DOI: 10.5061/dryad.sj300pb
title: Spatial structure affects phage efficacy in infecting dual-strain biofilms of Pseudomonas aeruginosa

description: Bacterial viruses, or phage, are key members of natural microbial
 communities. Yet much research on bacterial-phage interactions has been
 conducted in liquid cultures involving single bacterial strains. Here we
 explored how bacterial diversity affects the success of lytic phage in
 structured communities. We infected a susceptible Pseudomonas aeruginosa
 strain PAO1 with a lytic phage Pseudomonas 352 in the presence versus
 absence of an insensitive P. aeruginosa strain PA14, in liquid culture
 versus colonies on agar. We found that both in liquid and in colonies,
 inter-strain competition reduced resistance evolution in the susceptible
 strain and decreased phage population size. However, while all sensitive
 bacteria died in liquid, bacteria in colonies could remain sensitive yet
 escape phage infection, due mainly to reduced growth in colony centers. In
 sum, spatial structure can protect bacteria against phage infection, while
 the presence of competing strains reduces the evolution of resistance to
 phage.
description: Data shown in Figure
 1Bacteria grown in mono-
 or co-culture in the presence or absence of phage. Each of the first 3
 sheets contains growth curve data over 48 hours for each of the bacterial
 species (PAO1 GFP and PA14) and the phage. The fourth sheet shows final
 population size of one strain (PAO1 GFP) in the presence of phage, as a
 function of initial population size. The fifth sheet shows final
 population size of both strains (PAO1 GFP and PA14) in co-culture in the
 presence of phage, as a function of initial population size of
 PA14.Figure1.xlsxData shown in Figure 2 and Supplementary Figure 1Growth curves of two bacterial strains (PAO1 GFP and PA14) and phage in mono- and co-culture colonies over 48 hours. Each of the first 6 sheets contains data for a different bacterial strain, either alone or mixed together at one of two ratios: 1:10 or 1:100 PAO1:PA14. The last three sheets show the phage growth curves in these same cultures. For all organisms, we quantify abundance in the whole colony or in the sampled center of the colony. For PAO1, we also quantify the number of resistant cells in those two locations. For phage, we also quantify them in the agar below the colony.Figure2.xlsxData shown in Figure 4C and 4DThe data represents a quantification of bacterial and phage density carried out by image analysis. The details of this analysis are outlined in the Methods section of the main text. The first two sheets quantify PAO1 bacteria and phage coming from the same samples of PAO1 mono-culture colonies in four different positions in 10 different replicates. For example, position 1, replicate 1 bacterial density corresponds to position 1, replicate 1 phage density. They are shown on the same plot in Figure 4C. The third sheet contains a measure of the density of resistant bacteria, again corresponding to the same samples as in the first two sheets. The fourth and fifth sheet show data from a different set of samples: where PA14 was also present, and correspond to one another. They correspond to the plot in Figure 4D.Figure4.xlsxData shown in Figure 5 and Supplementary Figures 13 and 14Growth curves of four bacterial strains (PAO1, PAO1 DsRed, PAO1 res1 and PAO1 res2) and phage in mono- and co-culture colonies over 48 hours. Each of the first 4 sheets contains data for a different bacterial strain in mono-culture. The fifth shows the abundance of phage in each of these mono-cultures. The next three sheets (6-8) show data for co-culture colonies of PAO1 DsRed together with PAO1 res1 in a 1:10 ratio, while ,the last three sheets show co-culture colonies of PAO1 DsRed together with PAO1 res2 in a 1:10 ratio. Each group of three sheets shows bacteria 1 abundance, bacteria 2 abundance and phage abundance in the same colony. For all organisms, we quantify abundance in the whole colony or in the sampled center of the colony. For PAO1, we also quantify the number of resistant cells in those two locations. For phage, we also quantify them in the agar below the colony.Figure5.xlsxData shown in Supplementary Figure 2Growth curves of two bacterial strains (PAO1 and PA14) and phage in mono- and co-culture colonies over 48 hours with a high phage dose of ~10^9. Sheets 1, 2, 4 and 5 contain data for the different bacterial strains, either alone or mixed together at a 1:10 ratio of PAO1:PA14. Sheets 3 and 6 show the phage growth curves in these same cultures. For all organisms, we quantify abundance in the whole colony or in the sampled center of the colony. For PAO1, we also quantify the number of resistant cells in those two locations. For phage, we also quantify them in the agar below the colony.FigureS2.xlsxData shown in Supplementary Figure 5Growth curves of two bacterial strains (PAO1 GFP and PA14 mCherry) and phage in mono- and co-culture colonies over 48 hours. These data correspond to the images shown in Figure 2B. Each of the first 6 sheets contains data for a different bacterial strain, either alone or mixed together at one of two ratios: 1:10 or 1:100 PAO1:PA14. The last three sheets show the phage growth curves in these same cultures. For all organisms, we quantify abundance in the whole colony or in the sampled center of the colony. For PAO1, we also quantify the number of resistant cells in those two locations. For phage, we also quantify them in the agar below the colony.FigureS5.xlsxData shown in Supplementary Figure 7These data were collected to assess the ability of phage to adsorb to different bacterial strains. The two sheets correspond to data shown in the two panels of the figure. Each shows three technical replicates sampled immediately and after 5 and 10 minutes for 2 (sheet 1) and 3 (sheet 2) different strains.FigureS7.xlsxData shown in Supplementary Figure 11The data represents a quantification of bacterial and phage density carried out by image analysis. The details of this analysis are outlined in the Methods section of the main text. These data were a first attempt at collecting data for Figure 4, but in Figure 4 we repeated it with more replicates. The first two sheets quantify PAO1 bacteria and phage coming from the same samples of PAO1 mono-culture colonies in four different positions in 10 different replicates. For example, position 1, replicate 1 bacterial density corresponds to position 1, replicate 1 phage density. The third sheet contains a measure of the density of resistant bacteria, again corresponding to the same samples as in the first two sheets. The fourth and fifth sheet show data from a different set of samples: where PA14 was also present, and correspond to one another.FigureS11.xlsxData shown in Supplementary Figure 15These data were used to determine the ratio of PAO1 and PA14 at which they would co-exist. Data come from a plate reader (see Methods). In yellow, the initial conditions are outlined, then each row contains either the time, and below it the fluorescence intensity in a given well. Each treatment was conducted in technical triplicates.FigureS15.xlsxData shown in Supplementary Figure 10Data used to generate a standard curve to analyse how densities used in Figure 4, e.g. correspond to CFUs. We did this once for bacteria and once for phage. We used 9 different dilutions, and show the corresponding numbers in adjacent columns.FigureS10.xlsxImages shown in Figure 2BFluorescence microscopy images used to make Figure 2B.Figure2B_pics.zipImages shown in Figure 3Transmission Electron Micrographs used to make Figure 3.Figure3_pics.zipImages shown in Supplementary Figure 3Fluorescence microscopy images used to make Figure S3.FigureS3_pics.zipImages shown in Supplementary Figure 8Images of densities of bacteria and phage used to make Supplementary Figure 8, and for the data in Figure 4.FigureS8_pics.zipImages shown in Supplementary Figure 9Images of densities of bacteria and phage used to make Supplementary Figure 9.FigureS9_pics.zipImages shown in Supplementary Figure 10Images of densities of bacteria and phage used to generate the standard curve in Supplementary Figure 10.FigureS10_pics.zip