In nature, microorganisms grow mainly in biofilms, complex and dynamic ecosystems that form on a variety of environmental surfaces, from industrial conduits and water treatment pipelines to rocks in river beds. Biofilms are not restricted to solid surface substrates, however. Almost any surface in a liquid environment containing some minimal nutrients will eventually develop a biofilm. Microbial mats that float on water, for example, are biofilms that contain large populations of photosynthetic microorganisms. Biofilms found in the human mouth may contain hundreds of bacterial species. Regardless of the environment where they occur, biofilms are not random collections of microorganisms; rather, they are highly structured communities that provide a selective advantage to their constituent microorganisms.

Biofilm Structure

Observations using confocal microscopy have shown that environmental conditions influence the overall structure of biofilms. Filamentous biofilms called streamers form in rapidly flowing water, such as freshwater streams, eddies, and specially designed laboratory flow cells that replicate growth conditions in fast-moving fluids. The streamers are anchored to the substrate by a “head” and the “tail” floats downstream in the current. In still or slow-moving water, biofilms mainly assume a mushroom-like shape. The structure of biofilms may also change with other environmental conditions such as nutrient availability.

Detailed observations of biofilms under confocal laser and scanning electron microscopes reveal clusters of microorganisms embedded in a matrix interspersed with open water channels. The extracellular matrix consists of extracellular polymeric substances (EPS) secreted by the organisms in the biofilm. The extracellular matrix represents a large fraction of the biofilm, accounting for 50%–90% of the total dry mass. The properties of the EPS vary according to the resident organisms and environmental conditions.

EPS is a hydrated gel composed primarily of polysaccharides and containing other macromolecules such as proteins, nucleic acids, and lipids. It plays a key role in maintaining the integrity and function of the biofilm. Channels in the EPS allow movement of nutrients, waste, and gases throughout the biofilm. This keeps the cells hydrated, preventing desiccation. EPS also shelters organisms in the biofilm from predation by other microbes or cells (e.g., protozoans, white blood cells in the human body).

Biofilm Formation

Free-floating microbial cells that live in an aquatic environment are called planktonic cells. The formation of a biofilm essentially involves the attachment of planktonic cells to a substrate, where they become sessile (attached to a surface). This occurs in stages, as depicted in Figure. The first stage involves the attachment of planktonic cells to a surface coated with a conditioning film of organic material. At this point, attachment to the substrate is reversible, but as cells express new phenotypes that facilitate the formation of EPS, they transition from a planktonic to a sessile lifestyle. The biofilm develops characteristic structures, including an extensive matrix and water channels. Appendages such as fimbriae, pili, and flagella interact with the EPS, and microscopy and genetic analysis suggest that such structures are required for the establishment of a mature biofilm. In the last stage of the biofilm life cycle, cells on the periphery of the biofilm revert to a planktonic lifestyle, sloughing off the mature biofilm to colonize new sites. This stage is referred to as dispersal.

Within a biofilm, different species of microorganisms establish metabolic collaborations in which the waste product of one organism becomes the nutrient for another. For example, aerobic microorganisms consume oxygen, creating anaerobic regions that promote the growth of anaerobes. This occurs in many polymicrobial infections that involve both aerobic and anaerobic pathogens.

The mechanism by which cells in a biofilm coordinate their activities in response to environmental stimuli is called quorum sensing. Quorum sensing—which can occur between cells of different species within a biofilm—enables microorganisms to detect their cell density through the release and binding of small, diffusible molecules called autoinducers. When the cell population reaches a critical threshold (a quorum), these autoinducers initiate a cascade of reactions that activate genes associated with cellular functions that are beneficial only when the population reaches a critical density. For example, in some pathogens, synthesis of virulence factors only begins when enough cells are present to overwhelm the immune defenses of the host. Although mostly studied in bacterial populations, quorum sensing takes place between bacteria and eukaryotes and between eukaryotic cells such as the fungus Candida albicans, a common member of the human microbiota that can cause infections in immunocompromised individuals.

The signaling molecules in quorum sensing belong to two major classes. Gram-negative bacteria communicate mainly using N-acylated homoserine lactones, whereas gram-positive bacteria mostly use small peptides (Figure). In all cases, the first step in quorum sensing consists of the binding of the autoinducer to its specific receptor only when a threshold concentration of signaling molecules is reached. Once binding to the receptor takes place, a cascade of signaling events leads to changes in gene expression. The result is the activation of biological responses linked to quorum sensing, notably an increase in the production of signaling molecules themselves, hence the term autoinducer.

