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Biofilms are commonly defined as accumulations of microbes, embedded in a self-secreted, polysaccharide-rich extra-cellular matrix. This study aimed to characterize specific morphological changes that occur in Bacillus subtilis biofilms under nutrient-limiting growth conditions. Under varying levels of nutrient depletion, colony-type biofilms were found to exhibit different rates of spatial expansion and green fluorescent protein production. Specifically, colony-type biofilms grown on media with decreased lysogeny broth content exhibited increased spatial expansion and more stable GFP production over the entire growth period. By modeling the surface morphology of colony-type biofilms using confocal and multiphoton microscopy, we analyzed the appearance of distinctive folds or "wrinkles" that form as a result of lysogeny broth content reduction in the solid agar growth media. When subjected to varying nutritional conditions, the channel-like folds were shown to alter their morphology; growth on nutrient-depleted media was found to trigger the formation of large and straight wrinkles connecting the colony core to its periphery. To test a possible functional role of the formed channels, a fluorescent analogue of glucose was used to demonstrate preferential native uptake of the molecules into the channels' interiors which supports their possible role in the transport of molecules throughout biofilm structures.

Biofilms are commonly defined as accumulations of microbes, embedded in a self-secreted, polysaccharide-rich extra-cellular matrix. This study aimed to characterize specific morphological changes that occur in Bacillus subtilis biofilms under nutrient-limiting growth conditions. Under varying levels of nutrient depletion, colony-type biofilms were found to exhibit different rates of spatial expansion and green fluorescent protein production. Specifically, colony-type biofilms grown on media with decreased lysogeny broth content exhibited increased spatial expansion and more stable GFP production over the entire growth period. By modeling the surface morphology of colony-type biofilms using confocal and multiphoton microscopy, we analyzed the appearance of distinctive folds or "wrinkles" that form as a result of lysogeny broth content reduction in the solid agar growth media. When subjected to varying nutritional conditions, the channel-like folds were shown to alter their morphology; growth on nutrient-depleted media was found to trigger the formation of large and straight wrinkles connecting the colony core to its periphery. To test a possible functional role of the formed channels, a fluorescent analogue of glucose was used to demonstrate preferential native uptake of the molecules into the channels' interiors which supports their possible role in the transport of molecules throughout biofilm structures.

The purpose of the present study was to investigate the mechanism of action of our newly developed green sanitizer formulation comprising a natural phenolic compound, gallic acid (GA), strengthened by the Generally Recognized as Safe (GRAS) materials hydrogen peroxide (H2O2) and DL-lactic acid (LA). Combining 8 mM GA with 1 mM H2O2 resulted in an abundant generation of reactive oxygen species (ROS) and a bactericidal effect towards Gram-negative (Escherichia coli, Pseudomonas syringae, and Pectobacterium brasiliense) and Gram-positive (Bacillus subtilis) bacteria (4 to 8 log CFU mL−1 reduction). However, the exposure to this dual formulation (DF) caused only a modest 0.7 log CFU mL−1 reduction in the Gram-positive L. innocua population. Amending the DF with 20 mM LA to yield a triple formulation (TF) resulted in the efficient synergistic control of L. innocua proliferation without increasing ROS production. Despite the inability to grow on plates (>7 log CFU mL−1 population reduction), the TF-exposed L. innocua maintained high intracellular ATP pools and stable membrane integrity. The response of L. innocua to TF could be qualified as a "viable but nonculturable" (VBNC) phenomenon, while with the other species tested this formulation caused cell death. This research system may offer a platform for exploring the VBNC phenomenon, a critical food safety topic. [ABSTRACT FROM AUTHOR]

Biofilm is commonly defined as accumulation of microbes, embedded in a self-secreted extra-cellular matrix, on solid surfaces or liquid interfaces. In this study, we analyze several aspects of Bacillus subtilis biofilm formation using tools from the field of image processing. Specifically, we characterize the growth kinetics and morphological features of B. subtilis colony type biofilm formation and compare these in colonies grown on two different types of solid media. Additionally, we propose a model for assessing B. subtilis biofilm complexity across different growth conditions. GFP-labeled B. subtilis cells were cultured on agar surfaces over a 4-day period during which microscopic images of developing colonies were taken at equal time intervals. The images were used to perform a computerized analysis of few aspects of biofilm development, based on features that characterize the different phenotypes of B. subtilis colonies. Specifically, the analysis focused on the segmented structure of the colonies, consisting of two different regions of sub-populations that comprise the biofilm - a central "core" region and an "expanding" region surrounding it. Our results demonstrate that complex biofilm of B. subtillis grown on biofilm-promoting medium [standard lysogeny broth (LB) supplemented with manganese and glycerol] is characterized by rapidly developing three-dimensional complex structure observed at its core compared to biofilm grown on standard LB. As the biofilm develops, the core size remains largely unchanged during development and colony expansion is mostly attributed to the expansion in area of outer cell sub-populations. Moreover, when comparing the bacterial growth on biofilm-promoting agar to that of colonies grown on LB, we found a significant decrease in the GFP production of colonies that formed a more complex biofilm. This suggests that complex biofilm formation has a diminishing effect on cell populations at the biofilm core, likely due to a combination of reduced metabolic rate and increased levels of cell death within this region.

