Biofilms Research

By Jeremy Aguinaldo
April 27, 2015

In the summer of 1998, a large outbreak of Escherichia coli O157:H7 occurred in Alpine, Wyoming [1]. Investigators concluded that surface water containing deer and elk feces seeped into the aquifer that provided the town’s drinking water. One hundred fifty seven cases of illness were reported from Wyoming and 14 neighboring states. Those treated included three children and one adult who had hemolytic uremic syndrome, with all four eventually recovered.

The town’s water-collection system consisted of a series of perforated pipes that collected water 7 to 10 feet below ground level and carried to an underground tank. From the tank, pipes delivered unchlorinated water to the town.

The incident confirms the potential infection with E.coli O157:H7 in unprotected and unchlorinated water systems.

 

Intro to Biofilms:

The case series presented showed the results of a bacterial outbreak due to contaminated drinking water. With the usage of pipes and drains to distribute potable water as early as 2700 B.C. in ancient civilizations, microorganisms have adapted to utilize this system to its advantage. Water plumbing and drainage are essential and important system in all developed countries, yet the quality and safety is still a major issue around the world.

The purpose of this article is to highlight the mechanisms of how certain bacteria species form biofilms. These mechanisms include: hydrophobic and hydrophilic interactions on inanimate surfaces; symbiotic relationships with other species; and attachment on living tissues; and how it affect the safety of drinking water. I excluded the roles of parasites, viruses, and funguses, and strictly focus on bacteria species. Because of the diverse characteristics of multiple species of bacteria, I can only cover basic simplified mechanisms for each pathogen. It should also be noted that information on this particular defense mechanism for specific individual species is very limited. In addition, I also discuss the pathogenesis of infection and treatment.

Biofilms are a special adaptation of bacteria to facilitate colonization. Biofilm can be found in surgical appliances including artificial valves or indwelling catheters [2]. Native valve endocarditis, dental cavities, cystic fibrosis, otitis media, and periodontitis have also shown to be caused by biofilm-associated microorganisms.

 

Steps of Biofilm Formation:

  1. Bacteria attach to surface due to changes in gene expression, usually from external stresses. They usually take up a spore-like form.
  2. Bacteria are connected within a matrix of polysaccharide that binds the cells together and to the surface of the medium it is attached to.
  3. Production of biofilms requires sufficient numbers of bacteria (quorum). When the colony size is large enough (quorum sensing), biofilms are produced.
  4. This adaptation protects the bacteria from external stresses and antibiotics. Nutrients diffuse into the matrix. Other secondary bacterial species incorporate themselves within the biofilm. The close proximity of the species facilitates the exchange of genetic material.
  5. Shear force causes the release of bacteria.

Steps in Biofilm Formation

Role in Public Health:

Biofilms is an important issue for public health, due to the numerous potentially pathogenic bacteria [2]. These organisms are hardly found in bulk water, but in pipes where they are more protected against adverse environmental conditions, which includes disinfection measures. Drinking water associated biofilms may cause aesthetic problems including degradation of color, odor, and taste [3]. In addition, they can lead to biofouling in which there is an accumulation of pathogenic bacteria, viruses, fungi, protozoa, and algae. Developing countries are currently facing serious issues regarding safe drinking water, and the transmission of via fecal-oral route causing severe water related illnesses and death, particularly in infants.

Access to clean drinking water and sanitation facilities is a major priority for public health. Even in well developed countries, tap water quality assurance is facing new challenges, which includes biofouling and emerging waterborne pathogens. A major current issue involves the role of opportunistic pathogens for immunocompromised individuals: malnourished, immunosuppressed, diabetic, burn, cancer, AIDS, etc. [4]. Other populations sensitive to this issue are young children, elderly persons, or pregnant women [5]. Opportunistic infections are becoming a major threat, usually found in patients with indwelling cannula and catheters, implant devices, and contact lenses wearer [6].

