Claudia Picozzi

Genetic diversity in Italian Lactobacillus sanfranciscensis strains assessed by multilocus sequence typing and pulsed-field gel electrophoresis analyses.

Lactobacillus sanfranciscensis is a lactic acid bacterium that characterizes the sourdough environment. The genetic differences of 24 strains isolated in different years from sourdoughs, mostly collected in Italy, were examined and compared by PFGE and multilocus sequence typing (MLST). The MLST scheme, based on the analysis of six housekeeping genes (gdh, gyrA, mapA, nox, pgmA and pta) was developed for this study. PFGE with the restriction enzyme ApaI proved to have higher discriminatory power, since it revealed 22 different pulsotypes, while 19 sequence types were recognized through MLST analysis. Notably, restriction profiles generated from three isolates collected from the same firm but in three consecutive years clustered in a single pulsotype and showed the same sequence type, emphasizing the fact that the main factors affecting the dominance of a strain are correlated with processing conditions and the manufacturing environment rather than the geographical area. All results indicated a limited recombination among genes and the presence of a clonal population in L. sanfranciscensis. The MLST scheme proposed in this work can be considered a useful tool for characterization of isolates and for in-depth examination of the strain diversity and evolution of this species.

Comparison of surface sampling methods and cleanability assessment of stainless steel surfaces subjected or not to shot peening

Abstract The cleanability of AISI 304 stainless steel surfaces, indicated by the removal of Escherichia coli cells or Aspergillus niger spores was assessed by controlled inoculation and washing treatment of samples in standardised conditions. Two systems of recapture (Rodac plate technique and swabbing technique) were compared. Four industrial finishes, subjected or not to shot peening, contaminated at low concentration (1–10 cfu/cm 2 ), and then washed with distilled water or alkaline detergent, were examined. The Rodac plate technique detected most of microorganisms inoculated (80% for E. coli cells and 67% for A. niger spores), whereas the swabbing technique recovered only 1% of the E. coli cells and 26% of the A. niger spores. Using the Rodac plate technique E. coli cells proved to be easily detachable from samples either with distilled water (98%) or alkaline detergent (>99%). For the surfaces contaminated with A. niger spores, the cleanability increased from 34% with distilled water to 77% with alkaline detergent. In these working conditions type of finish (shot treated or not) had no significant effect on cleanability of stainless steel.

Assessment of the Brettanomyces bruxellensis metabolome during sulphur dioxide exposure

Brettanomyces bruxellensis displays a high degree of genotypic and phenotypic polymorphism and is the main yeast species involved in wine spoilage. The innate resistance of 108 B. bruxellensis strains to the antimicrobial agent SO2 used in winemaking was investigated. Nineteen strains (17.6%) were sensitive to SO2 , failing to grow at the lowest concentration tested (0.1 mg L(-1) molecular SO2). Twenty-nine strains (26.8%) grew at 0.1 mg L(-1), 42 strains (38.9%) grew at 0.2 mg L(-1) , and 16 strains (14.8%) were able to grow as high as 0.4 mg L(-1) mol. SO2. Two strains able to grow in the presence of 0.6 mg L(-1) mol. SO2 were further studied by GCMS-TOF analysis to define the metabolic response to SO2 treatment. Two hundred and fifty-three intracellular metabolites were detected. The main effect observed was a decrease in cytoplasmic levels of polyols and an increase in levels of some amino acids, alanine, glutamic acid, glycine, proline, 5-oxoproline, serine and valine, which were significantly accumulated in the presence of SO2. No alteration in the pentose phosphate pathway was observed, suggesting NADPH usage could be diverted to other pathways. Finally, a change in metabolites involved in the glycerophospholipid pathway (glycerol-3-phosphate and myo-inositol) was also found.

The role of teat skin contamination in the epidemiology of Staphylococcus aureus intramammary infections

Knowledge of the epidemiological pattern and the potential sources of infections is important to control Staphylococcus aureus in dairy herds. This paper reports the results of a study applying both pulse field gel electrophoresis (PGFE) and the assessment of a selected number of virulence genes to investigate the role of teat skin on Staph. aureus transmission among cows and on the contamination of milk. Overall 61 isolates were considered, 23 from teat skin, 33 from milk samples and 5 from curd samples. Teat swabs were taken in five herds, but in only three of them could Staph. aureus be isolated. Curd was sampled in three herds, but Staph. aureus could be isolated in only two herds. The distribution of isolates among herds confirmed the presence of herd-specific Staph. aureus strain in most of the herds. The same pattern was observed in teat skin samples, in quarter milk samples, and in the curd samples. Our findings are consistent with other studies showing the role of teat skin as a potential reservoir. Moreover, Staph. aureus was isolated from teat skin of confirmed Staph. aureus-negative cows that were segregated from infected ones. Our findings also suggest that some strains have higher chances to survive on teat skin and therefore to increase the risk for contamination of milk and milk products due to the persistence of intramammary infections.

Survey on indigenous Oenococcus oeni strains isolated from red wines of Valtellina, a cold climate wine-growing Italian area.

Spontaneous MLF in high acidity wines produced in cool-climate regions remains problematic though indispensable for the development of sensory characteristics. Genetic aspects and phenotypic traits of thirty-six Oenococcus oeni strains, most of them isolated from Valtellina wines over three consecutive years, were investigated. Molecular typing achieved by RAPD PCR and PFGE analyses allowed 27 different genotypes to be discriminated, whereas from the comparison of results arising by physiological tests (sugar fermentation, alcohol resistance, growth at low temperatures, biogenic ammines production) 28 different phenotypic profiles were obtained. Particularly, 69% of Valtellina isolates were able to develop at 5 degrees C in cultural broth. Micro-vinification experiments allowed the selection of strains with potential oenological performances and an interesting capability to grow in cold conditions was confirmed. Some O. oeni strains formed phenylethylamine (up to 47 mg/L) and tyramine (up to 36 mg/L) both in cultural broth and wine.

