Figure 4
NBD-UBIdend labels bacteria in situ in an ex vivo ovine lung model of pneumonia. (A) Panels show representative images of positive control of GFP S. aureus (upper) or PKH stained S. aureus (lower) instilled into an ovine pulmonary segment and imaged demonstrating a characteristic punctate fluorescent bacterial signal. (B) Representative images of OEM imaging where control segments (PBS vehicle control) and S. aureus segment with monomer NBD-UBI do not show characteristic punctate bacterial signal, whereas S. aureus, K. pneumoniae, E. coli and P. aeruginosa with NBD-UBIdend (2 μM) demonstrate in situ bacterial labelling when the SmartProbe is applied topically in the distal alveolar space (see also Videos 1–3). (C) Image analysis algorithm of entire datasets following the removal of redundant frames. Bars represents the combined mean (+/− SEM) of the percentage of frames with over 80 detected dots per frame over the imaging sequence for individual experiments. White bars represent NBD-UBI (monomer) with no significant increase for S. aureus (n = 6) over PBS control segments (n = 6), ns = not significant. Grey bars demonstrates a significantly higher number of detected dots per frame for the bacterial segments (S. aureus n = 8, E. coli n = 6, K. pneumoniae n = 8, P. aeruginosa n = 8) when compared to control segment (n = 9) for NBD-UBIdend, *p < 0.05, **p < 0.01, ***p < 0.001 Students t-test. (D) Performance characteristics to distinguish control segments (PBS instilled, n = 9) or bacterial segments (comprising of S. aureus, E. coli, K. pneumoniae or P. aeruginosa, n = 30) with NBD-UBIdend demonstrating an area under the curve (95% confidence interval) of 0.9259 (0.8169 to 1.035), p = 0.0001280. (E) BALF from ovine segments demonstrating clinically relevant bacterial yields, n = 4 for all segments except dilution of P. aeruginosa (PA) where n = 3. Bars represent mean (+/− SEM).
