Biofilm

Bacterial Biofilm and Dentistry

Justin Merritt; Maxwell H. Anderson, DDS, MS, MEd; No-Hee Park, DDS, PhD; and Wenyuan Shi, PhD

Copyright 2001 Journal of the California Dental Association.

Bacterial biofilms are ubiquitous in nature. Recent studies have demonstrated many unique qualities previously unknown to bacteria and have yielded new insights into relevant dental issues.

A biofilm is commonly accepted as a community of bacteria that is adherent to a particular surface. These communities have been observed in nature since the early days of microbiology. Dating back to the mid 17th century, Antony van Leeuwenhoek was making observations about the various organisms he witnessed in tooth plaque. For the many decades following Leeuwenhoek’s initial observations, biofilms were typically a phenomenon studied by environmental microbiologists, who usually studied them in the context of various aquatic and marine habitats. By the 1970s, pioneers in the field were beginning to recognize that biofilms were more than an aberration seen in certain river beds, but were actually a mode of bacterial survival common to many different environments.¹ In some instances, biofilm bacteria were recovered from the most unthinkable of locations. For example, a report in 1998 described the isolation of several sulfate-reducing and acid-producing species of bacteria from the storage basin for spent nuclear fuels. These communities persisted in extremely nutrient poor deionized water under constant stress from α-, β-, and γ-emitting radioactive material.² By the 1990s, it was very clear that sessile biofilm communities were the dominant populations of bacteria inhabiting every sampled (and imaginable) aquatic environment.³

Bacterial biofilms are exceptionally well-organized and typically form mushroom-like structures.³ Within the biofilm communities are numerous water channels that deliver nutrients and remove toxic waste products. These structures are so well-ordered that they have been compared to higher levels of eukaryotic organization.⁴

Mechanisms for Formation

Attachment of bacteria to a particular surface has long been associated with biofilm growth,⁵ but the steps leading to the biofilm development process have only recently been elucidated. A layer of exopolysaccharide typically surrounds wild-type free-floating bacterial cells (called planktonic cells). These provide the first mechanisms of potential attachment to a surface or tissue.⁶ Once the planktonic cells have sensed that they have attached to a surface, they quickly respond by expressing a new set of genes that enable them to form biofilms. Mutagenesis experiments in different biofilm-producing bacteria have shown many common themes in the production of biofilms. As might be expected, genes involved with the production of exopolysaccharide are a common requirement for normal biofilm development. In a recent mutagenesis study on Vibrio cholerae,⁷ it was shown that exopolysaccharide-defective mutants failed to form normal biofilms. It also appears that some sort of motility and/or adhesion apparatus is required for biofilm formation.^7-10 This makes sense given that motility can help bacteria get close to a surface, while adhesion apparatus such as pili enable bacteria to attach to the surface.

A less-obvious biofilm-forming mechanism involves bacterial cell signaling. In Pseudomonas aeruginosa, a mutant in the lasI gene produced flat, undifferentiated biofilms, which were also much more susceptible to treatment with biocide.¹¹ The lasI gene is a critical factor associated with intercellular signaling in P. aeruginosa. This provided the first proof that biofilm production is actually a signaling event involved in regulating genes responsible for proper biofilm development. More recently, it has also been shown in the oral bacterium Streptococcus gordonii that mutations in its intercellular signaling system disrupt proper biofilm development.¹⁰

Physiological Differences

It is only recently that microbiologists have begun to treat biofilm bacteria as distinct entities from their planktonic counterparts.⁶ Recent studies show that biofilm formation induces many phenotypic changes in bacteria, and large portions of the entire gene expression profile are affected. A screen for differential expression of genes in Escherichia coli found that 38 percent of its genes had changes in expression as a consequence of biofilm development.¹² In P. aeruginosa, biofilm cells were found to have 30 percent to 40 percent of their cell envelope proteins expressed in only the biofilm phenotype.⁶ The metabolic capacity of biofilm bacteria also undergoes a dramatic change, with bacterial doubling time being markedly increased.¹³ Cells in these communities also have an extraordinary ability to modulate their metabolic needs based upon their particular niche within the biofilm.¹⁴

It has been demonstrated that bacteria within a biofilm have a strong propensity to share genetic material as well. In an experiment measuring genetic material exchange through conjugation within a biofilm between E. coli and Alcaligenes eutrophus, the rates of conjugation were observed to be as much as a 1,000-fold higher than those obtained from typical plating methods.¹⁵ Certainly, this presents a scenario that is ripe for rapid and efficient adaptation to any number of environmental challenges. Taken together, it becomes increasingly clear why there has been such a strong selective pressure for biofilm growth in many different species of bacteria. Biofilms afford bacteria a protected community in which to grow, allow for great flexibility in metabolic needs, and facilitate rapid adaptation to an unimaginable number of environments.