http://www.cdc.gov/pulsenet/pathogens/ecoli.html

CDC: http://www.cdc.gov/pulsenet/pathogens/ecoli.html

E. coli O157:H7:

Escherichia coli (E. coli) are rod-shaped, gram-negative, lactose fermenting, facultative anaerobes [8]. These species are commonly found in the lower intestines of warm-blooded organisms. Most strains are harmless, but some serotypes can cause serious illnesses due to different types of virulence factors. These include fimbriae (cystitis and pyelonephritis), K capsule (pneumonia and neonatal meningitis), and LPS endotoxin (septic shock). E. coli causes an estimated 73,000 cases of infection and 61 deaths in the United States each year. Infection often leads to bloody diarrhea, vomiting, and occasionally to kidney failure.

E. coli produced exopolysacharide (EPS), which helps facilitate cell adhesion to hydrophilic surfaces [7]. EPS is the best indicator of fecal pollution and the possible presence of pathogens. This substance affects the attachment of cells on the surface of stainless steel. In addition, E. coli O157:H7 produces curli, a thin, coiled fimbraei-like extracellular structure, with an occasional point mutation on the csgD promoter. This protein enhances attachment of cells on the surface of polystyrene. Curli does not affect attachment of cells to stainless steel but does promote biofilm production.

O157:H7 is the most common serotype of enterohemorrhagic Escherichia coli (EHEC). Unlike the other E. coli species, EHEC does not ferment sorbitol. They produce Shiga-like toxin which causes Hemolytic-uremic syndrome (HUS) (triad of anemia, thrombocytopenia, and acute renal failure). The endothelium swells and narrows lumen, leading to mechanical hemolysis and reduced renal blood flow and the damaged endothelium consumes the platelets. The toxin is a protein composed of A subunit responsible for the toxic action of the protein, and five molecules of the B subunit responsible for binding to a specific cell type. The A subunit of the Shiga toxin modifies the RNA portion of the ribosome that leads to its inactivation and halting of protein synthesis, which leads to cell death. This will cause the necrosis and inflammation associated with dysentery.

Non-specific supportive therapy, including hydration, is important [8]. Antibiotics should not be used to treat this infection. There is no evidence that treatment with antibiotics is helpful, and taking antibiotics may increase the risk of HUS as well as taking antidiarrheal agents.

http://blog.gwcollegedemocrats.com/picsvqzp/mycobacterium-avium-complex

http://blog.gwcollegedemocrats.com/picsvqzp/mycobacterium-avium-complex

 

Mycobacterium avium complex (MAC):

Mycobacteria are gram positive but due to the high lipid Mycolic acid content in the cell wall and they are detected by carbolfuchsin in acid-fast stain [10]. This includes M. tuberculosis and M. leprae. M avium complex (MAC) is the most common of the nontuberculous mycobacteria that causes disease in humans and is most commonly composed of M. avium and M. intracellulare. Disseminated infections are usually seen with HIV patients. In HIV infected patients, manifestations include night sweats, weight loss, abdominal pain, fatigue, diarrhea, and anemia. In children, the most common syndrome is cervical lymphadenitis.

Adhesive properties of mycobacteria are related to chain length of their mycolic acids [9]. M. avium and M. intracellulare adhere to particulate matter in soil and water. This property also allows them to be resistant to heavy metals and chlorine. The combination of high hydrophobicity and resistance to chlorine and heavy metals suggests that M. avium and M. intracellulare form biofilms on copper and galvanize pipes.

Route of transmission is unclear, but most commonly environmentally acquired. Tumor necrosis factor (TNFα) and interferon (IFNγ) are important in the defense against mycobacterial diseases. Defect in these cytokines have been linked to increased susceptibility to MAC infections. Abnormalities of the lung protection barrier, such as injury to respiratory mucosa have also increase the risk of infection with MAC. MAC penetrates the gastrointestinal mucosa by unknown mechanisms and is phagocytosed by macrophages in the lamina propria. Macrophages fail to kill the bacteria, and it spreads through the submucosal tissue. Lymphatic drainage transports organisms to abdominal lymph nodes and then they enter the bloodstream, which can spread to other sites. Spleen, bone marrow, and liver are the most common areas of infection.

