Improving Phage-Biofilm In Vitro Experimentation

Abstract

Bacteriophages or phages, the viruses of bacteria, are abundant components of most ecosystems, including those where bacteria predominantly occupy biofilm niches. Understanding the phage impact on bacterial biofilms therefore can be crucial toward understanding both phage and bacterial ecology. Here, we take a critical look at the study of bacteriophage interactions with bacterial biofilms as carried out in vitro, since these studies serve as bases of our ecological and therapeutic understanding of phage impacts on biofilms. We suggest that phage-biofilm in vitro experiments often may be improved in terms of both design and interpretation. Specific issues discussed include (a) not distinguishing control of new biofilm growth from removal of existing biofilm, (b) inadequate descriptions of phage titers, (c) artificially small overlying fluid volumes, (d) limited explorations of treatment dosing and duration, (e) only end-point rather than kinetic analyses, (f) importance of distinguishing phage enzymatic from phage bacteriolytic anti-biofilm activities, (g) limitations of biofilm biomass determinations, (h) free-phage interference with viable-count determinations, and (i) importance of experimental conditions. Toward bettering understanding of the ecology of bacteriophage-biofilm interactions, and of phage-mediated biofilm disruption, we discuss here these various issues as well as provide tips toward improving experiments and their reporting.

Keywords: bacteriophage therapy; biocontrol; biofilm; biofouling; biological control; chronic infection; microcolony; phage ecology; phage therapy.

Figure 1

Different aspects of phage-biofilm interactions. Across the bottom: Biofilms begin with bacteria attaching to surfaces and to each other. This is followed by bacterial transition from planktonic to sessile lifestyles (colonization) and then to increases in biofilm bulk, i.e., as a consequence of a combination of biofilm-bacteria replication and extracellular matrix production (growth). Net growth ceases in association with biofilm maturation. Biofilms also can display various forms of cell dispersion, allowing for colonization of new surfaces. Across the top: (1) Phage prevention of bacterial surface colonization (mustard-colored virions), (2) phage-associated control of growth in biofilm bulk (gray-colored virions), and (3) phage-mediated removal of existing biofilm material (red-colored virions). In addition, (4) phages can produce new virions (propagation, purple-colored virions). This figure has been adapted from “Polymicrobial biofilm” by BioRender.com (2020) and retrieved from https://app.biorender.com/biorender-templates.

Figure 2

Control of biofilm growth vs. removal of existing biofilm. The extent of phage impact on biofilms can be contingent on when phages are added as well as what is used as a negative-treatment control. Toward illustration, two hypothetical experiments with different zero time points are presented: prior to biofilm maturation and after biofilm maturation (downward-pointing arrows). Mock treatment is shown as a solid-green curve ending in the “Mature biofilm” label. Negative controls consist of determinations of biofilm properties either at zero times (arrows) or post-treatment (corresponding to vertical gray lines). “Partial Control” indicates that biofilm growth might be possible even in phage presence, though less growth than with mock treatment. Created with BioRender.com.

Figure 3

What phage titer ultimately is required to clear biofilms? Solid line: biofilm presence. Dashed lines: phage titer and increase due to in situ phage propagation, as starting with an MOI of somewhat less than 1. Scales for y axes to the left and to the right are not necessarily equivalent. Illustrated is the idea that there is little possibility of understanding phage-bacterial population dynamics without quantifying phage titers over time in combination with at what point in time biofilm amounts start to be reduced (arrow). Toward increasing clarity, depiction of synchronous phage replication, e.g., as seen in Figure 1 of [80], is intentionally not provided in the figure. Created with BioRender.com.

Figure 4

Possible impacts of different phage titers on biofilms. Pharmacologically, it is typical that the application of greater drug amounts will result in greater impacts on target tissues. Dosing with greater numbers of phages, as added in the figure at the phage icon, therefore should generally be attempted toward enhancing anti-biofilm activities, particularly when desired anti-biofilm activity is not otherwise achieved. This should especially be rather than employing as maximum doses phage MOIs of approximately 1 or lower. The middle dashed line, unlabeled, is meant to describe the results of some in-between phage dose. Created with BioRender.com.

Figure 5

End-point analyses tend to overly simplify population dynamics. Many studies take their first and often only time point at 24 h following phage application. Though it is possible that bacteria killing or biofilm disruption is still ongoing after this length of time (C), it also is possible that bacterial populations or biofilm presence instead is recovering (A), or neither declining nor recovering (B). Possible underlying mechanisms for the shape of the presented curves are: (A) Phage-mediated reductions in biofilm presence followed by grow back of less biofilm-forming-capable, phage-resistant mutants. (B) Incomplete phage-mediated reductions in biofilm presence. (C) Ongoing but slow phage-mediated reductions in biofilm presence. Created with BioRender.com.

Figure 6

Illustration of greater susceptibility of bacteria to phage adsorption given biofilm disruption without virucides present (or without sufficient dilution; top) or with virucides present (or with sufficient dilution; bottom). Bacteria adsorbed by phages are indicated to the right. Created with BioRender.com.