Quality of silages from Italian farms as attested by number and identity of microbial indicators.

AIMS: This study evaluated the quality and possible hygiene risks related to farm-made silages by analysing the presence and number of micro-organisms that influence the preservation and safety in samples from four Italian regions. METHODS AND RESULTS: Lactic acid bacteria, clostridia, lactate-fermenting yeasts and propionibacteria (PAB) were isolated and identified by random amplified polymorphic DNA PCR, sequencing of the V2-V3 16S rRNA gene region, 5.8S-ITS rDNA RFLP and species-specific PCR. The Lactobacillus plantarum cluster was the most numerous and comprised strains mostly isolated from alfalfa silage. The Lactobacillus buchneri cluster, second in number, comprised isolates from both alfalfa and maize silage. Anaerobic spore formers were assigned to the species Clostridium baratii, Clostridium beijerinkii, Clostridium butyricum, Clostridium perfringens, Clostridium saccharolyticum, Clostridium tyrobutyricum and Paenibacillus macerans. Yeast isolates were identified as Candida apicola, Candida mesenterica and Pichia fermentans. PAB strains, detected only in unifeed, were all identified as Propionibacterium acidipropionici. CONCLUSIONS: The occurrence of spoiling micro-organisms was frequent and the possibility of contamination by potentially pathogenic clostridia was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest the need for improved ensiling practices and appropriate control measures to safeguard the hygienic and nutritional quality of silages produced in farms.

Detection of Staphylococcus aureus and enterotoxin genotype diversity in Monte Veronese, a Protected Designation of Origin Italian cheese.

AIMS: To evaluate the risk associated with the load and enterotoxigenicity of Staphylococcus aureus in Monte Veronese, a PDO (Protected Designation of Origin) cheese of the Lessinia area (Verona, Italy). METHODS AND RESULTS: Staphylococcus aureus was quantified by a conventional culture method and by a nucA targeted real-time PCR assay developed in this study. Staphylococcus aureus numbers in cheese were higher than the limit tolerated by the Italian food legislation in 78% instances, according to both detection methods. Multiplex PCR tests for 17 Staph. aureus enterotoxin (SE) genes were applied to nucleic acids extracted from curds, cheeses and Staph. aureus isolates. The SE gene diversity appeared reduced after ripening. The gene encoding SED was found most frequently in dairy samples and the enterotoxin genes ser, sed, seg and sem predominated in the isolates. CONCLUSIONS: The occurrence of enterotoxigenic Staph. aureus strains with complex SE genotypes in this PDO cheese at numbers often exceeding the Italian tolerance threshold represents an important risk factor. SIGNIFICANCE AND IMPACT OF THE STUDY: The high frequency of contamination of Monte Veronese PDO cheese and, expectedly, similar typical productions from raw milk, by enterotoxigenic Staph. aureus imposes a tighter hygienic control in the earlier manufacturing phases.

Reclassification of Lactobacillus thermotolerans Niamsup et al. 2003 as a later synonym of Lactobacillus ingluviei Baele et al. 2003.

The relatedness of the species Lactobacillus ingluviei and Lactobacillus thermotolerans was investigated by comparing partial sequences of the 16S rRNA gene (99.9 % similarity over 1504 bp), the hsp60 gene (98.8 % similarity over 954 bp) and the recA gene (98.5 % similarity over 452 bp) and by determining DNA-DNA binding levels (79+/-3 %) and genomic DNA G+C contents (50 and 49 mol%, respectively). These data, in addition to their similar biochemical characteristics, suggest that the two taxa constitute a single species. According to Rules 38 and 42 of the Bacteriological Code, they should be united under the name Lactobacillus ingluviei, with the name Lactobacillus thermotolerans as a later heterotypic synonym.

Characterization of Saccharomyces cerevisiae strains isolated from must of grape grown in experimental vineyard.

