Excretions/secretions Research Articles

Maggot metabolites and their combinatory effects with antibiotic on Staphylococcus aureus

BackgroundMaggot therapy has been in practice for effective debridement, disinfection and healing of chronic wounds. Due to their antiseptic action during wound healing, their metabolites have been investigated in the past for antibacterial activity. They have been particularly useful for treatment of wounds infected with multi-drug resistant Staphylococcus aureus. Antibiotics, on the other hand, can predispose bacteria to develop resistance. Substances that are able to modulate or delay the occurrence of resistance in bacteria are under investigation by many researchers around the world. In the present study, antibacterial activity in excretions/secretions (ES) from maggots of Lucilia cuprina blowfly was demonstrated. The extracts were also screened in combination with antibiotic, ciprofloxacin.MethodsL. cuprina blowfly maggots were reared for extraction of its metabolites. The ES extracted was screened against S. aureus, alone and in combination with ciprofloxacin, both for short term and long term exposure analysis. A microchannel-based device and system was used for experiments instead of conventional techniques.ResultsThe original ES had shown partial bacterial growth inhibition. However, in combination with ciprofloxacin, at sub-inhibitory concentrations, certain combinations revealed anti-staphylococcal activity, with bacterial reduction of up to 50%, after 24 hours. The six day study on S. aureus exposed to ES-ciprofloxacin combination suggested a potential delay in development of adaptive resistance as opposed to when ciprofloxacin was used as single agent.ConclusionsThe combination effect of ES and ciprofloxacin at sub-MIC levels showed enhanced antibacterial activity compared to the effect of ES and ciprofloxacin as single agents. Based on the results of ES-ciprofloxacin combinations, a more effective means of treatment for S. aureus can be proposed.

Combinations of maggot excretions/secretions and antibiotics are effective against Staphylococcus aureus biofilms and the bacteria derived therefrom

Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Previously we reported that maggot excretions/secretions (ES) break down Staphylococcus aureus biofilms but do not kill the bacteria. As many antibiotics are not effective against biofilms we assessed the effect of combinations of ES and antibiotics on S. aureus biofilms and on the survival of the bacteria released from the biofilms. Effects of ES, antibiotics (vancomycin, daptomycin or clindamycin) and combinations thereof on S. aureus ATCC 29 213 biofilms and bacterial viability were determined using microtitre plates and in vitro killing assays. Vancomycin and daptomycin dose-dependently enhanced biofilm formation, whereas clindamycin reduced S. aureus biofilm size. Adding ES to antibiotic incubations caused a complete biofilm breakdown. After a lag time the bacteria derived from biofilms became susceptible to vancomycin and clindamycin, provided that the medium was refreshed. Daptomycin dose-dependently eliminated the biofilm-derived bacteria immediately. Furthermore, it was significantly more effective against bacteria derived from ES-exposed biofilms than those from control biofilms. ES did not affect the activity of the antibiotics against log-phase S. aureus. Combinations of maggot ES and antibiotics eliminate S. aureus biofilms and the bacteria derived therefrom.

Quorum-sensing-regulated virulence factors in Pseudomonas aeruginosa are toxic to Lucilia sericata maggots

Maggot debridement therapy (MDT) is widely used for debridement of chronic infected wounds; however, for wounds harbouring specific bacteria limited effect or failure of the treatment has been described. Here we studied the survival of Lucilia sericata maggots encountering Pseudomonas aeruginosa PAO1 in a simple assay with emphasis on the quorum-sensing (QS)-regulated virulence. The maggots were challenged with GFP-tagged P. aeruginosa wild-type (WT) PAO1 and a GFP-tagged P. aeruginosa ΔlasR rhlR (ΔRR) QS-deficient mutant in different concentrations. Maggots were killed in the presence of WT PAO1 whereas the challenge with the QS mutant showed a survival reduction of ∼25 % compared to negative controls. Furthermore, bacterial intake by the maggots was lower in the presence of WT PAO1 compared to the PAO1 ΔRR mutant. Maggot excretions/secretions (ES) were assayed for the presence of QS inhibitors; only high doses of ES showed inhibition of QS in P. aeruginosa. Thus P. aeruginosa was shown to be toxic to L. sericata maggots. This, coupled to the preferential feeding by the maggots and reduced ingestion of P. aeruginosa, could explain MDT failure in wounds colonized by P. aeruginosa. Wounds heavily colonized with P. aeruginosa should be a counterindication for MDT unless used in combination with a pre-treatment with other topical therapeutics targeting P. aeruginosa.

