Basics of biofilm in clinical otolaryngology.

Bacterial attachment to surfaces has been recognized for more than 50 years. By the 1970s, microbiologist became aware that probably most bacteria in nature exist as biofilms, even though most of the classic infectious disease doctrine has been based on the study of free floating planktonic bacteria. In the simplest sense, biofilms are created when free-floating bacteria or fungi sense a surface, attach to it, and then elaborate chemical signals to coordinate differentiation and structure formation, including the development of a protective polysaccharide coating.

A major breakthrough in our understanding of biofilms occurred in the 1980s with the discovery of protein "inducers" that are responsible for "quorum sensing." (1) The 1990s witnessed an explosion of new knowledge about the genetics of cell-to-cell signaling and coordinated gene translocation for defense and virulence factors. (2) Possibly all prokaryotes are capable of cell-to-cell communication, and biofilm production has profound implications for human disease. Two public health examples are the original outbreak of Legionella pneumonia caused by biofilms on air-conditioning ducts and the outbreak of Pseudomonas infection caused by biofilms created during the production of albuterol; the latter resulted in approximately 100 deaths in the early 1990s. (3) Other biofilm-induced disease states include dental plaque and caries, catheter sepsis, bacterial endocarditis, and cystic fibrosis pneumonia. Specific otolaryngology disease states include tympanostomy tube otorrhea, bacterial colonization of tracheostomy tubes and voice prostheses, chronic middle ear effusion, and possibly chronic sinusitis.

Cell-to-cell communication

Examples of coordinated bacterial behavior, such as the "swarming" movements of enteric bacteria, have been observed for almost 100 years. However, the notion that virtually all prokaryotes are capable of communication for enhanced survival is a truly revolutionary concept in our understanding of infectious disease. While studying the symbiotic relationship between Vibrio fischeri bacteria within light organs and their host squid and fish, Engelbrecht and Silverman first began to unlock some of the mysteries of cell-to-cell signaling. (1) They noted that bioluminescence was triggered suddenly when bacterial numbers reached a certain threshold. They subsequently identified two quorum-sensing systems: luxI and luxR. The lnxI regulatory protein is responsible for induction of the homoserine lactone-signaling molecule ("auto-inducer"), which, at a critical concentration threshold, triggers the luxR transcriptional activator protein in the colony of bacteria that is responsible for transcription of to luciferase and thus bioluminescence. The luxI and luxR systems have been identified in de almost all gram-negative bacteria, and cell-to-cell signaling molecules (peptides) have been identified in more than 30 species of gram-positive bacteria. The lux systems have been extensively studied in Pseudomonas spp and Escherichia coil, and they have been found to be necessary for both biofilm maturation and activation of numerous virulence factor genes. (4-6) Presumably, the ability of pathogenic organisms to "wait" for a buildup of large numbers before releasing virulence factors confers a survival advantage by increasing the chances of overwhelming host defenses. (7)

Biofilm formation, structure, and properties

In terms of human disease, biofilms tend to develop on inert surfaces (e.g., tympanostomy tubes and ventriculoperitoneal shunts), necrotic tissue (as in temporal bone osteomyelitis), or relatively ischemic normal tissue (e.g, heart valve cartilage and bone), Bacteria can sense a surface and form an active, energy-consuming bond, sometimes via appendages such as pill. Surface characteristics influence binding, and binding properties vary among species. For example, An and Friedman demonstrated that Pseudomonas spp bind relatively well to hydrophobic plastics (e,g., polyethylene), moderately well to hydrophilic metals, and poorly to hydrophilie substances that have a negative charge (e.g., glass and oxidized plastics). (8)

Once a critical number of bacteria have formed a surface attachment, quorum sensing allows for specific differentiation and for production of an extracellular protective glycocalyx (polysaccharide "slime"). The biofilm assumes a physical structure and develops channels for diffusion of water and nutrients. Living in a more efficient "societal" structure allows for a reduction in the metabolic rate of individual cells.

