This month yet another paper was published whose results confirm that the Petri dish is a thing of the past. The study, which was published in BMC Microbiology, used a series of molecular techniques to identify the species of biofilm bacteria present inside several different kinds of wounds.

Bioflims are formed when a complex and varied group of bacteria aggregate together inside a protective and adhesive protein matrix. The bacteria inside a biofilm cooperate to promote their own survival and the chronic nature of an infection. While dentists have long realized that bacteria in the mouth often reside inside biofilms – they form much of the goo that they remove from teeth – researchers are just starting to investigate bioflim communities that form in other areas of the body.

Since few studies have performed extensive surveys of the bacterial populations within different types of chronic wound biofilms, the BMC team, comprised of individuals from the United States Department of Agriculture, the Medical Biofilm Research Institute in Texas, and the Center for Biofilm Engineering at Montana State University, used several advanced molecular techniques to survey the major populations of bacteria in the pathogenic biofilms of three types of chronic wounds:

• diabetic food ulcers
• venous leg ulcers
• pressure ulcers

The researchers used several different molecular methods – ribosomal amplification and cloning, Sanger sequencing (FRACS), partial ribosomal amplification, density gradient gel electrophoresis (DGGE), and Sanger sequencing (PRADS) – to provide a comprehensive survey of microbial populations.

Together, these molecular techniques revealed that some specific populations of bacteria were evident in the biofilms of all chronic wound types. These bacteria included Staphylococcus, Pseudomonas, Peptoniphilus, Enterobacter, Stenotrophomonas, Finegoldia, and Serratia.

But there were also marked difference between the bacterial populations in each of the wounds. For example, in pressure ulcers, 62% of the populations identified were from a class of bacteria called obligate anaerobes – bacteria that do not require oxygen for growth and may even die in its presence.

In fact, every wound that the research team tested with new molecular technology was also tested for the presence of bacteria using traditional based culture methods. When the results of molecular analysis were compared to the bacteria obtained using these traditional methods, the team found that culture methods were only able to correctly identify the primary bacterial population in one wound type.

“Here we show that culturing failed to identify major contributing populations, especially strict anaerobes, within the given wound types,” state the team, “Standard culturing techniques are inherently biased as they only examine only the 1% of all microorganisms which are able to grow fairly rapidly in pure culture.”

The team also commented on the fact that it takes several days for bacteria grown by culturing methods to be identified, whereas molecular methods such as PCR can typically be completed within several hours. They also stress the fact that some forms of bacteria can simply not grow under the conditions required for standard cultivation, stating that “In addition, certain of the isolates that we have shown to be primary populations within a wound type may never be cultured in the laboratory due to reduced metabolic activity, obligate cooperation with other bacteria, requirements for specialized nutrients, or growth in specific environmental conditions.”

They also argue that molecular methods, unlike culture methods, have more potential to provide quantitative data, stating “arguably, we have shown that molecular methods will allow populations residing within biofilms to be more fully characterized.”

Furthermore, they make it clear that doctors will greatly benefit by knowing the exact composition of bacteria that each of their individual patients harbors. “The continued development of molecular methods may lead to vastly improved tools or diagnostics that will identify and provide quantification of the diverse species potentially present in chronic wounds thereby allowing physicians to better tailor their treatment to each patient’s unique pathogenic biofilm populations,” states the team.

Thus, much like other teams who are using molecular methods to detect bacteria, the researchers make an urgent call for the standardized use of improved diagnostic methods. Not only that, but they also hope the results of their study might actually “foster the pioneering, and development of new diagnostic tools.”

One of the study’s findings that also bears pertinence to the Marshall Protocol is the fact that the bacterial species Staphylococcus was found inside all chronic wound types. Two of the other largest studies to date to seek bacteria using molecular technology (one which looking at bacteria removed prosthetic joints, and another that looked at bacterial populations in patients with Cystic Fibrosis) also found Staphylococcus to be a common pathogen in their samples. Since minocycline is one of the antibiotics that best targets Staphpylococcus, these findings suggest that the decision to use minocycline as the base antibiotic for the Marshall Protocol was well warranted.

Also interesting is that researchers at the University of Turku in Finland just released the results of a study which found that infants who harbor the Staphylococcus aureus are more likely to become overweight and obese later in life. Again, this suggests that Staphylococcus is a common cause of Th1 symptoms, and that obesity should respond to the MP antibiotic regimen that allows the bacterial species to be killed.

The team analyzed 490 skeletons from a London cemetery for Black Death victims – the name given to those people who succumed to the plague epidemic of 1347 to 1351. Black Death – named after the black spots the bubonic form of the plague caused on the skin – was one of the deadliest recorded in human history, killing about 75 million people, according to some estimates, including more than a third of Europe’s population.

Experts have long believed that the Black Death killed indiscriminately regardless of age, sex or level of health because it was so virulent. But anthroplogists Sharon deWitte and James Wood, who led the Penn State team, have demonstrated that the infection did not affect everyone equally.

The anthropologists found that while many perfectly healthy people certainly were cut down, those already in poor health prior to the arrival of the plague were more likely to have perished.

