Phage "delay" towards enhancing bacterial escape from biofilms: a more comprehensive way of viewing resistance to bacteriophages

Abstract

In exploring bacterial resistance to bacteriophages, emphasis typically is placed on those mechanisms which completely prevent phage replication. Such resistance can be detected as extensive reductions in phage ability to form plaques, that is, reduced efficiency of plating. Mechanisms include restriction-modification systems, CRISPR/Cas systems, and abortive infection systems. Alternatively, phages may be reduced in their "vigor" when infecting certain bacterial hosts, that is, with phages displaying smaller burst sizes or extended latent periods rather than being outright inactivated. It is well known, as well, that most phages poorly infect bacteria that are less metabolically active. Extracellular polymers such as biofilm matrix material also may at least slow phage penetration to bacterial surfaces. Here I suggest that such "less-robust" mechanisms of resistance to bacteriophages could serve bacteria by slowing phage propagation within bacterial biofilms, that is, delaying phage impact on multiple bacteria rather than necessarily outright preventing such impact. Related bacteria, ones that are relatively near to infected bacteria, e.g., roughly 10+ µm away, consequently may be able to escape from biofilms with greater likelihood via standard dissemination-initiating mechanisms including erosion from biofilm surfaces or seeding dispersal/central hollowing. That is, given localized areas of phage infection, so long as phage spread can be reduced in rate from initial points of contact with susceptible bacteria, then bacterial survival may be enhanced due to bacteria metaphorically "running away" to more phage-free locations. Delay mechanisms-to the extent that they are less specific in terms of what phages are targeted-collectively could represent broader bacterial strategies of phage resistance versus outright phage killing, the latter especially as require specific, evolved molecular recognition of phage presence. The potential for phage delay should be taken into account when developing protocols of phage-mediated biocontrol of biofilm bacteria, e.g., as during phage therapy of chronic bacterial infections.

Keywords: abortive infection systems; bacteriophage therapy; biofilm; central hollowing; dissemination; extracellular polymeric substances; microcolony; native dispersion; phage resistance; phage therapy; reduced infection vigor; seeding dispersal.

Figure 1.. Summary of possible mechanisms of delay of virion movement, phage penetration, or phage propagation into bacterial biofilms, with a biofilm represented as a labelled, multi-colored, partial oval. Note that the shape of the biofilm as presented is not intended to provide meaning, nor the direction of phage contact or movement, except relative to the interior and surface of the biofilm. Thus, for example, phage virions could first come into contact with the top of biofilms, along the sides of “mushroom”-like projections, or along the walls of water channels which pass within a biofilm. In all cases, however, virions first make contact with a biofilm “surface” as defined in Section 3.3., i.e., the interface between extracellular matrix and overlying fluid lacking in intact extracellular matrix. Phages then, as indicated in the figure, proceed past that surface into a biofilm's “interior”, with “interior” defined as being beneath surface bacteria (also Section 3.3.). Discussion of various impediments to the penetration of phages to biofilm interiors, as indicated in Figure 1, can be found in Section 1.1. Note that the shown phage virion is not drawn to scale, i.e., it is shown as much larger than would typically be the case relative to biofilm dimensions.

Figure 2.. Illustration of mechanisms of bacterial escape from microcolonies. Seeding dispersal (a.k.a., central hollowing or native dispersion) is an active process, that is, following a biofilm/microcolony developmental process, as too can be erosion (a.k.a., detachment), though the latter also can and perhaps more likely occurs as a consequence of passive processes. Both processes give rise to the release of individual bacteria, but erosion also can give rise to the release of clumps of bacteria. Release can be imposed also by enzymatic action, fluid flow, abrasion, and grazing by consumer organisms (protists or animals). Sloughing is a more extensive release than simply the release of smaller cell clumps, involving instead a much more substantial portion of the biofilm. As so released, sloughing however is not shown in the figure. Though also not indicated in the figure, seeding dispersal typically will involve the escape of flagellated dispersal bacteria. Release can be more or less continuous (e.g., erosion) or instead episodic (seeding dispersal, sloughing, or as caused by grazing or abrasion).

Figure 3.. Illustration of mechanisms of bacterial resistance to phages. The inner circle of orange ovals represents phage infections of bacteria. Beyond that circle is infection outcome. This, starting at the top and going clockwise, includes (i) normal burst size and latent period, (ii) delayed lysis, (iii) no burst and bacterium survival (the latter as represented by a green oval), (iv) no burst along with lack of bacterium survival (abortive infection, sensu stricto), and (v) normal lysis timing but diminished burst size. Also shown (upper right) is delayed phage access to bacteria, i.e., reduced virion access to host receptors. In normal, lytic infections (top), phages display full infection vigor, meaning an outcome consisting of a typical latent period and burst size. In infections displaying reduced infection vigor, rates of phage population growth slow due to smaller burst sizes (v, left) and/or due to extended latent periods (ii, right). Alternatively, infection vigor may be reduced to zero (iii or iv, bottom). This figure is derived in part from as published in .

