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14.3C: Biofilms and Infections - Biology

14.3C: Biofilms and Infections - Biology



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Biofilms will form on virtually every non-shedding surface in a non-sterile aqueous (or very humid) environment.

Learning Objectives

  • Discuss the importance of biofilms in the biomedical community

Key Points

  • Biofilms have been found to be involved in a wide variety of microbial infections in the body.
  • Microbes form a biofilm in response to many factors, which may include cellular recognition of specific or non-specific attachment sites on a surface and nutritional cues.
  • Bacterial biofilms may impair cutaneous wound healing and reduce topical antibacterial efficiency in healing or treating infected skin wounds.

Key Terms

  • biofilm: A thin film of mucus created by and containing a colony of bacteria and other microorganisms.
  • sterile: unable to reproduce (or procreate)

A biofilm is an aggregate of microorganisms in which cells adhere to each other on a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS).

Microbes form a biofilm in response to many factors, which may include cellular recognition of specific or non-specific attachment sites on a surface, nutritional cues, or in some cases, by exposure of planktonic cells to sub-inhibitory concentrations of antibiotics. When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in behavior in which large suites of genes are differentially regulated.

Biofilms are ubiquitous. Nearly every species of microorganism, not only bacteria and archaea, have mechanisms by which they can adhere to surfaces and to each other. Biofilms will form on virtually every non-shedding surface in a non-sterile aqueous (or very humid) environment.

Biofilms have been found to be involved in a wide variety of microbial infections in the body, by one estimate in 80% of all infections. Infectious processes in which biofilms have been implicated include common problems such as urinary tract infections, catheter infections, middle-ear infections, formation of dental plaque, gingivitis, and coating contact lenses. Biofilms have also been implicated in less common but more lethal processes such as endocarditis, infections in cystic fibrosis, and infections of permanent indwelling devices such as joint prostheses and heart valves.

More recently it has been noted that bacterial biofilms may impair cutaneous wound healing and reduce topical antibacterial efficiency in healing or treating infected skin wounds. It has recently been shown that biofilms are present on the removed tissue of 80% of patients undergoing surgery for chronic sinusitis. The patients with biofilms were shown to have been denuded of cilia and goblet cells, unlike the controls without biofilms who had normal cilia and goblet cell morphology. Biofilms were also found on samples from two of 10 healthy controls mentioned. The species of bacteria from interoperative cultures did not correspond to the bacteria species in the biofilm on the respective patient’s tissue. In other words, the cultures were negative though the bacteria were present.

Biofilms can also be formed on the inert surfaces of implanted devices such as catheters, prosthetic cardiac valves, and intrauterine devices. New staining techniques are being developed to differentiate bacterial cells growing in living animals, e.g. from tissues with allergy-inflammations.

Pseudomonas aeruginosa biofilms

The achievements of medical care in industrialized societies are markedly impaired due to chronic opportunistic infections that have become increasingly apparent in immunocompromised patients and the aging population. Chronic infections remain a major challenge for the medical profession and are of great economic relevance because traditional antibiotic therapy is usually not sufficient to eradicate these infections.

Pseudomonas aeruginosa is not only an important opportunistic pathogen and causative agent of emerging nosocomial infections but can also be considered a model organism for the study of diverse bacterial mechanisms that contribute to bacterial persistence. In this context the elucidation of the molecular mechanisms responsible for the switch from planktonic growth to a biofilm phenotype and the role of inter-bacterial communication in persistent disease should provide new insights. It should help researchers learn about the pathogenicity of P. aeruginosa, contribute to a better clinical management of chronically infected patients, and lead to the identification of new drug targets for the development of alternative anti-infective treatment strategies.