“A lot of people have assumed that the Black Death killed indiscriminately, just because it had such massive mortality,” states DeWitte. “People already in poor health often are more vulnerable in epidemics. “But there’s been a tradition of thinking that the Black Death was this unique case where no one was safe and if you were exposed to the disease that was it. You had three to five days, and then you were dead.”

DeWitte analyzed skeletons unearthed from the East Smithfield cemetery in London, dug especially for plague victims and excavated in the 1980s, for bone and teeth abnormalities that would show that people had health problems before they died of plague.

She found such abnormalities in many skeletons, suggesting these people had experienced malnutrition, iron deficiencies and infections well before succumbing to the Black Death.

Today we understand that, due to the chronic nature of bone deterioration and dental problems, these infections were almost certainly caused by L-form bacteria.

Bone loss results after L-form bacteria create substances that block the Vitamin D Receptor – preventing it from transcribing an enzyme that keeps the level of the hormone/cytokine 1,25-D in check. As 1,25-D rises above a certain range (around 43 pg/ml), it stimulates bone osteoclasts, or cells that remove minerals from the bone. Stimulated osteoclasts dissolve bone material, causing it to be reabsorbed into the bloodstream – leading to osteoporosis and osteopenia.

We are also familiar with the “anemia of chronic disease”, or the fact that people with Th1 disease are frequently deficient in iron. This deficiency is the direct result of the fact that L-form and biofilm bacteria secrete iron-binding complexes called siderophores that remove iron from host proteins, making it available for use by the pathogens.

Futhermore, tooth decay is almost certainly impacted by L-form bacteria, as a wide range of treatment resistant bacteria, including those that persist in bioflims, have been detected in the mouth, not to mention the fact that dental immunopathology and subsequent improvement of dental issues is common among patients on the Marshall Protocol.
Thus it comes as no surprise that DeWitte and team found that the proportion of people with such signs of Th1 disease in the cemetery, compared to those who appeared to have been of robust health before the epidemic, indicate that Black Death was somewhat selective in who it killed.

“The Black Death was highly virulent and undoubtedly killed many otherwise healthy people who would have been unlikely to die under normal-mortality conditions,” they wrote. But people already in poor health were more likely to die.”

Those people in poor health were certainly immunosuppressed, thanks to the fact that as L-form and other stealth bacteria accumulate, the pathogens produce substances that block the VDR, causing the innate immune system to become increasingly compromised. It’s no wonder their lives were claimed by the Black Death, just as today people with Th1 disease are easy victims for the HIV virus.

Several months ago, two international trials aimed at testing an experimental AIDS vaccine were stopped after it became clear that the vaccine did not prevent infection with the AIDS virus. The trials were conducted in the United States, Peru, Brazil, Dominican Republic, Haiti, Jamaica, Australia and South Africa. Today, the researchers conducting the trial are faced with another problem. Earlier this month they reported “worrying” indications that the thousands of people who received the vaccine are now at greater risk for infection. They have already begun counseling volunteers about the fact that they could be at higher risk for acquiring HIV – the fatal and incurable virus that causes AIDS.

To test vaccines and new drugs, researchers always aim for what are called placebo-controlled, double-blinded trials. This means that neither the researchers nor the volunteers know who gets a placebo and who gets an active ingredient – the goal being to minimize any biases in determining whether the treatment works.

But Merck and the academic researchers who conducted the vaccine trial are planning to “unblind” the study – meaning that participants will find out who got an active shot and who got a dummy injection.

“All study volunteers will be encouraged to continue to return to their study sites on a regular basis for ongoing risk reduction counseling and study-related tests,” the researchers said in a statement.

Were the vaccines contaminated with L-form bacteria? It’s quite probable. Especially since L-form bacteria are now known to create ligands that bind and block the Vitamin D Receptor (VDR). Since the VDR controls the activity of the innate immune system and the antimicrobial peptides, people who acquire L-form bacteria begin to suffer from immune dysfunction – much like the study participants in the trial described above.

Ensuring that the vaccines, injections, and blood transfusions we receive are not contaminated by L-form bacteria only strengthens the reality that the pathogens need to be brought into the spotlight immediately. In the meantime, people such as those unlucky enough to receive the actual vaccine in the Merck trial will continue to get sick after taking a measure ironically aimed at preventing disease.

And just who is able to describe the process of culturing L-form bacteria so eloquently? She’s an Associate Professor at the Department of Pathogenic Bacteria Institute of Microbiology at the Bulgarian Academy of Sciences, who’s worked with L-form bacteria for the last 15 years. Meet Nadya Markova.

1. What led you to become interested in L-form bacteria?

I graduated as a medical doctor, but my interest in microbiology led me to the Bulgarian Academy of Sciences (where I defended my PhD thesis in the field of medical microbiology). Researchers already working there at the time had great experience in L-form research and had made many interesting observations about the bacteria, all of which sparked my interest. They were my teachers, who inspired me to continue their research in the same field.

2. How long have you been working with L-form bacteria? How many people do you currently work with and how to they contribute to the research environment?

