In order to better understand horizontal gene transfer, I spoke with Dr. Peter Gogarten at the University of Connecticut and Dr. James Lake at UCLA, both of whom are leaders in the field of gene transfer. Both of them were extremely friendly and seemed excited to speak with me about the phenomenon. I asked them the same questions. Here is how they responded:

Can you tell me a little about why horizontal DNA transfer (HGT) is so important?

Lake:   Well, without taking horizontal gene transfer into account, how do we explain the fact that prokaryotes (bacterial organisms) continue to trade genes even though they have no means of sexual reproduction? The only way that new bacterial species can form, and populations of bacteria can adapt and conform to new circumstances, is if they exchange DNA or genes during their lifetimes.

Dr. James Lake

We now realize that organisms with similar characteristics find it much easier to swap DNA. But on occasion, a group of organisms, such as a species of bacteria, can trade DNA with a class of organisms that have very different characteristics. If this does happen, it means that through the process of horizontal gene transfer, a bacterial species can acquire a host of new characteristics, even from organisms that are quite different from them. These new acquired characteristics may or may not offer them a survival advantage, but if the acquired traits do endow them with an advantage, they may be able to survive in a new environment or infect a new species – anything along those lines.

So, in my opinion, it turns out that the exchange of genes among prokaryotes is more fundamental than we’ve ever thought it to be in the past. Because of horizontal gene transfer, the evolution of many species, many different types of bacteria, and also multi-celled organisms are all entangled. Our genetic histories are definitely the result of our DNA mixing with the DNA of other species, including bacteria. Clearly this phenomenon plays a significant role in the body.

Gogarten:   Horizontal gene transfer allows us to understand how organisms such as bacteria, that don’t have sex, are able to exchange genetic material and create genetic diversity amongst their populations. Human beings and most mammals reproduce via sex in what is called vertical gene transfer. In the case of vertical gene transfer, two sets of different chromosomes (that contain different genes), one from each parent, combine, so that the offspring has a combination of genes from both mother and father. So horizontal gene transfer, the type of gene transfer that occurs between bacteria, viruses etc, is another mode of sharing DNA that still fosters diversity. If bacteria and other organisms couldn’t trade genes via horizontal gene transfer, then there would be no recombination at all, bacteria would not be able to change or acquire new characteristics from generation to generation. Species that have more in common are likely to trade genes more often. However even species that are not of the same lineage can trade genetic material. For example, research has shown that over the past million years, species of bacteria picked up DNA from the domain Archea, prokaryotes that are very different than bacteria. These exchanges may be rare, but still occur.

The Marshall Protocol puts forth the idea that diseases of unknown cause are bacterial illnesses. How do you feel about this hypothesis?

Lake:   I am open to the idea that bacteria may be behind diseases of unknown cause. Lately, I have been fascinated by many of the studies which have found that certain species of bacteria in the gut affect an individual’s tendency to gain weight. Even before these studies came out, I’d been thinking about such a possibility for years. I thought a connection would be found. I started to think about the possibility after taking a trip to Japan about four years ago. I was helping advise a steel plant which had just built two refineries for their waste (waste can easily be infected by bacteria). They were identical – they had the exact same design, were the exact same size, and had the exact same content inside (remnants of waste). Yet one of the refineries worked perfectly well and the second refinery simply didn’t work. We ended up taking the bacteria from the refinery that didn’t work and transferring the populations over to the refinery that did work – much like the researchers in these obesity studies take bacteria from obese mice and implant them into thin mice. In the case of the refineries, once we did the bacterial transfer, the second refinery stopped working in the exact same manner as the first. So clearly, certain species of bacteria determined whether each refinery was able to function. I went away thinking, “If this can happen in a reactor mill, it’s got to be able to happen in the body!”

Dr. Peter Gogarten

Gogarten:   I do believe that in the future we will discover that many more diseases of unknown cause have a microbial component. I believe the fact that we have not implicated bacteria in more diseases is related to our inability to correctly culture so many different forms of pathogens. Current culturing mechanisms are obviously very poor at identifying the presence of bacteria. Once molecular technology becomes used more frequently, we will probably be able to detect more pathogens and recognize their association with disease. I also suspect that we will be hearing more about how an imbalance of bacteria in the body can cause disease.

I’m very interested in this question. At what rate do you think horizontal gene transfer occurs in the body? Is it happening constantly? Does it only happen on occasion?

