Effects of <i>ymcA</i>, <i>ylbF</i>, <i>degU</i>, <i>yqxM</i>, <i>hag</i> and <i>abrB</i> mutations on macrocolony and swimming phenotypes.

<p>Macrocolonies grown on TSA medium (1.5% agar) for 72h at 30°C. The left-hand column depicts macrocolony morphologies. The scale bar is 5mm. The middle column depicts aerial views of the centers of macrocolonies from confocal image series using IMARIS software. The scale bar is 0.5mm. The right-hand column shows the swimming plate (9 cm diameter, TSB+0.25% agar) after 24h of incubation at 30°C.</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

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

Effect of mutations on the three-dimensional structure of biofilms.

<p>Effects of mutations on immersed biofilm structures of the biofilms obtained with different GFP-carrying mutant strains and the corresponding reference wild-type (WT) strain from the confocal image series using the IMARIS software. Images depict an aerial view of 48h-biofilms in the microplate system. The scale bar is 50µm.</p

Biofilm biovolumes of the 14 <i>B.subtilis</i> mutant strains.

<p>Effects of mutations on the biofilm biovolumes of the 48h-biofilms obtained with the 14 mutants and wild-type strain (GM2812) (GFP-carrying strains). Biovolumes were normalized to wild-type (GM2812) and classified in ascending order. The error bars indicate the standard error and the statistically significant difference in biovolume obtained with the wild-type (<i>P</i><0.05) is indicated by a star (*).</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

Architecture of <i>B. subtilis</i> communities.

<p>(A) Three-dimensional projection of <i>B. subtilis</i> 168 GFP and ND_medical GFP biofilms obtained from xyz confocal images series using IMARIS software. Images present an aerial view of biofilm structure with the virtual shadow projection on the right. Scale bar correspond to 50 µm. (B) SEM images of 24 h-biofilms. (C) Dye binding properties of <i>B. subtilis</i> macrocolonies grown for 72 h on Congo red indicator plates.</p

Real-time visualization of PAA (0.05%) activity in B. subtilis biofilms.

<p>(A) Quantification of Chemchrome V6 fluorescence intensity in B. subtilis 168 (white squares) and ND_medical (black squares) biofilms during PAA treatment at 500 ppm. Values shown corresponds to the average of the fluorescence at five depths with standard deviation in the biofilm for a representative experiment. Inactivation rate k_max was obtained after fitting GinaFIT “log linear + tail” inactivation model on experimental data. The means of three experiments ± standard deviation are presented in the inset. (B) Visualization of Chemchrome V6 fluorescence loss (membrane permeabilisation) in B. subtilis 168 and ND_medical biofilms at 0. 15 and 30 secondes and 0. 5 and 10 minutes respectively during PAA treatment (0.05%). Each image corresponds to the superimposition of a green fluorescence image of a representative experiment on a greyscale image of initial fluorescence at the same location. Scale bars correspond to 20 µm.</p

Bactericidal activity of water and 0.35% PAA on single and mixed species biofilms after 5 min of treatment.

<p>Data shown are mean of three experiments ± standard deviation. Samples from which no survivor were recovered are represented by (−). Minimum detection of 2 logs CFU/well.</p

Strains used in this study.

a<p><i>spec</i> and <i>ery</i> stand for spectinomycin and erythromycin resistance markers respectively.</p