Characteristics of the <i>Streptococcus thermophilus</i> natural isolates used in this study.

<p>^a, Sequence typing [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128099#pone.0128099.ref018" target="_blank">18</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128099#pone.0128099.ref019" target="_blank">19</a>];</p><p>^b, (-) poor, (+/-) moderate, (+) strong biofilm production;</p><p>^c, (-) absence and (+) presence of STH8232_0171, STH8232_0714 and STH8232_1361 orthologs;</p><p>ND, not determined;</p><p>NA, not available.</p><p>Characteristics of the <i>Streptococcus thermophilus</i> natural isolates used in this study.</p

Differences in biofilm formation between natural <i>S</i>. <i>thermophilus</i> isolates.

<p>Biofilm formation was evaluated by quantitative microtiter plate assays based on the staining of 18-hour-old biofilms with crystal violet (CV). The columns show the mean values for CV absorbance (at 590 nm) of at least four independent experiments performed in duplicate, and the error bars indicate standard deviations. Statistical analyses included one-way ANOVA and the Bonferroni post hoc test, comparing the mean value for each strain with that of the strain with the lowest mean value (CNRZ1595). Asterisks denote statistically significant differences (<i>P</i> < 0.0001).</p

Biofilm formation by <i>S</i>. <i>thermophilus</i> JIM8232 and its isogenic mutants.

<p>The biomass of 18-hour-old biofilms from the IS<i>S1</i> transposon mutants (STH8232_0171::IS<i>S1</i>, STH8232_0714::IS<i>S1</i>, STH8232_1361::IS<i>S1</i>) and the corresponding reconstructed mutants (STH8232_0171 [JIM9171], STH8232_0714 [JIM9169], STH8232_1361 [JIM9170]) was measured in a crystal violet (CV) assay. The columns show mean CV absorbance values for at least four independent experiments performed in triplicate, and the error bars indicate standard deviations. The significance of the differences between JIM8232 and its isogenic mutants was determined in Student’s paired <i>t</i>-tests (****, <i>P</i> < 0.0001).</p

Schematic diagram of the STH8232_1361, STH8232_0714 and STH8232_0171 loci.

<p>(A) The STH8232_1361 region <i>in S</i>. <i>thermophilus</i> LMD-9, JIM8232, ND03, CNRZ1066-like (CNRZ1066, TH985, TH982, TH1436, DGCC7710, 1F8CT), LMG18311, M17PTZA496-like (M17PTZA496, TH1435) and <i>S</i>. <i>salivarius</i> JIM8777. (B) The STH8232_0714 region in <i>S</i>. <i>thermophilus</i> JIM8232, DGCC7710, LMD-9, ND03-like (ND03, MN_ZLW_002), CNRZ1066-like (CNRZ1066, LMG18311) and 1F8CT. (C) The STH8232_0171 region in <i>S</i>. <i>thermophilus</i> JIM8232, M17PTZA496 and TH1477. The orthologous genes in each panel are represented by pentagons of the same color, except for mobile elements, which are shown in white. The lengths of genes and intergenic regions are drawn to scale.</p

Role of STH8232_0171, STH8232_0714 and STH8232_1361 in biofilm formation.

