Exploring Listeria monocytogenes in Ewe Milk: ssrA Gene-based Real-time PCR Identification, Phylogenetic Analysis, and Antibacterial Assessment of Magnesium Oxide Nanoparticles Synthesized with Myrtus communis Leaf Extract

Abstract

Background: Listeria monocytogenes, a zoonotic pathogen affecting humans and animals, exhibits a global distribution, including Iraq. This study focused on the rapid identification and phylogenetic analysis of L. monocytogenes in freshly collected ewe milk samples from various farms in Al-Qadisiyah province, Iraq.Methods: The study was conducted with care and precision, involving 150 milk samples. These samples were subjected to traditional bacterial isolation and identification using the enrichment culture method and biochemical tests, with the PCR technique confirming the results. The antibacterial activity of MgONPs was then assessed using the disc diffusion method, ensuring a comprehensive and reliable approach to the study.Result: The results show that 150 ewe milk samples underwent real-time PCR (RT-PCR) targeting the ssrA gene, followed by partial 16S rRNA gene sequencing (PSGS) of purified conventional PCR products. Furthermore, the study entails the biosynthesis of magnesium oxide nanoparticles using Myrtus communis leaf extract, followed by a comprehensive characterization utilizing UV-spectra, FTIR, SEM, and TEM techniques. The Agar well diffusion method assessed the antibacterial efficacy of these Biosynthesized nanoparticles against L. monocytogenes. The RT-PCR results revealed the presence of L. monocytogenes in 36 out of 150 samples (24%). Subsequent PCR analysis confirmed the presence of the pathogen in 30 out of these 36 positive samples (83.33%). Sequencing of two purified PCR products demonstrated 100% nucleotide identity with global isolates from Iraq and Turkey. Furthermore, the study demonstrated that L. monocytogenes exhibited substantial sensitivity (24.66 ± 0.3) to the biosynthesized magnesium oxide nanoparticles. These findings underscore the speed and precision of the RT-PCR method for detecting L. monocytogenes in fresh ewe milk samples.Conclusion: This comprehensive investigation enhances our understanding of L. monocytogenes prevalence in ewe milk and highlights the potential of Myrtus communis -derived nanoparticles for combating this pathogen.Keywords: Antibacterial nanoparticles; Listeria monocytogenes; Magnesium oxide; Myrtus communis sheep milk; ssrA gene