In nature, there are microbial communities, called biofilms, in which individual cells are attached to a surface by means of extracellular polymeric substances (EPS). Bacteriophages (also called phages) are natural “predators” of bacteria and represent the biological entity that seems to be more widespread on planet Earth. The interactions between bacteria and phages are indeed complex and peculiar, and indeed the role of phages in shaping bacterial diversity in these microenvironments is still not well understood. However, in vitro or ex vivo infection tests, coupled with computational simulations, may be useful to better understand these natural interactions.
The mechanisms underlying the phage-bacteria relationship within biofilms are very different, as are the study methods useful for investigating the potential applications of phages for biofilm control. In this article, we will explore the above-mentioned aspects.
Phages and biofilms are present in all kinds of environments. The phage-bacteria relationship has certainly been fundamental for obtaining the enormous phenotypic and genotypic diversity of the microbial world. This particular link can lead to effects that we can define as negative, due to the defense that bacteria must develop against the attack of phages, but also to the positive effects that these can have on bacterial hosts.
In Figure 1 we show an example of a structure characterized by phages and bacteria.
The light blue halo in the figure represents the adhesive substance used by bacteria to adhere to the surface (EPS) and create a favorable environment for the development of the community. Three types of cells that make up the microbial community are also represented: cells colored in light green are metabolically active, those colored in dark green can be defined as dormant and, finally, those colored in red are resistant to phage attack.
Moreover, are represented some molecules released by bacteria, outer membrane vesicles, also called OMV (Outer Membrane Vesicles), able to deceive phages that will bind these molecules rather than attacking bacterial cells.
The form of attack most used by phages for bacterial infection is that linked to the encoding of enzymes capable of degrading polymeric substances including capsular polysaccharides, exopolysaccharides, or lipopolysaccharides. These enzymes produced by phages, called depolymerases, are responsible for the destruction of bacterial capsules and thus allow the entry of the phage into the cell. In addition, they can influence matrix rupture.
This rupture phenomenon facilitates the spread of phages within the biofilm and results in an increased likelihood of attack by target bacteria. In essence, these enzymes have antibiotic properties, which can also be enhanced by other enzymes, called endolysins. Endolysins, as observed in a study of Staphylococcus aureus biofilms, are produced by the phages themselves and are responsible for the degradation of peptidoglycans.
How do bacteria escape phage predation?
To date, genotypic analysis has revealed the presence of bacteria that have modified their phage-associated receptor structures in order to avoid infection. However, signaling or extraordinary systems such as CRISPR-Cas are certainly present.
Bacterial communication is known to rely on various molecules including some referred to as autoinducers. These regulate gene expression in response to changes in population density by a mechanism called quorum sensing (QS, Fig. 2). The latter can modulate the expression of phage receptors in the bacterial cell surface, thus it can also modulate and reduce phage uptake, as has been observed in Vibrio anguillarum.
CRISPR-Cas systems, on the other hand, provide bacteria with a second defense system and thus adaptive immunity against invasive genetic elements, including phages.
Phage-resistant and phage-sensitive bacteria coexist in the biofilm. When phage-resistant cells are rare in the biofilm, susceptible cells are eliminated by phages and, consequently, the number of phage-resistant cells will increase. Then the remaining susceptible cells will be protected from phage exposure by immobilization of phages in the resistant cell clusters.
Biofilm study methods
There are no standardized and appropriate protocols for simulating real biofilms under laboratory conditions. Methods “commonly used to form biofilms” are recognized.
In vitro models
Microtiter plates are used that guarantee good results at a low cost. The problem with this method lies in the difficulty of relating the data obtained to reality, i.e. to biofilm formation in precise environmental, clinical, food, or veterinary contexts. In fact, biofilms formed in real conditions are associated with various types of stresses, so air/liquid flows that laboratory devices are not able to imitate them faithfully.
In reference to this, the use of more advanced devices such as flow cells, drip reactors, modified Robbins devices and rotating biofilm devices are preferred. This leads to a significant difference in the quality of the results: under static conditions thin and poorly structured biofilms are observed, while when biofilms grow in dynamics they are highly organized with microcolonies forming a dense network.
Another important feature is inherent in the effect of culture media on biofilm structure and the cells that characterize them. Most studies are performed on bacteria growing in rich media. In some studies on Proteus mirabilis formed in Luria-Bertani broth and in artificial urine, it was observed that biofilms formed on rich media showed the typical mushroom structure with water/nutrient channels, while those formed using artificial urine showed a flat structure with almost no channels.