AIMS: Isolation and characterization of indigenous Saccharomyces cerevisiae strains from 12 grape varieties grown in an experimental vineyard of Apulia. METHODS AND RESULTS: Thirty to 40 colonies from each of the 12 fermentations were obtained at the end stage of spontaneous fermentation. By using morphological and physiological methods and by the PCR analysis of internal transcribed ITS1-5,8S-ITS2, the isolates belonging to Saccharomyces genus were identified. These isolates were further characterized by amplification with S. cerevisiae species- and delta element-specific primers, thus allowing the identification of S. cerevisiae strains selected from each of the 12 fermentations. By means of RFLP analysis of mtDNA, each S. cerevisiae population isolated from a single fermentation appeared to constitute a genetically homogenous group. The comparison of the 12 cultivar-specific mtDNA RFLP patterns, allowed classifying the 12 S. cerevisiae populations into three genetically homogenous groups. The isolated strains fermented vigorously in synthetic and grape juice medium and showed high alcohol and sulphur dioxide (SO(2)) resistance and low hydrogen sulphite (H(2)S) production. CONCLUSIONS: The molecular analysis, in conjunction with the traditional morphological and physiological methods, was useful in discriminating at strain level the indigenous population of S. cerevisiae present in a vineyard of Apulia. The dominant S. cerevisiae strains identified in the 12 fermented musts showed potentially important oenological characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of natural S. cerevisiae strains from several typical Italian grapes grown in a restricted experimental vineyard is an important step towards the preservation and exploitation of yeast biodiversity of Apulia, a relevant wine-producing region. The close relationship between the S. cerevisiae strains from different grapes grown in the same vineyard indicated that the occurrence of native strains is representative of the area rather than of the variety of grapes.

Typical metabolic traits of two Oenococcus oeni strains isolated from Valpolicella wines.

AIMS: Physiological comparison of two indigenous Oenococcus oeni strains, U1 and F3 isolated in the same area (Valpolicella, Italy) in order to select a performant starter for MLF in wine. METHODS AND RESULTS: Growth rate, sugar and malate metabolism in FT80 media at pH 5.3 and 3.5 were analysed. The amount of total protein synthesized and the level of expression of the small Hsp Lo18 were evaluated by radiolabelling and immunodetection experiments after heat (42 degrees C), acid (pH 3.5) and ethanol (12% v/v) stresses. Strain U1 showed significantly lower specific growth rate and growth yield in acid conditions than strain F3. However, strain U1 had a higher malate consumption capacity at pH 3.5 than strain F3, in relation with an higher malolactic activity determined on whole cells. Strain U1 exhibited about half the total protein synthesis level than strain F3, but both strains expressed Lo18 similarly. Evaluation of malolactic fermentation (MLF) performance by microvinification trials was carried out. Strain U1 was able to complete MLF, whereas strain F3 degraded malic acid partially when inoculated in Amarone wine. CONCLUSIONS: Considering its performances in microvinifications experiments, strain U1 could be a good candidate for malolactic starter as an alternative to deficient commercial starters.

Rapid identification and differentiation of Saccharomyces cerevisiae, Saccharomyces bayanus and their hybrids by multiplex PCR.

AIMS: To develop a multiplex PCR assay for the specific identification and differentiation of Saccharomyces cerevisiae, S. bayanus and their hybrids. METHODS AND RESULTS: Two sets of primers with sequences complementary to the region YBR033w were used. A single amplicon of 1710 bp or 329 bp was obtained with species S. cerevisiae and S. bayanus, respectively, while the presence of both bands was observed in S. pastorianus because of its hybrid nature. Both amplification products were also obtained after amplification from DNA of several laboratory S. cerevisiae x S. bayanus hybrid strains. CONCLUSIONS: Multiplex PCR was optimized for the rapid and reliable identification of S. cerevisiae, S. bayanus and their hybrids. SIGNIFICANCE AND IMPACT OF THE STUDY: The procedure may be used for routine detection of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes, overcoming the problems of conventional techniques.

Characterization of sourdough lactic acid bacteria based on genotypic and cell-wall protein analyses.

