Background

Biofilms are considered the rule in nature rather than the exception. If you have chronic infection, biofilms may be an underlying cause. Many, if not most, microorganisms form and persist in cohesive community structures termed biofilms. These cells secrete a gelatinous intracellular substance consisting of an extracellular polysaccharide (sugar), DNA, and protein matrix. Biofilms are often found attached to living and inert stable surfaces that have a constant liquid flow that brings nutrients and removes waste products from the biofilms. Biofilms often are not composed of a single organism but contain two or more organisms making significant contributions to the biological stability, characteristics, and behavior of the resulting biofilm. Organisms found within biofilms have distinct genetic expression and functional behavior compared to individual organisms subsisting in an individual planktonic state. The establishment and life cycle of biofims on surfaces typically proceed through four main stages: 1) Initial Attachment, 2) Irreversible Attachment, 3) Various Maturation Phases, 4) Active Dispersion or Blebbing/Fragmenting. Many microorganisms spend most of their life cycle in a persistent biofilm state switching to free-living or planktonic phases only during brief periods when environmental conditions are favorable. As Dr. Bill Costerton, the dean of Biofilms succinctly says in his text, biofilms are “- a structured community of bacterial cells enclosed in a self-produced polymer matrix and attached to an inert or living surface.”[1].

Biofilms are observed on most stable non-sterile surfaces in an aquatic environment. Biofilms are found in natural environments such as hot springs, rivers and streams, lakes, subterranean stromatolites, and tide pools, to man-made and industrial environments such as water and drainage pipes, sanitation systems, house-hold sinks, toilets, and showers, and even in the water tanks of nuclear power plants. Lastly, biofilms are ubiquitous and present in normal and diseased states in humans. It is estimated that 80% of all human infections have biofilm involvement. These infections range from urinary tract infections, middle-ear infections, cystic fibrosis, dental plaque, chronic skin infections, many chronic diseases, and coatings on indwelling devices such as joint replacement, catheters, cardiac valves, and contact lenses [1-15].

Dental Health

The involvement of biofilms in the dental industry has long been studied and recognized as important in improving dental health. The dental films and plaque are primarily polymicrobial biofilms. Typically these biofilms consist of bacteria that are considered normal oral flora and usually are kept in balance by normal dental hygiene and the other cohabitating bacteria, however the population dynamics and members of these biofilms may change and induce conditions where tooth decay, gingivitis, and other oral health problems may arise. Where dental health is concerned, it is synonymous with biofilms.

Chronic Wounds

Another important impact of biofilms in human health is cutaneous and skin related biofilm infections. Many infections are easily treated with antibiotics or resolve spontaneously through the body’s natural immune response, however biofilms complicate this picture. When certain species of bacteria and/or fungi are present, they may produce a biofilm-based infection that is exceptionally difficult to treat. These chronic wounds are typified by open wounds that fail to heal, are recalcitrant to antibiotic treatment, produce a profuse seepage, and represent a legitimate risk to patient health. All too often, invasive biofilm-based infections result in the removal of the afflicted area or limb! Recent advances in treatments for biofilm-based chronic wounds include multi-drug therapies, rotational strategies, routine cleaning, and direct topical application of antibiofilm agents.

Chronic Sinus and Inner Ear Infections

One additional recognized involvement of biofilms in human health is its role in chronic rhinosinusitis (sinus infections) and otitis media (inner ear infections). In both cases the symptoms may be due to a persistent biofilm-based infection that the body fails to fully eliminate. Continual aggravation by these infections may produce lasting, disrupting, and damaging effects. Treatment options informed by biofilm research for ear, nose, and throat (ENT) are currently an area of great interest and it is likely future treatments will be guided by this emerging information.

