Chapter 1 : New Technologies for Studying Biofilms

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The results of recent biofilm characterizations have helped reveal the complexities of these surface-associated communities of microorganisms. The activities of the cells and the structure of the extracellular matrix material demonstrate that biofilm bacteria engage in a variety of physiological behaviors that are distinct from planktonic cells ( ). For example, bacteria in biofilms are adapted to growth on surfaces, and most produce adhesins and extracellular polymers that allow the cells to firmly adhere to the surfaces or to neighboring cells ( ). The extracellular material of biofilms contains polysaccharides, proteins, and DNA that form a glue-like substance for adhesion to the surface and for the three-dimensional (3D) biofilm architecture ( ). The matrix material, although produced by the individual cells, forms structures that provide benefits for the entire community, including protection of the cells from various environmental stresses ( ). Biofilm cells form a community and engage in intercellular signaling activities ( ). Diffusible signaling molecules and metabolites provide cues for expression of genes that may benefit the entire community, such as genes for production of extracellular enzymes that allow the biofilm bacteria to utilize complex nutrient sources ( ). Biofilm cells are not static. Many microorganisms have adapted to surface-associated motility, such as twitching and swarming motility ( ). Cellular activities, including matrix production, intercellular signaling, and surface-associated swarming motility suggest that biofilms engage in communal activities. As a result, biofilms have been compared to multicellular organs where cells differentiate with specialized functions ( ). However, bacteria do not always cooperate with each other. Biofilms are also sites of intense competition. The bacteria within biofilms compete for nutrients and space by producing toxic chemicals to inhibit or kill neighboring cells or inject toxins directly into neighboring cells through type VI secretion ( ). Therefore, biofilm cells exhibit both communal and competitive activities.

Citation: Franklin M, Chang C, Akiyama T, Bothner B. 2015. New Technologies for Studying Biofilms, p 1-32. In Ghannoum M, Parsek M, Whiteley M, Mukherjee P (ed), Microbial Biofilms, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MB-0016-2014
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Figure 1

Examples of methods for biofilm cultivation under static conditions. (A) Biofilm cultured at the air-water interface, forming a pellicle. Published with permission from reference . (B) Biofilm cultured on a glass coupon under static conditions. Published with permission from reference . (C) Example of biofilm growth as a colony biofilm. Published with permission from reference .

Citation: Franklin M, Chang C, Akiyama T, Bothner B. 2015. New Technologies for Studying Biofilms, p 1-32. In Ghannoum M, Parsek M, Whiteley M, Mukherjee P (ed), Microbial Biofilms, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MB-0016-2014
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Figure 2

Examples of continuous-flow reactors for biofilm cultivation. (A) CDC reactor with medium inlet and outlet ports. Biofilms form on coupons arranged on removable Teflon rods. Published with permission from reference . (B) Drip-flow reactor with medium inlet and outlet ports and air exchange ports. Biofilms form on removable slides. Published with permission from reference . (C) Capillary flow cell for imaging biofilms. Published with permission from http://centerforgenomicsciences.org/research/biofilm_flow.html.

Citation: Franklin M, Chang C, Akiyama T, Bothner B. 2015. New Technologies for Studying Biofilms, p 1-32. In Ghannoum M, Parsek M, Whiteley M, Mukherjee P (ed), Microbial Biofilms, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MB-0016-2014
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Figure 3

Examples of microfluidics approaches applied to biofilm research. (A) Example of a microfluidics device for precise control of fluids. Published with permission from reference . (B) Biofilm streamers forming within a microfluidics flow channel. Published with permission from reference . Microdroplet biofilm reactor showing phenotypic switching of cells and simultaneous change in expression from cyan fluorescent protein to the yellow fluorescent protein Published with permission from reference . (D) Schematic representation of microfluidics flow cell. Published with permission from reference .

Citation: Franklin M, Chang C, Akiyama T, Bothner B. 2015. New Technologies for Studying Biofilms, p 1-32. In Ghannoum M, Parsek M, Whiteley M, Mukherjee P (ed), Microbial Biofilms, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MB-0016-2014
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Figure 4

Example of a biofilm vertical transect, showing GFP–gene expression heterogeneity. Areas were cut from different biofilm strata and captured for transcriptomics analyses. Published with permission from reference .

Citation: Franklin M, Chang C, Akiyama T, Bothner B. 2015. New Technologies for Studying Biofilms, p 1-32. In Ghannoum M, Parsek M, Whiteley M, Mukherjee P (ed), Microbial Biofilms, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MB-0016-2014
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Figure 5

Gene expression heterogeneity, demonstrated by translational fusions of target proteins to the yellow fluorescent protein (YFP). (A) Translational fusion of the IbpA protein to YFP, showing uniform distribution of IbpA throughout the biofilm. Cells were counterstained with mCherry fluorescent protein (mCFP). (B) Translational fusion of Rmf protein to YFP, showing that most Rmf production occurs in cells at the top of the biofilm. Cells counterstained with mCFP (M.J. Franklin, unpublished data).

Citation: Franklin M, Chang C, Akiyama T, Bothner B. 2015. New Technologies for Studying Biofilms, p 1-32. In Ghannoum M, Parsek M, Whiteley M, Mukherjee P (ed), Microbial Biofilms, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MB-0016-2014
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Figure 6

(A) Images of biofilms, where the cells constitutively express the GFP. (B) Extracellular matrix material stained with Bodipy 630/650-X NHS from Life Technologies. (C) Combined image showing GFP-fluorescent bacteria and Bodipy-stained matrix (M.J. Franklin, unpublished data).

Citation: Franklin M, Chang C, Akiyama T, Bothner B. 2015. New Technologies for Studying Biofilms, p 1-32. In Ghannoum M, Parsek M, Whiteley M, Mukherjee P (ed), Microbial Biofilms, Second Edition. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MB-0016-2014
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