Dental plaque

Dental plaque is a biofilm that adheres to teeth surfaces and consists of bacterial cells, salivary polymers, and bacterial extracellular products. This accumulation of microorganisms subject the teeth and gingival tissues to high concentrations of bacterial metabolites which results in dental disease. The biofilms attached to the surfaces of some dental alloys, impression materials, dental implants, restorative and cement materials play an essential role concerning the biofilms establishment dynamics toward the physical-chemical properties of the materials which biofilms are attached to.

Legionellosis

Legionella bacteria are known to grow under certain conditions in biofilms, in which they are protected against disinfectants. Workers in cooling towers, persons working in air conditioned rooms, and people taking a shower are exposed to Legionella by inhalation when the systems are not well designed, constructed, or maintained. Neisseria gonorrhoeae is an exclusive human pathogen. Recent studies have demonstrated that it utilizes two distinct mechanisms for entry into human urethral and cervical epithelial cells involving different bacterial surface ligands and host receptors. In addition, it has been demonstrated that the gonococcus can form biofilms on glass surfaces and over human cells. There is evidence for the formation of gonococcal biofilms on human cervical epithelial cells during natural disease. Evidence also suggests that the outer membrane blebbing by the gonococcus is crucial in biofilm formation over human cervical epithelial cells.


Biofilm

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Biofilm, aggregate of bacteria held together by a mucuslike matrix of carbohydrate that adheres to a surface. Biofilms can form on the surfaces of liquids, solids, and living tissues, such as those of animals and plants. Organisms in biofilms often display substantially different properties from the same organism in the individual, or free-living (planktonic), state. Communities form when individual organisms, which may be of the same or different species, adhere to and accumulate on a surface this process is called adsorption. Following a period of growth and reproduction, the organisms produce an extracellular matrix consisting of carbohydrates called polysaccharides. This matrix serves to hold the bacteria together and to irreversibly bind them to the surface.

Bacteria that have aggregated into biofilms can communicate information about population size and metabolic state. This type of communication is called quorum sensing and operates by the production of small molecules called autoinducers, or pheromones. The concentration of quorum-sensing molecules—most commonly peptides or acylated homoserine lactones (AHLs special signaling chemicals)—is related to the number of bacteria of the same or different species that are in the biofilm and helps coordinate the behaviour of the biofilm.

Biofilms are advantageous to bacteria because they provide a nutrient-rich environment that facilitates growth and because they confer resistance to antibiotics. Biofilms can cause severe infections in hospitalized patients the formation of biofilms in these instances is typically associated with the introduction into the body of foreign substrates, such as artificial implants and urinary catheters. Biofilms also form on the thin films of plaque found on teeth, where they ferment sugars and starches into acids, causing the destruction of tooth enamel. In the environment, biofilms fill an important role in the breakdown of organic wastes by filtering wastes from water and by removing or neutralizing contaminants in soil. As a result, biofilms are used to purify water in water treatment plants and to detoxify contaminated areas of the environment.

This article was most recently revised and updated by Robert Lewis, Assistant Editor.


How fatal biofilms form

By severely curtailing the effects of antibiotics, the formation of organized communities of bacterial cells known as biofilms can be deadly during surgeries and in urinary tract infections. Yale researchers have just come a lot closer to understanding how these biofilms develop, and potentially how to stop them.

Biofilms form when bacterial cells gather and develop structures that bond them in a gooey substance. This glue can protect the cells from the outside world and allow them to form complex quasi-organisms. Biofilms can be found almost everywhere, including unwashed shower stalls or the surfaces of lakes. Because the protective shell can keep out potential treatments, biofilms are at their most dangerous when they invade human cells or form on sutures and catheters used in surgeries. In American hospitals alone, thousands of deaths are attributed to biofilm-related surgical site infections and urinary tract infections.

"Biofilms are a huge medical problem because they are something that makes bacterial infections very difficult to deal with," said Andre Levchenko, senior author of the study, which was published Oct. 5 in Nature Communications.

Fighting biofilms has been particularly difficult because it hasn't been well understood how bacteria cells make the transition from behaving individually to existing in collective structures. However, the researchers in the Levchenko lab, working with colleagues at the University of California-San Diego, recently found a key mechanism for biofilm formation that also provides a way to study this process in a controlled and reproducible way.

