Article

Maximising the secondary beneficial effects of larval debridement therapy

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Laboratory-based clinical investigations have shown that maggots and their secretions promote, among other activities, fibroblast motogenesis and angiogenesis. These events would contribute to re-granulation if translated to the wound environment. Maggot secretions also have ascribed antibacterial actions and may exhibit anti-inflammatory effects. Many of these biological events would be lost in the presence of necrotic tissue, making debridement a prerequisite for the release of larval-secreted secondary beneficial effects on the wound. We argue that Larval Debridement Therapy (LDT) should be considered as a primary and secondary treatment in wound management, with the primary application designed to debride the wound, and with subsequent applications to the debrided wound targeted to cellular events that promote healing. This review lends support to a re-evaluation of larval application protocols, in order to optimally harness the potential secondary beneficial clinical effects of larval therapy.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Ammonia is produced that increases pH (Gottrup and Jorgensen 2011). This results in the eradication and inhibition of biofilm formation (Nigam et al. 2010, Pritchard andNigam 2013). 3. Promotion of tissue growth by stimulating regeneration: this involves stimulation of growth of granulation tissue (Pritchard and Nigam 2013, Nigam and Morgan 2016, Bazalinski et al. 2019. ...
... This results in the eradication and inhibition of biofilm formation (Nigam et al. 2010, Pritchard andNigam 2013). 3. Promotion of tissue growth by stimulating regeneration: this involves stimulation of growth of granulation tissue (Pritchard and Nigam 2013, Nigam and Morgan 2016, Bazalinski et al. 2019. Significantly higher levels of hepatocyte growth factor (HGF), a critical molecule in cutaneous wound healing, have been associated MT treatments (Honda et al. 2011). ...
... 5. Consider the potential for application of differentially prepared larvae in a multi-stage treatment plan. Pritchard and Nigam (2013) suggest repeated application of maggots is critical in successful MT: the first batch should conduct rapid debridement, and once their enzymatic activity has been exhausted, either a second batch or their sterile secretions should be applied to the wound bed to stimulate angiogenesis. 6. Conduct randomized clinical trials of MT that will produce a robust body of data to encourage general acceptance by medical professionals. ...
Article
Blowfly larvae of Lucilia sericata (Meigen) (Diptera: Calliphoridae) are well established as debridement agents in nonhealing wounds. Maggot therapy (MT) experienced reduced application following adoption of Penicillin and other antibiotics, but the advent of antibiotic resistance and the growing global wound burden have boosted demand for new therapies. The mechanisms of action are well accepted, with debridement, disinfection, biofilm destruction, and inhibition, as well as the stimulation of tissue growth uniformly acknowledged as a remarkable biotherapy. The mechanisms of action, while well-recognized, are still being examined. The efforts to understand isolated aspects of a complex system, have resulted in a tendency to approach the field from simplified viewpoints that remove the holistic system of the larvae. Furthermore, clinical studies have conflated wound debridement and healing in definitions of ‘success’. Thus, both in vitro and clinical studies have reported mixed results, presenting some uncertainty regarding the utility of MT that prohibits routine clinical adoption. This review builds from the generally accepted basic mechanisms to justify a future for MT that encompasses larval-bacterial symbioses as the basis to a holistic system. Symbioses are well documented in the Insecta, and literature in MT supports the existence of established symbiotic associations that provide enhanced debridement action. The future of MT requires consideration of a biological system that confers enhanced antimicrobial action on larvae when selective pre-exposure to carefully selected symbionts is adopted. In treating contemporary infections, there is much to be gained from reflecting on the natural biology of the organism, as MT was used with success long before we sterilized the system.
... In addition, the composition of larval products may change in response to local clinical conditions in the case of larval therapy, which is not the case when isolated materials are used in laboratory experiments. Thus, although there is an indication of medical benefit beyond debridement (30,31), clear proof of the clinical relevance of these effects is still required. ...
... In summary, the production and composition of wound fluid can be considered as a secondary beneficial effect of LDT (30), as the normalisation of exudate is connected with the removal of dead tissue and its associated microbial bioburden. As the debridement process progresses, the wound bed regenerates by laying down healthy granulation tissue. ...
Article
Full-text available
Wound bed preparation (WBP) is an integral part of the care programme for chronic wounds. The acronym TIME is used in the context of WBP and describes four barriers to healing in chronic wounds; namely, dead Tissue, Infection and inflammation, Moisture imbalance and a non-migrating Edge. Larval debridement therapy (LDT) stems from observations that larvae of the blowfly Lucilia sericata clean wounds of debris. Subsequent clinical studies have proven debriding efficacy, which is likely to occur as a result of enzymatically active alimentary products released by the insect. The antimicrobial, anti-inflammatory and wound healing activities of LDT have also been investigated, predominantly in a pre-clinical context. This review summarises the findings of investigations into the molecular mechanisms of LDT and places these in context with the clinical concept of WBP and TIME. It is clear from these findings that biotherapy with L. sericata conforms with TIME, through the enzymatic removal of dead tissue and its associated biofilm, coupled with the secretion of defined antimicrobial peptides. This biotherapeutic impact on the wound serves to reduce inflammation, with an associated capacity for an indirect effect on moisture imbalance. Furthermore, larval serine proteinases have the capacity to alter fibroblast behaviour in a manner conducive to the formation of granulation tissue. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.
... These methods are also less stressful for patients [61,64,73]. Biobags and commercially available specialist dressings are not effective in penetrating wounds because larvae should have unobstructed access to the necrosis [26,76]. If the relevant area is particularly sensitive, or if the patient is afraid of larvae, they should only be kept in the wound for up to 24-48 h (this recommendation only applies to free-range larvae; the biobag should remain for 72 h). ...
... These methods are also less stressful for patients [61,64,73]. Biobags and commercially available specialist dressings are not effective in penetrating wounds because larvae should have unobstructed access to the necrosis [26,76]. If the relevant area is particularly sensitive, or if the patient is afraid of larvae, they should only be kept in the wound for up to 24-48 hours (this recommendation only applies to free-range larvae; the biobag should remain for 72 hours). ...
Article
Full-text available
The process of successful wound healing depends on effective debridement and infection control. One method of wound debridement, known since antiquity, is based on the use of fly larvae. Solid scientific evidence proves that maggot debridement therapy (MDT), like surgical intervention, can be effectively and safely used to remove necrotic tissue. Based on a review of the related literature, this study was designed to assess the effectiveness of chronic wound cleansing with the use of larvae of Lucilia sericata (Phaenicia sericata). Maggot therapy, applied in wound debridement and treatment, is a safe and effective method. Its benefits are associated with debridement, disinfection and faster tissue growth. MDT may reduce the duration of antibiotic therapy and the need for hospitalization, or it may decrease the number of outpatient visits required. It is a relatively cost-effective method, and, in addition to financial gains, it may reduce the frequency of inpatient treatment. In the literature, an increasing amount of scientific evidence confirms that such treatment can effectively reduce the biofilm and bacterial load in a wound.
... Additionally, an abundance of published literature, including large clinical trials, supports the view that medicinal maggots work extremely effectively to clear away dead, unhealthy tissue [2][3][4]. Scientific studies also show that maggots exhibit unique and effective antimicrobial and wound healing properties and can be of great benefit to the wound due to their considerable secondary benefits, which include the ability to destroy antibioticresistant strains of bacteria [5,6]. Maggots do not harm living tissue; this selectivity is believed to be due to the inability of their enzymes to digest healthy, living tissue [6]. ...
... Scientific studies also show that maggots exhibit unique and effective antimicrobial and wound healing properties and can be of great benefit to the wound due to their considerable secondary benefits, which include the ability to destroy antibioticresistant strains of bacteria [5,6]. Maggots do not harm living tissue; this selectivity is believed to be due to the inability of their enzymes to digest healthy, living tissue [6]. However, given its efficiency and cost effectiveness , Maggot Therapy has a surprisingly low uptake and is increasingly regarded as an underutilised, last resort clinical treatment for chronic wounds [2,7]. ...