Biofilm Research

Fry Laboratories couples our clinical diagnostics with a robust research and development lab charged with the singular task to investigate novel infections, develop cutting edge and valuable assays, and execute basic science research with the explicit task to inform and assist in patient treatment. Our involvement with biofilms started in the Fall of 2008 when we were researching techniques to improve the existing detection methods for Bartonella and other epierythrozoan bacterial species in the blood of afflicted patients. Simply, we observed hematologic biofilm forming processes and neutrophil extracellular traps (NETs) in patients with chronic infections and illness [16-32]. Our laboratory is one of the first to report biofilm forming protozoan communities [33]. This ultimately resulted in the refinement of the Advanced Stains test that places the power of a generalized morphological screening test in the hands of physicians and health care professionals. Now intense research is focused on the nature, contribution, and involvement of blood-borne biofilm infections. Fry Laboratories has made great strides in the molecular identification of the organisms involved, testing, treatment options, and understanding the contributions of biofilms make to some of the most intractable health concerns currently faced.

1. Costerton, J.W., P.S. Stewart, and E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections. Science, 1999. 284(5418): p. 1318-22. 2. Al-Mutairi, D. and S.J. Kilty, Bacterial biofilms and the pathophysiology of chronic rhinosinusitis. Curr Opin Allergy Clin Immunol, 2010. 3. Busscher, H.J., et al., Biofilm formation on dental restorative and implant materials. J Dent Res, 2010. 89(7): p. 657-65. 4. Cernohorska, L. and P. Slavikova, [Antibiotic resistance and biofilm formation in Pseudomonas aeruginosa strains isolated from patients with urinary tract infections]. Epidemiol Mikrobiol Imunol, 2010. 59(4): p. 154-7. 5. Crawford, R.W., et al., Gallstones play a significant role in Salmonella spp. gallbladder colonization and carriage. Proc Natl Acad Sci U S A, 2010. 107(9): p. 4353-8. 6. Cushion, M.T., M.S. Collins, and M.J. Linke, Biofilm formation by Pneumocystis spp. Eukaryot Cell, 2009. 8(2): p. 197-206. 7. Hall-Stoodley, L. and P. Stoodley, Evolving concepts in biofilm infections. Cell Microbiol, 2009. 11(7): p. 1034-43. 8. Haussler, S. and M.R. Parsek, Biofilms 2009: new perspectives at the heart of surface-associated microbial communities. J Bacteriol, 2010. 192(12): p. 2941-9. 9. Hoa, M., et al., Biofilms and chronic otitis media: an initial exploration into the role of biofilms in the pathogenesis of chronic otitis media. Am J Otolaryngol, 2010. 31(4): p. 241-5. 10. Jarvensivu, A., et al., Candida yeasts in chronic periodontitis tissues and subgingival microbial biofilms in vivo. Oral Dis, 2004. 10(2): p. 106-12. 11. Klotz, S.A., Fungal adherence to the vascular compartment: a critical step in the pathogenesis of disseminated candidiasis. Clin Infect Dis, 1992. 14(1): p. 340-7. 12. Miller, V.M., et al., Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves. Am J Physiol Heart Circ Physiol, 2004. 287(3): p. H1115-24. 13. Tang, H. and Y. Xu, [Bacterial biofilms and chronic osteomyelitis]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2010. 24(1): p. 108-11. 14. Tapiainen, T., et al., Biofilm formation by Streptococcus pneumoniae isolates from paediatric patients. Apmis, 2010. 118(4): p. 255-60. 15. Tunpiboonsak, S., et al., Role of a Burkholderia pseudomallei polyphosphate kinase in an oxidative stress response, motilities, and biofilm formation. J Microbiol, 2010. 48(1): p. 63-70. 