Investigation of whole body extract metabolites of Lucilia sericata larvae and potential antibacterial effects

Öz

Aim: Complementary medicinal techniques have gainedfocus by modern medicine, recently. Maggot Debridement Therapy is a widely-used method worldwide. It is especially recommended for chronic wounds, and has serious advantages such as low cost, easily-applicability and rare adverse effects, but its effect mechanisms remains unclear. The aim of this study is to detect components and to investigate potential antibacterial effects of whole body extract metabolites of Lucilia sericata larvae.

Material anf Methods: Due to potential antibacterial effects, agar well diffusion and flowcytometry methods were used against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis to do evaluation on whole body extracts of previously-cloned maggots in specialized climate room. After this antibacterial effect evaluation, 2-D PAGE analysis was done for protein investigation.

Results: Inhibition zones were observed for S.aureus (16mm), E.coli (22mm) and E.faecalis (14mm), but for P.aeruginosa, the extract could not provide any inhibiton zone. In flow cytometry, different killing rates were detected in different extract dilutions, and for the lowest (1/64) dilution, killing rates were 51.9%, 75%, 80% and 98.7% for P.aeruginosa, E.faecalis, E.coli and S.aureus, respectively. 2-D PAGE showed various proteins with different molercular mass (<10-260kDa) and pI (3-9).

Conclusion: Antibacterial effects of maggot whole body extracts on tested strains are obviously detected. Many protein spots with widely variable molecular mass and isoelectric points were observed. As a result, this antibacterial effects may be caused by these proteins, but it is necessary that these proteins must be further evaluated via mass spectrometry and protein databases.

Anahtar Kelimeler

• Lucilia sericata; Chronic Wound Care; Larval Debridement; Biosurgery; Maggot Debridement Therapy

Lucilia sericata larvalarının tüm vücut ekstrakt metabolitlerinin araştırılması ve potensiyel antibakteriyel etkileri

Öz

Amaç: Yakın dönemde, tamamlayıcı tıp uygulamaları modern tıbbın ilgi alanına girmiştir. Maggot Debritman Tedavisi dünya çapında yaygın olarak kullanılan bir yöntemdir. Bu yöntem, özellikle kronik yaraların tedavisinde tavsiye edilmektedir ve düşük maliyet, kolay uygulanabilirlik ve nadir yan etkiler gibi avantajları bulunmaktadır, ancak yöntemin etki mekanizması henüz tam olarak ortaya konulamamıştır. Bu çalışmanın amacı, Lucilia sericatalarvalarının tüm vücut ekstraktının metabolitlerini ortaya koymak ve bunların potensiyel antibakteriyel niteliğini araştırmaktır.

Gereç ve Yöntemler: Antibakteriyel etkinliği araştırmak için, önceden özel iklim odalarında üretilmiş larvalarının tüm vücut ekstraktları, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coliveEnterococcus faecalisbakterileri için agar difüzyon ve akan hücreölçer ile test edilmiştir. Antibakteriyel incelemeyi takiben, iki boyutlu elektroforez ile protein araştırılması yapılmıştır.

Bulgular: S.aureus (16mm), E.coli (22mm) ve E.faecalis (14mm) için inhibisyon alanı gözlenmiş ancak P.aeruginosaiçinalan oluşmamıştır. Hücreölçer ile farklı dilüsyonlarda farklı öldürme oranları gözlenmiş ve en düşük dilüsyonda (1/64), P.aeruginosa, E.faecalis, E.coliveS.aureusiçin sırasıyla%51,9, %75, %80ve %98,7 oranları alınmıştır. İki boyutlu elektroforezde farklı moleküler ağırlık (<10-260kDa) ve izoelektrik noktada (3-9) proteinler tespit edilmiştir.

Sonuç: Maggot ekstraklarının test edilen suşlar üzerine antibakteriyel etkisi net olarak gözlenmiştir. Farklı moleküler ağırlık ve izoelektrik noktada proteinler tespit edilmiştir. Antibakteriyel etkinin bu proteinler tarafından sağlanması muhtemel olsa da, proteinlerin kütle spektrometrisi ve protein veri bankaları ile ayrıca araştırılması gerekir.

