Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Original Paper Skin Pharmacol Physiol 2007 20:112���115 DOI: 10.1159/000097983 In vitro Antibacterial Activity of Lucilia sericata Maggot Secretions G. Daeschlein a K.Y. Mumcuoglu b O. Assadian c B. Hoffmeister a A. Kramer a a Institute for Hygiene and Environmental Medicine, Ernst Moritz Arndt University, Greifswald , Germany b Department of Parasitology, Hebrew University-Hadassah Medical School, Jerusalem , Israel c Department of Hygiene and Medical Microbiology, Medical University Vienna, Vienna , Austria Introduction Maggot debridement therapy, the intentional treat- ment of non-healing wounds or chronic osteomyelitis with maggots of calliphorid flies, was scientifically and therapeutically introduced by William S. Baer in the 1920s [1] , became popular in the early 1930s [2���3] , but because of the introduction of antibiotics and improved surgical procedures was abandoned in the 1940s. How- ever, the method re-emerged in 1988 for the treatment of chronic wounds [4���7] . The use of maggots for the treatment of wounds has three beneficial effects: debridement of necrotic tissue, promotion of tissue granulation [8] , and wound-antisep- sis due to antibacterial secretions [9] . In 1931, Baer al- ready noted that maggots augment the healing of chron- ic wounds. Shortly thereafter, the presence of an antibac- terial substance in the body and secretions of Lucilia sericata was demonstrated by Weil et al. [10] , Robinson and Norwood [11] , and Simmons [12] . Furthermore, de- struction of ingested bacteria in the intestinal tract of the maggots was demonstrated by Robinson and Norwood [13] and confirmed by Mumcuoglu et al. [14] . In recent years, several reports described the presence of two specific peptides with antibacterial activity, either in the body or the secretions of maggots ��� one peptide with a molecular weight of 2���10 kDa, and the other with a molecular weight of less than 1 kDa [15���18] . Key Words Maggot Lucilia sericata Chronic wound Antibacterial activity Quantitative suspension test Methicillin-resistant Staphylococcus aureus strain Abstract Maggots of the green blowfly, Lucilia sericata , are used as an alternative to surgical intervention and long-term antiseptic therapy for the treatment of chronic wounds. 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 quantitative suspension test (EN 1040) was developed, in which a co-culture of maggots and bacteria ( Micrococcus luteus, Escherichia coli, methicillin-sensitive Staphylo-coccus aureus ) in tryptic soy broth was tested. The numbers of bacterial colonies with and without maggot ex- posure were compared after 24, 48 and 72 h of exposure. The mean log 10 reduction factor (RF) for bacterial elimination per maggot was 1 4 at all examined times for all tested bacteria. Thus, maggot secretion fulfilled the required definitions of an antiseptic. In addition, the maggots��� ability to ingest bac- teria was also evaluated. Maggots contained viable bacteria after 48 h of contact with the respective organisms. These maggots also continued excreting bacteria. Therefore, mag- gots should be disposed of after use as they must be regard- ed as medical waste. Copyright �� 2007 S. Karger AG, Basel Received: July 17, 2006 Accepted: October 5, 2006 Published online: December 13, 2006 Univ.-Prof. Dr. Ojan Assadian Department of Hygiene and Medical Microbiology, Medical University Vienna Waehringer G��rtel 18���20, AT���1090 Vienna (Austria) Tel. +43 1 40400 1904, Fax +43 1 40400 1907 E-Mail ojan.assadian@meduniwien.ac.at �� 2007 S. Karger AG, Basel 1660���5527/07/0202���0112$23.50/0 Accessible online at: www.karger.com/spp

Antibacterial Activity of Maggot Secretions Skin Pharmacol Physiol 2007 20:112���115 113 The aim of this work was to study the bactericidal ac- tivity of L. sericata maggot secretions using a modifica- tion of the European standardized test (EN 1040) for as- sessment of antiseptic compounds. In addition, their ability to ingest bacteria was also evaluated. Materials and Methods Blowfly Maggots Sterile maggots of the species Lucilia sericata (Diptera: Calli- phoridae) were purchased from Biomonde GmbH (Barsb��ttel, Germany) and kept for 24 h on Columbia agar until they reached the third larval stage before being used for the experiments. Bacteria Bacteria used in this study included Micrococcus luteus (ATCC 9341), a colonizer of chronic wounds Staphylococcus aureus, both a methicillin-sensitive strain (MSSA, ATCC 6538) and a methicil- lin-resistant strain (MRSA, isolated from a patient with an in- fected chronic wound) and Escherichia coli (ATCC 11229), also typically found in wound infections. With the exception of MRSA, all organisms