In Vivo and in Vitro Detection of Porphyrin-producing Wound Pathogens with Real-time Bacterial Fluorescence Imaging

Background: Porphyrins are naturally occurring, red fluorescing intermediates of the heme synthesis pathway, essential to bacterial survival. Fluorescence imaging is able to visualize polymicrobial populations in chronic wounds based on porphyrin fluorescence.

Purpose: This study investigated prevalent chronic wound pathogens and their ability to produce detectable red fluorescence in vitro and in vivo.  

Methods: (In vitro) 12 bacterial pathogens and one yeast were plated on Remel Porphyrin Test Agar (containing required intrinsic substrates for porphyrin production) in triplicate and imaged for fluorescence after 24 and 40 hours using a fluorescence imaging device [2]. (In vivo) Mice (n=3/group) were given full thickness wounds inoculated with one of three pathogens (Staphylococcus, E. coli, or Streptococcus, 10^7 CFU/wound) or vehicle control. Wounds were longitudinally imaged over 11 days. 

Results: All porphyrin producing bacterial species (10/12) exhibited red fluorescence by 24 hours in vitro (e.g. Staphylococcus, Proteus, Acinetobacter, Klebsiella, Enterobacter) and by day 2 post-inoculation in vivo. Yeast and non-porphyrin producing bacteria (Streptococcus, Enterococcus [3]) did not produce detectable red fluorescence in vitro, nor did Streptococcus in vivo. Interestingly, a 4:1 Enterococcus: Staphylococcus in vitro plating, mimicking clinical polymicrobial growth, fluoresced bright red. 

Conclusions: Red bacterial fluorescence is specific to porphyrin-producing bacteria, which represents the vast majority of wound pathogens [4]. Plating 4:1 Enterococcus: Staphylococcus suggests that detectable red fluorescence would also be present in wounds with predominately non-porphyrin producing species; these species exist monomicrobially in < 1% of chronic wounds [4]. Overall, results support the clinical rationale for using fluorescence imaging to detect a wide array of pathogenic bacteria which would not otherwise be visible.