In the present work we developed a MPN quantitative real-time PCR (MPN-qPCR) method for a fast and reliable detection and quantification of Listeria monocytogenes and Escherichia coli O157:H7 in minimally processed vegetables. In order to validate the proposed technique, the results were compared with conventional MPN followed by phenotypic and biochemical assays methods. When L. monocytogenes and E. coli O157:H7 were artificially inoculated in fresh-cut vegetables, a concentration as low as 1 CFU g−1 could be detected in 48 hours for both pathogens. qPCR alone allowed a limit of detection of 101 CFU g−1 after 2 hours of enrichment for L. monocytogenes and E. coli O157:H7. Since minimally processed ready-to-eat vegetables are characterized by very short shelf life, our method can potentially address the consistent reduction of time for microbial analysis, allowing a better management of quality control. Moreover, the occurrences of both pathogenic bacteria in mixed salad samples and fresh-cut melons were monitored in two production plants from the receipt of the raw materials to the early stages of shelf life. No sample was found to be contaminated by L. monocytogenes. One sample of raw mixed salad was found positive to an H7 enterohemorrhagic serotype.

To enhance the productivity of mixed microbial cultures for fermentative bio-hydrogen production, chemical-physical pre-treatments of the original seed are needed to suppress the activity of hydrogen (H2)-consuming microbes. This approach might influence negatively the composition and diversity of the hydrogen-producing community with consequences on the functional stability of the H2-producing systems in case of perturbations. In this study, we aimed at investigating the effect of different types of pre-treatment on the performance of hydrogen production systems in the presence of an inhibitor, such as 5-hydroxymethylfurfural (HMF). The efficiency and the microbial community structure of batch reactors amended with HMF and inoculated with non-pretreated and pretreated (acid, heat shock, and aeration) anaerobic sludge were evaluated and compared with control systems. The type of pre-treatments influenced the microbial community assembly and activity in inhibited systems, with significant effect on the performance. Cumulative H2 production tests showed that the pre-aerated systems (control and HMF inhibited) were the most efficient, while the difference of the lag phase of the pre-acidified control and HMF-added test was negligible. Analyses of the structure of the enriched microbial community in the systems through PCR-denaturing gradient gel electrophoresis (DGGE) followed by band sequencing revealed that the differences in performance were mostly related to shifts in the metabolic pathways rather than in the predominant species. In conclusion, the findings suggest that the use of specific inoculum pre-treatment could contribute to regulate the metabolic activity of the fermentative H2-producing bacteria in order to enhance the bio-energy production.

Listeria to 10 g of food were incubated in Fraser broth at 30°C for 48 h for standard MPN analysis. After incubation, broth samples were taken from each tube and DNA was extracted. DNA from enrichment tubes was used as template in a qPCR assay targeting a 64 bp hlyA gene sequence of L. monocytogenes. Results of this assay were than compared with those of standard MPN analysis and a complete accordance was observed. Furthermore, we tested an enrichment free approach using the same qPCR assay. Samples were prepared as described for MPN-qPCR while DNA extraction was performed prior to enrichment of inoculated salads. This approach allowed us to identify L. monocytogenes in samples spiked with 10-10 5 CFU/g. The whole process, including DNA extraction, required less than four hours, thus providing a fast and reliable tool for detection of L. monocytogenes in fresh cut vegetables.

Leafy vegetables are consumed fresh after harvest with bland washes, not always efficient in removing pathogenic bacteria that may be attached to the external skin or surfaces. In this work, an optimized ELISA based method developed in our laboratory was used to detect the presence of Listeria monocytogenes in fresh-cut rocket leaves. From a certain quantity (5 g) of fresh rocket leaves contaminated with Listeria monocytogenes, bacteria were isolated and the pathogen was detected using an ELISA protocol. The preliminary results are promising in the use of antibody-antigen interaction to verify the presence of L. monocytogenes in the minimally processed vegetable distribution chain.