Anthocyanin-Incorporated Chromogenic Agar for Rapid, Selective Detection of <i>Streptococcus pneumoniae</i> via Hydrogen Peroxide-Mediated Oxidation

The rapid and cost-effective identification of <i>Streptococcus pneumoniae</i> (<i>S. pneumoniae</i>) is crucial for the prompt treatment of pneumonia and meningitis. However, conventional culture methods are time-consuming and molecular techniques are prohibitively expensive in resource-limited settings. To address this challenge, we developed a novel anthocyanin chromogenic agar as a diagnostic tool for the selective and rapid detection of <i>S. pneumoniae</i>. Unlike traditional pH-based indicators, this method exploits the unique oxidative capability of <i>S. pneumoniae</i>, which releases excessive amounts of hydrogen peroxide (H₂O₂). This results in the oxidative degradation of the blue anthocyanin pigment to a colorless form, creating distinct gray zones. The detection efficiency was evaluated as a function of bacterial concentration and incubation time. Upon inoculation at concentrations ranging from 1 to 1000 CFU/mL, color changes were monitored visually over 24 h. Notably, an inoculum of 1000 CFU/mL produced visible gray spots within just 7 h of incubation. The anthocyanin chromogenic agar demonstrated high selectivity against common coexisting pathogens, which caused a pH-dependent color change from blue to pink, whereas only <i>S. pneumoniae</i> induced the specific gray signal. Furthermore, colorimetric results were analyzed using digital image processing for objective detection and differentiation. In conclusion, anthocyanin chromogenic agar presents a clinically applicable alternative that significantly reduces both incubation time and cost compared to other chromogenic agars. While the standardization of natural anthocyanin stability remains a consideration for commercial upscaling, this method offers a promising perspective for decentralized testing in resource-limited settings.