Lake:   Right now we are only able to estimate and guess about the exact rate of horizontal gene transfer that occurs between organisms. As I mentioned before, it is much easier for similar organisms to trade genes, so HGT happens more frequently between such organisms rather than in organisms with different characteristics. I can’t tell you an exact rate, but I do believe that gene transfer occurs very frequently among similar organisms because of the fact that such transfer happens relatively easily and there are several different ways for DNA transfer to occur. Among all these options, it’s probable that transfer happens quite often. There are three fundamental ways that organisms exchange DNA:

The first is called conjugation. This process simply involves introducing new genetic material into a different organism. Whether the genetic material is actually incorporated by the new organism is something that’s harder to track. It used to be thought that conjugation was a way to explain how bacteria like E. coli might have “sex” or foster new organisms with different genetic characteristics. But researchers soon realized that E.coli can also exchange genetic material with organisms with very different characteristics (like cyanobacteria) through conjugation. So it’s not technically sex if it can happen between very different organisms. Still, this is one of the easiest ways to exchange DNA and can be performed in the lab.

The second way that organisms exchange DNA is through a process known as transformation. I’m really interested in transformation. Several decades ago it was mistakenly thought that organisms actually feared strange DNA, or DNA from organisms not like themselves. However, we now realize that this is definitely not the case. Now we know that, in the lab, it’s possible to take organisms with very different DNA, put them in solution, and electrically shock the plate. During the shock, the organisms in the plate trade much of their DNA. This suggests that under stressful situations in particular, organisms are more likely to engage in gene transfer. So, in the body, gene transfer may be particularly common under situations of stress or starvation. If an organism finds itself in a cell that isn’t getting adequate nutrients, it’s logical that it would try to swap DNA with another nearby organism in the chance that the DNA swap might offer it some sort of survival advantage. It’s quite possible that the DNA swap would have no effect, but then again, maybe the swap could give the bacterial species an enzyme that would allow it to use an alternate energy source still available in the cell. This may be how bacteria remain alive when they are forced to go into “survival mode.”

Last, but not least, gene transfer can occur through transduction – a process in which virus infect bacteria or humans and in the process integrate their DNA into the organism they have infected. So it’s perfectly likely that if a person is infected with both bacteria and viruses, the two forms of organisms can swap DNA.

Gogarten:   This is a difficult question. It’s very hard to estimate the rate of HGT. But several studies have been eye-openers to me, suggesting that horizontal gene transfer happens quite frequently. I remember a study in which researchers looked at three different E. coli genomes. Basically, they just took the three genomes and sequenced their DNA. Without taking HGT into consideration one would expect the genes of each E. coli in each genome to be identical because they are all the same species. But, after using a molecular technique, the researchers found that only 40% of the genes that each E. coli harbored were identical. 60% of the genes differed between each E.coli genome sequenced. This suggests that among E. coli, and other similar bacteria, an enormous amount of horizontal gene transfer is taking place during just a short period of time. A really amazing amount of transfer.

One must understand that when bacteria and other organisms swap DNA through HGT, most of the changes that occur when the DNA is swapped are not important or fail to give an organism that acquires new DNA a survival advantage. But in some cases the swap results in a situation that endows another bacterium with a plasmid, or a protein that does provide an advantage, allowing the organism to live in a new ecological niche or survive new environmental conditions. This is how bacteria end up adapting to new challenging circumstances. But for the most part, it is hit or miss. I’d say the occasions on which organisms are actually conferred a serious survival advantage thanks to HGT are rare. They are, for the most part, the exceptions. But when they do occur, the organism involved in the transfer can really benefit.

Also take, for example, the amount of HGT that probably goes on inside bacterial biofilms. Biofilms are definitely environments that foster HGT. I remember a study where researchers took several plasmids and stained them with a fluorescent dye. They did this so that if they were taken up by another organism, you could see them glowing inside and know the plasmid DNA had indeed been incorporated. In this particular case, the researchers started with two bacterial biofilms and introduced several of these plasmids. Soon, both biofilms were glowing with light. So it was obviously quite easy for the organisms in the biofilms to pick up new genes.

Right now, I am very interested in studying xenobacteria, single-celled organisms that live in oceans. There are many strains of these bacteria, and all of them differ because, over time, each acquired different genes. Some of the genes that all xenobacteria picked up allowed them to change from normal bacteria to organisms that are able to perform photosynthesis under the water. Even now, the different strains of xenobacteria still shuttle genes back and forth. It’s almost like trying to study and classify Darwin’s finches. Each bird acquired (through vertical gene transfer) genes that allowed it to adapt to its own niche and find unique sources of food. In the same sense, HGT has allowed xenobacteria to do the same thing, and tracking their diversity is just as interesting as looking at the differences among Darwin’s finches. Studying xenobacteria has convinced me of the tremendous amount of horizontal gene transfer that takes place in the ocean.

Do you think that researchers currently underestimate the amount of horizontal gene transfer occurring in the body, or is the concept of horizontal gene transfer taken adequately into consideration in the studies you read?

Lake:   It’s hard to tell because the phenomenon is so complex. For the most part, it seems that researchers are making an effort to account for horizontal gene transfer but some of the genetic changes that occur due to the phenomenon may definitely be too subtle for us to pick up. The whole process is likely too complex to be fully accounted for in the average study.