Biofilms and Human Health

The human body harbors many types of biofilms, some beneficial and some harmful. For example, the layers of normal microbiota lining the intestinal and respiratory mucosa play a role in warding off infections by pathogens. However, other biofilms in the body can have a detrimental effect on health. For example, the plaque that forms on teeth is a biofilm that can contribute to dental and periodontal disease. Biofilms can also form in wounds, sometimes causing serious infections that can spread. The bacterium Pseudomonas aeruginosa often colonizes biofilms in the airways of patients with cystic fibrosis, causing chronic and sometimes fatal infections of the lungs. Biofilms can also form on medical devices used in or on the body, causing infections in patients with in-dwelling catheters, artificial joints, or contact lenses.

Pathogens embedded within biofilms exhibit a higher resistance to antibiotics than their free-floating counterparts. Several hypotheses have been proposed to explain why. Cells in the deep layers of a biofilm are metabolically inactive and may be less susceptible to the action of antibiotics that disrupt metabolic activities. The EPS may also slow the diffusion of antibiotics and antiseptics, preventing them from reaching cells in the deeper layers of the biofilm. Phenotypic changes may also contribute to the increased resistance exhibited by bacterial cells in biofilms. For example, the increased production of efflux pumps, membrane-embedded proteins that actively extrude antibiotics out of bacterial cells, have been shown to be an important mechanism of antibiotic resistance among biofilm-associated bacteria. Finally, biofilms provide an ideal environment for the exchange of extrachromosomal DNA, which often includes genes that confer antibiotic resistance.

Recall that biofilms are microbial communities that are very difficult to destroy. They are responsible for diseases such as infections in patients with cystic fibrosis, Legionnaires’ disease, and otitis media. They produce dental plaque and colonize catheters, prostheses, transcutaneous and orthopedic devices, contact lenses, and internal devices such as pacemakers. They also form in open wounds and burned tissue. In healthcare environments, biofilms grow on hemodialysis machines, mechanical ventilators, shunts, and other medical equipment. In fact, 65 percent of all infections acquired in the hospital (nosocomial infections) are attributed to biofilms. Biofilms are also related to diseases contracted from food because they colonize the surfaces of vegetable leaves and meat, as well as food-processing equipment that isn’t adequately cleaned.

Biofilm infections develop gradually; sometimes, they do not cause symptoms immediately. They are rarely resolved by host defense mechanisms. Once an infection by a biofilm is established, it is very difficult to eradicate, because biofilms tend to be resistant to most of the methods used to control microbial growth, including antibiotics. Biofilms respond poorly or only temporarily to antibiotics; it has been said that they can resist up to 1,000 times the antibiotic concentrations used to kill the same bacteria when they are free-living or planktonic. An antibiotic dose that large would harm the patient; therefore, scientists are working on new ways to get rid of biofilms.

When treating bacterial infections of the skin and eyes, it is important to consider that few such infections can be attributed to a single pathogen. While biofilms may develop in other parts of the body, they are especially relevant to skin infections (such as those caused by S. aureus or P. aeruginosa) because of their prevalence in chronic skin wounds. Biofilms develop when bacteria (and sometimes fungi) attach to a surface and produce extracellular polymeric substances (EPS) in which cells of multiple organisms may be embedded. When a biofilm develops on a wound, it may interfere with the natural healing process as well as diagnosis and treatment.

Because biofilms vary in composition and are difficult to replicate in the lab, they are still not thoroughly understood. The extracellular matrix of a biofilm consists of polymers such as polysaccharides, extracellular DNA, proteins, and lipids, but the exact makeup varies. The organisms living within the extracellular matrix may include familiar pathogens as well as other bacteria that do not grow well in cultures (such as numerous obligate anaerobes). This presents challenges when culturing samples from infections that involve a biofilm. Because only some species grow in vitro, the culture may contain only a subset of the bacterial species involved in the infection.

