Biofilms: Microbial Life on Surfaces

Rodney M. Donlan

Disclosures

Emerging Infectious Diseases. 2002;8(9) 

In This Article

Biofilm Structure

Biofilms are composed primarily of microbial cells and EPS. EPS may account for 50% to 90% of the total organic carbon of biofilms[38] and can be considered the primary matrix material of the biofilm. EPS may vary in chemical and physical properties, but it is primarily composed of polysaccharides. Some of these polysaccharides are neutral or polyanionic, as is the case for the EPS of gram-negative bacteria. The presence of uronic acids (such as D-glucuronic, D-galacturonic, and mannuronic acids) or ketal-linked pryruvates confers the anionic property.[39] This property is important because it allows association of divalent cations such as calcium and magnesium, which have been shown to cross-link with the polymer strands and provide greater binding force in a developed biofilm.[38] In the case of some gram-positive bacteria, such as the staphylococci, the chemical composition of EPS may be quite different and may be primarily cationic. Hussain et al.[40] found that the slime of coagulase-negative bacteria consists of a teichoic acid mixed with small quantities of proteins.

EPS is also highly hydrated because it can incorporate large amounts of water into its structure by hydrogen bonding. EPS may be hydrophobic, although most types of EPS are both hydrophilic and hydrophobic.[39] EPS may also vary in its solubility. Sutherland[39] noted two important properties of EPS that may have a marked effect on the biofilm. First, the composition and structure of the polysaccharides determine their primary conformation. For example, many bacterial EPS possess backbone structures that contain 1,3- or 1,4-β-linked hexose residues and tend to be more rigid, less deformable, and in certain cases poorly soluble or insoluble. Other EPS molecules may be readily soluble in water. Second, the EPS of biofilms is not generally uniform but may vary spatially and temporally. Leriche et al.[41] used the binding specificity of lectins to simple sugars to evaluate bacterial biofilm development by different organisms. These researchers' results showed that different organisms produce differing amounts of EPS and that the amount of EPS increases with age of the biofilm. EPS may associate with metal ions, divalent cations, other macromolecules (such as proteins, DNA, lipids, and even humic substances).[38] EPS production is known to be affected by nutrient status of the growth medium; excess available carbon and limitation of nitrogen, potassium, or phosphate promote EPS synthesis.[39] Slow bacterial growth will also enhance EPS production.[39] Because EPS is highly hydrated, it prevents desiccation in some natural biofilms. EPS may also contribute to the antimicrobial resistance properties of biofilms by impeding the mass transport of antibiotics through the biofilm, probably by binding directly to these agents.[42]

Tolker-Nielsen and Molin noted that every microbial biofilm community is unique[43] although some structural attributes can generally be considered universal. The term biofilm is in some ways a misnomer, since biofilms are not a continuous monolayer surface deposit. Rather, biofilms are very heterogeneous, containing microcolonies of bacterial cells encased in an EPS matrix and separated from other microcolonies by interstitial voids (water channels).[44] Figure 3 shows a biofilm of P. aeruginosa, Klebsiella pneumoniae, and Flavobacterium spp. that has developed on a steel surface in a laboratory potable water system. This image clearly depicts the water channels and heterogeneity characteristic of a mature biofilm. Liquid flow occurs in these water channels, allowing diffusion of nutrients, oxygen, and even antimicrobial agents. This concept of heterogeneity is descriptive not only for mixed culture biofilms (such as might be found in environmental biofilms) but also for pure culture biofilms common on medical devices and those associated with infectious diseases. Stoodley et al.[45] defined certain criteria or characteristics that could be considered descriptive of biofilms in general, including a thin base film, ranging from a patchy monolayer of cells to a film several layers thick containing water channels. The organisms composing the biofilm may also have a marked effect on the biofilm structure. For example, James et al.[46] showed that biofilm thickness could be affected by the number of component organisms. Pure cultures of either K. pneumoniae or P. aeruginosa biofilms in a laboratory reactor were thinner (15 µ and 30 µ, respectively), whereas a biofilm containing both species was thicker (40 µ). Jones et al. noted that this could be because one species enhanced the stability of the other.

Polymicrobic biofilm grown on a stainless steel surface in a laboratory potable water biofilm reactor for 14 days, then stained with 4,6-diamidino-2-phenylindole (DAPI) and examined by epifluorescence microscopy. Bar, 20 µm. Photograph by Ricardo Murga and Rodney Donlan, CDC.

Biofilm architecture is heterogeneous both in space and time, constantly changing because of external and internal processes. Tolker-Nielsen et al.[47] investigated the role of cell motility in biofilm architecture in flow cells by examining the interactions of P. aeruginosa and P. putida by confocal laser scanning microscopy. When these two organisms were added to the flow cell system, each organism initially formed small microcolonies. With time, the colonies intermixed, showing the migration of cells from one microcolony to the other. The microcolony structure changed from a compact structure to a looser structure over time, and when this occurred the cells inside the microcolonies were observed to be motile. Motile cells ultimately dispersed from the biofilm, resulting in dissolution of the microcolony.

Structure may also be influenced by the interaction of particles of nonmicrobial components from the host or environment. For example, erythrocytes and fibrin may accumulate as the biofilm forms. Biofilms on native heart valves provide a clear example of this type of interaction in which bacterial microcolonies of the biofilm develop in a matrix of platelets, fibrin, and EPS.[48] The fibrin capsule that develops will protect the organisms in these biofilms from the leukocytes of the host, leading to infective endocarditis. Biofilms on urinary catheters may contain organisms that have the ability to hydrolyze urea in the urine to form free ammonia through the action of urease. The ammonia may then raise the pH at the biofilm-liquid interface, resulting in the precipitation of minerals such as calcium phosphate (hydroxyapatite) and magnesium ammonium phosphate (struvite).[49] These minerals can then become entrapped in the biofilm and cause encrustation of the catheter; cases have been described in which the catheter became completely blocked by this mineral build-up. Minerals such as calcium carbonate, corrosion products such as iron oxides, and soil particles may often collect in biofilms of potable and industrial water systems, providing yet another example of particle interactions with biofilms.[50]

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