Bacterial Toxins and Wound Healing

Bacteria and Wounds Bacteria are ubiquitous in the geography of the human body. In the skin, the average human being harbors at least 200 species of bacteria, totaling more than 10¹² organisms.1 Therefore, when the skin is broken by trauma or disease, bacteria are also ubiquitous in wounds. When discussing the presence of bacteria in an open wound of a human host, three conditions are noted with respect to their presence on or in the tissue, their impact on the healing of the wound, and the associated immune response from the host. The first condition is bacterial contamination or the simple existence of bacteria on the surface of the wound. Contamination is specifically defined as the presence of non-proliferating organisms on the superficial tissues. Contaminating bacteria do not elicit an immune response from the host and do not impact the healing process. The second condition, bacterial colonization, is differentiated from contamination in that it refers to proliferating organisms on the wound surface - bacteria that have adhered to the superficial tissues and have begun to form colonies. Colonization is also characterized by a lack of immune response from the host and generally is not believed to impact or interfere with the healing process.2 Wounds that contain nonviable tissue (ie, slough and/or eschar) offer a particularly hospitable environment for colonization because the dead tissues provide a ready source of nutrients for the growing bacterial colonies. In the third condition, bacterial infection, proliferating bacteria are not only present on the surface of the wound or in nonviable tissue, but have also invaded healthy, viable tissue to such a depth and extent that they elicit an immune response from the host. Local clinical signs of tissue redness, pain, heat, and swelling generally characterize this immune response, along with an increase in exudate production or purulence. Bacterial infection delays and may even halt the healing process. The mechanism of this healing delay involves competition between host cells and bacterial cells for oxygen and nutrients and increased host cell production of inflammatory cytokines and proteases in response to the bacteria and their associated toxins.3 Critical Colonization In chronic wound management, clinicians have generally accepted that colonization of most chronic wounds is common - perhaps even unavoidable - and have primarily focused their attention on avoiding or managing wound infection due to its impact on healing as well as on overall patient morbidity. However, focus has recently shifted due to recognition that healing progress may be impaired by another bacterial condition - "critical colonization" - that occurs before invasive infection of the wound tissues.4,5 A relatively new concept, critical colonization is not yet validated in terms of distinct criteria or data. This indistinct condition represents a transition state between surface colonization by bacteria that does not impair the healing process and invasion of those bacteria into viable tissues. Critical colonization may eventually be better defined by local biochemical values than by bacterial numbers and, at present, is based on clinical observations. The current working definition of critical colonization is the presence of bacteria - still on the surface of the wound, not yet into the deeper tissues - but to such an extent that the build-up of their secreted toxins, as well as host cell cytokines and proteases, impairs the healing progress. Because critical colonization does not involve the invasion of bacteria into viable tissues, local clinical signs of redness pain, heat, and swelling usually will not manifest. However, other more subtle clinical signs are present that may indicate superficial bacterial levels may be delaying healing. The critically colonized wound can appear healthy, with an absence of nonviable tissue. However, the granulation tissue may have a characteristic color and texture - where healthy granulation tissue is often described as beefy red, the granulation tissue in a critically colonized wound may appear bright red in color, foamy in appearance, and may bleed quite easily. Critically colonized wounds also may exhibit new areas of tissue breakdown, bridging of epithelium, and sudden odor and increased exudate production.5 Yet in many cases, the only sign of critical colonization of the wound surface may be a plateau in healing progress.4 Bacterial Toxins and Wound Healing Bacterial toxins are thought to play a key role in delayed healing due to critical colonization. Toxins, by definition, are chemicals that produce a toxic result or adverse health effect (eg, poisons such as snake venom or strychnine); therefore, bacterial toxins are chemicals produced by bacteria that enable them to cause an adverse health effect in their host. These toxins also may be considered determinants of bacterial virulence. In other words, bacteria that produce potent toxins are more virulent or pathogenic than those that produce weaker toxins and, therefore, are able to cause disease at lower concentrations. Although some bacteria species are almost always associated with disease, the manifestation of that disease will depend on several variables, including the quantity or number of the bacteria present in the host, the strength or virulence of the bacteria (eg, the potency of their toxins), and the immune status or strength of the host. Many bacterial toxins have specific systemic effects in the human host that are well characterized and recognized as diseases, such as diptheria (caused by the diptheria toxin secreted from Corynebacterium diptheriae) and cholera (caused by cholera toxin secreted from Vibrio cholerae). Bacterial toxins also may have specific local