Persistent Bacterial Infection

It has only been within the past decade that much attention has been directed toward the study of biofilms. This delay in attention was largely due to the misconception that pathogenic processes are a phenomenon associated with planktonic bacteria. Certainly, this was a bias that was firmly established in the days of the golden age of microbiology. Without any perceived clinical relevance, biofilms were simply left to be studied more as an academic discipline. Ironically, improvements in medical technology created new niches that were exceptionally well-suited for biofilm growth. With the advent of implanted medical devices and a growing population that was immunocompromised, there began an increasing trend in patients to become afflicted with baffling chronic infections.^6,16 What was even more puzzling for doctors was the fact that treatment by conventional methods such as antibiotic therapy would yield only temporary relief of symptoms. The same infections would inevitably recur in a seemingly endless cycle of treatment and reinfection. To complicate matters further, when organisms were cultured from patients and plated as planktonic cells, they were typically found to be readily susceptible to conventional therapy.

Costerton recounted a case in which a patient was stricken with a recurrent bacteremia. The patient was treated for three weeks using 16g of cloxacillin per day. Inevitably, the treatment regimen would eliminate the bacteremia. However, upon termination of antibiotic therapy, there would be another cycle of infection. It was known that the patient had an old pacemaker, and therefore it was decided that the device would be removed. Upon removal, it was discovered that the pacemaker contained a thick biofilm of Staphylococcus aureus.¹⁷ The intense antibiotic therapy provided very little challenge to the cells growing on the device, and certainly the patient’s own immune system was not capable of clearing the infection in the protective biofilm.

This type of scenario was increasingly becoming more common as various medical devices such as catheters and heart valves were implanted into patients. Also, many different chronic infections of tissues within the body were discovered to be biofilm-related as well.¹⁸ In vitro biofilm experiments readily demonstrated the remarkable capability of biofilm bacteria to resist antibacterial therapies. In a comparison of planktonic P. aeruginosa with its biofilm counterpart, it was found that a treatment regimen of 50 μg/ml of tobramycin for eight hours was certain overkill for the planktonic bacteria. However, when the biofilm phenotype P. aeruginosa was given 1,000 μg/ml for an even longer time period, there was no significant reduction in biofilm viability.¹⁷

If the body is viewed as just one of many aquatic environments inhabited by biofilms, it makes sense that they are so resistant to different treatments. In the body, bacteria are able to employ the same mechanisms that protect them from microbial predation and chemical antagonism in the wild. In fact, it is commonly held that about 1,000 to 1,500 times more of specific antimicrobial agents are required to treat a biofilm-living bacteria as opposed to its planktonic form.⁶

Certain conditions such as cystic fibrosis are known to be biofilm-mediated diseases. In this particular disease, the immune system is unable to clear a biofilm infection, and the host’s own immune system mechanisms are the cause of major tissue damage. This is largely a result of immune complexes accumulating on the outside of the biofilm.¹⁸

What properties permit such exquisite resistance to otherwise harsh environments? There are several explanations that are probably all factors contributing to the success of biofilm bacteria. The first is more of a physical limitation. Biofilms are surrounded by a thick, viscous layer of exopolysaccharide that makes penetration of antibacterial agents very difficult for a variety of reasons.^19,20 The exopolysaccharide is analogous to a bullet-proof vest. If the bullets are not able to effectively penetrate the vest, then no real damage will occur.

