Growth and Detachment of Cell Clusters from Mature Mixed-Species Biofilms

Abstract

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...

Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm

“…This mixed-species biofilm has previously been shown to be reproducible with respect to structure and viable cell counts (Jackson et al, 2001;Stoodley et al, 2001). In addition, these three species are commonly found in soil habitats and are reported to coexist in metalworking fluids, where the growth of P. protegens is a prerequisite for the growth of P. aeruginosa and K. pneumoniae (Chazal, 1995).…”

Viscoelasticity in Biological Systems: A Special Focus on Microbes

“…A biofilm is a surfaceassociated three dimensional consortia of microbial communities, surrounded by a matrix of protective biopolymers, macromolecular debris, sediment and precipitate, making it a highly complex mechanical, compliant, and viscoelastic structure. Biofilms can also be highly heterogeneous consisting of mixed populations of bacteria, fungi, protozoa [112] with interspersed pores and channels. Biofilms exhibit enormous resistance to external stress factors, with exopolysaccharides (EPS) contributing to their overall mechanical stability and enabling them to withstand external forces.…”

“…A decade ago it was demonstrated that biofilms are effectively viscoelastic, a property that plays a major role in the various stages of biofilm growth [109,112,[114][115][116][117]. Biofilms, all of which exhibit classic viscoelastic behaviours [109,115,116,118,119], can grow in a wide variety of environments including laboratory conditions, under flow and even in hot springs, demonstrating their ability to absorb elevated changes in shear stresses.…”

“…Biofilms, all of which exhibit classic viscoelastic behaviours [109,115,116,118,119], can grow in a wide variety of environments including laboratory conditions, under flow and even in hot springs, demonstrating their ability to absorb elevated changes in shear stresses. Biofilms are elastic for shorter periods, can resist shear stresses and detachment, and over longer periods of time they flow as viscous fluids and become streamlined [109,112,114]. Viscoelasticity of the biofilm matrix has been shown to determine its structural integrity, resistance to stress, and ease of dispersion [120].…”

Biofilms and hygiene on dairy farms and in the dairy industry: sanitation chemical products and their effectiveness on biofilms - a review

“…(3) Release of microbial cells from the biofilm structures or from the surface (Stoodley et al 2001a); the process of active release is physiologically controlled. Separate cells and small clusters are released more often, but large pieces are also released; the latter constitute a health hazard regarding the infectious dose (Stoodley et al 2001b). The released bacteria or their clusters drif within the liquid environment or are attached to damp semi-finished products and ready-made foodstuffs and can become sources of microbial contamination of other surfaces (Zottola & Sasahara 1994) or the products of both animal and vegetable origins (Rayner et al 2004).…”