Christine A. Cochrane

The role of endogenous and exogenous enzymes in chronic wounds: a focus on the implications of aberrant levels of both host and bacterial proteases in wound healing.

Cutaneous wound healing is orchestrated by a number of physiological pathways that ultimately lead to reformation of skin integrity and the production of functional scar tissue. The remodeling of a wound is significantly affected by matrix metalloproteinases (MMPs), which act to control the degradation of the extracellular matrix (ECM). Regulation of MMPs is imperative for wound healing as excessive levels of MMPs can lead to disproportionate destruction of the wound ECM compared to ECM deposition. In addition to human MMPs, bacterial proteases have been found to be influential in tissue breakdown and, as such, have a role to play in the healing of infected wounds. For example, the zinc‐metalloproteinase, elastase, produced by Pseudomonas aeruginosa, induces degradation of fibroblast proteins and proteoglycans in chronic wounds and has also been shown to degrade host immune cell mediators. Microbial extracellular enzymes have also been shown to degrade human wound fluid and inhibit fibroblast cell growth. It is now being acknowledged that host and bacterial MMPs may act synergistically to cause tissue breakdown within the wound bed. Several studies have suggested that bacterial‐derived secreted proteases may act to up‐regulate the levels of MMPs produced by the host cells. Together, these findings indicate that bacterial phenotype in terms of protease producing potential of bacteria should be taken into consideration during diagnostic and clinical intervention of infected wound management. Furthermore, both host MMPs and those derived from infecting bacteria need to be targeted in order to increase the healing capacity of the injured tissue. The aim of this review is to investigate the evidence suggestive of a relationship between unregulated levels of both host and bacterial proteases and delayed wound healing.

Silver resistance in MRSA isolated from wound and nasal sources in humans and animals

Methicillin‐resistant Staphylococcusaureus (MRSA) colonises skin, nasal passages and dermal wounds. Methods used to manage wounds infected and colonised with MRSA often include the use of topical antiseptics such as ionic silver and iodine. The objectives of this study were to determine the prevalence of silver‐resistance (sil) genes in MRSA and methicillin‐resistant coagulase‐negative staphylococci (MR‐CNS) isolated from wounds and nasal cavities of humans and animals, and also to determine the susceptibility of sil‐positive and sil‐negative MRSA isolates to a silver‐containing Hydrofiber® (SCH) wound dressing, on planktonic silE‐positive and silE‐negative MRSA. Polymerase chain reaction was used to determine the presence of three silver‐resistance (sil) genes, silE, silP and silS in 33 MRSA and 8 methicillin‐resistant staphylococci (MR‐CNS). SilP and silS genes were absent in all isolates tested; however, two MRSA strains were found to contain the silE gene, together with one isolate of MR‐CNS. Phenotypic resistance of the silE‐positive strains and their susceptibility to the SCH dressing was evaluated using the zone of inhibition test on Mueller Hinton agar, and confocal laser microscopy using a live/dead fluorescent stain. Results confirmed that the SCH dressing was effective in killing all MRSA strains with and without the silE gene. First, this study showed that the prevalence of sil genes was low in the isolates investigated; and secondly, that the presence of a silver‐resistance gene (silE) in MRSA and MR‐CNS did not afford protection to the organism in the presence of a SCH wound dressing. The use of topical antiseptics in chronic wound care should be considered before the use of antibiotics that can result in their overuse and the risk of further resistance.

Prevalence of silver resistance genes in bacteria isolated from human and horse wounds.

