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L-form bacteria

As a part of their natural life cycle, bacteria can transform into a variety of forms. One of those phases is the L-form. L-form bacteria, also known as cell wall deficient bacteria, are a phase of bacteria that are very small and lack cell walls.

Though the subject of a great deal of research over the last 100 years and implicated in a variety of diseases, L-forms remain largely misunderstood - or at the very least, underappreciated - by the medical research community. According to the Marshall Pathogenesis, L-forms are part of a metagenomic microbiota responsible for chronic disease.

Species capable of L-form transformation

Thus far, researchers have identified over 50 different species of bacteria capable of transforming into the L-form and it is likely that more species will be found in the coming years. “Probably most bacterial species can be converted into L-forms if treated with the antibiotics that inhibit cell wall synthesis,” states researcher Josep Casadesus. 1

Some of the species of L-form bacteria that have been implicated in chronic disease include Bacillus anthracis, Treponema pallidum, Mycobacterium tuberculosis, Helicobacter pylori, Rickettsia prowazekii, and Borrelia burgdorferi. Not all species cause disease.

Size and shape

L-form bacteria are pleomorphic, that is, they can change size and shape. During much of their lifetimes they are tiny, about 0.01 microns in diameter.

Since they are smaller than viruses or fungal particles, they cannot be seen with a normal optical microscope. The small, individual forms of L-form bacteria are often referred to as coccoid bodies. Coccoid bodies sometimes group together, assuming the appearance of a string of pearls

Occasionally L-form bacteria break out of the cells. In the lab they can grow into long, thin biofilm filaments that can reach 60-70 microns in length. The biofilm filaments are composed of L-form bacteria and a protective protein sheath. For reasons still unknown, L-forms can also grow into large “giant” bodies.

L-form bacteria lack flagella, long slender appendages that allow some forms of bacteria to propel themselves forward by using a whip-like motion. Instead they glide to their destinations in a snail-like fashion.

Groups of L-form bacteria are often encased inside tubules. They are also separated from the environment inside the cell by a membrane or exoskeleton that keeps them from being digested by the cell.

Resistance to certain antibiotics

Cell wall deficient variants of bacteria cannot be killed by many commonly used antibiotics. Multiple studies, from many years ago2 to the present, have also shown that when one of the Beta-lactam antibiotics, a class of antibiotics that includes penicillin, are applied to wild-type bacteria in a Petri dish, small colonies of L-form bacteria form on the edges of the plate. According to Casadesus:3

Treatment with penicillin does not merely select for L-forms (which are penicillin resistant) but actually induces L-form growth.

Josep Casadesus

Replication and reproduction

L-form bacteria replicate in various ways, including budding, filamentous growth and binary fission. Some species of L-forms such as Proteus can form large bodies that replicate by division. In other instances, granules bud from the body of the bacterium and give rise to small L-form colonies.

Survival mechanisms

Classical forms of most bacterial species can be found in the bloodstream. However L-form bacteria have figured out how to successfully infect and live inside the very cells of the immune system whose role is to kill bacteria. Once inside these cells, they can no longer be detected by the immune system and are able to persist in the body over long periods of time. L-form bacteria can infect many types of cells but prefer to infect white blood cells called macrophages.

Several very recent studies have confirmed the fact that bacteria can live inside the cells of the immune system. In a paper published in the Journal of Immunology by a team at the University of Michigan Medical School, Gabreil Nunez, senior author of the paper, stated “In our study, the presence of bacterial microbes inside the cell is what triggers the immune response.”4

Similarly, a team of researchers at the Bacterienne Institute in France released a paper detailing how the bacteria E. coli is able to live inside the cells of the immune system.

[E. coli are] true invasive pathogens, able to invade intestinal epithelial cells and replicate intracellularly. Strains also survive and replicate within the macrophages.

Nathalie Rolhion et al. 5

Markova has provided evidence that E. coli can survive lethal treatments such as boiling or autoclaving (subjecting equipment to high pressure steam at 121 °C or more) by transitioning into the L-form.6

Culturing and detection

Once bacteria have transformed into the L-form they can no longer be detected by many standard laboratory procedures. Although scientists have known about L-form bacteria for over a century, many of them have not detected them in tissue and blood samples because they are very difficult to culture.