AIMS: To evaluate the effectiveness of two independent methods in differentiating a large population of lactic acid bacteria (LAB) isolated from wheat flours and sourdoughs and to correlate eventual differences/similarities among strains with their geographical origin and/or process parameters. METHODS AND RESULTS: One hundred fifty strains belonging to Lactobacillus spp. and Weissella spp., plus eight type strains, one for each species, and two unidentified isolates, were characterized by randomly amplified polymorphic DNA (RAPD) and SDS-PAGE of cell-wall proteins. The RAPD analysis separated the eight type strains but did not always assign all the strains of a species to the same group, while SDS-PAGE cell-wall protein profiles were species-specific. Frequently, strains isolated from sourdoughs of the same geographical origin or produced by similar raw material/process parameters showed similar RAPD and/or cell-wall profiles. CONCLUSIONS: The combined use of the RAPD and cell-wall protein analysis represents a useful tool to classify large adventitious microbial populations and to discriminate the diversity of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a typing of a large collection of flour/sourdough LAB and provides evidence of the advantage of using two independent methods in the classification and traceability of microorganisms.

Rapid identification of Enterococcus durans and Enterococcus hirae by PCR with primers targeted to the ddl genes.

Species-specific PCR assays with primers targeted to D-alanine:D-alanine ligase (ddl) encoding genes were developed for the identification of Enterococcus durans and E. hirae. The specificity of the primers was validated in a multiplex PCR on well characterised E. durans (n=30) and E. hirae (n=16) strains, all of which were identified correctly. This PCR procedure offers a reliable and rapid alternative to conventional phenotypic methods for speciation of these enterococci of growing clinical importance.

Phenotypic and genetic diversity of enterococci isolated from Italian cheeses.

In the present study, 124 enterococcal strains, isolated from traditional Italian cow, goat and buffalo cheeses, were characterized using phenotypic features and randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). The RAPD-PCR profiles obtained with four primers and five different amplification conditions were compared by numerical analysis and allowed an inter- and intraspecific differentiation of the isolates. Whole cell protein analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used as a reference method for species identification. The strains were identified as Enterococcus faecalis (82 strains), E. faecium (27 strains), E. durans (nine strains), E. gallinarum (four strains) and E. hirae (two strains). Species recognition by means of RAPD-PCR was in agreement with the SDS-PAGE results except for eight strains of E. faecium that clustered in separated groups. On the other hand, phenotypic identification based on carbohydrate fermentation profiles, using the rapid ID 32 STREP galleries, gave different results from SDS-PAGE in 12.1% of the cases. The majority of the strains had weak acidifying and proteolytic activities in milk. One E. faecium strain showed vanA (vancomycin resistance) genotype while four strains showed a beta-haemolytic reaction on human blood. Several strains showed antagonistic activity towards indicator strains of Listeria innocua, Clostridium tyrobutyricam and Propionibacterium freudenreichii subsp. shermanii.

Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers.

In this study, we succeeded in differentiating Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum by means of recA gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the neighbor-joining model, were coherent and clearly separated the three species. The validity of the recA gene and RecA protein as phylogenetic markers is discussed. Based on the same sequences, species-specific primers were designed, and a multiplex PCR protocol for the simultaneous distinction of these bacteria was optimized. The sizes of the amplicons were 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for L. paraplantarum. This strategy permitted the unambiguous identification of strains belonging to L. plantarum, L. pentosus, and L. paraplantarum in a single reaction, indicating its applicability to the speciation of isolates of the L. plantarum group.

Rapid detection of viable yeasts and bacteria in wine by flow cytometry.

The potential of using flow cytometry (FCM) in combination with fluorescent dyes for rapidly estimating counts of yeasts and malolactic bacteria in laboratory media and wines was examined. In general, there was a good correlation (regression coefficient, 0.94) between viable counts of yeasts determined by FCM and by standard plate assay. The FCM detection limit of yeasts in YPDE medium and in Pinot noir must was 10(3) cells/ml. The lowest bacterial concentration detected by FCM was 10(4) cells/ml. When yeast and malolactic bacteria populations were simultaneously analysed in wine by FCM without any previous sample treatment, difficulties were encountered in the count of bacterial cells due to their size, which is similar to natural debries present in wine. However, after the optimisation of the sample preparation, the technique appeared promising in determining the presence of such microorganisms in wine with one single measurement. Because it is rapid and easy to use, flow cytometry can be considered a useful method for microbiological quality control in wineries and for the investigation of the growth dynamics of microorganisms in wine.

