1. Antimicrobial effects of Maggot Secretion Charlie Kerr Central Catholic High School

2. Infections Bacterial and Fungal infect specific area of the body and rapidly reproduce. They release toxins into the body and can cause illness Commonly treated by pharmaceutical antifungals or antibiotics A CDC estimate from 2001 suggests that approximately 290,000 SSIs occur every year, 70% are related to skin infections

3. Rising Problem Bacteria and other microbes that cause infections are remarkably resilient and have developed several ways to resist antibiotics and other antimicrobial drugs – Antibiotic resistance can be exchanged between strains and species of bacteria by the process of horizontal gene transmission (HGT).

4. MRSA (Methicillin Resistant Staphylococcus aureus) People who have open wounds, invasive devices, or weakened immune system are at a great risk of contracting the infection. resistant to most Beta-Lactams which include methicillin, dicloxacillin, penicillin, nafcillin, and oxacillin In 2007 the Center for Disease Control and Prevention reported that the number of MRSA infection treated in hospital from 127,000 cases in 1999 to 227,000 in 2005

5. Maggot Therapy Fly larvae are specialized decomposers When placed in infected skin wound: 1.consume only necrotic tissue 2.disinfect the wound by killing bacteria (extracorpeal digestion) and secretion 3.promote wound healing (micro-massage) Secretes a substance containing: 1.broad spectrum of enzymes 2.Anti-microbial properties such as allantoin, urea, phenylacetic acid, phenylacetaldehyde, and calcium carbonate

6. Escherichia coli One of the most common forms of bacteria found in many environments including the intestinal tracts of many mammals. Gram Negative bacterium There are many of different strains of E.coli, most of which are non-pathogenic.

7. Saccharomyces cerevisiae Reproduce by budding Eukaryotic model organism

8. Purpose: To determine if maggot secretions have antimicrobial properties Null Hypotheses There will be no significant effect of the concentrations of the maggot extract on Yeast survivorship. All concentrations of maggot extract will decrease Yeast survivorship. All concentrations of maggot extract will decrease E. coli survivorship. Hypotheses There will be no significant effect of the concentrations of the maggot extract on E. coli survivorship.

9. Materials 32 LB agar plates (1 % tryptone, 5 % yeast extract, 1% NaCl, 1.5 % agar) LB media (1 % tryptone, 5 % yeast extract, 1% NaCl) 32 YEPD agar plates ( YEPD agar media Klett spectrophotometer Sterile pipette tips Micropipettes Vortex Incubator Sidearm flask Spread Turn Table DJ master 3000 Spreader bar Ethanol 1 mL sterile capped micro-tubes E.coli B Sterile dilution fluid 2.1 grams of Musca domestica larvae (House Fly) Ultraviolet shield Sterile syringe with filter Blender 50 mL of SDF Sterile plates

10. Procedure for Maggot Extract 1.2.1g of house fly larvae were blended in 40 ml of SDF to create the 5% maggot extract 2.For sterilization of maggot extract, the maggot extract was evenly pipetted into 2 sterile plates and the plates were placed in an ultraviolet shield for 45 minutes 3.The extract was pipetted into the sterile syringe. The extract was pushed through a sterile filter into a 10 mL test tube 4.The concentrations of 0%,.1%, 1%, and 2% of the maggot extract were created in sterile micro-tubes

11. Maggot Concentrations 0%0.10%1%2% 5% Maggot Extract0 mL.005 mL.05 mL.1 mL SDF1 mL.995 mL.95 mL.9 mL E. coli/ Yeast.01 mL

12. Procedure 1.Yeast was grown overnight in sterile YEPD agar media and E. coli was grown in LB agar media. 2.Samples of the overnight yeast and E. coli cultures were added to fresh media in a sterile sidearm flask. 3.The cultures were placed in a shaking water bath (30°C Yeast; 37°C E. coli) until a density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10 7 cells/mL (yeast culture) and 10 9 cells/mL(E. coli culture) 4.The cultures were diluted in sterile dilution fluid to a concentration of approximately 10 5 cells/ml (yeast culture) and 10 3 cells/ml (E. coli culture) 5.The selected experimental variables were diluted with sterile dilution fluid to the chosen concentrations to a total of 1 mL 6.0.1 mL of cell cultures were then added to the micro-tubes, yielding a final volume of 1 mL. and a cell density of approximately 10 3 cells/mL 7.The solutions were mixed by vortexing and allowed to sit at room temperature for 15 minutes 8.After vortexing to evenly suspend cells, 0.1 mL aliquots were removed from the tubes and spread on 64 plates 9.The plates were incubated at 36°C for 24 hours. 10.The resulting colonies were counted. Each colony is assumed to have arisen from one cell.

14. Dunnett’s Test ConcentrationT ValueT CriticalT value > T critical 0.10%3.62.88 Significant 1%3.3 2.88Significant 2%5.54.0Significant

16. Dunnett’s Test ConcentrationT valueT CriticalT value > T critical 0.10%4.34.0Significant 1%6.34.0Significant 2%6.34.0Significant

17. Conclusion Null Hypotheses – There will be no significant effect of the concentrations of the maggot extract on Yeast survivorship. REJECTED – There will be no significant effect of the concentrations of the maggot extract on E. coli survivorship. REJECTED Hypotheses – All concentrations of maggot extract will decrease Yeast survivorship. ACCEPTED – All concentrations of maggot extract will decrease E. coli survivorship. REJECTED

18. Extensions Using – gram positive and gram negative bacteria – Different species of maggots Directly extracting maggot secretions – Infusing maggot secretions directly into agar Comparing antimicrobial effects of secrections with antibiotics and antifungals

19. Limitations Maggot extract may have had beneficial growth media for E. coli and not Yeast Lack of trials

20. References http://www.nlm.nih.gov/medlineplus/bacterialinfections.html http://www.cdc.gov/ncidod/dhqp/FAQ_SSI.html