Do genetic defects cause the vast majority of chronic diseases? Not according to evolutionary biologist Paul Ewald, who teaches biology at University of Louisville. If chronic diseases were genetic in origin, he argues, “A disease-causing gene that reduces survival and reproduction would normally eliminate itself over a number of generations.” He contends that the thinking underlying today’s “Human Genome Mania” often violates the fundamental principle of biology, Darwin’s Theory of Natural Selection.

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.^[1][2]

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.

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.

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.^[3] 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.^[4]

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. ^[5] 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, 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.^[6]

Then, of course, humans also 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. ^[7]

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 when it comes to the pathogens that cause chronic disease, it appears we are dealing with a chronic, intraphagocytic, metagenomic microbiota - a microbiota that has been largely ignored because they are so difficult to culture ex-vivo.

The second 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.”

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 biomedical researcher Trevor 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.

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.^[8]

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 is driven by bacterial microbes.

REFERENCES

1. Berman, J. (2007). Human skin harbors completely unknown bacteria. EurekaAlert! [↩]
2. Gao, Z., Tseng, C., Pei, Z., & Blaser, M. J. (2007). Molecular analysis of human forearm superficial skin bacterial biota. Proceedings of the National Academy of Sciences, 104(8), 2927-2932. [↩]
3. Mattman, L. (2000). Cell Wall Deficient Forms: Stealth Pathogens. CRC Press. [↩]
4. Domingue GJ, S., & Woody, H. (1997). Bacterial persistence and expression of disease. Clin. Microbiol. Rev., 10(2), 320-344. [↩]
5. Casadesús, J. (2007). Bacterial L-forms require peptidoglycan synthesis for cell division.BioEssays : news and reviews in molecular, cellular and developmental biology, 29(12), 1189-91. [↩]
6. Marshall, T. G. (2006e). Immunity & Ageing. Comments: Is inflammaging an auto[innate]immunity subclinical syndrome? [↩]
7. Hall-Stoodley, L., Costerton, J. W., & Stoodley, P. (2004). Bacterial biofilms: from the Natural environment to infectious diseases. Nat Rev Micro, 2(2), 95-108. [↩]
8. Dempsey, K. E., Riggio, M. P., Lennon, A., Hannah, V. E., Ramage, G., Allan, D., et al. (2007). Identification of bacteria on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties by 16S rRNA gene sequencing and by microbiological culture. Arthritis research & therapy, 9(3), R46. [↩]