On Feasibility of Fluorescence-based Bacteria Presence Quantification

Abstract Body: Introduction: Wound healing normally occurs in the presence of bacteria, at levels ranging from contamination to colonization to infection. The role of bacteria in wound healing depends on their concentration, species composition, and host response. Thus, determination of bacterial load is of great importance. However, existing clinical bacteria load assessment methods (biopsy or swabbing in combination with culture methods) are slow, labor- and time-consuming. Pseudomonas aeruginosa is a known pathogen implicated in numerous healthcare-associated infections and is known to express fluorescent metabolites during proliferation. In particular, the siderophore pyoverdine produces a fluorescent emission between 420-520 nm when excited at 400nm, which can be measured quantitatively. The goal of the current project is to investigate the possibility of quantification of bacteria presence using fluorescence measurements. Methods: Cultures of P.aeruginosa (PA01) were grown at various temperatures (36-43°C) and inoculum starting condition (5*107-5*108 CFU/mL) in Tryptic Soy Broth media. Media optical density (OD, as a proxy of bacterial concentration) and fluorescence (ex. 400nm, em. 420-520nm) were measured hourly for 10 hours. Results: Cultures remained metabolically active in the whole temperature range, producing pyoverdine fluorescence (emission max at 455nm). We correlated optical density with a fluorescent signal to establish dependence between fluorescence and growth stage. Noticeable pyoverdine accumulation started approximately 3 hours after the beginning of the log growth phase and experienced saturation at the beginning of the stationary phase. Three distinct regimes (a sigmoid curve) were observed: linear dependence of fluorescence on OD for low concentrations, more rapid nonlinear dependence, and saturation when approaching the stationary phase. Conclusions: The sigmoid dependence of bacterial fluorescence on their concentration persisted through variations in temperature and inoculum starting condition; thus, it may have the potential for determining culture growth phase progression. These results, combined with classical knowledge on disease progression, could also lead to an advanced infection diagnosis prior to current pathogenesis observation techniques.