Detection of Antibacterial Properties of Musca domestica, Drosophila melanogaster, and Sarcophaga nodosa Using Resazurin as A Growth Indicator in Bacterial Cells

Document Type : Original Research

Authors
M. Moshrefi 1
K. Ahmadi 1
A. Purhematy 1
M. Jajarmi 2
Y. SarveAhrabi 3
1 Plant Protection, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran. 
2 Department of Pathobiology, Faulty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran.
3 Department of Biology, Central Tehran Branch, Islamic Azad University, Tehran, Iran.
10.29252/iem.6.3.201
Abstract
Background: Due to the side effects of chemical and synthetic antibiotics and the increase in bacterial resistance, extensive research has been conducted to obtain natural compounds without side effects from natural sources such as insects, especially Diptera order, because these insects require to live and survive on rotting food and be in direct contact with pathogenic and dangerous microorganisms because of their special diet.

Materials & Methods: In this study, hemolymphs were extracted from Musca domestica, Drosophila melanogaster, and Sarcophaga nodosa, and then the anti-bactericidal activity of these extracts against important pathogenic bacteria was investigated separately by the minimum inhibitory concentration (MIC) method using resazurin indicator.

Findings: S. nodosa and M. domestica larvae were not able to prevent the growth of any of the bacteria. D. melanogaster larvae extract completely inhibited the growth of Escherichia coli and Pseudomonas aeruginosa bacteria at all densities, while Staphylococcus aureus was completely resistant to all concentrations. The minimum inhibitory concentration of D. melanogaster larvae extract against two bacteria of Listeria monocytogenes and Salmonella typhimurium was determined as 125 and 500 μL/mL, respectively.

D. melanogaster pupae extract was unable to inhibit the growth of E. coli and S. typhimurium but prevented the growth of P. aeruginosa at all concentrations. Also, the minimum inhibitory concentration of this extract against both S. aureus and L. monocytogenes was determined as 1000 μL/mL.

Conclusion: These outcomes show that D. melanogaster holds a high potential of antibacterial effects, and the purification and evaluation of this extract active substances are recommended for future utilization as antibacterial agents and food preservatives to fight pathogenic and toxigenic microorganisms.

