In vitro reduction of Escherichia coli biofilm mass by platelet-rich plasma lysate (PRP-L)

Abstract

Placentitis-induced, pre-term fetal loss has caused hardships and financial losses to the equine breeding industry for decades. While traditional multi-modal treatments including a combination of gentamicin, penicillin, altrenogest, firocoxib, and pentoxifylline can sometimes prolong gestation and neonatal survival, treatment failures still occur and antibiotic therapy can be quite lengthy. Understanding potential biofilms in these reproductive infections uncovers a pitfall to the success of traditional treatment options: antimicrobials are ineffective at penetrating and killing biofilms in vitro and in vivo. Platelet-rich plasma lysate (PRP-L) is increasingly accepted as an adjunctive treatment option for biofilm associated infections across species. The aim of this study was to evaluate the reduction in biofilm formation of 10 isolates of Escherichia coli collected from the equine uterus. Isolates were stored in glycerol at -80°C; each isolate including a positive (ATCC 25922) and negative (ATCC BAA 2469) strain, was cultured for 18-24 h at 33-37°Cin ambient air until bacterial colonies were visible. A suspension of each isolate, adjusted to a 0.5 McFarland value, was used to inoculate a tissue culture treated, round-bottom 96-well microtiter plate incubated under microaerophilic orbital shaking conditions for 18-24 h at 33-37°C. Following incubation, with or without PRP-L, the supernatant was removed, wells were stained with 0.03% crystal violet for 15 minutes at room temperature with orbital shaking, rinsed to remove excess stain, solubilized with ethanol, and absorbance optical density (OD570) read with a plate reader. The range in reduction of biofilm mass with PRP-L treatment was 48 – 84%, with an average of 60%. In conclusion, the results suggest that comparison of a traditional multi-modal therapy for placentitis with a multi-modal therapy augmented with PRP-L has the potential to finally improve placentitis outcomes and reduce the length of antibiotic treatment, but further in vivo evaluation of this approach is required.