I started thinking about how L-form bacteria change form in the beginning of the 90s, and my interest in them has risen ever since. Unfortunately, our research team is comprised of only 5 people. One of them must be mentioned, my teacher Professor Lilia Michailova. She is an excellent electron microscopist and, without her, our achievements wouldn’t have been possible. I’m really glad that she is still active and that we continue to work together.

3. Is your team working (or in the past has focused on) any particular species of L-form bacteria?

L-form bacteria as seen under an electron microscope; this photo and others taken by Nadya Markova

We have conducted various experiments with L-forms of Staphylococcus aureus, Streptococcus pyogenes, and Listeria monocytogenes. At present we are very interested in, and work exclusively with, L-forms of Mycobacterium tuberculosis.

4. How do you grow the L-form bacteria that you observe in the lab? What techniques do you use? What type of microscopes do you use to observe them?

We grow L-forms on specific media (semisolid and liquid) with supplements specifically designed to facilitate L-form growth. These media are well known in the literature and are widely used by other researchers. We observe L-forms using electron and light microscopes. However, in order to correctly observe L-form bacteria using a light microscope, a person must have a great deal of experience with L-forms.

5. What aspects of L-form bacteria has your research focused on?

We focus on bacterial host cell interactions, especially interactions of bacterial L-forms with peritoneal and alveolar macrophages in experimental infections.

6. Could you explain that to me in simple terms?

Samples showing L-forms multiplying in number

There are two types of experimental infections. The first involves dividing experimental animals in two groups. We inject the first group of animals with normal pathogenic bacteria and the other group with L-form strains. We grow the L-forms under specific laboratory conditions prior to the experiment.

During the experimental infection, we collect samples of white blood cells called alveolar macrophages in the animal’s lungs, and white blood cells called peritoneal macrophages from the abdomen of each animal. Macrophages are cells of the immune system that are supposed to be the first to react to infections. Their job is to engulf and digest the pathogens and then, in turn, create an antigen, a special type of molecule that provokes a response from the host’s immune system.

Phagocytosis is the process of engulfing, digesting, and transforming pathogenic bacteria into antigens. In the case of normal pathogenic bacteria, macrophages easily recognize the bacteria and the process of phagocytosis goes smoothly. But when macrophages are faced with L-form bacteria, they have difficulty recognizing the bacteria as pathogenic, and the normal process of phagocytosis is impeded.

L-forms stay longer on the surface of macrophages without being engulfed. Even when they are engulfed, L-forms are not properly digested and they continue to persist inside the macrophage. In short, the whole process of destroying bacteria is distorted.

It is important to understand that these phenomena happen in vivo, or inside animals that are alive.

7. What is one of the hardest parts about culturing L-form bacteria?

Nadya Markova and colleague, Professor Lilia Michailova

The hardest part is not so much cultivating the bacteria, but rather developing the skills to recognize which bacteria are L-forms. The importance of a teacher passing down this knowledge to a student is critical – this cannot be learned from books or photographs; one has to see it in person and have it explained.

8. What role to you think L-form bacteria play in causing certain chronic diseases? What, if any, diseases do you think they might cause?

In my opinion, long-lasting persistent irritation of the immune system by these unusual bacterial L-forms is the cause of most chronic diseases. I wouldn’t speculate and mention specific diseases, but I support the opinion that L-forms could be the reason for latent, chronic, and relapsing infections, as well as diseases of unknown infectious-allergic or autoimmune origin.

9. What do you feel are some of the most fascinating aspects of L-form bacteria?

L-form bacteria are so unusual, so it is hard for me to decide what I feel is most fascinating about them. I’m always amazed at how they can drastically change their life style. By life style, I mean that each microbe has its own shape and size, or its own morphology. Microbes are mainly shaped like rods or, shaped like spheres (called cocci). Usually microbes multiply by simple division, that is, each microbe divides into two new microbes which are morphologically identical.

But when it comes to L-forms, the process of division does not occur in the same, usual way. L-forms stop producing cell walls and start creating morphological units such as large bodies, elementary bodies, filaments, filterable granules, empty bodies, vesicles and membranous structures. When these diverse populations of L-forms get into an organism, they do not behave like other populations of normal pathogenic bacteria. They no longer interact in a normal manner with cells from the immune system.

Large bodies of the L-form Staphylococcus

Perhaps another one of the most important aspects about the pathology of L-form characteristics is that they are exceptionally resistant to many factors, including most antibacterial drugs and bactericidal host defense mechanisms. Simply speaking, the bactericidal host defense mechanism is the ability of macrophages to engulf and destroy pathogens and in the process create an antigen. It is the only way that the organism can produce a normal immune response to the bacterial threat.

10. What would you say has been your greatest or most interesting discovery about L-forms?

We are fascinated by, and focused on, observing and documenting the atypical behavior of L-forms in vivo, or in the body rather than the lab.

11. What do you feel are some of the most misunderstood aspects of L-form bacteria?

One of the main misconceptions about L-form bacteria is that they are considered to be an artificially provoked phenomenon capable of existing only in laboratory conditions. But a number of studies have shown that L-forms can exist in vivo (inside the body) as well.

12. Over the years, how has your work been received?

It’s hard to get our papers published, because few people actually understand and want to accept our investigations concerning bacterial L-form transformations. Unfortunately, this continues to this very day.