Amino acid derivatives from Lucilia sericata excretions/secretions may contribute to the beneficial effects of maggot therapy via increased angiogenesis

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.

Maggot excretions/secretions induces human microvascular endothelial cell migration through AKT1

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.

Do maggots have an influence on bacterial growth? A study on the susceptibility of strains of six different bacterial species to maggots of Lucilia sericata and their excretions/secretions

Purpose The maggots of Lucilia sericata are successfully used as a treatment for infected wounds. Many articles are published about possible direct antibacterial properties of maggots and their excretions/secretions (ES), but with different results. The present study reinvestigates the susceptibility of six bacterial strains to maggots and their ES. Methods Live maggots were added to a bacterial suspension. After incubation for 16 h, the bacterial growth in this suspension was compared with the growth in a suspension without maggots. We tested Instar-1 and Instar-3 maggots and compared nutrient broths. A turbidimetric assay investigated the antibacterial activity of ES. Finally, we compared the bacterial growth of Gram-positive and Gram-negative bacteria. Results The test with live maggots showed an increase of bacterial growth. Instar-1 maggots stimulated more bacterial growth than Instar-3 maggots, as well as the use of a more nutritious broth. The turbidimetric assay showed no inhibition of bacterial growth. For all bacteria, except Pseudomonas aeruginosa, an increase in bacterial growth was shown. Conclusion There is no direct antibacterial effect of maggots and/or ES in vitro, however in clinical observations maggot therapy is successful. More research is needed to focus on possible indirect antibacterial activity, such as an immune-related effect.

Maggot excretions/secretions are differentially effective against biofilms of Staphylococcus aureus and Pseudomonas aeruginosa

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.

Maggot excretions/secretions inhibit multiple neutrophil pro-inflammatory responses

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 2O 2 production by fMLP-activated neutrophils; maximal inhibition was seen with 5–50 μg of ES/ml. In contrast, ES did not affect phagocytosis and intracellular killing of Candida albicans by neutrophils. Furthermore, 0.5 μg 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.

Promotion of Human Dermal Fibroblast Migration, Matrix Remodelling and Modification of Fibroblast Morphology within a Novel 3D Model by Lucilia sericata Larval Secretions

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.

Failure of acid secretion during infection of the ovine abomasum by the nematode parasite Teladorsagia circumcincta

Elevation of the abomasal pH of ruminants is a well known consequence of nematode parasitism. Many have argued that the nematodes seek to increase pH to promote their survival. Indeed, there is limited evidence that substances released by the parasites are capable of directly influencing parietal cell activity. In one experimental series, excretions/secretions (ES) of Teladorsagia circumcincta reduced the apparent activity of rabbit gastric glands. Worm ES were shown to contain ammonia, probably accounting for the observed activity, and the effects of ES/ammonia are almost certainly an in-vitro phenomenon only. An alternative hypothesis contends that the loss of acid secretion arises as a consequence of the profound host response elicited by the presence of the parasites. Invariably, presence of parasites is accompanied by an inflammatory response in which eosinophils are conspicuous. Recent research has shown that exudates from inflamed tissues were also inhibitory to gastric gland activity and that during infection, expression of the pro-inflammatory cytokine interleukin 1-beta, the most potent known inhibitor of acid secretion by parietal cells, was increased. Further, the elevation of abomasal pH may prove to have adverse effects on the ability of the parasites to maintain their position within the abomasum.