were obtained from American Type Culture Collection (ATCC, Manassas, Va., USA). Tests in Liquid Medium Following the recommendations of the European phase 1 sus- pension test (EN 1040) [19] , test bacteria were cultured aerobi- cally on tryptone soy agar (TSA) for 24 h and transferred to fresh TSA for another 24 h of incubation. To simulate conditions fre- quently found on wounds, the proposed phase 1 standardized test suspension was modified. Instead of standardized sterile hard water, casein peptone-soybean flour-peptone solution (CSS) broth (Oxoid Ltd., Basingstoke, UK) was used. Bacterial cells were sus- pended in CSS broth to an optical density at 620 nm correspond- ing to a concentration of 1.2 ! 10 7 to 3.4 ! 10 9 CFU ml ���1 (initial contamination). 20 ml of this test suspension was placed on a semirecumbent sterile Petri dish containing 10 maggots. This as- say was incubated at 34 �� C for 1���3 days. Samples were taken 24, 48, and 72 h after inoculation. Immediately thereafter, a neutral- izer was added, which consisted of 10 ml of a buffer containing 34 g of KH 2 PO 4 per liter adjusted to pH 7.2 with NaOH, 3 g of lecithin from soybeans per liter, 30 ml of Tween 80 per liter, 5 g of Na 2 S 2 O 3 per liter, and 1 g of L -histidine per liter. The same assay as above was prepared without maggots and served as control. All experiments were performed at least in quadruplicate at the same time using the same bacterial suspension. In order to determine the number of surviving bacteria, 100 l of each sample was plated on TSA. The plates were incu- bated at 37 �� C for 24 h and thereafter the colony forming units (CFU) were counted. The log 10 reduction of CFU (reduction fac- tor, RF) after exposure to maggots was calculated as log 10 CFU control minus log 10 CFU test for each exposure time per maggot. The standard deviation ( 8 SD) was calculated using the log 10 re- duction factors obtained in the independent experiments. Viability of Bacteria Ingested by Maggots One maggot was placed at 37 �� C for 24 h on Columbia agar plates containing confluent MSSA or MRSA. The maggot was re- moved, transferred to sterile Columbia agar and left for 24 h at 37 �� C. Subsequently, the maggot was removed from the agar, its surface was wrapped in a gauze fleece saturated with 70% ethyl alcohol, disinfected by rolling back and forth for 5 min, dried with a second sterile gauze fleece, and finally dried over a period of 10 min in a laminar air flow sterile box (class II bench). The next steps were performed under sterile conditions in the box. To test maggots for contamination, they were rolled on sterile agar using slight pressure. Then, the agar plate was incubated at 37 �� C for 24 h. 15 l of sterile physiological saline solution (heated to 37 �� C) was injected under the cuticula of the maggots and approximate- ly 5 l of the fluid was re-aspirated. The aspirate was microbio- logically examined and the maggots were then left for a further 24 h on sterile Columbia agar. Disinfected maggots placed on Co- lumbia agar without MSSA or MRSA culture were kept under the same experimental conditions as controls. Viability of Bacteria until Pupation Maggots were kept on Columbia agar with confluent MSSA ( 1 300 CFU/plate) at 37 �� C until pupation. This was visible when maggots became immobile and their color changed from whitish to brown, which occurred within 9���13 days. The surface of the pupa was disinfected with 70% ethyl alcohol. After 6 h, 15 l of a sterile NaCl solution was injected into the pupa and 6 l of fluid was re-aspirated, which was examined for the presence of bacte- ria. Two maggots, which were kept under the same conditions but without exposure to MSSA served as control. Results Elimination Rate of Bacteria in Liquid Medium After 24, 48 and 72 h of exposure, the mean RFs ( 8 SD) for M. luteus were 6.7 8 0.9, 7.3 8 1.2 and 6.1 8 1.5, respectively, for MSSA 7.5 8 2.2, 4.8 8 3.9 and 4.6 8 5.4, respectively, and for E. coli 8.7 8 1.4, 3.8 8 4.5 and 4.51 8 3.9, respectively. In all three tested organisms a mean RF of 1 4 log per maggot was found. The highest RF was observed for E. coli , with a mean RF of 8.7 8 1.4 log after 24 h. The ca- pacity of maggots to reduce E. coli and MSSA decreased over time. In contrast, the RFs for M. luteus remained stable over time ( fig. 1 ). Ingestion of MSSA and MRSA Maggots which first were kept on agar plates with MSSA or MRSA for 24 h and thereafter transferred to sterile agar plates for another 24 h without MSSA/MRSA contained viable bacteria after 48 h of contact with the respective organisms. Viable MSSA (mean 1.1 8 0.3 CFU/ l) or MRSA (mean 0.75 8 0.1 CFU/ l) were detected in the aspirates obtained from the maggots after