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.

No seasonal effect on culturable pseudomonads in fresh milks from cattle herds.

Freshly drawn raw milk from 37 single herds on farms manufacturing raw cow cheese under the Protected Designation of Origin (PDO) label were sampled over 13 mo for pseudomonad counts. Coliforms, somatic cells, and coagulase-positive staphylococci were counted and total fat and protein contents measured. For pseudomonad counts, the overall mean value was 3.60×10(3) cfu/mL. We observed very high variation between different producers and within the same producers (average standard deviation 1.30×10(4) cfu/mL), but we did not detect a seasonal effect. The only statistical correlation with other milk quality parameters was with coliforms. A survey of milking practices and milking machine sanitation together with environmental and milk sampling for pseudomonad counts in 7 cheese workshops showed that no real negligence or error could be imputed to producers. The main problems were the presence of non-aeruginosa pseudomonads in potable water and a few isolated failures during the cleaning and rinsing phases of sanitation.

Genetic studies of a thermoregulated gene in the psychrotrophic bacterium Pseudomonas fluorescens.

In the psychrotrophic bacterium Pseudomonas fluorescens, some genes are thermoregulated: they are maximally expressed at a particular temperature within the broad range of temperatures that allow growth of this bacterium. To study this regulation, random transcriptional insertion fusions were obtained by means of mini-Tn5lacZ1 or mini-Tn5luxAB transposition. One fusion was studied in which beta-galactosidase production was maximal at a low-growth temperature. The mutated gene (that we call xsf) was highly homologous to xseA from Escherichia coli (and from other bacteria) which encodes the large subunit of exonuclease VII. Genetic tools were constructed in order to analyse and manipulate this fusion: a plasmid derived from R68.45 was used for chromosome transfer and a replacement vector was constructed to allow in situ marker exchange of the mini-Tn5lacZ1 by an Hg(r) interposon. This vector was used to make double mutants and hence to study the effect of the insertion in xsf on the expression of other fusions. Six genes were thereby identified with a decreased expression in an xsf- background and with different characteristics of thermoregulation.

Effect of growth temperature on several exported enzyme activities in the psychrotrophic bacterium Pseudomonas fluorescens.

In accordance with previous results, the activity of extracellular proteases from Pseudomonas fluorescens MF0 is maximal at a growth temperature of 17.5 degrees C, well below the optimal growth temperature. In addition, the activities of three periplasmic phosphatases display the same growth temperature optimum. Chemostat experiments have shown that it is the growth temperature itself and not the value of the growth rate that regulates these activities. In contrast, a foreign periplasmic phosphatase, expressed under the control of its own promoter, displays a different sensitivity toward temperature. We conclude that in the psychrotrophic strain P. fluorescens MF0, growth temperature exerts a specific control upon the activity of certain enzymes. The critical temperature (17.5 degrees C) is within the range of normal growth, suggesting that this control is probably different from a cold shock or heat shock response.