Biofilms are dense aggregates of bacterial colonies embedded inside a self-produced polymeric matrix. Biofilms have received increasing attention in medical, industrial, and environmental settings due to their enhanced survival. Their characterization using microscopy techniques has revealed the presence of structural and cellular heterogeneity in many bacterial systems. However, these techniques provide limited chemical detail and lack information about the molecules important for bacterial communication and virulence. Mass spectrometry imaging (MSI) bridges the gap by generating spatial chemical information with unmatched chemical detail, making it an irreplaceable analytical platform in the multi-modal imaging of biofilms. In the last two decades, over 30 species of biofilm-forming bacteria have been studied using MSI in different environments. The literature conveys both analytical advancements and an improved understanding of the effects of environmental variables such as host surface characteristics, antibiotics, and other species of microorganisms on biofilms. This review summarizes the insights from frequently studied model microorganisms. We share a detailed list of organism-wide metabolites, commonly observed mass spectral adducts, culture conditions, strains of bacteria, substrate, broad problem definition, and details of the MS instrumentation, such as ionization sources and matrix, to facilitate future studies. We also compared the spatial characteristics of the secretome under different study designs to highlight changes because of various environmental influences. In addition, we highlight the current limitations of MSI in relation to biofilm characterization to enable cross-comparison between experiments. Overall, MSI has emerged to become an important approach for the spatial/chemical characterization of bacterial biofilms and its use will continue to grow as MSI becomes more accessible.

Biofilm formation is an important survival strategy commonly used by bacteria and fungi, which are embedded in a protective extracellular matrix of organic polymers. They are ubiquitous in nature, including humans and other animals, and they can be surface- and non-surface-associated, making them capable of growing in and on many different parts of the body. Biofilms are also complex, forming polymicrobial communities that are difficult to eradicate due to their unique growth dynamics, and clinical infections associated with biofilms are a huge burden in the healthcare setting, as they are often difficult to diagnose and to treat. Our understanding of biofilm formation and development is a fast-paced and important research focus. This review aims to describe the advancements in clinical biofilm research, including both in vitro and in vivo biofilm models, imaging techniques and techniques to analyse the biological functions of the biofilm.

CONCLUSIONS: The development of an imaging technique to accurately identify biofilm regions on tissues and in wounds is crucial for the implementation of precise surface-based treatments, leading to better patient outcomes and reduced chances of infection.
OBJECTIVE: The goal of this study was to develop an imaging technique that relies on selective trypan blue (TB) staining of dead cells, necrotic tissues, and bacterial biofilms, to identify biofilm regions on tissues and wounds.
METHODS: The study explored combinations of ambient multi-colored LED lights to obtain maximum differentiation between stained biofilm regions and the underlying chicken tissue or glass substrate during image acquisition. The TB imaging results were then visually and statistically compared to fluorescence images using a shape similarity measure.
RESULTS: The comparisons between the proposed TB staining method and the fluorescence standard used to detect biofilms on tissues and glass substrates showed up to 97 percent similarity, suggesting that the TB staining method is a promising technique for identifying biofilm regions.
CONCLUSIONS: The TB staining method demonstrates significant potential as an effective imaging technique for the identification of fluorescing and non-fluorescing biofilms on tissues and in wounds. This approach could lead to improved precision in surface-based treatments and better patient outcomes.

Biofilms are cell assemblies embedded in an exopolysaccharide matrix formed by microorganisms of a single or many different species. This matrix in which they are embedded protects the bacteria from external influences and antimicrobial effects. The biofilm structure that microorganisms form to protect themselves from harsh environmental conditions and survive is found in nature in many different environments. These environments where biofilm formation occurs have in common that they are in contact with fluids. The gene expression of bacteria in complex biofilm differs from that of bacteria in the planktonic state. The differences in biofilm cell expression are one of the effects of community life. Means of quorum sensing, bacteria can act in coordination with each other. At the same time, while biofilm formation provides many benefits to bacteria, it has positive and negative effects in many different areas. Depending on where they occur, biofilms can cause serious health problems, contamination risks, corrosion, and heat and efficiency losses. However, they can also be used in water treatment plants, bioremediation, and energy production with microbial fuel cells. In this review, the basic steps of biofilm formation and biofilm regulation in the model organism Escherichia coli were discussed. Finally, the methods by which biofilm formation can be detected and monitored were briefly discussed.

Bacterial biofilms consist of surface-attached communities that secrete polymeric substances to form a biofilm matrix, generating a local microenvironment which helps protect from external factors. One such matrix component produced by a diverse list of microorganisms is the polysaccharide poly-β-1,6-N-acetylglucosamine (PNAG). Dispersin B is a PNAG-specific glycosyl hydrolase, which by leveraging its unique specificity, can be used to design a macromolecular fluorescent PNAG binding probe. An active site mutant of Dispersin B was fused to a fluorescent protein, to generate a probe that bound PNAG but did not hydrolyze its polysaccharide target. The ease and versatility of this strategy has made it possible to study PNAG in the context of maturing biofilms, as the probe tends to sequester in regions of high PNAG density. In this chapter, typical workflows from probe construction to cell-binding and imaging experiments are described.