Treatment regimen should contain either clarithromycin with ethambutol as a second drug [10]. Many clinicians have added Rifampin in some situations. Pyrazinamide and Isoniazid are not effective for the therapy of MAC infections.

http://www.cibsejournal.com/cpd/2012-07/

http://www.cibsejournal.com/cpd/2012-07/

Legionella pneumophila:

Legionella species are gram negative rods [12]. They grow on charcoal yeast extract culture with iron and cysteine. The most common presentation of Legionella pneumophila is acute pneumonia (legionellosis); potentially any species of Legionella can cause the disease. Extrapulmonary disease (e.g., pericarditis and endocarditis) is rare. Less often, disease presents as a nonpneumonic epidemic, influenzalike illness called Pontiac fever.

L. pneumophila can be influenced by protozoa [11]. The pathogen replicate intracellularly due to co-evolution with multiple species of protozoa. In biofilm communities, several amoeba species have been found to be associated with L. pneumophila. Protozoans often graze on bacteria presence in multispecies biofilms that L. pneumophila exploits in order to replicate. They can grow off the debris from dead amoeba. Protozoa is a risk factor for L. pneuophila outbreaks. L. pneumophila may attach to protozoa in floating biofilms in the absence of available abiotic surfaces.

Legionella bacilli reside in surface and drinking water and are usually transmitted to humans in aerosols. Infection begins in the lower respiratory tract. Alveolar macrophages, which are the primary defense against bacterial infection of the lungs, engulf the bacteria; however, Legionella is a facultative intracellular bacteria that multiplies in macrophages. The bacteria bind to alveolar macrophages and are engulfed into a phagosome vesicle. However, the bacteria block the fusion of the lysosomes with the phagosome and prevent the normal acidification of the phagolysosome. The bacilli then multiply within the phagosome. Eventually, the cell will be lysed and will release additional bacteria to infect other cells. Person-to-person transmission has never been reported.

Legionnaires’ disease requires treatment with Erythromycin or other respiratory Fluoroquinolones such as Levofloxacin, Moxifloxacin, or Gemifloxacin [12]. Pontiac fever goes away without specific treatment and antibiotics provide no benefit for a patient with Pontiac fever.

http://www.agencia.fiocruz.br/incid%C3%AAncia-de-leptospirose-representa-alto-custo-social-para-o-rio-de-janeiro

http://www.agencia.fiocruz.br/incid%C3%AAncia-de-leptospirose-representa-alto-custo-social-para-o-rio-de-janeiro

Leptospira interrogans:

Leptospira bacteria are spirochetes, which are spiral-shaped with axial filaments [13]. Infection can lead to Leptospirosis, which includes flu-like symptoms, jaundice, photophobia with conjunctivitis. This is prevalent among surfers and in the tropics (i.e. Hawaii). Weil’s disease (icterohemorrhagic leptospirosis) is a severe form with jaundice and azotemia from liver and kidney dysfunction; fever, hemorrhage, and anemia.

Spread of leptospires can occur due to contact with urine, blood or tissues from infected persons. The organisms enter the body through the breaks in the skin or through mucous membranes. The organisms can also be acquired by drinking contaminated water. The organisms multiply in the blood and tissues of the body. Without treatment, Leptospirosis can lead to kidney damage, meningitis (inflammation of the membrane around the brain and spinal cord), liver failure, respiratory distress, and even death. There is evidence that leptospiras form a biofilm during kidney colonization in the proximal renal tubule lumen of certain rabbit species [14].

Leptospirosis is treated with antibiotics, such as doxycycline or penicillin, which should be given early in the course of the disease [13]. Intravenous antibiotics may be required for persons with more severe symptoms.

 

Conclusions:

Biofilms in drinking water are associated with bacterial pathogens and have an impact on its quality. The contaminated water supply may be a threat to human health. In developing countries, access to safe clean water continues to be a major global issue. Even in developed nations, high quality drinking water continues to be problematic with the risk of the emergence of opportunistic infections.

