Effect of Maggot (Larva) Secretions on Bacterial Growth

ISSN: 1812–1217

Effect of Maggot (larva) Secretions on Bacterial Growth

Wisam M Al-Saeed Department of Dental Basic Science BVMS, MSc, PhD (Asst. Prof.) College of Dentistry, University of Mosul

Hind J Mahmood Mosul Technical Institute BSc (Asst.Prof.)

ABSTRACT Aim: To evaluate the antimicrobial properties of maggots in- vitro. The secretions of maggots are known to have antibacterial properties. To quantify the bactericidal effect of secretions from larvae of L. sericata, an in vitro test model based on the modified European qualitative test. Material and me- thod: In this study, the activity of the maggots was demonstrated against Staphylococcus aureus, Pseu- domonas aeruginosa, Streptococcus group A and group B, and a clinical isolate of MRSA. The num- bers of bacterial colonies with and without maggot exposure were compared after 24, 48 and 72 h of exposure. Results: Maggots applied in the center of the bacterial culture showed a clear zone of inhibition of the bacterial growth in addition the maggot contained viable bacteria after 48 hrs of contact with the respective organisms. Thus, the maggot secretions regarded as an antiseptic against different types of bacteria. In addition, the maggots ability to ingest bacteria was also evaluated. These maggots also continued excreting bacteria. Therefore, maggots should be disposed after use, as they must be regarded as medical wastes. Conclusions: Complete lyses of the bacteria in the area of maggots application indicated the antimicrobial properties of maggots. Key words: Maggot, antimicrobial secretions, larva secretion

Al-Saeed WM, Mahmood HJ . Effect of Maggot (larva) Secretions on Bacterial Growth. Al– Rafidain Dent J. 2012; 12(1): 160-164. Received: 29/12/2010 Sent to Referees: 29/12/2010 Accepted for Publication: 10/2/2011

INTRODUCTION wound-antisepsis due to antibacterial se- Live maggots were clinically suggested to cretions.(5) The presence of an antibacterial kill or inhibit the growth of a wide range substance in the body and secretions of of pathogenic bacteria, especially Methi- Lucilia sericata was demonstrated by Ker- cillin resistant Staphylococcus aureus ridge et al, (6) Furthermore, the destruction (MRSA) , group A and B Streptococci and of ingested bacteria in the intestinal tract Pseudomonas aeruginosa.(1,2,3,4) They of the maggots was demonstrated by showed clinical activity against Pseudo- Mumcuoglu et al. (7) In recent years, sever- monas species, although no formal pros- al reports have described the presence of pective experimental study was arranged two specific peptides with antibacterial in the past. (1) The use of maggots for the activity, either in the body or the secre- treatment of wounds has three beneficial tions of maggots – one peptide with a mo- effects: Debridement of necrotic tissue, lecular weight of 2–10 kDa, and the other promotion of tissue granulation, and with a molecular weight of less than 1

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kDa.(3,6,8) The objective of this work is to caught from the environment using 1x1 study the bactericidal activity of L. serica- foot cage houses > 50 flies, which live on ta maggot secretions on different types of water, dry sugar, and occasional meat. bacteria and assessment of antiseptic com- Every 3 days, eggs were collected from the pounds, as well as to evaluate their abili- underside of meat. Clusters were sepa- ties to ingest bacteria. rated in 0.5% sodium hypochlorite, steri- lized in 1% Lysol for 5 minutes, and hatch MATERIALS AND METHODS on chicken liver (Figure 1). Lucillia sericata ( Green-bottle flies) family: Calliphoridae (9) are readily

Figure 1: Maggots (Larvae) of Lucilia sericata (green bottle fly) on chiken liver.

