stoodley, P. (2004). Bacterial biofilms: from the Natural environment to infectious diseases. Nat Rev Micro, 2(2), 95-108. [] [] []
    11. Lewis, K. (2001). Riddle of biofilm resistance. Antimicrobial agents and chemotherapy, 45(4), 999-1007. [] []
    12. Parsek, M. R., & Singh, P. K. (2003). Bacterial biofilms: an emerging link to disease pathogenesis. Annual review of microbiology, 57, 677-701. []
    13. Trampuz, A., Piper, K. E., Jacobson, M. J., Hanssen, A. D., Unni, K. K., Osmon, D. R., et al. (2007). Sonication of Removed Hip and Knee Prostheses for Diagnosis of Infection. N Engl J Med, 357(7), 654-663. []
    14. Ristow, P., Bourhy, P., Kerneis, S., Schmitt, C., Prevost, M., Lilenbaum, W., et al. (2008).Biofilm formation by saprophytic and pathogenic leptospires. Microbiology, 154(5), 1309-1317. []
    15. Moreau-Marquis, S., Stanton, B. A., & O’Toole, G. A. (2008). Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway. Pulmonary pharmacology & therapeutics. []
    16. Hall-Stoodley, L., Hu, F. Z., Gieseke, A., Nistico, L., Nguyen, D., Hayes, J., et al. (2006).Direct Detection of Bacterial Biofilms on the Middle-Ear Mucosa of Children With Chronic Otitis Media. JAMA, 296(2), 202-211. []
    17. Imamura, Y., Chandra, J., Mukherjee, P. K., Lattif, A. A., Szczotka-Flynn, L. B., Pearlman, E., et al. (2008). Fusarium and Candida albicans Biofilms on Soft Contact Lenses: Model Development, Influence of Lens Type, and Susceptibility to Lens Care Solutions. Antimicrob. Agents Chemother., 52(1), 171-182. []
    18. James, G. A., Swogger, E., Wolcott, R., Pulcini, E. D., Secor, P., Sestrich, J., et al. (2008).Biofilms in Chronic Wounds. Wound Repair and Regeneration, 16(1), 37-44. []
    19. Marshall, T. G. (2006b). A New Approach to Treating Intraphagocytic CWD Bacterial Pathogens in Sarcoidosis, CFS, Lyme and other Inflammatory Diseases. [] []
    20. Marshall, T. G., & Marshall, F. E. (2004). Sarcoidosis succumbs to antibiotics–implications for autoimmune disease. Autoimmunity reviews, 3(4), 295-300. [] []
    21. Sr, G. J. D., & Woody, H. B. (1997). Bacterial persistence and expression of disease. Clinical Microbiology Reviews, 10(2). []
    22. Marshall, T. G. (2007). Bacterial Capnine Blocks Transcription of Human Antimicrobial Peptides. Nature Precedings. []
    23. Morrison, H. I., Ellison, L. F., & Taylor, G. W. (1999). Periodontal disease and risk of fatal coronary heart and cerebrovascular diseases. Journal of cardiovascular risk, 6(1), 7-11. []
    24. Stewart, R., & Hirani, V. (2007). Dental Health and Cognitive Impairment in an English National Survey Population. Journal of the American Geriatrics Society, 55(9), 1410-1414. []
    25. Falkinham Iii, J. O., Iseman, M. D., Haas, P. D., & Soolingen, D. V. (2008). Mycobacterium avium in a shower linked to pulmonary disease. Journal of water and health, 6(2), 209-13. []
    26. Lewis, K. (2001). Riddle of biofilm resistance. Antimicrobial agents and chemotherapy, 45(4), 999-1007. []
    27. Starner, Timothy D et al. 2008. Subinhibitory Concentrations of Azithromycin Decrease Nontypeable Haemophilus influenzae Biofilm Formation and Diminish Established Biofilms.Antimicrobial agents and chemotherapy 52(1):137-45. []
    28. Cogan, N. G., Cortez, R., & Fauci, L. (2005). Modeling physiological resistance in bacterial biofilms. Bulletin of mathematical biology, 67(4), 831-53. []
    29. Marshall, T. G. (2006). VDR Nuclear Receptor Competence is the Key to Recovery from Chronic Inflammatory and Autoimmune Disease. []
  • Comments Off on Understanding Biofilms
  • Filed under: biofilms, featured articles
  • Patients with diabetic neuropathy may not notice minor injuries due to loss of feeling in their lower extremities. Since the Vitamin D Receptor is inactivated by bacterial ligands, a small cut or sore can become infected, and flare into a limb- or life-threatening condition in as little as three days. These wounds are so difficult to heal that most of medicine considers them a lost cause and treats them with amputation. Amputations are often considered to be the beginning of the end for patients with diabetes.