Gogarten:   I think the pendulum swings back and forth. In the 1940s, scientists had largely given up on the idea that we could create a tree of life – a chart that would show us the lineage of organisms on Earth. Researchers figured because so much HGT was going on, it would be impossible to separate species into distinct lineages. Then, over the past decades, researchers like myself have given thought to the possibility that we may indeed be able to classify organisms, at least to some extent, despite the fact that they so frequently trade DNA. Yet rather than a tree of life, I think what we have to envision when we think of connections between species is more like a web, a network – where there are main lines of ancestry, yet some species that don’t fall strictly into any category between them. In this area, I think we are just scratching the surface of what we will find when we really start to learn more about how HGT has affected the evolution of organisms over the course of history.

Any parting thoughts on HGT?

Lake:   These are very exciting times. I feel that in the next five years our whole view of the evolution of life may change as we continue to take this phenomenon into account. I’m glad you’re taking a close look at the characteristics of bacteria on your site.

Gogarten:   My work has showed me that HGT is capable of endowing organisms with dramatically new traits and completely change the capabilities of microorganisms.

Just this month, researchers led by John H. Werren at the University of Rochester in New York elucidated yet another way that bacterial DNA is likely passed from person to person. This demonstrates just how easy it is for bacterial DNA to become incorporated into human DNA – a reality that is central to biomedical researcher Trevor Marshall’s model of chronic disease in which pathogens are constantly swapping genetic material with each other and their host.

The study describes how due to horizontal gene transfer – or the reality that once inside the body, organisms swap genetic material with each other, and also with the host – bacterial DNA often ends up integrated into human DNA. This integrated genetic material is then passed from generation to generation, and it is very likely that many of these acquired segments of DNA may help bacteria survive more easily in the body. “Our data are indicating that [DNA transfer] is going on all the time,” says Werren.

“The mechanism therefore provides an alternative to mutation of existing DNA as a way for the species to acquire new genetic traits,” states Patrick Barry of Science News. “The transfer of DNA from bacteria means that an individual could acquire and pass on genes that it had not inherited.”

Warren’s team looked at several species of insects and roundworms infected by a parasitic bacterium called Wolbachia pipientis. The bacterium lives inside the animals’ cells, including their egg cells, giving it ready access to the chromosomes that are passed on to the animals’ offspring.

When the researchers compared the genetic code of the bacterium with the code of 11 other species: four roundworms, four fruit flies, and three wasps, they found that all but three of the fruit fly species had segments of the bacterium’s genetic code embedded in their DNA.

The team also scanned an archive of published genomes for 21 other invertebrate species and found bacterial genes in nine of them – proving that bacterial DNA can indeed be passed from mother to child. Whether this occurs in humans has not yet been demonstrated, but in principle, seems quite possible.

But this process has been taking place for centuries. Why hasn’t it been analyzed sooner?

“Such bacterial genetic code is routinely ignored during the sequencing of animals’ genomes because most scientists have assumed that the foreign DNA is a sign of contamination, Werren says. However, the new research rules out the possibility of contamination, says the scientist.

Paul Ewald

One example of this is schizophrenia; patients with this mental illness rarely reproduce. Ewald posits that if schizophrenia were a genetic illness, the genes that cause the disease would have gradually been eliminated from the population. And what about identical twins who share the exact same DNA? When one identical twin develops breast cancer the other twin has only a 10% – 20% chance of also developing the disease. Ewald argues that “for the common damaging chronic diseases, the evidence considered in light of evolutionary principles implicates infection” and that “adding infectious causation into the mix can best explain the documented epidemiological patterns, and does so in accordance with evolutionary principles.”

For Ewald, the conclusion is inescapable. In the book The Next Fifty Years: Science in the First Half of the Twenty-First Century, he states that, “Given the implications of evolutionary theory, the march of medical research, and the accumulated evidence, I expect that the common and highly damaging chronic diseases– atherosclerosis, diabetes, Alzheimer’s disease, most cancers, and most fertility problems– will, in the next fifty years, be accepted as caused by infection….” According to the molecular biologist, this scenario is supported by history, which has demonstrated that the most widespread and deadly diseases such as AIDS, malaria, and syphilis have already been identified as infectious. Ewald makes a provocative statement, but where are these microbes and how can they be identified?

Ewald’s above prediction is gaining strength as research teams are beginning to use powerful molecular techniques in order to better identify the presence of diverse and novel forms of bacteria in human subjects. Several recent studies by researchers at New York University have revealed that the most numerous cells in the human body are bacterial, outnumbering our cells 10 to 1. The latest study by the research team focuses on bacteria in the skin. “The skin is home to a virtual zoo of bacteria,” reports Dr. Martin J. Blaser, one of the authors of the study. The research is part of an emerging effort to study bacteria. The team has previously examined bacterial populations in the stomach and esophagus.

