What makes up biofilm

Cations have been shown to cross-link the anionic groups of polymers such as polysaccharides , resulting in contraction. Beech and Gaylarde 31 found that lectins inhibited but did not prevent attachment. Glucosidase and N-acetylglucosaminidase reduced attachment for P. Lectins preferentially bind to polysaccharides on the cell surface or to the EPS.

Binding of lectins by the cells would minimize the attachment sites and affect cell attachment if polysaccharides were involved in attachment. Zottola 32 confirmed the role of polysaccharides in attachment in studies with Pseudomonas fragi. Korber et al. Nonmotile strains also do not recolonize or seed vacant areas on a substratum as evenly as motile strains, resulting in slower biofilm formation by the nonmotile organisms.

Flagella apparently play an important role in attachment in the early stages of bacterial attachment by overcoming the repulsive forces associated with the substratum. In light of these findings, cell surface structures such as fimbriae, other proteins, LPS, EPS, and flagella all clearly play an important role in the attachment process.

Cell surface polymers with nonpolar sites such as fimbriae, other proteins, and components of certain gram-positive bacteria mycolic acids appear to dominate attachment to hydrophobic substrata, while EPS and lipopolysaccharides are more important in attachment to hydrophilic materials. Flagella are important in attachment also, although their role may be to overcome repulsive forces rather than to act as adsorbents or adhesives. The attachment of microorganisms to surfaces is a very complex process, with many variables affecting the outcome.

An increase in flow velocity, water temperature, or nutrient concentration may also equate to increased attachment, if these factors do not exceed critical levels. Properties of the cell surface, specifically the presence of fimbriae, flagella, and surface-associated polysaccharides or proteins, also are important and may possibly provide a competitive advantage for one organism where a mixed community is involved.

Table 1 summarizes the variables important in cell attachment and biofilm formation. Evidence is mounting that up- and down-regulation of a number of genes occurs in the attaching cells upon initial interaction with the substratum.

Davies and Geesey 34 demonstrated algC up-regulation in individual bacterial cells within minutes of attachment to surfaces in a flow cell system. This phenomenon is not limited to P. Prigent-Combaret et al.

Becker et al. A recent study by Pulcini 37 also showed that algD , algU, rpoS, and genes controlling polyphosphokinase PPK synthesis were up-regulated in biofilm formation of P. Biofilms are composed primarily of microbial cells and EPS. 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 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 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. EPS is also highly hydrated because it can incorporate large amounts of water into its structure by hydrogen bonding. 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. 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. EPS may associate with metal ions, divalent cations, other macromolecules such as proteins, DNA, lipids, and even humic substances 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 Slow bacterial growth will also enhance EPS production 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 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 Figure 3 shows a biofilm of P.

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. The organisms composing the biofilm may also have a marked effect on the biofilm structure.

For example, James et al. Pure cultures of either K. Polymicrobic biofilm grown on a stainless steel surface in a laboratory potable water biofilm reactor for 14 days, then stained with 4,6-diamidinophenylindole DAPI and examined by epifluorescence microscopy. Biofilm architecture is heterogeneous both in space and time, constantly changing because of external and internal processes.

Tolker-Nielsen et al. 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 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 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 The basic structural unit of the biofilm is the microcolony. Proximity of cells within the microcolony or between microcolonies Figure 4A and B provides an ideal environment for creation of nutrient gradients, exchange of genes, and quorum sensing.

Since microcolonies may be composed of multiple species, the cycling of various nutrients e. Polymicrobic biofilms grown on stainless steel surfaces in a laboratory potable water biofilm reactor for 7 days, then stained with 4,6-diamidinophenylindole DAPI and examined by epifluorescence microscopy.

Biofilms also provide an ideal niche for the exchange of extrachromosomal DNA plasmids. Conjugation the mechanism of plasmid transfer occurs at a greater rate between cells in biofilms than between planktonic cells 51 — Ghigo 54 has suggested that medically relevant strains of bacteria that contain conjugative plasmids more readily develop biofilms. He showed that the F conjugative pilus encoded by the tra operon of the F plasmid acts as an adhesion factor for both cell-surface and cell-cell interactions, resulting in a three-dimensional biofilm of Escherichia coli.

Plasmid-carrying strains have also been shown to transfer plasmids to recipient organisms, resulting in biofilm formation; without plasmids these same organisms produce only microcolonies without any further development. The probable reason for enhanced conjugation is that the biofilm environment provides minimal shear and closer cell-to-cell contact.

Since plasmids may encode for resistance to multiple antimicrobial agents, biofilm association also provides a mechanism for selecting for, and promoting the spread of, bacterial resistance to antimicrobial agents. Cell-to-cell signaling has recently been demonstrated to play a role in cell attachment and detachment from biofilms. Xie et al. Davies et al. At sufficient population densities, these signals reach concentrations required for activation of genes involved in biofilm differentiation.

Mutants unable to produce both signals double mutant were able to produce a biofilm, but unlike the wild type, their biofilms were much thinner, cells were more densely packed, and the typical biofilm architecture was lacking.

In addition, these mutant biofilms were much more easily removed from surfaces by a surfactant treatment. Addition of homoserine lactone to the medium containing the mutant biofilms resulted in biofilms similar to the wild type with respect to structure and thickness. Stickler et al. Yung-Hua et al. Transformational frequencies were 10— times higher in biofilms than planktonic cells. Bacteria within biofilms may be subject to predation by free-living protozoa, Bdellovibrio spp.

Murga et al. Predation has also been demonstrated with Acanthamoeba spp. James et al. Stewart et al. Apparently P. Several frank bacterial pathogens have been shown to associate with, and in some cases, actually grow in biofilms, including Legionella pneumophila 59 , S. Although all these organisms have the ability to attach to surfaces and existing biofilms, most if not all appear incapable of extensive growth in the biofilm.

