Resumo

Introduction: Studies demonstrated that food matrices, temperature and other physiological conditions found in the host to which L. monocytogenes (LM) is exposed can alter its virulence potential. Objective: To quantify gene expression of intA in LM isolates after exposure to adverse conditions. Methods: A total of 10 isolates of LM from different sources were grown in Brain Heart Infusion Broth (BHI) at 37°C/24h and 8ml were centrifuged at 13,000 rpm/5min. Pellets were ressupended in 1ml of each broth at following conditions for 1 hour: a)BHI 25°C b) BHI 4°C c) BHI plus 2% sucrose; d) BHI plus 5% NaCl; e) BHI plus 0.3% Oxgall; f) BHI pH 4.5 (adjusted with HCl) and f) BHI plus 50% (v/v) of extract from bacteriocin-producing cultures of Lactobacillus sakei 1, Leuconostoc mesenteroides 11A or Enterococcus faecium 130. RNA was extracted and converted to cDNA. The 16S and RpoB primers were used as reference genes and used for amplification in parallel with the target gene (intA). For each sample, 12 μL PCR reaction was prepared as follow: 1.5 µl of cDNA, 6.3 µl AbsoluteM SYBR Green QPCR Master Mix (ABgene, Surrey, UK), 0.6 µl of each primer solution (0.25 mM of each primer) and 3 µl of purified water. The conditions for PCR reaction comprised an initial denaturation step at 95ºC for 15 min, 45 cycles of 95ºC for 2 s, 55ºC for 10s, and 72ºC for 10s, performed with an instrument Mini-Opticon (BioRad, USA). Results and discussion: The vast majority of the conditions applied in this study were significant in down regulation of intA gene expression. However, LM HU 471 isolate presented up to 8 times higher intA expression in presence of sucrose 2%. Conclusion: The present study shows that stress factors are capable of changing the relative transcription of an important virulence gene in LM.