<p>(A-B), Properties of biofilms formed by <i>S</i>. <i>thermophilus</i> JIM8232 and its isogenic mutants JIM9169 (STH8232_0714), JIM9170 (STH8232_1361) and JIM9171 (STH8232_0171). Panel (A) shows 3-D reconstructions of representative confocal images of biofilms and panel (B) shows biofilm biovolumes (Biofilm biovol.), substrate coverages (Subst. cover.) and mean thicknesses (Mean thick.), normalized against those for JIM8232, for each strain, as measured in nine different CLSM image stacks from three independent experiments. Scale bar, 50 μm. (C), Bacterial adhesion (after 1 h) to polystyrene wells. Adherent bacteria were counted as colony-forming units on M17 solid agar plates and the results are expressed as percentages of the input inoculum. The columns show the mean values for five independent experiments, each performed in triplicate, and the error bars indicate the standard deviations. (D), Percentage of bacteria bound to hexadecane solvent. The columns show the mean values for four independent experiments performed in duplicate, and the error bars indicate standard deviations. (E), Scanning electron microscopy of <i>S</i>. <i>thermophilus</i> JIM8232 and its isogenic mutants. Arrows indicate the cell wall polysaccharide layer. Scale bar, 0.2 μm. The significance of the differences between JIM8232 and its isogenic mutants was determined in Student’s paired <i>t</i>-tests (****, <i>P</i> < 0.0001; ***, <i>P</i> < 0.001; **, <i>P</i> < 0.01).</p

Adhesion to HT-29 cells of several <i>S</i>. <i>thermophilus</i> and <i>S</i>. <i>salivarius</i> strains.

<p>Adherent bacteria were counted as colony-forming units on M17 solid agar plates and the results are expressed as percentages of the input inoculum. The columns show the mean values for three independent experiments, each performed in triplicate, and the error bars indicate the standard deviations. Student’s paired <i>t</i>-test was used for statistical analysis (**** <i>P</i> < 0.0001 for comparisons with JIM8232 or JIM8777).</p

Biofilm biovolumes and the initial adhesion levels of the seven <i>B.subtilis</i> strains.

<p>(A) The biovolumes (µm³) in ascending order of the 48h-biofilms obtained with the seven <i>B. subtilis</i> strains in microtiter plates from confocal image series using the PHLIP tool. (B) Number of cells adhering per cm² after 1h30 of adhesion in the microtiter plate. The error bars indicate the standard error and the statistically significant difference observed with strain 168 (<i>P</i><0.05) is indicated by a star (*).</p

Spatial organization of <i>S</i>. <i>thermophilus</i> LMD-9 and JIM8232 biofilms, from series of confocal images.

<p>(A) Three-dimensional confocal projection of 18-hour-old biofilms produced by strains LMD-9 and JIM8232, before (upper panel) and after (lower panel) washing, obtained from <i>xyz</i> confocal image series with IMARIS software. The images show a representative and aerial view of biofilm structures, with the virtual shadow projection on the right. Scale bars correspond to 50 nm. (B) Mean biofilm biovolume, (C), substrate coverage, and (D) biofilm thickness of the 18-hour-old biofilms produced by strains LMD-9 and JIM8232 before (gray bars) and after (black bars) washing, extracted from confocal images with the PHLIP Matlab tool. The columns show the means of three independent experiments performed in triplicate, and the error bars indicate the standard deviation. Statistical analysis included Student’s paired <i>t</i>-tests (**** <i>P</i> < 0.0001, **, <i>P</i> < 0.01 for comparisons with JIM8232 after washing).</p

Three-dimensional biofilm structures obtained with the seven <i>B.subtilis</i> strains.

<p>These images present a representative, aerial, 3D view of the 48h-biofilm structures obtained with the seven <i>B. subtilis</i> strains using a microplate system, obtained from confocal image series using IMARIS software (including the shadow projection on the right). One iso-surface representation of a particular “beanstalk-like” structure is also shown for <i>B. subtilis</i> ND_medical.</p

<i>Bacillus subtilis</i> strains used in this study.

a<p><i>spec</i>, <i>cat</i>, <i>neo</i>, <i>kan</i> and <i>ery</i> stand for spectinomycin, chloramphenicol, neomycin, kanamycin and erythromycin resistance markers, respectively.</p>b<p>CTSCCV, centre technique de la salaison, de la charcuterie et des conserves de viandes; ISHA, Institut Scientifique d'Hygiène et d'Analyse; BSFA strains were constructed during the “<i>Bacillus subtilis</i> functional analysis programme” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016177#pone.0016177-Kobayashi4" target="_blank">[56]</a>.</p