Article
Maggot Therapy is an established, effective treatment for chronic infected wounds. Despite its worldwide success, it suffers from poor public regard and acceptance. In 2019, the primetime BBC Medical Drama, Casualty, decided to run a Maggot Therapy storyline over four episodes of its recent series (series 33). Our study focusses on an evaluation of the impact of this storyline on changes in public awareness and acceptability of Maggot Therapy. The evaluation comprised an online questionnaire (administered through an independent private research company). Our results showed that exposure to the BBC Casualty maggot storyline was associated with a significant increased awareness of Maggot Therapy. Additionally, this resulted in a more positive perception and general acceptability of the treatment, and a decrease in negative responses towards it. Post-wave participants were also more likely to find Maggot Therapy acceptable for their own wound. Our findings suggest that television storylines and narratives are a useful route to raise awareness, inform and educate viewers about important health-related issues. Our study supports the notion that for effective treatments like Maggot Therapy, which often evoke feelings of disgust and reluctance, the persuasive effects of entertainment education could help to transform perception and acceptability.
... There are three major benefits of WBP by MDT for CLI patients [19][20][21]. First, selective and efficient debridement can be performed. Even for wounds with unclear margins with the surrounding normal tissues, maggots selectively englobe and liquefy only necrotic tissues [22]. ...
... Maggot secretions promote the cellular processes, which is related to increased healing activity. Such processes include activation of fibroblast migration, angiogenesis (the formation of new blood vessels from pre-existing vessels) within the wound bed and an enhanced production of growth factors within the wound environment [19,20]. These are believed to have been part of the success of WBP with our patient although his skin perfusion pressure (SPP) in the dorsum of the foot was only 15 mmHg before MDT. ...
Article
Full-text available
Ischaemic skin ulcer occurred on the foot of a 73-year-old man who had a history of fulminant myocarditis with severe cardiac dysfunction. We attempted wound bed preparation by maggot debridement therapy and salvaged his limb. It can be one of the adjuvant treatment strategies for critical limb ischaemia.
... Assistant Professor of Podiatric Medicine, Surgery and Biomechanics, Western University of Health Sciences College of Podiatric Medicine, Pomona, CA, USA The beneficial effects of maggots are evidenced in the historical paintings of Mayans, Burmese, Chinese and aboriginal people in Australia (Pritchard and Nigam, 2013). Maggots were used by Napoleon's chief surgeon and by confederate medical officers in the Civil War to enhance tissue granulation and shorten the healing process (Larrey, 1829). ...
Article
Full-text available
Maggot debridement therapy is used extensively in the UK in both community and hospital situations, but remains a potentially under-used modality in many wound care markets. It promotes wound healing by performing three key processes: debridement, disinfection and growth-promoting activity. It can be used for the debridement of non-healing necrotic skin and soft tissue wounds, including pressure ulcers, venous stasis ulcers, neuropathic foot ulcers and non-healing traumatic of post-surgical wounds. With the increase in chronic diabetic foot wounds, maggot debridement therapy is a promising tool for health professionals dealing with difficult wounds. This article presents an overview of the research evidence surrounding maggot debridement therapy that serves as a guide to health professionals who may be users of this form of treatment now and in the future.
... It has even been suggested that MDT be considered both primary and secondary treatment in wound management, the primary action (debridement and disinfection) being a necessary prerequisite for the secondary effects on wound healing. 13 An emerging trend in the management of very chronic wounds in humans (e.g., diabetic, ischemic, or decubital ulcers) is "maintenance MDT," where treatment of 2-3 days' duration is repeated weekly until wound healing is advanced. 10 ...
Preprint
Full-text available
The first two articles in this 4-part series explored the question, Why do some infections persist and progress despite seemingly appropriate treatment?, as it pertains to wounds involving joints and other synovial structures. 1,2 Of the many possible reasons, most serious wound infections involve at least one, and usually a combination, of these factors: 3 1. extensive contamination, or bacterial burden that overwhelms the patient's resources 2. refugia which protect the bacteria from host defenses and antibiotic drugs 3. immunocompromise 4. poor perfusion 5. antibiotic insensitivity of the wound pathogen(s) These same factors, often in combination, also contribute to the persistence of wound infections that involve bone. In horses, wounds that involve bone range in severity, complexity, and long-term impact from those containing a thin sequestrum on the surface of the third metacarpus/tarsus that resolve with routine wound care after sequestrum removal, to septic osteomyelitis at the site of internal fixation that results in failure of the fracture repair and potentially in euthanasia. Yet in all cases, the principles of successful treatment are the same: • debride the devitalized or irreparably damaged bone and soft tissue • preserve and protect the vascular supply to bone and soft tissue • maintain or restore structural integrity at the site • control infection through appropriate local/regional and systemic antibiotic therapy • protect the wound from further contamination, desiccation, maceration, and trauma There are a number of review articles on the management of wounds involving bone in horses. 4-7 So, rather than plowing the same ground, this article examines some advances in wound care over the past 15 years as they relate to wound infections involving bone.
... [17] However, Pritchard and Nigam argue in favor of harnessing benefi t of subsequent applications to cellular events like fi broblast motogenesis and angiogenesis which will promote healing. [18] Medical-grade maggots are commercially available since 2004 [19] and today there is a resurgence of interest in MDT with 12 laboratories in 20 countries dispensing them at low cost. [20] There have been several studies attempting to identify how the maggots increase granulation in the wound bed. ...
Article
Full-text available
An ancient remedy, Maggots debridement therapy was reintroduced by William. S. Baer, an orthopedic surgeon who worked at John Hopkins Hospital at Baltimore, Maryland. Maggot debridement therapy is a popular technique for wound debridement. It is considered cheap and safe form of therapy to prepare healthy wound bed. Major constraints are unavailability of sterile/medical grade maggots. A 56-year-old male patient presented with a wound on leg containing maggots. This acted as a trigger to review and search literature about the present status of maggot therapy. Although, this form of therapy is a commonly practiced in Europe and North America, others needed to have a relook at this modality in view of the increasing prevalence of antibiotic resistance to infections.
... 11,12 In addition, other noted benefits of larval therapy have been suggested, including reperfusion, reduction in inflammation, and antifungal properties. 13,14 The ability of medicinal maggots to debride is primarily attributed to the way in which they feed. Being necrophagous by nature, the larvae break down and consume necrotic tissue enzymatically by a process of extracorporeal digestion. ...
Article
The effective use of larvae of the greenbottle fly, Lucilia sericata, in wound debridement requires a working knowledge of how feeding changes over time. Using a laboratory assay and bagged larval dressings, the effect of incubation time on larval feeding rates and body mass was investigated for up to 120 hours at 32°C. The mass of tissue digested increased significantly in incremental 24‐hour periods up to 72 hours, with no significant consumption occurring afterwards. Larval mass increased only up to 48 hours. A further test comparing the efficacy of a single 96‐hour application of larvae against two consecutive 48‐hour applications found that the mass of tissue digested in the latter was 14.3% higher than the former, a difference that was statistically significant. Current clinical guidance suggests a 4‐day application period for bagged larvae. Based on these results, an incubation time of 72 hours (3 days) for bagged larvae would be the most effective at the study temperature. However, it is acknowledged that wound temperature can vary, whereby feeding rates would likely differ. In view of this, we conclude that a period of 3 to 4 days is optimum for the application of larvae, and current guidelines should be adhered to.
... 23 Clinical studies have demonstrated maggot therapy to be safe and effective in patients both with and without diabetes and for many problematic wounds, including pressure ulcers, venous stasis leg ulcers, wound bed preparation prior to surgical closure, and a variety of other traumatic, infectious, and vascular wounds. [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] ...
Article
Cutaneous ulcers tend to become chronic and have a profound impact on quality of life. These wounds may become infected and lead to greater morbidity and even mortality. In the past, larvae (ie, maggots) of certain common flies (Lucilia sericata and Lucilia cuprina) were considered useful in ulcer management because they only remove necrotic tissue while promoting healthy tissue in the wound bed, thus helping wounds heal faster. Recently, maggots from several other fly species (Calliphora vicina, Calliphora vomitoria, Phormia regina, Chrysomya albiceps, Sarcophaga carnaria, and Hermetia illucens) have been shown in vitro to possess characteristics (ie, debridement efficacy and putative antimicrobial potentialities) that make them suitable candidates for possible use in clinical practice. This review presents a historical analysis of larval debridement and speculates future directions based on the literature presented.
