Enhanced avidity from a multivalent fluorescent antimicrobial peptide enables pathogen detection in a human lung model

Abstract

Rapid in situ detection of pathogens coupled with high resolution imaging in the distal human lung has the potential to provide new insights and diagnostic utility in patients in whom pneumonia is suspected. We have previously described an antimicrobial peptide (AMP) Ubiquicidin (fragment UBI 29–41 ) labelled with an environmentally sensitive fluorophore that optically detected bacteria in vitro but not ex vivo . Here, we describe further chemical development of this compound and demonstrate that altering the secondary structure of the AMP to generate a tri-branched dendrimeric scaffold provides enhanced signal in vitro and ex vivo and consequently allows the rapid detection of pathogens in situ in an explanted human lung. This compound (NBD-UBI dend ) demonstrates bacterial labelling specificity for a broad panel of pathogenic bacteria and Aspergillus fumigatus . NBD-UBI dend demonstrated high signal-to-noise fluorescence amplification upon target engagement, did not label host mammalian cells and was non-toxic and chemically robust within the inflamed biological environment. Intrapulmonary delivery of NBD-UBI dend , coupled with optical endomicroscopy demonstrated real-time, in situ detection of bacteria in explanted whole human Cystic Fibrosis lungs.

Exploratory Use of Fluorescent SmartProbes for the Rapid Detection of Microbial Isolates Causing Corneal Ulcer

“…WITH OCULAR PATHOGENS: BAC One and BAC Two have previously been evaluated against a panel of lung pathogens. 23,24 Here they were evaluated against clinically isolated ocular pathogens; bacterial isolates Staphylococcus epidermidis (gram-positive) and Pseudomonas aeruginosa (gram-negative); and fungal isolates Aspergillus flavus and Fusarium spp. Colonies were sampled from solid media with a toothpick and smeared onto a glass slide within 20 mL saline.…”

“…Clinical-grade BAC One and BAC Two were synthesized and characterized in-house at the University of Edinburgh as previously described. 23,24 ASSESSMENT OF BAC ONE AND BAC TWO: TRAINING SET STATISTICAL ANALYSIS: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy scores, including 95% confidence intervals were calculated using Prism 8.4 (GraphPad Software, San Diego, CA, USA). The same software was used for plotting the graphical representations of the data.…”

“…The purpose of this exploratory study was to assess whether fluorescence microscopy and optical molecular imaging approaches with microbe-specific SmartProbes [18][19][20][21] could be an alternative and additive approach to conventional direct microscopy and/or microbial culture for cases of suspected MK. Optical molecular imaging of infection is an emerging field, and here we have investigated the utility of 2 activatable peptide-based fluorescent imaging probes that have been previously developed and evaluated for in situ point-of-care pulmonary applications: BAC One is able to report on the presence of microbes (bacterial and fungal) through an antimicrobial peptide binding fragment, ubiquicidin (UBI 29-41 ) 22,23 ; and BAC Two is able to report on the presence of gram-negative bacteria; utilizing a derivative of polymyxin B to bind to lipid A of the gram-negative cell wall. 24 In both cases, the fluorophore incorporated into the compound is 7-nitrobenz-2-oxa-1,3-diazole (NBD), an environmentally sensitive dye that self-quenches in aqueous environments but fluoresces strongly in hydrophobic environments, 25 such as the microbial membrane, thus enabling wash-free detection of positive samples, providing ease of sample preparation and a high signal-to-noise ratio ( Figure 1).…”

Frugal filtering optical lenses for point-of-care diagnostics

“…M0678), 0.5% glycerol (Merck, catalogue G5516) and sodium pyruvate (Merck, catalogue 5280) at 37°C and stored at 4°C. A single colony was smeared directly into an 'all microbe', NBD-based SmartProbe (30 µL, 5 µM) [22] on a clean glass microscope slide and mounted with a coverslip. Images were captured using the smartphone-IFL device setup as described above, or by wide-field microscopy (EVOS FL Imaging System, Thermo Scientific AMF4300) with GFP LED filter cube and 20x objective.…”

“…Furthermore, the associated costs of multiple components can be prohibitively high [11]. In terms of sample preparation, recent chemical innovation has led to targeted 'smart' fluorescent stains that need little processing, acting as a switch when they come into contact with a bacterial cell membrane [22][23][24]. These labels may have major utility in settings where time and resources are limited such as in rural LMICs.…”