13. If you could briefly talk to the leader of the World Health Organization about L-form bacteria, what would you tell him?

Filamentous L-forms of M. tuberculosis

I would tell him what an extremely important role L-form bacteria play in latent, chronic, and relapsing infections, as well as in diseases of unknown infectious-allergic or autoimmune origin. I would also tell him, if possible, to support and sponsor the researchers working with L-form bacteria, as they are mostly ignored.

14. In Bulgaria, what is the prevailing opinion about the cause of chronic disease?

Unfortunately, physicians in Bulgaria, and probably in other countries, don’t pay enough attention to the role of L-form bacteria in chronic diseases, and accept them with difficulty.

15. Are you in contact with other researchers who work with L-form bacteria?

The founder of L-form research in Bulgaria – Professor Toshkov – had great contacts and collaboration projects with L-form research teams in France, Germany and Russia, as well as contacts with famous scientists like Lida Mattman. Nowadays, there are relatively few people with whom I am in contact. However, no matter how hard it is, I’m doing my best to get in touch with other scientists working with modern molecular biological tools. I want to spark their enthusiasm so that they too will work in L-form research, which will allow this area of medicine to achieve real success.

16. If people don’t accept your research, what reasons do they give for saying you are incorrect? Does this frustrate you?

Spherical-shaped L-forms of M. tuberculosis

When reviewers don’t accept our papers, one of the main reasons they give is that our results are not supported with standard molecular-microbiological methods, which is why they refuse to accept them as real. It is very hard to explain to them that those standard methods for classical bacteria are not valid for L-form cells, and that new methods and approaches need to be developed in order to identify them using DNA- based technology.

17. If you could change one aspect of the current medical climate,
what would it be?

I think that it is very important to establish better cooperation and contacts between practicing physicians and those microbiologists working in laboratories that specialize on the isolation, cultivation, and identification of L-forms.

18. Have you heard about the Marshall Protocol? If so, how do you feel about the treatment?

Yes, I have heard about the Marshall Protocol and I am aware of the basic principles of the treatment. I feel that the approach of the Marshall Protocol, which first acts on the immune system and then continues the healing process with an extended etiotropic low-dose antibiotic therapy, is correct. In my opinion, at this stage of our understanding of L-form behavior, minocycline is one of the best antibiotics that is best able to suppress the activity of L-forms. However, I feel that all researchers working in the field of L-forms should be concentrating all their energy on developing new drugs that can specifically target and block the process of L-form conversion.

19. Does your team have any leads or ideas on how to go about creating a medicine that would block L-form conversion?

In order to create an effective medicine we have to profoundly investigate and fully understand the mechanism of L-form conversion (how normal pathogenic bacteria turn into L-forms) on the molecular and genetic level. Then we will be able to determine the exact level at which we can act in order to stop the process.

PAPERS

Michailova L., Kussovski V., Radoucheva T., Jordanova M. & Markova N. (2007). Persistence of Staphylococcus aureus L-form during experimental lung infection in rats. FEMS Microbiol Lett 268: 88-97.

Michailova L., Kussovski, V., Radoucheva, T., Jordanova M., Berger W., Rinder H. & Markova N. (2005). Morphological variability and cell wall deficiency in Mycobacterium tuberculosis ‘heteroresistant’ stains. The Int. J. Tuberc. Lung. Dis. 9: 907-914.

Michailova, L, Markova N., Radoucheva T., Stoitsova S., Kussovski V. & Jordanova M.. (2000). Atypical behaviour and survival of Steptococcus pyogenes L-forms during intraperitoneal infection in rats. FEMS Immunology and Medical Microbiology 28: 55-65.

Michailova, L., S. Stoitsova, N.Markova, T. Radoucheva, V.Kussovski, M. Jordanova, & Dimova. I. (2000). Interaction of alveolar macrophages with Staphylococcus aureus and induction of microbial L-forms during infection in rats. Int. J. Med. Microbiol. 290:259-267.

Markova N., Mihailova L., Vesselinova A. , Kussovski V., Radoucheva T., Nikolova S., & Paskaleva I. (1997). Cell wall deficient forms (L-forms) of Listeria monocytogenes in experimentally infected rats. Zbl. Bakt. 286: 46-55.

Mihailova, L., Markova, T., Radoucheva, D., Veljanov, S., & Radoevska. (1993). Cell interaction of Listeria monocytogenes L-forms and peritoneal exudative cells in rats. Can. J. Microbiol. 39: 1014-1021.

Gerald Domingue

Gerald Domingue is a medical researcher and academic who served as Professor of Urology, Microbiology and Immunology in the Tulane University School of Medicine and Graduate School for thirty years and also as Director of Research in Urology. He is currently retired and resides in Zurich, Switzerland where he is engaged in painting and creative writing. At retirement he was honored with the title of Professor Emeritus at Tulane. Prior to Tulane, he served on the faculty of St. Louis University, was a lecturer at Washington University and director of clinical microbiology in St. Louis City Hospital, St. Louis, MO.