... Maggot debridement therapy (MDT) or the use of maggots to treat wounds is one such therapy that has been in use for centuries. 5 Larvae of the green bottle fly Lucilia sericata are used in MDT. An important step in assisting chronic non-healing wounds progress through healing is wound bed preparation. ...
Article
Full-text available
Gurudutt Naik, Keith G Harding Welsh Wound Innovation Centre, Cardiff University, Cardiff, UK Abstract: Chronic wounds remain a challenge to most healthcare systems worldwide despite the technological advances we have seen to date. Many chronic non-healing wounds require alternative approaches, in addition to standard conventional therapies. Maggot debridement therapy (MDT) or the use of maggots to treat wounds is one such therapy that has been in use for centuries. We conducted a review of articles published in PubMed, NICE evidence documents, and linked literature with the aim of providing a brief perspective on the evolution of MDT, and the context in which maggot therapy is currently used along with evidence behind such methods. Keywords: wound healing, maggot debridement therapy, debridement, Lucilia sericata, chronic disease, larva
... On the other hand, the only study done on human models indicated no difference in bacterial count over time and only the studies performed on ex vivo models favoured the use of L. sericata secretions to reduce bacteria. Additionally, it might be difficult to control the right concentration levels of the secretions in in vivo studies ( Pritchard and Nigam, 2013). Thus, the results cannot be generalised for all individuals with leg ulcers. ...
Article
Full-text available
Maggot debridement therapy (MDT) is an effective method for debriding wounds such as leg ulcers, supporting the concept of wound bed preparation (Dumville et al, 2009; Soares et al, 2009; Mudge et al, 2014). New evidence is emerging to suggest that maggots might contribute to wound healing in other ways. For example, the reduction of biofilms and disinfection of wounds (Van Der Plas et al, 2008; Brown et al, 2012; Pritchard and Brown, 2015) plus regulation of protease levels (Chambers et al, 2003; Van Der Plas et al, 2009a; Van Der Plas et al, 2009b). This review will discuss research exploring new benefits of maggots in the management of individuals with chronic leg ulcers.
... 53,54 Larval debridement therapy has been used around the world to promoted wound healing. 55 Generally, larval debridement therapy has used the disinfected fly larvae of Lucilia sericata in the treatment of wounds resistant to conventional therapy. 56,57 However, large controlled clinical trials assessing benefits and risks of this therapy have not been performed. ...
Article
Full-text available
Animals in Healthcare Facilities: Recommendations to Minimize Potential Risks - Volume 36 Issue 5 - Rekha Murthy, Gonzalo Bearman, Sherrill Brown, Kristina Bryant, Raymond Chinn, Angela Hewlett, B. Glenn George, Ellie J.C. Goldstein, Galit Holzmann-Pazgal, Mark E. Rupp, Timothy Wiemken, J. Scott Weese, David J. Weber
... The proficiency of larval therapy in debriding wounds is well documented and has been proven clinically (Sun et al., 2014). Although the therapy is reputed to aid in various other aspects of wound treatment, such as disinfection (Kerridge et al., 2005) and the destruction of biofilm (van der Plas et al., 2008), and to accelerate wound healing (Pritchard & Nigam, 2013), it is primarily used for debridement. ...
Article
Larval therapy, the therapeutic use of blowfly larvae to treat chronic wounds, is primarily used in debridement. There are, however, gaps in current knowledge of the optimal clinical application of the therapy and mechanisms of action in the debridement process. Using an artificial assay, two studies were undertaken to investigate these aspects of larval debridement by Lucilia sericata Meigen (Diptera: Calliphoridae); the first studied the effects of the density of larvae on tissue digestion and larval mass, and the second considered the effects on the same parameters of incorporating protease inhibitors into the feeding substrate. The total mass of tissue digested increased with larval density until saturation was observed at 5.0-7.5 larvae/cm(2) . This range was considered optimal as lower doses resulted in the removal of less tissue and higher doses offered no additional tissue removal and appeared to exacerbate competition for feeding. In the second study, increased protease inhibitor concentration led to significant decreases in tissue digestion and larval mass, suggesting that serine proteases, particularly trypsin, may play major roles in larval digestion. Such information is important in elucidating the main constituents that make up larval digestive products and may be significant in the development of new therapies.
Article
The upward trend of diabetes and its complications had taken a big toll on developing countries where big budgets are allocated to manage it. Diabetes-based foot ulcerations has become a nightmare for patients and equally the clinicians due to its chronicity and devastating complications. Diabetic foot ulcers (DFU) take a long time to heal and generally resistant to conventional methods. DFU are commonly associated with high numbers of foot complications such as infection, gangrene and lower limb amputations. Despite the technological advancement in chronic wound management, the numbers of preventable foot complications especially amputations of toes and limb are still in the upward trend as the number of diabetics increases across the globe. Alternative method using sterile maggots of Lucilia spp has been much talked about for the past few decades to improve wound healing outcomes and ultimately reduce foot complications. Maggot debridement therapy, commonly known as MDT has been widely used as an alternative tool in the debridement of chronic wounds to remove slough, necrotic tissue from the wound bed. The usage of MDT has produced significant debridement and healing outcomes in diabetic foot ulcers and has been shown to reduce infections and stimulate healing. Despite numerous findings pointing to the relevance of MDT in the treatment protocol of chronic wounds especially DFU, MDT remains as the last resort in the process of salvaging limbs.
Article
With the introduction of the Nursing and Midwifery Council's (NMC) (2015a) revalidation directive, nurses are required to demonstrate an ongoing commitment to providing safe and effective care by continually combining sound empirical evidence with reflective practice (Sackett et al, 1996; Rolfe et al, 2011). Using Gibbs' (1998) model, I will reflect on an episode of care undertaken while I was on a recent placement. This reflective account will discuss the clinical use of honey and larvae therapy in the treatment of foot gangrene following meningococcal septicaemia. The psychosocial impact of ill health will also be considered. The use of newly acquired nursing skills and knowledge will be evaluated and the nurse-patient therapeutic relationship explored.
Chapter
The process of debridement currently includes the removal of skin, soft tissue, tendon, and bone, and can even include amputation of digits. This chapter discusses different debridement techniques: mechanical debridement; biological debridement; enzymatic debridement; autolytic debridement; and wound cleansing. Two additional wound treatments discussed are WF10 and hydrogen peroxide. Surgical debridement is still considered the gold standard; however, as mentioned earlier, there are multiple forms of debridement. Sharp, surgical excision of debris and necrotic tissue is the fastest and most accessible method. The choice of a specific type of debridement predominantly depends on clinicians’ experiences and preferences, but also on the co‐morbidities and the desires of the patient. There are several developments in the approaches that could have a significant impact on the future of debridement.
Article
General purpose: To present an overview of the advantages of maggot debridement therapy as a treatment for chronic wounds through the review of several larval properties. Target audience: This continuing education activity is intended for physicians, physician assistants, nurse practitioners, and nurses with an interest in skin and wound care. Learning objectives/outcomes: After participating in this educational activity, the participant will be able to:1. Summarize the use, process, and precautions for maggot debridement to treat chronic wounds.2. Synthesize the results of the bibliographic review of the use of maggot debridement to treat chronic wounds. Abstract: Maggot debridement therapy (MDT) is effective for ulcer debridement, achieving it in less time than other therapies. It offers a benefit to healing. However, it is unclear whether maggots reduce treatment time and there is considerable controversy around the treatment's potential antimicrobial action and cost-effectiveness. Nevertheless, it can be effective in preventing amputations and reducing the need for systemic antibiotics. This bibliographic review assesses the advantages of MDT as a treatment for chronic wounds through the review of several larval properties. The review was carried out by consulting biomedical databases including CINAHL, MEDLINE (PubMed), and Scopus, and concludes that MDT is an effective debridement and potential technique to facilitate healing. However, more data is needed on the wound type application frequency and the efficacy of treatment.