The aim of this study was to investigate the prevalence of silver resistance genes in 172 bacterial strains which had been isolated from both human and equine wounds. PCR screening for 8 currently named genes in 3 silver resistance transcriptional units, silE, silRS and silP, silCBA and silF was performed on total DNA extracted from all clinical isolates. Plasmids were isolated from sil-positive strains to determine if the genes were present on the chromosome. MICs and zone of inhibition assays were utilised to examine phenotypic resistance to silver nitrate and ionic silver. Evidence of silver resistance genes was demonstrated in six strains of Enterobacter cloacae, an organism rarely implicated as a primary pathogen in chronic wounds. MIC data showed that all strains were inhibited at silver nitrate concentrations > or =5mg/L. When tested against a silver-containing absorbent wound dressing all strains showed inhibition of growth after 24h. In MIC and zone of inhibition studies, inhibition was evident but reduced in strains which contained sil genes. Although sil genes were found in six of the wound isolates studied, the genes were consistently associated with a non-pathogenic bacterium. Furthermore, investigation of phenotypic resistance in sil-positive isolates showed that silver continued to be effective.

Microbiology of equine wounds and evidence of bacterial biofilms

Horse wounds have a high risk of becoming infected due to their environment. Infected wounds harbour diverse populations of microorganisms, however in some cases these microorganisms can be difficult to identify and fail to respond to antibiotic treatment, resulting in chronic non-healing wounds. In human wounds this has been attributed to the ability of bacteria to survive in a biofilm phenotypic state. Biofilms are known to delay wound healing, principally due to their recalcitrance towards antimicrobial therapies and components of the innate immune response. This study describes the presence of bacterial biofilms within equine wounds. Thirteen 8-mm diameter tissue samples were collected from (n=18) chronic wounds. Following histological staining, samples were observed for evidence of biofilms. Fifty one wounds and control skin sites were sampled using sterile swabs. Control skin sites were on the uninjured side of the horse at the same anatomical location as the wound. The isolated bacteria were cultured aerobically and anaerobically. The biofilm forming potential of all the isolated bacteria was determined using a standard crystal violet microtitre plate assay. Stained tissue samples provided evidence of biofilms within 61.5% (8 out of 13) equine wounds. In total 340 bacterial isolates were identified from all the equine wound and skin samples. Pseudomonas aeruginosa and Enterococcus faecium were the most predominantly isolated bacterial species from equine wound and skin samples respectively. Staphylococcus was the most commonly isolated genus in both environments. Bacteria cultured from chronic and acute wounds showed significantly (P<0.05) higher biofilm forming potential than bacteria isolated from skin. This paper highlights preliminary evidence supporting the presence of biofilms and a high microbial diversity in equine chronic wounds. The presence of biofilms in equine wounds partly explains the reluctance of many lower limb wounds to heal. Non-healing limb wounds in horses are a well documented welfare and economic concern. This knowledge can be used to shape future treatments in order to increase the healing rate and decrease the costs and suffering associate with equine wounds.

Application of an in vitro model to evaluate bioadhesion of fibroblasts and epithelial cells to two different dressings

The cellular component of a healing wound consists of many cell types and the environment in which these cells grow is important to the rate and quality of healing which can be influenced by the type of dressing used. The most commonly used dressings are traditional gauze-type dressings. In many cases these dressings may adhere to the wound surface, and subsequent removal is often traumatic, causing pain and tissue reinjury. Some modern gelling dressings have been developed to overcome this adherence problem. In order to evaluate in more detail cell-dressing interactions, an in vitro model has been developed utilising wound fibroblasts and epithelial cells. Quantitative evaluation of adherence of cells cultured with a traditional gauze or a new gelling dressing has been undertaken using radiolabel and manual counting techniques. Scanning electron microscopy has been used to visualise the cells adherent to dressings allowing evaluation of their adhesion-morphology. The results show differential attachment of cells to viscose and gelling fibres of the dressings; considerably reduced cell adhesion to the gelling fibre was evident, and it was apparent that cells adhered predominantly to the viscose component of the dressing. This model can be used to investigate and compare the adhesion of cells to different dressings and their components.

