Biofilms Tutorial

introduction Journal of the American Society for Microbiology (April, 2002)

Some bacteria “hibernate”—sometimes for centuries— until conditions are right to awaken. Biologists are deterred from studying such microbes because of false positive results that have tainted their efforts to recover ancient DNA. Scientists have developed a database describing these elusive bacteria not only as a resource for evolutionary research but also to study the potential for using these ancient organisms in the industrial production of chemicals.

During their state of suspended animation, these bacteria assume different forms, often in clusters called biofilms. More than 99 percent of all bacteria types can live in biofilm communities.

Some are beneficial, such as those used in sewage treatment plants to remove contaminants. Biofilms are found wherever surfaces are in contact with water. Examples are slime on river stones, insides of household water pipes, swimming pool walls and filters, and plaque on teeth. These bacteria can adhere to clean stainless steel within 30 seconds of exposure. Copper, Zinc and Silver surfaces are less susceptible because these metals have antimicrobial properties. Biofilms growing on implants have been found to cause the immune system to react to the infection, leading to rejection of the medical implants. Biofilms are the source of much of the free-floating (planktonic form) bacteria found in drinking water. The common Pseudomonas aeruginosa biofilm bacteria can infect animals with suppressed immune systems.

Although a biofilm cluster can spread by ordinary cell division, it will also shed cells with the express purpose of starting new colonies. Other microorganisms within a colony act symbiotically with biofilm bacteria, sharing nutrients and providing mutual protection for community survival. Biofilms have been called “communal slime cities.” Members of a colony can also interchange genetic recipes via plasmid messengers. In this way the resistance to antibiotics can be learned by different microbes in a colony.

The use of water purification systems causes bacteria to alter their cell wall structure in order to increase their ability to adhere to surfaces. Biofilm resistance to biocides is remarkable according to CDC experiments. The biofilm colony surrounds itself with a protective shield of polysaccharides and polymers. A disinfectant’s oxidizing power is depleted before it reaches the interior cell responsible for forming the biofilm. Free-floating organisms are more vulnerable.

Because biofilm bacteria anchor themselves to surfaces with exuded sticky polymers, simple flushing is inadequate to remove them. Chlorinated reverse osmosis water systems, copper piping, and water filters on all house taps can limit biofilm contamination but cannot completely eradicate it. Colonies can also be found in aerators and spray nozzles attached to water faucet outlets. This has been the source of pathogenic infections in hospitals.

The human body is about 60 percent water. Could it be that bacteria in our bodies’ organic “pipes” exhibit the same behavior as biofilms? Is the protective coating that biofilm bacteria secrete to ward off attack from disinfectants analogous to the way antigens try to thwart the immune system’s antibodies?

Future research will hopefully answer these questions and lead to better understanding of these pathogenic microorganisms. Although biofilms have been with us for eons, their behavior suddenly became a topic of intensive research in the early 1990s. Bacteriologists had persistently assumed that bacteria are simple unicellular microbes. This was because the hunt for disease-causing organisms traditionally began by isolating a single cell of the suspected pathogen.

Many existing theories of bacterial behavior are based on extrapolations made from this early research. New studies have revealed that bacteria build complex communities, differentiate into various cell types, hunt prey cooperatively in groups, and secrete chemical trails to direct movement of others in the colony.

There is still much to be done to fully understand the immune system and microbial infections. Biofilms harboring Cryptosporidium may be the cause of recent cruise ship sickness outbreaks.

Katherine is on the board of directors of the Arthroplasty Patient Foundation which has produced several videos explaining how biofilms must be specially treated to make antibiotic treatments more effective.

See: Arthroplasty Patient Foundation's Videos: Why am I still sick?

Also see: GOOGLE [how to dissolve biofilms in vivo]

Biofilms are persistent microbiome colonies

Our medical tactics need to manage the biofilms that live inside us. We need to promote biofilm microbiomes that are healthy and not pathogenic. The human microbiome project is working to organize the known information about the tens of thousands of microbes and strains that live inside us and our food sources.