Article
Objective: Patients with critical limb ischaemia (CLI) lack sufficient blood flow in to the limb, which leads to difficulties in the normal wound healing process. Therefore, maggot debridement therapy (MDT) has not generally been recommended for CLI patients. We evaluated the effectiveness of wound bed preparation by MDT in CLI patients who had undergone mid-foot amputation. Methods: Patients who underwent mid-foot amputation after angioplasty between April 2014 and October 2016 were retrospectively investigated by classifying them into an MDT group or a conventional treatment group. The primary outcome was defined as achievement of wound healing. Secondary outcomes were the proportions of amputation-free survival (AFS) and successful ambulatory improvement. Propensity scores were used to evaluate treatment outcomes based on five factors: ankle-brachial index, skin perfusion pressure of the foot, nutritional status, experience with dialysis and age. Results: A total of 39 patients (39 legs) were included, seven within the MDT group and 32 in the conventional treatment group. Clinical backgrounds of the two groups showed no significant differences except for higher albumin levels for the MDT group (3.5±0.4g/dl; p=0.014). The wound healing proportion was significantly higher in the MDT group (86%) than in the control group (38%) (p=0.035). At 6 months after amputation, no significant differences were found between the two groups for AFS (71% versus 47%; p=0.41) or ambulatory capability (43% versus 28%; p=0.65). This result was also similar to the propensity score adjustment analysis. Conclusions: The efficacy of MDT with favourable wound bed preparation was shown in our CLI patients based on effective debridement and granulation formation by maggots, avoiding the loss of their heels. Wound-healing rates after MDT were higher for patients than for those receiving conventional treatment. MDT is considered a valid adjuvant treatment strategy for patients with CLI after revascularisation treatment is conducted. More favourable wound bed preparation and successful graft take were achieved in the MDT group, suggesting the effectiveness of MDT for wound healing in CLI patients.
Article
Full-text available
Staphylococcus aureus and Staphylococcus epidermidis biofilms cause chronic infections due to their ability to form biofilms. The excretions/secretions of Lucilia sericata larvae (maggots) have effective activity for debridement and disruption of bacterial biofilms. In this paper, we demonstrate how chymotrypsin derived from maggot excretions/secretions disrupts protein-dependent bacterial biofilm formation mechanisms.
Article
Full-text available
A novel homologue of insect defensin designated lucifensin (Lucilia defensin) was purified from the extracts of various tissues (gut, salivary glands, fat body, haemolymph) of green bottle fly (Lucilia sericata) larvae and from their excretions/secretions. The primary sequence of this peptide of 40 residues and three intramolecular disulfide bridges was determined by ESI-QTOF mass spectrometry and Edman degradation and is very similar to that of sapecin and other dipteran defensins. We assume that lucifensin is the key antimicrobial component that protects the maggots when they are exposed to the highly infectious environment of a wound during the medicinal process known as maggot therapy. We also believe that lucifensin is that long-sought larger molecular weight antimicrobial factor of the Lucilia sericata excretions/secretions believed to be effective against pathogenic elements of the wound microbial flora.
Article
Full-text available
The spatial organization of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds was investigated in the present study. Wound biopsy specimens were obtained from patients diagnosed as having chronic venous leg ulcers, and bacterial aggregates in these wounds were detected and located by the use of peptide nucleic acid-based fluorescence in situ hybridization and confocal laser scanning microscopy (CLSM). We acquired CLSM images of multiple regions in multiple sections cut from five wounds containing P. aeruginosa and five wounds containing S. aureus and measured the distance of the bacterial aggregates to the wound surface. The distance of the P. aeruginosa aggregates to the wound surface was significantly greater than that of the S. aureus aggregates, suggesting that the distribution of the bacteria in the chronic wounds was nonrandom. The results are discussed in relation to our recent finding that swab culturing techniques may underestimate the presence of P. aeruginosa in chronic wounds and in relation to the hypothesis that P. aeruginosa bacteria located in the deeper regions of chronic wounds may play an important role in keeping the wounds arrested in a stage dominated by inflammatory processes.
Article
Full-text available
Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. As monocytes may contribute to the excessive inflammatory responses in such wounds, this study focussed on the effects of maggot secretions on the pro-inflammatory activities of these cells. Freshly isolated monocytes were incubated with a range of secretions for 1 h and then stimulated with lipopolysaccharides (range 0-100 ng/ml) or lipoteichoic acid (range 0-5 microg/ml) for 18 h. The expression of cell surface molecules, cytokine and chemokine levels in culture supernatants, cell viability, chemotaxis, and phagocytosis and killing of Staphylococcus aureus were measured. Maggot secretions dose-dependently inhibited production of the pro-inflammatory cytokines TNF-alpha, IL-12p40 and macrophage migration inhibitory factor by lipopolysaccharides- and lipoteichoic acid-stimulated monocytes, while enhancing production of the anti-inflammatory cytokine IL-10. Expression of cell surface receptors involved in pathogen recognition remained unaffected by secretions. In addition, maggot secretions altered the chemokine profile of monocytes by downregulating macrophage inflammatory protein-1beta and upregulating monocyte chemoattractant protein-1 and IL-8. Nevertheless, chemotactic responses of monocytes were inhibited by secretions. Furthermore, maggot secretions did not affect phagocytosis and intracellular killing of S. aureus by human monocytes. Finally, secretions induced a transient rise in the intracellular cyclic AMP concentration in monocytes and Rp-cyclic AMPS inhibited the effects of secretions. Maggot secretions inhibit the pro-inflammatory responses of human monocytes through a cyclic AMP-dependent mechanism. Regulation of the inflammatory processes by maggots contributes to their beneficial effects on chronic wounds.
Article
Full-text available
Sharp debridement is the most clinically and cost-effective way of physically removing and suppressing a biofilm. Continued debridement, as part of a multifaceted treatment strategy, will keep the biofilm in a weakened state.
Article
Full-text available
To compare the clinical effectiveness of larval therapy with a standard debridement technique (hydrogel) for sloughy or necrotic leg ulcers. Pragmatic, three armed randomised controlled trial. Community nurse led services, hospital wards, and hospital outpatient leg ulcer clinics in urban and rural settings, United Kingdom. 267 patients with at least one venous or mixed venous and arterial ulcer with at least 25% coverage of slough or necrotic tissue, and an ankle brachial pressure index of 0.6 or more. Loose larvae, bagged larvae, and hydrogel. The primary outcome was time to healing of the largest eligible ulcer. Secondary outcomes were time to debridement, health related quality of life (SF-12), bacterial load, presence of meticillin resistant Staphylococcus aureus, adverse events, and ulcer related pain (visual analogue scale, from 0 mm for no pain to 150 mm for worst pain imaginable). Time to healing was not significantly different between the loose or bagged larvae group and the hydrogel group (hazard ratio for healing using larvae v hydrogel 1.13, 95% confidence interval 0.76 to 1.68; P=0.54). Larval therapy significantly reduced the time to debridement (2.31, 1.65 to 3.2; P<0.001). Health related quality of life and change in bacterial load over time were not significantly different between the groups. 6.7% of participants had MRSA at baseline. No difference was found between larval therapy and hydrogel in their ability to eradicate MRSA by the end of the debridement phase (75% (9/12) v 50% (3/6); P=0.34), although this comparison was underpowered. Mean ulcer related pain scores were higher in either larvae group compared with hydrogel (mean difference in pain score: loose larvae v hydrogel 46.74 (95% confidence interval 32.44 to 61.04), P<0.001; bagged larvae v hydrogel 38.58 (23.46 to 53.70), P<0.001). Larval therapy did not improve the rate of healing of sloughy or necrotic leg ulcers or reduce bacterial load compared with hydrogel but did significantly reduce the time to debridement and increase ulcer pain. Current Controlled Trials ISRCTN55114812 and National Research Register N0484123692.
