Biofilms

Bacterial Biofilms: A general viewBlgo. Carlos A. Fernández Miñ[email protected] – Marzo, 2014Segunda Especialidad en Microbiología ClínicaHospital Regional LambayequeUniversidad Nacional Pedro Ruiz Gallo

mailto:[email protected]

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What are Biofilms?• Biofilms are communities of surface-associated

microorganisms encased in a self-produced extracellular matrix.• Biofilm formation is a nearly universal bacterial trait,

and biofilms are found on almost all natural and artificial surfaces• Some examples of biofilms are the plaque that forms on

teeth and the slime that forms on surfaces in watery areas.

Biofilms are everywhere

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Biofilms appear early in the fossil record. There is evidence of biofilm formation early in the fossil record, particularly in hydrothermal environments.

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High-resolution scanning electron microscopy image of a community-associated methicillin-resistant Staphylococcus aureus biofilm embedded in extracellular matrix, formed on implanted catheter tubing during experimental biofilm infection.

M. Otto, D. Dorward, E. Fischer and A. Mora, National Institute of Allergy and Infectious Diseases, USA.

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Human Infections linked to biofilms

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A simple view: B. subtilis biofilm formation

Vlamakis, H., Chai, Y., Beauregard, P., Losick, R., Kolter, R. Sticking together: building a biofilm the Bacillus subtilis way. Nature Reviews Microbiology, 11: 157-168 (2013).

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Vlamakis, H., Chai, Y., Beauregard, P., Losick, R., Kolter, R. Sticking together: building a biofilm the Bacillus subtilis way. Nature Reviews Microbiology, 11: 157-168 (2013).

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A closer look at biofilms Extracellular Matrix

In situ observations of biofilm structure using confocal laser microscopy showed sessile bacteria growing in heterogeneous matrix-enclosed microcolonies interspersed with open water channels.

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The complex structure of bacterial biofilms

McDougald, D., Rice, S., Barraud, N., Steinberg, P., Kjelleberg, S. Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal . Nature Reviews Microbiology, 10: 39-50 (2010).

ΔPf4

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Oral bacterial colonization

Kolenbrander, P.E., Palmer, R.J., Periasamy, S., Jakubovics, N.S. Oral multispecies biofilm development and the key role of cell–cell distance. Nature Reviews Microbiology, 8: 471-481 (2010).

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There is a strong relationship between the cell’s environment and its physiology and structure.

Science News

Biofilms form Nutrient levels decrease towards the

center

Variety of environmental niches form

Biofilm formation and growth

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• Quorum sensing and chemical signaling• Microcolonies with extracellular matrix• Network of channels• Cells experience different environmental conditions• Multi-species

Biofilm formation and growth

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Stages of Biofilm Formation

Bacteria switch from a free-floating (planktonic) state where they function as individuals to a sessile state where they function as communities.

Watnick, P., Kolter, R. Biofilm, City of microbes. J. Bacteriol. 182 (10): 2675-2679 (2000).

Biofilm formation in Vibrio cholerae

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Candida albicans biofilm structure in vitro and in vivo

Scanning electron micrograph (SEM) of an in vitro Candida albicans biofilm. The biofilm sample was sliced to show three layers in a cross-sectional view.

SEM of an in vivo C. albicans biofilm from the rat catheter model.

Images of J. Suhan (Carnegie Mellon University, Pittsburgh, Pennsylvania, USA), and J. Nett and D. Andes (University of Wisconsin–Madison, USA).

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Singh, P.K., Schaefer, A.L., Parsek, M.R., Moningerk, T.O., Welsh, M.J., Greenberg, E.P. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 407: 762-764 (2000).

In the microscopic examination of sputum from patients colonizated by P. aeruginosa reveled that they are encased in a densely stained matrix. This appearance is consistent with previous microscopic observations and consistent with the hypothesis that in CF sputum, P. aeruginosa exists in biofilms.

a, Low magnification; b, high magnification.

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In P. aeruginosa, differentiation is cued by one of two quorum-sensing signals N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and N-butyryl-LHSL (C4-HSL). The signal required for microcolony differentiation in biofilms is 3OC12-HSL).


C4-HSL as a fraction of the total C4-HSL plus 3OC12-HSL. From left to right, P. aeruginosa in early logarithmic (A600 = 0.5), mid-logarithmic (A600 = 1), late logarithmic (A600 = 2), stationary (A600 = 4) and late stationary phase (A600 = 6).

C4-HSL 1 C4-HSL 3 C4-HSL 663OC12-HSL 3 3OC12-HSL 1 3OC12-HSL 1A/(A+B) = 0.25 A/(A+B) = 0.75 A/(A+B) = 0.99

Incremento de 3OC12-HSL

C4-HSL 1 C4-HSL 3 C4-HSL 663OC12-HSL 3 3OC12-HSL 1 3OC12-HSL 1A/(A+B) = 0.25 A/(A+B) = 0.75 A/(A+B) = 0.99

Incremento de C4-HSL

QUORUM SENSING

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The findings are consistent with the hypothesis that P. aeruginosa in CF lungs exist as a biofilm.

