Biofilm formation (BF) in wounds and on biomaterials is a severe complication in trauma and orthopaedic surgery. Maggot therapy is successfully applied in wounds, that are suspected for BF. This study investigated BF by Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella oxytoca, Enterococcus faecalis and Enterobacter cloacae on polyethylene, titanium and stainless steel and tested the effect on BF by maggot excretions/secretions (ES). Comb-forming models of the biomaterials were made to fit into a 96-well microtiter plate. In the wells, a suspension of 2.5 x 105 bacteria/ml and nutrient medium was pipetted. Combs were placed in the wells and incubated for 3, 5, 7, and 9 days at 37°C. The formed biofilms were stained in crystal-violet and eluted in ethanol. The optical density (OD 595 nm) was measured to quantify BF. Then, maggots excretions/secretions (ES) were collected according to a standardized method, added in different concentrations to (non-stained) mature biofilms (7 days), incubated another 24 hours and at last stained and measured. The results showed biofilm reduction by ES on all biomaterials. Biofilms formed by S. aureus were reduced to minima of 40% on PE and SSS (p< 0.001) and 50% on TI (p=0.005). The biofilm reduction for S. epidermidis was even greater on PE, SSS and TI with respectively minima of 8% (p< 0.001), 32% (p< 0.001) and 38% BF (p< 0.001). The quantity of BF by S. aureus and S. epidermidis had a comparable strength (p=ns) and was for both bacteria the greatest on polyethylene and the lowest on titanium (p< 0.001). Klebsiella oxytoca, Enterococcus faecalis and Enterobacter cloacae formed weak biofilms on all materials. Mature BF was reached between 5 to 7 days by S. epidermidis and between 7 to 9 days by S. aureus. Our previous research showed biofilm inhibition and breakdown of Pseudomonas aeruginosa by ES. This study showed that maggot ES also reduce biofilms formed by S. aureus and S. epidermidis which are frequently isolated from biomaterial-associated infections. There may be pharmacologic agents that could be developed from maggot ES. While BF on orthopaedic materials is an increasing problem, this experimental study could indicate a new treatment for BF on infected biomaterials

In the literature a lot is written about the antibacterial properties that maggots and their secretions are thought to possess, with inconsistencies among different studies. This study investigates the mechanism of the successful clearance of infections in wounds by maggots through examining living maggots and their excretions.

To test the excretions a turbidometric assay was carried out. The sterile excretions were pipetted in different dilutions into a microtiter plate and had bacteria added. Five bacteria were tested, viz. S. aureus, S. pyogenes, E. faecalis, P. aeruginosa and K. oxytoca. After 20 hours the wells were checked. Clear wells presented bacteriolytic activity, whereas cloudy wells presented bacterial growth.

Putting maggots in tubes together with bacterial suspension tested the effect of living maggots. Control tubes contained bacteria only. Same bacteria as ascribed above were used, except for E. faecalis that was changed for coagulase-negative Streptococ. Young maggots (Instar-1) and full-grown maggots (Instar-3) were used, and as a medium for the suspension, Muller Hinton (MH) was used with and without 5% sheep blood. The tubes were horizontally incubated for 16 hours, and every two hours a sample was taken and put onto an agar plate. After 24 hours, the bacterial colonies were counted.

The turbidometric assay showed cloudy wells for all bacteria and dilutions with the less diluted excrete showing the highest stimulation of bacterial growth.

The test with living maggots showed increased bacterial growth as compared to the controls (p=0.001 using the S. aureus). Young maggots stimulated growth more than full-grown maggots (p=0.002 using the S. aureus). Using a more nutritious medium, viz. MH with 5% sheep blood, no difference in growth of bacteria was observed between the tubes with maggots and the controls (p=0.271 using the S. aureus). The other bacteria gave similar results.

This study shows that other mechanisms must be accounted for the clearance of infections in wounds by maggots than their proposed antibacterial properties.

Maggot therapy as an ancient method is succesfully used for treatment of acute and chronic wound infections in traumatology and orthopaedics. In this study, for the first time, the influence of sterile maggot excretions of Lucilia sericata on Pseudomonas aeruginosa (PAO1) biofilm formation on three common used orthopaedic materials was investigated.

Sterile maggot excretions were collected according to a standardized method and the protein concentration was measured. The influence of the excretions on PAO1-biofilm formation was tested on comb-like devices, especially designed for these experiments, made from polyethylene, titanium and stainless steel. These combs were made to fit into a flat-bottom 96-wells microtiter plate. In the wells a suspension of PAO1-bacteria, nutrient medium and maggot excretions were pipetted. In the control wells, no excretions were added. Combs were placed in the wells and incubated for 24 hours at 37°C. The formed biofilms were stained in crystal violet and eluted with ethanol. The Optical Density (OD 595 nm) was read to quantify biofilm formation. The experiments were conducted with excretions from young maggots (Instar-1 maggots) and full grown maggots (Instar-3 maggots). All experiments were done in quadruplicate.

The following can be concluded: PAO1-biofilm formation is the strongest on polyethylene and the weakest on stainless steel. Sterile maggot excretions are effective at preventing initial biofilm formation (p≤0.013) as well as preventing additional accumulation after its initiation (p≤0.038). The excretions even cause a significant breakdown of an existing biofilm (p≤0.028). Excretions from full grown maggots are more effective than those from young maggots.

This study shows for the first time that sterile maggot excretions of Lucilia sericata inhibit biofilm formation, prevent its further grow and break down existing biofilms. While biofilm formation on orthopaedic materials is a severe complication, this experimental study could indicate a new treatment for biofilm formation on infected biomaterials.