Introduction Traditionally, microbiologists have studied bacterial structure, function, and susceptibility using cells that have been cultured in liquid medium. In this state, bacteria exist as free-floating planktonic cells. However, it is increasingly being recognized that in their natural habitat, most bacteria grow attached to a surface.[1] The growth of large aggregates of cells on a surface encased within a three-dimensional matrix of extracellular polymers (otherwise known as extracellular polymeric substance or EPS) produced by the sessile bacteria is known as a biofilm.[2] In man, the surfaces that are available for attachment are many and varied and include skin, teeth, the respiratory tract, and intestinal mucosa (Table 1). Given the right conditions, all bacteria can grow a biofilm. Most species of bacteria that cause infection are members of the normal microflora of humans and form biofilms at sites where they exist as harmless commensals. In this situation, biofilms are considered to play a protective and beneficial role in the host. For example, biofilms in the vagina prevent colonization by exogenous pathogens—a phenomenon known as colonization resistance—and this process is synonymous with vaginal health.[3] However, due to a selection of endogenous and exogenous factors, the microbial composition of such “healthy” biofilms can become disturbed to produce a pathogenic biofilm. This has been documented to lead to diseases, such as dental caries.[4] Staphylococci, which are members of the normal microflora of the skin, frequently form biofilms on implantable medical devices, such as intravenous catheters and hip and knee joint prostheses.[5,6,7] Similarly, Pseudomonas aeruginosa is an environmental organism that regularly causes infections in burns and other wounds and constitutes a major concern for immunocompromised individuals.[8] Pseudomonas aeruginosa is very adept at biofilm formation and readily forms such structures in the lungs of individuals with cystic fibrosis; this is associated with shortened life span of the patient.[9] Most biofilms, specifically urinary and oral biofilms, are comprised of a variety of organisms, i.e., polymicrobial. In fact, in dental plaque, more than 350 different bacterial species have been identified[10] by traditional microbiological methods, although it is likely that culturable organisms may represent as little as one percent of the total microbial population.[11] Why can Biofilms be a Problem? It has been estimated that biofilms are associated with 65 percent of nosocomial infections[12] and that treatment of these biofilm-associated infections costs greater than $1 billion annually in the United States.[1,13] The estimated cost of a hip replacement in the UK is £3,500, but the hospital costs associated with a subsequent infection can be as high as £30,000.[5] So why are biofilm-related infections such a problem to treat? The challenge arises as a consequence of the following several factors: 1. Biofilm bacteria are less susceptible to our immune defense system, and consequently, a biofilm-associated infection can persist for a long period of time (i.e., progress from an acute to a chronic infection). Phagocytic cells have difficulty ingesting bacteria within a biofilm due to antiphagocytic properties of the biofilm matrix.[14,15] In the absence of specific antibodies, the polysaccharide component of the biofilm matrix also blocks complement activation. If antibodies are present, the polymeric matrix generally renders them ineffective. It has been shown that the biofilm matrix is also able to inhibit chemotaxis and degranulation by polymorphonucleocytes (PMNs) and macrophages and also depress the lymphoproliferative response of monocytes to polyclonal activators.[15,16] Not only are host defenses unable to deal effectively with biofilms, but their persistence can cause tissue damage (e.g., lung tissue in cystic fibrosis). Contact with a surface triggers the expression of a panel of bacterial enzymes that catalyze the formation of sticky polymers that promote colonization and protection. The structure of biofilms is such that immune responses may be directed only at those antigens found on the outer surface of the biofilm, and antibodies and other serum proteins often fail to penetrate into the biofilm. In addition, PMNs are unable to effectively engulf bacteria growing within a complex polymer matrix attached to a solid surface. This causes the PMNs to release large amounts of pro-inflammatory enzymes and cytokines, leading to chronic inflammation and destruction of nearby tissues (i.e., chronic inflammation). Bacteria that may be embedded within the wound biofilm matrix are likely to be resistant to both immunological and non-specific defense mechanisms of the body. 2. Biofilms display innate resistance to antimicrobial agents, thus protecting associated bacteria. The reasons for this are not clear, but it is likely that antimicrobial agents are readily inactivated or fail to penetrate into the biofilm. Bacteria within biofilms may be up to 1,000 times more resistant to antimicrobial agents than those in a planktonic state.[17] 3. Biofilms increase the opportunity for gene transfer between and among bacteria. This is important, since bacteria resistant to antibiotics may transfer the genes for resistance to neighboring susceptible bacteria. Also, gene transfer could convert a previous avirulent commensal organism into a highly virulent pathogen.[18] Antibiotic Resistance Antibiotics are used to treat bacterial infections. However, biofilm-related infections do not succumb so easily to this form of treatment, because they provide a protective mechanism that renders bacterial cells less susceptible to both antibiotics and biocides. However, on removal of these cells from the matrix of the biofilm, they are equally susceptible to biocides.[19] There have been a number of models used to determine resistance in biofilms, and the results of these studies have highlighted a number of the factors thought to contribute to the ability of a biofilm to tolerate high concentrations of antibiotics. These include: 1. Impaired penetration of an antibiotic into the biofilm matrix.[20,21] Many researchers have investigated the possible lack of antibiotic/biocide penetration as an explanation of biofilm resistance. It was suggested that the antimicrobial agent either reacted chemically with the extracellular components of the biofilm or attached to the anionic polysaccharides. However, since the exopolymer matrix does not form a complete impenetrable barrier to antimicrobial agents, other mechanisms must exist within biofilms aiding bacterial survival. 