Figure 4.. Physiological as well as physical impediments to phage propagation into microcolonies. Downward in the figure corresponds to deeper penetration into a microcolony (that is, from a microcolony's surface into a microcolony's interior, whether that surface is found on the top or side of a microcolony, or instead is found lining a water channel found within a biofilm; see Section 3.3. for clarification). Green cells (circles, top) are more metabolically active, yellow (middle) less metabolically active, and orange (bottom) even less. Arrows refer to some number of infection-released virions moving in the indicated directions. So long as bacteria are sufficiently numerous and sufficiently adsorbable then those bacteria can serve as “barriers” to deeper movement (sorptive scavenging; Section 6.2.2.), delaying phage movement in the course phage infection rather than allowing phage virions to diffuse past these bacteria. Movement may be further diminished in rate due to longer phage latent periods. Further interference with rates of phage penetration into microcolonies may include smaller burst sizes, virion diffusion other than towards the microcolony interior, and diffusion impediments imposed by EPS, the latter imposed potentially especially by more mature EPS. Reduced infection vigor can be solely a consequence of reduced bacterium metabolic rates (less infection-conducive cell physiologies) or can be due to “imperfect” expression of abortive infection-like mechanisms, or both. Strictly abortive infections (indicated with the black cell, center-bottom), even if not seen with all infections, also can have the effect of reducing progeny phage survival, thereby lowering phage effective burst sizes as well as rates of virion penetration further into microcolonies. So too can phage adsorption to already phage-infected bacteria reduce phage virion-progeny survival. The latter, though, is not explicitly indicated in the figure.

Figure 5.. Consideration of differential phage susceptibility within a mature microcolony. To indicate all potential layers as discussed in the main text, the microcolony is depicted on its left (“With Central Hollowing”) as producing seeding-dispersal cells internally (central hollowing). Microcolonies not undergoing central hollowing (right) will not possess this additional layer. Regions consist of outer layers (green) that are other than the microcolony foundation, an interior region associated with central hollowing (also green though only as indicated on the left), a region found between these external and internal regions that potentially can serve to some degree as a phage-propagation barrier or “wall” (orange and which should be considered to continue to the center of the microcolony absent central hollowing, i.e., as indicated on the right), and also an external layer serving as the microcolony foundation (bottom, red fading into orange). As indicated previously, the overall shape of the depicted microcolony is not intended to provide information but instead solely depicts distinctions between microcolony surfaces and interiors (as clarified in Section 3.3.). Here this surface is seen as the outside of the shape especially as in contact with the outer layer of green. Microcolony interiors by contrast are seen in the figure as the inside of the shape, especially as surrounded by or instead below the outer layer of green. Surfaces found on a microcolony's top, covering its sides, or lining water channels otherwise should be considered to be equivalent—from the standpoint of this study—in terms of phage susceptibility, with microcolony interiors defined in terms of distance from any of these surfaces.

Figure 6.. Summary of phenomena potentially interfering with phage penetration or propagation towards microcolony interiors. These phenomena are discussed as follows: (1) Mechanisms with specific molecular targets are often not present or at least not fully active against specific phage types, but may block phage propagation altogether if present. (2) Viruses that are released from infections even if displaying full vigor nevertheless likely are not available solely for penetration into microcolony interiors. (3) If present, resistance mechanisms may display less than full activity against phages, acting probabilistically and potentially resulting in infection vigor that is reduced though not to zero. (4) To the extent that infection of bacteria can impose delays on virion movement towards microcolony interiors, then phage adsorption of these bacteria would be expected to be encouraged by microcolony bacteria rather than intentionally bypassed. (5) With reduced metabolic activity, then reduced infection vigor may be manifest via less specific or otherwise not genetically encoded means, resulting in some cases in phage burst sizes reduced to zero (effectively abortive infections) or phage infection progression which is reduced to the point that bacteria survive (effectively representing restrictive infections). In addition, it is conceivable that some genetically encoded resistance mechanisms may be enhanced in less metabolically active bacteria, e.g., with burst size reductions being more pronounced. (6) Phage adsorption to bacteria that are already infected with the same phage type will result, effectively, in the loss of one of the two (or more) phages, that is, since bacteria generally at best will support only a single phage burst. (7) Biofilm matrix material to a degree may interfere with virion diffusion, and it is conceivable that this interference is enhanced in the course of EPS maturation, i.e., as associated with less recently replicated bacterial cells.