Trevor Marshall Protocol Knowledge Base Biofilm Bacteria Colonies.

Wild elephants already know this gut-health secret and human doctors’ in protocol trials recently proved that: a fecal transplant is 3x better than Vancomycin in treating a pathogenic gut infection. Flora from healthy persons used as a drug, proved 85% to 90% effective in eliminating C. difficile chronic gut infections.

We have had personal experience in use of antibiotics, chiropractic treatment, vitamin C, and rebooting the gut to overcome arthritic pain in lower back. Gut Health Case Histories Vinegar is a powerful gut-cleaning saturated fatty acid; it is useful in cases of food poisoning. It is mentioned in the material below in fighting gut biofilms.

Biofilm colonies in the body can cause inflammation that is mistakenly diagnosed as an autoimmune response. Part of the problem is bacteria that generate toxins, part may be in retention of toxic metals: lead, mercury, nickel, chromium, copper, aluminum, selenium.

Vitamin C as a universal antitoxin and chelator can help reduce the toxic effects of biofilms, without eliminating them.

The immune system vs. Pseudomonas aeruginosa biofilms

By Peter Østrup Jensen, Michael Givskov, Thomas Bjarnsholt, Claus Moser

FEMS Immunology & Medical Microbiology Volume 59, Issue 3, August 2010

Abstract:

“Ilya Metchnikoff and Paul Ehrlich were awarded the Nobel prize in 1908. Since then, numerous studies have unraveled a multitude of mechanistically different immune responses to intruding microorganisms. However, in the vast majority of these studies, the underlying infectious agents have appeared in the planktonic state. Accordingly, much less is known about the immune responses to the presence of biofilm-based [colony] infections (which is probably also due to the relatively short period of time in which the immune response to biofilms has been studied). Nevertheless, more recent in vivo and in vitro studies have revealed both innate as well as adaptive immune responses to biofilms. On the other hand, measures launched by biofilm bacteria to achieve protection against the various immune responses have also been demonstrated. Whether particular immune responses to biofilm infections exist remains to be firmly established. However, because biofilm infections are often persistent (or chronic), an odd situation appears with the simultaneous activation of both arms of the host immune response, neither of which can eliminate the biofilm pathogen, but instead, in synergy, causes collateral tissue damage. Although the present review on the immune system vs. biofilm bacteria is focused on Pseudomonas aeruginosa (mainly because this is the most thoroughly studied), many of the same mechanisms are also seen with biofilm infections generated by other microorganisms.”

Properties and Treatment for Biofilm Colonies

The following notes describe biofilms in more detail, how they build themselves protective structures to form a chronic infection colony. Biofilms live within the body’s tubular structures; penetrating epithelial protective mucosa; invading epithelial cells for replication; and attaching the colonies to the epithelium. Structures and encapsulating gels protect the microbes from antibiotics with a shield, requiring more than 1000 times higher antibiotic concentration to reach killing range. Because the colonies do not shed microbe contents unless dissolved by lysing agents, culture tests for the microbes in the biofilm often show false negative results. The colonies may contain pathogenic bacterial members that are exo-/endo-toxin generators, storage reservoirs for toxic/allergenic heavy metals and occasional sources of planktonic microbe cells that can infect other body sites.

1. Visualizing Microfilm Colonies:

Source: Imaging Biofilms at University of California at Berkeley

Lead Researcher: Veysel Berk, a postdoctoral fellow in the UC Department of Physics

The news release contains color pictures and also contains a Video.

By … “employing super-resolution light microscopy, the researchers were able to examine the structure of sticky plaques called bacterial biofilms that make these infections so tenacious. They also identified genetic targets for potential drugs that could break up the bacterial community and expose the bugs to the killing power of antibiotics.”

“He [Dr. Veysel Berk] discovered that, over a period of about six hours, a single bacterium laid down a glue to attach itself to a surface, then divided into daughter cells, making certain to cement each daughter to itself before splitting in two. The daughters continued to divide until they formed a cluster – like a brick and mortar building – at which point the bacteria secreted a protein that encased the cluster like the shell of a building.”