Over the course of his thirty-nine year career, Domingue received funding from the National Institutes of Health, Veterans Administration, and a variety of national and international research foundations. He enjoys international recognition as an authority on the basic biology and medical significance of atypical bacterial organisms and is considered an expert on the role of these bacteria in the persistence and expression of kidney and urological infectious diseases.

A picture taken with scanning electron microscope, showing elongated L-form bodies harvested from filtered blood

He first became interested in the role of atypical bacterial forms after noting that a large number of patients with urinary tract infections suffer from continual relapsing illness. Using a direct phase microscope, he examined the urine specimens of several patients with urinary tract infections and found L-form bacteria in his sample.

He began to investigate L-form bacteria, striving to better understand their biology and the role they play in causing disease. Over the course of the next 30 years, he was able to explain much of the mystery behind how the bacteria are able to persist in the body, and published a wide array of clinical and experimental studies on the subject.

Freshly passed urine from a patient with renal Fanconi syndrome, showing large L-form granules with dense cores

Domingue worked with a team that included pre and post doctoral students and fellows along with faculty colleagues and laboratory assistants. Together they discovered that L-form bacteria are able to form tiny dense bodies within parent cells that already lack cell walls. They noted that the forms, which they called electron dense bodies were so small that they could pass through bacterial filters that normally withheld ordinary bacteria with cell walls.

The electron dense bodies could persist inside tissue culture cells in the laboratory. After applying this data to the human condition, Domingue reasoned that in some patients who suffer from chronic bacterial infections, the disease process could be related to the fact that bacteria are able to differentiate into the resistant electron dense bodies that he observed in tissue cultures.

An L-form in which a bacterium-like cell is evolving in its core

In 1974, he and his graduate student, Mary Green, along with Paul Heidger, a faculty collaborator, published two landmark companion papers in the prestigious journal Infection and Immunity. The papers detail how L-form bacteria inside an experimental human embryonic kidney tissue culture system are able to persist in cells and explains how they are able to revert into the cell wall-containing parent bacterial form. They also proposed a detailed reproductive cycle for L-form bacteria, followed by electron microscopy of the microorganisms.

These papers set the stage for Domingue and his team to delve even further into the role that cryptic atypical bacteria play in causing persistent and recurrent infections.

In 1997, he and a colleague, the late Hannah Woody published an invited extensive review article on chronic bacterial infection in Clinical Microbiological Reviews. Among their conclusions was the claim that “difficult to culture and dormant bacteria are involved in the latency of infection and that these persistent bacteria may be pathogenic.”

Domingue in 1992 at the Tulane University School of Medicine

He implicated L-form bacteria in several kidney-related diseases including pyelonephritis, glomerulonephritis, idiopathic hematuria, and interstitial cystitis. He also speculated about their role in other diseases such as rheumatic fever, tuberculosis, syphilis, and rheumatoid arthritis.

In the review Domingue stated, “Clearly, any patient with a history of recurrent infection and persistent disability is sending the signal that the phenomenon (infection with L-form bacteria) could be occurring. The so-called autoimmune diseases in which no organism can be identified by routine testing techniques are particularly suspect.”

A stain of a renal cell in the urine of a patient with kidney disease. Note the yellow-green fluorescence of the L-form

He went on to conclude, “Bacteriologic advances, which include special culture media and stains, electron microscopy and molecular techniques such as PCR (polymerase chain reaction), have revealed an increasing number of previously unidentifiable organisms in a variety of pathologic conditions. It is unwise to dismiss the pathogenic capacities of any microbe in a patient with a mysterious disease.”

Over the course of his thirty-nine year career Domingue published 160 papers, monographs, and book chapters; 65 devoted to L-form research. He was invited to deliver over fifty international and national lectures about L-form bacteria and wrote a book on the subject, Cell Wall-Deficient Bacteria: Basic Principles and Clinical Significance. His papers are filled with photos of cultures of L-form bacteria taken with an electron microscope. They show the microbes inside human and animal cells.

Although Domingue’s primary research focused on bacterial L-forms, he also published extensively on the relationship between a molecule that stimulates the immune response called the Entobacterial Common Antigen (CA) and certain types of bacteria. He detailed the structure of the antigen and explained how it is able to elicit antibodies in humans and in animal models. He also detailed how the antigen could serve as a possible vaccine against urinary tract infections. He also studied the effects that a vasectomy might have on the immune system and performed studies on the relationship between the host and various species of bacteria in the disease pyelonephritis.

An L-form within a kidney cell containing two forms, one a large dense body and one a smaller body

He delved into the effects of antibiotic therapy and chemotherapy on patients with urinary tract infections, and performed several studies on bacteria that produce a substance called chorionic gonadotropin-like hormone, detailing the way the bacteria might be involved in an experimental tumor model. He was even the co-author of a publication that characterized the oral microbial flora of alligators in order to develop better therapy for alligator bites.

When asked recently about his work Domingue replied, “I worked in a controversial research area for decades, and I found that sticking to the facts and hard data are the best ways to make progress in a field. Meaningful experimental designs and careful interpretation and discussion of the results are of prime importance in science. The ultimate aim was always to seek the truth about the problem at hand. Unfortunately, in the area of L-form or cell wall-defective bacteriology, too often there have been conclusions (anecdotal) drawn without supporting scientific data. In my opinion, many of these studies have hampered progress in the field and especially the role of these cryptic organisms in bacterial persistence and expression of disease. Sometimes the controversial issues have become political, which is unfortunate.