Forms of bacteria with cell walls can be easily grown outside the body (grown in vitro). However L-form bacteria have great difficulty surviving in a foreign environment. In order to grow them successfully in the lab, conditions must be similar to those in the human body (grown in vivo). Consequently they can be cultured on a medium called blood agar at very specific temperatures and at a certain pH.

The concept that some bacteria cannot grow in vitro is not new. Scientists have known for decades that neither (Syphilis Treponema pallidum ) nor leprosy (Mycobacterium leprae) cannot be easily cultivated outside the body.

L-form bacteria take several measures to ensure they can survive for as long as possible inside a cell. They are able to infect all types of white blood cells, but seem to prefer to infect macrophages, the type of white blood cell, which at 45 days, has the longest life span.

Several studies have shown that once inside a macrophage, L-form bacteria are able to delay the process of apoptosis, or programmed cell death, allowing them to thrive inside the cell for a period of time even longer than 45 days.

PCR testing

Classical bacterial forms can be detected by a lab test called Polymerase Chain Reaction (PCR). PCR identifies and amplifies the proteins and DNA of bacteria that have been killed. However since L-form bacteria are able to persist inside the macrophages for such extended periods of time, few of them die and only tiny amounts of L-form bacterial proteins and genetic material reach the bloodstream at any given time; an amount so small that the PCR test cannot pick them up.

Even if a few small fragments from L-forms that have been killed are identified by PCR testing, the remains are often not from the bacterial species causing the most harm to the patient. This is because the most well adapted, persistent bacterial species are the ones who have developed the most effective survival mechanisms and are consequently least likely to die.

Antibody testing

L-forms can also not be detected with antibody testing. Antibodies are Y-shaped proteins that are found in blood. They are used by the immune system to identify and neutralize foreign objects including bacteria.

However, antibodies only form in response to bacteria that have died. Since L-form bacteria are able to persist for such long periods of time inside the cells, very few antibodies are created in response to their presence.

Constant rediscovery

In 1915, Löhnis authored a paper entitled “Life cycles of the bacteria” in which they marveled at the complexity of the bacterial life cycle but also his failure to recognize its potential significance earlier:

The senior author also did not know that for more than 11 years he had in his collection, patiently waiting to be photographed, that fine prepárate now shown in figure 6 (PI. A) until, as stated before, he decided to take down his “theoretical blinders.” We have already mentioned that a careful study of the illustrations contained in our daily used textbooks will now reveal several things which we were so very well trained not to see. Certainly the German philosopher Lichtenberg made a very wise remark when he said: Was jederman für ausgemacht hält, verdient am meisten untersucht zu werden. [What everyone thinks identified deserves to be studied the most]

More recently, scientists such as Lida Mattman at Wayne State University, Gerald Domingue, and Nadya Markova have worked extensively with the L-form and figured out new ways to grow and view the pathogens. These techniques include a variety of special staining techniques. Nevertheless, many doctors and researchers still question whether the L-forms actually exist.

Mattman and others have spent decades figuring out how to correctly culture the L-form. Applying their techniques correctly requires rigorous adherence to specific guidelines. Mattman has said that, over and over again, researchers misinterpreted just one of the steps required to correctly grow the bacteria. They then report to the medical community that no L-forms appear in their samples.

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.

Gerald Domingue, professor emeritus, Tulane University School of Medicine

“Features of a number of important but poorly explained human clinical syndromes strongly indicate a microbial etiology,” states Relman. “In these syndromes, the failure of cultivation-dependent microbial detection methods reveals our ignorance of microbial growth requirements.” 7

Select diseases linked to l-forms

  • psoriasis – In a 2009 study, Wang investigated the carriage rate of cell wall deficient bacteria in the tonsil or pharynx of psoriasis patients. CWDB were isolated from 74.2% of psoriasis patients, 23.5% of chronic tonsillitis patients and only 6.3% of controls. Meanwhile, our study showed that CWDB and wide-type bacteria significantly enhanced the production of IFN-gamma, in vitro, and peripheral blood mononuclear cells proliferation.8

Koch's postulates

Another problem with fully understanding the role of L-forms rests with the fact that many researchers rely on a series of rules called Koch’s postulates when interpreting research data. The postulates state that only one pathogen can cause a given disease. But research has shown each chronic disease is the result of infection with multiple species of L-forms.