These studies are prime examples of how new molecular techniques are able to identify bacteria that many scientists and clinicians never realized existed in their samples. Traditionally, bacteria are grown in the lab in setups such as Petri dishes, which contain nutrients that foster the growth of bacteria. But Dr. Zhan Gao, lead author of the NYU study, argues that these methods lead to inaccuracies because only a fraction of bacteria in any given sample actually grow in the medium. So his team used powerful molecular techniques to identify and analyze the bacteria on the forearms of six healthy subjects – three men and three women. “This is essentially the first molecular study of the skin,” says Blaser. “There are probably fewer than ten labs in the U.S. looking at this question. It’s very intensive work.” In fact, the research took three years to complete.

Molecular models showing the structure of 16s RNA

After rubbing a swab on each individual’s forearms, the researchers used special tools known as primers to extract a unit of genetic material from each sample. The sequence extracted was 16S ribosomal DNA, a conserved gene present in every known species of bacteria. 16S ribosomal DNA is of particular value to researchers in that it is species-specific. Blaser and team compared their samples to a database that lists individual species of bacteria along with their unique sequence of 16S ribosomal DNA. When two sequences matched up, they were able to determine the type of bacteria present. Some sequences in the database represent the DNA of bacteria that have yet to be named and identified. These sequences are known as SLOTUs, or species-level operational taxonomic units.

Roughly half or 54.4% of the bacteria identified in the samples represented the genera Propionibacteria, Corynebacteria, Staphylococcus and Streptococcus, or species of bacteria which have long been considered more or less permanent residents of human skin. But about 100 species of bacteria were present that had never been previously detected in the skin, causing the team to conclude that “cultivation methods [growing in Petri dishes etc] substantially under-represent the extent of bacteria diversity.”

In fact, 8% of the 16S ribosomal DNA sequences corresponded to unknown bacterial species that have never yet been described. The paper states, “Previously uncharacterized [types of bacteria] were common in this study, some displaying >10% sequence dissimilarity from published sequences.” The bacteria observed differed substantially among the six subjects. Only 2.2% of the SLOTUs and 6.6% of the identified bacteria were found in all six subjects, indicating that the populations of bacteria in the skin are highly diverse.

Not all the bacteria in a given sample can be culture in a petri dish.

So why might 8% of the 16S ribosomal sequences detected by Blaser and team correspond to bacteria yet to be characterized?

Besides the fact that there are almost certainly species of bacteria yet to be discovered, it’s doubtful that researchers will ever fully characterize the bacteria in their samples without developing an understanding of L-form bacteria. Over the past few decades, researchers such as Emmy Nobel, Emil Wirotsko, Gerald Domingue, and Andy Wright have published studies which demonstrate that part of the life cycles of many bacteria (including those detected by Blaser and team) include phases where they transform into small forms that lose their cells walls. These bacteria are called cell wall deficient (CWD) or L-form bacteria. L-form bacteria live inside human cells, including cells of the immune system called macrophages.

In a recent BioEssay, Dr. Josep Casadesus at the University of Sevilla argues that every species of bacteria is capable of transforming into the L-form. L-form bacteria have been implicated in a wide array of diseases including sarcoidosis, Alzheimer’s, cardiovascular disease, rheumatoid arthritis and multiple sclerosis. Nearly everyone acquires substantial levels of L-form bacteria as they age. In fact, according to biomedical researcher Trevor Marshall of Autoimmunity Research Foundation, the diseases generated by L-form bacteria are far more common than currently realized, and are often only noticed as subtle signs of aging, such as osteoporosis, obesity, fatigue and arthritis.

Then, research indicates that most humans also appear to harbor myriad bacterial biofilm communities. Biofilm bacteria are able to evade the immune system by living inside self-created polymeric matrices. They are also very difficult to culture without the use of molecular technology. In 2004 paper in Nature Reviews, Paul Stoodley of the Center for Biofilm Engineering at Montana State University describes many reasons why biofilms are extremely difficult to culture, such as the fact that the diffusion of liquid through a biofilm and the fluid forces acting on a biofilm must be carefully calculated if it is to be cultured correctly. According to Stoodley, the need to master such difficult detection techniques has deterred many scientists from attempting to work with biofilms.

Nevertheless, in just a short period of time, researchers using molecular technology to study internal biofilms have already pegged them as the cause of numerous chronic infections and diseases, and the list of illnesses attributed to these bacterial colonies continues to grow rapidly. According to a recent public statement from the National Institutes of Health, more than 65% of all microbial infections are caused by biofilms. So, according to Marshall, when it comes to the pathogens that cause chronic disease, we may be dealing with a chronic, intraphagocytic, metagenomic microbiota – a microbiota that has been largely ignored because they are so difficult to culture ex-vivo.