Another resistance mechanism of biofilm bacteria stems from their metabolic capabilities. As was mentioned before, bacteria in the biofilm can tailor their metabolic needs based upon their own niche within the biofilm. Consequently, not all the bacteria in the biofilm are going to be particularly metabolically active. So, assuming an agent such as an antibiotic can actually penetrate the biofilm in any appreciable quantity, it may not be very effective against a metabolically inactive bacterium.¹⁸ In fact, many antibiotics are directed at some stage of bacterial metabolism.

Finally, there is some speculation that part of the biofilm developmental process includes differentiation into a phenotypically resistant organism. This is distinct from the metabolic changes that take place in response to nutrient availability. This theory is still largely speculative, but there has been some evidence suggesting a possible phenotypic resistance to tobramycin in the bacteria of younger biofilms.¹⁹

Nosocomial Infection through Medical Devices

In the hospital setting, it has been well-documented that biofilm bacterial species are a common source of nosocomial infection.^17,18 This is not surprising given the extraordinary potential for adaptability seen in biofilm bacteria. In hospitals, there has been continued difficulties with the pathogens P. aeruginosa, Legionella pneumophila, and nontuberculosis Mycobacteria species, with P. aeruginosa alone accounting for 9 percent to 11 percent of all reported nosocomial infections in the United States.¹⁶ These bacteria are found primarily as environmental species, but all are well-suited for growth wherever there is water and a supply of nutrients.

Even with the constant attempts at cleanliness and sterility, there will always be some minute niche that will be overlooked or not properly sanitized. For instance, in 1991, the University of Wisconsin reported a 36 percent increase in nosocomial upper gastrointestinal infections following endoscopy at its hospital.²¹ Subsequent investigative culturing of the endoscopes revealed heavy contamination with gram-negative bacilli, mainly P. aeruginosa. Further investigation proved that the source of the contamination was actually the automated endoscope washer -- the machine designed to clean and disinfect endoscopes after usage. The machine was new and routinely cleaned and disinfected according to manufacturer’s protocol.

Dental Plaque

While much effort has been devoted to the study of medically relevant single-species biofilms, one of the most commonly observed multispecies biofilms in humans has gone largely overlooked. This is dental plaque, which in many ways is proving to be a useful model for the study of multispecies biofilm interactions. While much of the knowledge gained as a result of single species biofilm research is also applicable to multispecies biofilms, there are many complexities that make multispecies biofilms unique.

An interesting phenomenon of oral bacteria is their tendency to consistently associate with a defined set of partners. When this association occurs in suspension, it is referred to as coaggregation, while a similar association in a biofilm setting is referred to as coadhesion.²² The ability of oral bacteria to associate with each other in a specific, defined manner becomes very important in the development of a dental biofilm. It appears that the formation of a multispecies dental biofilm is a process that occurs in discrete steps with certain groups of bacteria joining the biofilm at specific stages of biofilm development.

Many of the different Streptococcus species constitute the typical early colonizers of the tooth surface. This is generally thought to occur via saliva-specific receptors on the surface of oral Streptococcus species.²³ Fusobacterium nucleatum constitutes the next major colonizer of the dental biofilm through direct interactions with the early colonizing Streptococcus species. F. nucleatum is generally thought to be the microbial "glue" that can coaggregate with numerous oral bacteria and allow for many other late colonizers to join in the advanced stages of plaque development.²² Interestingly, F. nucleatum seems to be extremely important in the survival of different obligate anaerobic oral bacteria in both the planktonic and biofilm environments during aeration.²⁴ It is an intriguing possibility that in a dental plaque this function could be exploited to bridge the gap between the oxygen-tolerant facultative anaerobes and the much less tolerant obligate anaerobes.

Indeed, there are numerous examples of complex interdependency among the different species in dental plaque. More defined interactions have been elucidated in numerous examples of metabolic cooperation between oral bacteria, and this cooperativity seems to be intimately associated with specific coaggregations and coadhesions.²² One of the reasons this may occur is for metabolic efficiency of the biofilm community as a whole. For instance, many oral Streptococcus species produce fermentable carbohydrates as a product of host serum glycoprotein degradation.²⁵ These carbohydrates can then be fermented by other neighboring species of oral bacteria.²²

Other examples of metabolic communication may be even more fundamental. The ScaA lipoprotein in S. gordonii functions both to scavenge for Mn⁺² ions under limiting conditions and to act as a specific adhesin for various other oral bacteria. It has been proposed that ScaA may provide a source of Mn⁺² for different bacteria unable to scavenge the divalent ion, and that this cooperativitity may be mediated through specific coaggregations utilizing ScaA.²⁶ It would seem that the community aspects of multispecies biofilms are much more intricate than those of single species. In nature, it is thought that the majority of biofilms exist in a multispecies setting²⁷ and therefore, future work on dental biofilms will likely yield new insights into the convoluted metabolic and signaling events occurring in other multispecies biofilms.