Biofilms is just one example as a defense mechanism for bacteria. This article discussed basic mechanisms on specifically a few species of bacteria that can form biofilms in their own unique ways. Due to the very limited resources of information, there are still additional questions. There are countless species that have their own unique ways of forming their own biofilms; it would be difficult to cover them all in a single article. Various studies showed that the ecology of biofilms is influenced by a series of abiotic and biotic factors. Pipes of different material have distinctly different bacterial communities. Bacterial evolution has shown adaptation to further infect human populations.

Further public health research is needed to understand biofilm formation, prevention, and control in the drinking water industry. With microorganisms constantly evolving bypass safety measures that were initially set for their specific prevention, and a growing rise of antibiotic resistance around the world, it important to remain vigilant and anticipate what to be expected.

 

Sources:

[1] Olsen, S. J., Miller, G., Breuer, T., Kennedy, M., Higgins, C., Walford, J., . . . Mead, P. (2002). A Waterborne Outbreak of. Emerging Infectious Diseases, 8(4), 370-375.

a. Donlan, M. R. (2002). Biofilms: Microbial Life on Surfaces. Emerging Infectious Diseases, 8(9), 881-890.

[2] Rao, V., Ghei, R., & Chambers, Y. (2005). Biofilms Research- Implications to BIosafety and Public Health. Applied Biosafey, 10(2), 83-90.

[3]2002). Health Risks from Microbial Growth and Biofilms. Office of Ground Water and Drinking Water Standards and Risk Management Division, U.S. Environmental Protection Agency. Retrieved from http://www.epa.gov/safewater/disinfection/tcr/regulation_revisions.html

[4] Rusin, P., Rose, J., Haas, C., & Gerba, C. (1997). Risk assessment of opportunistic. Rev Environ Contam T, 152, 57-83.

[5] Reynolds, K., Mena, K., & Gerba, K. (2008). Risk of Waterborne Illness Via Drinking Water in the United States. Rev Environ Contamin T, 192, 23-30.

[6] Glasmacher, A., Engelhart, S., & Exner, M. (2003). Infections from HPC organisms in drinking-water amongst the immunocomprised. In J. Bartram, J. Cotruvo, M. Exner, C. Fricker, & A. Glasmacher, Heterotrophic Plate Counts and Drinking-water Safety (pp. 137-145). London, UK: IWA Publishing.

[7] Ryu, J.-H., & Beuchat, L. R. (2005). Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel: Effect of Exopolysaccharide and Curli Production on Its Resistance to Chlorine. Applied and Environmental Microbiology, 71(1), 247-254.

[8] E.coli (Escherichia coli). (2014, December 1). Retrieved from Centers for Disease Control and Prevention: http://www.cdc.gov/ecoli/general/index.html/

[9] Bendinger, B., Huub, R., Altendorf, K., & Zehnder, A. (1993). Physicochemical Cell Surface and Adhesive Properties of Coryneform Bacteria Related to the Presence and Chain Length of Mycolic Acids. Applied and Environmental Microbiology, 59(11), 3973-3977.

[10] Mycobacterium avium Complex. (2005, October 12). Retrieved from Centers for Disease Control and Prevention: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/mycobacteriumavium_t.htm

[11] Taylor, M., Ross, K., & Bentham, R. (2009). Legionella, Protozoa, and Biofilms: Interactions Within Complex Microbial Systems. Microbiology Ecology, 58, 538-547.

[12] Legionella (Legionnaires’ Disease and Pontiac Fever). (2013, February 5). Retrieved from Centers for Disease Control and Prevention: http://www.cdc.gov/legionella/index.html

[13] Leptospirosis. (2014, November 18). Retrieved from Centers for Disease Control and Prevention: http://www.cdc.gov/leptospirosis/

[14] Goncalves-de-Albuerque, C., Burth, P., Silva, A., Younes-Ibrahum, M., Castro-Faria-Neto, H., & Castro-Faria, M. (2012). Leptospira and Inflammation. Mediators of Inflammation.

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