The Klebsiella pneumoniae (KPN) strain of Escherichia coli (E. coli) is a multidrug-resistant pathogen responsible for severe infections, including urinary tract infections, bloodstream infections, gastrointestinal diseases, and surgical site complications. Beyond its clinical relevance, E. coli-KPN contamination in the environment, particularly in wastewater, drinking water sources, and agricultural runoff, raises serious public health concerns. In biological fluids, toxic levels of E. coli-KPN have been reported as 10^5–10^6 CFU/mL in urine, indicating urinary tract infections (UTIs), while concentrations exceeding ~10^3 CFU/mL in blood, in cerebrospinal fluid (CSF), even ~10^2 CFU/mL are associated with bacteremia and sepsis, requiring immediate medical attention. The presence of E. coli-KPN at concentrations exceeding 10^6 CFU/mL in environmental water bodies is considered hazardous, contributing to the spread of antimicrobial resistance and posing risks of widespread infections. The emergence of antibiotic resistance in E. coli-KPN poses a significant clinical challenge, making early detection crucial for effective treatment and infection control. Conventional detection techniques, such as culture-based assays and polymerase chain reaction (PCR), are time-consuming, require laboratory infrastructure, and are not suited for on-site monitoring. To address these challenges, we aimed to develop a portable, highly sensitive, and specific biosensing platform integrating molecular beacon (MB)-based fluorescence detection and electrochemical sensing. The MB-based fluorescence detection achieved a limit of detection (LOD) of 0.5 pg/μL or 50 CFU/L in 10-15 minutes, with negligible cross-reactivity against non-target bacterial strains such as Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa). The electrochemical detection module, utilizing screen-printed electrodes, enabled cost-effective and quantitative DNA detection of 10 ng/ul concentration. We also performed Raman spectral analysis to verify probe functionalization and DNA hybridization on the gold surface to corroborate the electrochemical results for the successful hybridization and E. coli DNA detection. This dual-modality approach offers a robust, field-deployable diagnostic tool suitable for wastewater surveillance, food safety monitoring, and point-of-care clinical diagnostics.

Muhammad Waseem Akram

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