Another factor that may account for unclassified sequences is that of horizontal DNA transfer. Pathogens, including classical bacteria, biofilm bacteria, and L-form bacteria behave much differently inside the body (in vivo) than in the lab (in vitro). Scientists now realize that inside the body, bacteria and other pathogens exchange genetic material, a process that is known as horizontal gene transfer. James Lake, a researcher at the Molecular Biology Institute at the University of California, puts it, “Increasingly, studies of genes and genomes are indicating that considerable horizontal gene transfer has occurred between bacteria.” In fact, due to increasing evidence suggesting the importance of the phenomenon in organisms that cause disease, molecular biologists such as Peter Gogarten at the University of Connecticut have described horizontal gene transfer as “a new paradigm for biology.”

A plasmid. Bacteria engage in horizontal gene transfer by passing each other plasmids.

Bacteria often engage in horizontal gene transfer by passing each other plasmids, circular molecules of DNA that can replicate independently of a pathogen’s other genetic material. This means that every analysis of bacteria is likely to turn up species of pathogens that can’t be classified, since some species may have traded parts of their 16S ribosomal DNA sequences with other pathogens in the body.

For Dr. Blaser and team, the next step is to investigate the bacteria in diseased skin, which they are now doing. “We plan to ask the question: Are the microbes in diseased skin, in certain diseases like psoriasis or eczema, different than the microbes in normal skin?” says the researcher.

The answer to Dr. Blaser’s question is likely to be yes, and as noted above, the sequences identified may very well correspond to DNA from biofilm, L-form, or other chronic persistent bacterial species. Are the 16S RNA sequences of L-form bacteria unique from those of their acute counterparts? Nobody knows for sure, since the genetic material of L-form bacteria has yet to be sequenced with molecular technology. But if bacteria in the L-form do have unique genetic sequences, as Blaser and team proceed to collect samples from the skin of people with eczema, psoriasis, and other chronic diseases, they may discover even more unknown 16S RNA sequences. That’s because the above diseases have been linked to the presence of cell wall deficient pathogens.

Then again, since the L-form is part of the natural lifecycle of many bacterial species, 16S RNA sequences may be conserved when classical bacteria transform into their cell wall deficient counterparts.

Blaser’s work seems to validate Marshall’s pathogenesis for chronic inflammatory disease. Marshall argues that every human accumulates what he describes as “pea soup” – a unique mix of pathogens that vary from person to person depending on what microbes they have encountered during various stages of life. Marshall has argued that these bacteria, the ones that have escaped most researchers’ attention until the advent of molecular techniques, are precisely the ones that cause people to fall ill with various forms of chronic disease.

16s RNA can be isolated using new molecular techniques

In fact, the six subjects of the study were found to share only four species of bacteria: Propionibacterium acnes, Corynebacterium tuberculostearicum, Streptococcus mitis, and Finegoldia AB109769. Almost three-quarters, or 71.4%, of the total number of bacterial species were unique to individual subjects, suggesting that the skin surface harbors a highly diverse mix of bacteria.

When it comes to the bacteria that cause chronic disease, Marshall argues, “I believe we have a totally different microbiota in play, one involving bacterial families never dreamed to exist in man.” He points to another study in which researchers from the Infection and Immunity Research Group in England isolated bacteria attached to the surface of prosthetic hip joints of ten subjects. As with the NYU study, the 16S RNA genes of the bacteria were sequenced, identified, and amplified using PCR.

The team identified bacteria such as Lysobacter, Gamma proteobacterium N4-7, Methylobacterium and Staphylococcus epidermidis. However, 7% of the samples once again represented bacteria that could not be identified. The team stated that “evidence exists that highly fastidious or non-cultivable organisms have a role in implant infections.” As mentioned above, the organisms they are having trouble identifying probably include biofilm, L-form, and other persistent bacterial species, as well as new strains and/or species created by horizontal gene transfer. Then, of course, some may simply correspond to species yet to be discovered.

“The tables in that paper give a list of DNA from species which boggle the mind,” says Marshall. “What we are dealing with [in people with chronic disease], is not bacteria such as Streptococcus or Staphalococcus. We are dealing with an ancient microbiota, one which has lived in harmony with man for millennia, but which has become dominant during the last few decades due to ill-advised changes in man’s lifestyle, and some aspects of modern medicine.”

The convergence of what is implied by evolutionary biology and what is revealed by molecular techniques is at hand. Sequencing the DNA of the individually infected cells of chronically ill people will go a long way towards showing that, as Ewald predicted, chronic disease may be driven by bacterial microbes.