Biofilms confer many advantages to the resident bacteria. For example, biofilms can facilitate attachment to surfaces on or in the host organism (such as wounds), inhibit phagocytosis, prevent the invasion of neutrophils, and sequester host antibodies. Additionally, biofilms can provide a level of antibiotic resistance not found in the isolated cells and colonies that are typical of laboratory cultures. The extracellular matrix provides a physical barrier to antibiotics, shielding the target cells from exposure. Moreover, cells within a biofilm may differentiate to create subpopulations of dormant cells called persister cells. Nutrient limitations deep within a biofilm add another level of resistance, as stress responses can slow metabolism and increase drug resistance.

Using Microscopy to Study Biofilms

A biofilm is a complex community of one or more microorganism species, typically forming as a slimy coating attached to a surface because of the production of an extrapolymeric substance (EPS) that attaches to a surface or at the interface between surfaces (e.g., between air and water). In nature, biofilms are abundant and frequently occupy complex niches within ecosystems (Figure 2.25). In medicine, biofilms can coat medical devices and exist within the body. Because they possess unique characteristics, such as increased resistance against the immune system and to antimicrobial drugs, biofilms are of particular interest to microbiologists and clinicians alike.

Because biofilms are thick, they cannot be observed very well using light microscopy; slicing a biofilm to create a thinner specimen might kill or disturb the microbial community. Confocal microscopy provides clearer images of biofilms because it can focus on one z-plane at a time and produce a three-dimensional image of a thick specimen. Fluorescent dyes can be helpful in identifying cells within the matrix. Additionally, techniques such as immunofluorescence and fluorescence in situ hybridization (FISH), in which fluorescent probes are used to bind to DNA, can be used.

Electron microscopy can be used to observe biofilms, but only after dehydrating the specimen, which produces undesirable artifacts and distorts the specimen. In addition to these approaches, it is possible to follow water currents through the shapes (such as cones and mushrooms) of biofilms, using video of the movement of fluorescently coated beads (Figure 2.26).

Figure 2.25 A biofilm forms when planktonic (free-floating) bacteria of one or more species adhere to a surface, produce slime, and form a colony. (credit: Public Library of Science)

Figure 2.26 In this image, multiple species of bacteria grow in a biofilm on stainless steel (stained with DAPI for epifluorescence miscroscopy). (credit: Ricardo Murga, Rodney Donlan)

Do you know what’s in your mouth? It’s home to about 700 species of microbes. These include germs like bacteria, fungus, and more.

“Everybody has these microbes in their mouth,” says Dr. Robert Palmer, an NIH expert on oral microbes.

Some microbes are helpful. Others can cause problems like tooth decay and gum disease. Troubles begin when microbes form a sticky, colorless film called plaque on your teeth.

Brushing and flossing help to keep your mouth clean. But after you brush and floss, germs grow again and more plaque forms. That’s why you need to clean your mouth regularly.

Community Growth

Different microbes grow in different places. Some stick to your teeth. Others prefer your tongue. Some lurk in the tiny pockets between tooth and gum. Once they’ve found their homes, they form diverse communities with the other germs.

Mouth microbes work together to protect themselves with a slimy, sticky material called a matrix. The matrix in plaque makes it harder to remove it.

The communities within the matrix include both helpful and disease-causing microbes. The good microbes help keep the growth of bad microbes in check. Good microbes also help you digest food and can protect against harmful microbes in food.

Certain things you may be doing can help bad microbes grow better than the good ones. Sugary foods and drinks feed some microbes and help them increase in number and spread out.

Some of these sugar-loving microbes can turn sugar into matrix and acid. The acid destroys the surface of your teeth. The more sugar in your diet, the more fuel is available for these microbes to build up plaque and damage teeth.

“It’s more productive to think about the community than it is to think about the single microbe that causes disease,” Palmer explains.

You can’t stop tooth decay by getting rid of just one type of acid-making microbe. There are several different types of microbes in the plaque that make acid.

The good news is that limiting sweets and brushing and flossing regularly can help prevent bad microbes from growing out of control.

Helpful Neighbors

“Many bacteria in our mouths depend on help from other members of their community to survive and prosper,” says Dr. Floyd Dewhirst, a dental expert who studies microbes at the Forsyth Institute.

Because microbes grow in communities, it’s important to understand how both helpful and harmful microbes work. Dewhirst’s team is trying to identify all the different germs living in the mouth and what they do.

