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1. Spatial ecology of microbial colonization intensity and behavior in rice rhizoplane biofilms analyzed by CMEIAS Bioimage Informatics software

   Dazzo, F.B.; Yanni, Y.G. (August 2017, Journal of soil science and plant nutrition)

   CMEIAS (Center for Microbial Ecology Image Analysis System) Bioimage Informatics software is designed to strengthen microscopy-based approaches for understanding microbial ecology at spatial scales directly relevant to ecological functions performed by individual cells and microcolonies. Copyrighted software components are thoroughly documented and provided as free downloads at . The software tools already released include CMEIAS-Image Tool v. 1.28, CMEIAS Color Segmentation, CMEIAS Quadrat Maker and CMEIAS JFrad Fractal Dimension analysis. The spatial ecology module of the next CMEIAS upgrade currently being developed (version 4.0) is designed to extract data from images for analysis of plotless point-patterns, quadrat-lattice patterns, geostatistical autocorrelation and fractal geometry of cells within biofilms. Examples presented here illustrate how selected CMEIAS attributes can be used to analyze the in situ spatial intensity, pattern of distribution, and colonization behavior of an indigenous population of a rhizobial strain on a sampled image of the rhizoplane landscape of a rice plant grown in field soil. The spatial ecology information gained can provide useful insights that help to predict the most likely performance of the biofertilizer test strain in relation to the growth response of the crop under field conditions. 

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2. Multispecies biofilm architecture determines bacterial exposure to phages

   https://doi.org/10.1371/journal.pbio.3001913  

   Winans, James B.; Wucher, Benjamin R.; Nadell, Carey D. (December 2022, PLOS Biology)

   Barr, Jeremy J. (Ed.)

   Numerous ecological interactions among microbes—for example, competition for space and resources, or interaction among phages and their bacterial hosts—are likely to occur simultaneously in multispecies biofilm communities. While biofilms formed by just a single species occur, multispecies biofilms are thought to be more typical of microbial communities in the natural environment. Previous work has shown that multispecies biofilms can increase, decrease, or have no measurable impact on phage exposure of a host bacterium living alongside another species that the phages cannot target. The reasons underlying this variability are not well understood, and how phage–host encounters change within multispecies biofilms remains mostly unexplored at the cellular spatial scale. Here, we study how the cellular scale architecture of model 2-species biofilms impacts cell–cell and cell–phage interactions controlling larger scale population and community dynamics. Our system consists of dual culture biofilms ofEscherichia coliandVibrio choleraeunder exposure to T7 phages, which we study using microfluidic culture, high-resolution confocal microscopy imaging, and detailed image analysis. As shown previously, sufficiently mature biofilms ofE.colican protect themselves from phage exposure via their curli matrix. Before this stage of biofilm structural maturity,E.coliis highly susceptible to phages; however, we show that these bacteria can gain lasting protection against phage exposure if they have become embedded in the bottom layers of highly packed groups ofV.choleraein co-culture. This protection, in turn, is dependent on the cell packing architecture controlled byV.choleraebiofilm matrix secretion. In this manner,E.colicells that are otherwise susceptible to phage-mediated killing can survive phage exposure in the absence of de novo resistance evolution. While co-culture biofilm formation withV.choleraecan confer phage protection toE.coli, it comes at the cost of competing withV.choleraeand a disruption of normal curli-mediated protection forE.colieven in dual species biofilms grown over long time scales. This work highlights the critical importance of studying multispecies biofilm architecture and its influence on the community dynamics of bacteria and phages. 

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3. Rheology of Candida albicans fungal biofilms

   https://doi.org/10.1122/8.0000427  

   Beckwith, Joanne K.; Ganesan, Mahesh; VanEpps, J. Scott; Kumar, Anuj; Solomon, Michael J. (July 2022, Journal of Rheology)

