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1. Comparative Biofilm Assays Using Enterococcus faecalis OG1RF Identify New Determinants of Biofilm Formation

   https://doi.org/10.1128/mBio.01011-21  

   Willett, Julia L.; Dale, Jennifer L.; Kwiatkowski, Lucy M.; Powers, Jennifer L.; Korir, Michelle L.; Kohli, Rhea; Barnes, Aaron M.; Dunny, Gary M. (June 2021, mBio)

   Cooper, Vaughn S. (Ed.)

   ABSTRACT Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar discs, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by six Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA , tig , OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions and identifies multiple new genetic determinants of biofilm formation. IMPORTANCE E. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions. 

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2. Heterogeneity formation within biofilm systems

   https://doi.org/10.1017/S0956792518000402  

   ARISTOTELOUS, ANDREAS C.; GRABOVSKY, YURY; KLAPPER, ISAAC (December 2018, European Journal of Applied Mathematics)

   Biofilms, and collections of embedded microbial communities, present structural heterogeneities with functional consequences for important processes, such as transport. The origin of such structures has been unclear. Here, we propose that they can arise as a consequence of diffusive transport limitation. To illustrate, a model allowing internal heterogeneity is developed. Linear analysis is applied to a simplified version of the model suggesting that heterogeneity forms on (or below) the active layer length, a length scale that may not be suitable for homogenization, with non-trivial implications for system scale properties such as reduction in system-wide diffusive transport efficiency. Numerics suggest that the simplified model provides useful insight into behaviour of the full model. We then show examples based on microcolony formation in host domains and argue that internal heterogeneity can be related to community function. 

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3. Isolating Microplastics from Biofilm Communities

   https://doi.org/10.1525/abt.2022.84.9.555  

   Meiller, Jesse; Sosa, Ana; May, Eva; Frederick, J. Adam (November 2022, The American Biology Teacher)

   Plastic debris in aquatic and marine environments often breaks up into fragments that are smaller than 5 millimeters, which are then classified as microplastics. While there is not yet a standardized and validated methodology for characterizing microplastics, the protocol developed in this study uses methods for isolating and observing microplastics and for the investigation of how they interact with organisms present in biofilms from urban waterways. Project-based learning (PBL) has been proven to be a successful strategy in K–12 science education; the implementation of PBL provides opportunities for student-driven inquiry and provides teachers with a means to integrate curriculum with current research and to consider the effects of human impacts on the environment. This paper describes the protocol developed for high school teachers to educate students about microplastics and how to successfully isolate and observe them. Teachers and students in Maryland successfully isolated microplastics from biofilm samples from the Inner Harbor, Baltimore, Maryland, and shared their results. International teachers and students in Barcelona, Spain, involved in a related project, had similar results and shared experiences through images, video, and online meetings. These collaborations provide important opportunities for student-driven inquiry and for them to engage in methods of current scientific research. 

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4. Visible Light-Activated Carbon Dots for Inhibiting Biofilm Formation and Inactivating Biofilm-Associated Bacterial Cells

   https://doi.org/10.3389/fbioe.2021.786077  

   Dong, Xiuli; Overton, Christopher M.; Tang, Yongan; Darby, Jasmine P.; Sun, Ya-Ping; Yang, Liju (November 2021, Frontiers in Bioengineering and Biotechnology)

   This study aimed to address the significant problems of bacterial biofilms found in medical fields and many industries. It explores the potential of classic photoactive carbon dots (CDots), with 2,2′-(ethylenedioxy)bis (ethylamine) (EDA) for dot surface functionalization (thus, EDA-CDots) for their inhibitory effect on B. subtilis biofilm formation and the inactivation of B. subtilis cells within established biofilm. The EDA-CDots were synthesized by chemical functionalization of selected small carbon nanoparticles with EDA molecules in amidation reactions. The inhibitory efficacy of CDots with visible light against biofilm formation was dependent significantly on the time point when CDots were added; the earlier the CDots were added, the better the inhibitory effect on the biofilm formation. The evaluation of antibacterial action of light-activated EDA-CDots against planktonic B. subtilis cells versus the cells in biofilm indicate that CDots are highly effective for inactivating planktonic cells but barely inactivate cells in established biofilms. However, when coupling with chelating agents (e.g., EDTA) to target the biofilm architecture by breaking or weakening the EPS protection, much enhanced photoinactivation of biofilm-associated cells by CDots was achieved. The study demonstrates the potential of CDots to prevent the initiation of biofilm formation and to inhibit biofilm growth at an early stage. Strategic combination treatment could enhance the effectiveness of photoinactivation by CDots to biofilm-associated cells. 

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5. A biophysical threshold for biofilm formation

   https://doi.org/10.7554/eLife.76380  

   Moore-Ott, Jenna A; Chiu, Selena; Amchin, Daniel B; Bhattacharjee, Tapomoy; Datta, Sujit S (June 2022, eLife)

   Bacteria are ubiquitous in our daily lives, either as motile planktonic cells or as immobilized surface-attached biofilms. These different phenotypic states play key roles in agriculture, environment, industry, and medicine; hence, it is critically important to be able to predict the conditions under which bacteria transition from one state to the other. Unfortunately, these transitions depend on a dizzyingly complex array of factors that are determined by the intrinsic properties of the individual cells as well as those of their surrounding environments, and are thus challenging to describe. To address this issue, here, we develop a generally-applicable biophysical model of the interplay between motility-mediated dispersal and biofilm formation under positive quorum sensing control. Using this model, we establish a universal rule predicting how the onset and extent of biofilm formation depend collectively on cell concentration and motility, nutrient diffusion and consumption, chemotactic sensing, and autoinducer production. Our work thus provides a key step toward quantitatively predicting and controlling biofilm formation in diverse and complex settings. 

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