Abstract: This paper reviews the potential role of bacteria living in an adhered matrix made of extracellular polysaccharide, a biofilm, in wound healing. Communication between bacterial cells or quorum sensing via transcription of certain genes within the biofilm will be discussed as it relates to the biofilm environment. Bacteria that live in the biofilm environment have been shown to be more resistant to antiseptics and antimicrobial agents. This knowledge may be important to healthcare providers who care for infected chronic wounds that are unresponsive to antimicrobial therapy. Several potential new ideas for controlling the formation of biofilm will be discussed, since enhancing lactoferrin, a component of innate immunity, may disrupt biofilms. The use of enzymes for the removal of biofilms has been successful on surfaces, such as soft contact lenses, and may have a role in removal of biofilms from a wound bed. Additional work is needed to substantiate our hypothesis that biofilms play a significant role in delayed wound healing and to investigate possible removal of bacteria living in this protected environment.

Free living bacteria (planktonic cells) behave in an identical way to environmental conditions. However, it has been known for many years that adhesion of a bacterium to a surface alters its phenotype and its pattern of behavior to environmental conditions.[1] Adhered microorganisms that live in a self-synthesized matrix are a biofilm. The biofilm is a three-dimensional structure containing one or more species. Areas within a fully formed biofilm may have both anaerobic and aerobic environments. Bacterial cells living in a biofilm are exposed to gradients in nutrients and physicochemical factors, including water channels. Nutrients flow to the bacterial cell through the water channels within the extracellular polysaccharide (EPS) matrix, which change the pattern of gene expression.[2] This change in gene expression is dependent upon a phenomenon known as quorum sensing. Quorum sensing involves the production by low molecular mass auto-inducers, which can activate the transcription of certain genes. Virulence factor secretion is dependent upon the bacterial population number and the cell-to-cell communication within the biofilm structure.[3]

This intercellular communication helps the biofilm community of organisms resist chemicals and other threats from the environment. When bacteria are placed in adverse conditions, quorum sensing signals are sent to force a slow-growing state.[4] This slower growing state makes the cells less susceptible to antimicrobial attack. For example, in the case of infections of the gastric mucosa with Helicobacter pylori (H. Pylori), the organism in a planktonic state is sensitive to most common antibiotics, but these infections have been proven difficult to cure.[5] This suggests the organisms have an environmental protective mechanism know as a biofilm, and H. pylori has been shown to form biofilms.[6]

Biofilms can be made in vitro and stained with fluorescent stain so that an image can be made of the EPS with a counter-stain for bacteria (DNA). These in-vitro biofilms reveal that bacterial cells are clustered together within the EPS matrix and that there are many water channels.[7] Other investigators have demonstrated that while leukocytes are “trapped” in a biofilm they are ineffective at interacting with bacterial cells. There appears to be an antiphagocytic property in the biofilm matrix.[8]

Biofilm-associated infections have been shown unresponsive to antibiotic therapy. Conventional antibiotic susceptibilities of planktonic cells may not reflect the reduced susceptibilities that biofilm-living bacteria have. Ceri, et al., compared planktonic and biofilm Staphylococcus aureus (S. aureus) and found that S. aureus biofilms may be 50 to 1000 times more resistant than planktonic or free-floating bacterial cells.[9]

Although emphasis is placed on the detrimental influences of biofilms, they can also play a protective role by preventing colonization by exogenous pathogens. For example, it is believed the hair follicles of the skin support biofilm formation, and the normal cutaneous microflora living within the follicles offers some protection against invading pathogens. It is also interesting to note that the number of normal flora increases during occlusive film dressing therapy of acute wounds, but healing and epithelization are still enhanced by occlusive film dressings. The numbers of normal flora return to baseline after epithelization is complete and film dressings are discontinued.[10]

Biofilm formation is most often studied in vitro, and single-species biofilms are most often used. Using in-vitro models, the timing of the biofilm formation can be imaged as can the quantity of EPS produced.[11] The authors have developed a specific assay to quantify the number of free-floating planktonic cells versus the number of biofilm-living bacteria. This assay has been used in vitro and in vivo. Specific staining techniques using a modified Congo Red/Ziehl Carbol fuchsin have been used to visualize the development of biofilm. Using an in-vitro model, the author’s group has determined that a Pseudomonas aeruginosa (P. aeruginosa) wound isolate takes 7 to 10 hours to make a mature biofilm.[12]

Having evaluated an in-vitro model, the author’s group wanted to develop and study an in-vivo model. The author’s group used a well-studied swine wound model. Swine skin has been shown to be the most similar to human skin in structure and normal microflora. Using this model, they have shown that bacteria live as both free-floating planktonic cells and also adhere to the wound bed as a biofilm.[13] To the author’s knowledge, this is the first time biofilm formation has been demonstrated in wounds. The author’s group has also shown that bacteria living within the biofilm environments are more resistant to topical antiseptics and antimicrobials, which work more slowly in eradicating bacteria that are protected by a biofilm.[14] Finally, the author’s group has been able to image S. aureus biofilm formation in an acute wound using scanning electron microscopy (Figure 1).[15]

Figure 1

Pictured here is a scanning electron micrograph of a porcine wound surface with Staphylococcus aureus biofilm (6000X).