Anahtar Kelimeler

• Lucilia sericata; Kronik Yara Bakımı; Larval Debritman; Biyocerrahi; Maggot Debritman Tedavisi

Kaynakça

1. 1. Fischer FH, Lewith G, Witt CM et al. High prevalence but limited evidence in complementary and alternative medicine: guidelines for future research. BMC complementary and alternative medicine 2014; 14: 46.
2. 2. World Health Organization (WHO), WHO traditional medicine strategy: 2014-2023. Geneva, Switzerland: World Health Organization; 2013.
3. 3. Barnes PM, Bloom B, Nahin RL. Complementary and alternative medicine use among adults and children: United States, 2007. National health statistics reports; No: 12. Hyattsville, MD: National Center for Health Statistics, 2008.
4. 4. Sherman RA, Mumcuoglu KY, Grassberger M, Tantawi TI. Maggot Therapy. In: Grassberger M, Sherman RA, Gileva OS, Kim CMH, Mumcuoglu KY (eds). Biotherapy-history, principles and practice: A practical guide to the diagnosis and treatment of disease using living organisms. Springer Science & Business Media, Amsterdam 2013; 5-29.
5. 5. Sherman RA, Hall M, Thomas S. Medicinal maggots: an ancient remedy for some contemporary afflictions. Annu Rev Entomol 2000; 45: 55-81.
6. 6. Sherman RA, Pechter EA. Maggot therapy: a review of the therapeutic applications of fly larvae in human medicine, especially for treating osteomyelitis. Med Vet Entomol 1988; 2: 225-30.
7. 7. Sherman RA, Tran JMT, Sullivan R. Maggot therapy for venous stasis ulcers. Arch Dermatol 1996; 132: 254-56.
8. 8. Sherman RA., Wyle FA. Low-cost, low-maintenance rearing of maggots in hospitals, clinics, and schools. Am J Trop Med Hyg 1996; 54: 38-41.

