Listeriosis outbreaks indicate that L. monocytogenes contamination is an issue for various food types, such as unpasteurized (raw) cheese.... Show moreListeriosis outbreaks indicate that L. monocytogenes contamination is an issue for various food types, such as unpasteurized (raw) cheese. Current regulation prevents the interstate sale and distribution of raw cheese and mandates a ≥60 day aging period at ≥2˚C to ensure product safety; yet studies demonstrate that pathogens can persist during aging. Environmental stress can alter the transcriptomic profiles of pathogens; however, these surveys are rarely conducted in food matrices. This study aimed to assess the transcriptomic profiles of L. monocytogenes strain F2365 during environmental stressors inherent throughout cheesemaking. First, quantitative polymerase chain reaction (qPCR) was used to monitor transcription levels of nine L. monocytogenes genes involved in virulence, stress response, energy transport, and metabolism during osmotic stress (11% NaCl) and varying temperature/time (5˚C 24 h, 25˚C 24 h, 38˚C 30 min) conditions in Brain Heart Infusion (BHI) broth, raw milk, and pasteurized milk. Generally, the genes prfA, lmo1381, lmo0963, and lmo1875 were down-regulated, whereas the genes lmo1864, lmo0914, lmo0348, lmo1428, and lmo1264 were up-regulated. Virulence gene, prfA, was most down-regulated when L. monocytogenes was grown in raw milk with salt at 25˚C (4774.72±838.14 fold; relative to 24 h growth at 37˚C). The stress response gene lmo0914, encoding σB, was most up-regulated when L. monocytogenes was grown in BHI at 25˚C with salt (14.61±7.72 fold). Additionally, transcription levels of the nine genes were assessed at points during the laboratory-scale manufacture of Gouda cheese made with raw milk artificially-inoculated with L. monocytogenes via qPCR. Similar differential regulation for both prfA and lmo0914 in L. monocytogenes was observed during cheesemaking. The gene lmo1864, encoding a putative pore-forming hemolysin, was up-regulated throughout the cheesemaking process, but was most up-regulated after stirring the curd (449.81±432.53 fold). Ultimately, these results indicate that the lmo1864 gene may play a role in L. monocytogenes survival during cheesemaking. Methods developed in this study can be used to assess the risk of L. monocytogenes, not only during cheesemaking, but during the ≥60-day aging process. Overall, these results contribute to the understanding of L. monocytogenes survival mechanisms during the cheesemaking process. M.S. in Food Safety and Technology, May 2017 Show less