An L-form within a renal cell of a patient with kidney disease

As far as I am concerned, modern technological tools are presently at hand to support all of the above microbiological and immunological findings at the molecular level… which is really what present day medical scientists, clinicians, pathologists are willing to accept as proof (maybe) of the role of such aberrant bacteria in disease.”

Indeed, molecular modeling has revealed how L-form bacteria are able to persist in the body and disable the immune system. Over the past few years, L-form bacteria have been linked to a wide array of chronic diseases, many of them previously considered to be autoimmune in nature. In 2002, biomedical researcher Trevor Marshall created a medical treatment that effectively kills L-form bacteria.

Now that L-form bacteria are known to cause a wide array of chronic inflammatory diseases, Domingue’s work is of utmost importance in allowing researchers to correctly demonstrate and understand their behavior.

SOURCES

Domingue, G., Lloyd, K., & Schlegel, J. U. (1974). In vitro phagocytosis of transitional phase bacterial variants utilizing autoradiography. Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.), 146(2), 635-42.

Domingue, G. J. (1980). Filterable cell-associated cryptic bacterial forms in immunologic renal diseases. Urological survey, 30(1), 1-4.

Domingue, G. J., Ghoniem, G. M., Bost, K. L., Fermin, C., & Human, L. G. (1995). Dormant microbes in interstitial cystitis. The Journal of urology, 153(4), 1321-6.

Domingue, G. J., & Schlegel, J. U. (1970). The possible role of microbial L-forms in pyelonephritis. The Journal of urology, 104(6), 790-8.

Domingue, G. J., & Schlegel, J. U. (1977). Novel bacterial structures in human blood: cultural isolation. Infection and immunity, 15(2), 621-7.

Domingue, G. J., & Schlegel, J. U. (1978). Novel bacterial structures in human blood. II. Bacterial variants as etiologic agents in idiopathic hematuria. The Journal of urology, 120(6), 708-11.

Domingue, G. J., Thomas, R., Walters, F., Serrano, A., & Heidger, P. M. (1993). Cell wall deficient bacteria as a cause of idiopathic hematuria. The Journal of urology, 150(2 Pt 1), 483-5.

Domingue, G. J., Woody, H. B., Farris, K. B., & Schlegel, J. U. (1979). Bacterial variants in urinary casts and renal epithelial cells. Archives of internal medicine, 139(12), 1355-60.

Domingue, G., & Woody, H. (1997). Bacterial persistence and expression of disease. Clin Microbiol Rev, 10(2), 320-344.

Domingue, G. J. (1982). Cell-wall Deficient Bacteria: Basic Principles and Clinical Significance. Reading, MA: Addison-Wesley Publishing Co.

Green, M. T., Heidger, P. M., & Domingue, G. (1974a). Demonstration of the phenomena of microbial persistence and reversion with bacterial L-forms in human embryonic kidney cells. Infection and immunity, 10(4), 889-914.

Green, M. T., Heidger, P. M., & Domingue, G. (1974b). Proposed reproductive cycle for a relatively stable L-phase variant of Streptococcus faecalis. Infection and immunity, 10(4), 915-27.

Ponig, B., Domingue, G., & Schlegel, J. (1972). The role of in vitro induced microbial L-forms in experimental hematogenous pyelonephritis. Investigative urology, 9(4), 282-5.

L-forms of various shapes and sizes inside the cells of a patient with breast cancer, photo taken by Alan Cantwell

Over the past century researchers have identified over 50 different species of bacteria capable of transforming into the L-form and it is likely that even more species will be discovered in the coming years. The vast majority of researchers and doctors are not aware that L-form bacteria are present in their patients because the pathogens will not grow under standard laboratory conditions and must be cultured in a different medium and at a different temperature than classical bacteria.

In 2005, a team of researchers at the Royal Brompton Hospital in London published a paper that reviewed the clinical significance of the L-form as an infectious agent. The review discussed the work of hundreds of researchers who have cultivated the L-form and implicated it in a wide array of diseases.

This piece describes several of the doctors and researchers discussed in the review who have perfected the lab methods needed to correctly culture L-form bacteria and have consequently been able to observe and document their behavior. L-forms have also been studied extensively in veterinary literature but those findings are not presented here.

In 1895 a scientist named Richard Pfeiffer described an altered form of the bacteria Vibrio cholerae that was difficult to see with a light microscope. Other workers in his lab confirmed that the bacteria lacked cell walls and were difficult to grow using standard laboratory techniques.

Around the same time, Ernest Alnquist, a friend of Louis Pasteur, began to culture the L-form. Alnquist was the first to suggest how extensive and diverse L-forms are. He once commented that “nobody can pretend to know the complete life cycle and all the varieties of even a single bacterial species. It would be an assumption to think so.”

Emmy Klieneberger-Nobel at a 1965 medical conference

In 1941, German scientist Emmy Klieneberger-Nobel began to study the L form at the Lister Institute in England. A meticulous lab worker, Klieneberger-Nobel, perfected the method of growing the pathogens on serum (blood) agar.

