Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.

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Biofilms contain bacterial cells that are in a wide range of physiological states. Within a biofilm population, cells with diverse genotypes and phenotypes that express distinct metabolic pathways, stress responses and other specific biological activities are juxtaposed. The mechanisms that contribute to this genetic and physiological heterogeneity include microscale chemical gradients, adaptation to local environmental conditions, stochastic gene expression and the genotypic variation that occurs through mutation and selection. Here, we discuss the processes that generate chemical gradients in biofilms, the genetic and physiological responses of the bacteria as they adapt to these gradients and the techniques that can be used to visualize and measure the microscale physiological heterogeneities of bacteria in biofilms.

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Chronic wounds including diabetic foot ulcers, pressure ulcers, and venous leg ulcers are a worldwide health problem. It has been speculated that bacteria colonizing chronic wounds exist as highly persistent biofilm communities. This research examined chronic and acute wounds for biofilms and characterized microorganisms inhabiting these wounds. Chronic wound specimens were obtained from 77 subjects and acute wound specimens were obtained from 16 subjects. Culture data were collected using standard clinical techniques. Light and scanning electron microscopy techniques were used to analyze 50 of the chronic wound specimens and the 16 acute wound specimens. Molecular analyses were performed on the remaining 27 chronic wound specimens using denaturing gradient gel electrophoresis and sequence analysis. Of the 50 chronic wound specimens evaluated by microscopy, 30 were characterized as containing biofilm (60%), whereas only one of the 16 acute wound specimens was characterized as containing biofilm (6%). This was a statistically significant difference (p<0.001). Molecular analyses of chronic wound specimens revealed diverse polymicrobial communities and the presence of bacteria, including strictly anaerobic bacteria, not revealed by culture. Bacterial biofilm prevalence in specimens from chronic wounds relative to acute wounds observed in this study provides evidence that biofilms may be abundant in chronic wounds.

Much of what makes life in a microbial biofilm different from life in a free aqueous suspension can be explained by invoking the phenomenon of diffusion. This article discusses the profound influence of the physics of the diffusion process on the chemistry and biology of the biofilm mode of growth. I have framed the discussion in the form of answers to five important questions. WHY IS DIFFUSION AN IMPORTANT PROCESS IN BIOFILMS?When microorganisms are grown in planktonic culture, diffusion is usually of little consequence. There are two reasons for this. The first reason is that planktonic cultures are generally agitated, and the resulting fluid flow transports solutes rapidly, resulting in a well-mixed system. Transport that occurs as a solute is carried by the bulk flow of a fluid (convection) is generally much faster than the transport resulting from random molecular motion (diffusion). Of course, there is no net convective flow of fluid into or out of the microbial cell. At some point close to the cell, diffusion becomes critical for moving the solute toward or away from the cell surface. The reason that diffusion does not limit this step is that the diffusion distance is small and diffusion is rapid over such short distances.Diffusion limitation arises readily in biofilm systems because fluid flow is reduced and the diffusion distance is increased in the biofilm mode of growth. The biofilm and the substratum to which it is anchored impede flow in the vicinity of the biofilm, throttling convective transport. Inside cell clusters, the locally high cell densities and the presence of extracellular polymeric substances arrest the flow of water. Diffusion is the predominant transport process within cell aggregates (7,36). Whereas the diffusion distance for a freely suspended microorganism is of the order of magnitude of the dimension of an individual cell, the diffusion distance in a biofilm becomes the dimension of multicellular clusters. This can easily represent an increase in the diffusion distance, compared to a single cell, of 2 orders of magnitude. As is explained in the next section, diffusive equilibration time scales as the square of the diffusion distance. In other words, a biofilm that is 10 cells thick will exhibit a diffusion time 100 times longer than that of a lone cell. HOW FAST DO SOLUTES DIFFUSE INTO OR OUT OF A BIOFILM?Suppose a stain is added to the medium bathing a biofilm. How long will it take this dye to permeate, by diffusion, to the interior of a cell cluster or to the bottom of the biofilm? Because I aspire in this article to avoid overwhelming the reader with mathematics, I will define a single, simple measure of diffusive penetration time. There are two versions of this measure, depending on the geometry of the system. The time required for a solute added to the fluid bathing a biofilm to attain 90% of the bulk fluid concentration at the base of flat slab (uniformly thick) biofilm is given very simply byHere, L is the biofilm thickness, and D e is the effective diffusion coefficient i...

Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.

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