Background

Both in basic research and in the pharmaceutical industry, there is a high demand for in vitro and in vivo models that can provide quick and reproducible results when testing antimicrobial formulations. Availability of bioluminescent bacteria and advanced imaging systems capable of acquiring bioluminescence originating from in vitro experiments or experimental animal models enable us to quickly and longitudinally evaluate the efficacy of antimicrobials. Conventional in vitro approaches, among many others, include radial diffusion and viable count assays (Balouiri et al., 2016). For in vivo evaluation, CFU (colony-forming unit) enumeration of the animal tissue is the standard practice. Although all these approaches have their own advantages, they are time-consuming as microbiological culturing protocols require 24 to 72 h before results are known. In particular, in vivo evaluation requires that animals be sacrificed for each experimental time point, which renders this process laborious and longitudinal analysis unattainable.

Bioluminescence imaging does not require an external excitation light source. Therefore, for in vivo applications, it is preferred over fluorescence imaging, which often generates a high background, as tissues in the body produce autofluorescence upon excitation with an external source (Choy et al., 2003).

The additional significant advantage is that in vivo bioluminescence imaging gives vital information on spatial distribution of the infection throughout the body (Karimi et al., 2016). Infection development from the original inoculation site can be tracked and its spread can be visualized and quantified. If required, this method can also be combined with fluorescently labeled drugs, so that signals from bioluminescent bacteria and fluorescent drugs can be acquired simultaneously and co-registered, to study their interactions (van Oosten et al., 2013; Puthia et al., 2020). This method can easily be adapted for different antimicrobial products such as gels, aqueous solutions, dressings, and implants. We have successfully used bioluminescent versions of the clinically important pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa for the initial in vitro screening of antimicrobial formulations (Puthia et al., 2020). Employing IVIS imaging, these bioluminescent bacteria were further used by us to evaluate the in vivo efficacy of an antimicrobial formulation in a mouse model of infection using a subcutaneous implant (Strömdahl et al., 2021).

Here, using bioluminescent P. aeruginosa, we describe an in vitro and an in vivo approach to rapidly evaluate the antimicrobial efficacy of the host-defense peptide TCP-25 (thrombin-derived C-terminal peptide). TCP-25 is a thrombin-derived C-terminal peptide (TCP-25, GKYGFYTHVFRLKKWIQKVIDQFGE) that shows strong antimicrobial (Puthia et al., 2020) and antiendotoxic properties (Saravanan et al., 2018). In vivo, TCP-25 protects against P. aeruginosa-induced sepsis and LPS-mediated shock (Papareddy et al., 2010; Kalle et al., 2012). Bioluminescent P. aeruginosa (P. aeruginosa Xen41) is derived from the isolate PAO1 and contains the luxCDABE operon of Photorhabdus luminescens, which is constitutively expressed (Winson et al., 1998; Dusane et al., 2017). Using bioluminescence imaging, we performed longitudinal analysis in both in vitro and in vivo assays, to obtain rapid and valuable results.