    Dr. Randall Wolcott

    70% of diabetics who undergo an amputation die within five years due to the stress placed on their hearts from their altered circulatory system. During those five years they are likely to have more amputations and to rate their quality of life worse than cancer patients, according to some studies.

    Nationally, an estimated 82,000 people with diabetes had lower-limb amputations in 2002, according to the Centers for Disease Control. But thanks to a doctor at the Southwest Regional Wound Care Center in Lubbock, Texas, who has teamed up with researchers from Montana State University’s Center for Biofilm Engineering, this situation is changing. After sending samples of the sludge on his patient’s wounds to the Center, Dr. Randall Wolcott was informed that his samples were largely composed of bacterial biofilms.

    This discovery eventually led to a paper on the findings published in the October issue of Wound Repair and Regeneration, an important step in convincing the medical community of biofilms’ importance in chronic wounds.

    In the meantime, with the help of other scientists, Wolcott created a series of treatments that allow him to successfully kill the biofilm bacteria that have taken over his patients’ wounds, saving most of them from the horrors of amputation.

    Before treating the biofilms on his patients’ wounds, Wolcott admitted patients for an estimated 10 to 15 amputations a month. Now, he’s gone months without one of his patients receiving an amputation. He can confidently look patients in the eye and say he’s 80% certain that their wound is going to heal.

    Since patients with diabetes and a host of inflammatory diseases are also killing biofilm bacteria thanks to the Marshall Protocol, Dr. Wolcott’s work is yet another wake-up call as to the massive role these communities of bacteria play in causing all stages of chronic disease. I was lucky enough to speak with Dr. Wolcott and his laboratory research coordinator, Dan Rhoads. We spoke about their work, the importance of biofilm research, and the characteristics of biofilms in general.

    Could you explain what a biofilm is?

    Well, that’s a big question but I’ll do my best! Biofilms have been around for at least 3 million years. They are essentially how organisms protect themselves from environmental attack – from chemicals, phages (viruses that infect bacteria), UV light, or other challenges. We now understand that early on, bacteria learned to act as a community. By doing so, they allowed their existence to become much more secure. When bacteria first started to be studied about 150 years ago, the idea of a bacterial biofilm was simply too complex for scientists at the time to grasp. Consequently, early microbiologists were only able to study single bacterial organisms, one at a time.

    However, today we have an array of new molecular tools that have opened up a whole new world when it comes to understanding how bacteria survive. We now realize that bacteria are hardly ever found individually (in what is referred to as a planktonic state), but instead frequently join communities. These communities are then able to secrete substances that allow them to grab substances from the surrounding environment in order to create a matrix that protects all the bacteria inside.

    Planktonic bacteria produce certain proteins, but once they join a biofilm, the biofilm community expresses vastly different proteins and genes. For example, studies have shown that when a single bacterium becomes part of a biofilm the expression of over 800 genes can change.

    When a bacterium is in its planktonic state (on its own), it’s generally able to be cultured in a laboratory. It can also usually be killed by antibiotics. But since biofilms are entities that form under specific conditions in the human body, it is often difficult or impossible to grow them in a laboratory setting. They can no longer be killed by the standard high-dose antibiotics that easily target most single, planktonic bacteria because the community works to protect its members.

    So planktonic bacteria and biofilms are as different as caterpillars and butterflies. The organisms have the same genotype, but totally different phenotypes.