The hatching maggot (larvae) were 48 and 72 hours after application of mag- then transferred to sterile vials. The col- gots. Degree of lyses in bacterial cultures lected sterile maggots kept for 24 hrs on in the area of maggot application (at the Columbia agar until they reached the third center of the cultured plate were estimated larval stage before being used for the ex- and Gram staining of both areas (centre & periments. Bacteria used in this study in- periphery of the bacterial culture) was per- cluded methicillin- resistant S. aueaus formed. Grams stain of smears made from (MRSA), isolated from a patient with an the hemolymph and guts of the maggots infected chronic wound, Streptococcus were done to detect if any bacteria present group A and B isolated from nasopharyn- after maggot use. geal region and tonsils and Pseudomonas aerogenosa.typically found in wound in- RESULTS fections. Application of live maggots, lar- Complete lyses of the bacterial culture vae of Lucilia (Phaenicia) sericata to total in the area of maggot application was ob- of 48 hour culture plates of Methicillin served after 24, 48, and 72 hours after resistant Staphyloccocus aureus (MRSA), application of live maggots in all culture Pseudomonas aeruginosa, (12 plates in plates of the different types of bacteria each group). The maggots were covered used in the experiment and confirmed by by a small plate inside the big plate with Gram staining (Figures: 2, 3, 4). the pathogen. All plates were incubated in the standard incubator and examined 24,

Figure (2): Maggot in the center of cillin resistant Staphylococcus. aureus (MRSA). Showing the central inhibition zone.

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Figure (3): Maggot applied in culture of Figure (4): Streptococcus spp. Culture, Pseudomonas aerugenosa & clear zone of the central inhibition zone is the area inhibition in the center of the culture (area of maggot application. of maggot application).

This complete lysis was persistent for been studied intensely over the years. Sev- more than 5 days after the maggot applica- eral mechanisms have been suggested, tion. Grams staining of hemolymph and including simple mechanical irrigation of gut revealed no bacteria in the hemolymph the wound by increased exudates, the pro- and hind gut of larvae , but a good amount duction of which is stimulated by larvae of bacteria was seen in the foregut and mid ingesting liquefied necrotic tissue, or by gut ,where as the hindgut was free from dilution of wound discharge following any type of bacteria. Culturing of the con- wound lavage by the maggots’ own secre- tents of the fore & mid gut revealed tions/excretions.(4,5) The excretion of a growth of bacteria after 24 hours of incu- waste product, ammonia, by Lucilia seri- bation, but no bacterial growth detected in cata was also believed to be responsible culturing material from hindgut. This con- for combating bacterial infections, since firmed that the secretions of hindgut have ammonia increases wound pH, resulting in specificity of antimicrobial substance. alkaline conditions unfavorable to many bacterial species. (6, 7) In addition, larvae of DISCUSSION L. sericata carry in their midgut a com- Combating of wound infection by mensal, Proteus mirabilis. These commen- maggots is not entirely clear and several sals produce agents such as phenyl acetic explanations have been suggested. In this acid (PAA) and phenyl acetaldehyde study, the bactericidal activity of maggots (PAL), with known antibacterial proper- was effective against all tested bacteria. ties.(8) The pH of maggot secretions is This is in agreement with in vitro findings known to be between 8–8.5.(10-12) Friedman on the antibacterial effect of maggot secre- et al., (1998), revealed that at alkaline pH tion on E. coli , M. luteus , P. aeruginosa, the antibacterial potential of PAA is low, Salmonella spp ., MSSA, MRSA , S. epi- while PAL is unstable and therefore li- dermidis and Listeria spp. (1) Ammonia in mited as a bactericide. (13) A more likely maggot secretions may partly account for explanation of how maggots combat this antimicrobial effect by raising wound wound infection is that larvae ingest pH. One report suggested that phenyl ace- wound bacteria, which are killed as they tate and phenyl acetaldehyde may exert pass through the maggot’s digestive tract, antimicrobial effect. Direct ingestion of such destruction of ingested microbes was bacteria along with semi liquid food by the reported by Mumcuoglu,(7) who noted that maggots and subsequent lyses in their gut while the stomach and crop were heavily is also possible explanation. How maggots contaminated with viable bacteria the combat clinical infection in wounds has hindgut was sterile. (14) The clinical find-