Keywords

Subjects

1. Celiktas OY, Kocabas EH, Bedir E, Sukan FV, Ozek T, Baser KH. Antimicrobial activities of methanol extracts and essential oils of Rosmarinus officinalis, depending on location and seasonal variations. Food Chemistry. 2007 Jan 1;100(2):553-9.
2. Pfaller M, Wenzel R. Impact of the changing epidemiology of fungal infections in the 1990s. European Journal of Clinical Microbiology and Infectious Diseases. 1992 Apr 1;11(4):287-91.
3. Zare P, Mahmoudi R, Ehsani A. Biochemical and antibacterial properties of essential oil from Teucrium polium using Resazurin as the indicator of bacterial cell growth. Pharm Sci. 2011;17(3):183-8.
4. Geesi MH, BAKHT MS, Anwar F, Khamis EH, Gilani MA. Volatiles composition, physicochemical properties, kinetic study and antioxidant potential of endemic artemisia (Artemisia Judaica L.) essential oil.
5. Davis MM, Engström Y. Immune response in the barrier epithelia: lessons from the fruit fly Drosophila melanogaster. Journal of innate immunity. 2012;4(3):273-83.
6. Fearon DT. Seeking wisdom in innate immunity. Nature. 1997 Jul;388(6640):323-4.
7. Gardiner EM, Strand MR. Hematopoiesis in larval Pseudoplusia includens and Spodoptera frugiperda. Archives of Insect Biochemistry and Physiology: Published in Collaboration with the Entomological Society of America. 2000 Apr;43(4):147-64.
8. Lavine MD, Strand MR. Insect hemocytes and their role in immunity. Insect biochemistry and molecular biology. 2002 Oct 1;32(10):1295-309.
9. Zare P, Mahmoudi R, Ehsani A. Biochemical and antibacterial properties of essential oil from Teucrium polium using Resazurin as the indicator of bacterial cell growth. Pharm Sci. 2011;17(3):183-8.
10. Teh CH, Nazni WA, Norazah A, Lee HL. Determination of antibacterial activity and minimum inhibitory concentration of larval extract of fly via Resazurin-based turbidometric assay. BMC microbiology. 2017 Dec;17(1):36.
11. Murtaugh MP, Steiner AL, Davies PJ. Localization of the catalytic subunit of cyclic AMP-dependent. Protein kinase in cultured cells using a specific antibody. The Journal of cell biology. 1982 Oct;95(1):64-72.
12. Hultmark D, Engström A, Andersson K, Steiner H, Bennich H, Boman HG. Insect immunity. Attacins, a family of antibacterial proteins from Hyalophora cecropia. The EMBO journal. 1983 Apr 1;2(4):571-6.
13. Tzou P, Ohresser S, Ferrandon D, Capovilla M, Reichhart JM, Lemaitre B, Hoffmann JA, Imler JL. Tissue-specific inducible expression of antimicrobial peptide genes in Drosophila surface epithelia. Immunity. 2000 Nov 1;13(5):737-48.
14. Tingvall TÖ, Roos E, Engström Y. The imd gene is required for local Cecropin expression in Drosophila barrier epithelia. EMBO reports. 2001 Mar 1;2(3):239-43.
15. Ryu JH, Nam KB, Oh CT, Nam HJ, Kim SH, Yoon JH, Seong JK, Yoo MA, Jang IH, Brey PT, Lee WJ. The homeobox gene Caudal regulates constitutive local expression of antimicrobial peptide genes in Drosophila epithelia. Molecular and cellular biology. 2004 Jan 1;24(1):172-85.
16. Meister M, Hetru C, Hoffmann JA. The antimicrobial host defense of Drosophila. InOrigin and Evolution of the Vertebrate Immune System 2000 (pp. 17-36). Springer, Berlin, Heidelberg.
17. Rabel D, Charlet M, Ehret-Sabatier L, Cavicchioli L, Cudic M, Otvos L, Bulet P. Primary structure and in vitro antibacterial properties of the Drosophila melanogaster attacin C Pro-domain. Journal of Biological Chemistry. 2004 Apr 9;279(15):14853-9.
18. Hoffmann JA. The immune response of Drosophila. Nature. 2003 Nov;426(6962):33-8.
19. Hultmark D. Drosophila immunity: paths and patterns. Current opinion in immunology. 2003 Feb 1;15(1):12-9.
20. Lemaitre B, Reichhart JM, Hoffmann JA. Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proceedings of the National Academy of Sciences. 1997 Dec 23;94(26):14614-9.
21. Benghezal M, Fauvarque MO, Tournebize R, Froquet R, Marchetti A, Bergeret E, Lardy B, Klein G, Sansonetti P, Charette SJ, Cosson P. Specific host genes required for the killing of Klebsiella bacteria by phagocytes. Cellular microbiology. 2006 Jan;8(1):139-48.
22. Rabel D, Charlet M, Ehret-Sabatier L, Cavicchioli L, Cudic M, Otvos L, Bulet P. Primary structure and in vitro antibacterial properties of the Drosophila melanogaster attacin C Pro-domain. Journal of Biological Chemistry. 2004 Apr 9;279(15):14853-9.
23. Apidianakis Y, Rahme LG. Drosophila melanogaster as a model host for studying Pseudomonas aeruginosa infection. Nature protocols. 2009 Sep;4(9):1285.
24. Kaës R. Un singulier pluriel. Paris, Dunod. 2007;14:119.
25. Popova AA, Koksharova OA, Lipasova VA, Zaitseva JV, Katkova-Zhukotskaya OA, Eremina SI, Mironov AS, Chernin LS, Khmel IA. Inhibitory and toxic effects of volatiles emitted by strains of Pseudomonas and Serratia on growth and survival of selected microorganisms, Caenorhabditis elegans, and Drosophila melanogaster. BioMed Research International. 2014;2014.
26. Scott JG, Warren WC, Beukeboom LW, Bopp D, Clark AG, Giers SD, Hediger M, Jones AK, Kasai S, Leichter CA, Li M. Genome of the house fly, Musca domestica L., a global vector of diseases with adaptations to a septic environment. Genome biology. 2014 Oct 1;15(10):466.
27. Dang XL, Wang YS, Huang YD, Yu XQ, Zhang WQ. Purification and characterization of an antimicrobial peptide, insect defensin, from immunized house fly (Diptera: Muscidae). Journal of medical entomology. 2014 Dec 1;47(6):1141-5.
28. Fu P, Wu J, Guo G. Purification and molecular identification of an antifungal peptide from the hemolymph of Musca domestica (housefly). Cellular & molecular immunology. 2009 Aug;6(4):245-51.
29. Reed MO, Ai Y, Leutcher JL, Jane JL. Effects of cooking methods and starch structures on starch hydrolysis rates of rice. Journal of food science. 2013 Jul;78(7):H1076-81.
30. Hou DY, Muller AJ, Sharma MD, DuHadaway J, Banerjee T, Johnson M, Mellor AL, Prendergast GC, Munn DH. Inhibition of indoleamine 2, 3-dioxygenase in dendritic cells by stereoisomers of 1-methyl-tryptophan correlates with antitumor responses. Cancer research. 2007 Jan 15;67(2):792-801.
31. Ai GX, Shi HL, Wu HT, Yan YH, Bian YJ, Hu YH, Li ZG, Guo J, Cai XD. A positioning system based on communication satellites and the Chinese Area Positioning System (CAPS). Chinese Journal of Astronomy and Astrophysics. 2008 Dec;8(6):611.
32. Chu YF, Wise ML, Gulvady AA, Chang T, Kendra DF, Van Klinken BJ, Shi Y, O'Shea M. In vitro antioxidant capacity and anti-inflammatory activity of seven common oats. Food chemistry. 2013 Aug 15;139(1-4):426-31.
33. Chen CF, Huang J, Li H, Zhang C, Huang X, Tong G, Xu YZ. MicroRNA-221 regulates endothelial nitric oxide production and inflammatory response by targeting adiponectin receptor 1. Gene. 2015 Jul 10;565(2):246-51.
34. Jiang C, Xiong Q, Gan D, Jiao Y, Liu J, Ma L, Zeng X. Antioxidant activity and potential hepatoprotective effect of polysaccharides from Cyclina sinensis. Carbohydrate polymers. 2013 Jan 2;91(1):262-8.
35. Okada M, Natori S. Primary structure of sarcotoxin I, an antibacterial protein induced in the hemolymph of Sarcophaga peregrina (flesh fly) larvae. Journal of Biological Chemistry. 1985 Jun 25;260(12):7174-7.
36. Matsuyama K, Natori S. Purification of three antibacterial proteins from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina. Journal of Biological Chemistry. 1988 Nov 15;263(32):17112-6.
37. Nanbu R, Nakajima Y, Ando K, Natori S. Novel peature of expression of the sarcotoxin IA gene in development of Sarcophaga peregrina. Biochemical and biophysical research communications. 1988 Jan 29;150(2):540-4.
Infection Epidemiology and Microbiology
Volume 6, Issue 3
August 2020
Pages 201-209