Direct and Rapid Detection and Quantification of Oenococcus oeni Cells in Wine by Cells-LAMP and Cells-qLAMP.

Fast detection and enumeration of Oenococcus oeni in winemaking are necessary to determine whether malolactic fermentation (MLF) is likely to be performed or not and to decide if the use of a commercial starter is needed. In other wines, however, performing MLF can be detrimental for wine and should be avoided. The traditional identification and quantification of this bacteria using culture-dependent techniques in wine-related matrices require up to 14 days to yield results, which can be a very long time to perform possible enological operations. Loop-mediated isothermal amplification (LAMP) is a novel culture-independent technique that amplifies nucleic acid sequences under isothermal conditions with high specificity and efficiency in less than 1 h with inexpensive equipment. We designed LAMP primers for the specific detection and quantification of O. oeni cells. The developed LAMP method allows O. oeni to be detected directly from both grape musts and wines within 1 h from the time that the LAMP reaction begins, and without DNA extraction and purification requirements. The high sensitivity of LAMP methodology is achieved by previous mechanical cells lysis with no further purification by detecting one single cell per reaction in culture media, and in white/red grape musts and wines by avoiding reaction inhibition by ethanol, polyphenols, and other wine inhibitors. Cells can be concentrated prior to the LAMP reaction to further increase this sensitivity. Moreover, the LAMP method does not require expensive equipment and can be easily operated. The developed method is both economic and fast and offers high sensitivity and specificity.

Cells-qPCR as a direct quantitative PCR method to avoid microbial DNA extractions in grape musts and wines.

A novel quantitative PCR assay called Cells-qPCR has been developed for the rapid detection and quantification of yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) directly from grape must and wine that does not require DNA extraction. The assay was tested on Brettanomyces bruxellensis, Saccharomyces cerevisiae, Lactobacillus plantarum, Oenococcus oeni, Acetobacter aceti and Gluconobacter oxydans in culture media, and in white and red grape musts and wines. Standard curves were constructed from DNA and cells for the six target species in all the matrices. Good efficiencies were obtained for both when comparing DNA and cells standard curves. No reaction inhibition was observed between matrices for each species. Cells quantification was linear over a range of cell concentrations (7, 5 or 4 orders of magnitude) and detected as few as one cell per reaction in all the matrices. The developed Cells-qPCR assay is a robust, reliable, fast and specific method to detect and quantify different yeasts, LAB and AAB species in grape must and wine that avoids DNA extraction and overcomes the presence of inhibitors like polyphenols and ethanol.

Characterization of Enterococci from Food and Food-Related Settings.

Enterococcus species are ubiquitous in nature, exist at high levels in food, and can cause severe diseases in humans. Thus, surveillance of enterococci harboring antibiotic resistance and virulence factors in food and food-related environments is needed. In the present study, 89 samples from food and food processing surfaces were collected in a cheese factory, a swine slaughterhouse, and a supermarket, and 132 Enterococcus isolates were recovered. Most isolates were identified as E. faecalis, which is considered the most pathogenic member of this genus. Safety analysis covering antibiotic resistance revealed that all isolates were resistant to sulfamethoxazole-trimethoprim and sensitive to amoxicillin-clavulanate, ampicillin, ciprofloxacin, gentamicin, levofloxacin, linezolid, nitrofurantoin, or teicoplanin. More than half of the isolates were resistant to quinupristin-dalfopristin, tetracycline, and bacitracin, and less than half were resistant to the other antibiotics evaluated. Regarding virulence factors, 52% the isolates were beta-hemolytic, 39% were gelatinase producers, and 45% contained the gelE gene. For the remaining genes evaluated, efaAfs was detected in more than half of the isolates, and agg, esp, and efaAfm were found in less than half of the isolates. The present investigation revealed that food-related enterococci obtained from very different settings have multidrug resistance and virulence factors, highlighting the importance of effective surveillance networks to avoid the spread of putative pathogenic enterococci.