Direct observations have clearly shown that biofilm bacteria predominate, numerically and metabolically, in virtually all nutrient-sufficient ecosystems. Therefore, these sessile organisms predominate in most of the environmental, industrial, and medical problems and processes of interest to microbiologists. If biofilm bacteria were simply planktonic cells that had adhered to a surface, this revelation would be unimportant, but they are demonstrably and profoundly different. We first noted that biofilm cells are at least 500 times more resistant to antibacterial agents. Now we have discovered that adhesion triggers the expression of a sigma factor that derepresses a large number of genes so that biofilm cells are clearly phenotypically distinct from their planktonic counterparts. Each biofilm bacterium lives in a customized microniche in a complex microbial community that has primitive homeostasis, a primitive circulatory system, and metabolic cooperativity, and each of these sessile cells reacts to its special environment so that it differs fundamentally from a planktonic cell of the same species.

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Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process. By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ. The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 m. Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent. Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass. These results have significance in the establishment of an infectious dose and public health risk assessment.The detachment of bacterial cells from biofilms is of fundamental importance to the dissemination of infection and to contamination in both clinical (12) and public health (13,18,19) settings. However, detachment is the least-studied biofilm process and remains poorly understood (10,14). The spontaneous detachment of cells from bacterial biofilms has been divided into two processes, erosion and sloughing, based on the magnitude and frequency of the detachment event (3, 4). Erosion is the continual detachment of single cells and "small portions of the biofilm," whereas sloughing is the "rapid, massive loss of biofilm" (4). However, the size distribution and detachment frequency of biofilm particulates have not been quantified. Information on biofilm detachment is usually inferred from monitoring the cell concentration in the liquid phase after sample homogenization. This process provides a temporally and spatially averaged detachment rate, but information on the size distribution and detachment frequency is lost. A minimum infectious dose of cells concentrated in a biofilm particulate may be overlooked if such an aggregate is disrupted and effectively diluted in the test tube.Digital time-lapse microscopy (DTLM) has been used to track single cells moving between biofilm cell clusters (5) and to quantify the dynamic viscoelastic behavior and movement of mature biofilms over solid surfaces in situ (15, 16). In previous investigations on the influence of hydrodynamics and nutrient concentration on the structure of a four-species laboratory biofilm, it was reported that an increase in C and N concentrations resulted in a change in architecture from a thin layer of migratory ripples (regularly spaced ridges running perpendicular to the flow direction) and filamentous streamers (cell clusters elongated in the downstream direction with "tails" rapidly oscillating in the flow) to one of mushroom-and mound-shaped cell clusters (17). DTLM suggested that these cell clusters were continually growing and detaching from the biofilm.The goal of the work presented here was to examine localized growth and detachment events at the microscopic scale and...

BackgroundBiofilm formation enhances the capacity of pathogenic Salmonella bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir for the contamination of food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most gene expression studies of Salmonella have focused on the effect of infection-relevant stressors on virulence or the comparison of mutant and wild-type bacteria. However little is known about the physiological responses taking place inside a Salmonella biofilm.ResultsWe have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% of the S. Typhimurium genome (433 genes) showed a 2-fold or more change in the biofilm compared with planktonic cells. The genes that were significantly up-regulated implicated certain cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), and that a functional SPI2 secretion system regulator (ssrA) was required for S. Typhimurium biofilm formation. We identified STM0341 as a gene of unknown function that was needed for biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to decreased bacterial attachment and this biofilm defect was restored by exogenous tryptophan or indole.ConclusionsBiofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation and reveal a link between SPI2 expression and surface-associated growth in S. Typhimurium.