    Do you believe that bacteria in biofilms could be causing what are now referred to as diseases of unknown cause?

    I believe that they could be. Once bacteria have joined into biofilm communities, they can no longer be effectively targeted by the immune system. This means that after a biofilm is created, it persists as a chronic infection. Like patients who suffer from chronic inflammatory disease, people with biofilm infections find that high-dose antibiotics or steroids may offer them temporary relief, yet their infection never actually goes away.

    Dan and I have been reading several review articles that link autoimmune disease to chronic inflammation, and the more we’ve read, the clearer it’s become that chronic inflammation is a result of bacterial infection. So we think there is a clear link between chronic inflammatory diseases and bacteria, and when we think, “chronic inflammation” we believe we are typically dealing with biofilm infections.

    How did you become interested in studying biofilms?

    I attended a lecture about biofilms in 2002 which piqued my interest in the subject. Then I used Google to search for further information on biofilms and came upon the Montana State University’s Center for Biofilm Engineering – the place that is, in my opinion, the keeper of all knowledge about biofilms. I called them and told them about what I was observing on the wounds of my diabetic patients. I highly suspected that much of the sludge that I was removing from the wounds was biofilm. The center agreed to work with our office, and we proceeded to send them 50 samples of material scraped off our patients’ chronic wounds. Their molecular techniques confirmed that the majority of the samples did contain bacterial biofilms.

    At this point, let me pause to say that diabetic foot ulcers kill tens of thousands of people. Over 100,000 limb amputations happen every year because of infected wounds. The suffering is tremendous and, if the infection from a wound spreads or if the limb is amputated, the patient has a high risk of death. So finding a way to quell the bacterial infections and to heal diabetic wounds is a matter of life or death. So when we realized that we had discovered a previously unrecognized bacterial cause that explains the chronicity of diabetic wounds – wounds that cause patients to lose their limbs – we went after the whole hog.

    How do you treat the biofilms on your patient’s wounds?

    First we use diagnostic tools to determine that biofilms are indeed present on the wound. The techniques also help us identify the species of bacteria in a particular biofilm.

    Oh- so you are using molecular tools, that was something I was going to ask you about later. Later I want to hear how you feel about standard culturing methods…

    Well, now that you’ve brought that up, let me address the question now. The agar cultures that most scientists still use today in order to grow bacteria in the lab are 150 years old. A century ago, Robert Koch first discovered that planktonic bacteria could grow on a plate of agar. He used agar because you can manipulate the plate, scrape out the contents, thin out the contents enough, and finally end up with just one single bacterial species growing on the plate. Koch is the founding father of medical microbiology, and we are now standing on his shoulders. However, our understanding of science and medicine has changed a lot in the last century. Based on his pure-culture techniques, he created a series of postulates which state that only one single species of bacteria can cause any one disease. His postulates also state that a disease pathogenesis can only be considered legitimate if the single bacterium connected to the disease can again be isolated alone on an agar plate.

    Of course, Koch did not isolate bacteria and try to grow them on a medium that wasn’t agar. This is because if he did – let’s say he had tried to grow a bacterial species on a potato or an egg – the single bacterium would have surely congregated with other bacteria in the environment to form a biofilm – a biofilm that Koch could not isolate and study. So growing bacteria on anything besides an agar plate meant dealing with a situation that was too difficult for Koch to understand. So it seems he chose not to deal with such matters.

    Unfortunately, Koch’s postulates caught on among other scientists and eventually became the rule of thumb for growing bacteria and accepting organisms as disease-causing agents. Agar was, and still is seen by many, as the only appropriate bacterial growth medium. Even today, doctors still rigorously adhere to Koch’s postulates, which I believe has significantly impeded their ability to study and understand how bacteria actually survive and cause disease in the body where they are seldom found as single entities.