   Fungi such as Candida albicans exist in biofilm phenotypes, which present as viscoelastic materials; however, a method to measure linear viscoelastic moduli, yield stress, and yield strain is lacking. Characterization methods for fungal materials have been limited to techniques specific to particular industries. Here, we present a method to measure the shear stress, strain amplitude, and creep of C. albicans BWP17 biofilms. Our method includes features tailored to the analysis of fungi including an in vitro growth protocol attuned to the slow growth rates of C. albicans biofilms and a resultant cultured biofilm that has sufficient integrity to be transferred to the rheometer tooling without disrupting its structure. The method's performance is demonstrated by showing that results are insensitive to gap, evaporative sealant, length of experiment, and specimen radius. Multiscale imaging of the fungal biofilm showed complex entanglement networks at the hundred-micrometer scale. For a wild-type strain cultivated for 14 days, using small-amplitude oscillatory rheology, we found that the elastic (G′) and viscous (G″) moduli were nearly independent of frequency over the range 0.1–10 s −1 , with magnitudes of [Formula: see text] and [Formula: see text], respectively. The yield stress was approximately [Formula: see text]. We modeled the linear creep response of the fungal biofilm and found that C. albicans has a characteristic relaxation time of [Formula: see text] and a viscosity of [Formula: see text]. We applied this method to probe the effects of altered chitin deposition in the C. albicans cell wall. Differences between the biofilm's phenotypic cell shape and rheological properties in mutants with altered chitin synthase activity were resolved. Discovering how genotypic, phenotypic, and environmental factors impact the material properties of these microbial communities can have implications for understanding fungal biofilm growth and aid in the development of remediation strategies. 

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4. Breakdown of clonal cooperative architecture in multispecies biofilms and the spatial ecology of predation

   https://doi.org/10.1073/pnas.2212650120  

   Wucher, Benjamin R.; Winans, James B.; Elsayed, Mennat; Kadouri, Daniel E.; Nadell, Carey D. (February 2023, Proceedings of the National Academy of Sciences)

   Biofilm formation, including adherence to surfaces and secretion of extracellular matrix, is common in the microbial world, but we often do not know how interaction at the cellular spatial scale translates to higher-order biofilm community ecology. Here we explore an especially understudied element of biofilm ecology, namely predation by the bacteriumBdellovibrio bacteriovorus. This predator can kill and consume many different Gram-negative bacteria, includingVibrio choleraeandEscherichia coli.V. choleraecan protect itself from predation within densely packed biofilm structures that it creates, whereasE. colibiofilms are highly susceptible toB. bacteriovorus. We explore how predator–prey dynamics change whenV. choleraeandE. coliare growing in biofilms together. We find that in dual-species prey biofilms,E. colisurvival underB. bacteriovoruspredation increases, whereasV. choleraesurvival decreases.E. colibenefits from predator protection when it becomes embedded within expanding groups of highly packedV. cholerae. But we also find that the ordered, highly packed, and clonal biofilm structure ofV. choleraecan be disrupted ifV. choleraecells are directly adjacent toE. colicells at the start of biofilm growth. When this occurs, the two species become intermixed, and the resulting disordered cell groups do not block predator entry. Because biofilm cell group structure depends on initial cell distributions at the start of prey biofilm growth, the surface colonization dynamics have a dramatic impact on the eventual multispecies biofilm architecture, which in turn determines to what extent both species survive exposure toB. bacteriovorus. 

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5. Thresholding Approach for Automatic Detection of Pseudomonas aeruginosa Biofilms from Fluorescence in situ Hybridization Images

   Zonglin Yang, Tatsuya Akiyama (January 2021, International Journal of Biomedical and Biological Engineering)

   null (Ed.)

   Pseudomonas aeruginosa is an opportunistic pathogen that forms surface-associated microbial communities (biofilms) on artificial implant devices and on human tissue. Biofilm infections are difficult to treat with antibiotics, in part, because the bacteria in biofilms are physiologically heterogeneous. One measure of biological heterogeneity in a population of cells is to quantify the cellular concentrations of ribosomes, which can be probed with fluorescently labeled nucleic acids. The fluorescent signal intensity following fluorescence in situ hybridization (FISH) analysis correlates to the cellular level of ribosomes. The goals here are to provide computationally and statistically robust approaches to automatically quantify cellular heterogeneity in biofilms from a large library of epifluorescent microscopy FISH images. In this work, the initial steps were developed toward these goals by developing an automated biofilm detection approach for use with FISH images. The approach allows rapid identification of biofilm regions from FISH images that are counterstained with fluorescent dyes. This methodology provides advances over other computational methods, allowing subtraction of spurious signals and non-biological fluorescent substrata. This method will be a robust and user-friendly approach which will enable users to semi-automatically detect biofilm boundaries and extract intensity values from fluorescent images for quantitative analysis of biofilm heterogeneity. 

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