Visualization and quantification of the cellular and extracellular components of Salmonella Agona biofilms at different stages of development

Abstract

Salmonella is a major food-borne pathogen able to persist in food processing environments because of its ability to form biofilms. A Salmonella enterica serotype Agona isolate from poultry (S24) was grown at 37°C in biofilms for up to 144 hours (H144) in attachment to polystyrene surfaces. Biofilm structures were examined at different stages in their development (H3, H24, H48, H72, H96 and H144) using confocal laser scanning microscopy (CLSM) in conjunction with fluorescent dyes for live cells (SYTO 9), dead cells (propidium iodide), proteins (fluorescein isothiocyanate isomer I), lipids (DiD’oil), α-polysaccharides (concanavalin A, tetramethylrhodamine conjugate), and β-polysaccharides (calcofluor white M2R). Strain S24 developed a robust biofilm at H72 (biovolume of 166,852.5 ± 13,681.8 μm3 in the observation field of 16,078.2 μm2). The largest biovolume of live cells was also detected at H72 (128,110.3 ± 4,969.1 μm3), decreasing thereafter, which was probably owing to the detachment of cells prior to a new phase of colonization. The percentage of dead cells with regard to total cells in the biofilms increased throughout the incubation, ranging from 2.3 ± 1.1% (H24) to 44.2 ± 11.0% (H144). Proteins showed the greatest biovolume among the extracellular components within the biofilms, with values ranging from 1,295.1 ± 1,294.9 μm3 (H3) to 19,186.2 ± 8,536.0 μm3 (H96). Maximum biovolume values of 15,171.9 ± 660.7 μm3 (H48), 7,055.3 ± 4,415.2 μm3 (H144), and 2,548.6 ± 1,597.5 μm3 (H72) were observed for β-polysaccharides, α-polysaccharides and lipids, respectively. A strong (P < 0.01) positive correlation was found between the total biovolume of biofilm and the biovolume of live cells, proteins and β-polysaccharides, which may serve as useful markers of biofilm formation. The present work provides new insights into the formation of S. Agona biofilms. Our findings may contribute to the designing of reliable strategies for preventing and removing these bacterial communities.

Architecture and Viability of the Biofilms Formed by Nine Listeria Strains on Various Hydrophobic and Hydrophilic Materials

“…The study of the formation and structure of the biofilms was carried out by CLSM using a previously described procedure [16]. CLSM combined with a quantitative analysis of the images that were obtained enabled numerical data to be collected for the structural parameters of biofilms, such as biovolume, facilitating the comparison of different biofilms by means of statistical analyses.…”

“…Thus, a total of 324 CLSM images were obtained (9 strains × 4 surface materials × 3 replicates × 3 surfaces in each well). The original Zeiss files (CZI format) were imported into the IMARIS 9.1 software package (Bitplane, Zurich, Switzerland) to analyse the images [16].…”

“…Cells in biofilms become more resistant than planktonic cells to different environmental stress factors such as sanitizing agents, facilitating bacterial survival. Furthermore, biofilms can be a continuous source of contamination when in contact with foodstuffs, which can trigger human disease [15,16].…”

The Flagellar Gene Regulates Biofilm Formation and Mussel Larval Settlement and Metamorphosis

“…Biofilms were stained following the method of González-Machado et al [57]. Details of used fluorescent dyes are listed in Table S3.…”

Visualizing Microbial Community Dynamics via a Controllable Soil Environment

“…Previously, the spatiotemporal dynamics of microbial community development and growth at microscale resolution have been studied using confocal microscopy (9)(10)(11)(12). In addition, advanced chemical imaging techniques, like mass spectrometry imaging (MSI) methods, have been employed to examine complex molecular architectures of mammalian tissues (13,14) and microbial communities on agar surfaces (15)(16)(17)(18).…”