    Of course, growing some strains on agar has helped us better understand diseases such as strep throat, but we’ve pretty much knocked such diseases out. What we are only starting to realize today is that at least 80% of all the infections we treat are caused by biofilm bacteria, not planktonic bacteria. Now the playing field has changed. We’ve taken care of the planktonic bacteria that cause infections. Now we need to start treating polymicrobial diseases – those caused by combinations of bacteria. Continuing to culture on agar and adhering to Koch’s postulates is going to hinder that line of research because it impedes us from looking at the real thing, or what actually happens in the body. In the body, bacteria group together in communities. Changing the way we look at infection will require a paradigm shift in the way doctors think about bacterial populations and the potential of biofilm bacteria to cause disease.

    Happily, PCR [polymerase chain reaction] has allowed us to detect many of the bacteria in the biofilms we have studied. There are also many other molecular tools that exist or are being created that will allow for better detection of biofilm bacteria and bacteria in general. Once our team started using some of these sensitive, DNA-based technologies to identify the composition of bacteria in wound biofilms, we detected hundreds of different species, most of which would never grow on an agar plate. And every time we run the tests over again, it seems like we come across even more sequences of DNA that indicate the existence of new pathogens. So, the more we use these molecular diagnostic tools, the more we are realizing what highly diverse populations are inside wound biofilms.

    Consider this. In one of our latest studies, we found that it is common for at least 10 bacterial species to comprise at least 1% of each wound’s microbiota. And there were over 40 different species of bacteria that comprised at least 1% of the population in one sample or another. When you look deeper at that 1%, you see that there can be 40, 50, 60 species of bacteria on every wound – an incredible amount of diversity.

    Our next step is to determine which of the bacteria we have identified are important and which are not? Perhaps it will turn out that all species detected are important contributors to the virulence of each biofilm, or maybe we will discover that some are key species that cause more harm than others. By continuing to identify the bacterial species in the biofilms of as many of our patients’ wounds as possible, we also hope to determine possible correlations between the component bacterial species in the biofilm population and wounds’ severity. For example, the existence of some species may be found on wounds that are more difficult to treat. Based on this information we may decide to treat different wounds in different ways.

    There are a lot of people in the biofilm community who argue about the importance of particular species of biofilm bacteria, and many different research groups, each of which usually has its own opinion on which bacteria in a biofilm may be causing more harm than others. But our stance is that all the bacteria in a biofilm are important because they may act synergistically. Biofilms represent entire ecosystems, just like a forest. A forest isn’t made up of just squirrels, or just trees. Rather, all the entities that make up a forest work together and all are important to the survival of the community.

    This brings me to the concept of functional equivalence – a phenomenon that explains why biofilms are able to resist so many sources of stress. Let’s say a single bacterial species such as Staphylococcus aureus is floating around as a single entity. It can be easily identified and attacked by the immune system. Similarly, if it attaches to a surface and starts to form a protective protein matrix around itself – a biofilm – the biofilm is still relatively easy to break down because Staphylococcus aureus has limited defense mechanisms on its own.

    But lets say that when Staphylococcus aureus starts to form a biofilm, 10 other nearby bacteria develop the ability to attach to the biofilm as well. Now, if the biofilm is attacked again (by the immune system or other chemicals) it will be much harder to break down. That’s because each different species of bacteria in the biofilm possesses its own characteristics and its own strengths to combat the attack. If one species goes down, four others may still be able to fight and remain functional. A different form of challenge may take down those four species, but then some of the species that were not as effective against the first challenge may rise up and have the capability to deal with the new attack. I think this phenomenon – functional equivalence – is a very, very, important concept in chronic inflammatory infection.

    Functional equivalence has been documented in vaginal biofilms. Most vaginal biofilms seem to be composed of only one species of bacteria called Lactobacillus. These biofilms adjust the vaginal environment so that it has a pH of around 4.5, a pH that is most conducive to their survival and the woman’s good health. But it has been found that functionally equivalent biofilms develop that mimic these Lactobacillus biofilms. In the functionally equivalent biofilms, subgroups of different bacterial species come together and interact in order to allow the environment to create the same acidic environment. The difference is that the functionally equivalent biofilms, composed of many different species of bacteria, can perform similarly to the lactobacillus biofilm. These two genotypically different biofilms are phenotypically equivalent. They are functional equivalents.

