Improved detection of Escherichia coli and coliform bacteria by multiplex PCR.

BACKGROUND: The presence of coliform bacteria is routinely assessed to establish the microbiological safety of water supplies and raw or processed foods. Coliforms are a group of lactose-fermenting Enterobacteriaceae, which most likely acquired the lacZ gene by horizontal transfer and therefore constitute a polyphyletic group. Among this group of bacteria is Escherichia coli, the pathogen that is most frequently associated with foodborne disease outbreaks and is often identified by ß-glucuronidase enzymatic activity or by the redundant detection of uidA by PCR. Because a significant fraction of essential E. coli genes are preserved throughout the bacterial kingdom, alternative oligonucleotide primers for specific E. coli detection are not easily identified. RESULTS: In this manuscript, two strategies were used to design oligonucleotide primers with differing levels of specificity for the simultaneous detection of total coliforms and E. coli by multiplex PCR. A consensus sequence of lacZ and the orphan gene yaiO were chosen as targets for amplification, yielding 234 bp and 115 bp PCR products, respectively. CONCLUSIONS: The assay designed in this work demonstrated superior detection ability when tested with lab collection and dairy isolated lactose-fermenting strains. While lacZ amplicons were found in a wide range of coliforms, yaiO amplification was highly specific for E. coli. Additionally, yaiO detection is non-redundant with enzymatic methods.

Salt influence on surface microorganisms and ripening of soft ewe cheese.

The effect of different brining treatments on salt uptake and diffusion during the first 30 d of ripening was determined in soft ewe cheese. Additionally, salt influence on surface microorganisms and physicochemical parameters was evaluated. Cheeses were placed into different brine solutions (14, 18 and 24°Bé) at 5 and 10 °C for 1, 2 or 3 h. Samples from rind, outer core and inner core were analysed at 0, 7, 15 and 30 d. Complete salt diffusion from rind to the inner core took about 15 d. The resulting salt gradient favoured the development of a pH gradient from the surface to the inner core. Salt concentration also had a significant effect on the growth of surface microorganisms (mesophiles, pseudomonads and halotolerants). However, mould and yeasts were not affected throughout ripening by the salt levels achieved. Brine salting by immersion for 3 h at 10 °C in 24°B brine was found to be the most suitable treatment to control pseudomonads in cheese rind, as spoilage microorganism.

A low-cost procedure for production of fresh autochthonous wine yeast.

A low-cost procedure was designed for easy and rapid response-on-demand production of fresh wine yeast for local wine-making. The pilot plant produced fresh yeast culture concentrate with good microbial quality and excellent oenological properties from four selected wine yeasts. The best production yields were obtained using 2% sugar beet molasses and a working culture volume of less than 60% of the fermenter capacity. The yeast yield using 2% sugar grape juice was low and had poor cell viability after freeze storage, although the resulting yeast would be directly available for use in the winery. The performance of these yeasts in commercial wineries was excellent; they dominated must fermentation and improved its kinetics, as well as improving the physicochemical parameters and the organoleptic quality of red and white wines.

Genetic instability of heterozygous, hybrid, natural wine yeasts.

We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae. Spontaneous cyh2(R)/cyh2(R) mutants resistant to high levels of cycloheximide can be directly isolated from cyh2(S)/cyh2(S) wine yeasts. Heterozygous cyh2(R)/cyh2(S) hybrid clones vary in genetic instability as measured by loss of heterozygosity at cyh2. There were two main classes of hybrids. The lawn hybrids have high genetic instability and generally become cyh2(R)/cyh2(R) homozygotes and lose the killer phenotype under nonselective conditions. The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype. The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported. Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity. This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations.