Mimicking biofilm formation and development: Recent progress in in vitro and in vivo biofilm models

Abstract

Biofilm formation in living organisms is associated to tissue and implant infections, and it has also been linked to the contribution of antibiotic resistance. Thus, understanding biofilm development and being able to mimic such processes is vital for the successful development of antibiofilm treatments and therapies. Several decades of research have contributed to building the foundation for developing in vitro and in vivo biofilm models. However, no such thing as an "all fit" in vitro or in vivo biofilm models is currently available. In this review, in addition to presenting an updated overview of biofilm formation, we critically revise recent approaches for the improvement of in vitro and in vivo biofilm models.

Keywords: Microbiofilms; Microbiology.

Graphical abstract

Figure 1

Schematic representation for single bacterial species biofilm formation on a solid surface The schematic depicts the five main steps for the formation and spreading of biofilms.

Figure 2

Schematic representation for the main nonspecific and specific interactions between bacteria and surfaces (A) Some physicochemical interactions include the attractive van der Waals forces; attractive or repulsive electrostatic interactions, which depend on the microenvironment conditions, where the presence of a conditioning film may contribute to reducing repulsion; and the attractive/repulsive acid-base interactions (Kimkes and Heinemann, 2020). (B: Left side) Pili-mediated temporal attachment. Pili elongation allows attachment to the surface, whereas pili retraction may cause the bacterium to be tugged toward the surface, reach different directions, change from horizontal to vertical (and vice versa) orientations by using different types of motility, or it may be released back. (B: Right side) Flagella-mediated temporal attachment may be caused because of their hydrophobic nature, as well as by some of the flagellar motor components. When flagella become anchored to the surface, the polarly attached cells spin around, often leading to detachment of bacteria. (C) Specific temporal attachment may be mediated by binding of adhesins, expressed onto the bacterial surface or at the tip of certain pili appendages, to particular host receptors.

Figure 3

Schematic representation for the functional steps involved in bacteria adhesion to a solid surface

Figure 4

Potential routes for the formation of microcolonies Microcolony formation arises from the accumulation of cells in continuous growth and division and may be enhanced by the incorporation of planktonic cells from the bulk fluid or product of cell division and the integration of bacterial clusters. (1) Once bacteria become strongly attached, the colonization of the surface takes place by means of cell growth and division. One of the daughter cells may remain attached, and the other may be released from the surface (3A), where it becomes free to colonize other sites (2) by landing onto target surfaces (3B) or, it may become part of recently formed bacterial clusters either on the surface (3C) or the bulk fluid (3D). Bacterial clusters formed in the absence of a solid substrate may colonize new surfaces or land onto biofilms under development (3E). During cell proliferation and biofilm formation, QS signals and production of EPS matrix occur. As cell density increases, some of the bacteria slide along each other (3F) leading to the formation of small bacterial aggregates, which correspond to “immature” biofilms known as microcolonies (4).

Figure 5

QS influences biofilm formation and physiology AIs are continuously being produced by bacteria; they constantly move in and out of the cell by diffusion, active transport, or into vesicles, depending on their chemical nature. When the concentration of AIs reaches a threshold level as consequence of increased cell density, the detection response is enabled, and they bound to the corresponding membrane-associated or intracellular receptors, activating these response regulator proteins. As a consequence, signal processing leads to collective transcription of target genes to produce a communal behavior of the bacterial population. One of the potential responses is the expression of a series of beneficial traits involved in biofilm physiology.

Figure 6

Morphology and architecture of mature biofilms Molecular and structural components contribute to the 3D spatial organization and physiology of biofilms. Phenotypically different bacteria are found along the gradients observed in mature biofilms.

Figure 7

Biofilm dispersal Bacterial regulatory mechanisms drive biofilm disruption for the release of individual cells and bacterial clusters into the bulk fluid where they become available for further spread and colonization.

Figure 8

Biofilm mechanisms of immune evasion Some reported strategies utilized by different biofilms to overcome and modulate host innate and adaptive immune responses are shown.

Figure 9

Schematic depiction for the overall steps involved in the current standard in vitro protocols of biofilm formation Blue heading boxes show the general methodology followed in studies of biofilm development; same procedure is utilized when investigating the efficacy of biofilm eradication strategies (red box). Moreover, if prevention of biofilm formation is the goal of the study (green box) or if microcosm models are required (yellow box), functionalization/modification of materials and development of the appropriate disease models are respectively needed. After this, the materials under evaluation and the disease models may be combined with the chosen in vitro model for biofilm formation or with a preformed biofilm. Evaluation and characterization of the biofilm may be done directly on the model or detachment may be required for processing and analysis, depending on the chosen methods. Representative examples of the methods and techniques used for characterizing biofilms are shown; some of them are also used to evaluate the effect of antibiofilm strategies. SEM, scanning electron microscopy; FESEM, field emission SEM; TEM, transmission electron microscopy; AFM, atomic force microscopy; OCT, optical coherence tomography; qPCR, quantitative real-time PCR; MS, mass spectrometry; MSI, MS imaging; ToF-SIMS, time-of-flight secondary ion MS.

Figure 10

Schematic depiction for the overall steps involved in the design of in vivo protocols of biofilm formation and characterization Blue heading boxes show the general methodology followed in studies of in vivo biofilm development. The red heading box highlights the methods that can be used to characterize biofilm progression. If prevention of biofilm formation is the goal of the study (green box) or if disease or implanted medical device models are required (yellow box), functionalization/modification of materials and development of the appropriate disease models are respectively needed. After this, the materials under evaluation and the disease models may be combined to generate an appropriate in vivo model for biofilm formation. Evaluation and characterization of the biofilm can be done in situ using advanced in vivo techniques, ex situ through the analysis of swabs and excised tissue samples, and ex vivo (i.e., histological analysis of tissue at end point of study).

Figure 11

Suggested decision-making system for selecting a biofilm model

Figure 12

Schematic representation for the limitations and advantages of in vitro and in vivo biofilm models