Phage-biofilm interactions are usually studied on dynamic biofilms using simulated body fluids. Simulations performed on Foley catheter sections with phage cocktails showed that biofilm-forming devices, conditions used and culture media strongly interfere with biofilm structure/composition both in terms of phages and bacteria present.
Ex vivo and in vivo models
Ex vivo models involve the use of tissues derived from a living organism in an artificial environment and thus allow for greater control of the starting parameters. The main obstacles are the lack of host (human or animal) response and the short duration of the experiments.
The in vivo approach is useful to identify the pathology of the infection by comparing various aspects such as the different routes of administration of phages and the effect of dosage.
Methods for studying phage-biofilm interaction
These methods are useful in assessing biofilm biomass and/or cell viability. They are divided into culture-based, molecular, physical, chemical, microscopic, computational, and mathematical models.
Those related to cultures are based on the determination of the number of colony-forming units (CFU). This method is based on serial dilutions of bacterial suspensions and is the most widely used technique to assess the efficacy of phage killing in biofilms (Fig. 3).
In contrast, PCR-based or molecular methods can be used to study biofilm communities and allow quantification of the number of viable cells. Typically, quantitative PCR (qPCR) is used. Unlike CFU determination, qPCR (Fig. 3) often overestimates the number of viable cells, as the results are affected by the presence of eDNA and dead cells.
To improve biofilm cell counting in terms of accuracy, flow cytometry in combination with bacterial cell staining with fluorophores can be considered (Fig. 4). In addition to a count, this approach also allows an assessment of the physiological state of the cells and the phage-biofilm interactions taking place in real-time.
Chemical methods allow indirect measurement of biofilm characteristics through the use of dyes or fluorochromes that can adsorb or bind to cells or matrix components. For example, resazurin and XTT have been used to determine precisely the effect of phages against biofilms.
There are a number of solutions associated with microscopy to analyze biofilms, and many of these approaches have already been used to examine phage-biofilm interactions: epifluorescence microscopy, CLSM, scanning electron microscopy (SEM), and atomic force microscopy.
Lastly, mathematical models are potentially useful for quantitative description. Using such models in conjunction with a computational framework, simulations have been developed that have defined an equilibrium state of phage-biofilm interaction that is closely influenced by the availability of biofilm cell nutrition, the probability of infection, and the ability of phages to spread through biofilms.
In addition, the biofilm matrix can also impact these interactions by regulating the coexistence of prey and predator in the biofilm microenvironment. Recently, “BiofilmQ“, an innovative image cytometry software tool that allows, even without programming skills, the visualization of various biofilm-related properties, was born.
Using phages to limit the development of hazardous biofilms
We often read about the application of cocktails composed of phages that target different cellular receptors. These are used with antibiotics and have been described to be highly effective against biofilms. Synergy occurs because bacterial lysis associated with phages releases useful nutrients to reactivate the metabolism of dormant cells that become sensitive to antibiotics. Simultaneously, cell lysis also causes EPS dispersion, which improves antibiotic diffusion into the innermost layers of the biofilm matrix.
Phages may play a crucial role in the prevention or even control of infections in healthcare equipment; in fact, their usage considerably lowers biofilm development. The ability of phages to prevent or regulate biofilms in catheters has been widely explored. In this context, many studies have been conducted on Proteus mirabilis, a major cause of urinary tract infections when using these devices.
A final growing trend is the use of phages to target bacterial pathogens in various foods and food contact surfaces. The most recent tests have been performed on food and not yet on biofilms, but it is very likely that antibiotic activity can control biofilm growth as well. Many phage preparations exist for this purpose: ListShieldTM, ListexTM P100, EcoShieldTM, SalmoFreshTM, FinalyseTM. These have been awarded the “Generally Recognized as Safe by the Food and Drug Administration” designation for use as food additives and/or food processing agents against many foodborne pathogens.
Conclusions and future perspectives
Phage-biofilm interactions are difficult to study and understand because of the variability of many factors and the lack of standardized methods. Undoubtedly, phages can be considered powerful weapons to combat unwanted and dangerous biofilms, but they have limitations. Indeed, the efficacy of phages in controlling biofilms depends on the inherent biological properties of the phages and the biofilm.
In light of this variable efficacy, further studies on this subject represent an important step in order to evaluate new strategies in combination with chemical, enzymatic, physical treatments or genetically modified phage design. The new knowledge could then lead to the definition of phage treatments for therapeutic and/or industrial purposes.
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