In the host, biofilm provokes both cellular and humoral immune responses, but these responses are often relatively ineffective because of poor penetration through the glycocalyx and because of other genetically triggered specific defense factors. In fact, the ineffective host inflammation can be responsible for a large part of the host disease burden. Cystic fibrosis is an example of a chronic biofilm state that leads to a significant degree of chronic lung disease (e.g., pneumonia and bronchitis) caused by the host's immune response. (5) Antimicrobial medications are likewise ineffective because of poor penetration, the slow metabolic rate of the biofilm cells, and specific genetriggered enhancements of defense mechanisms (e.g., multidrug resistance pumps). (8)

At times, a certain percentage of biofilm cells may be released from the structure. Although these free-floating planktonic cells then become susceptible again to host immune defenses and to antimicrobial agents, they can also form new biofilms at distant sites. One example of such an occurrence is recurring sepsis in a patient with an infected shunt or catheter.

Biomaterials in otolaryngology

Tympanostomy tubes are perhaps the most common foreign-material devices implanted in human hosts. The influences of surface and material physical properties on biofilm formation have been studied to a limited degree with electron and confocal laser scanning microscopy. During experiments with Pseudomonas aeruginosa and Staphylococcus aureus, the smoother surfaces (such as ion-bombarded silicon and tetrafluoroplastic) have been found to be less conducive to biofilm development. (9,10) Likewise, the changing surface hydrophobicity of voice prostheses has been shown to alter biofilm formation. (11)

Bacterial accumulation on endotracheal tubes in intensive care units has been documented since the 1970s. The buildup of gram-negative biofilms on endotracheal tubes has been correlated with nosocomial pneumonia, which is presumably spread mechanically during suctioning. (12,13) At the Children's Hospital of Seattle, we recently completed a study of biofilm development on pediatric tracheotomy tubes. (14) Using bacterial fluorescent DNA staining and confocal laser scanning microscopy, we identified bacterial biofilms in the distal lumens of 10 of the 11 tubes we studied. Because accidental decannulation and "mucus plugging" are the two most common causes of tracheostomy-related death, strategies to reduce biofilm formation have an obvious potential for improving outcomes.

Otitis media with effusion 

The behavior of pathogens in acute otitis media fits in fairly well with classic microbiologic tenets; bacteria are culturable in most cases, and antinficrobial therapy has a significant effect on acute symptoms of pain and fever. In otitis media with effusion, on the other hand, pathogen behavior is not so clear cut. Cultures often show no growth, antibiotic therapy has little or no effect on resolution, and the duration of the disease is often prolonged. Evidence of biofilm formation in chronic middle ear effusion is beginning to shed light on many of these mysteries.

Bacterial DNA has been identified in culture-negative middle ear effusions in the past. However, questions remain regarding the viability and pathogenicity of those bacteria. Recent studies of bacterial messenger RNA (short-lived and necessary for DNA transcription) and bacterial endotoxins in nonculturing middle ear effusions implicate biofilms in the pathogenesis of these infections. (15,16) An animal model of middle ear biofilm formation has also been created. (17) If biofilms are involved in chronic middle ear disease, it is logical to presume that they also play a role in other chronic upper respiratory diseases, such as sinusitis, tonsillitis, and adenoiditis.

Future directions: Strategies for biofilm attack

The recognition that biofilms are responsible for a significant portion of human disease opens up the possibility for numerous novel approaches to treatment and prevention. Examples of inhibiting attachment by altering surface and material characteristics have already been mentioned. Other approaches to inhibiting attachment to surfaces include using chelating agents to restrict iron, which is necessary for the attachment of Pseudomonas spp pill. (18) Xylitol (a natural alcohol sugar in beech trees) has shown some clinical effectiveness in preventing caries and otitis media, possibly via a mechanism of reduced bacterial binding. (19) Competitive inhibition of signaling proteins and peptides via analogues also holds great promise, and several strategies are being studied. (20) Interfering with biofilm structure by dissolving matrix polymers is another avenue of attack. It is also interesting that macrolide antibiotics appear to inhibit polysaccharide synthesis and thereby degrade biofilm surface protection. (21) Finally, changing the surface environment through competitive inhibition from other bacteria (e.g., alpha streptococci) or increasing oxygen tension (in patients with tympanostomy tubes) are two other promising strategies.

In summary, bacteria and fungi do not always act in isolation. Density-dependent signaling of specific gene transcription (quorum sensing) allows for the development of biofilms and for the elaboration of more potent virulence factors. Biofilms no doubt play a role in most chronic respiratory tract diseases. Strategies directed against biofilms will become increasingly important in infectious disease therapy.

Finally, I wish to acknowledge Jonathon Perkins, DO, for his collaboration with me in preparing this presentation.

References

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