Detection and partial characterisation of two antibacterial factors from the excretions/secretions of the medicinal maggot Lucilia sericata and their activity against methicillin-resistant Staphylococcus aureus (MRSA)

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.

In vitro effects of Fasciola hepatica on the main functions of polymorphonuclear leukocytes: Chemotaxis and free radical generation induced by phagocytosis

The effects of adult fluke excretions-secretions (ES) on two major functions of circulating bovine polymorphonuclear (PMN) cells were investigated. Under agarose, PMN chemotaxis was not affected by the ES. ES preincubated with PMN for 15, 30 or 60 min at 37°C, before a chemoluminescence assay, inhibited phagocytosis and/or free radical generation in a dose and time dependent manner.

Origin, kinetics of circulation and fate in vivo of the major excretory-secretory product of Acanthocheilonema viteae.

The excretions-secretions (E-S) of Acanthocheilonema viteae consist mainly of one product, molecular weight 62kDa. This molecule is synthesized during the vertebrate phase of the parasite life-cycle and is first detectable in the E-S of L4 parasites. It is cross-reactive with E-S of human filarial parasites as a consequence of possessing a phosphorylcholine (PC) moiety. The 62 kDa molecule has been employed as a model for the study of the origin and fate of filarial E-S. Immunohistological analysis has shown the molecule to be located predominantly in the parasite gut. Transplantation of adult female [35S] methionine pulsed worms into uninfected jirds resulted in the radio-labelled secreted 62 kDa antigen being detected in the bloodstream within 4 h by SDS-PAGE/immunoprecipitation analysis. The systemic half-life of the molecule as estimated by clearance of injected, purified 125I-labelled material was measured in naive and infected jird hosts. It was reduced from 2-7 h in naive animals to less than 30 min in 4-10 week infected rodents, a finding which correlated with clearance of antigen by antibody in the infected group. In animals infected for longer time periods the serum half-life returned to the values observed in naive jirds. The idea that this change in half-life may reflect differences in the nature of 62 kDa antigen containing circulating immune complexes as infection progresses is discussed. The 125I-labelled antigen is predominantly removed from the circulation via the liver and ultimately excreted in the urine in a non-antigenic form. This work provides the first description of the origin, kinetics of circulation and fate of a defined filarial E-S product and may aid in determining the function and assessing the diagnostic utility of PC-bearing E-S components.

Evaluation of the potential of excretions-secretions (E-S) ofLitomosoides carinii to substitute for human filarial E-S

The antigenic cross-reactivity of the excretions-secretions (E-S) of Litomosoides carinii was investigated. Immunoprecipitation using pooled sera from a number of human filarial infections in combination with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed variations in the cross-reactivity of individual molecules. Some were specific to L. carinii, the major examples in this category being two E-S components of 140 and 160 kDa released by day 40- to 42-day-old female worms. Another, a high-molecular-weight product of 26- to 28-day E-S, was broadly cross-reactive. A third group appeared to exhibit reactivity to antibody to some but not all human filarial parasites. The most striking examples of this were two distinct 14-kDa products that bound solely to antibodies in an onchocerciasis serum pool. These results are discussed in relation to the use of cross-reacting molecules in investigating the immunisation potential, defining the function, and evaluating the diagnostic utility of human filarial E-S.

Partial protection of lambs against Haemonchus contortus by vaccination with a fractionated preparation of the parasite

Florida Native lambs, <6 months of age, were successfully vaccinated against Haemonchus contortus with a high mol. wt fraction (>30 000 daltons) derived from a somatic extract of H. contortus larvae (SEL) and excretions secretions (ES) of larvae isolated during in vitro development from the infective 3rd to 4th stage. A 59% reduction in adult worm numbers was obtained in vaccinates compared to naive lambs following challenge. The protection in vaccinated lambs was similar to that seen in lambs exposed to a primary infection of H. contortus larvae which had been cleared with anthelmintic prior to the challenge infection. The unfractioned SEL/ES preparation and a low mol. wt fraction gave no significant protection against challenge infection.