    The same thing happens in wound biofilms. We see some that are predominantly colonized by single, well-known pathogens. But then we also see clinically similar biofilms made up of many different species of bacteria, and it is usually these diverse biofilms that display interesting growth patterns, interesting characteristics, and probably the best survival mechanisms.

    Since many diabetic patients end up with wounds covered in bacterial biofilms, do you think that biofilm bacteria might play a role in the entire pathogenesis of diabetes?

    We don’t know, although Dan and I have had several conversations about it. It’s mostly conjecture at the moment. We do know that a delayed immune response is what allows wound biofilms to become established. When wound biofilms start to form, they do so very quickly. Sometimes, they can even be detected after 20 minutes of growth. In other words, bacteria begin to form biofilms as soon as possible. If the immune system of people with diabetes were working up to par, they would be able to delay or retard such quick establishment of the biofilm, which is what a healthly individual can do.

    But you are usually able to kill the bacteria on your patient’s wounds. What procedures do you use?

    Yes, most of our patients’ wounds heal by using various treatments to wear away at the biofilms that cover them. These treatments include putting lactoferrin and xylitol on the wound. Lactoferrin occurs naturally in tears, mucus and breast milk and appears to attack the bacteria from multiple angles. It is used commercially in meat packing plants to prevent biofilms from growing on carcasses. Xylitol occurs in fruits, vegetables and other plants. It is also produced as part of normal human metabolism. It is used in toothpaste and chewing gum because of its anti-biofilm properties.

    An invaluable first step to treating a wound is debridement, or scraping the biofilm — a yellow-greenish sludge — along with dead tissue off the top of the wound with a curette. For some patients, this can be painful even with an anesthetic. Others feel nothing as diabetes has destroyed the nerve endings in their feet and legs. We also use five hyperbaric chambers where patients spend hours in a super-oxygenated environment that’s good for healthy tissue and bad for biofilms. We also use an arsenal of antibiotics and a new lipid-based gel. We recently finished a study in which we used a bacteriophage (viruses that infect bacteria) cocktail to fight the biofilms.

    How has your novel approach to treating diabetic wounds been accepted by your peers?

    I don’t mean to sound arrogant, but we know we’re right. We know that diabetic wounds are covered in biofilms and that it is biofilm growth that causes them to deteriorate to the point where most other doctors usually just cut them off. Once we realized that our patients’ wounds were covered with biofilm bacteria, we just knew, “This is it!” So we intend to spread the word about our discoveries ASAP. There are just a drastic number of medical issues that stem from biofilm infection. 500,000 people suffer from sinus infections caused by biofilms ever year. There are dozens and dozens of chronic infections that are biofilm related – infections that are now left uncured and thus force people to have heart valves put in, or lead to the removal of entire colons, or lead to tubes in the ears – all kinds of things. It’s bad, and we need a different answer to the way we treat so many conditions.

    Yet we are still often met with skepticism. I try to take the approach that nothing is fun if it isn’t controversial. Once the presence of biofilms on diabetic wounds is accepted as truth, the excitement and ambition of working in the area will dwindle. What we want now is confrontation. We want to push our ideas. It’s up to us to prove this is the real thing. It’s a vetting process, but we can win.

    What we do need is for researchers and doctors to be open minded. Instead of brushing us off, they need to look at the evidence we are presenting, absorb it, and at least argue with it if they think it’s wrong. When it comes to chronic biofilm infections, we are dealing with life and death situations, so it’s important that others take note of the facts and reasonable arguments that are currently on the table.

    Dr. Wolcott treating a wound

    But since your patients are still immunocompromised after you have treated their wound, don’t you find that many come back with new wounds that you must treat again?

    Well, when diabetic patients develop an infected wound that causes a limb to turn black, the trauma serves as a major wake-up call. Many of our patients start taking their illness much more seriously. Some buy insulin pumps. Others are careful to buy special diabetic shoes that offer better foot care, or finally make regular visits to their podiatrist to have difficult-to-cut nails sawed off. All these measures reduce the likelihood that they will develop another wound.