Bacterial biofilms are defined as complex aggregates of bacteria that grow attached to surfaces or are associated with interfaces. Bacteria within biofilms are embedded in a self-produced extracellular matrix made of polysaccharides, nucleic acids, and proteins. It is recognized that bacterial biofilms are responsible for the majority of microbial infections that occur in the human body, and that biofilm-related infections are extremely difficult to treat. This is related with the fact that microbial cells in biofilms exhibit increased resistance levels to antibiotics in comparison with planktonic (free-floating) cells. In the last years, the introduction into the market of novel compounds that can overcome the resistance to antimicrobial agents associated with biofilm infection has slowed down. If this situation is not altered, millions of lives are at risk, and this will also strongly affect the world economy. As such, research into the identification and eradication of biofilms is important for the future of human health. In this sense, this article provides an overview of techniques developed to detect and imaging biofilms as well as recent strategies that can be applied to treat biofilms during the several biofilm formation steps.

Multidrug-resistant bacteria (MRB) and their biofilms, both of which develop high levels of drug tolerance, cause severe threats to global health. This study demonstrates that biocompatible fluorescent silicon-containing nanodots can be a multifunctional platform for simultaneously imaging and eliminating MRB and their biofilms. Ultrasmall epoxy group (oxirane)-functionalized organosilica nanodots (OSiNDs) with a high photoluminescence quantum yield of ≈31% are synthesized via a simple one-step hydrothermal treatment of an epoxy group-containing silane molecule, 3-glycidoxypropyltrimethoxysilane, and an organic dye, rose bengal. The resultant OSiNDs can be employed as a universal imaging reagent for visualizing various bacteria/biofilms, including MRB and their biofilms. Moreover, the epoxy group-terminated OSiNDs can be conjugated with amine-containing reagents only via the simple stirring of the mixtures at an elevated temperature (e.g., 60 °C) for several hours (e.g., 3 h) without the addition of activating reagents. The amine-containing antibiotic vancomycin (Van) can thus be easily conjugated with the OSiNDs, and the obtained OSiNDs-Van can successfully inhibit the growth of MRB and even eliminate their biofilms. Collectively, the present work may give new impetus to the development of novel antibacterial and anti-biofilm agents for overcoming the drug resistance of bacteria.

Imaging of biofilm systems is a prerequisite for a better understanding of both structure and its function. The review aims to critically discuss the use of optical coherence tomography (OCT) for the visualization of the biofilm structure as well as its dynamic behavior. A short overview on common and well-known, established imaging techniques for biofilms such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), Raman microscopy (RM), and magnetic resonance imaging (MRI) paves the way to imaging biofilms at the mesoscale, which is perfectly covered by means of OCT. Principle, resolution, imaging velocity, and limitations of OCT are subsequently presented and discussed in the context of biofilm applications. Examples are provided showing the strength of this technique with respect to the visualization of the mesoscopic biofilm structure as well as the estimation of flow profiles and shear rates. Common and new structural parameters derived from OCT datasets are presented. Additionally, the review shows the importance of OCT with respect to a better description of mechanical biofilm properties. Finally, the implementation of multi-dimensional OCT datasets in biofilm modelling is shown by several examples aiming on an improved understanding of mass transfer at the bulk-biofilm interface. Biotechnol. Bioeng. 2017;114: 1386-1402. © 2017 Wiley Periodicals, Inc.

Imaging and modeling are two major approaches in biofilm research to understand the physical and biochemical processes involved in biofilm development. However, they are often used separately. In this study we combined these two approaches to investigate substrate mass transfer and mass flux. Cross-sectional biofilm images were acquired by means of optical coherence tomography (OCT) for biofilms grown on carriers. A 2D biofilm model was developed incorporating OCT images as well as a simplified biofilm geometry serving as structural templates. The model incorporated fluid flow, substrate transfer and biochemical conversion of substrates and simulated the hydrodynamics surrounding the biofilm structure as well as the substrate distribution. The method allowed detailed analysis of the hydrodynamics and mass transfer characteristics at the micro-scale. Biofilm activity with respect to substrate fluxes was compared among different combinations of flow, substrate availability and biomass density. The combined approach revealed that higher substrate fluxes at heterogeneous biofilm surface under two conditions: pure diffusion and when high flow velocity along the biofilms surface renders the whole liquid-biofilm interface to be highly active. In-between the two conditions the substrate fluxes across the surface of smooth biofilm geometry were higher than that of the heterogeneous biofilms.