Before the team can study a microbe, they have to figure out how to grow it. The challenge is that some microbes don’t like to grow anywhere but in your mouth. About 30% of the 700 species haven’t been grown in the lab yet.

Dewhirst’s team is working on growing those microbes in the lab that no one has grown before. They’re using genetic and other information to identify each one and learn more about them.

“The question is,” he says, “once you know who is there and have a quick way of identifying them, what are all of these bacteria doing?”

Dewhirst’s studies have shown that some microbes make certain substances that help their neighbors grow. His team is trying to identify what those substances are.

They also want to find out how these microbes may affect people’s health. Being able to grow microbes in the lab lets scientists run tests to figure out how they’re involved in health and disease. This information could one day help scientists come up with better ways of preventing and treating oral diseases.

Partners in Decay

An important health problem caused by mouth microbes is early childhood tooth decay. “In the U.S., about 23% of our children between the ages of 1 and 5 are affected by this disease,” says Dr. Hyun (Michel) Koo, a dental researcher and oral health expert at the University of Pennsylvania.

Tooth decay can get worse very fast. The microbe matrix and acid from bacteria are thought to be the main cause of tooth decay in young kids.

Koo’s team has found that there’s also fungus in the plaque of kids with rampant tooth decay. The fungus partners with the matrix- and acid-making bacteria to worsen tooth decay.

“Bacteria by itself can cause tooth decay,” Koo explains. “But when fungus is there, it boosts up the entire machinery.”

Koo’s team has shown that some fungus can get energy from sugar that bacteria release while making acid. The fungus then releases substances that feed the bacteria’s growth. This helps the bacteria form an even tougher matrix and make more acid.

Busting Plaque

Koo’s team is looking for new ways to fight plaque buildup and tooth decay. They’ve developed tiny substances, called nanoparticles, that are small enough to get inside and destroy the matrix that protects microbes. The nanoparticles can also kill the acid-making bacteria without harming good bacteria in the mouth.

Koo’s team has shown that these tiny substances can reduce acid damage to the tooth surface. The researchers hope to test the approach in people in the future.

Nanoparticles are just one approach now being studied to prevent or treat mouth diseases.

Future technologies may help keep our mouths healthier. But there are many things you can do to keep bad mouth microbes in check now. See the Wise Choices box for some tips. You can’t have a healthy body without a healthy mouth.

Keep Mouth Microbes in Check

These tips can help prevent tooth decay and mouth infections:

• Brush your teeth with a toothpaste that contains fluoride.
• Floss away the plaque between your teeth.
• Don’t forget to brush your tongue.
• Limit sugary foods and drinks.
• Drink fluoridated water. Learn about fluoride levels in your water (CDC).
• Get regular dental exams and professional cleanings.

As noted earlier, normal oral microbiota can cause dental and periodontal infections. However, there are number of other infections that can manifest in the oral cavity when other microbes are present.

Herpetic Gingivostomatitis

As described in Viral Infections of the Skin and Eyes, infections by herpes simplex virus type 1 (HSV-1) frequently manifest as oral herpes, also called acute herpes labialis and characterized by cold sores on the lips, mouth, or gums. HSV-1 can also cause acute herpetic gingivostomatitis, a condition that results in ulcers of the mucous membranes inside the mouth (Figure). Herpetic gingivostomatitis is normally self-limiting except in immunocompromised patients. Like oral herpes, the infection is generally diagnosed through clinical examination, but cultures or biopsies may be obtained if other signs or symptoms suggest the possibility of a different causative agent. If treatment is needed, mouthwashes or antiviral medications such as acyclovir, famciclovir, or valacyclovir may be used.

(a) This cold sore is caused by infection with herpes simplex virus type 1 (HSV-1). (b) HSV-1 can also cause acute herpetic gingivostomatitis. (credit b: modification of work by Klaus D. Peter)

Oral Thrush

The yeast Candida is part of the normal human microbiota, but overgrowths, especially of Candida albicans, can lead to infections in several parts of the body. When Candida infection develops in the oral cavity, it is called oral thrush. Oral thrush is most common in infants because they do not yet have well developed immune systems and have not acquired the robust normal microbiota that keeps Candida in check in adults. Oral thrush is also common in immunodeficient patients and is a common infection in patients with AIDS.