After growing colonies of the bacterium Streptobacillus monliforme she confirmed that several of the pathogens in her Petri dish did indeed lack cell walls. She named the wall-less variants L-forms after the Lister Institute where she worked. In the years that followed she studied other species of L-forms and published several papers describing their characteristics and behavior. She once said that “the L-form is an entity of its own as different from bacteria as the tadpole from the frog.”

Louis Dienes next to incubator used to grow L-form bacteria

A few years later, Harvard Medical School researcher Louis Dienes began to work with the L-form. He applied penicillin to various species of classical bacteria such as Salmonella typhosacoule and discovered that some of the pathogens transformed into cell wall-less variants of the same species. They also found that exposing the original strains to other antibiotics in the same class as penicillin, chemical injury, high levels of amino acids, lithium, calcium, chromatin and mercuric salts could cause L-form variants to form as well.

In many of his experiments Dienes noted that the individual L-forms in his samples often swelled into large round bodies. He also found that small colonies of L-form bacteria such as S.moniliformis are able to revert back to the classical form. He published numerous papers detailing his discoveries and was one of the first scientists to warn the medical community that some antibiotics such as penicillin can actually precipitate the formation of L-form bacteria.

Dienes kept his lab open at all times so that anyone interested could see his L-form cultures and ask questions about how to grow them correctly. Nevertheless, few of the other researchers at Harvard took note of his work. He once remarked that he only became known to the medical staff after a hospital art show displayed some of his watercolors.

Virginia Livingston

Around the same time, a team of doctors under Virginia Wuerthele-Caspe Livingston cultured L-form bacteria from patients with the skin disease scleroderma. Livingston noted that some of the L-forms she observed were as small as viruses. But others were the size of classical bacteria and some were larger forms resembling spores of fungi and yeasts

Livingston and her colleagues injected the L-form bacteria they collected from patients with scleroderma into chicks and guinea pigs. The chicks died. Some of the guinea pigs developed hardening of the skin like scleroderma, and some developed cancer. In the years that followed Livingston was also able to grow L-forms from various human cancer tumors.

Her work was published in the American Journal of Medical Sciences and in the years that followed she wrote several books on the subject.

In 1975, H.M Butler and team wrote a review of L-forms, describing their resistance to penicillin and ability to change form. They concluded that “such organisms may be clinically significant in cases of chronic and recurrent infection.”

At the same time, Bisset and Barlett identified L-form variants of Bacillus licheniformia during different stages of its life cycle. They hypothesized that the wall-less variants of the bacteria they observed had previously been wrongly classified as other species of pathogens.

Emil Wirostko’s photo of L-form bacteria inside a white blood cell

A decade later, a doctor working at Columbia University by the name of Emil Wirostko began to culture and photograph L-form. In a series of related experiments, he took white blood cells from the liquid inside the eyes of patients with sarcoidosis, juvenile rheumatoid arthritis and Crohn’s disease and observed them under an electron microscope.

He detected L-forms in many of the specimens and noticed that the bacteria were grouped into colonies and encased inside tubuoles. He also noted that they were separated from the environment inside the cell by a membrane or exoskeleton that kept them from being digested by the cell.

Wirostko published three papers that detailed his findings and took many pictures of the L-forms he observed, 20 of which are dispersed throughout his papers.

L-form bacteria inside the tissues of a patient with prostate cancer, photo by Cantwell

Around the same time, researcher Alan Cantwell took great interest in Livingston’s work and proceeded to study the L-form. He applied a technique called acid fast staining to the tissue sections of the skin and lymph nodes of patients with the lung disease sarcoidosis and found L-forms in his samples. Cantwell later isolated the L-form of Streptococcus B from the lymph nodes and blood of patients with HIV. He noted that the L-forms he observed could grow into extremely large forms and determined that they were what are known as “Russell bodies.”

In the years that followed, Cantwell took samples from the lymph nodes, skin tumors and other organs from the corpses of patients who had died from Hodgkin’s disease and cancer. He found copious amounts of L-form bacteria in his samples, including round forms resembling the bacterial species staphylococci and rod-shaped bacteria known as corynebacteria.

Over the course of his career Cantwell published over 30 papers and wrote several books that implicate the L-forms in chronic disease, including a book about Livingston and three other women who had worked on her research team.

Lida Mattman at her microscope

Around the same time, researcher Lida Mattman began to study the L-form. After working as senior bacteriologist at the University of Massachusetts, she became the director of a laboratory that evaluated specimens sent by mail from doctors around the world. Mattman, who would study and photograph the L-from over the course of decades, confirmed that the pathogens could vary widely in size and shape.

Mattman’s success in growing L-forms was due in part to a relentless drive to perfect the laboratory techniques which allowed her to culture the pathogens. She used fluorescent antibodies and a variety of staining techniques to view the various cell wall deficient forms. She even figured out how to grow them directly on slides.

Mattman studied patients with Tuberculosis and found that in every patient tested, the blood was saturated with a variety of L-forms.