“The clusters are separated by microchannels that may allow nutrients in and waste out,” Berk said.

“If we can find a drug to get rid of the glue protein, we can move the building as a whole. Or if we can get rid of the cement protein, we can dissolve everything and collapse the building, providing antibiotic access,” Berk said. “These can be targets for site-specific, antibiotic [anti-biofilm structure destroying] medicines in the future.”

2. Specific Biofilm Eradication Methods

Commentary by KFP, including additional data from other sources.

Biofilms on surfaces are a great problem where people congregate. In hospitals, where sick persons import pathogenic microbes this is a real problem. Shopping cart handles during flu season can be a source of infection.

Newly developed synthetic hydrogels can kill surface contaminating biofilms, which are the seed-source for patient and medical staff infections. IBM with the Singapore Institute of Bioengineering & Nanotechnology developed new Antimicrobial Hydrogel to fight Superbugs (MRSA) and drug-resistant protective biofilms. See Plastic Ninjas These new hydrogels dissolve the protective biofilms and the MRSA microbes without any added antibiotics targeted to the planktonic form of the bacteria.

Biofilm dissolving is discussed below. Use of these hydrogels as medicines is being researched. They may be injected into artificial joints to assist in clearing metal surfaces of the biofilm infection.

Biofilms in the body:

Wherever the body has a tube or cavity, biofilms can colonize. Fibromyalgia may be a biofilm infection of the lymph system. Epithelial cells line the tubes and may be infected. The gut is filled with biofilms. Circulatory system plaques are biofilms, some of the same microbes are found there and in dental plaques. The respiratory tract, urinary tract, ear system cavities, sinus, tonsils and adenoids, vagina, uterus and fallopian tubes may be infected by biofilm colonies. Amniotic fluid during pregnancy often is not sterile; it can be infected by biofilms. The plaques of Alzheimer’s might have a biofilm brain-infection cause. Spinal stenosis, bone spurs, TB and Sarcoidosis lesions (granulomas), arthritic calcifications, and benign tumors (–omas) are biofilms. The biofilms block the effect of antibiotics by attenuation factors of over 1000, protecting the colony residents. Lysing enzymes can attack the structures, but the IBM hydrogels offer an exciting alternative to be researched.

For successful treatment, the biofilm must be dissolved.

End KFP commentary.

Using Chelators and Enzymes Against Gut Biofilms

Source: Dr Anju Usman Biofilm Notes by A Rainville

With added material by KFP

1- Dissolve and Detach the Biofilm: Use enzymes, mucus liquefying agents (Guaifenesin and Serrapeptase) and chelators [EDTA] on empty stomach to liquefy the biofilm. Oral EDTA is not well absorbed which keeps it in the gut where you want it. Enzymes are serrapeptase, bromelain, papain, nattokinase, Lumbrokinase. Guaifenesin liquefies respiratory phlegm and unclogs the lymph system biofilms; it also has neural effects that may reduce pain. Nattokinase also dissolves clots and thins the blood. Serrapeptase has similar effects and lyses scar tissue and breaks down bacterial calcifications. Serrapeptase works to dissolve ear-system and urinary tract biofilms.

A prebiotic sugar-alcohol, Xylitol, causes over 90% of mouth plaque bacteria die off , if given with Ketonic diet. (Atkins induction, no sugar diet) The remaining survivor bacteria do a gene-shift to bacteria that no-longer form dental plaques and have reduced systemic pathogenicity (perhaps in the gut of making less endotoxin). The result of this gene shift in the microbiome is less-severe ear and urinary tract infections. Xylitol and Ketonic state shifts the microbiome away from harboring yeasts; they cannot use this sugar; humans can metabolize it for energy.

Proanthocyanidins (PACs) in cranberry juice prevent E. coli, from sticking to urinary tract epithelial cells by changing the bacteria’s surface properties. The effect stops, unless you drink the juice every day. But it can synergize the antibiotics taken at the same time.

