Underestimated Survival of in Raw Milk Highlighted by Viability Real-Time PCR and Growth Recovery

Raw milk is a frequent vehicle for transmission of thermophilic , leading to reported outbreaks. Milk is a challenging food matrix for pathogen detection, due to its high protein and lipid content. Limited detection of colony-forming unit (CFU) in raw milk might underestimate the pathogen's infectious potential. We optimized a viability real-time PCR (qPCR) for application with raw milk. The procedure was robust against variations of milk lots and different strains. Various DNA-intercalating dyes were evaluated for their ability to reduce the PCR signal of dead cells. Only propidium monoazide (PMA) and PMAxx qualified for diagnostic use. Different sedimentation properties of viable and dead and strains in 10-fold diluted milk enhanced viable/dead differentiation. The new method enabled to review survival of spp. in raw milk based on viable cells harboring an intact cell membrane. The data were compared to culturability according to ISO10272-2:2017. A difference of up to 4.5 log between viable counts and CFU values became apparent. Relevance of viability qPCR values was corroborated by full recovery of CFU under extremely reduced oxygen concentration in the presence of hydrogen. Recovery of CFU was limited, however, upon prolonged exposure in raw milk. The data confirm that survival in raw milk can be largely underestimated when relying on CFU data only. We conclude that raw milk led to oxidative stress-induced growth arrest in thermophilic , which was reversible by reduction of the oxygen partial pressure in a time-limited way.