Fate of Listeria monocytogenes on fully ripened Greek Graviera cheese stored at 4, 12, or 25 degrees C in air or vacuum packages: in situ PCR detection of a cocktail of bacteriocins potentially contributing to pathogen inhibition.
The behavior of Listeria monocytogenes on fully ripened Greek Graviera cheese was evaluated. Three batches (A, B, and C) were tested. Batches A and C were prepared with a commercial starter culture, while in batch B the starter culture was combined with an enterocin-producing Enterococcus faecium Graviera isolate. Cheese pieces were surface inoculated with a five-strain cocktail of L. monocytogenes at ca. 3 log CFU/cm2, packed under air or vacuum conditions, stored at 4, 12, or 25 degrees C, and analyzed after 0, 3, 7, 15, 30, 60, and 90 days. L. monocytogenes did not grow on the cheese surface, regardless of storage conditions. However, long-term survival of the pathogen was noted in all treatments, being the highest (P
Five grams of seafood products were inoculated with one to 500 viable or 10(9) heat-killed cells of Listeria monocytogenes. The presence of the pathogen was detected by the polymerase chain reaction (PCR) with primers specific for fragments of the listeriolysin O (hly) gene (two sets) and for the invasion-associated protein (iap) gene (one set). For DNA preparation, boiling, either alone or in combination with lysozyme and proteinase K treatment, was not always sufficient to lyse L. monocytogenes, while treatment with Triton X-100 produced consistently good DNA suitable for amplification. To avoid false-negative and false-positive results, 48 h incubations were necessary and a subculturing step after an initial 24 h incubation greatly improved the results. The primers that amplified regions of the listeriolysin O gene gave clearer and stronger products than primers for the invasion-associated protein gene. Using this method we were able to detect one to five L. monocytogenes cells in 5 g of product in a total of 55 h.