High prevalence and diversity of mesophilic bacteria detected in pre-enrichment raw milk from Ecuador using two multiplex TaqMan qPCR assays

The consumption of raw cow’s milk remains common and traditional in Ecuador, especially due to its sale in informal markets, despite its recognized potential to act as a vehicle for multiple zoonotic pathogens and chemical substances. This study aimed to validate a multiplex quantitative PCR (qPCR) assay for the simultaneous detection and semi-quantitative assessment of eight mesophilic bacteria of food and public health relevance [ Listeria monocytogenes , Staphylococcus aureus (S. aureus) , Salmonella spp., Campylobacter spp., group B Streptococcus , Escherichia coli ( E. coli ), Shigella spp. and E. coli O157:H7], and to apply this tool to characterize their prevalence and distribution in raw milk from different production contexts in Ecuador. A total of 450 milk samples were analyzed, including 400 raw milk samples from cows without clinical mastitis, 40 samples from cows with confirmed mastitis, and 10 milk samples treated with ultra-high temperature (UHT) as negative controls. The multiplex assays showed amplification efficiencies between 95 and 105%, correlation coefficients ≥ 0.99 and limits of detection and quantification of 1 genomic copy/µL, with high analytical specificity and repeatability. In raw milk, at least one target bacterium was detected in almost all samples, with the highest prevalences observed for S. aureus (90.5%), E. coli (81.8%) and E. coli O157:H7 (70.8%), followed by Campylobacter spp. (46.3%), L. monocytogenes (34.0%), Salmonella spp. (28.8%), Shigella spp. (5.8%) and group B Streptococcus (1.8%). Mastitis milk showed 100% positivity for E. coli , high frequencies of S. aureus and group B Streptococcus , as well as absence of Salmonella spp. and L. monocytogenes , whereas all UHT samples were negative. Quantitative analysis revealed high bacterial loads for several pathogens, and the stratified evaluation showed significant differences in contamination patterns across provinces and producer size, although no significant seasonal (climatic) effect was observed. Overall, the findings confirm a substantial microbiological risk associated with raw milk consumption and support the use of multiplex qPCR as a robust tool for surveillance and risk-based control in dairy production systems. The consumption of raw milk poses a risk to public health, and given the results obtained here which indicate that there is little oversight in the region the public is urged to consume only pasteurized milk.