    We do realize that even when we effectively save a patient’s wound, our patients are still at risk for new wounds because they are immunocompromised. However, when a patient comes in with a wound on one limb we can demonstrate that the same comorbidities are present in the other limb as well, but it does not have a wound. The other limb appears to be intact and generally healthy. If we can work to heal the wounded limb, it should be just as healthy as the patient’s unwounded limb.

    But yes, we do have patients that we have treated once for a wound who come back two or three years later with another wound. The good thing is that, based on their first experience, these patients know to come see us as quickly as possible. The sooner we can treat the wound the less likely it is that the infection will spread to the bone where it is much harder to manage.

    Do you believe biofilms are actively studied to the extent they should be? If not, why are they not getting more attention?

    Oh, don’t get me started! Here’s my favorite example. I went to a biofilm conference in 2005. The first speech I heard was given by researchers from Proctor and Gamble. They had developed Compound 227 that works to prevent biofilm growth in the mouth and thus prevent the accumulation of dental plaque. They had literally spent millions and million of dollars on this research, just tremendous amounts of money so that they could use any discoveries to create a more effective toothpaste. Others spend millions to identify chemicals that can better remove and prevent biofilms that often accumulate on toilets—you know, those ugly rings.

    The presentation that followed the dental and industrial presentations was about biofilms and medicine. There was next to nothing to report and practically no spending whatsoever in the area. I came away understanding that right now we are spending way more money on preventing biofilm growth on teeth and toilets than on finding ways to effectively treat the dozens of different serious (many life or death) medical conditions that result from biofilm infection.

    Why aren’t more doctors like you – searching for a curative treatment option rather than content to simply palliate symptoms?

    Well, as we’ve moved forward with our work, we’ve taken our share of hits from all different types of regulatory agencies as well as funding agencies. So for some doctors, it may seem like this kind of scrutiny is not worth the extra hassle.

    But I feel many other doctors care. They are tired of telling their patients, “That’s just the way it is. I can’t make you better,” but often they don’t know how to get started. For example, a physician from England recently came to visit the clinic. She is interested in starting to treat the biofilms that she encounters most often: chronic and recurring bladder infections. However, in order to take this new approach, she is required to begin to work more independently from the National Health Service.

    I take it you are familiar with evidence-based medicine? It’s the increasingly accepted approach for making clinical decisions about how to treat a patient. Basically, doctors are trained to make a decision based on the most current evidence derived from research. But what such thinking boils down to is that I am supposed to do the same thing that has always been done – to treat my patient in the conventional manner – just because it’s become the most popular approach. However, when it comes to chronic wound biofilms, we are in the midst of a crisis – what has been done and is accepted as the standard treatment doesn’t work and doesn’t meet the needs of the patient.

    Thus, evidence-based medicine totally regulates against innovation. Essentially doctors suffer if they step away from mainstream thinking. Sure, there are charlatans out there who are trying to sell us treatments that don’t work, but there are many good therapies that are not used because they are unconventional. It is only by considering new treatment options that we can progress.

    Have you conducted a double-blind placebo control trial on your findings? If not, why not?

    We know without a doubt that chronic diabetic wounds can be saved if the biofilm bacteria that cover them are eliminated. So we are simply unwilling to use a control group as guinea pigs when we know we’ve got the methods to save most of their limbs as well. Granted, we are using some medications for off-label purposes, but they are all approved by the FDA. This is not just experimental stuff. We know that what we are doing is right for the patient. So we simply refuse to do a study where the control group is not treated. The only double-blinded trial we’ve done tested the effect of bacteriophages on wound biofilms, but in that case, the control group still got treated with everything else in our arsenal except the bacteriophages.

    We hope we can come to a compromise. We have plenty of data, and even though it’s retrospective, it’s still very valuable. So we hope that the medical community will take this evidence as proof that we are doing the right thing, in lieu of a blinded trial. Right now, rather than focusing on a blinded trial, we are simply focusing on what is best for the patient. We are trying to heal as many patients’ wounds as possible. That’s our main priority – treating patients right here and right now. If you take time to look at the retrospective evidence, it is solid. Our patients do very well.

