Cristina Giusto

Classical and molecular analyses to characterize commercial dry yeasts used in wine fermentations

Aims:  The aim of the work was to apply PCR‐temperature gradient gel electrophoresis (PCR‐TGGE) and restriction enzyme analysis (RE) assays to identify commercially available starters of Saccharomyces cerevisiaesensu stricto complex.

A molecular method to detect Bacillus cereus from a coffee concentrate sample used in industrial preparations.

Aims: The aim of this work was to develop specific primers which are able to detect Bacillus cereus in a coffee concentrate sample.

Molecular methods to evaluate biodiversity in Bacillus cereus and Bacillus thuringiensis strains from different origins.

The spore-forming genus Bacillus includes species of industrial, clinical and environmental significance. The possibility of differentiating between Bacillus cereus and Bacillus thuringiensis, toxin producers associated with illness, is a real need in monitoring potentially contaminated foods to understand the real distribution of B. cereus/B. thuringiensis in different outbreak cases. As the use of DNA comparison obtains clearer results than classical microbiological methods in distinguishing B. cereus from B. thuringiensis in this work PCR-TTGE (Temporal Temperature Gradient gel Electrophoresis), rep-PCR and RAPD-PCR methods have been compared to assess the intra- and inter-specific variability of B. cereus and B. thuringiensis. 80 strains of B. cereus and B. thuringiensis isolated from food, patients and pesticides were analyzed using a gyrB gene DNA sequence in TTGE; primer M13 in the RAPD-PCR and primers REP1DT and REP2DT in the rep-PCR methods. A widespread distribution of the electrophoretic profiles was obtained either for B. cereus or for B. thuringiensis using TTGE. rep-PCR and RAPD-PCR were not always able to group strains from the same origin or belonging to the same species. The fingerprints obtained with the rep- and RAPD-PCR methods confirm the high intraspecific variability present in B. cereus and B. thuringiensis indicating the difficulty to discriminate between these two species in outbreak cases.

Utilization of Denaturing Gradient Gel Electrophoresis (DGGE) to evaluate the Intestinal Microbiota of Brown Trout Salmo trutta fario

Utilization of Denaturing Gradient Gel Electrophoresis (DGGE) to evaluate the Intestinal Microbiota of Brown Trout Salmo trutta fario The microbial flora present in the intestine of the fish plays an important role, such as to break down ingested foods, or the inhibition of the colonization of the fish intestine by pathogens. Because only a small percentage of microorganisms in seawater and freshwater can be cultured and cultivated in the laboratory, molecular techniques based on 16S rDNA amplification have been developed to detect and identify culturable and nonculturable bacteria. Many studies have combined traditional methods with molecular methods to provide more detailed informations on microbial communities. The aim of this work was to evaluate the microbiota of Salmo trutta fario intestine, using PCR-Denaturing Gradient Gel Electrophoresis (DGGE), to assess the influence of a diet on the population composition.

Nested PCR for the detection of Candidatus arthromitus in fish.

Rainbow trout gastroenteritis has been related to the accumulation of segmented filamentous bacteria in the digestive tract of fish, which presents lethargy, reduced appetite and accumulation of mucoid faeces. Some authors associate the comparison of illness with the presence of viable filaments, which produce and release strings of endospores in the lumen of the gut. The segmented filamentous bacteria that could not be cultured in vitro have been related to Clostridium group I, and they have been named Candidatus arthromitus. Despite the various strategies that have been used to detect unculturable microorganisms, molecular methods have facilitated studies on culture-independent microorganisms. Direct DNA extraction from samples and subsequent study of 16S rRNA genes represent a tool for studying unculturable microbial flora. As direct detection of specific microorganisms is possible through the utilization of primers or probes annealing specific DNA sequences, the aim of this work was to design specific primers for the direct detection of C. arthromitus in fish using a nested PCR.