16. Aulik, N.A., et al., Mannheimia haemolytica and its leukotoxin cause neutrophil extracellular trap formation by bovine neutrophils. Infect Immun, 2010. 78(11): p. 4454-66. 17. Behrendt, J.H., et al., Neutrophil extracellular trap formation as innate immune reactions against the apicomplexan parasite Eimeria bovis. Vet Immunol Immunopathol, 2010. 133(1): p. 1-8. 18. Dolgushin, II and S. Andreeva Iu, [Neutrophil extracellular traps: method of detection and assessment of bacterial trapping efficacy]. Zh Mikrobiol Epidemiol Immunobiol, 2009(2): p. 65-7. 19. Ermert, D., A. Zychlinsky, and C. Urban, Fungal and bacterial killing by neutrophils. Methods Mol Biol, 2009. 470: p. 293-312. 20. Gupta, A.K., et al., Neutrophil NETs: a novel contributor to preeclampsia-associated placental hypoxia? Semin Immunopathol, 2007. 29(2): p. 163-7. 21. Gupta, A.K., et al., Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death. FEBS Lett, 2010. 584(14): p. 3193-7. 22. Hakkim, A., et al., Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation. Nat Chem Biol, 2010. 23. Hakkim, A., et al., Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc Natl Acad Sci U S A, 2010. 107(21): p. 9813-8. 24. Jann, N.J., et al., Neutrophil antimicrobial defense against Staphylococcus aureus is mediated by phagolysosomal but not extracellular trap-associated cathelicidin. J Leukoc Biol, 2009. 86(5): p. 1159-69. 25. Li, P., et al., PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med, 2010. 207(9): p. 1853-62. 26. Marcos, V., et al., CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation. Nat Med, 2010. 16(9): p. 1018-23. 27. Pilsczek, F.H., et al., A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol, 2010. 185(12): p. 7413-25. 28. Urban, C.F., et al., Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol, 2006. 8(4): p. 668-76. 29. von Kockritz-Blickwede, M., et al., Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation. Blood, 2008. 111(6): p. 3070-80. 30. Wang, Y., et al., Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol, 2009. 184(2): p. 205-13. 31. Wartha, F., et al., Neutrophil extracellular traps: casting the NET over pathogenesis. Curr Opin Microbiol, 2007. 10(1): p. 52-6. 32. Wartha, F. and B. Henriques-Normark, ETosis: a novel cell death pathway. Sci Signal, 2008. 1(21): p. pe25. 33. Ellis, J.E., M. Prochazka, and S.E. Fry, Evidence for In-Vivo Hematologic Biofilm Communities in 3 Patients with ALS. 5th ASM Conf. on Biofilms, 2009. 158.Selected References 1. Costerton, J.W., P.S. Stewart, and E.P. Greenberg, Bacterial biofilms: a common cause of persistent infections. Science, 1999. 284(5418): p. 1318-22. 2. Al-Mutairi, D. and S.J. Kilty, Bacterial biofilms and the pathophysiology of chronic rhinosinusitis. Curr Opin Allergy Clin Immunol, 2010. 3. Busscher, H.J., et al., Biofilm formation on dental restorative and implant materials. J Dent Res, 2010. 89(7): p. 657-65. 4. Cernohorska, L. and P. Slavikova, [Antibiotic resistance and biofilm formation in Pseudomonas aeruginosa strains isolated from patients with urinary tract infections]. Epidemiol Mikrobiol Imunol, 2010. 59(4): p. 154-7. 5. Crawford, R.W., et al., Gallstones play a significant role in Salmonella spp. gallbladder colonization and carriage. Proc Natl Acad Sci U S A, 2010. 107(9): p. 4353-8. 6. Cushion, M.T., M.S. Collins, and M.J. Linke, Biofilm formation by Pneumocystis spp. Eukaryot Cell, 2009. 8(2): p. 197-206. 7. Hall-Stoodley, L. and P. Stoodley, Evolving concepts in biofilm infections. Cell Microbiol, 2009. 11(7): p. 1034-43. 8. Haussler, S. and M.R. Parsek, Biofilms 2009: new perspectives at the heart of surface-associated microbial communities. J Bacteriol, 2010. 