She identified two different species of L-form bacteria in patients with Parkinson’s Disease. The L- form species of Borrellia burgdoferi was detected in patients with Lyme disease. She cultured serum from forty patients with multiple sclerosis and found a different species of the borrelia L-form present in her samples. Soon after, she detected Chlamydia pneumonia in the blood of patients who had suffered a pulmonary thrombosis. She also found bacteria that resembled M. tuberculosis in the blood of patients with the lung disease sarcoidosis.

In the end, Mattman detected dozens of species of L-form bacteria and was able to culture these wall-less forms of bacteria from the blood samples of patients with over 20 incurable illnesses. She published numerous papers throughout her career and in authored an entire medical textbook in which she details her findings.

In 1997 a team of researchers at Tulane University under Gerald Domingue published an extensive review article on chronic bacterial infection in Clinical Microbiological Reviews. Among their conclusions was the claim that ”the difficult to culture and dormant bacteria are involved in latency of infection and that these persistent bacteria may be pathogenic.”

The review also detailed how L-form bacteria are able to form electron dense bodies within previously infected cells. Domingue implicated L-form bacteria in several kidney-related diseases including pyelonephritis, glomerulonephritis, idiopathic hematuria, and Interstitial cystitis. He also speculated about their role in other diseases such as rheumatic fever, tuberculosis, syphilis, and rheumatoid arthritis.

In the review Domingue states “Certainly, any patient with a history of recurrent infection and persistant disability is sending the signal that the phenomenon (infection with L-form bacteria) is occurring. The so called autoimmune diseases in which no organism can be identified by routine testing techniques are particularly suspect.”

Over the course of his thirty-nine year career Domingue published 65 papers, monographs, and book chapters about L-form bacteria. He was invited to deliver over fifty international and national lectures about atypical forms of bacteria and wrote a book on the subject called Cell Wall-Deficient Bacteria: Basic Principles and Clinical Significance.

Rickettsia helvetica inside the cell of a patient with sarcoidosis, image from Kenneth Nilsson

Several years later, Kenneth Nilsson, a researcher at Uppsala University Hospital in Sweden, published photos of the bacteria Rickettsia helvetica living inside the white blood cells of patients with sarcoidosis. The fact that the bacteria was able to persist inside the cells suggested that something was very wrong with the patient’s immune systems. Dozens of other researchers have also implicated other species of L-forms in sarcoidosis.

A decade later, researchers at the Academy of Science in Bulgaria infected rats with the L-form of Staphylococcus aureus and found that the pathogen were able to “internalize, replicate and persist “ in the lungs of the infected rats. They concluded that “cell wall deficient bacterial forms may be involved in the pathogenesis of chronic and latent lung infections.”

Long thin L-forms emerging from a cell as shown in a photo taken by Andy Wright

A team of researchers and doctors in the United Kingdom are currently studying the L-form in patients with Chronic Fatigue Syndrome (CFS). The microbiology team, lead by CFS clinician Dr. Andy Wright has detected L-forms in every single one of the CFS patients they have tested (about 600 to date).

Wright has developed a method of taking pinprick blood (usually from the ear) and allowing it to degrade for 6-36 hours. The process causes the L-form bacteria to break out of the cells and they can subsequently be observed with a dark field microscope. The bacteria can be stained with fluorescent dye. If the L-forms are alive they will stain green, while dying/dead L-forms stain orange. Wright has created several videos of L-forms under the microscope in which the pathogens can be seen quite clearly.

In the videos, the bacteria often lengthen into long filamental forms that look thin and snakelike. They can be seen weaving in between infected cells. Sometimes “giant” L-forms, which are more rectangular in shape, begin to grow inside the cells.

Danish researcher Marie Kroun has also taken several videos of L-form bacteria under a high-resolution microscope.

Trevor Marshall, creator of the Marshall Protocol

However it’s quite possible that L-form bacteria might have remained in relative obscurity if a scientist from Adelaide Australia by the name of Trevor Marshall hadn’t taken an interest in their ability to persist in the body. Marshall wasn’t a medical doctor – he was an biomedical engineer with an impressive grasp of molecular modeling software. Whereas a doctor can look at a patient and infer a mechanism for disease based only on symptom presentation, a biomedical researcher can take the actual compounds created by L-form bacteria and mathematically determine how they affect the body’s receptors and enzymes.

Thus, after decades of research, Marshall was able to succeed in two areas where the L-form researchers before him had failed. By combining precise molecular modeling data with previous research on stealth bacteria, he was able to create a model that explained exactly how L-form bacteria are able to dysregulate the immune system and persist in the body. Secondly, he was able to use his model in order to create a treatment that effectively kills L-form bacteria. The Marshall Protocol was born. Patients on the treatment use pulsed, low-dose antibiotics, along with a medication that activates the immune system to eliminate L-form bacteria over a period of several years.

Patients with a wide array of chronic diseases are using the treatment. Most are reporting symptomatic improvement and a number of patients have claimed complete resolution of symptoms.

Marshall has since written several papers and given numerous presentations that detail the pathogenesis of chronic disease.

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Butler, H.M., & Blakey, J.L. (1975). A review of bacteria in L-phase and their possible clinical significance. The Medical journal of Australia, 2(12), 463-7.

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