The investigators designed and built microfluidic devices and novel gels that housed uropathogenic E. coli cells, which are often the cause of urinary tract infections. These devices mimicked the environment inside human cells that host the invading bacteria during infections. The scientists found that the bacterial colonies would grow to the point where they would be squeezed by either the walls of the chamber, the fibers, or the gel. This self-generated stress was itself a trigger of the biofilm formation.

"This was very surprising, but we saw all the things you would expect from a biofilm," said Levchenko, the John C. Malone Professor of Biomedical Engineering and director of the Yale Systems Biology Institute. "The cells produced the biofilm components and suddenly became very antibiotic-resistant. And all of that was accompanied by an indication that the cells were under biological stress and the stress was coming from this mechanical interaction with the environment."

With this discovery, Levchenko said, researchers can use various devices that mimic other cellular environments and explore biofilm formation under countless environments and circumstances. They can also use the devices introduced in this study to produce biofilms rapidly, precisely, and in high numbers in a simple, inexpensive, and reproducible way. This would allow screening drugs that could potentially breach the protective layer of the biofilms and break it down.

"Having a disease model like this is a must when you want to do these kinds of drug-screening experiments," he said. "We can now grow biofilms in specific shapes and specific locations in a completely predictable way."


Attacking biofilms that cause chronic infections

A clever new imaging technique discovered at the University of California, Berkeley, reveals a possible plan of attack for many bacterial diseases, such as cholera, lung infections in cystic fibrosis patients and even chronic sinusitis, that form biofilms that make them resistant to antibiotics.

By devising a new fluorescent labeling strategy and employing super-resolution light microscopy, the researchers were able to examine the structure of sticky plaques called bacterial biofilms that make these infections so tenacious. They also identified genetic targets for potential drugs that could break up the bacterial community and expose the bugs to the killing power of antibiotics.

"Eventually, we want to make these bugs homeless," said lead researcher Veysel Berk, a postdoctoral fellow in the Department of Physics and the California Institute for Quantitative Biosciences (QB3) at UC Berkeley.

Berk and his co-authors, including Nobel laureate and former UC Berkeley professor Steven Chu, report their findings in the July 13 issue of the journal Science.

"In their natural habitat, 99.9 percent of all bacteria live as a community and attach to surfaces as biofilms according to the National Institutes of Health, 80 percent of all infections in humans are related to biofilms," Berk said.

The researchers were able to employ new techniques that allowed them to zoom into a street-level view of these biofilms, where they learned "how they grow from a single cell and come together to form rooms and whole buildings," Berk said. "Now, we can come up with a logical approach to discovering how to take down their building, or prevent them from forming the building itself."

Combining super-resolution microscopy with the technique Berk developed, which allows continuous labeling of growing and dividing cells in culture, biologists in many fields will be able to record stop-motion video of "how bacteria build their castles," he said.

"This work has led to new insights into the development of these complex structures and will no doubt pave the way to new approaches to fighting infectious disease and also bacteriological applications in environmental and industrial settings," said Chu, a former UC Berkeley professor of physics and of molecular and cell biology and former director of the Lawrence Berkeley National Laboratory.

Bacteria are not loners

The popular view of bacteria is that they are free-living organisms easily kept in check by antibiotics, Berk said. But scientists now realize that bacteria spend most of their lives in colonies or biofilms, even in the human body. While single bacteria may be susceptible to antibiotics, the films can be 1,000 times more resistant and most can only be removed surgically.

Implants, such as pacemakers, stents and artificial joints, occasionally become infected by bacteria that form biofilms. These biofilm sites periodically shed bacteria -- adventurers, Berk calls them -- which can ignite acute infections and fever. While antibiotics can knock out these free-swimming bacteria and temporally calm down the infection, the biofilm remains untouched.The only permanent solution is removal of the biofilm-coated device and replacement with a new sterilized implant.