Article
Full-text available
As part of a major clinical trial, sequential biopsies were taken from the margins of venous leg ulcers during their healing. The changing patterns of tissue architecture and extracellular matrix synthesis during healing were documented histologically and immunocytochemically. Initial biopsies were similar in appearance: prominent fibrin cuffs, variable inflammation, hemosiderin, and red blood cell extravasation. So called "fibrin cuffs" were highly organized structures composed of laminin, fibronectin, tenascin, and collagen as well as trapped leukocytes and fibrin. Fibronectin was absent from the ulcer tissue although collagen was abundant. Major histologic changes were observed after 2 weeks' pressure bandage therapy; hemosiderin, acute inflammation, and granulation tissue with the deposition of fibronectin had all increased and epithelial migration had commenced. Complete epithelialization was frequent by the fourth week of treatment, but the basement membrane was incomplete. At this time, hemosiderin and red blood cell extravasation had decreased and "fibrin cuffs" were virtually absent although chronic inflammation remained. The complex organization of the so-called "fibrin cuffs" may inhibit angiogenesis (but offer protection against increased venous pressure) in addition to their previously ascribed role in causing tissue ischemia.
Article
Full-text available
Lucilia sericata larvae, or green bottle fly maggots are applied to chronic wounds to aid healing. Previously, our laboratory has characterized the enzymatic activities present within maggot excretions/secretions (ES). Since then, we have related these to the degradation of extracellular matrix components, alteration of human, dermal fibroblast adhesion to surfaces and the stimulation of fibroblast migration within a two-dimensional in vitro assay. In this study, we developed a novel three-dimensional in vitro assay in which to observe fibroblast migration and morphology in response to maggot ES. Here, primary human foreskin fibroblasts were embedded within collagen gels containing fibronectin. Phase contrast and confocal microscopy were used in conjunction with image analysis software to examine and quantify aspects of fibroblast behavior. Our results showed that maggot ES stimulated fibroblast migration through the matrix and induced altered cell morphologies. Remodelling of the extracellular matrix located between individual fibroblasts was also induced, providing a mechanism by which cells may detect each other's presence over considerable distances. Thus, mechanisms by which maggots enhance tissue formation within wounds may be via the promotion of fibroblast motility, acceleration of extracellular matrix remodelling and coordination of cellular responses.
Article
Full-text available
Lucilia sericata maggots are successfully used for treating chronic wounds. As the healing process in these wounds is complicated by bacteria, particularly when residing in biofilms that protect them from antibiotics and the immune system, we assessed the effects of maggot excretions/secretions (ES) on Staphylococcus aureus and Pseudomonas aeruginosa biofilms, the clinically most relevant species. We assessed the effects of ES on biofilms using microtitre plate assays, on bacterial viability using in vitro killing and radial diffusion assays, and on quorum sensing systems using specific reporter bacteria. As little as 0.2 microg of ES prevented S. aureus biofilm formation and 2 microg of ES rapidly degraded biofilms. In contrast, ES initially promoted P. aeruginosa biofilm formation, but after 10 h the biofilms collapsed. Degradation of P. aeruginosa biofilms started after 10 h and required 10-fold more ES than S. aureus biofilms. Boiling of ES abrogated their effects on S. aureus, but not on P. aeruginosa, biofilms, indicating that different molecules within ES are responsible for the observed effects. Modulation of biofilms by ES did not involve bacterial killing or effects on quorum sensing systems. Maggot ES are differentially effective against biofilms of S. aureus and P. aeruginosa.
Article
There is growing evidence to suggest that the resident microflora of chronic venous leg ulcers impairs cellular wound-healing responses, thereby playing an important role in maintaining the non-healing phenotype of many of these wounds. The significance of individual species of bacteria will remain unclear until it is possible to characterize fully the microflora of such lesions. The limitations and biases of culture-based microbiology are being realized and the subsequent application of molecular methods is revealing greater diversity within mixed bacterial populations than that demonstrated by culture alone. To date, this approach has been limited to a small number of systems, including the oral microflora. Here, for the first time, the comprehensive characterization of the microflora present in the tissue of a chronic venous leg ulcer is described by the comparison of 16S rDNA sequences amplified directly from the wound tissue with sequences obtained from bacteria that were isolated by culture. The molecular approach demonstrated significantly greater bacterial diversity than that revealed by culture. Furthermore, sequences were retrieved that may possibly represent novel species of bacteria. It is only by the comprehensive analysis of the wound microflora by both molecular and cultural methods that it will be possible to further our understanding of the role of bacteria in this important condition.
Article
REPORT OF A CASE A 74-year-old man with a history of recurrent venous stasis ulcers presented to the clinic with a 1-month history of an enlarging, erythematous, tender, right pretibial wound. He recalled no trauma. Despite bedrest and oral dicloxacillin as prescribed, the patient returned to the clinic the following week with a larger wound, now described as a central black eschar with surrounding cellulitis. He was admitted for therapy with intravenous antibiotics (piperacillin and tazobactam) and frequent bedside surgical débridement. He did not tolerate a trial of hydrotherapy because of symptomatic hypotension. Plastic surgery consultants recommended split-thickness skin grafting after wound débridement; the wound was débrided two to three times weekly, limited primarily by the patient's pain and by concerns about the great depth of the wound and the patient's poor healing ability. Between débridements, his wound was dressed with hydrocolloid pads to aid in autolysis or with a
Article
The larvae of Lucilia sericata (the medicinal maggot) have been exploited to undertake many applications, from their use in the management of chronic, non-healing wounds (larval therapy), to their assistance in forensic investigations and analysis. This current review describes these various applications and discusses the recent scientific and clinical developments supporting their use in wound healing and management. The review includes aspects such as their role in wound debridement, disinfection properties and biofilm disruption, as well as their potential role in the acceleration of wound healing. Also considered is the study of novel therapeutic compounds and biological therapies derived from this species. From a clinical perspective, recent studies that have been concerned with the efficacy and value of maggot debridement therapy are summarised and discussed.
Article
Venous leg ulcer slough is unpleasant to the patient and difficult to manage clinically. It harbours infection, also preventing wound management materials and dressings from supporting the underlying viable tissues. In other words, slough has significant nuisance value in the tissue viability clinic. In this study, we have sought to increase our knowledge of slough by building upon a previous but limited analysis of this necrotic tissue. In particular, slough has been probed using Western blotting for the presence of proteins with the capacity to engage microbial surface components recognising adhesive matrix macromolecules. Although the samples were difficult to resolve, we detected fibrinogen, fibronectin, IgG, collagen, human serum albumin and matrix metalloproteinase-9. Furthermore, the effect of a maggot-derived debridement enzyme, chymotrypsin 1 on macromolecules in slough was confirmed across seven patient samples. The effect of chymotrypsin 1 on slough confirms our thesis that this potential debridement enzyme could be effective in removing slough along with its associated bacteria, given its observed resistance to intrinsic gelatinase activity. In summary, we believe that the data provide scientists and clinicians with further insights into the potential molecular interactions between bacteria, wound tissue and Lucilia sericata in a clinically problematic yet scientifically interesting wound ecosystem.
Article
The complement system plays an important role in the activation of the inflammatory response to injury, although inappropriate complement activation (CA) can lead to severe tissue damage. Maggot therapy is successfully used to treat infected wounds. In this study, we hypothesized that maggot excretions/secretions influence CA in order to modulate the host's inflammatory response. Therefore, the effect of maggot excretions on CA was investigated in preoperatively and postoperatively obtained sera from patients. Our results show that maggot excretions reduce CA in healthy and postoperatively immune-activated human sera up to 99.9%, via all pathways. Maggot excretions do not specifically initiate or inhibit CA, but break down complement proteins C3 and C4 in a cation-independent manner and this effect proves to be temperature tolerant. This study indicates a CA-reducing substrate that is already successfully used in clinical practice and may explain part of the improved wound healing caused by maggot therapy. Furthermore, the complement activation-reducing substance present in maggot excretions could provide a novel treatment modality for several diseases, resulting from an (over)active complement system.
Article
In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre-formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non-healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata.