2- Kill the Microbe: (30-60 minutes after step 1) use targeted antimicrobial antibiotics (against Lyme, yeast, bacteria)

Natural antiviral foods include Vitamin A (antiviral), Vitamin C, Vitamin E (ascorbic acid and Liposomal AA), vinegar (acetic acid), Palmitic acid (lung surfactant and POPG precursor molecule), Coconut Oil (Lauric acid is antiviral anti bacterial), caprylic and caproic acids (from goat butter and goat cheese is anti-yeast).

3- Neutralize toxins: 1-2 hours later (or at night) take toxin neutralizers: Vitamin C (AA), Vitamin E and antioxidants. ARB blockers moderate toxin ability to cause cytokine inflammation cascade. High and frequent AA intake is essential to stop toxemia. See How Much Vitamin C. See Dr Thom Levy Curing the Incurable, AA Antitoxin Chapter 3. With high toxemia you can run out of AA overnight. Take several grams of AA at bedtime, when awakening, before breakfast, and whenever you feel sick from the toxins. Every 1-4 hours between meals, if you have sickness symptoms. Also take Liposomal vitamin C.

4- Export chemical debris using binding substances: With surface area to attract toxins: Fiber (warning psyllium allergies), Chitosan (warning shellfish allergy), Clays & Zeolites, Chlorella (may be moldy), Modifilan, Apple pectin, Butyrate (butyric acid is antimicrobial), Activated Charcoal (works with killing yeast). Mercola’s detox diet Dr Weil's view of clays and detox.

5- Rebuild: Prebiotics, Probiotics, fermented foods [yogurt fermented ground/shredded cabbage, sauerkraut, Kim-chi, vitamins, minerals: Calcium, potassium, magnesium, sunflower seed/butter for minerals, helps build up immune system. Continue to supply coconut/palm oils in diet. CoQ10 helps improve energy functions in cells mitochondria; it helps increase exercise tolerance and blood oxygen flow. Xylitol

6- Exercise: Endotoxin levels are higher among persons that live a sedentary life style. This could be explained by the fact that sick persons exercise less. It is not clear that forcing sick persons to exercise will cure their illnesses without first killing off the microbes that generate endotoxins. Endotoxins (lipopolysacarides) promote insulin generation, which could lead to conversion of sugars to adipose fats, if insulin resistance is a result. Dietary intervention, loss of weight and exercise can improve health and reduce endotoxin production. See Study Predisposition not to exercise may depend on thyroid activity; hormone imbalance should be checked out by hormone tests.

Enzymes: The specific enzymes to break down the biofilm are still a work in progress. The key component so far may be xylanase,

Some products being used:

- SPS 30 by Theramedix (www.theramedix.net)

- Mucostop by Enzymedica (www.enzymedica.com)

- Apple cider vinegar Acetic acid is a 2nC carbon chain saturated fatty acid, where n=1. These saturated fatty acids (n=1, 2, 3, 4, 5…etc. ) dissolve microbes lipid envelopes. Butyric acid (Butyrate n=2 gives butter its flavor and is antimicrobial) POPG is a lung surfactant that makes breathing easier and has anti-inflammatory actions.

Antimicrobial fatty acids: See

Natural Tropical Oils.

A Revolution in Thinking: Oils and Nutrition

Heart Disease and Saturated Fats: Old Myths and A New Reality

3. Overview of Clinical Methods to Defeat Biofilm Colonies:

Clinical Pearls on How to Conquer Stubborn Biofilms

The reference, above, is an image of Web Page that contains an overview/tutorial description of biofilms well worth reading. Nutrients from whole food fermentation help destroy pathogenic biofilms. Acetic acid, sauerkraut, lactose fermentation probiotics, yogurt, buttermilk, doogh, kefir, Kim chi, pickles. Biofilms also contain microbes that invade epithelial cells to avoid antibiotics, meaning that long-term anti-biofilm treatments are needed, or repeated treatments are needed to reduce resurgent re-colonization. Prebiotic nutrients and probiotic microbes in fermented food cultures can help maintain a protective microbiome.