effects in the environment of the superficial wound tissues that impair healing progress. Bacteria produce two primary types of toxins, distinguished by their chemical makeup, their source, and the mechanism of their release from the bacteria: exotoxins and endotoxins. Exotoxins. Exotoxins are soluble proteins produced by both Gram-positive and Gram-negative bacterial species during active proliferation or exponential growth. Secreted extracellularly by the bacteria and subsequently diffused into the host environment, exotoxins are somewhat similar to enzymes in that 1) once released from the bacteria, they bind to and degrade specific target substrates and 2) they may be denatured by heat. Exotoxins may be either very narrow or very broad in their substrate specificity. Human substrates for bacterial exotoxins include cells, tissues, and fluids. Examples of well-known exotoxins with very narrow substrate specificity include tetanus toxin (secreted by Clostridium tetani) and botulism toxin (secreted by Clostridium botulinum), both of which attack only neuron cells (see Table 1). Exotoxins produced by common wound bacteria (eg, Staphylococci, Streptococci, Pseudomonas) tend to be broader in terms of their substrate specificity, attacking many types of cells and tissues, resulting in generalized tissue necrosis at the wound surface. Endotoxins. Bacterial endotoxins are composed of lipopolysaccharide. In general, they are much less potent than exotoxins and because they are not protein in composition, they are heat stable. Toxicity is usually due to the lipid portion of the toxin, while immunogenicity is usually associated with the polysaccharide portion. Endotoxins are associated with Gram-negative bacterial species only. They serve as a constituent of the Gram-negative bacteria's cell wall and are thought to play a role in cell wall permeability as well as in the ability of the bacteria to form biofilms. Although proliferating bacterial cells actively release exotoxins, endotoxins are only released in small amounts during cell proliferation. However, they are freed from their location in the cell wall in significant amounts when the Gram-negative bacterial cell is lysed, either by host phagocytic cells or by antimicrobial chemicals. Endotoxins in the wound environment have been found experimentally to stimulate the production of inflammatory mediators such as TNF-alpha and the interleukins, which in turn induce the production of endogenous matrix metalloproteases (MMPs).6,7 Increased levels of MMPs are known to exist in many types of nonhealing wounds and are believed to contribute to the local destruction of growth factors, receptors, and tissue components.8 Clinical and research data demonstrate that bacterial endotoxins have a detrimental effect on wound tensile strength. Endotoxins have been found to decrease collagen deposition and cross-linking and are associated with surgical wound dehiscence.9 Controlling Bacterial Toxins in the Local Wound Environment Controlling bacterial exotoxins and endotoxins in the local wound environment is not straightforward. Lowering bacterial counts using topical antimicrobials may reduce the amount of exotoxins being produced, but antimicrobials do not address the exotoxins that have already been produced and released. In fact, destroying bacteria is what releases endotoxins from Gram-negative bacteria's cell wall, so topical antimicrobials may actually contribute to an increase in local endotoxin levels. Absorbent dressings such as alginates, hydrofibers, and foams will absorb wound fluids in bulk and may take up some of the toxins, but they are not selective for toxin reduction. However, one material - activated charcoal - specializes in the binding and removal of chemicals such as bacterial toxins. Activated charcoal is well known for its ability to selectively adsorb chemical poisons. In order to understand how activated charcoal works, it is important to first understand exactly what it is. Activated charcoal. Regular charcoal is the by-product of burning organic (or carbon-based) matter to leave behind only the carbon component. Activated charcoal is a variation of charcoal produced by burning the organic matter in a superoxygenated environment to yield what might be called the "Thomas' English Muffin" version of charcoal - that is, charcoal that has an extremely high density of pores or "nooks and crannies." The increased porosity of activated charcoal creates a tremendous increase in its surface area relative to its volume. For example, 1 g of activated charcoal may contain up to 1,000 square meters of surface area. This surface area component is what defines the "activation" of the charcoal. Once produced, activated charcoal is a fine powder that can be further processed into grains, pellets, fibers, cloths, and papers. Activation equals adsorption. The extremely large surface area of activated charcoal makes it one of the most efficient adsorptive agents available. Adsorption is a process in which molecules physically adhere to a surface with which they come into contact - almost like molecular "gravity." Adsorption or the binding of molecules or particles to a surface must be distinguished from absorption, the filling of pores in a solid. Adsorption is a surface phenomenon while absorption is a volume phenomenon. Because activated charcoal's increased surface area is created via the production of pores in the carbon material, the pores also could be filled - ie, activated charcoal is also an absorptive agent. However, consider the amount of hollow space or pores in the aforementioned gram of activated charcoal relative to the amount of surface area created by those same pores (1,000 square meters) - adsorption is the dominant process. The adsorption capacity of activated charcoal