Article
Background: Through clinical observation, Lucilia sericata (greenbottle fly) larvae are credited with exerting the following beneficial effects upon a chronic nonhealing wound: removal of necrotic tissue ('debridement'), disinfection of the wound and active promotion of granulation tissue formation. As a major cellular component of granulation tissue, fibroblasts play an extensive role in healing. The composition of extracellular matrix (ECM) located in the wound is another important factor, partaking in a dynamic feedback loop with the fibroblasts that produce it. Fibroblast-ECM interactions therefore exert considerable influence upon new tissue formation. Objectives: To investigate the effects of L. sericata larval excretory/secretory products (ES) upon the behaviour of fibroblasts, seeded upon ECM component surfaces. Methods: ES were collected by washing freshly hatched larvae in phosphate-buffered saline. Human dermal neonatal fibroblast cells were seeded upon fibronectin- or collagen-coated surfaces, together with untreated (or 'native') ES, heat-treated ES, or no ES (ES blank). Following incubation, fibroblast adhesion was determined using an adenosine triphosphate assay and, for confirmation, a total nucleic acid content assay. Cell spreading was observed using microscopy. The effect of ES upon fibronectin structure was observed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Peptide sequencing of suspected fibronectin fragments was performed using an 'electrospray' type time of flight mass spectrometer and 'Peptident' database search. Results: ES significantly reduced fibroblast adhesion to both fibronectin and, to a lesser extent, collagen. Cell spreading was also reduced, yet cells remained viable. For both spreading and adhesion, heat-treated ES exerted significantly less activity than native, untreated ES. However, they still exhibited significant activity when compared with the ES blank. ES appeared to modify fibroblast adhesion indirectly via proteolytic fragmentation of the fibronectin protein surface. Conclusions: L. sericata larval secretions modify fibroblast adhesion and spreading across ECM protein surfaces, while keeping cells viable. Proteolytic activity of the ES played a significant role. If transferred to the wound situation, such alteration of fibroblast-ECM interactions may enhance new tissue formation.
Article
To study the efficacy of bagged larvae on wound debridement compared with conventional treatment. Randomized, multicenter, controlled, prospective phase 3 trial with blinded assessment of outcome measures by a single observer. Two hospital referral centers in Caen and Lyon, France. Random sampling of 119 patients with a nonhealing, sloughy wound 40 cm(2) or smaller, less than 2 cm deep, and an ankle brachial index of 0.8 or higher. During a 2-week hospital stay, patients received either maggot debridement therapy (MDT) or conventional treatment. At discharge, conventional dressings were applied and a follow-up visit occurred at day 30. Percentage of slough in wounds at day 15. There was a significant difference between groups at day 8 (54.5% in the MDT group and 66.5% in the control group) (P = .04). The mean percentage of slough at day 15 was 55.4% in the MDT group and 53.8% in the control group (P = .78). Although MDT shows no significant benefit at day 15 compared with conventional treatment, debridement by MDT is significantly faster and occurs during the first week of treatment. Because there is no benefit in continuing the treatment after 1 week, another type of dressing should be used after 2 or 3 applications of MDT. clinicaltrials.gov Identifier: NCT01211236.
Article
Maggot debridement therapy (MDT) is effective for treating intractable wounds, but its precise molecular mechanism, including the association between MDT and growth factors, remains unknown. We administered MDT to nine patients (66.3 ± 11.8 yr, 5 male and 4 female) with intractable wounds of lower extremities because they did not respond to conventional therapies. Significant increases of hepatocyte growth factor (HGF) levels were observed in femoral vein blood during 48 h of MDT (P < 0.05), but no significant change was found for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-β1 (TGF-β1), or tumor necrosis factor-α (TNF-α). We conducted NIH-3T3 cell stimulation assay to evaluate the relation between HGF and protease activity in excretion/secretion (ES) derived from maggots. Compared with the control group, HGF was significantly higher in the 0.05 μg/ml ES group (P < 0.01). Furthermore, protease inhibitors suppressed the increase of HGF (P < 0.05). The HGF expression was increased in proportion to the ES protein concentration of 0.025 to 0.5 μg/ml. In fact, ES showed stronger capability of promoting HGF production and less cytotoxicity than chymotrypsin or bromelain. HGF is an important factor involved in cutaneous wound healing. Therefore, these results suggest that formation of healthy granulation tissue observed during MDT results from the increased HGF. Further investigation to identify molecules enhancing HGF expression by MDT will contribute greatly to drug target discovery for intractable wound healing therapy.
Article
Recently, we identified a new insect defensin, named lucifensin that is secreted/excreted by the blowfly Lucilia sericata larvae into a wound as a disinfectant during the medicinal process known as maggot therapy. Here, we report the total chemical synthesis of this peptide of 40 amino acid residues and three intramolecular disulfide bridges by using three different protocols. Oxidative folding of linear peptide yielded a peptide with a pattern of disulfide bridges identical to that of native lucifensin. The synthetic lucifensin was active against Gram-positive bacteria and was not hemolytic. We synthesized three lucifensin analogues that are cyclized through one native disulfide bridge in different positions and having the remaining four cysteines substituted by alanine. Only the analogue cyclized through a Cys16-Cys36 disulfide bridge showed weak antimicrobial activity. Truncating lucifensin at the N-terminal by ten amino acid residues resulted in a drop in antimicrobial activity. Linear lucifensin having all six cysteine residues alkylated was inactive. Circular dichroism spectra measured in the presence of α-helix-promoting compounds showed different patterns for lucifensin and its analogues. Transmission electron microscopy revealed that Bacillus subtilis treatment with lucifensin induced significant changes in its envelope.
Article
Larval biotherapy is a debridement tool used in wound management. The mechanism of action involves degradation of eschar by serine proteases including chymotrypsin within the alimentary fluids of first instar Lucilia sericata. With the rationale of obviating some limitations of biotherapy, including cost, complexity of use, and patient reticence, the present study describes a mobile hydrogel formulation containing freeze-dried recombinant L. sericata chymotrypsin designed for topical application. Neither freeze-drying nor formulation into the hydrogel significantly attenuated the measured activity of released enzyme compared to fresh-frozen enzyme in aqueous solution. Gel electrophoresis confirmed qualitatively that the chymotrypsin/hydrogel formulation both with and without supplementary urea at 10% (w) /(v) degraded human chronic wound eschar ex vivo. Mindful that the hallmark of intractability of chronic wounds is aberrant biochemistry, the pH activity profile for the enzyme/hydrogel formulation was compared with exudate pH in chronic wounds of mixed aetiology in a cohort of 48 hospital in-patients. Five patients' wounds were acidic, however, the remainder were predominantly alkaline and coincided with the pH optimum for the insect enzyme. Thus, a recombinant L. sericata chymotrypsin and hydrogel formulation could represent a pragmatic alternative to larval therapy for the management of chronic wounds.
Article
A chymotrypsin found in the secretions of Lucilia sericata and manufactured as a recombinant enzyme degrades chronic wound eschar ex vivo. To characterize the inhibition profile of the L. sericata recombinant chymotrypsin I. Activity of recombinant chymotrypsin I and its sensitivity to endogenous inhibitors were determined enzymatically using the fluorogenic substrate succinyl-alanyl-alanyl-prolyl-phenylalanyl-aminomethyl coumarin. We report the presence of high concentrations of two endogenous inhibitors, α1-antichymotrypsin and α1-antitrypsin, in wound eschar and a trace of a third, α2-macroglobulin, with the potential to inhibit this debridement process. However, the addition of a soluble and inhibitor-containing extract of chronic wound eschar to chymotrypsin I did not affect activity of the enzyme, neither did the addition of purified native α1-antichymotrypsin or α1-antitrypsin, although chymotrypsin I was inhibited by α2-macroglobulin. Conversely, the mammalian equivalent, α-chymotrypsin, was inhibited by the purified native α1-antichymotrypsin, α1-antitrypsin and α2-macroglobulin and by the soluble extract of wound eschar. The data suggest that the maggot-derived chymotrypsin I is biochemically distinct from human α-chymotrypsin and the lack of inhibition by wound eschar suggests a means by which chymotrypsin I activity survives within the wound to contribute towards debridement during maggot biotherapy.