4. One Prebiotic Food-based Biofilm Modifier

Source: Modulation of Gut Mucosal Biofilms

Conclusion:

“Non-digestible inulin-type fructans, such as oligofructose and high-molecular-weight inulin, have been shown to have the ability to alter the intestinal microbiota composition in such a way that members of the microbial community, generally considered as health-promoting, are stimulated. Bifidobacteria and lactobacilli are the most frequently targeted organisms. Using rats inoculated with a human faecal flora as an experimental model we have found that inulin-type fructans in the diet modulated the gut microbiota by stimulation of mucosa-associated bifidobacteria as well as by partial reduction of pathogenic Salmonella enterica subsp. enterica serovar Typhimurium and thereby benefit health. In addition to changes in mucosal biofilms, inulin-type fructans also induced changes in the colonic mucosa stimulating proliferation in the crypts, increasing the release of mucins, and altering the profile of mucin components in the goblet cells and epithelial mucus layer. These results indicate that inulin-type fructans may stabilise the gut mucosal barrier. Dietary supplementation with these prebiotics could offer a new approach to supporting the barrier function of the mucosal biofilm”

Other prebiotics with anti biofilm actions are Serrapeptase proteolytic enzyme and Xylitol, a sugar alcohol that forces a die off of bacteria that cannot use it for energy. See http://www.ra-infection-connection.com/CaseHistories.htm#SerraPep

5. Campylobacter Study of Microbial Reseeding

Source: Investigation of motility and biofilm formation by intestinal Campylobacter concisus strains

“Evidence suggests that both animals and the oral cavity of humans provide a reservoir of C. concisus that could pass into the intestinal tract of humans following ingestion. Based on the results of this study, we hypothesize that strains with higher motility have a greater chance to swim through the intestinal mucus layer and reach the epithelial surface. Once adhered to the epithelium through their flagellum, strains with the proper pathogenicity factors such as the exotoxin 9, which has been associated with the invasive potential of C. concisus, can invade into the host cell, induce an inflammatory response, and subsequently, cause disease.”

Biofilms bind cooperating microbes together in a slimy matrix. They are protected from antibiotics. They share genetic recipes via exchange of plasmid messenger bodies. The pathogenic microbe components are not shed from the biofilm, thus cultures are often falsely negative for their presence.

6. Excerpt of Biofilm End Notes from our second book.

1. Jaenisch, R, Bird, A. "Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals." Nature genetics 33 Suppl (3s): 245–254 (2003)

2. Find a wealth of information on biofilms at http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/primer.html and http://www.biofilmcommunity.org/ and http://home.swipnet.se/isop/biofilms.htm and http://www.wellnessresources.com/tag/biofilms and overview at http://bacteriality.com/2008/05/26/biofilm/

3. EDSTROM. Biofilm: Understanding and Controlling Growth. White paper at www.edstrom.com/documents (2011)

4. The way biofilms develop, thrive, and spread are described in detail at the homepage of the National Science Foundation’s Center for Biofilm Engineering, Montana State University, at www.erc.montana.edu.

5. O'May GA, et al. “Effect of pH and antibiotics on microbial overgrowth in the stomachs and duodena of patients undergoing percutaneous endoscopic gastrostomy feeding.” J Clin Microbiol. 2005 Jul;43(7):3059-65.