gives it the ability to selectively remove small molecules such as the bacterial toxins or other poisons from biological environments. Activated charcoal has a long history of medical use as a universal poison or toxin antidote. Nontoxic, odorless and tasteless, it can be taken orally as a slurry in water or sorbitol. Used increasingly in emergency rooms as the first-line method of decontamination in accidental or intentional poisonings or overdoses, activated charcoal effectively binds and neutralizes most poisons (exceptions are alcohols, strong acids, or alkalis and heavy metals such as gold, lithium, iron, potassium, and mercury). Activated charcoal is also widely used as a deodorizing agent - many odor molecules are adsorbed onto the large surface area of the activated charcoal, which prevents the volatile odor molecules from reaching receptors in the nose. Activated charcoal also has been shown to adsorb various bacterial toxins as well as the bacteria themselves.10-12 In a cloth format, activated charcoal has demonstrated the ability to adsorb bacteria from solution.13 Theoretically, the placement of an activated charcoal layer at the surface of a wound could result in physical adsorption of bacteria as well as their freely secreted exotoxins from the wound surface and from the wound fluid. Bacteria adsorbed to the surface of activated charcoal alone would remain viable; however, a combination of activated charcoal with an antimicrobial component could not only bind but also kill the bacteria. In the case of Gram-negatives, their released endotoxins also would be adsorbed by the charcoal component. Activated charcoal and silver: synergies in topical wound management. A topical wound dressing consisting of an activated charcoal cloth impregnated with 0.15% metallic silver (Actisorb Silver 220; Johnson + Johnson Wound Management, Somerville, NJ) provides just such a combination of technologies - bacterial adsorption and killing with toxin adsorption. This technology has been associated with in vitro reductions in colony forming units of common wound pathogens as well as with in vivo improvements in wound size and epithelialization. In a single center, randomized controlled study of 40 patients with venous leg ulcers, the silver-impregnated activated charcoal dressing was compared to conventional therapies and demonstrated a three-fold higher healing rate as well as statistically significant improvements in epithelialization and wound area reduction.14 The results of five observational studies conducted in Europe aggregated the experience of 12,444 wounds of various etiologies that were treated with the silver dressing.15 All studies included 6-week follow-up assessment and collected most of the same efficacy and safety data, allowing the results to be pooled. During these studies, some patients also may have received other complimentary therapies, such as systemic antibiotics. These wounds were persistent (average duration of wound was 9 months before activated charcoal/silver dressing treatment) and had been unresponsive to previous treatment. Wounds included in these studies included leg ulcers (7,798), diabetic foot ulcers (1,493), pressure ulcers (2,435), and other wounds (718). Some of the key findings from these studies include a healing rate of 40.7%, an 88.5% reduction in wounds showing signs of infection, and significant reduction in wound odor and wounds requiring antibiotic treatment. The dressing also has documented effects on toxin binding, which may be related to the mechanism of action for its clinical effects.16 In in vitro studies, the dressings were pre-wet and exposed to Pseudomonas aeruginosa solutions of differing bacterial concentrations (10⁴-10⁵, 10⁵-10⁶, and 10⁶-10⁷ cfu/mL). Aliquots of bacterial solutions after incubation with dressings were sampled. Bacterial counts and endotoxin content of the aliquots were determined and showed significant log reductions in bacterial count after only 3 hours of exposure without inducing significant levels of endotoxin to be released into solution. The dressings were also exposed to Escherichia coli endotoxin standard to determine endotoxin-binding capability. Pre-wet dressings were exposed to solutions of known endotoxin concentration over 24 hours (37 degrees C). Aliquots of the endotoxin solution were taken after 0.5 hours, 3 hours, and 24 hours of contact with the dressing and analyzed for toxin content. The dressings bound increasing levels of bacterial endotoxin over a 24-hour period with significant differences in binding (P = 0.002) compared to controls after 3 hours and 24 hours. The total binding capacity after 24 hours was more than 4.5 times greater than gauze. Conclusion Recent recognition of the negative impact of local biochemical imbalances present in nonhealing wounds has re-focused attention on the role of bacteria in delaying wound healing. Bacteria present in the wound environment secrete exotoxins during active proliferation, and Gram-negative species release endotoxins upon cell lysis or death. These bacterial toxins have direct effects on tissue necrosis and stimulate the production of local inflammatory mediators such as cytokines and proteases, which are known to impair healing progress at elevated levels. The use of topical antimicrobial agents such as antiseptic solutions, antibiotics, and sustained released antiseptic dressings can play a role in reducing bacterial numbers and exotoxin release; however, they do not address the endotoxin release that follows Gram-negative cell lysis. High surface area adsorptive agents such as activated charcoal, which can specifically bind and reduce endotoxin levels, may be helpful adjuncts to topical wound management and infection control. - OWM

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