9. 9. Stoddard S, Sherman R., Mason B, Pelsang D, Sherman R. Maggot debridement therapy. An alternative treatment for nonhealing ulcers. J Am Podiatr Med Assoc 1995; 85: 218-21.
10. 10. Fleischmann W, Grassberger M, Sherman RA. Maggot therapy: A handbook of maggot-assisted wound healing. Thieme Publification, London, 2004.
11. 11. Game FL, Apelqvist J, Attinger C et al. IWGDF Guidance on use of intervantions to enhance healing of chronic ulcers of the foot in diabetes. International Working Group on Wound Healing Document, Amsterdam, 2015.
12. 12. Andersen AS, Sandvang D, Schnorr KM, Kruse T, Neve S, Joergensen B, Karlsmark T, Krogfelt KA. A novel approach to the antimicrobial activity of maggot debridement therapy. J Antimicrob Chemother 2010; 65: 1646-54.
13. 13. Kruglikova A, Chernysh S. Antimicrobial compounds from the excretions of surgical maggots, Lucilia sericata (Meigen) (Diptera, Calliphoridae). Entomological Review 2011; 91: 813-19.
14. 14. Chernysh SI, Gordja NA, Simonenko NP. Diapause and immune response: induction of antimicrobial peptides synthesis in the blowfly, Calliphora vicina R.-D. (Diptera: Calliphoridae). J Entomol Sci 2000; 3: 139-44.
15. 15. Čeřovský V, Žďárek J, Fučík V, Monincová L, Voburka Z, Bém R. Lucifensin, the long-sought antimicrobial factor of medicinal maggots of the blowfly Lucilia sericata. Cell Mol Life Sci 2010; 67: 455-66.
16. 16. El Shazely B, Veverka V, Fučík V, Voburka Z, Žďárek J, Čeřovský V. Lucifensin II, a defensin of medicinal maggots of the blowfly Lucilia cuprina (Diptera: Calliphoridae). J Med Entomol 2013; 50: 571-78.
17. 17. Valachova I, Majtan T, Takac P, Majtan J. Identification and characterisation of different proteases in Lucilia sericata medicinal maggots involved in maggot debridement therapy. J Appl Biomed 2014; 12: 171-77.
18. 18. Valachova I, Takac P, Majtan J. Midgut lysozymes of Lucilia sericata–new antimicrobials involved in maggot debridement therapy. Insect Mol Biol 2014; 23: 779-87.
19. 19. Pöppel AK, Koch A, Kogel KH et al. Lucimycin, an antifungal peptide from the therapeutic maggot of the common green bottle fly Lucilia sericata. Biol Chem 2014; 395: 649-56.
20. 20. Pöppel AK, Vogel H, Wiesner J, Vilcinskas A. Antimicrobial peptides expressed in medicinal maggots of the blow fly Lucilia sericata show combinatorial activity against bacteria. Antimicrob Agents Chemother 2015; 59: 2508-14.
21. 21. Tanyuksel M, Araz E, Dundar K et al. Maggot debridement therapy in the treatment of chronic wounds in a military hospital setup in Turkey. Dermatol 2004; 210: 115-18.
22. 22. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard — Eleventh Edition, CLSI document M02-A11, 2012.
23. 23. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). Antimicrobial susceptibility testing EUCAST disk diffusion method, version 5.0, Jan 2015.
24. 24. Dogandemir G. Investigation of antimicrobial activities of Lucilia sericata against mikroorganisms colonising on chronic wounds. Medical Microbiology Thesis, Gulhane Military Medical Academy, Ankara, Turkey, 2010.
25. 25. Michelsen CF, Christensen AMJ, Bojer MS, Høiby N, Ingmer H, Jelsbak L. Staphylococcus aureus alters growth activity, autolysis, and antibiotic tolerance in a human host-adapted Pseudomonas aeruginosa lineage. J Bacteriol 2014; 196: 3903-11.
26. 26. Faria‐Ramos I, Espinar MJ, Rocha R, Santos‐Antunes J, Rodrigues AG, Cantón R, Pina‐Vaz C. A novel flow cytometric assay for rapid detection of extended‐spectrum beta‐lactamases. Clin Microbiol Infect 2013; 19: 8-15.
27. 27. Nuding S, Zabel LT. Detection, identification, and susceptibility testing of bacteria by flow cytometry. J Bacteriol Parasitol 2013; S5:005.
28. 28. Becton Dickinson (BD) BioSciences. Application Note: Bacterial Detection and Live/Dead Discrimination by Flow Cytometry [Available at http://www.bdbiosciences.com/us/home, Date of Access: 15 May 2016].
29. 29. Friedman DB, Hoving S, Westermeier R. Isoelectric focusing and two-dimensional gel electrophoresis. Methods in Enzymology 2009; 463: 515-40.
30. 30. Garfin DE. Gel electrophoresis of proteins. In: Davey J, Lord M. (eds) Essential Cell Biology Volume 1: Cell Structure, A Practical Apporach. Oxford University Press, Oxford, UK 2003; 197-268.
