Isolation, Identification, and Characterization of Listeria from Municipal and Dairy Farm Wastewater

Wastewater systems can serve as reservoirs and dissemination pathways for Listeria spp., yet comparative data from municipal and agricultural wastewater remain limited. The objective of this study was to isolate Listeria monocytogenes from municipal wastewater and dairy-farm runoff for use as enrichment hosts in a subsequent and separate study on the isolation of bacteriophage from the same wastewater streams. During this process, additional Listeria spp. were recovered and characterized along with L. monocytogenes. Identification of Listeria sp. was accommodated using multilocus sequencing, LIPI-1-targeted PCR, and phenotypic confirmation. Wastewater samples were filtered, enriched with Demi-Fraser broth, treated with anti-Listeria magnetic beads, and plated on MOX agar. Bead-captured Listeria were later plated on Listeria Brilliance Agar to differentiate phospholipase-positive L. monocytogenes. Putative isolates were characterized by sequencing of 16S rRNA, sigB, and iap genes, and by PCR detection of four Listeria Pathogenicity Island 1 (LIPI-1) markers (hlyA, inlA, actA, prfA). Using this combined approach, 27 Listeria isolates were recovered from 21 wastewater samples during a 6-week sampling period: 13 samples of municipal wastewater yielded 5 isolates of L. monocytogenes (38% prevalence rate) and 11 other Listeria sp. (85% overall prevalence rate; 1 L. innocua, and 5 Listeria sp.); 8 samples of dairy wastewater yielded 2 isolates of L. monocytogenes (25% prevalence) and 9 other Listeria sp. (100% overall prevalence rate; 8 L. innocua and 1 Listeria sp.). Seven isolates from municipal and dairy wastewater were confirmed as L. monocytogenes based on concordant sigB and iap gene sequence identities, LIPI-1 gene presence, and phospholipase-positive reactions on chromogenic agar. Six isolates yielded discordant results across the multilocus sequencing markers and could not be confidently assigned to a species and were designated Listeria sp. These findings highlight the value of integrating chromogenic media with multilocus genetic markers for accurate identification of L. monocytogenes in complex environmental matrices.