2. Reduced growth rate of bacteria in biofilms, which renders them less susceptible to antibiotics (they change from being physiologically active in the planktonic state to sessile in the biofilm state). Antibiotics are more effective in killing cells when they are growing actively. Antibiotics, such as ampicillin and penicillin, are not able to kill nongrowing cells.22 Cephalosporins and fluoroquinolones, however, are able to kill nongrowing cells but are nonetheless more effective in killing cells that are rapidly growing and dividing. Therefore, evidence of bacteria that are growing slowly in a biofilm may contribute to reduced susceptibility to antibiotics.[22] 3. Altered micro-environment within the biofilm (e.g., pH, oxygen content), which reduces the activity of an antimicrobial agent. There is evidence of gradients of physiological activity within a biofilm in response to antibiotic treatment.[23] This would suggest that the response to antibiotics will vary according to the location of specific cells within a biofilm ecosystem. 4. Altered gene expression. Altered gene expression by organisms within a biofilm or a general stress response of a biofilm have been documented as factors known to reduce susceptibility to antibiotics.[24] 5. Quorum sensing (QS). QS has been documented as being involved in antibiotic resistance, but its role is currently unclear, which justifies the need for additional research in this area.[25] QS involves the production of signalling molecules within a bacterial population that enables bacteria to communicate with each other and initiate a response to their surrounding environment once a critical population density (quorum) has been reached. In a wound environment, many different bacteria live together in often dense populations, and this provides an ideal situation for QS to occur. Communication in this way allows bacteria to coordinate their behavior and, if necessary, change physiologically to enable them to adapt to a new environment (e.g., a wound). Adaptive responses of bacteria within a wound environment may be associated with nutrient availability, competition with other microorganisms, and the avoidance of host defense mechanisms. Adaptation to a wound environment via QS may involve bacteria secreting protective EPS and increasing the production of enzymes that facilitate their tissue invasion. 6. Reduced biofilm-specific phenotype. It has been suggested that a biofilm-specific phenotype may be induced in a subpopulation of the biofilm.[26] These subpopulations have been shown to express active mechanisms to reduce the efficacy of antibiotics.[26,27] The Potential Significance of Biofilms in Wounds Based on the authors’ hypothesis, it is likely that a wound environment is able to support the development of bacterial biofilms, although at the present time, there is very little clinical evidence to support this. Wounds have been shown to possess many of the characteristics that suggest the existence of biofilms. In fact, an investigation by Serralta and colleagues[28] provided evidence that biofilms may form in wounds, and it is probable that biofilms could have a significant effect on inflammation, infection, and healing. Wounds are susceptible to microbial contamination from both exogenous and endogenous sources including the nose, skin, mouth, and the gut, and it is likely that such organisms are involved in the evolution of microbial communities in wounds. The development of microbial communities and their variation over time previously has been demonstrated in an acute full-thickness porcine wound model.[29] In this study, the authors found that the microbial ecology changed from a predominantly Gram-positive bacterial population during the early days after wounding to a predominantly Gram-negative population by Day 6 with anaerobes becoming evident between Days 6 and 8. It is likely that a similar microbial progression and community development occurs in human cutaneous wounds, and biofilms are likely to play an important role in this. Microbial progression within a wound is thought to consist of a number of stages (Figure 1) and if this process is not controlled, the probability of infection increases.[30] It is proposed by the authors that this model is in fact a model of a progressing biofilm. Early contaminants on a wound surface are most likely to be skin flora (e.g., Staphylococcus epidermidis) that adhere to the wound, proliferate, synthesize EPSs, and form a “healthy” biofilm. It is at this point that the host initiates a normal immune response and maintains a “homeostasis” at the site of contamination. It is conceivable, based on documented evidence,[29] that Gram-negative bacilli may then colonize the biofilm—these organisms utilize available oxygen and provide growth factors that enable anaerobes to establish within the biofilm thus forming a complex but stable polymicrobial “climax” community[31] often termed microbial homeostasis.[32] A climax community is a collection of microorganisms within a “quasi” steady state, implying stable associations and integrations of function between microbial populations. In this situation, the progression to wound healing may become compromised by the biofilm community, i.e., the microbial to host balance is weighted in favor of the microorganisms, and the term critically colonized has been used to describe this state. At this stage, the microorganisms, while interfering with the wound healing process, may not necessarily induce any clinical signs of infection, although there may be subtle signs that indicate bacterial imbalance (e.g., change in wound color or odor together with the presence of devitalized tissue and ischemia). It may be appropriate to consider the use of a broad-spectrum topical antimicrobial agent at this stage to control the microbial challenge. The combined effects of the antimicrobial agent and the host immune response are likely to improve conditions for healing where bacterial imbalance is evident. Without control of microbial progression, a transition from an early “healthy” biofilm to a “pathogenic” wound biofilm may develop and ultimately lead to clinical infection. While the net pathogenic effect of the biofilm community exceeds the host’s immune response, wound healing is likely to be compromised. Scientific and clinical research in the area of wound biofilms and the associated bacterial interactions is now warranted to better understand the impact on wound healing.