Claudia Picozzi

Publications (18) View all

• Article: Intron splice site PCR analysis as a tool to discriminate Dekkera bruxellensis strains

  Ileana Vigentini, Claudia Picozzi, Roberto Foschino
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  ABSTRACT: Dekkera bruxellensis yeast species can develop off-flavours in wine through a specific reductive metabolism. In particular, volatile phenols are often produced in amounts that are higher than the perception threshold with a loss in product quality. Recent observations suggest that “brett spoilage” is strictly strain-dependent, and therefore, a rapid and reliable identification at strain level of D. bruxellensis becomes strategic for an efficient prevention. Among the techniques used to analyse DNA regions with high rate of sequence evolution, intron splice site PCR amplification (ISS-PCR) has allowed the detection of polymorphisms in commercial strains of S. cerevisiae. Recently, the genome of a D. bruxellensis isolated from wine has been sequenced and the results have shown that about 2% of the genes, a value similar to the ones found in other hemiascomycetes (1% in D. hansenii, 4% in S. cerevisiae) contain introns. Moreover, D. bruxellensis introns have 5′, 3′ and branch motifs that are very similar to the consensus motif in S. cerevisiae. Although the use of the 5′ intron–exon splice site as a target for ISS-PCR in D. bruxellensis did not allow the discrimination at strain level, an optimisation of primers could permit the development of a consistent tool for the typing of the species. In the present study, 17 D. bruxellensis strains belonging to the international CBS collection have been investigated for the ISSs, employing specific oligonucleotides containing different 5’ consensus sequences: GTATGT (S. cerevisiae) and GTAAGT (D. bruxellensis). Results have shown that almost the whole yeast collection was discriminated at strain level using different combinations of primers. Therefore, to simplify the approach, a multiplex PCR protocol able to generate stable genetic profiles was developed. Keywords Dekkera bruxellensis –Yeast typing–Introns
  Annals of Microbiology 04/2012; 61(1):153-157. · 0.69 Impact Factor
• Article: Intraspecific variations of Dekkera/Brettanomyces bruxellensis genome studied by capillary electrophoresis separation of the intron splice site profiles.

  Ileana Vigentini, Gabriella De Lorenzis, Claudia Picozzi, Serena Imazio, Annamaria Merico, Silvia Galafassi, Jure Piškur, Roberto Foschino
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  ABSTRACT: In enology, "Brett" character refers to the wine spoilage caused by the yeast Dekkera/Brettanomyces bruxellensis and its production of volatile phenolic off-flavours. However, the spoilage potential of this yeast is strain-dependent. Therefore, a rapid and reliable recognition at the strain level is a key point to avoid serious economic losses. The present work provides an operative tool to assess the genetic intraspecific variation in this species through the use of introns as molecular targets. Firstly, the available partial D./B. bruxellensis genome sequence was investigated in order to build primers annealing to introns 5' splice site sequence (ISS). This analysis allowed the detection of a non-random vocabulary flanking the site and, exploiting this feature, the creation of specific probes for strain discrimination. Secondly, the separation of the intron splice site PCR fragments was obtained throughout the set up of a capillary electrophoresis protocol, giving a 94% repeatability threshold in our experimental conditions. The comparison of results obtained with ISS-PCR/CE versus the ones performed by mtDNA RFLP revealed that the former protocol is more discriminating and allowed a reliable identification at strain level. Actually sixty D./B. bruxellensis isolates were recognised as unique strains, showing a level of similarity below 79% and confirming the high genetic polymorphism existing within the species. Two main clusters were grouped at similarity levels of about 46% and 47%, respectively, showing a poor correlation with the geographic area of isolation. Moreover, from the evolutionary point of view, the proposed technique could determine the frequency of the genome rearrangements that can occur in D./B. bruxellesis populations.
  International journal of food microbiology 03/2012; 157(1):6-15. · 3.01 Impact Factor
• Article: Assessment of transduction of Escherichia coli Stx2-encoding phage in dairy process conditions.

  Claudia Picozzi, Giorgio Volponi, Ileana Vigentini, Silvia Grassi, Roberto Foschino
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  ABSTRACT: 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.
  International journal of food microbiology 12/2011; 153(3):388-94. · 3.01 Impact Factor
• Article: Genetic diversity in Italian Lactobacillus sanfranciscensis strains assessed by multilocus sequence typing and pulsed-field gel electrophoresis analyses.

  Claudia Picozzi, Gaia Bonacina, Ileana Vigentini, Roberto Foschino
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  ABSTRACT: 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.
  Microbiology 04/2010; 156(Pt 7):2035-45. · 3.06 Impact Factor
• Article: Survey on indigenous Oenococcus oeni strains isolated from red wines of Valtellina, a cold climate wine-growing Italian area.

  Ileana Vigentini, Claudia Picozzi, Antonio Tirelli, Anna Giugni, Roberto Foschino
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  ABSTRACT: 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.
  International journal of food microbiology 11/2009; 136(1):123-8. · 3.01 Impact Factor