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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Combinations of maggot ES and antibiotics eliminate S. aureus biofilms and the bacteria derived therefrom.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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(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.

Release of calcium from intracellular stores and subsequent uptake by mitochondria are essential for the candidacidal activity of an N-terminal peptide of human lactoferrin.

OBJECTIVES: Earlier studies showed that mitochondrial damage is a hallmark of the candidacidal activity of an N-terminal peptide of human lactoferrin, further referred to as hLF(1-11). Since uptake of Ca(2+) by mitochondria may be essential for their activation, the aim of this study was to define the role of Ca(2+) in killing of Candida albicans by the hLF(1-11) peptide. METHODS: The effect of compounds interfering with Ca(2+) homeostasis on the hLF(1-11)-induced candidacidal activity, changes in mitochondrial membrane potential, and reactive oxygen species production were evaluated using a killing assay, rhodamine 123 staining, and 2',7'-dichlorofluorescein diacetate, respectively. The increase in cellular Ca(2+) content was measured using (45)Ca(2+). RESULTS: Our results revealed that Ruthenium Red, which inhibits the mitochondrial Ca(2+)-uniporter and the voltage-sensitive Ca(2+) release from internal stores, blocked (P<0.05) the hLF(1-11)-induced candidacidal activity as well as changes in the membrane potential of mitochondria, and reactive oxygen species production. Oxalate, which precipitates Ca(2+) in intracellular organelles, decreased (P<0.05) the peptide-induced changes in the membrane potential of mitochondria, reactive oxygen species production, and candidacidal activity. Furthermore, the Ca(2+) ionophore ionomycin combined with high CaCl(2) concentrations enhanced the hLF(1-11)-induced candidacidal activity. Moreover, hLF(1-11) caused an influx of Ca(2+) from the extracellular medium into C. albicans reaching a three-fold increase at 2 h, whereas no increase was found in unexposed cells. In agreement, the Ca(2+)-chelator EGTA blocked the peptide-induced candidacidal activity. CONCLUSIONS: Ca(2+) release from intracellular stores, probably through subsequent mitochondrial Ca(2+) uptake, is essential for the hLF(1-11)-induced candidacidal activity.

Synergistic activity of the N-terminal peptide of human lactoferrin and fluconazole against Candida species.

In light of the need for new antifungal regimens, we report that at noncandidacidal concentrations, the lactoferrin-derived peptide hLF(1-11), which is highly active against fluconazole-resistant Candida albicans, acts synergistically with fluconazole against this yeast and a fluconazole-sensitive C. albicans strain as well as C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis. When these yeasts were exposed to hLF(1-11) for 5 min and then incubated with fluconazole, they were killed effectively, while no candidacidal activity was observed when they were incubated first with fluconazole and then exposed to the peptide, indicating that the candidacidal activity is initiated by the peptide while fluconazole is only required during the effector phase. Investigations of the effect of azide, which inhibits mitochondrial respiration, on the activity of combinations of hLF(1-11) and fluconazole against fluconazole-resistant C. albicans revealed that it inhibits this activity, even when added during the effector phase only. As expected, azide inhibited the accumulation of rhodamine 123 in mitochondria and the production and release of ATP by C. albicans that occurred upon exposure to the combination of hLF(1-11) and fluconazole. Accordingly, oxidized ATP (oATP), an antagonist of ATP receptors, completely blocked the candidacidal activity of the hLF(1-11)-fluconazole combination, whereas oATP did not block the activity when its presence was restricted to the effector phase. The candidacidal activity of combinations of hLF(1-11) and fluconazole, which is initiated by the peptide through the involvement of energized mitochondria, renders fluconazole-resistant C. albicans sensitive to this azole.