Interspecies collective resistance.

<p>Still images (overlay of phase contrast and fluorescence microscopy) of a time-lapse experiment of <i>S</i>. <i>pneumoniae</i> Cm^S, cocultivated with a strain of the pneumococcal niche competitor <i>S</i>. <i>aureus</i> (strain LAC pCM29) that expresses CAT and GFP, growing on a semi-solid surface supplemented with 3 μg ml⁻¹ Cm. Scale bar 10 μm.</p

Population dynamics of bacterial communities.

<p>(<b>a</b>) Simulated growth trajectories for Cm^R and Cm^S populations subject to antibiotic stress and resource competition. (<b>b</b>) Dynamic of intracellular Cm (<i>y</i>_r and <i>y</i>_s) and growth-limiting resource (<i>z</i>). Simulation time is scaled relative to the mean residence time of cells in a chemostat, which is equal to the generation time at steady state. At low population densities, the Cm^R strain can grow, whereas Cm^S cannot, due to a high concentration of Cm. However, the invasion of Cm^R lowers antibiotic stress, generating permissive conditions for the growth of Cm^S cells. The chemostat is then rapidly colonized by both strains (shortly after <i>t</i> = 180) until the resource becomes limiting. From that moment onwards, total cell density changes little, while the relative frequencies of the two strains continue to shift. Eventually, a stable equilibrium is reached, at which the cost and benefit of CAT expression (i.e., reduced growth rate efficiency for Cm^R cells versus their lower intracellular Cm concentration) balance out. Inset (<b>c</b>), The dark-red dot pinpoints the parameter set used in the simulation shown in <b>a</b> and <b>b</b>: <i>r</i> = 20.0, <i>η</i> = 0.9, <i>k</i>_z = 4.0, <i>c</i> = 1.0, <i>p</i> = 50.0, <i>h</i>_Y = 0.25/<i>Y</i>₀, <i>k</i>_Y = 2.5/<i>Y</i>₀, <i>d</i> = 30.0/<i>Y</i>₀ and <i>Y</i>₀ = 0.8. These parameters were selected to lie in a restricted area of parameter space (highlighted in red) where stable coexistence between Cm^S and Cm^R cells is observed Alternative model outcomes, which were identified by a numerical bifurcation analysis (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000631#pbio.2000631.s007" target="_blank">S1 Text</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000631#pbio.2000631.s004" target="_blank">S4 Fig</a>), include establishment of Cm^S only (area S), establishment of Cm^R only (area R), no bacterial growth (area N), and competition-induced extinction (area E, where Cm^S bacteria first outcompete Cm^R bacteria and subsequently are cleared by the antibiotic; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000631#pbio.2000631.s005" target="_blank">S5 Fig</a>).</p

Experimental setup to determine passive resistance.

<p>Antibiotic-susceptible cells (Ab^S) constitutively expressing <i>luc</i> are grown together with antibiotic-resistant cells (Ab^R, which do not express <i>luc</i>). Only when the concentration of the antibiotic in the medium is reduced by enzymatic deactivation of resistant cells will the genetically antibiotic-susceptible cells be able to grow and produce light.</p

Cross-protection in a mouse pneumonia model.

<p>(<b>a</b>) Eight-wk-old female CD1 mice were infected intratracheally with Cm^S pneumococci or an equivalent amount of Cm^S + Cm^R pneumococci in a one-to-one ratio. One h post infection, mice were treated with one intraperitoneal injection of Cm 75 mg kg⁻¹ followed by two additional doses spaced 5 h apart. Control mice received an injection of the vehicle alone. <i>n</i> = 14 for Cm^S control; 13 for Cm^S Cm-treated; 13 for Cm^S + Cm^R control; and 14 for Cm^S + Cm^R Cm-treated. Data plotted as average and s.e.m. of two independent experiments combined. Dashed line ‘inoc’ denotes the initial inoculum. <i>*p < 0</i>.<i>05</i>; one-way ANOVA with Tukey's multiple comparison post-test. (<b>b</b>) Bacterial colonies recovered from the Cm^S + Cm^R control and Cm^S + Cm^R Cm-treated mice were individually picked and used to inoculate THY media in 96-well plates. These 96-well plates were then used to inoculate 96-well plates with THY media containing either 15 μg ml⁻¹ Cm or 100 μg ml⁻¹ kanamycin to determine whether or not the original bacterial colony was Cm^S or Cm^R. n = 9 for Cm^S + Cm^R control and 14 for Cm^S + Cm^R Cm-treated. Data plotted as average and s.e.m. of two independent experiments combined. <i>*p</i> = 0.04; Mann–Whitney <i>U</i> test (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000631#pbio.2000631.s008" target="_blank">S1 Data</a>).</p

Cm deactivation during mixed population assays.

<p>(<b>a</b>) Plate reader assay sets in quadruplicate (average and standard error of the mean [s.e.m.]) measuring luminescence (symbols with color outline) and cell density (corresponding grey symbols) of <i>S</i>. <i>pneumoniae</i> Cm^S growing in the presence of 3 μg ml⁻¹ Cm, in presence (+) or absence (−) of Cm^R cells. (<b>b</b>) Development of the count of viable Cm^S cells (colony-forming units per ml [CFUs ml⁻¹]) during the cultivation assay presented in <b>a</b>, determined via plating in the presence of kanamycin; average values of duplicates are shown. (<b>c</b>) Culture supernatant (S) samples after 0, 1, 2, and 4 h of Cm^R cultivation (inoculation at optical density OD 0.001) in the presence of 5 μg ml⁻¹ Cm, analyzed for Cm content by high-performance liquid chromatography (HPLC) separation and ultraviolet (UV) detection at 278 nm. (<b>d</b>) Luminescence and cell density profiles of Cm^S cells treated with 3 μg ml⁻¹ Cm (inoculation at OD 0.001) in dependency of the inoculum size of Cm^R cells. (<b>e</b>, <b>f</b>), Cm^S luminescence and growth analysis (<b>e</b>) in Cm-supplemented medium (3 μg ml⁻¹) that was pretreated with Cm^R cell pellet (P), S, and culture lysate (L), and controls without (C⁻) and with Cm (C⁺); (<b>f</b>) schematic overview of the assay (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000631#sec010" target="_blank">Methods</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000631#pbio.2000631.s008" target="_blank">S1 Data</a>).</p

<i>Bacillus subtilis</i> Biosensor Engineered To Assess Meat Spoilage

Here, we developed a cell-based biosensor that can assess meat freshness using the Gram-positive model bacterium <i>Bacillus subtilis</i> as a chassis. Using transcriptome analysis, we identified promoters that are specifically activated by volatiles released from spoiled meat. The most strongly activated promoter was P<i>_sboA</i>, which drives expression of the genes required for the bacteriocin subtilosin. Next, we created a novel BioBrick compatible integration plasmid for <i>B. subtilis</i> and cloned P<i>_sboA</i> as a BioBrick in front of the gene encoding the chromoprotein amilGFP inside this vector. We show that the newly identified promoter could efficiently drive fluorescent protein production in <i>B. subtilis</i> in response to spoiled meat and thus can be used as a biosensor to detect meat spoilage