In situ analysis of Bacillus licheniformis biofilms: amyloid-like polymers and eDNA are involved in the adherence and aggregation of the extracellular matrix.

AIMS: This study attempts to determine which of the exopolymeric substances are involved in the adherence and aggregation of a Bacillus licheniformis biofilm. METHODS AND RESULTS: The involvement of extracellular proteins and eDNA were particularly investigated using DNase and proteinase K treatment. The permeability of the biofilms increased fivefold after DNase I treatment. The quantification of the matrix components showed that, irrespective to the enzyme tested, eDNA and amyloid-like polymers were removed simultaneously. Size-exclusion chromatography analyses supported these observations and revealed the presence of associated nucleic acid and protein complexes in the biofilm lysates. These data suggest that some extracellular DNA and amyloid-like proteins were closely interlaced within the matrix. Finally, confocal laser scanning microscopy imaging gave supplementary clues about the 3D organization of the biofilms, confirming that eDNA and exoproteins were essentially layered under and around the bacterial cells, whereas the amyloid-like fractions were homogeneously distributed within the matrix. CONCLUSION: These results confirm that some DNA-amyloid complexes play a key role in the modulation of the mechanical resistance of B. licheniformis biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights the need to consider the whole structure of biofilms and to target the interactions between matrix components. A better understanding of B. licheniformis biofilm physiology and the structural organization of the matrix will strengthen strategies of biofilm control.

Quantification of biofilm exopolysaccharides using an in situ assay with periodic acid-Schiff reagent.

A novel approach to the quantification of extracellular polysaccharides in miniaturized biofilms presenting a wide variety of extracellular matrices was developed. The assay used the periodic acid-Schiff reagent and was first calibrated on dextran and alginate solutions. Then it was implemented on 24-h and 48-h biofilms from three strains known to produce different exopolymeric substances (Pseudomonas aeruginosa, Bacillus licheniformis, Weissella confusa). The assay allowed quantification of the total exopolysaccharides, taking into account possible interferences due to cells or other main expolymers of the matrix (eDNA, proteins).

Epicocconone, a sensitive and specific fluorescent dye for in situ quantification of extracellular proteins within bacterial biofilms.

Biofilms are ecosystems of closely associated bacteria encapsulated in an extracellular matrix mainly composed of polysaccharides and proteins. A novel approach was developed for in situ quantification of extracellular proteins (ePNs) in various bacterial biofilms using epicocconone, a natural, fluorescent compound that binds amine residues of proteins. Six commercial proteins were tested for their reaction with epicocconone, and bovine serum albumin (BSA) was selected for assay optimization. The optimized protocol, performed as a microassay, allowed protein amounts as low as 0.7 µg to as high as 50 µg per well to be detected. Addition of monosaccharides or polysaccharides (glucose, dextran or alginate) to the standard BSA solutions (0 to 250 µg ml(-1)) showed little or no sugar interference up to 2000 µg ml(-1), thus providing an assessment of the specificity of epicocconone for proteins. The optimized protocol was then applied to three different biofilms, and in situ quantification of ePN showed contrasted protein amounts of 22.1 ± 3.1, 38.3 ± 7.1 and 0.3 ± 0.1 µg equivalent BSA of proteins for 48-h biofilms of Pseudomonas aeruginosa, Bacillus licheniformis and Weissella confusa, respectively. Possible interference due to global matrix compounds on the in situ quantification of proteins was also investigated by applying the standard addition method (SAM). Low error percentages were obtained, indicating a correct quantification of both the ePN and the added proteins. For the first time, a specific and sensitive assay has been developed for in situ determination of ePN produced by bacterial cells. This advance should lead to an accurate, rapid tool for further protein labelling and microscopic observation of the extracellular matrix of biofilms.

Removal of microbial multi-species biofilms from the paper industry by enzymatic treatments.

This study aimed to characterize biofilms from the paper industry and evaluate the effectiveness of enzymatic treatments in reducing them. The extracellular polymeric substances (EPS) extracted from six industrial biofilms were studied. EPS were mainly proteins, the protein to polysaccharide ratio ranging from 1.3 to 8.6 depending on where the sampling point was situated in the paper making process. Eight hydrolytic enzymes were screened on a 24-h multi-species biofilm. The enzymes were tested at various concentrations and contact durations. Glycosidases and lipases were inefficient or only slightly efficient for biofilm reduction, while proteases were more efficient: after treatment for 24 h with pepsin, Alcalase® or Savinase®, the removal exceeded 80%. Savinase® appeared to be the most adequate for industrial conditions and was tested on an industrial biofilm sample. This enzyme led to a significant release of proteins from the EPS matrix, indicating its potential efficiency on an industrial scale.

Assessment of the genotoxicity of Cu and Zn in raw and anaerobically digested slurry with the Vicia faba micronucleus test.

Genotoxicity of Cu and Zn was assessed by use of the micronucleus (MN) test on Vicia faba roots. Plants were exposed to various leachates of raw and anaerobically digested pig slurry, with maximum total concentrations of 200microM Cu and 600microM Zn. The results indicated stabilisation of the organic matter during anaerobic digestion of the slurry and bioconversion of some phytotoxic organic compounds (e.g. phenols or p-cresol), but did not show a relationship between Cu and Zn concentrations and MN frequency. Exposure of Vicia plants to binary inorganic solutions of Cu and Zn (CuSO(4)/ZnSO(4), 1:3) showed a significant micronucleus induction at concentrations of 40microM Cu and 120microM Zn and higher. When MN frequency was plotted against dissolved Cu (<0.45microm), applied as slurry or as CuSO(4), a single curve was obtained. At concentrations lower than 10microM, modulation of the genotoxic effect of Cu was found. At concentrations up to 150microM, MN induction increased significantly, while phytotoxic symptoms appeared at higher concentrations.