Molecular Profiling of Foodborne Pathogens in Ready-to-Eat Foods, Al-Madinah Al-Munawarah, Saudi Arabia

Foodborne pathogens remain a global public health concern, and antimicrobial resistance increases their impact. In mass-gathering cities such as Al-Madinah Al-Munawarah, contaminated ready-to-eat (RTE) fast foods can contribute to both local transmission and international spread. In this study, 300 RTE fast food samples, including shawarma, burgers, fried chicken, sandwiches, and salads, were collected from international franchises, local restaurants, and street vendors. Pathogens were identified using conventional culture combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility testing followed CLSI guidelines, and real-time PCR confirmed species identity and screened resistance determinants. Principal component analysis (PCA) and dendrogram clustering were used to assess diagnostic discrimination. Among the 300 samples, 129 (43.0%) were culture positive. The most common pathogens were <i>Staphylococcus aureus</i> (14.3%) and <i>Escherichia coli</i> (13.0%), followed by <i>Salmonella</i> spp. (9.0%) and <i>Acinetobacter baumannii</i> (6.7%). About 35% of <i>S. aureus</i> isolates were methicillin resistant (MRSA), and 85% of <i>A. baumannii</i> carried OXA-type carbapenemase genes. MALDI-TOF MS achieved 96.1% score-based identification and, with PCA, showed strong interspecies separation. PCR confirmed species identity and detected widespread resistance genes, with genotype-phenotype concordance of at least 80%. Overall, 60.5% of isolates were multidrug resistant. RTE fast foods in Al-Madinah represent reservoirs of MDR pathogens, including carbapenemase-producing <i>A. baumannii</i>. The combined use of MALDI-TOF MS and real-time PCR established a rapid and scalable workflow that provided reliable identification and resistance profiling in less than 24 h, compared with 48 to 72 h for conventional methods. This approach supports One Health surveillance in high-risk food settings and strengthens preparedness for mass gatherings.