6. Amir S, et al. “Acanthamoeba castellanii an environmental host for Shigella dysenteriae and Shigella sonnei.” Archives of Microbiology. Volume 191, Number 1, 83-88. Online at http://www.springerlink.com/content/l76jh3kh552042x2/

7. See www.erc.montana.edu Center for Biofilm Engineering

8. Puttamreddy S, Minion FC. “Linkage between cellular adherence and biofilm formation in Escherichia coli.” FEMS Microbiol Lett. 2011 Feb;315(1):46-53. Online at http://www.ncbi.nlm.nih.gov/pubmed/21166710. Also see Xicohtencatl-Cortes J, et al. “Intestinal adherence associated with type IV pili of enterohemorrhagic Escherichia coli.” J Clin Invest. 2007 Nov;117(11):3519-29. Online at www.ncbi.nlm.nih.gov/pubmed/17948128

9. Dr. Art Ayers. “Biofilms as Human Gut Mycorrhizals.” http://coolinginflammation.blogspot.com/2009/11/biofilms-as-human-gut-mycorrhizals.html

10. Shayan R, Achen MG, and Stacker, SA. “Lymphatic vessels in cancer metastasis: bridging the gaps.” Carcinogenesis vol.27 no.9 pp.1729–1738, 2006. Online at carcin.oxfordjournals.org/content/27/9/1729.full

11. www.atcc.org/CulturesandProducts/Microbiology/BacteriaandPhages/tabid/176/Default.aspx

12. http://www.stanford.edu/~amatin/MatinLabHomePage/Biofilm.htm

13. http://www.medicalnewstoday.com/articles/184972.php (2010)

14. Presentation by Dr. Sandra Macfarlane at the 2011 International IFM conference in Bellevue, WA. Also Macfarlane S, Bahrami B, Macfarlane GT. “Mucosal biofilm communities in the human intestinal tract.” Adv Appl Microbiol. 2011;75:111-43.

15. An expert interview with Dr. Scot Dowd of PathoGenius labs (Lubbock, TX) explaining biofilms and testing methods can be found at www.biofilmcommunity.org/f6/dr-scot-dowd-pathogenius-102/

16. Murphy TF, Kirkham C. “Biofilm formation by nontypeable Haemophilus influenzae: strain variability, outer membrane antigen expression and role of pili.” BMC Microbiology 2002, 2:7 at www.biomedcentral.com/1471-2180/2/7/

17. http://www.columbia.edu/itc/hs/medical/pathophys/id/2006/PeriodontalDisease.pdf

18. Hajishengallis G, et al. “Low-Abundance Biofilm Species Orchestrates Inflammatory Periodontal Disease through the Commensal Microbiota and Complement.” Cell Host & Microbe. Volume 10, Issue 5, 17 November 2011, Pages 497–506.

19. Socransky SS and Haffaje AD. “Dental biofilms: difficult therapeutic targets.” Periodontology 2000. Vol. 28, No. 1, pages 12–55, January 2002. Online at http://onlinelibrary.wiley.com/doi/10.1034/j.1600-0757.2002.280102.x/full

20. http://genome.cshlp.org/content/15/6/820.full.pdf (2005)

21. Sivagnanam S and Deleu D. “Red Man Syndrome.” Crit Care. 2003; 7(2): 119–120. Online at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC270616/

7. Additional biofilms tutorial References:

Our Experience and Qualifications

(Katherine Poehlmann, PhD and Karl Poehlmann)

Inflammation, chronic infections, nutrition and immunity are topics we have researched broadly in our studies of worldwide medical knowledge, documented on the Internet and in the historical archives of medicine. We have spent over ten calendar years reading about these inter-related subjects, attending postgraduate medical conferences. We have read countless medical texts, abstracts, and papers, online in the National Library of Medicine and contained at various authoritative medical, nutritional and biological websites. The mass of the available information worldwide is tremendous. Search engines can reach much of it, so it can be correlated productively.

Disclaimer

Nothing herein or referenced herein should be considered prescriptive for any medical condition. This information is for study and education purposes only. The readers are advised to find and consult well-educated, trained and licensed medical and nutritional practitioners who shall evaluate the many circumstances and conditions of each of their patients and will devise appropriate treatments and nutritional plans for them. It is recognized that each person has the right and duty to be well informed about the best foods, nutrition and medical practices available that will promote their own good health. The opinions expressed herein are those of the author(s) and the sources cited and there are many divergences of opinions on many topics. The readers must resolve the conflicts, in their own minds, after careful consideration of all the details and after any further necessary research and study.