Whole-genome sequencing and prevalence of Listeria spp. in wastewater, ruminant farms, natural, and food environments in Spain.

Listeria monocytogenes is a foodborne pathogen capable of persisting in different environmental settings. This study investigates the Listeria spp. genetic diversity in different ecological niches in southeastern Spain. A total of 8,885 samples were collected (from 2019 to 2023) from wastewater, ruminant farms, natural environments, and food and food processing environments, selectively enriched in Fraser broth, and analyzed for the presence of Listeria spp. plating in RAPID´L.mono or ALOA, yielding 800 isolates. Whole-genome sequencing (WGS) was performed to characterize all obtained isolates. DNA extraction was conducted using manual and automated methods, and DNA libraries were prepared using the Nextera XT DNA Sample Kit (Illumina, USA). Sequencing was carried out on NextSeq 500 (2 × 150 bp) and MiSeq (2 × 300 bp) platforms.