Occurrence of Listeria monocytogenes in Raw Bovine Milk from Ecuador: Culture-Based Isolation and Molecular Identification.

Listeria monocytogenes is one of the main foodborne pathogens associated with milk and dairy products due to its ability to survive under refrigeration conditions and persist in dairy production environments. The aim of this study was to determine the occurrence of L. monocytogenes in raw bovine milk samples collected from dairy farms in the provinces of Pichincha and Manabí, Ecuador, using culture-based and molecular methods. A total of 633 raw milk samples were collected between September 2022 and July 2023 from small-, medium-, and large-scale producers. Detection was performed by qPCR targeting the hly gene after pre-enrichment, followed by Bacterial culture and molecular confirmation through 16S rRNA gene analysis. Overall, 27.33% (173/633) of the samples tested positive for L. monocytogenes. Positive samples were distributed among small-scale producers (35.84%), medium-scale producers (36.99%), and large-scale producers (27.17%). Statistically significant differences were observed between provinces (p < 0.0001). Multivariable logistic regression analysis showed that samples obtained in Pichincha had considerably greater probabilities of being positive compared to those from Manabí (OR = 2.37; 95% CI: 1.61-3.48; p < 0.001). However, during the dry season, samples collected in Manabi showed significant probabilities of being positive compared to the rainy season (OR = 0.31; 95% CI: 0.21-0.45; p < 0.001). Producer size was not significantly associated with positivity after adjustment. Agreement between qPCR and culture-based methods was moderate (κ ≈ 0.6). Relative diagnostic performance analysis demonstrated that qPCR showed a relative sensitivity of 92.35%, relative specificity of 96.54%, positive predictive value (PPV) of 90.75%, and negative predictive value (NPV) of 97.17% compared with Bacterial culture. These findings confirm the occurrence of L. monocytogenes in raw bovine milk from the studied regions and highlight the importance of maintaining hygienic practices and microbiological surveillance during milk production and handling.