Real-time monitoring of bacterial dynamics and interactions and early biofilm formation

Biofilms are complex dynamic microbial communities that colonize and grow on surfaces. The transition from the individual planktonic state (single bacterial cell floating in solution) to a biofilm improves the tolerance of bacteria to antibiotics and their growth even under unfavorable conditions1. In the biofilm, the bacteria are enclosed in a self-produced extracellular matrix, composed of extracellular polymeric substances (EPS) which, together with carbohydrate-binding proteins, adhesive structures such as pili and flagella and extracellular substances, act as a scaffold stabilizer for the three-dimensional structure of the biofilm. This structure provides bacteria with the appropriate amount of nutrients needed to support their growth and reproduction, enhancing cell-cell interactions, DNA exchange, and protecting biofilm components from desiccation, predation, and other external harmful agents.2.

The mechanisms of bacterial adhesion and biofilm formation are governed by several physical, chemical and biological factors. The first phase of biofilm formation involves the individual attachment of a single bacterium to a surface. Individual bacterial cells are transported to the surface either by physical forces or by intrinsic locomotion ability. Motile bacteria use structures, such as flagella, to approach the surface, guided by chemotactic, aerotactic, or phototactic responses. Motility promotes both initial interaction with the surface and movement along it. On the other hand, non-motile cells are delivered to the surface by diffusion and sedimentation processes or by the flow of the fluid in which they are suspended.3. Once a bacterium has approached the surface, initial attachment is regulated by: attractive and repulsive forces, primarily Van der Waals and electrostatic interactions; surface properties such as texture, roughness and hydrophobicity; and, solution properties such as pH and temperature4. However, attached bacteria can detach from the surface and rejoin the planktonic state in a process called reversible adhesion, as a result of hydrodynamic forces, repulsive forces, or in response to nutrient availability.5. If environmental conditions are favorable, a bacterium attaches permanently to the surface and begins to secrete EPS, establishing a permanent bond with the surface (called irreversible adhesion) and promoting the attachment of additional bacteria6. Irreversibly fixed bacteria continue to secrete EPS, forming a micro-niche favorable to their survival, proliferation and cohesion. The presence of two-dimensional microcolonies of interacting bacteria attached to a surface represents the early phase of the process of biofilm formationseven. Bacterial biofilm grows from a two-dimensional monolayer of irreversibly attached bacteria to a multi-layered three-dimensional colony that continues to grow, projecting into the surrounding medium hundreds of microns. This biofilm structure acts as a primitive circulatory system, connecting individual cells and allowing the exchange of nutrients and the elimination of wastes. In this last stage, the biofilm is finally broken by the pressure caused by the ever increasing number of bacteria or by the action of external forces, such as shear or abrasion from fluids. The bacteria are then again dispersed in the surrounding environment, able to colonize and infect a new substrate (Fig. 1)8.

Figure 1

Schematic representation of the five stages of biofilm formation8.

Bacterial biofilms account for up to 80% of hospital-acquired infections in the United States, and due to their adaptability and greater resistance to antibiotics, bacterial biofilms can easily form on medical devices and human tissues, leading to chronic and life-threatening infections9. Therefore, methodologies aimed at preventing biofilm formation or activating the immune system to eradicate these communities are essential for effective control of biofilm-related diseases.

Several strategies have been investigated previously to prevent bacterial biofilm formation and subsequent infection, with the development of a range of so-called antimicrobial surfaces. The majority of these surfaces are designed to kill bacteria on or near contact through the release of antimicrobial substances (biocidal surfaces)ten.

Despite significant efforts to characterize and prevent biofilm formation and due to the variety of forces involved, a clear understanding of preliminary bacteria-surface interaction and subsequent early-stage biofilm formation is needed to develop strategies. effective antimicrobials and to support their translation into in vivo and real-world scenarios. In this study, we followed in real time the dynamics and surface interactions of E.coli and P. aeruginosa bacteria exposed to glass surfaces, with or without antimicrobial coating, without bacteria labelling. E.coli is a Gram-negative, rod-shaped, non-spore-forming bacterium considered a model organism for studies in biological engineering and industrial microbiology. More E.coli The strains are harmless to the human body and are found in the intestines of warm-blooded organisms. However, several E.coli the strains are pathogenic and responsible for infections in various medical devices, such as urethral and intravascular catheters, joint prostheses, shunts and grafts11. E.coli biofilm can also be responsible for skin and soft tissue infections12. P. aeruginosa is a Gram-negative, rod-shaped, asporogenous, monoflagellate bacterium known to cause serious infections in immunocompromised cancer patients and patients with severe burns and cystic fibrosis13.

Bacterial imaging is commonly performed using fluorescence microscopy because it improves on the low resolving power and low contrast achievable with common brightfield microscopy. However, given the limitations of fluorescence microscopy, including photobleaching and phototoxicity, and the lack of knowledge about the effect on bacterial functions and processes caused by exposure to fluorescent dyes, strategies have been developed to perform label-free monitoring of bacterial cells and their initial adhesion to a surface. Among other techniques, phase contrast microscopy is one of the most effective markerless optical techniques in terms of resolution and contrast of the acquired image, allowing the three-dimensional tracking of a bacterial cell in simple and complex environments.14. However, phase contrast microscopy requires a specialized and expensive optical assembly, equipped with a specific condenser with a condenser ring coupled to objectives characterized by the presence of phase plates responsible for the delay of the diffracted rays.15. Another optical technique complementary to phase contrast microscopy, and capable of producing high contrast images of biological organisms, is differential interference contrast microscopy. High resolution and contrast are achieved through the use of two interfering coherent beams. The technique is able to visualize the human16 and bacterial cells17; however, it requires a specialized and expensive optical setup, fitted with a series of polarizers and prisms.

In this study, imaging was performed by generating caustic signatures of bacterial populations, allowing their monitoring in a common inverted optical microscope with only a few simple adjustments and without any requirement for fluorescent labeling. The high resolving power of the caustic-based optical setup allowed the qualitative characterization of the dynamics of E.coli and P. aeruginosa bacteria and the detection of preliminary bacteria-surface interactions, providing real-time information about bacteria forming a biofilm and proliferating on the target surface.

The aim of the study was to investigate the mechanism of attachment of bacteria to surfaces and to qualitatively characterize the potential relationship between the dynamics exhibited by bacteria when interacting with the surface and their viability or ability to start a biofilm. The results provide evidence for relationships between bacterial dynamics and biofilm formation that has direct implications for characterizing the effectiveness of antimicrobial surfaces as well as for assessing early biofilm formation. The technology described and employed in this study can be used to generate real-time image data to facilitate cost-effective, label-free studies of the effectiveness of antimicrobial surfaces in vitro.

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