Direct Comparison of Physical Properties of Bacillus subtilis NCIB 3610 and B-1 Biofilms

Abstract

Many bacteria form surface-attached communities known as biofilms. Due to the extreme resistance of these bacterial biofilms to antibiotics and mechanical stresses, biofilms are of growing interest not only in microbiology but also in medicine and industry. Previous studies have determined the extracellular polymeric substances present in the matrix of biofilms formed by Bacillus subtilis NCIB 3610. However, studies on the physical properties of biofilms formed by this strain are just emerging. In particular, quantitative data on the contributions of biofilm matrix biopolymers to these physical properties are lacking. Here, we quantitatively investigated three physical properties of B. subtilis NCIB 3610 biofilms: the surface roughness and stiffness and the bulk viscoelasticity of these biofilms. We show how specific biomolecules constituting the biofilm matrix formed by this strain contribute to those biofilm properties. In particular, we demonstrate that the surface roughness and surface elasticity of 1-day-old NCIB 3610 biofilms are strongly affected by the surface layer protein BslA. For a second strain,B. subtilis B-1, which forms biofilms containing mainly γ-polyglutamate, we found significantly different physical biofilm properties that are also differently affected by the commonly used antibacterial agent ethanol. We show that B-1 biofilms are protected from ethanol-induced changes in the biofilm's stiffness and that this protective effect can be transferred to NCIB 3610 biofilms by the sole addition of γ-polyglutamate to growing NCIB 3610 biofilms. Together, our results demonstrate the importance of specific biofilm matrix components for the distinct physical properties of B. subtilis biofilms.

FIG 1

mRNA production of genes for biofilm matrix proteins in B. subtilis strain B-1. Gene expression analyses were performed for biofilms generated by wild-type strain B-1, at 10 h (A) and 18 h (B) of biofilm growth. The mRNA production of genes corresponding to matrix components described for wild-type strains NCIB 3610 and B-1, in comparison with the production of the control, 16S rRNA, was analyzed. C_T values represent the mean values obtained from 3 biological and 3 technical triplicates. The error bars denote standard deviations. P values from pairwise t tests for these data are given in Table S1 in the supplemental material. Additionally, an ensemble significance test (one-way ANOVA and Tukey's honestly significant difference test; MATLAB) was performed to confirm that the genes blsA, ywsC, epsH, and tasA were transcribed at significantly lower levels than the 16S rRNA control. *, ensemble significant differences.

FIG 2

Surface stiffness of different B. subtilis biofilms. Young's modulus values were obtained for 1-day-old biofilms of the wild-type strains B. subtilis B-1 (red) and NCIB 3610 (blue) and three mutant strains of NCIB 3610 (blue). Error bars are Gaussian errors resulting from the error in the method. Photographs above the graph are images of single biofilm colonies. Scale bars, 1 mm. White arrows point to the faint single biofilm colonies of BslA and EpsA-O mutant strains. P values for these data are given in Table S2 in the supplemental material.

FIG 3

Surface roughness of different B. subtilis biofilms. The root mean squared surface roughness was determined for 1-day-old biofilms of wild-type B. subtilis strains B-1 (red) and NCIB 3610 (blue) and three mutant strains of NCIB 3610 (blue). (A) Profilometer images of wild-type strain NCIB 3610, BslA mutant NCIB 3610, and wild-type strain B-1 biofilms. The color code indicates the sample heights, according to the key. (B) Initial roughness of untreated B. subtilis biofilms. (C) Decreases in the roughness of B. subtilis biofilms after 60 min of exposure to ethanol. Error bars denote the standard deviations for 9 samples from 3 different growth batches. P values for these data are given in Table S2 in the supplemental material. P values below 0.05 indicate significant differences from the data obtained for strain NCIB 3610 and are indicated with an asterisk.

FIG 4

Bulk viscoelasticity of B. subtilis biofilms. (A) Storage modulus values for 1-day-old biofilms of wild-type strains B. subtilis B-1 (red) and NCIB 3610 (blue) and three mutant strains of NCIB 3610 (blue); (B) storage modulus values for 1-day-old biofilms of wild-type B. subtilis strain NCIB 3610 grown in the presence or absence of γ-polyglutamate (PGA) and after different periods of exposure to ethanol (see Materials and Methods). Biofilms were not treated or were exposed to ethanol for 10 or 60 min, as indicated. Error bars denote the standard deviations for 9 samples from 3 different growth batches. P values for these data are given in Tables S2 and S3 in the supplemental material.

FIG 5

Comparison of normalized total and dry masses of biofilms formed by the wild-type strains NCIB 3610 and B-1. This bar plot shows the total produced masses of 1-day-old biofilms of the wild-type strains NCIB 3610 (blue) and B-1 (red), as well as the dry masses of biofilms of these two wild-type strains, as determined by lyophilization (see Materials and Methods). Biofilms were not treated, were treated with ethanol (EtOH) for 60 min, or were grown in the presence of γ-PGA and then treated with ethanol for 60 min, as indicated. The circle indicates that under these particular conditions, a subset of samples could not be successfully lyophilized, indicating that ethanol might be taken up into the B-1 biofilm. The data given here represent the dry weights of the samples for which lyophilization was successful. Error bars denote the standard deviations. P values for these data are given in Table S4 in the supplemental material. P values below 0.05 indicate that the data obtained in the presence of ethanol were significantly different from those obtained in the absence of ethanol for that strain and are indicated with an asterisk.

FIG 6

Images of plated B. subtilis biofilms immediately after the application of 99% ethanol. Images were obtained for the wild-type strains B. subtilis B-1 (red) and NCIB 3610 and three mutant strains of NCIB 3610 (blue). Scale bars, 1 mm.