This means that separate teams of researchers often detect different L-forms in patients with the same disease. For example both Borrelia burgdorferi and Rickettsia helvetica have been detected in patients with sarcoidosis. These findings make little sense to researchers still bent on adhering to Koch’s postulates.

Infection with L-form bacteria

People are exposed to L-form bacteria in many places. Because they cannot be killed by pasteurization or chlorination, L-form bacteria can be found in milk, food, and water. They can be transmitted via sperm, intimate contact, and can be passed from mother to child during childbirth. Since they are too small to be filtered during the purification processes used in pharmaceutical manufacturing procedures, they can be transmitted through injectable medicines. They have even been cultured from dry soil.

One 2007 trial for an AIDS vaccine had to be abruptly discontinued when scientists realized that the vaccine somehow raised the risk of infection. The researchers administering the trial stressed that the vaccine could not itself cause the infection. Although it was not mentioned in the news story about the incident, one very plausible hypothesis is that the vaccines were infected with L-form bacteria.

Once macrophages and other cells have been infected with L-form bacteria, the bacteria circulate in the blood and tissues. In some cases they cluster together in clumps called granulomas. In other cases, they accumulate in regions such as the joints.

After L-form bacteria have successfully invaded a cell, they begin to use the nutrients inside the cell to their own advantage, disturbing the cell’s delicate chemical balance. They are also able to take control of the host’s genetic material, which allows them to create proteins that enhance their ability to survive.

Inflammation as evidence of infection

L-form bacteria cause inflammation and painful symptoms by taking control of the protein known as Nuclear Factor Kappa B. They are able to activate proteins that increase the activity of Nuclear Factor Kappa B, which subsequently moves to the nucleus or center of the cell. Once there, it turns on a variety of genes that cause the release of inflammatory cytokines, proteins that generate pain and/or fatigue. These cytokines include interferon gamma and TNF alpha.

In this way, an inflammatory response is correlated with diseases caused by L-form bacteria.

An inflammatory immune response—one of the body’s primary means to protect against infection—defines multiple established infectious causes of chronic diseases, including some cancers.

David Relman, MD

Inflammation also drives many chronic conditions that are still classified as (noninfectious) autoimmune or immune-mediated (e.g., systemic lupus erythematosus, rheumatoid arthritis, Crohn’s disease). Both [the innate and adaptive immune systems] play critical roles in the pathogenesis of these inflammatory syndromes. Therefore, inflammation is a clear potential link between infectious agents and chronic diseases.

Siobhán M. O'Connor of the Centers for Disease Control and Prevention concurs, stating, “The epidemiologic, clinical, and pathologic features of many chronic inflammatory diseases are consistent with a microbial cause.”

Future research

There seems to be little incentive for scientists to study the L-form. Since the bacteria can be killed by simple low-dose antibiotic therapy, drug companies have little interest in investing money into related research. Researchers studying the L-form often find themselves with very little grant money but must still work long, tedious hours in the lab.

It is generally agreed among scientists that L-form bacteria are extraordinarily intriguing, interesting tools for biological study, yet the most neglected area of research has been on the role of these organisms in disease, particularly in host-pathogen interactions.

Gerald Domingue, PhD

Notes and comments

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These guys are looking at the same stuff Andy Wright was:

Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11358-63. Epub 2006 Jul 18. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Gorby YA, Yanina S, McLean JS, Rosso KM, Moyles D, Dohnalkova A, Beveridge TJ, Chang IS, Kim BH, Kim KS, Culley DE, Reed SB, Romine MF, Saffarini DA, Hill EA, Shi L, Elias DA, Kennedy DW, Pinchuk G, Watanabe K, Ishii S, Logan B, Nealson KH, Fredrickson JK.