QUORUM SENSING

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Whiteley, M., Bangera, M.G., Bumgarner, R.E., Parsek, M.R., Teitzel, G.M., Lory, S., Greenberg, E.P. Gene expression in Pseudomonas aeruginosa biofilms. Nature 413: 860-864 (2001).

Genes for synthesis of pili and flagella are repressed in biofilms. Pili and flagella are reported to be involved in the initial steps (attachment and microcolony formation) of development of P. aeruginosa biofilms. These appendages may not be required for maintenance of a mature biofilm. Once development has proceeded through these steps, pili and flagella are no longer required.

Diferential gene expression

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Coordinated cell-death is necessary to maintain the biofilm

A confocal image (400× magnification) showing a mixed population of live (green) and dead (red) Staphylococcus aureus cells within a 3-day-old biofilm. Image by Ethan Mann, University of Nebraska Medical Center, Nebraska, USA.

The cidABC and lrgAB operons of S. aureus regulate cell lysis during the stationary phase of growth and in response to antibiotic exposure. There are homologues of cid/lrg genes present in a wide range of Gram-positive and Gram-negative bacteria.

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Moving of bacteria in biofilms

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Active biofilm dispersal and variant formation

McDougald, D., Rice, S., Barraud, N., Steinberg, P., Kjelleberg, S. Should we stay or should we go: mechanisms and ecological consequences for biofilm dispersal . Nature Reviews Microbiology, 10: 39-50 (2010).

Each biofilm population becomes a mutator

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Hall-Stoodley, L., Costerton, J.W., Stoodley, P. Bacterial biofilms: from the natural environment to infectious diseases. Nature Reviews Microbiology, 2: 95-108 (2004).

Bacterial endocarditis shows how microorganisms on the skin or in the oral cavity that transiently enter the bloodstream can colonize abnormal or implanted valves, or altered endothelial surfaces in the heart.


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Progression of Staphylococcus aureus from an infected or contaminated source through the bloodstream to a metastatic target

Thwaites, G.E., Gant, V. Are bloodstream leukocytes Trojan Horses for the metastasis of Staphylococcus aureus? Nature Reviews Microbiology, 9: 215-222 (2011).

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Multiplicity of phenotypic states in biofilms

Stewart, P.S., Franklin, M.J., Physiological heterogeneity in biofilms. Nature Reviews Microbiology, 6: 199-210 (2008).

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• High diversity• Less parallelism• Each biofilm population

becomes a mutator

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Scanning electron micrograph of an untreated biofilm of S. epidermidis (a) and an identical biofilm exposed to vancomycin and rifampin for 72 h at concentrations exceeding the MIC and MBC for the organism (b). Despite obvious changes in the treated biofilm, viable organisms were recovered for which the MIC and MBC of both agents were unaltered. Photo: American Society for Microbiology.

Drug resistance in Biofilms

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Time- and dose-dependent killing of biofilms by metals

Harrison, J.J., Ceri, H., Turner, R.J., Multimetal resistance and tolerance in microbial biofilms. Nature Reviews Microbiology, 5: 928-938 (2007).

a) In vivo b) In vitro

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Drug resistance in Biofilms mediated by persister cells

Lewis, Kim. Persister cells, dormancy and infectious disease. Nature Reviews Microbiology, 5: 48-56 (2007).

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Formation of persister cells

Lewis, Kim. Persister cells, dormancy and infectious disease. Nature Reviews Microbiology, 5: 48-56 (2007).

The treatment of a population with an antibiotic results in cell death, leaving only persister cells or resistant mutants alive

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Multiresistant Bacteria• Horizontal gene transfer is promoted in biofilms;

therefore, it is possible that antibiotic resistance spreads more easily than in planktonic populations

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Existence in a biofilm induces moderate levels of resistance to all antimicrobial treatments. This could afford cells in a biofilm the opportunity to respond to an antibiotic by inducing genes more specific to that antibiotic.

We compared biofilms exposed to tobramycin with untreated biofilms.

Consistent with our hypothesis, 20 genes were differentially expressed in tobramycin-treated biofilms: 14 were activated and 6 were repressed by tobramycin (seven times the minimum

inhibitory concentration for planktonic cells). Of these 20 genes, 12 were classified as genes coding for hypothetical proteins of unknown function. As expected, treatment with tobramycin, which causes errors in protein synthesis, seemed to induce a stress response, with activation of dnaK and groES genes, for example. Tobramycin strongly induced several genes coding for hypothetical proteins.


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Survival of cells in wild-type and rpoS-mutant biofilms after treatment with the antibiotic tobramycin. Open symbols, planktonic cultures; filled symbols, biofilms; squares, parent strain PAO1; triangles, rpoS mutant. At tobramycin concentrations above 5 mg ml -1 there were 2 viable cells ml -1 in planktonic cultures.

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Taken together, the data indicate that the ability to form biofilms is an ancient and integral characteristic of prokaryotes. In the context of EVOLUTION AND ADAPTATION it is likely that biofilms provided homeostasis in the face of the fluctuating and harsh conditions of the primitive earth (extreme temperatures, pH and exposure to ultraviolet (UV) light.

Biofilms are not new

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Conceptualization of biofilm development and dynamic behaviours. Biofilms are structurally and dynamically complex biological systems


Biofilms

Science

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