192(12): p. 2941-9. 9. Hoa, M., et al., Biofilms and chronic otitis media: an initial exploration into the role of biofilms in the pathogenesis of chronic otitis media. Am J Otolaryngol, 2010. 31(4): p. 241-5. 10. Jarvensivu, A., et al., Candida yeasts in chronic periodontitis tissues and subgingival microbial biofilms in vivo. Oral Dis, 2004. 10(2): p. 106-12. 11. Klotz, S.A., Fungal adherence to the vascular compartment: a critical step in the pathogenesis of disseminated candidiasis. Clin Infect Dis, 1992. 14(1): p. 340-7. 12. Miller, V.M., et al., Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves. Am J Physiol Heart Circ Physiol, 2004. 287(3): p. H1115-24. 13. Tang, H. and Y. Xu, [Bacterial biofilms and chronic osteomyelitis]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2010. 24(1): p. 108-11. 14. Tapiainen, T., et al., Biofilm formation by Streptococcus pneumoniae isolates from paediatric patients. Apmis, 2010. 118(4): p. 255-60. 15. Tunpiboonsak, S., et al., Role of a Burkholderia pseudomallei polyphosphate kinase in an oxidative stress response, motilities, and biofilm formation. J Microbiol, 2010. 48(1): p. 63-70. 16. Aulik, N.A., et al., Mannheimia haemolytica and its leukotoxin cause neutrophil extracellular trap formation by bovine neutrophils. Infect Immun, 2010. 78(11): p. 4454-66. 17. Behrendt, J.H., et al., Neutrophil extracellular trap formation as innate immune reactions against the apicomplexan parasite Eimeria bovis. Vet Immunol Immunopathol, 2010. 133(1): p. 1-8. 18. Dolgushin, II and S. Andreeva Iu, [Neutrophil extracellular traps: method of detection and assessment of bacterial trapping efficacy]. Zh Mikrobiol Epidemiol Immunobiol, 2009(2): p. 65-7. 19. Ermert, D., A. Zychlinsky, and C. Urban, Fungal and bacterial killing by neutrophils. Methods Mol Biol, 2009. 470: p. 293-312. 20. Gupta, A.K., et al., Neutrophil NETs: a novel contributor to preeclampsia-associated placental hypoxia? Semin Immunopathol, 2007. 29(2): p. 163-7. 21. Gupta, A.K., et al., Activated endothelial cells induce neutrophil extracellular traps and are susceptible to NETosis-mediated cell death. FEBS Lett, 2010. 584(14): p. 3193-7. 22. Hakkim, A., et al., Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation. Nat Chem Biol, 2010. 23. Hakkim, A., et al., Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc Natl Acad Sci U S A, 2010. 107(21): p. 9813-8. 24. Jann, N.J., et al., Neutrophil antimicrobial defense against Staphylococcus aureus is mediated by phagolysosomal but not extracellular trap-associated cathelicidin. J Leukoc Biol, 2009. 86(5): p. 1159-69. 25. Li, P., et al., PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med, 2010. 207(9): p. 1853-62. 26. Marcos, V., et al., CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation. Nat Med, 2010. 16(9): p. 1018-23. 27. Pilsczek, F.H., et al., A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol, 2010. 185(12): p. 7413-25. 28. Urban, C.F., et al., Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol, 2006. 8(4): p. 668-76. 29. von Kockritz-Blickwede, M., et al., Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation. Blood, 2008. 111(6): p. 3070-80. 30. Wang, Y., et al., Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol, 2009. 184(2): p. 205-13. 31. Wartha, F., et al., Neutrophil extracellular traps: casting the NET over pathogenesis. Curr Opin Microbiol, 2007. 10(1): p. 52-6. 32. Wartha, F. and B. Henriques-Normark, ETosis: a novel cell death pathway. Sci Signal, 2008. 1(21): p. pe25. 33. Ellis, J.E., M. Prochazka, and S.E. Fry, Evidence for In-Vivo Hematologic Biofilm Communities in 3 Patients with ALS. 5th ASM Conf. on Biofilms, 2009. 158.