    Are you familiar with the Marshall Protocol?

    Only from what you’ve just told us, but we plan to investigate it further. The idea of pulsed antibiotics makes a lot of sense – essentially it may allow the antibiotics to target the growing or regenerating cells in the biofilm, which were previously persister cells. Please send us more information.

    Me: Great! I think that the Marshall pathogenesis will help you better understand why the diabetic patients you treat are so immunocompromised. As you know, we believe that the entire pathogenesis of diabetes is caused by L-form and biofilm bacteria, and that these bacteria are able to create substances that slow the Vitamin D Receptor and subsequently the activity of the innate immune system. Thus, we believe that restoring the competence of the Vitamin D Receptor is key to recovery to inflammatory disease. Activating the VDR, or putting your patients on the full Marshall Protocol, could help restore their innate immune function, which may go a long way in preventing them from developing new infected wounds. At least that’s my take!


    The following is an excerpt taken directly from an article on the Montana State University’s Center for Biofilm Engineering website. It describes the experience of just one of Dr. Wolcott’s patients.

    The fruits of this science can be seen in the story of Jerry Montemayor, a 38-year-old school administrator in Lubbock, who stubbed his toe on the corner of his bed one morning in December 2005 and nearly lost his foot.Initially, Montemayor ignored the bruise. A diabetic, Montemayor has poor blood circulation in his lower legs and feet. Three days later, his toe was discolored and he limped with discomfort. He went to an emergency room.Emergency room physicians told Montemayor his foot was severely infected and he must be admitted. He spent the next 12 days in the hospital. When his infection didn’t respond to treatment, Montemayor’s physicians told him his foot should be amputated, or he risked losing his entire leg, and possibly his life.

    “First they said it would be the top of my foot, then half of my foot, then my whole foot,” Montemayor said. “They kept telling me I needed to set a date and time for my amputation. Believe me, if it wasn’t for the power of prayer I don’t think I’d have gotten through this.”

    Montemayor sought a second opinion. The next day, two staff members from Wolcott’s center visited.

    “I’ll never forget that visit,” Montemayor said. “One of the girls said ‘We’ve seen worse. We suggest you do not get this amputated. We can treat this.’”

    It was Christmas Eve.

    Montemayor took their advice and began nearly a year’s worth of treatments at Wolcott’s clinic on Christmas Day. Today, he walks on both feet.

    “The clinic staff said they were going to do their best and they did,” Montemayor said. “I’m blessed to be walking.”

    “It’s hard to relive that experience in the hospital,” he said. “At the time I was thinking about my personal life. I was thinking how this would affect me meeting someone, or having a relationship with someone. Is she going to accept and support me? Is she going to be able to walk next to me and accept that I have a prosthetic limb?

    “I was thinking ‘If I have kids will I be able to run and play with them?’” Montemayor said. “I was thinking ‘Am I going to be a whole man?’”

    Update – May 24, 2009

    Since the time of this interview, Dr. Wolcott has continued to successfully treat his patients’ wounds. At around the time of this interview, Dr. Wolcott authored a paper, the abstract of which appears in PubMed: A study of biofilm-based wound management in subjects with critical limb ischaemia. Here is the money quote:

    When comparing the healing frequency in this study with a previously published study, [Biofilm-based wound care management] strategies significantly improved healing frequency. These findings demonstrate that effectively managing the biofilm in chronic wounds is an important component of consistently transforming ‘non-healable’ wounds into healable wounds.

    I am also including images Dr. Wolcott put online. Anyone who is interested can view the large PDF file which contains images of the patients Dr. Wolcott has treated according to his biofilm-based wound management strategy. Here’s a sample.

    wounds

  • Comments Off on Interview with Dr. Randall Wolcott, bacterial biofilm wound specialist
  • Filed under: biofilms, featured articles, interview (doctor/researcher)
  • About Amy Proal

    Amy and Zeus

    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.