Engineered colorimetric detection of Staphylococcus aureus extracellular proteases

Abstract

This work describes the engineering of a feasible, sensitive, and specific colorimetric assay for the detection of Staphylococcus aureus (S. aureus) extracellular protease production, using a tailored agar-based readout platform. Firstly, S. aureus protease production was evaluated using different culture media. Maximum proteolytic activity was achieved after 24 h of incubation in brain heart infusion (BHI) broth at 37 °C, as confirmed using azo-casein and Sigma's non-specific protease activity assays. Secondly, to enhance proteolysis detection, novel agar-based platform readouts for S. aureus proteases were developed. Clear proteolytic zones were observed after 48 h of incubation at 37 °C, using an agar-agar platform supplemented with 3% skim milk as a non-specific protease substrate. Thirdly, the colorimetric visualization of S. aureus proteolytic zones was evaluated using three different techniques, namely, the use of chromogenic media, dye flooding over the platform, and protease staining prior to testing. Colorimetric sensing of proteolysis was achieved by applying a Bromocresol Purple-colored protease solution on a skim milk plate count agar-PEG 4000 readout platform. Color change (yellow to burgundy) indicated a positive readout after 15 min of incubation. The lowest limit of S. aureus detection was 10² CFU mL^-1. Moreover, direct detection of S. aureus BHI culture was achieved by sensing the pH change because of protease production, using bromothymol blue dye. A light-green color represented a positive readout. A direct relationship between S. aureus culture concentration and color change was observed, with a detection limit as low as 10³ CFU mL^-1. Examination of blind samples of Escherichia coli and S. aureus showed the potential of this assay to specifically detect S. aureus in situ. Therefore, the developed approach is anticipated to help detect S. aureus for biomedical applications.

Keywords: Colorimetric assay; Extracellular proteases; Microorganism; PH indicators; Proteolytic activity; Staphylococcus aureus.