Article
Larvae of the greenbottle Lucilia sericata are used to debride nonhealing wounds and stimulate the production of fresh granulation tissue. Previous publications have shown that secretions from L. sericata contain a number of proteolytic activities including a chymotrypsin that degrades a number of extracellular matrix components such as fibronectin, laminin and collagen. To produce a recombinant L. sericata chymotrypsin (chymotrypsin I) and determine its effects on the degradation of patient wound eschar. An active recombinant chymotrypsin I from L. sericata was cloned and expressed in Sf9 cells and its subsequent effects ex vivo on eschar from venous leg ulcers were determined by two-dimensional electrophoresis. The recombinant enzyme had the attributes of a chymotrypsin, possessing sequence homology with other chymotrypsins and demonstrating attributes of the native enzyme including cleavage of the chymotrypsin substrate succinyl-alanyl-alanyl-prolyl-phenylalanyl-7-amino-4-methyl coumarin, inhibition by phenylmethylsulphonyl fluoride and lack of inhibition by amidinophenylmethylsulphonyl fluoride. Importantly, the recombinant chymotrypsin cleaved the majority of proteins from slough/eschar from venous leg ulcers in a superior manner to chymotrypsins from human and bovine sources. The ex vivo degradation of eschar from venous leg ulcers indicates the potential value of recombinant chymotrypsin I as a novel, stand-alone debridement agent.
Article
Chronic infections are commonly associated with biofilms formed by bacteria such as Staphylococcus epidermidis. With the increase in antibiotic resistant bacteria, maggot debridement therapy has been reintroduced for the treatment of chronic wounds. Studies have shown that the excretion/secretions (ES) of Lucilia sericata larvae (maggots) contain many bioactive compounds which may contribute to the efficacy of maggot therapy. The present study evaluates the effect of L. sericata ES on the formation and disruption of S. epidermidis 7457 and 5179-R1 biofilms. These strains employ either polysaccharide intercellular adhesin (PIA) or accumulation associated protein (Aap) for intercellular adhesion. A semiquantitative biofilm assay was used to measure the formation/disruption of S. epidermidis 7457 and 5179-R1 biofilms by ES. ES activity was characterized according to concentration, incubation time and temperature, thermal stability, and size. Immunofluorescence microscopy was used to ascertain the effect of ES on PIA and Aap. In the presence of ES, S. epidermidis 7457 and 5179-R1 nascent bio film formation was inhibited, and pre-formed biofilms disrupted. ES activity was temperature and time dependent, inactivated by heat treatment, and disruption depended on the mechanism of intercellular adhesion. The molecule(s) responsible was >10 kDa in size and appeared to have protease or glucosaminidase activity. ES interferes with S. epidermidis biofilm formation, specifically degrading factors employed in biofilm accumulation, which would increase bacterial susceptibility to antibiotics and the host's immune system. In purified form, ES-factors may have general applicability for the treatment or prevention of chronic biofilm infections caused by staphylococci.
Article
Maggot therapy, utilizing the larvae of Lucilia sericata, has been reported to reduce the bacterial load within wounds and also to enhance wound healing. Maggot excretions/secretions (ES) have been shown to have a role in the success of maggot therapy. While the protein content of ES has been investigated, to date little research has focused on the small metabolites present in ES and their potential contribution to the therapy. Study of the molecular composition of the secretions and the potential bioactivities present will allow for a more detailed evaluation of the efficacy of maggot therapy. We studied the amino acid-like compounds present in ES of L. sericata larvae in order to determine the compounds present and their potential role in the wound healing process. These included thin-layer chromatography/mass spectrometric analysis of ES to identify amino acid-like components, a turbidometric assay to investigate their potential antibacterial activity and cell proliferation studies to investigate their potential mitogenic ability. Three prominent compounds were detected and identified as histidine, valinol and 3-guanidinopropionic acid. While these amino acids were not shown to exhibit antibacterial activity, a proliferative effect on the growth of human endothelial cells, but not fibroblasts, was noted. The demonstrated proliferative effect, selectively on endothelial cells, suggests that the amino acid-like compounds present in maggot ES may have a role in wound healing, by stimulating angiogenesis.
Article
Maggot therapy is a simple and highly successful method for healing of infected and necrotic wounds. The increasing evidences indicate that Maggot excretions/secretions (ES) plays important roles in the wounds healing process. But the precise molecular mechanisms remain undefined. Herein, we investigated if ES induced cell migration during wound healing process using microvascular endothelial cells (HMEC-1) as model, and this effect was associated with the activation of AKT1 and ERK1/2. Wound healing and transwell migration assays were performed to study the effects of ES on HMEC-1 cell migration. Our data showed that ES significantly induced HMEC-1 cell migration in both wound healing and transwell assays, and time-dependently (P < 0.05) activated AKT1, but not ERK1/2. Moreover LY294002 (a PI3K inhibitor) partially attenuated (P < 0.05) ES-induced cell migration in wound healing assay while completely inhibited (P < 0.05) ES-induced AKT1 activation. These findings demonstrate that ES directly induces HMEC-1 cell migration and this event is partially mediated by the activation of AKT1.
Article
Forty-one biopsy specimens, taken from the ulcer-bearing skin of 41 legs of 21 patients attending the varicose vein clinic, were selectively stained for fibrin with phosphotungstic acid haemotoxylin before being blindly assessed,. Layers of fibrin were found surrounding the dermal capillaries in all 26 legs with lipodermatosclerosis. None of the specimens from the 15 legs with clinically normal skin contained fibrin. There was also an increased number of dermal capillaries cut in cross section per high powered field in 24 of the 26 legs with lipodermatosclerosis compared with two of the 15 legs with normal skin (p less than 0.001). The mean reduction in foot vein pressure during exercise was significantly less in the 26 limbs with pericapillary fibrin than in the other 15 limbs (p less than 10(-6). Lipodermatosclerosis is synonymous with pericapillary fibrin deposition and is associated with, and probably secondary to, both a persistently raised venous pressure and an increase in the size of the dermal capillary bed. This extravascular deposition of fibrin probably stimulates tissue fibrosis and blocks the diffusion of oxygen to the overlying epidermis, producing cellular death and venous ulceration.
Article
The pathogenesis of venous ulceration is unknown. We propose that macromolecules leaking into the dermis as a result of venous hypertension bind to or "trap" growth factors and matrix material, which then become unavailable for tissue repair and for the maintenance of tissue integrity.
Article
Maggot therapy has been used since the 1930s for treating soft-tissue wounds. Despite decades of experience with this therapy, selecting appropriate dressing materials continues to be a problem. Before initiating our maggot therapy service, we needed to develop a dressing that would (1) prevent the maggots from escaping, (2) permit oxygen to reach the maggots, (3) facilitate drainage, (4) allow inspection of the wound, (5) require minimal maintenance, and (6) be of low cost. The optimal dressing design proved to be a two-layered cagelike dressing, the bottom layer of which comprised a hydrocolloid pad, applied to the surrounding healthy skin and covered by a fine chiffon or nylon mesh. Liquefied necrotic tissue drained through the mesh and was absorbed in a top layer of gauze, which was replaced periodically. Thus it was possible to contain the maggots within the wound by means of readily available materials.
Article
The skin is colonized by an array of microorganisms which form its natural microflora. Disruption to the normal barrier function of the skin (due to trauma or disease) may result in invasion of the dermis by opportunistic bacteria. To date, these organisms, which may contribute to the chronicity of skin wounds, have been analyzed solely by culture methods. It is increasingly realized that standard culture methods of analysis do not accurately reflect the full diversity of complex microflora. This review discusses the limitations of traditional culture approaches and reviews recent advances in molecular microbiological techniques which facilitate a more comprehensive characterization of the microflora within clinical samples. The currently available technologies and techniques are described, as is their use in clinical practice and their potential for diagnostic screening. Chronic venous ulceration of the lower limbs is an important skin disorder in which the microflora invading the dermal tissues contribute to the observed delayed healing. Using chronic leg ulcers as a working example, we show how strict culture and molecular microbiological techniques may be employed, for the first time in combination, to definitively characterize the invading microbial community of the dermis.
