The possibility of using xenogeneic phagocytes in wound treatment.

Metamorphosis in the insect larva is associated with disintegration, engulf and digestion of larval tissues. These processes are accompanied by a significant shift in physiological parameters like high activity of hydrolytic enzymes and decrease of pH. In the way, the metamorphosing larva resembles the processes occurring in the wound at the stage of inflammation. Based on this thesis, we put forward the idea of the possibility of using insect phagocytes in the wound treatment. The search for a suitable insect cell line and the study of its properties were the purpose of the work. The abilities of insect phagocytes to retain viability and functional activity under conditions physiological for humans were also investigated. We found that blue blowfly Calliphora vicina larvae had histolysocytes, a specialized population of professional phagocytes involved in the histolysis. In vitro, histolysocytes possess high phagocytic activity to fragments of vertebrate soft tissues and debris. These cells retain viability and functional activity for a long time under conditions that are physiological for vertebrate cells. Moreover histolysocytes can realize the humoral control over the bacteria through the synthesis of antimicrobial peptides. So histolysocytes have the potential to be used as xenogeneic phagocytes in the wound treatment. The data obtained allow proceeding to experiments on laboratory animals for studying the effect of such therapy on the wound healing process.

Biofilm infections between Scylla and Charybdis: interplay of host antimicrobial peptides and antibiotics.

PURPOSE: The aim of this study is to improve the anti-biofilm activity of antibiotics. We hypothesized that the antimicrobial peptide (AMP) complex of the host's immune system can be used for this purpose and examined the assumption on model biofilms. METHODS: FLIP7, the AMP complex of the blowfly Calliphora vicina containing a combination of defensins, cecropins, diptericins and proline-rich peptides was isolated from the hemolymph of bacteria-challenged maggots. The complex interaction with antibiotics of various classes was studied in biofilm and planktonic cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii by the checkerboard method using trimethyl tetrazolium chloride cell viability and crystal violet biofilm eradication assays supplemented with microscopic analysis. RESULTS: We found that FLIP7 demonstrated: high synergy (fractional inhibitory concentration index <0.25) with meropenem, amikacin, kanamycin, ampicillin, vancomycin and cefotaxime; synergy with clindamycin, erythromycin and chloramphenicol; additive interaction with oxacillin, tetracycline, ciprofloxacin and gentamicin; and no interaction with polymyxin B. The interaction in planktonic cell models was significantly weaker than in biofilms of the same strains. The analysis of the dose-effect curves pointed to persister cells as a likely target of FLIP7 synergistic effect. The biofilm eradication assay showed that the effect also caused total destruction of S. aureus and E. coli biofilm materials. The effect allowed reducing the effective anti-biofilm concentration of the antibiotic to a level well below the one clinically achievable (2-3 orders of magnitude in the case of meropenem, ampicillin, cefotaxime and oxacillin). CONCLUSION: FLIP7 is a highly efficient host antimicrobial system helping antibiotics to overcome biofilm barriers through persisters' sensitization and biofilm material destruction. It is promising for the treatment of biofilm infections as an adjuvant of various small-molecule antibiotics.

Natural antimicrobial peptide complexes in the fighting of antibiotic resistant biofilms: Calliphora vicina medicinal maggots.

Biofilms, sedimented microbial communities embedded in a biopolymer matrix cause vast majority of human bacterial infections and many severe complications such as chronic inflammatory diseases and cancer. Biofilms' resistance to the host immunity and antibiotics makes this kind of infection particularly intractable. Antimicrobial peptides (AMPs) are a ubiquitous facet of innate immunity in animals. However, AMPs activity was studied mainly on planktonic bacteria and little is known about their effects on biofilms. We studied structure and anti-biofilm activity of AMP complex produced by the maggots of blowfly Calliphora vicina living in environments extremely contaminated by biofilm-forming germs. The complex exhibits strong cell killing and matrix destroying activity against human pathogenic antibiotic resistant Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii biofilms as well as non-toxicity to human immune cells. The complex was found to contain AMPs from defensin, cecropin, diptericin and proline-rich peptide families simultaneously expressed in response to bacterial infection and encoded by hundreds mRNA isoforms. All the families combine cell killing and matrix destruction mechanisms, but the ratio of these effects and antibacterial activity spectrum are specific to each family. These molecules dramatically extend the list of known anti-biofilm AMPs. However, pharmacological development of the complex as a whole can provide significant advantages compared with a conventional one-component approach. In particular, a similar level of activity against biofilm and planktonic bacteria (MBEC/MIC ratio) provides the complex advantage over conventional antibiotics. Available methods of the complex in situ and in vitro biosynthesis make this idea practicable.