Maggot excretions inhibit biofilm formation on biomaterials

Abstract

Background: Biofilm-associated infections in trauma surgery are difficult to treat with conventional therapies. Therefore, it is important to develop new treatment modalities. Maggots in captured bags, which are permeable for larval excretions/secretions, aid in healing severe, infected wounds, suspect for biofilm formation. Therefore we presumed maggot excretions/secretions would reduce biofilm formation.

Questions/purposes: We studied biofilm formation of Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella oxytoca, Enterococcus faecalis, and Enterobacter cloacae on polyethylene, titanium, and stainless steel. We compared the quantities of biofilm formation between the bacterial species on the various biomaterials and the quantity of biofilm formation after various incubation times. Maggot excretions/secretions were added to existing biofilms to examine their effect.

Methods: Comb-like models of the biomaterials, made to fit in a 96-well microtiter plate, were incubated with bacterial suspension. The formed biofilms were stained in crystal violet, which was eluted in ethanol. The optical density (at 595 nm) of the eluate was determined to quantify biofilm formation. Maggot excretions/secretions were pipetted in different concentrations to (nonstained) 7-day-old biofilms, incubated 24 hours, and finally measured.

Results: The strongest biofilms were formed by S. aureus and S. epidermidis on polyethylene and the weakest on titanium. The highest quantity of biofilm formation was reached within 7 days for both bacteria. The presence of excretions/secretions reduced biofilm formation on all biomaterials. A maximum of 92% of biofilm reduction was measured.

Conclusions: Our observations suggest maggot excretions/secretions decrease biofilm formation and could provide a new treatment for biofilm formation on infected biomaterials.

Fig. 1

An overview of the study design is shown. BF = biofilm formation; PE = polyethylene; TI = titanium; SSS = surgical stainless steel; ES = excretions and secretions.

Fig. 2A–B

The ODs reflect the quantities of 7-day-old biofilms produced by (A) S. aureus and (B) S. epidermidis on PE, SSS, and TI and answer the second and the third research questions, respectively, whether the quantity of BF differed among S. aureus and S. epidermidis and whether BF depended on the biomaterial surface. Both bacteria formed equal quantities of BF and showed PE had the highest BF followed by SSS and TI with the lowest quantity of biofilm. *For these comparisons all p values < 0.001.

Fig. 3A–B

The graphs show the amount of BF after various incubation times and answer the fourth research question, whether quantity of biofilms differed among 3, 5, 7, or 9 days. The 7-day-old BF was defined as 100%. (A) Biofilms by S. aureus kept on growing until Day 9 on PE, TI, and SSS. (B) The largest amount of biofilm by S. epidermidis was formed within 7 to 9 days on SSS and TI. The amount of biofilm on PE was similar at all incubation times. *For these comparisons all p values < 0.001; all other comparisons were similar.

Fig. 4A–C

These graphs compare the mean BF produced by S. aureus with ES and the mean control BF after 8 days without ES and answer the last research question whether ES reduced biofilms produced by S. aureus. The 7-day-old biofilm was defined as 100%. All values lower than the 8-day-old biofilm showed biofilm reduction. (A) The biofilm on SSS was decreased for all concentrations with a maximum of 59.5% for 0.31 μg ES per well. (B) The biofilm on PE also was reduced for all concentrations with a maximum of 60.8% for 0.93 μg ES per well. (C) On TI, only 8.33 μg ES per well showed biofilm reduction. *For these comparisons all p values < 0.001; all other comparisons were similar.

Fig. 5A–C

These graphs compare the mean BF produced by S. epidermidis with ES and the mean control BF after 8 days without ES and answer the last research question whether ES reduced biofilms produced by S. epidermidis. The 7-day-old biofilm was defined as 100%. All values lower than the 8-day-old biofilm showed biofilm reduction. (A) Biofilm on SSS was reduced for all concentrations with a maximum of 67.7% for 25 μg ES per well. (B) Biofilm on PE also was decreased for all concentrations with a maximum of 92.4% for 8.33 μg ES per well. (C) On TI, only 0.31 μg ES per well and 0.93 μg ES per well showed biofilm reduction. *For these comparisons all p values < 0.001; all other comparisons were similar.