Article
Larvae of the greenbottle fly Lucilia sericata are used routinely for the clinical treatment of difficult necrotic and infected wounds. Degradation by proteinases contained in larval excretory/secretory (ES) products is thought to contribute to wound debridement by removal of dead tissue. However, proteinase activity may also affect host tissue remodelling processes. To identify proteolytic enzymes derived from L. sericata ES products with activities against fibrin and extracellular matrix (ECM) components. Larval proteinase activities were assayed in vitro using class-specific substrates and inhibitors. Their action against fibrin and ECM components was examined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three classes of proteolytic enzyme were detected in the secretions using fluorescein isothiocyanate-labelled casein as a model substrate. The predominant activity belonged to serine proteinases (pH optima 8-9) of two different subclasses (trypsin-like and chymotrypsin-like), with a weaker aspartyl proteinase (pH 5) and a metalloproteinase (pH 9) with exopeptidase characteristics also present. Using skin-relevant ECM components as substrates L. sericata ES products solubilized fibrin clots and degraded fibronectin, laminin and acid-solubilized collagen types I and III. Hydrolysis of ECM macromolecules was inhibited by preincubating ES products with phenylmethylsulphonyl fluoride but not 4-amidinophenylmethylsulphonyl fluoride, indicating that degradation was due to the 'chymotrypsin-like' serine proteinase. These data suggest that a combination of L. sericata ES proteinases involving chymotrypsin-like and trypsin-like activities could potentially influence wound healing events when maggots are introduced into necrotic and infected wounds, with the chymotrypsin-like activity involved in the remodelling of ECM components.
Article
In chronic wounds, biofilms probably play a vital role in protecting bacteria from host defenses and antimicrobial medications by creating a barrier of exopolysaccharide that is difficult for the immune system and antibiotics to penetrate. A biofilm consists of an exopolysaccharide matrix that is produced and secreted by certain species of bacteria. The purpose of this study was to visualize and time the progressing growth of a biofilm by a wound-isolated Pseudomonas aeruginosa. P. aeruginosa that was initially isolated from a human burn wound was allowed to grow a biofilm in vitro. We used a modified Congo red staining technique to demonstrate the sequential development of a mature biofilm as examined by light microscopy. We show that the exopolysaccharide of the developing biofilm is visible in just 5 hours after inoculation and has the characteristics of a mature biofilm by 10 hours. The rapidity of biofilm growth suggests that bacteria in wounds possess the capacity of producing this shield against antibiotics and immune effector cells early in the infection process. Therefore, efforts to prevent or slow the proliferation of bacteria and biofilms should occur soon after a wound is created. Additionally, this staining technique can be used to demonstrate the ability of agents to slow biofilm growth or to interrupt formed biofilm and may be useful in future studies of chronically infected wounds.
Article
Maggot therapy is a simple and highly successful method for cleansing infected and necrotic wounds. The use of maggots has become increasingly important in the treatment of non-healing wounds, particularly those infected with the multidrug-resistant pathogen, methicillin-resistant Staphylococcus aureus (MRSA). The increasing challenge concerning the treatment of MRSA infections and the recent finding of vancomycin-resistant strains of MRSA have elicited the search for novel antibacterial compounds and, in particular, investigations into the potent antibacterial mechanism(s) behind maggot therapy. In this study, we report that excretions/secretions (ES) from the blowfly, Lucilia sericata, exhibit potent, thermally stable, protease resistant antibacterial activity against MRSA in vitro. We describe the initial characterisation of two antibacterial factors from native ES of L. sericata. A small, <500 Da factor with significant antibacterial activity against MRSA was partially isolated using ultrafiltration techniques. The potent activity of this factor was comparable to that of native excretions/secretions. A larger, 0.5-3-kDa factor with significant activity against S. aureus was also partially characterised.
Article
Lucilia sericata larvae, or greenbottle fly maggots, placed within chronic wounds have been observed to remove necrotic tissue and infection. They are also believed to actively promote granulation tissue formation. Interactions between fibroblasts and the surrounding extracellular matrix play a crucial role in tissue formation, influencing fibroblast proliferation, migration, and tissue remodeling. For example, the strength of cell adhesion to surfaces coated with extracellular matrix influences cell motility. L. sericata larval excretory/secretory products having previously been shown to modify fibroblast adhesion to collagen and particularly fibronectin, it was hypothesized that these products would alter fibroblast migration. This was investigated using a two-dimensional in vitro wound assay, time-lapse digital photography, enzyme class-specific substrates and inhibitors, and gel electrophoresis. Results showed that L. sericata excretory/secretory products promoted fibroblast migration upon a fibronectin-coated surface. This was related to the degradation of fibronectin by serine proteinases within maggot excretion/secretions. The presence of a metalloproteinase activity may also have played a role. Thus, a possible mechanism by which maggots enhance tissue formation within wounds may be via the promotion of fibroblast motility, providing for a wider distribution of viable fibroblasts.
Article
Postoperative wound infection is a rare, but major, complication of replantation. Failure to control infection can lead directly to vascular thrombosis and, in turn, to loss of the replanted extremity. The use of maggots for wound debridement has a long history and has been lately re-introduced for treatment of intractable wounds. In this report, the authors present the experience of successful debridement of a severely infected wound after forearm replantation, using maggot therapy. The results and mechanism of maggot therapy are discussed.
Article
The resurgence of larval biotherapy as a debridement tool in wound management has been accompanied by several clinical reports highlighting concomitant tissue regeneration. Studies employing in vitro cell motility assays have found that purified excretory/secretory (ES) products from Greenbottle larvae (blowfly, Lucilia sericata) are motogenic for human dermal fibroblasts when used as a supplement in culture media. The objective of the present study was to determine whether ES delivered using a prototype hydrogel wound dressing induced similar motogenic effects on fibroblastic (3T3) and epithelial cells (HaCaTs) comprising a scratched-monolayer wound model. Quantitative analysis by MTT assay failed to detect significant mitogenic effects of ES on either cell type. Quantitative image analysis revealed that ES exposure markedly accelerated wound closure through a motogenic effect on both fibroblasts and keratinocytes. Quantitative histochemical analysis detected significantly higher phosphotyrosine (pTyr) expression in ES-exposed cell cultures than in controls; moreover immunocytochemistry revealed conspicuously raised levels of pTyr expression in cells located at the wound margin. By attenuation with a panel of enzyme inhibitors these effects were attributed to the protease components of ES. The present results suggest that controlled delivery of ES as a follow-up to maggot debridement therapy may be an effective therapeutic option for stimulation of tissue regeneration in wound management.
Article
There is renewed interest in the use of maggots (Lucilia sericata) to aid in healing of chronic wounds. In such wounds neutrophils precipitate tissue damage rather than contribute to healing. As the molecules responsible for the beneficial actions of maggots are contained in their excretions/secretions (ES), we assessed the effects of ES on functional activities of human neutrophils. ES dose-dependently inhibited elastase release and H(2)O(2) production by fMLP-activated neutrophils; maximal inhibition was seen with 5-50 microg of ES/ml. In contrast, ES did not affect phagocytosis and intracellular killing of Candida albicans by neutrophils. Furthermore, 0.5 microg of ES/ml already inhibited neutrophil migration towards fMLP. ES dose-dependently reduced the fMLP-stimulated expression of CD11b/CD18 by neutrophils, suggesting that ES modulate neutrophil adhesion to endothelial cells. ES did not affect the fMLP-induced rise in [Ca(2+)](i) in neutrophils, indicating that ES act down-stream of phospholipase C-mediated activation of protein kinase C. In agreement, ES inhibited PMA-activated neutrophil functional activities. ES induced a rise in intracellular cAMP concentration in neutrophils and pharmacological activators of cAMP-dependent mechanisms mimicked their inhibitory effects on neutrophils. The beneficial effects of maggots on chronic wounds may be explained in part by inhibition of multiple pro-inflammatory responses of activated neutrophils by ES.
Article
The application of Lucilia sericata larvae to chronic, infected wounds results in the rapid elimination of infecting microorganisms, including MRSA. Previously, we demonstrated in vitro antibacterial activity of native excretions/secretions (nES) from L. sericata and partially purified two low mass antibacterial compounds with masses of 0.5-10kDa and <500Da. The present study reports the antibacterial effects of the <500Da fraction (ES<500) on the growth and morphology of a range of bacteria, including 12 MRSA strains. Distinct morphological changes were observed in Bacillus cereus and Escherichia coli following exposure to ES<500. Flow cytometry and confocal microscopy analyses, in conjunction with turbidometric and CFU assays, revealed bacteriostatic activity of nES against S. aureus and E. coli. ES<500 also demonstrated bacteriostatic activity against S. aureus, however, bactericidal activity and the induction of a viable but non-culturable state were observed with ES<500-treated E. coli.