31. 31. Righetti PG, Sebastiano R, Citterio A. Capillary electrophoresis and isoelectric focusing in peptide and protein analysis. Proteomics 2013; 13: 325-340.
32. 32. Barnes KM, Dixon RA, Gennard DE. The antibacterial potency of the medicinal maggot, Lucilia sericata (Meigen): variation in laboratory evaluation. J Microbiol Methods 2010; 82: 234-37.
33. 33. Bexfield A, Bond AE, Roberts EC et al. The antibacterial activity against MRSA strains and other bacteria of a< 500Da fraction from maggot excretions/secretions of Lucilia sericata (Diptera: Calliphoridae). Microbes Infect 2008; 10: 325-33.
34. 34. Bexfield A, Nigam Y, Thomas S, Ratcliffe NA. 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). Microbes Infect 2004; 6(: 1297-1304.
35. 35. Huberman L, Gollop N, Mumcuoglu KY, Block C, Galun R. Antibacterial properties of whole body extracts and haemolymph of Lucilia sericata maggots. J Wound Care 2007; 16: 123-27.
36. 36. Huberman L, Gollop N, Mumcuoglu KY et al. Antibacterial substances of low molecular weight isolated from the blowfly, Lucilia sericata. Med Vet Entomol 2007; 21: 127-31.
37. 37. Bexfield A, Bond AE, Morgan C et al. Amino acid derivatives from Lucilia sericata excretions/secretions may contribute to the beneficial effects of maggot therapy via increased angiogenesis. Br J Dermatol 2010; 162: 554-62.
38. 38. Kerridge A, Lappin‐Scott H, Stevens J. Antibacterial properties of larval secretions of the blowfly, Lucilia sericata. Med Vet Entomol 2005; 19: 333-37.
39. 39. Nunan R, Harding KG, Martin P. Clinical challenges of chronic wounds: searching for an optimal animal model to recapitulate their complexity. Dis Model Mech 2014; 7: 1205-13.
40. 40. Daeschlein G. Antimicrobial and antiseptic strategies in wound management. Int Wound J 2013; 10: 9-14.
41. 41. Mosiman VL, Patterson BK, Canterero L, Goolsby CL. Reducing cellular autofluorescence in flow cytometry: an in situ method. Cytometry 1997; 30: 151-56.
42. 42. Trøstrup H, Bjarnsholt T, Kirketerp-Møller K, Høiby N, Moser C. What Is New in the Understanding of Non Healing Wounds Epidemiology, Pathophysiology, and Therapies. Ulcers 2013; 8: 1-6.
43. 43. Cazander G, Van de Veerdonk MC, Vandenbroucke-Grauls CM, Schreurs MW, Jukema GN. Maggot excretions inhibit biofilm formation on biomaterials. Clin Orthop Relat Res 2010; 468: 2789-96.
44. 44. Cazander G, van Veen KE, Bouwman LH, Bernards AT, Jukema GN. The influence of maggot excretions on PAO1 biofilm formation on different biomaterials. Clin Orthop Relat Res 2009; 467: 536-45.
45. 45. Brown A, Horobin A, Blount DG et al. Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm. Med Vet Entomol 2012; 26: 432-39.
46. 46. Jiang KC, Sun XJ, Wang W e al. Excretions/secretions from bacteria-pretreated maggot are more effective against Pseudomonas aeruginosa biofilms. PloS one 2012; 7: 49815.
47. 47. Masiero FS, Aquino MFK, Nassu MP, Pereira DIB, Leite DS, Thyssen PJ.First Record of Larval Secretions of Cochliomyia macellaria (Fabricius, 1775)(Diptera: Calliphoridae) Inhibiting the Growth of Staphylococcus aureus and Pseudomonas aeruginosa. Neotrop Entomol 2017; 46: 125-29.
48. 48. Čeřovský V, Bém R. Lucifensins, the insect defensins of biomedical importance: the story behind maggot therapy. Pharmaceuticals 2014; 7: 251-64.
49. 49. Van Der Plas MJ, Jukema GN, Wai SW et al. Maggot excretions/secretions are differentially effective against biofilms of Staphylococcus aureus and Pseudomonas aeruginosa. J Antimicrob Chemother 2008; 61: 117-22.
50. 50. Daeschlein G, Mumcuoglu KY, Assadian O, Hoffmeister B, Kramer A. In vitro antibacterial activity of Lucilia sericata maggot secretions. Skin Pharmacol Physiol 2006; 20: 112-15.
51. 51. Clinical and Laboratory Standards Institute (CLSI). Methods for Determining Bactericidal Activity of Antimicrobial Agents; Approved Guideline CLSI document M26-A, 1999.
52. 52. Jaklič D, Lapanje A, Zupančič K, Smrke D, Gunde-Cimerman N. Selective antimicrobial activity of maggots against pathogenic bacteria. J Med Microbiol 2008; 57: 617-25.