Comparison of conventional plating, PMA-qPCR, and flow cytometry for the determination of viable enterotoxigenic Escherichia coli along a gastrointestinal in vitro model.

Recent technological advances for bacterial viability assessment using molecular methods or flow cytometry can provide meaningful interest for the demarcation between live and dead microorganisms. Nonetheless, these methods have been scarcely applied to foodborne pathogens and never for directly assessing their viability within the human digestive environment. The purpose of this study was to compare two methods based on membrane integrity (propidium monoazide (PMA) q-PCR and Live/Dead flow cytometry) and the classical plate-count method to determine the viability of a common foodborne pathogen, enterotoxigenic Escherichia coli (ETEC), during its transit trough simulated human gastrointestinal environment. Viable ETEC counts in the gastric and small intestinal compartments of the gastrointestinal TIM model indicated a consensus between the three tested methods (PMA-qPCR, flow cytometry, and plate counts). In a further step, flow cytometry analysis appeared as the preferred method to elucidate ETEC physiological states in the in vitro digestive environment by discriminating four subpopulations, while PMA-qPCR can only distinguish two. The defined viable/altered ETEC population was found during all in vitro digestions, but mainly in the gastric compartment. Being able to discriminate the particular physiological states of pathogenic microorganisms in the digestive environment is of high interest, because if some cells are not observable on culture media, they might keep their ability to express virulence functions.

Development of the recombinase-based in vivo expression technology in Streptococcus thermophilus and validation using the lactose operon promoter.

AIMS: To construct and validate the recombinase-based in vivo expression technology (R-IVET) tool in Streptococcus thermophilus (ST). METHODS AND RESULTS: The R-IVET system we constructed in the LMD-9 strain includes the plasmid pULNcreB allowing transcriptional fusion with the gene of the site-specific recombinase Cre and the chromosomal cassette containing a spectinomycin resistance gene flanked by two loxP sites. When tested in M17 medium, promoters of the genes encoding the protease PrtS, the heat-shock protein Hsp16 and of the lactose operon triggered deletion of the cassette, indicating promoter activity in these conditions. The lactose operon promoter was also found to be activated during the transit in the murine gastrointestinal tract. CONCLUSIONS: The R-IVET system developed in ST is relatively stable, functional, very sensitive and can be used to assay activity of promoters, which are specifically active in in vivo conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This first adaptation of R-IVET to ST provides a highly valuable tool allowing an exploration of the physiological state of ST in the GIT of mammals, fermentation processes or dairy products.