Dental Unit Water System

Many investigations have revealed numerous issues of safety concerning equipment, personnel, and patients in dental clinics. While the epidemiological data establishing the link between dental treatment and biofilm-related nosocomial infection is weak, there are reported cases of infection due to treatment.²⁸ In particular, biofilms present an ever-growing concern due to their prevalence in dental unit water systems. While there seems to be little direct evidence suggesting dental unit water system biofilms as sources of nosocomial infection, there is a general concern that they may serve as potential reservoirs of infection, especially in the elderly and immunocompromised.¹⁶

Groups such as the American Dental Association Council on Scientific Affairs point out that the public should expect the highest standards of safety and sanitation from the modern dentist regardless of risk.²⁹ For these reasons, in 1995 the ADA set forth a goal for dental unit water systems to deliver water with less than 200 colony-forming units/ml of unfiltered output water.²⁹ In essence, the goal was to effectively remove biofilms from dental unit water systems by the year 2000. It is worthy of note however, that having less than 200 cfu/ml can underestimate the number of bacterial counts in a water sample, simply due to the fact that only a very small fraction of cells in a sample are able to form colonies on agar plates.³⁰ In any case, this goal was to be the voluntary standard of acceptable cleanliness and was to be achieved through a combination of research into waterline-related issues as well as through a conscious effort to inform practitioners. In the five years since the statement, there has been a strong response from industry, and a wide array of products made specifically for dental units are available. These fall into one of four categories: independent water systems, chemical treatment methods, point-of-use filters, or sterile water delivery systems.²⁹ The ADA Council on Scientific Affairs evaluates many such products submitted for its Seal of Acceptance Program.²⁹

Despite the plethora of available products, some investigations into dental unit water systems in the clinical setting have yielded dismal results. In a recently published study in England, 55 water and tube samples were taken from actual dental surgeries. The aim was to assess relative cleanliness under real-use conditions as well to compare contamination from different types of dental unit water systems. In 95 percent of the samples taken, counts were higher than the ADA-recommended less than 200 cfu/ml standard. Of the 55 surgeries, waterline samples had an average microbial load of 2,900 cfu/ml, while air rotor waterlines contained an average of 3,300 cfu/ml. The surfaces of the waterlines contained an average biofilm coverage of 43 percent, while the corresponding air line value was 5.2 percent. Oral streptococci were identified in 7 percent of the samples, thus raising the possibility that some antiretraction devices may be inadequate. It is interesting to note that the authors were not able to show to statistical significance that there was any difference in microbial contamination between different types of dental unit water systems.³⁰ This contradicts the belief some dentists may have that certain units are inherently cleaner than others.

It is not surprising that sampling efforts of dental unit water systems have produced numbers much higher than recommended values. Despite the best intentions of many dentists, there is likely to be the assumption that typical decontamination protocols will be sufficient to provide clean water to their patients. When dealing with planktonic bacteria, surely these efforts would be sufficient. However, biofilm-dwelling bacteria present a unique situation that defies many of the classical microbiology dogmas.

Furthermore, it is an exceptional challenge to keep a closed system like the dental unit completely free of biofilm formation. Once a biofilm has been established, it is difficult to remove from the system. Indeed, biofilm removal has been a major issue plaguing other industries for years. It seems the most effective way of removing a biofilm is simply by using brute force and mechanically disrupting it. This is the same reason that scaling and root planning is so effective and why people benefit from brushing their teeth every day. In a closed system like a dental unit, mechanical disruption is not a time- and cost-effective method of biofilm removal. However, numerous recommendations have been made to help ameliorate the problem. Both the Centers for Disease Control and Prevention and ADA recommend the simplest of these.²⁹ They advise dentists to flush their waterlines for two to three minutes before treating the first patient of the day in an effort to eliminate suspended bacteria in the water. It was also proposed that dentists flush their lines for about 30 seconds between patients, which helps to remove any bacteria that may have entered the waterlines during treatment.^31,32 This should also prevent cross-contaminating bacteria between patients.

