Importance of the biofilm matrix for the erosion stability of Bacillus subtilis NCIB 3610 biofilms

Abstract

Production and secretion of biomolecules can provide new emergent functionalities to the synthesizing organism. In particular, the secretion of extracellular polymeric substances (EPS) by biofilm forming bacteria creates a biofilm matrix that protects the individual bacteria within the biofilm from external stressors such as antibiotics, chemicals and shear flow. Although the main matrix components of biofilms formed by Bacillus subtilis are known, it remains unclear how these matrix components contribute to the erosion stability of B. subtilis biofilms. Here, we combine different biophysical techniques to assess this relation. In particular, we quantify the importance of specific biofilm matrix components on the erosion behavior of biofilms formed by the well-studied Bacillus subtilis NCIB 3610. We find that the absence of biofilm matrix components decreases the erosion stability of NCIB 3610 biofilms in water, largely by abolishing the hydrophobic surface properties of the biofilm and by reducing the biofilm stiffness. However, the erosion resistance of NCIB 3610 biofilms is strongly increased in the presence of metal ions or the antibiotic ciprofloxacin. In the first case, unspecific ionic cross-linking of biofilm components or individual bacteria seems to be responsible for the observed effect, and in the second case there seems to be an unspecific interaction between the antibiotic and the biofilm matrix. Taken together, our results emphasize the importance of the biofilm matrix to reduce biofilm erosion and give insights into how the specific biomolecules interact with certain chemicals to fulfill this task.

Fig. 1. Erosion stability of NCIB 3610 biofilms. (A) Schematic representation of the erosion experiment. Left: a PTFE slide containing 10 biofilm-covered agar patches is inserted into a tube which is then filled with a testing solution (middle). Shear forces are induced by setting the tube into rotational motion using a lab shaker. Right: images and sketch of biofilm-covered agar patches before (0% biofilm removal) and during (63% biofilm removal) an erosion experiment. Sometimes folding of the biofilm over the edge during detachment was observed. Consequently, only the biofilm free area was calculated as a measure for biofilm removal. (B) Biofilm erosion of biofilms formed by the NCIB 3610 wild-type strain and mutant strains in water. (NCIB 3610 is depicted in blue, the tasA mutant in turquoise, the bslA deletion mutant in green and the epsA-O mutant in orange). (C) Time-point of 50% detachment for NCIB 3610 and mutant strains in water.

Fig. 2. Absence of biofilm matrix components affects wettability of the biofilm surface and biofilm stiffness. (A) Wetting behavior of NCIB 3610 wild-type and mutant strain biofilms. Turquoise +: corresponding contact angle right after application of water droplet (this value is not included in the significance analysis). (B) Biofilm stiffness given as the storage modulus obtained by macrorheology.

Fig. 3. Metal ions increase the erosion stability of NCIB 3610 biofilms. (A) Biofilm erosion of the NCIB 3610 wild-type and several mutant strains as mean fraction of removed biofilm from the agar patches compared to the fully covered patches at time t = 0 min. (B) Biofilm stiffness of the NCIB 3610 wild-type and several mutant strains. (A and B) Black line indicates value of corresponding water treated sample.

Fig. 4. Influence of pH on biofilm erosion. The pH of each tested ionic solution is given underneath the corresponding biofilm removal data of the wild-type strain.

Fig. 5. Treatment with the antibiotic ciprofloxacin increases erosion stability of NCIB 3610 biofilms. (A) Biofilm erosion of the NCIB 3610 wild-type in water and three different antibiotics. (B) Biofilm detachment for NCIB 3610 and mutant strains in the presence of ciprofloxacin. Black lines indicate the biofilm removal of each strain in water.