Pacific Northwest National Laboratory, Richland, WA 99352, USA. yuri.gorby@pnl.gov Abstract Shewanella oneidensis MR-1 produced electrically conductive pilus-like appendages called bacterial nanowires in direct response to electron-acceptor limitation. Mutants deficient in genes for c-type decaheme cytochromes MtrC and OmcA, and those that lacked a functional Type II secretion pathway displayed nanowires that were poorly conductive. These mutants were also deficient in their ability to reduce hydrous ferric oxide and in their ability to generate current in a microbial fuel cell. Nanowires produced by the oxygenic phototrophic cyanobacterium Synechocystis PCC6803 and the thermophilic, fermentative bacterium Pelotomaculum thermopropionicum reveal that electrically conductive appendages are not exclusive to dissimilatory metal-reducing bacteria and may, in fact, represent a common bacterial strategy for efficient electron transfer and energy distribution.

PMID: 16849424

So are these people:

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Listeria L-Forms: Discovery Of An Unusual Form Of Bacterial Life

http://www.sciencedaily.com/releases/2009/09/090912145843.htm

The Foundation has now put Lida Mattman's second-to-last presentation on YouTube. This was filmed at our 2005 Chicago conference.

http://www.youtube.com/watch?v=WozrCFW0mRM

http://www.discoverymedicine.com/Gerald-J-Domingue/2010/09/23/demystifying-pleomorphic-forms-in-persistence-and-expression-of-disease-are-they-bacteria-and-is-peptidoglycan-the-solution/

PLoS One. 2009 Oct 6;4(10):e7316. Insights into the molecular basis of L-form formation and survival in Escherichia coli.9

Glover WA, Yang Y, Zhang Y. Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America. Abstract L-forms have been shown to occur among many species of bacteria and are suspected to be involved in persistent infections. Since their discovery in 1935, numerous studies characterizing L-form morphology, growth, and pathogenic potential have been conducted. However, the molecular mechanisms underlying the formation and survival of L-forms remain unknown. Using unstable L-form colonies of Escherichia coli as a model, we performed genome-wide transcriptome analysis and screened a deletion mutant library to study the molecular mechanisms involved in formation and survival of L-forms. Microarray analysis of L-form versus classical colonies revealed many up-regulated genes of unknown function as well as multiple over-expressed stress pathways shared in common with persister cells and biofilms. Mutant screens identified three groups of mutants which displayed varying degrees of defects in L-form colony formation. Group 1 mutants, which showed the strongest defect in L-form colony formation, belonged to pathways involved in cell envelope stress, DNA repair, iron homeostasis, outer membrane biogenesis, and drug efflux/ABC transporters. Four (Group 1) mutants, rcsB, a positive response regulator of colanic acid capsule synthesis, ruvA, a recombinational junction binding protein, fur, a ferric uptake regulator and smpA a small membrane lipoprotein were selected for complementation. Complementation of the mutants using a high-copy overexpression vector failed, while utilization of a low-copy inducible vector successfully restored L-form formation. This work represents the first systematic genetic evaluation of genes and pathways involved in the formation and survival of unstable L-form bacteria. Our findings provide new insights into the molecular mechanisms underlying L-form formation and survival and have implications for understanding the emergence of antibiotic resistance, bacterial persistence and latent infections and designing novel drugs and vaccines. PMID: 19806199

Listeria L-Forms: Discovery Of An Unusual Form Of Bacterial Life

References

4 Kanneganti TD, Lamkanfi M, Kim YG, Chen G, Park JH, Franchi L, Vandenabeele P, Núñez G Pannexin-1-mediated recognition of bacterial molecules activates the cryopyrin inflammasome independent of Toll-like receptor signaling. Immunity. 2007;26:433-43.
5 Rolhion N, Darfeuille-Michaud A Adherent-invasive Escherichia coli in inflammatory bowel disease. Inflamm Bowel Dis. 2007;13:1277-83.
6 Markova N, Slavchev G, Michailova L, Jourdanova M. Survival of Escherichia coli under lethal heat stress by L-form conversion. Int J Biol Sci 2010; 6:303-315.
8 Wang GL, Li XY, Wang MY, Xiao DG, Zhang YY, Yuan XY, Wang QY, Song JJ Cell-wall-deficient bacteria: a major etiological factor for psoriasis? Chin Med J (Engl). 2009;122:3011-6.
Last modified: 10.03.2013
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