Numerous studies have been conducted to evaluate the effectiveness of accepted decontamination methods. High concentrations of disinfectants (such as formalin) have been shown to be effective at killing biofilm bacteria, however, they are largely ineffective at removing the biofilm matrix from the attached surface. In fact, there is some evidence they may even impede further cleaning efforts.³³ Given the extreme toxicity of such compounds, there is a large concern that the biofilm will serve as a reservoir of toxic disinfectant that can be transmitted to the patient. In some instances, disinfection is achieved through cleaning waterlines with enzyme detergents.³³ This can effectively remove the biofilm matrix from tubing, however, it should be noted that this can release many bacteria from the biofilm into suspension. In the previously mentioned study of 55 dental unit water systems, five of the units were recently decontaminated and all showed higher levels of bacterial contamination in the water samples.³⁰ Direct sampling of the biofilms on the dental unit water systems tubing did confirm the presence of fewer biofilm embedded bacteria, however. Therefore, careful flushing of the waterlines is advisable if such a cleaning strategy is to be used because there is likely to be a transient worsening of water quality.

The most extensively investigated option is the periodic treatment of waterlines with a 1:10 solution of sodium hypochlorite.²⁹ A recent study evaluated the effectiveness of such a treatment and concluded that weekly treatments with 1:10 NaOCl combined with the use of chlorinated water was sufficient to retard biofilm production and maintain the ADA recommended less than 200 cfu/ml.³⁴ Hypochlorite-based bleach has also been demonstrated to aid in the removal of biofilms.³³ It is not known whether such a treatment regimen has a detrimental effect on oral tissue or various dental procedures.³⁴

Clearly, there are numerous options dentists may employ to achieve better dental unit water systems water quality. As has been demonstrated before, it should not be assumed that any device or treatment protocol is going to perform to expectations. Consider the scenario presented with the University of Wisconsin automated endoscope washer. Therefore, the ADA Council on Scientific Affairs has wisely suggested a routine sampling of waterline quality for patient safety as well as preventing dentist liability. They advise water samples to be taken before routine disinfection in order to assess maximum potential exposure.²⁹ There are waterline testing kits available that are both cost-effective and easy to use. Their efficacy was demonstrated in a study that found a strong correlation between kit results and those obtained from laboratory methods.³⁵ Given the present lack of definitive options, this may be the optimal strategy to monitor the quality of water to which patients and staff are subjected.

Summary

Bacteria form complex communities (called biofilm) on particular surfaces. Bacteria in biofilm survive better and exhibit stronger resistance to various environmental factors than do planktonic cells. This unique physiological status causes persistent bacterial infection or contamination of medical devices. Similarly, biofilm is also responsible for various oral bacterial diseases and contamination of dental unit water systems. The dental field has much to gain from molecular investigations of biofilm production. Novel methods of prevention and treatment of tooth decay and periodontal disease are inevitable outcomes from the biofilm research under way in many laboratories throughout the world. Future research will also undoubtedly yield new biofilm-resistant materials and/or safe chemical additives that disrupt biofilm formation. However, it is unlikely that scientists and practitioners will ever again take sanitation for granted, given the relentless persistence of biofilm bacteria.

Acknowledgment

This work was supported by a NIH training grant T32-AI07323 to J. Merritt and a Washington Dental Service Grant to W. Shi.

Authors

Justin Merritt is a PhD student at the University of California at Los Angeles Molecular Biology Institute.

Maxwell H. Anderson, DDS, MS, MEd, is the vice president and dental director of Washington Dental Service.

No-Hee Park, DDS, PhD, is the dean and a professor of UCLA School of Dentistry.

Wenyuan Shi, PhD, is an associate professor at UCLA Molecular Biology Institute and School of Dentistry.

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To request a printed copy of this article, please contact/Wenyuan Shi, PhD, UCLA School of Dentistry, 10833 Le Conte Ave., Los Angeles, CA 90095, or at wenyuan@ucla.edu.