A permanent bacterial biofilm in the sinuses can ignite an immune response leading to chronic sinus infections, with symptoms including fever and cold-like symptoms. So far, the most effective treatment is to surgically remove the affected tissue.

Bacteria also form permanent, mostly lifelong, biofilms in the mucus-filled lungs of cystic fibrosis patients and are responsible for the chronic lung infections that lead to early death. Although long-lasting antibiotic treatment helps, it cannot eradicate the infection completely.

To study a biofilm formed by cholera bacteria (Vibrio cholerae), Berk built his own super-resolution microscope in the basement of UC Berkeley's Stanley Hall based on a 2007 design by coauthor Xiaowei Zhuang, Chu's former post-doctoral student who is now a professor at Harvard University. To actually see these cells as they divided to form "castles," Berk devised a new technique called continuous immunostaining that allowed him to track four separate target molecules by means of four separate fluorescent dyes.

He discovered that, over a period of about six hours, a single bacterium laid down a glue to attach itself to a surface, then divided into daughter cells, making certain to cement each daughter to itself before splitting in two. The daughters continued to divide until they formed a cluster -- like a brick and mortar building -- at which point the bacteria secreted a protein that encased the cluster like the shell of a building.

The clusters are separated by microchannels that may allow nutrients in and waste out, Berk said.

"If we can find a drug to get rid of the glue protein, we can move the building as a whole. Or if we can get rid of the cement protein, we can dissolve everything and collapse the building, providing antibiotic access," Berk said. "These can be targets for site-specific, antibiotic medicines in the future."

Super-resolution microscopy: painting with light

Berk is a biologist trained in physics and optics with expertise in imaging the structures of proteins: He was part of a team that a few years ago determined the atomic-scale structures of the ribosome, the cellular machine that translates genetic message into a finished protein.

He suspected that powerful new super-resolution light microscopy could reveal the unknown structure of biofilms. Super-resolution microscopy obtains 10 times better resolution than standard light microscopy -- 20 instead of 200 nanometers -- by highlighting only part of the image at a time using photo-switchable probes and compiling thousands of images into a single snapshot. The process is much like painting with light -- shining a flashlight beam on a dark scene while leaving the camera shutter open. Each snapshot may take a few minutes to compile, but for slow cellular growth, that's quick enough to obtain a stop-action movie.

The problem was how to label the cells with fluorescent dyes to continuously monitor their growth and division. Normally, biologists attach primary antibodies to cells, then flood the cells with fluorescent dye attached to a secondary antibody that latches onto the primary. They then flush away the excess dye, shine light on the dyed cells and photograph the fluorescence.

Berk suspected that a critically balanced concentration of fluorescent stain -- low enough to prevent background, but high enough to have efficient staining -- would work just as well and eliminate the need to flush out excess dye for fear it would create a background glow.

"The classical approach is first staining, then destaining, then taking only a single snapshot," Berk said. "We found a way to do staining and keep all the fluorescent probes inside the solution while we do the imaging, so we can continuously monitor everything, starting from a single cell all the way to a mature biofilm. Instead of one snapshot, we are recording a whole movie."

"It was a very simple, cool idea, but everyone thought it was crazy," he said. "Yes, it was crazy, but it worked."


Why are biofilms so hard to kill?

First there’s the slime, which antibiotics and chemicals have difficulty penetrating. In addition, electrical charges on the slime’s surface can form a barrier that keeps out antibiotics.

Because many cells deep within a biofilm are nutrient- and oxygen-starved, they grow fairly slowly — and are therefore less susceptible to antibiotics, which work best on actively dividing cells. To make matters worse, biofilms contain zombie-like “persister” cells which lie dormant when antibiotics are present but spring into action after antibiotic treatment ends.

Finally, cells within biofilms can organize themselves to pump drugs right out of cells — something Sauer called “a kind of bulimic behavior.”