Staphylococcus caprae is a Gram-positive, coagulase-negative staphylococci (CoNS), which appears as commensal in the skin, as well as a prevalent mastitis pathogen of goats. Occasionally, it is also associated with infections in humans. Biofilm formation has been identified as a putative virulence factor in S. caprae. Biofilms are multicellular communities protected by a self-produced extracellular matrix (ECM), which facilitates the resistance of bacterial cells to antimicrobial treatments. The ECM is constructed by exopolysaccharides, including the major exopolysaccharide—polysaccharide intercellular adhesion (PIA), regulated by the ica operon in Staphylococcus species. The aim of this study was to characterize the expression of the ica operon in relation to biofilm formation in S. caprae. Results showed that within a few hours of growth, S. caprae could adhere to polystyrene surfaces, start to accumulate, and form biofilm. Peak biofilm biomass and maturation were reached after 48 h, followed by a reduction in biomass after 72 h. Confocal laser scanning microscopy showed the expression of matrix-associated proteins and polysaccharides at various time points. The expression dynamics of the ica operon were investigated using real-time reverse transcriptase PCR (RT)-qPCR, which showed elevated expression during the early stages of biofilm formation and subsequent downregulation throughout the biofilm aging process. In conclusion, our results show that the ica operon is essential in regulating biofilm formation in S. caprae, similar to other Staphylococcus species. Furthermore, the robustness of the observed biofilm phenotype could account for the successful intramammary colonization and may explain disease persistence caused by this pathogenic bacterium. [ABSTRACT FROM AUTHOR]

Bacillus subtilis forms biofilms in response to signals that remain poorly defined. We report that biofilm formation is stimulated by sublethal doses of chlorine dioxide (Cl[O.sub.2]), an extremely effective and fast-acting biocide. Cl[O.sub.2] accelerated biofilm formation in B. subtilis as well as in other bacteria, suggesting that biofilm formation is a widely conserved response to sublethal doses of the agent. Biofilm formation depends on the synthesis of an extracellular matrix that holds the constituent cells together. We show that the transcription of the major operons responsible for the matrix production in B. subtilis, epsA-epsO and yqxM-sipW-tasA, was enhanced by Cl[O.sub.2], in a manner that depended on the membrane-bound kinase KinC. Activation of KinC appeared to be due to the ability of Cl[O.sub.2] to collapse the membrane potential. Importantly, strains unable to make a matrix were hypersensitive to Cl[O.sub.2], indicating that biofilm formation is a defensive response that helps protect cells from the toxic effects of the biocide. doi: 10.1128/JB.01025-10

Streptococcus mutans often adopts a sessile biofilm lifestyle that differs greatly from that of free-living cells. Biofilm formation represents a protected mode of growth that allows cells to survive in hostile environments. In this study, in vitro comparative transcriptome analysis was carried out to identify genes that are differentially expressed in biofilm of S. mutans compared with free-living cells. DNA-microarray analyses indicated that about 12% of genes showed significant differential expression: 139 were activated and 104 were repressed in biofilm vs the planktonic environment. The differential expression of 20 selected genes was confirmed by real-time RT-PCR. In addition, regulation of expression of these genes during biofilm development was tested in 100 and 400 [micro]m deep biofilms. Direct comparison of optical images consistently demonstrated that changes in biofilm thickness are accompanied by significant shifts in cell viability. From evaluation of gene expression patterns, it was shown that the majority of the genes tested were significantly down-regulated in 400 vs 100 [micro]m deep biofilms. This study provides a genome-scale synopsis and adds important insights into gene expression in biofilm development processes of S. mutans, which are strongly associated with the pathogenesis of dental diseases. DOI 10.1099/mic.0.2006/002030-0