Relative transcription of Listeria monocytogenes virulence genes in liver pâtés with varying NaCl content.

Quantitative real time polymerase chain reaction (qRT PCR) was used to compare the relative transcription of prfA, inlA, sigB and clpC for three Listeria monocytogenes strains after incubation in i) a standard liver pâté versus brain heart infusion (BHI) broth and ii) the standard liver pâté versus three liver pâtés with reduced NaCl content of which one also has been supplied with organic acids (Ca-acetate and Ca-lactate). The three strains (EGD-e: reference strain; O57: more NaCl sensitive; 6896: more NaCl tolerant) were selected out of twelve strains based on their growth in BHI broth adjusted to 6%, 8%, 10% (w/v) NaCl. The three strains were spiked into the liver pâtés (10(9) cfu/g) and the BHI (10(9) cfu/ml) and incubated for 48 h at 7 degrees C; all incubation conditions supported growth of the strains. Extraction of intact listerial RNA from the liver pâtés was complicated by the complexity of the liver pâté matrix. However, a method has been optimized and described, and the quality of RNA extracted from liver pâtés was equal to the quality of RNA extracted from BHI. The amplification efficiencies of the six genes used for the transcription analyses (the four target genes and two reference genes, gap and rpoB) were within the acceptable range from 90% to 110% for all three strains in both liver pâté and BHI. Comparison of the three strains after incubation in the standard liver pâté and BHI showed that the relative transcription of prfA for O57 and the relative transcription of inlA and sigB for both O57 and 6896 were significantly higher when the strains were grown in BHI compared to the standard liver pâté. Reducing the NaCl content of the standard liver pâté did not change relative transcription levels of prfA, inlA, sigB or clpC (except for prfA in O57 and sigB in 6896). However, the presence of Ca-acetate and Ca-lactate induced relative transcription of the stress response gene, clpC, for all three strains. This study demonstrates that relative microbial gene transcription can be measured in complex food matrices and points to the need for designing experimental set-ups in real food matrices to replace the laboratory model systems. With respect to L. monocytogenes, it seems that the NaCl content of liver pâté can be lowered within the investigated range without significant changes in relative virulence gene transcription while more caution should be taken when adding organic acids such as acetate and lactate.