Phages lyse cells at edges of single-strain sensitive biofilms. To simulate a setup where a biofilm first forms on a solid surface and is later exposed to phage, we first grew the bacteria for 12 hours in colonies growing on a membrane filter placed on agar. Our goal was to produce biofilms of comparable population sizes to those exposed to phage in the liquid experiment. This was done by transferring the filter with the 12-hour biofilm onto a new agar plate onto which we had placed a drop (approximately the diameter of the membrane filter) containing either phage or PBS as a control. The biofilm was then left to grow in the presence or absence of the phage for an additional 36 hours.
In PAO1 mono-culture biofilms, phage treatment reduced bacterial populations to 52.81\(\pm\)11.41% of their pre-treatment size (paired t-test CFUs at 12 and 48 hours, df=2, P=0.034). Fluorescence microscopy images taken immediately prior to infection and 36 hours later (48h total) showed that colonies treated with phage were smaller in diameter after treatment (data), with the surviving cells visible in the center of the colony. Following imaging, cells were harvested, counted and tested for phage resistance. In the colonies that had been treated with phage, resistant cells were recovered (1.36%, 1.63% and 15.74% of the population in the three replicate colonies).
To understand why so many sensitive cells survived and where in the colony resistance had occurred, before destroying the colonies, we touched an inoculation loop in the center of each colony, resuspended the cells in PBS and plated them on agar to calculate the number of resistant and sensitive cells, as well as phage. We found no resistant cells in the center of the colony, indicating that resistance arose at the colony edges where cells were growing. However, phages were detected in the center at a similar multiplicity of infection (MOI) to the initial dose (PFU/CFU of 0.99\(\pm\)0.27), suggesting that the phage could diffuse into the center of the colony, possibly infecting cells but not lysing them. This phenomenon, called "pseudolysogeny" has been observed for starved cells in stationary phase, where phage have been found to return to their lytic state once bacterial growth resumes \cite{Kokjohn1991,Abedon2008,Ripp1997,Los2003}. In agreement with this prediction, on plating bacteria from the center for quantification, only bacteria at low dilutions could grow, indicating increased phage lytic activity (supp. Fig).
In contrast, PA14 (the phage resistant strain) mono-culture colonies were indistinguishable with and without phage treatment (Fig. for pics, t-test CFUs with and without phage, df=2.6, P=0.87). On sampling the colony centers, no phage were detected, indicating that phage could not diffuse from the agar into the colony. Indeed, total phage populations fell to 11\(\pm\)2.8% of their original size in PA14 colonies over the 36 hours, which we suspect is due to toxicity of LB to phage \cite{Hadas1997} given that phage populations also fell to 8.1\(\pm\)5.4% in the absence of any bacteria.
Taken together, these data support a model whereby PAO1 death and the emergence of phage resistance occur mainly at the edges of the colony where cells are growing, whereas phage can diffuse into PAO1 colonies - but not PA14 - and adsorb to stationary-phase cells at the center through pseudolysogeny.
Phage efficacy is reduced in mixed sensitive-resistant biofilms. Knowing that phage do not diffuse into resistant PA14 colonies, we next asked how the presence of this resistant strain would impact the survival of the targeted pathogen (PAO1) within a mixed biofilm treated with phage. We repeated the previous experiment, where we grew cells - this time a mixture of both PAO1 and PA14 - on a filter for 12 hours, then transferred the biofilm onto a dried phage drop on a different agar plate and left to grow for a further 36 hours. In contrast to the decrease in population size observed in the PAO1 monoculture colony, despite phage treatment, PAO1 increased by 233.9\(\pm\)123.4% over 36 hours (paired t-test CFUs at 12 and 48 hours, df=2, P=0.025). Curiously, the population size of the phage increased concomitantly in the three replicates, suggesting that significant bacterial infection must also have occurred (Fig. xx). Although PAO1 cells were not significantly fewer with and without phage (t-test, df=2.01, P=0.16), microscopy showed that patches of PAO1 (labelled with GFP) were absent from the edges of the colonies treated with phage, indicating cell lysis at the growing edges.
These data suggest that as in PAO1 biofilms, phage could lyse growing cells at the colony edges, while PAO1 cells in the center were not killed. In contrast to the single-strain biofilms, however, PAO1 populations continued to increase. This suggests that pockets of PAO1 may be present that are not accessible to phages. In support of this hypothesis, we found a significantly lower MOI of PAO1 in centers of mixed compared to mono-culture colonies (t-test, df=3.16, P=0.013, see Fig. xD). This led us to consider whether some sensitive bacteria might be completely escaping phage infection, particularly in the colonies where they were mixed with the resistant PA14.
Resistant cells protect sensitive cells in biofilm. To quantify the extent to which sensitive cells could escape phage infection, we simulated a scenario where cells would have a chance to reseed a new environment and begin to grow, and asked how often regrowth occurred depending on the biofilm of origin. In particular, each colony was If cells were in pseudolysogeny in the biofilm, i.e., infected but not lysed because not enough energy as availabl for bacteria and phage to replicate, we expect them to lyse on reinoculation onto fresh media.
Discussion
In sum, we show that a targeted pathogen is more likely to survive a phage attack if growing on a solid surface, in the presence of a phage-resistant competitor. It is also most likely to develop resistance to the phage in the absence of competitors, where it can grow to a sufficiently large population size. Competing species thus reduce the likelihood of resistance evolution, but may create a haven for phage-sensitive pathogens that can spread if dispersed.
Presumably, cells that divided at the colony edges were killed by the phage, while cells that had divided prior to phage addition could not be killed by the phage because they were no longer dividing (phage require cellular division to replicate (REF)).