Recombinant shiga toxin-1 subunit B [His]

E. coli O157 is the most commonly identified serotype among Shiga like-toxin E. coli infections. Current reference methodologies to detect these bacteria are reliable but take several days to provide a definitive result, as they require selective enrichment, molecular detection of the bacteria, followed by plate confirmation. Even alternative methods need extensive sample enrichment in order to increase the amount of bacteria to detectable numbers, resulting in "next-day detection" methodologies, as it is not possible to reach the final result within the same day of the start of the analysis. In the current study, the sample treatment was optimized in such a way that after a short enrichment (3 h) followed by an enzymatic treatment, DNA extraction and amplification by real-time PCR, it was possible to detect down to 3.9 and 3.3 CFU/25 g of ground beef and leafy greens respectively, achieving a combined LOD95 of 3.6 CFU/25 g. This sample treatment optimization allowed us to develop a real "same-day detection" method, being this a major advantage over other previously published methodologies. Additionally, the analysis of spiked samples provided excellent results (90%) for the relative sensitivity, specificity and accuracy; along with the positive and negative predictive values. Finally, the Cohen's k also obtained a value of 1, indicating that the results obtained with the novel method were in "almost complete concordance" with the expected values. It is worth to highlight that the performance parameters fulfill the requirements of the NordVal regulation for the validation of alternative methods.

Bacteriophages, natural killers of bacteria, and plant secondary metabolites, such as condensed tannins, are potential agents for the control of foodborne pathogens. The first objective of this study evaluated the efficacy of a bacteriophage SA21RB in reducing pre-formed biofilms on stainless-steel produced by two Shiga toxin-producing Escherichia coli (STEC) strains, one from South Africa and the other from Canada. The second objective examined the anti-bacterial and anti-biofilm activity of condensed tannin (CT) from purple prairie clover and phlorotannins (PT) from brown seaweed against these strains. For 24-h-old biofilms, (O113:H21; 6.2 log(10) colony-forming units per square centimeter (CFU/cm(2)) and O154:H10; 5.4 log(10) CFU/cm(2)), 3 h of exposure to phage (10(13) plaque-forming units per milliliter (PFU/mL)) reduced (p <= 0.05) the number of viable cells attached to stainless-steel coupons by 2.5 and 2.1 log(10) CFU/cm(2) for O113:H21 and O154:H10, respectively. However, as biofilms matured, the ability of phage to control biofilm formation declined. In biofilms formed for 72 h (O113:H21; 5.4 log(10) CFU/cm(2) and O154:H10; 7 log(10) CFU/cm(2)), reductions after the same duration of phage treatment were only 0.9 and 1.3 log(10) CFU/cm(2) for O113:H21 and O154:H10, respectively. Initial screening of CT and PT for anti-bacterial activity by a microplate assay indicated that both STEC strains were less sensitive (p <= 0.05) to CT than PT over a concentration range of 25-400 mu g/mL. Based on the lower activity of CT (25-400 mu g/mL), they were not further examined. Accordingly, PT (50 mu g/mL) inhibited (p <= 0.05) biofilm formation for up to 24 h of incubation at 22 degrees C, but this inhibition progressively declined over 72 h for both O154:H10 and O113:H21. Scanning electron microscopy revealed that both SA21RB and PT eliminated 24 h biofilms, but that both strains were able to adhere and form biofilms on stainless-steel coupons at longer incubation times. These findings revealed that phage SA21RB is more effective at disrupting 24 than 72 h biofilms and that PT were able to inhibit biofilm formation of both E. coli O154:H10 and O113:H21 for up to 24 h.