Oral thrush is characterized by the appearance of white patches and pseudomembranes in the mouth (Figure) and can be associated with bleeding. The infection may be treated topically with nystatin or clotrimazole oral suspensions, although systemic treatment is sometimes needed. In serious cases, systemic azoles such as fluconazole or itraconazole (for strains resistant to fluconazole), may be used. Amphotericin B can also be used if the infection is severe or if the Candida species is azole-resistant.

Overgrowth of Candida in the mouth is called thrush. It often appears as white patches. (credit: modification of work by Centers for Disease Control and Prevention)

Mumps

The viral disease mumps is an infection of the parotid glands, the largest of the three pairs of salivary glands (Figure). The causative agent is mumps virus (MuV), a paramyxovirus with an envelope that has hemagglutinin and neuraminidase spikes. A fusion protein located on the surface of the envelope helps to fuse the viral envelope to the host cell plasma membrane.

Mumps virus is transmitted through respiratory droplets or through contact with contaminated saliva, making it quite contagious so that it can lead easily to epidemics. It causes fever, muscle pain, headache, pain with chewing, loss of appetite, fatigue, and weakness. There is swelling of the salivary glands and associated pain (Figure). The virus can enter the bloodstream (viremia), allowing it to spread to the organs and the central nervous system. The infection ranges from subclinical cases to cases with serious complications, such as encephalitis, meningitis, and deafness. Inflammation of the pancreas, testes, ovaries, and breasts may also occur and cause permanent damage to those organs; despite these complications, a mumps infection rarely cause sterility.

Mumps can be recognized based on clinical signs and symptoms, and a diagnosis can be confirmed with laboratory testing. The virus can be identified using culture or molecular techniques such as RT-PCR. Serologic tests are also available, especially enzyme immunoassays that detect antibodies. There is no specific treatment for mumps, so supportive therapies are used. The most effective way to avoid infection is through vaccination. Although mumps used to be a common childhood disease, it is now rare in the United States due to vaccination with the measles, mumps, and rubella (MMR) vaccine.

This child shows the characteristic parotid swelling associated with mumps. (credit: modification of work by Centers for Disease Control and Prevention)

Give them a suitable surface, some water and nutrients, and bacteria will likely put down stakes and form biofilms. Biofilms are communities made up of many individual bacterial cells held together and stuck to surfaces by a kind of biological glue. These sticky, slimy microbial metropolises wreak havoc when they clog medical devices implanted in the body, such as stents and catheters.

A recent study funded in part by the National Institutes of Health revealed how biofilms cause such clogs: The bacteria form streamers that tangle with each other and trap other passing bacteria, creating a full blockage in a surprisingly short period of time.

Researchers at Princeton University used time-lapse microscopy imaging to monitor fluid flow and biofilm formation in curvy, narrow tubes. Unlike many previous studies, these experiments more closely mimicked real-life conditions, using rough rather than smooth surfaces and pressure-driven flow rather than nonmoving or uniformly flowing fluid.

The researchers tagged bacteria known to be common contaminants of medical devices with a green fluorescent dye. As the bacterial cells flowed through the microscopic channel, they attached to the inner wall, where they began to multiply and form a biofilm (seen in green in the movie above). The buildup of this biofilm only modestly affected the rate of fluid flow through the channel.

Forty-three hours into the experiment, the researchers began flowing bacteria tagged with a red fluorescent dye into the channel. They found that, after about 50 hours total, bacteria on the walls of the channel shed some of the sticky substance that holds them together, creating thin streamers that rippled in the liquid and that eventually tangled to form a sievelike mesh in the channel. Some of the red-labeled bacterial cells got stuck in the mesh, creating an even-larger web of biofilm streamers (seen in red in the movie) that ensnared more cells. Within an hour after the streamers first formed, the entire tube became blocked and the fluid flow stopped.

Additional experiments revealed that some of the genes previously shown to be required for biofilm formation on smooth surfaces were not needed to form biofilm streamers in the flow system used in this study, underscoring the need to study biofilms in realistic environments. The studies also showed that biofilm streamers form in stents, water filters and porous materials similar to those used in wastewater treatment plants, suggesting that the streamers are the likely explanation for why biofilms block up these materials.

Together, these findings could help shape strategies to prevent clogging of medical devices and other materials that are prone to bacterial contamination.

By Elia Ben-Ari