However in 1867 a scientist named Robert Koch discovered that anthrax is able to cause disease and was able to successfully transfer the germ from cows to mice. Since that time, bacteria have been implicated in an ever greater range of diseases.

Over the past few decades, scientists such as Lida Mattman, Alan Cantwell and Trevor Marshall have provided great evidence for the hypothesis that chronic diseases ranging from arthritis to Alzheimers are the result of bacterial infection. Nevertheless, a great majority of the medical community still feel that these diseases are caused by toxins in the environment or are autoimmune in nature.

Koch’s Postulates

After working with anthrax, Koch developed a series of ground rules to determine whether a given organism can cause a given disease. These rules, known as “Koch’s Postulates” state that a scientist must find the same microbe in every person with a given disease. Furthermore, the specific microbe must be able to be grown on pure culture medium in the lab and when reintroduced into a healthy animal or person must produce the disease again.

Robert Koch in his laboratory

Many researchers still believe that Koch’s rules are universal and correct despite the fact that a massive body of research has shown that the principles are outdated and can no longer be applied to a modern understanding of disease.

For example, in the early 19th century researchers realized that viruses invalidate Koch’s Postulate because they require another living cell in order to replicate. According to TD Brock at the American Society of Microbiology, attempts to rigidly apply Koch’s postulates to the diagnosis of viral diseases may have significantly impeded the early development of the field of virology.

But the fact that scientists are still trying to apply Koch’s Postulates to bacteria is causing an even greater array of problems. For one, bacteria in the L-form cannot be easily grown in the lab and can only be studied in conditions that mimic those of the human body. As Gerald Domingue, Professor Emeritus at Tulane University states, “When it comes to L-form bacteria, Koch’s Postulates cannot be fulfilled because it is impossible to duplicate all the variables involved in disease expression.”

It is already widely accepted that some species of bacteria cause disease despite the fact that they do not fulfill Koch’s Postulates since Mycobacterium leprae and Treponema pallidum, (which are implicated in leprosy, and syphilis repectively) cannot be grown in pure culture medium.

Horizontal Gene Transfer

Another problem with the postulates is that they do not take into account a phenomenon called horizontal gene transfer. Horizontal gene transfer is a process in which organisms swap genetic material. The phenomenon is itself a challenge to Koch’s Postulates, which state that only one organism can be isolated, cultured, and held responsible for causing a single disease.

Over the past few decades, humans have entered into an age in which new technology has made it possible to sequence an organism’s genetic code. Scientists now know that bacteria can insert genetic material into the genomes of other pathogens or into the genome of their host. They often do this while in the form of plasmids, circular molecules of DNA that can replicate independently of a pathogen’s other genetic material.

For example, researchers at the Cancer Research Institute in Slovakia analyzed the bacterial DNA isolated from the intestinal tract of 11 American and 30 Slovak patients with HIV/AIDS. They found that the intestinal bacteria genes were more than 90% homologous to the corresponding sequence in HIV – suggesting that the bacteria and the HIV virus had traded a significant amount of genetic material.

A diagram of a plasmid, a circular molecule of DNA

James Lake, a researcher at the Molecular Biology Institute at the University of California, puts it, “Increasingly, studies of genes and genomes are indicating that considerable horizontal gene transfer has occurred between bacteria.” In fact, due to increasing evidence suggesting the importance of the phenomenon in organisms that cause disease, molecular biologists such as Peter Gogarten at the University of Connecticut have described horizontal gene transfer as “a new paradigm for biology.”

Gorgarten insists that horizontal gene transfer is “more frequent than most biologists could even imagine a decade ago” and that this reality turns the idea that we can classify organisms in a simple “tree of life” on its head.

Instead Gogarten suggests that biologists use the metaphor of a mosaic to describe the different histories combined in individual genomes and use the metaphor of a net to visualize the rich exchange of DNA among microbes.

A molecule of DNA

Trading plasmids

This transfer of DNA among pathogens means that once harmless microbes can acquire properties that allow them to cause problems for the host. “The mobile nature of..gene islands, transported between bacteria via plasmids or phages, creates the potential for acquired virulence in previously innocuous microbes,” states researcher Dave Relman of Stanford University. “This concept should inspire some reflection the next time one receives a culture report reading “normal flora.”

Take, for example, the bacterial species Bacillus anthracis, a species of bacteria that has two plasmids. One plasmid codes for genes that allow the pathogens to create toxins, the other codes for proteins that help it evade the immune system by living inside the white blood cells that kill and digest bacteria.

Bacillus anthracis can be found in soil, so people can pick it up relatively easily. Once inside the body it comes in contact with other species of bacteria. Let’s say it encounters Bacillus cereus, a species of bacteria that causes foodborne illness. The two bacteria may trade genetic material. If Bacillus cereus picks up the plasmids for creating toxins and evading the immune system from Bacillus anthracis, it will be much more successful at staying alive, persisting inside the cells, and ultimately causing problems for the host.