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ings are consistent with the observations of Maggot- Assisted Wound Healing. that maggots can combat infections in a Stuttgart, Georg Thieme, 2004. variety of wound types, including those 6. Kerridge A, Lappin-Scott H, Stevens infected with antibiotic-resistant strains.(5, JR. Antibacterial properties of larval 11, 15) In fact, the treatment of wounds in- secretions of the blowfly, Lucilia seri- fected with MRSA is likely to become a cata . Med. Vet. Entomol. 2005; 19: major indication for the use of maggot 333-337. therapy in the future.(3, 4, 16) More recently, 7. Mumcuoglu KY, Miller J, Mumcuoglu using high-performance liquid chromato- M, et al.Destruction of bacteria in the graphy, an antibacterial agent from mag- digestive tract of the maggot of Lucilia gots was partially purified using Micro- sericata (Diptera: Calliphoridae). J coccus luteus as the indicator bacteria.(14, Med. Entomol. 2001; 38: 162-166. 17) This factor, reported to possess a mole- 8. Huberman L, Mumcuoglu KY, Gollop cular weight of 6000 Da, was digested by N, Galun R: Antibacterial substances proteases, caused efflux of potassium ions excreted by the maggot of the green from bacterial cells, and exhibited a wide bottle fly, Lucilia sericata . Sixth inte- spectrum of antibacterial activity against rernational Conference on Biotherapy, many resident pathogenic strains including Cumhuriyet University, Sivas, Turkey, MRSA.(3,4,18) 2003, pp 27-28. 9. Richard, W. & Davies S. 2001. Veteri- CONCLUSIONS nary ectoparasites: Biology, Pathology, Complete lyses of the bacterial cul- and Control. 2nd edition, Blackwell tures in the area of maggot application Science Ltd. London England. provides convincing evidence for the an- 10. Wayman J, Nirojogi V, Walker A, So- timicrobial properties of maggots. The winski A, Walker MA. The cost effec- exact mechanism of antimicrobial property tiveness of larval therapy in venous of maggots requires further investigation. ulcers. J Tissue Viability. 2000 Jul;10(3):91-4. Erratum in: J Tissue REFERENCES Viability 2001. Jan;11(1):51. 1. Thomas S, Andrews A, Hay P, Bour- 11. Bonn D. Maggot therapy: an alterna- goise S. The anti-microbial activity of tive for wound infection. Lancet. 2000 maggot secretions: results of a prelimi- Sep 30; 356(9236): 1174. nary study. J Tissue Viability. 1999; 12. Vizioli J, Salzet M. Antimicrobial pep- 9:127-32. tides from animals: focus on inverte- 2. Jarvis A. Maggot therapy. Lancet. brates. Trends Pharmacol Sci. 2002; 2000 Dec 9; 356(9246): 2016. 23: 494–6. 3. Bexfield A, Nigam Y, Thomas S, Rat- 13. Friedman E, Shaharabany M, Ravin S, cliffe NA. Detection and partial charac- Golomb E, Gollop N. Partially Purified terization of two antibacterial factors Antibacterial Agent from Maggots from the excretions/ secretions of the Displays a Wide Range of Antibacterial medicinal maggot Lucilia sericata and Activity; Presented at 3rd International their activity against methicillin- Conference on Biotherapy; Jerusalem, resistant Staphylococcus aureus Israel. 1998. (MRSA). Microbes Infect. 2004; 6: 14. Zasloff M. Antimicrobial peptides of 1297-1304. multicellular organisms. Nature 2002; 4. Dissemond J, Koppermann M, Esser S, 415: 389-95. Schultewolter T, Goos M, Wagner 15. Schultz GS, Sibbald RG, Falanga V, SN. Treatment of methicillin-resistant Ayello EA, Dowsett C, Harding K, et Staphylococcus aureus (MRSA) as part al. Wound bed preparation: a systemat- of biosurgical management of a chronic ic approach to wound management. leg ulcer. Hautarzt. 2002 Sep; 53(9): Wound Repair Regen. 2003; 11: 1-28. 608-1. 16. Sherman RA. Maggot therapy for treat- 5. Fleischmann W, Grassberger M, Sher- ing diabetic foot ulcers unresponsive to man R. Maggot Therapy: A Handbook conventional therapy. Diabetes Care. 2003; 26: 446-51.

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