This may be because of their fastidious growth requirements or because of their inability to compete with indigenous organisms. The mechanism of interaction and growth apparently varies with the pathogen, and at least for L. Survival and growth of pathogenic organisms within biofilms might also be enhanced by the association and metabolic interactions with indigenous organisms.

Camper et al. The picture of biofilms increasingly is one in which there is both heterogeneity and a constant flux, as this biological community adapts to changing environmental conditions and the composition of the community.

Biofilm cells may be dispersed either by shedding of daughter cells from actively growing cells, detachment as a result of nutrient levels or quorum sensing, or shearing of biofilm aggregates continuous removal of small portions of the biofilm because of flow effects.

The mechanisms underlying the process of shedding by actively growing cells in a biofilm are not well understood. Gilbert et al. Hydrophobicity was lowest for the newly dispersed cells and steadily increases upon continued incubation and growth.

Alginate is the major component of the EPS of P. Boyd and Chakrabarty 70 studied alginate lyase production in P. Inducing alginate lyase expression substantially decreased the amount of alginate produced, which corresponded with a significant increase in the number of detached cells.

The authors suggested that the role of algL the gene cassette for alginate lyase production in wild type P. Polysaccharidase enzymes specific for the EPS of different organisms may possibly be produced during different phases of biofilm growth of these organisms.

Detachment caused by physical forces has been studied in greater detail. A biofilm forms when certain microorganisms for example, some types of bacteria adhere to the surface of some object in a moist environment and begin to reproduce. The microorganisms form an attachment to the surface of the object by secreting a slimy, glue-like substance. Biofilms can form on just about any imaginable surface: metals, plastics, natural materials such as rocks , medical implants, kitchen counters, contact lenses, the walls of a hot tub or swimming pool did you ever notice that the sides of a hot tub or swimming pool seemed slightly slimy?

Indeed, wherever the combination of moisture, nutrients, and a surface exists, biofilms will likely be found as well. A biofilm community can be formed by a single kind of microorganism, but in nature biofilms almost always consist of mixtures of many species of bacteria, as well as fungi, algae, yeasts, protozoa, and other microorganisms, along with non-living debris and corrosion products. For example, over bacterial species have been identified in typical dental plaque biofilms!

Biofilms can be so thin as to avoid detection by the naked eye—just a few cell layers thick. The biofilms that almost certainly exist on your kitchen counter, for instance, are generally undetectable to the eye unless, like some college students, you don't wash your counters very often. They can also grow to become many inches thick; probably not on a countertop at least we hope not , but certainly as algae on rocks in a streambed.

Engineers and scientists have discovered that biofilms are held together by sugary molecular strands, collectively termed "extracellular polymeric substances" a mouthful of a term that essentially means "compounds or substances that form outside the walls of cells" or "EPS.

Free-floating, or planktonic , bacteria encounter a submerged surface and within minutes can become attached. They begin to produce slimy extracellular polymeric substances EPS and to colonize the surface. EPS production allows the emerging biofilm community to develop a complex, three-dimensional structure that is influenced by a variety of environmental factors.

Biofilm communities can develop within hours. Biofilms can propagate through detachment of small or large clumps of cells, or by a type of "seeding dispersal" that releases individual cells. Either type of detachment allows bacteria to attach to a surface or to a biofilm downstream of the original community. Now here we get to the crux of the biofilms issue.

Read this little section carefully, because when you get the point here, you will understand why the study of biofilms is so radical and important, and the rest of this hypertextbook will make sense to you.

You see, the effective treatment i. That's right. Harmful microorganisms were studied and still are, unfortunately, to a large extent in isolation, not as members of a biofilm colony, where they actually normally reside. Let's discuss this by way of an example.

Sitting still takes less energy, and bacteria that sit in the right spot can wait for food to come to them. Sitting still is the first step in making a bacterial biofilm Figure 2. The first bacterium that sits still might be joined by others, or it might reproduce and make many more bacteria that are copies of itself.

When more and more bacteria get together, they start to make sticky substances called extracellular polymeric substances , that they cover themselves with. This sticky community of bacteria is called a biofilm. The bacteria in a biofilm live happily eating whatever food comes along and can communicate with each other by releasing special molecules. Biofilms are very common [ 2 ]. The icky gunk in the bathroom drain is a biofilm. Scum covering a rock in a river is a biofilm. The mossy feeling your teeth get when you have not brushed them in a while?

That is a biofilm on your teeth! Biofilms form on any surface that is wet and that has food for bacteria to eat. The human body makes special cells that find and destroy bacteria.

These special cells, as well as antibiotics prescribed by a doctor to fight an infection, are very good at fighting bacteria that are swimming around inside your body. If these bacteria are exposed to an antibiotic for long enough, the bacteria will die, and you will be cured of the infection.

On the other hand, bacteria living in a biofilm cannot be killed by antibiotics. These bacteria are safe inside their protective biofilm. When the antibiotic comes along, it gets caught up in the sticky extracellular polymeric substances, and does not even reach the bacteria! Some bacteria on the outside of the biofilm may be killed by the antibiotic, but the bacteria on the inside of the biofilm are safe.

The bacteria inside the biofilm are not all exactly the same, even if they are all copies of the first bacterium to settle down.

This means, even if antibiotic gets inside a biofilm, it does not kill all the bacteria. In fact, it would take times more antibiotic to kill all the bacteria in a biofilm community than is needed to kill bacteria while they are swimming on their own [ 3 ]. That would be so much antibiotic that it would kill the patient along with the bacterial infection!

So, if antibiotics do not work, what can we do about chronic infections? The best way to get rid of a biofilm is to scrub it off.

That is why we like to brush our teeth so much!