Some bacteria have more than 20 plasmids. Also it should be noted that other types of pathogens such as viruses can and do engage in horizontal gene transfer. This activity must be accounted for. Consider this situation envisioned by biomedical researcher Trevor Marshall.

Actual plasmids as seen under an electron microscope

“If you take the 21 plasmids of Borrelia, they can transfer DNA in 21! (21 factorial) combinations with other species, which is a VERY large number. Then you have to add in the DNA in the plasmids of the other key species – Staph, Rickettsia, Strep, Treponema, E.coli, Bacillus, and then add all of their chromosomes, add in the remaining non-plasmid bacterial species (like Mycobacteria), add the viruses, stir the soup together, accumulating new components for a few decades, and the number of combinations of pathogenic DNA in our cells becomes virtually infinite.”

DNA “Soup”

The image of people acquiring their own unique “soup” or mix of pathogens is a good way to visualize chronic disease. Marshall’s molecular models indicate that chronic disease results when individuals begin to accumulate different species of L-form, biofilm or other choronic bacteria. Biofilm bacteria create proteins such as capnine which are able to bind and inactivate the Vitamin D Receptor (VDR), the fundamental receptor of the immune system. Thus, as an individual accumulates more and more disease-causing bacteria, their immune systems start to shut down.

A molecular model by Dr. Trevor Marshall showing a molecule of capnine bound into the vitamin D receptor.

When this occurs, people have a very hard time keeping other pathogens under control. They often find that childhood viral infections reactivate, or that they acquire Candida (pathogenic yeast) and mycoplasma as well. Thus, according to Marshall, every person who falls ill with chronic disease has a different mix of pathogens to kill depending on what microbes they have encountered during various stages of life.

It does seem that only one species of bacteria is involved in each disease, then patients with the same illness would manifest with much more similar symptoms. On the contrary, patients with the same chronic disease often report very different aches and pains.

This isn’t to say that people with the same diseases might not all have some species of bacteria in common, or that specific bacteria don’t generate specific symptoms. It simply means that a combination of pathogens all likely contribute to different aspects of an illness. Consequently, the idea that only one of the pathogens can be isolated, cultured in the lab, and blamed for the entire disease seems increasingly implausible.

Bacteria growing on pure culture medium in the lab

Furthermore, as a result of horizontal DNA transfer, the mix of pathogens in the body is constantly evolving. Because plasmids can be swapped, new species or strains of bacteria with new characteristics can emerge at any time.

Horizontal DNA transfer can also make it easier for some of the most harmful and difficult to kill pathogens to survive. As mentioned before, bacteria often trade genes that help them effectively evade the immune system. As Marshall says, “The longer they have persisted in the host, the more the opportunity for horizontal DNA transfer”, meaning that the bacteria which are hardest to kill have more chances to pass on the genes that helped them evade the immune system.

Antibiotic resistant bacteria

Hua Wang, a researcher at Ohio State University has shown that pathogenic bacteria have the ability to engage in horizontal DNA transfer with various commensal bacteria and even beneficial bacteria, including those from the food chain.

Wang writes, “We have demonstrated not only that organisms carrying such intrinsic mechanisms have the potential to become an important reservoir for antibiotic resistance genes but, more importantly, that these intermediate organisms can disseminate antibiotic resistance genes in subsequent events much more effectively than the parental donor strain.

Once we no longer limit ourselves to foodborne pathogens and look at commensal bacteria, we will find that the magnitude of antibiotic-resistant bacterial contamination in the food chain is tremendous.”

Clearly, identifying the pathogens behind chronic disease is more complex than conventional wisdom would have it. It is a shame then that many researchers are still adhering to the idea that only one organism can cause a specific disease. As Marshall says, “Koch’s Postulates make little sense in the era of the Genome.”

SOURCES

Evans, A. S. (1976). Causation and disease: the Henle-Koch postulates revisited. The Yale journal of biology and medicine, 49(2), 175-95.

Gogarten, P. (2000). Horizontal Gene Transfer – A New Paradigm for Biology, Esalen Center for Theory & Research.

Grimes, D. (2006). Koch’s Postulates–Then and Now. Microbe Magazine.

Jain, R., Rivera, M. C., & Lake, J. A. (1999). Horizontal gene transfer among genomes: The complexity hypothesis. PNAS, 96(7), 3801-3806.

Marshall, T. (2006). Molecular genomics offers new insight into the exact mechanism of action of common drugs – ARBs, Statins, and Corticosteroids. FDA CDER Visiting Professor presentation.

Marshall, T. (2007). Bacterial Capnine Blocks Transcription of Human Antimicrobial Peptides. Nature Precedings.