Molecular Methods to Detect Bacillus cereus and Bacillus thuringiensis in Foods

Numerous types of foods have been associated with food poisoning and a lot of cases have been linked to heat treated foods. Bacillus cereus group is involved in many outbreaks due to the consumption of cooked food. B. cereus is present in most raw foods and spores have been found also in packaging materials representing a contamination source for treated foods. Storage temperature is a critical point for processed foods in relation with spore germination. While vegetative cells die during heat treatment, spores can survive and germinate under not limiting conditions. B. cereus food poisoning is principally associated with temperature abuse during the storage of cooked foods. B. cereus may cause illness through the production of a high variety of toxins and enzymes, including a necrotizing enterotoxin, an emetic toxin, phospholipases, proteases and haemolysin inducing also non-gastro-intestinal (e.g. systemic or pulmonary) infections. Enterotoxins could also be produced from B. circulans, B. lentus and B. mycoides, B. thuringiensis and B. anthracis closely related to B. cereus. Many cases are confused with those caused by other pathogens. An accurate evaluation of potentially contaminated foods lead to the utilization of fast and sensitive methods in food monitoring to satisfy safety requirements. Classical methods which use enrichment methods due to the usual small number of B. cereus present in food, are time consuming. Moreover, recent data showed proteases and chitinases contribute to virulence leading to the need for suitable means of differentiating members of the B. cereus group during the monitoring of potentially contaminated foods. Some authors experienced difficulties distinguishing strains considered to be members of the “B. cereus group” such as B. mycoides, B. thuringiensis and B. anthracis either using classical or molecular methods. Identification by classical microbiological methods fails due to horizontal gene transfer from B. cereus group members. Characters responsible for some phenotypic characteristics can be lost from one species and can be acquired from another one causing confusion in taxonomy based upon phenotypic characters evaluation. Different molecular approaches were able to differentiate B. anthracis from B. cereus but failed to differentiate. B. cereus from B. thuringiensis. Even with recent improvements in the molecular methods used for microbial phylogeny, B. cereus, B. mycoides and B. thuringiensis were referred to as B. cereus when 16S rRNA sequences were used for the differentiation. Genetic similarity between B. cereus, B. thuringiensis and B. anthracis has been investigated by means of DNA-DNA reassociation, multilocus enzyme electrophoresis (MEE) which compare the allozyme patterns of 10–20 genes, Pulsed Field Gel Electrophoresis (PFGE), Randomly Amplified Polymorphic DNA (RAPD)-PCR, repetitive extragenic palindromic (rep)-PCR, Real Time PCR, Microarrays. Genotypic approaches tend to be less dependent on bacterial growth variables, less time consuming and useful for determining phylogenetic reltionships between microbial isolates and for assigning strains in specific groups. Molecular methods using PCR are useful in specific detection of B. cereus from plate isolates and for PCR application on microbial DNA extracted from food. Species identification can be achieved by specific couple of primers annealing gyrB gene sequence. To decrease the detection limit of B. cereus in food the type of treatment applied before DNA extraction is fundamental, as it allows the increase of the amplification reaction efficiency. Detection limit for artificially contaminated foods was 50 cells g−1 for boiled rice, 5 × 102 cells g−1 for minced meat, 30 cells g−1 for salad, 20 cells mL−1 for pasteurized milk and <10 cells mL−1 for concentrated coffee. RE technique applied onto amplification products allowed differentiation between B. cereus and B. thuringiensis for the 81 sample tested using Hinf I endonuclease. RAPD-PCR and rep-PCR used for microbial typing gave different results. Cluster analyes for rep-PCR was more effective in discriminating between B. cereus and B. thuringiensis strains than RAPD-PCR. Strains were grouped in 14 clusters, from A to P using 70% similarity by RAPD-PCR, and in ten clusters from A to L by rep-PCR using 80% similarity in fingerprinting analysis. PCR-TTGE and PCR-DGGE are methods used for single point mutations detection among DNA sequences. Amplicons obtained for B. cereus and B. thuringiensis strains were analyzed by this molecular technique to differentiate strains among the two species identified previously by the PCR-RE method. B. cereus strains were divided in seven levels whereas B. thuringiensis strains in two levels. The distribution of PCR products in each gel indicate high variability either within B. thuringiensis or B. cereus. No clear differentiation was obtained by TTGE between the two species considered. Horizontal gene transfer between strains of these different “species” makes it impossible to delineate discrete levels. A desirable goal is to differentiate the organisms detected in food products and patients to understand the real contribution of B. cereus and B. thuringiensis to human infections, either due to food consumption or to hospitalization, using fast and sensitive protocols.

Application of PCR-DGGE for the identification of lactic acid bacteria in acitve dry wine yeasts

In this work a Polymerase Chain Reaction (PCR)-Denaturing Gradient Gel Electrophoresis (DGGE) protocol was used to identify the Lactic Acid Bacteria (LAB) contaminants in enological active dry yeasts routinely used in the wine production. The method is based on the PCR amplification of a DNA fragment from the region V1 of 16S rDNA gene followed by a DGGE technique. The main contaminant wasLactobacillus spp. andPediococcus spp.

EFFECT OF ESSENTIAL OIL ON BIOFILM PRODUCTION BY DIFFERENT LISTERIA MONOCYTOGENES STRAINS

The effects of different essential oil (hexanal, 2-(E)-hexenal, carvacrol, citron, red orange, thymol and limonene) on biofilm production of some Lmonocytogenes strains are evaluated. The formation of biofilm on certain surfaces or on the food, seems to be related with cross-contamination during processing or with the contamination of the final product, with potential risk for the consumer. Many studies were done on the antimicrobial activity of essential oils and their components, but not too much is known about their capacity to influence and reduce the microbial production of biofilm. Our data showed that essential oils can inhibit or limit the biofilm production.