Exploring chronic disease
13 Apr 2008
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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?’”
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.
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.