Heat inactivation of wine spoilage yeast Dekkera bruxellensis by hot water treatment

Cell suspensions of four Dekkera bruxellensis strains (CBS 2499, CBS 2797, CBS 4459 and CBS 4601) were subjected to heat treatment in deionized water at four different temperatures (55·0, 57·5, 60·0 and 62·5°C) to investigate their thermal resistance. The decimal reduction times at a specific temperature were calculated from the resulting inactivation curves: the D‐values at 55·0°C ranged from 63 to 79·4 s, at 57·5°C from 39·6 to 46·1 s, at 60·0°C from 19·5 to 20·7 s, at 62·5°C from 10·2 to 13·7 s. The z‐values were between 9·2 and 10·2°C, confirming that heat resistance is a strain‐dependent character. A protocol for the sanitization of 225 l casks by immersion in hot water was set up and applied to contaminated 3‐year‐old barrels. The heat penetration through the staves was evaluated for each investigated temperature by positioning a thermal probe at 8 mm deep. A treatment at 60°C for an exposure time of 19 min allowed to eliminate the yeast populations up to a log count reduction of 8.

Polymorphisms of Saccharomyces cerevisiae Genes Involved in Wine Production

The setting up of new molecular methods for Saccharomyces cerevisiae typing is valuable in enology. Actually, the ability to discriminate different strains in wine making can have a benefit both for the control of the fermentation process and for the preservation of wine typicity. This study focused on the screening of single-nucleotide polymorphisms in genes involved in wine production that could evolve rapidly considering the selective pressure of the isolation environment. Preliminary screening of 30 genes in silico was performed, followed by the selection of 10 loci belonging to 8 genes. The sequence analysis showed a low polymorphism and a degree of heterozygosity. However, a new potential molecular target was recognized in the TPS1 gene coding for the trehalose-6-phosphate synthase enzyme involved in the ethanol resistance mechanism. This gene showed a 1.42% sequence diversity with seven different nucleotide substitutions. Moreover, classic techniques were applied to a collection of 50 S. cerevisiae isolates, mostly with enologic origin. Our results confirmed that the wine making was not carried out only by the inoculated commercial starter because indigenous strains of S. cerevisiae present during fermentation were detected. In addition, a high genetic relationship among some commercial cultures was found, highlighting imprecision or fraudulent practices by starter manufacturers.

Assessment of transduction of Escherichia coli Stx2-encoding phage in dairy process conditions.

In the environment, bacteriophages are regarded as natural vector for the transmission of Shiga-toxin genes among Shiga-toxin Escherichia coli strains. The possibility of transduction has been noticed in intestinal tract of various animals but experimental observations on this phenomenon in food processes are lacking. To investigate the transduction in milk at different temperature profiles and cell concentrations, an experimental plan including two different Stx(2)-phages (ϕGV2412 and ϕL34), induced respectively from E. coli O157:H7 181181/2 and E. coli O157:H7 EC34, and two recipient E. coli strains (CNCTC 6896, WG5) was performed. The donor strains were generated by lysogenization of CNCTC 6896 with ϕGV2412 and ϕL34 respectively. Spectinomycin resistance gene (aadA) was inserted into stx(2) operon in order to select transduced cells. Transductants were never observed at 4°C up to 24 h, whereas after a treatment at 37°C for 2 h and at 25°C for 22 h they were detected in 67% of the trials with a ratio of transduction varying from 1.13 10(-6) to 7.87 10(-8). A treatment at 48°C for 2 h followed by a second step at 25°C for 22 h showed an occurrence of transduction events in only 19% of cases with a ratio of transduction varying from 2.22 10(-7) to 2.67 10(-8). The generation of transductants and the spontaneous induction of phages in milk were not affected by initial or final concentration of the donor or recipient strains. The results show that transduction phenomenon occurs when the cells are metabolically active and it does not take place at low temperatures. Therefore, the maintenance of the chilling chain proved to be a main factor to prevent the spread of Stx-genes in dairy processes.

Use of the tna Operon as a New Molecular Target for Escherichia coli Detection

ABSTRACT A quantitative real-time PCR targeting the tnaA gene was studied to detect Escherichia coli and distinguish E. coli from Shigella spp. These microorganisms revealed high similarity in the molecular organization of the tna operon.

Development of a Type I gluten‐free sourdough

The aim of this study was the setting up of a gluten‐free sourdough from selected lactobacilli and yeasts isolated from a traditional wheat‐based Type I sourdough. A gluten‐free matrix was inoculated with Lactobacillus sanfranciscensis and Candida humilis, fermented to pH 4·0, and constantly propagated for ten times. A stable association between micro‐organisms was observed from the second refreshment with mean values of 9·08 ± 0·25 log CFU g−1 for lactobacilli and 7·81 ± 0·07 log CFU g−1 for yeasts. In order to have a good workability of the dough, a 230 BU consistency was considered. Rheofermentographic indices remained constant over the ten refreshments, showing an average value of 23·2 mm dough height in about 7·5 h. The CO2 production and retention volumes reached average values of 1430 and 1238 ml respectively. The microbiological and technological data obtained highlighted that a GF sourdough was effectively developed.