Shiga toxin production by sausage-borne Escherichia coli O157:H7 in response to a post-processing in vitro digestion challenge

In order to study the effects of the fermentation-drying procedure and subsequent in vitro digestion on Shiga toxins (Stx) production by Escherichia coli O157:H7, dry sausages were inoculated during the formulation step with pure cultures of strains 5-1 and ATCC 43895. The inoculated sausages were submitted to a minimum (30 min, pH between 3.1 and 3.5) or a maximum (120 min at stepwise adjusting the pH downward) gastric challenge followed by a 240-min pancreatic challenge at pH 8.0 and 37 °C. Production of toxins by the overnight culture controls, assessed using the Vero cell assay, was dependent on the pathogen cell concentration. The effect of cell concentration was not relevant in sausage samples and data showed: (a) higher Stx production in contaminated sausage samples than in overnight cultures; (b) the lowest Stx levels were detected with undigested sausage samples; (c) the maximum gastric challenge enhanced Stx production, compared to minimally digested and undigested samples. Reverse transcriptase polymerase chain reaction (RT-PCR) performed on extracts from inoculated, digested (4.5-6 h process) and undigested sausages produced amplicons for both stx1 and stx2 mRNA, suggesting that post-stress expression of stx genes had occurred. Our data suggest that sub-lethal stresses imposed by the fermentation-drying procedure and subsequent digestion of ingested food (i.e. contaminated sausages) may affect the degree to which the surviving E. coli O157:H7 cells express their virulence in vivo.