Woese, C. R. (2004). A New Biology for a New Century. Microbiol. Mol. Biol. Rev., 68(2), 173-186.

According to the CDC, infectious agents likely determine more cancers, immune-mediated syndromes, neurodevelopmental disorders, and other chronic conditions than currently appreciated. In fact, they argue that the potential to avoid or minimize chronic disease by preventing or treating infections may yet be substantially underestimated. Those of us familiar with the Marshall Protocol know that they are absolutely correct.

The same can be said for Dave Relman, PhD, assistant professor of medicine and of microbiology and immunology at Stanford University in California who argues, “The list of chronic inflammatory diseases with possible microbial etiologies is extensive; it includes sarcoidosis, various forms of inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, Wegener granulomatosis, diabetes mellitus, primary biliary cirrhosis, tropical sprue, and Kawasaki disease….. the concept of pathogenic mechanism should be viewed broadly.”

Granuoles of Borellia burgdorferi, Kersten 1995

Fortunately, the stealth pathogens responsible for causing the vast majority of chronic diseases have already been identified.

Almost all of us have suffered from a bacterial infection. Sometimes the forms of bacteria causing our symptoms can be killed by antibiotics that work by targeting their cell walls.

However, part of the life cycles of many bacteria include phases where they transform into small forms that lose their cell walls. This means that they can no longer be killed by many commonly used antibiotics. These bacteria are called cell wall deficient (CWD) or L-form bacteria.

Multiple studies 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. “Treatment with penicillin does not merely select for L-forms (which are penicillin resistant) but actually induces L-form growth,” states researcher Josep Casadesus in a paper about L-form bacteria published last month in the medical journal BioEssays.

B. hermsii after exposure to penicillin, image taken by Agbarbour

L-form bacteria are pleomorphic, a term that refers to their ability to change in size and shape. During much of their lifetimes they are tiny, about 0.01 microns in diameter, and can be found clustered together inside the cells of the immune system.

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.

T. pallidum inside a cell, Ovcinnikov 1971

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.

L-form bacteria also 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.

Long L-form biofilm filaments between infected cells, picture by Andy Wright

Researchers have currently 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 Casadesus.

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. However an increasing body of research has shown that these bacteria are responsible for causing a wide array of chronic diseases including rheumatoid arthritis, Chronic Fatigue Syndrome, Lyme disease, sarcoidosis, and Crohn’s disease.

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 burgdorgeri.

A macrophage

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 job 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.

The life cycle of Sprirochaeta gallinerum, Hindle 1912

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 Jounal 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.”

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. The researchers state that E.coli are “true invasive pathogens, able to invade intestinal epithelial cells and replicate intracellularly. Strains also survive and replicate within the macrophages.”

Infection with L-form bacteria

People are exposed to L-form bacteria in many places. Not all species cause disease.

A granuloma

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.

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.

Once 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.

A diagram of Nuclear Kappa Factor B entering the nucleus

L-form bacteria cause inflammation and painful symptoms by taking control of a protein called 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.

Thus, 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,” argues Relman. “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.

The CDC concurs, stating, “The epidemiologic, clinical, and pathologic features of many chronic inflammatory diseases are consistent with a microbial cause.”

Detecting L-form bacteria

Once bacteria have transformed into the L-form they can no longer be detected by many standard laboratory procedures.

Regular forms of bacteria 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 prefer to infect macrophages, the type of white blood cell with the longest life span (about 45 days.)

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.

T. pallidum inside a fibroblast, Lauderdale 1972

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.

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.

Granuoles of B. burdorferi, Hayes 1993

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.

Gaining acceptance

Scientists such as Lida Mattman at Wayne State University 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.

British clinician Andy Wright and Danish researcher Marie Kroun have used a Dark Field Bradford Microscope to view L-forms in the bloodstream.

Nevertheless, many doctors and researchers still question whether the L-forms actually exist.

Mattman and other researchers 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.

As Gerald Domingue, a (retired) professor at the Tulane University School of Medicine stated, “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.”

“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.”

There is also 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.

As Domingue states, “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.”

A neutrophil infected with L-form bacteria, picture by Andy Wright

Another problem 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.

Hopefully as the medical community begins to better understand the role of the L-form in chronic disease, more and more researchers will take the time to learn how to correctly culture and interpret these forms of bacteria.

Most importantly, now that L-form bacteria can be effectively killed by the Marshall Protocol, the opportunity to curb chronic disease is groundbreaking. According to the CDC, chronic diseases represent the major health burden of established economies (>90 million people in the United States) and are a rapidly growing burden in developing economies.

“If a mere 5% of chronic disease is attributable to infectious agents, in the United States alone 4.5 million of the 90 million people living with chronic disease might benefit from strategies designed to prevent or appropriately treat selected infections. Worldwide, the impact could be far greater,” states the 2006 CDC report.

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