Gene Regulation by Attached Cells

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. aeruginosa. Prigent-Combaret et al. (35) found that 22% of these genes were up-regulated in the biofilm state, and 16% were down-regulated. Becker et al. (36) showed that biofilms of Staphylococcus aureus were up-regulated for genes encoding enzymes involved in glycolysis or fermentation (phosphoglycerate mutase, triosephosphate isomerase, and alcohol dehydrogenase) and surmised that the up-regulation of these genes could be due to oxygen limitation in the developed biofilm, favoring fermentation. 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. aeruginosa. Prigent-Combaret et al. (35) opined that the expression of genes in biofilms is evidently modulated by the dynamic physicochemical factors external to the cell and may involve complex regulatory pathways.


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The Role of Bacterial Biofilms in Ocular Infections

There is increasing evidence that bacterial biofilms play a role in a variety of ocular infections. Bacterial growth is characterized as a biofilm when bacteria attach to a surface and/or to each other. This is distinguished from a planktonic or free-living mode of bacterial growth where these interactions are not present. Biofilm formation is a genetically controlled process in the life cycle of bacteria resulting in numerous changes in the cellular physiology of the organism, often including increased antibiotic resistance compared to growth under planktonic conditions. The presence of bacterial biofilms has been demonstrated on many medical devices including intravenous catheters, as well as materials relevant to the eye such as contact lenses, scleral buckles, suture material, and intraocular lenses. Many ocular infections often occur when such prosthetic devices come in contact with or are implanted in the eye. For instance, 56% of corneal ulcers in the United States are associated with contact lens wear. Bacterial biofilms may participate in ocular infections by allowing bacteria to persist on abiotic surfaces that come in contact with, or are implanted in the eye, and by direct biofilm formation on the biotic surfaces of the eye. An understanding of the role of bacterial biofilm formation in ocular infections may aid in the development of future antimicrobial strategies in ophthalmology. We review the current literature and concepts relating to biofilm formation and infections of the eye.


Biofilm formation

Biofilm formation begins when free-floating microorganisms such as bacteria come in contact with an appropriate surface and begin to put down roots, so to speak. This first step of attachment occurs when the microorganisms produce a gooey substance known as an extracellular polymeric substance (EPS), according to the Center for Biofilm Engineering at Montana State University. An EPS is a network of sugars, proteins and nucleic acids (such as DNA). It enables the microorganisms in a biofilm to stick together.

Attachment is followed by a period of growth. Further layers of microorganisms and EPS build upon the first layers. Ultimately, they create a bulbous and complex 3D structure, according to the Center for Biofilm Engineering. Water channels crisscross biofilms and allow for the exchange of nutrients and waste products, according to the article in Microbe.

Multiple environmental conditions help determine the extent to which a biofilm grows. These factors also determine whether it is made of only a few layers of cells or significantly more. "It really depends on the biofilm," said Robin Gerlach, a professor in the department of chemical and biological engineering at Montana State University-Bozeman. For instance, microorganisms that produce a large amount of EPS can grow into fairly thick biofilms even if they do not have access to a lot of nutrients, he said. On the other hand, for microorganisms that depend on oxygen, the amount available can limit how much they can grow. Another environmental factor is the concept of "shear stress." "If you have a very high flow [of water] across a biofilm, like in a creek, the biofilm is usually fairly thin. If you have a biofilm in slow flowing water, like in a pond, it can become very thick," Gerlach explained.

Finally, the cells within a biofilm can leave the fold and establish themselves on a new surface. Either a clump of cells breaks away, or individual cells burst out of the biofilm and seek out a new home. This latter process is known as "seeding dispersal," according to the Center for Biofilm Engineering.


Footnotes

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Financial & competing interests disclosure

This work was supported by a grant from the National Institutes of Health, National Institute of Allergy and Infectious Disease P01 AI083211. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.