Effect of growth factors on the characteristics of cells associated with equine wound healing and sarcoid formation

Wound healing in equidae is delayed and more complicated than in other species. These complications arise from a condition known as exuberant granulation tissue formation. The lower limb of the horse is frequently slower to heal than other parts of the body and has a particular tendency to produce excess (exuberant) granulation tissue. Sarcoids are tumor‐like lesions of the skin which often appear at the site of wounds. This study compared the growth characteristics of the sarcoid and granulation tissue‐derived cells with normal dermal fibroblasts grown from primary cell cultures. All three cell types had distinct morphologic differences. Growth rate studies showed that the sarcoid and granulation tissue‐derived cells grew at a slower rate than the normal cells. The addition of the growth factors epidermal growth factor, acidic fibroblast growth factor, and basic fibroblast growth factor selectively stimulated the replication of normal and sarcoid‐derived cells but inhibited the growth of granulation tissue‐derived cells. In contrast, transforming growth factor‐β was not preferentially inhibitory for the granulation tissue‐derived cells. The addition of growth factors to the medium also produced distinct alterations in the morphology of the cells.

Biofilms and Role to Infection and Disease in Veterinary Medicine

Biofilms play an increasing role within the medical and veterinary community. Due to the increased resistance of a biofilm, they can have direct and indirect effects upon a range of infections and diseases including chronic non-healing wounds, implant/prosthesis infection and mastitis. These problems can have significant effects on other industries, for example mastitis can have a detrimental effect on milk yield in the dairy industry. The degree of severity biofilms can cause increases the pressure on the veterinary industry to diagnose and treat infections and diseases quicker and with more effective results. With maturity, biofilms may become more resistant to the effects of antimicrobials which make the infection harder to treat. As elaborated on in previous chapters, many antibiotherapy treatments currently used to treat bacterial infections are aimed at planktonic bacterial cells as opposed to cells encased in a biofilm; this makes their treatment increasingly problematic. Without adequate diagnostic and treatment protocols to treat veterinary biofilms, their impact will remain a significant challenge.

The role of polyphosphates in the sequestration of matrix metalloproteinases

This study outlines the potential of a novel therapeutic dressing for the management of chronic wounds. The dressing incorporates polyphosphate, a non toxic compound with a number of beneficial characteristics in terms of wound healing, in a foam matrix. The aim of this study was to identify the potential of polyphosphate incorporated in the foam dressing to sequester the activity of matrix metalloproteinases (MMPs) and proteases derived from Pseudomonas aeruginosa. Methods used included gelatin zymography and milk‐casein agar plate analysis. Results have shown that this dressing is effectively capable of reducing the levels of MMP‐2 and MMP‐9 in both their active and latent forms using an in vitro model. The dressing also demonstrated the compounds potential in the regulation of P. aeruginosa derived proteases.

In vitro study of the effectiveness of different dressings for debriding fibrin in blood clots from horses.

Franz diffusion cells containing uniformly dehydrated equine blood clots to simulate fibrinous eschar were used to measure the rate of debridement of fibrin by novel and traditional wound dressings, under standardised conditions of temperature, pH and humidity. Significant increases in protein breakdown occurred within 24 hours with all the dressings, but not thereafter. In general, dressings hydrated in normal saline were better as debriding agents than dressings hydrated in water. Autolytic debriding agents were 47 per cent more effective than chemical debriding agents; specifically, hydrofibre and gauze dressings hydrated in saline broke down more than 3500 μg/ml of protein whereas dressings impregnated with proteolytic enzymatic agents digested less than 1400 μg/ml.

Wound Infection and Biofilms

The existence of biofilms in wounds is now clearly evident, with their role being significant in inhibiting the healing process. All chronic wounds are colonised by bacteria encased within a polymicrobial biofilm contributing to a prolonged inflammatory response in the host. Proteases released by sessile bacteria, possibly in synergy with human proteases, may destroy human growth factors and other tissue proteins thought to be essential to the wound-healing process. Subsequently a better understanding of the physiology of biofilms in wounds may aid in the development of more effective methods of treatment and, ultimately, better prognoses for patients.