The role of bacterial biofilms in chronic wounds is not well understood. Drs. Bello and Ricotti of the author’s group have demonstrated that chronic wounds contain bacteria biofilms using two staining techniques. First, light microscopy was used to visualize EPS on a smear taken from human chronic wounds that was stained with a modified Congo Red/Ziehl Carbol Fuchsin.[16] Secondly, they were able to detect a wound biofilm using Calcofluor white to visualize the polysaccharide matrix by epifluorescent microscopy.[17] This biofilm formed in vivo differs from biofilms that are formed in vitro because it is polymicrobial. In Figure 2, a Calcofluor white stain of a P. aeruginosa biofilm made in vitro is double stained with Ethidium bromide to demonstrate the bacterial DNA.

Figure 2

Pictured here is a double stain of an in-vitro formed biofilm of P. aeruginosa. Calcofluor white stains the EPS (purple haze) and Ethidium bromide stains the bacterial DNA (pink).

Although the role of biofilm formation in wounds is not well understood, it is possible that certain therapeutics may be helpful in preventing biofilm formation within the wound bed. One such idea was put forth by Singh and his collaborators in Nature.[18] They hypothesized that a component of innate immunity may prevent bacterial biofilm development. They demonstrated that lactoferrin, an ubiquitous and abundant substance found in human secretions, may block biofilm development by P. aeruginosa. Lactoferrin stimulates a form of cellular motility, which encourages the bacterial cells to wander instead of forming biofilms. A lactoferrin preparation could conceivably disrupt already existing biofilms, and this disruption might provide wound protection from infection.

Another possible approach for controlling biofilm formation is that of interfering with quorum sensing, which controls the expression of various virulence factors of bacteria living within biofilms. Recently, scientists from the University at Buffalo have described a promising new drug that can inhibit the sophisticated bacterial communication system or quorum sensing.[19] By evaluating the structure of the quorum-sensing molecules, the researchers were able to discover a subset of molecules that act as a key in quorum sensing. Using the subset of molecules that participate in quorum sensing, they then synthesized a library of “quorum sensing antagonists” and anticipate that these antagonists will restore the potency of antibiotic treatments. It is possible that these antagonists would be useful in chronic wounds.

Enzymes have been used to remove bacterial biofilms from surfaces, such as stainless steel, polypropylene, and soft contact lenses.[20] The enzymes used for these studies were not bactericidal but did remove the bacterial cells from the biofilm matrix. They were effective against both Gram-positive and Gram-negative biofilm bacteria, including S. aureus and P. aeruginosa, two common wound pathogens. To the author’s knowledge no one has tried to remove biofilms from chronic wounds with enzymes. However, it is possible that enzymatic debridement agents do remove biofilms. This hypothesis has not been studied.

In addition to the EPS that forms around bacteria in a biofilm, it has been shown that DNA also may serve as biofilm glue. Researchers Mattick and his colleagues reported that bacteria exposed to DNase I, which breaks down DNA, could not form a matrix.[21] This suggests that this enzyme may be useful in preventing and/or reducing bacterial biofilm formation.
Recently, low doses of the macrolide antibiotic, erythromycin, have been noted to be effective for treatment of chronic, lower-respiratory tract, biofilm infections. Using experimental models, Mitsuya and his colleagues found that erythromycin may have an inhibitory effect on the formation of biofilms.[22] This approach was further studied by Yamasaki and colleagues in an in-vitro and in-vivo mouse model of S. aureus infections. Their study demonstrated that a combination of roxithromycin, a macrolide antibiotic, and imipenem, a thienamycin (Beta-lactam antibiotic), is a potentially effective treatment for S. aureus biofilm-associated skin infections.[23] In addition, they demonstrated that the treatment can induce the invasion of polymorphonuclear leukocytes into the biofilm. Further studies are necessary to elucidate the precise mechanisms of these macrolide antibiotics either acting alone or in concert with other antibiotics.

It is certainly intriguing to hypothesize that biofilms play a role in the chronic nature of chronic wounds. Additional work is needed to substantiate the author’s hypothesis that biofilms play a significant role in delayed wound healing. Several potential new approaches for preventing, eliminating, or controlling biofilms are being investigated and hold the potential for aiding chronic wound therapy and soft-tissue infection. This area of biofilms in chronic human disease is receiving attention from the National Institute of Heath as evidenced by their recent request for applications for research support in the area of biofilm research.

Acknowledgments

The author wishes to thank Esperanza Welsh, MD, Carlos Ricotti, MD, and Alejandro L. Cazzaniga for the images used as figures for this article and William H. Eaglstein, MD, for his editorial assistance.