Development of the Specific and Random Amplification (SARA)-PCR for both species identification of enterococci and detection of the vanA gene.

A rapid and reliable polymerase chain reaction (Specific and Random Amplification (SARA)-PCR) has been developed to identify enterococcal species and to detect the vanA gene in one single reaction. This technique was based on the use of the primer set previously designed to amplify a part of the vanA gene (Dutka-Malen et al., J. Clin. Microbiol., 33 (1995) 24-27). In the chosen low stringency conditions complex patterns were obtained, with a sharp band of approximately 700 bp in cases where the vanA gene was present. Discrimination at the species and strain level was achieved by applying the SARA-PCR assay to a collection of 55 enterococcal isolates and type strains. Simultaneously the vanA gene was detected in all strains showing high resistance to vancomycin.

Comparative sequence analysis of a recA gene fragment brings new evidence for a change in the taxonomy of the Lactobacillus casei group.

The taxonomic positions of species of the Lactobacillus casei group have been evaluated by sequencing and phylogenetic analysis of a 277 bp recA gene fragment. High sequence similarity between strain ATCC 393T, currently designated as the type strain of L. casei, and the type strain of Lactobacillus zeae, LMG 17315T, has been established, while L. casei ATCC 334 and Lactobacillus paracasei NCDO 151T form a single phylogenetic group. The taxonomic status of species and strains at issue is discussed.

Vector-free cloning of a bacterial endo-1,4-beta-glucanase in Lactobacillus plantarum and its effect on the acidifying activity in silage: use of recombinant cellulolytic Lactobacillus plantarum as silage inoculant.

In this research, the advantage of use of cellulolytic recombinant Lactobacillus plantarum as microbial inoculants for alfalfa silage fermentation was evaluated. To such purpose, two L. plantarum strains, one (L. plantarum Lp80) currently commercialised and the other (L. plantarum B41) suitable as silage microbial additive, were genetically modified by integration of celA gene, encoding an alkaline endo-1,4-beta-glucanase from Bacillus sp., in the chromosome, by means of a vector-free cloning technique. The heterologous gene was cloned in two fashions: preceded by two promoters (AC1 modification) or in translational coupling with a partial upstream ORF (AC2 modification). Therefore two different genetically modified organisms (GMOs) per each wild-type (WT), producing 43-59 U/l cellulase in 16 h, were examined. Thirty-five micro-ensiling experiments were carried out by inoculating the WT or the derived GMOs. L. plantarum B41AC1 cellulolytic clone exhibited significantly increased acidification capacity in silage samples incubated at 37 degrees C. No advantage of use was evident for the other GMOs.

Differentiation of Lactobacillus plantarum, L. pentosus and L. paraplantarum species by RAPD-PCR and AFLP.

Two high-resolution genotypic techniques (RAPD-PCR and AFLP) were evaluated for their possibility to discriminate the species Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum and to type these taxa at the infra-species level. In total 23 strains of L. plantarum, three strains of L. pentosus, two strains of L. paraplantarum and two related strains for which the species assignment was not clear, were studied. For RAPD-PCR, suitable oligonucleotides and amplification conditions were selected and tested. For AFLP, a double digest of total genomic DNA was used and a subset of restriction fragments was selectively amplified and visualised using different primer combinations. Both methodologies generated, species-specific electrophoretic profiles. Moreover, the presence of distinct subgroups was revealed within the species L. plantarum.

Genomic DNA fingerprinting of Oenococcus oeni strains by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA-PCR.

Genetic diversity of 60 Oenococcus oeni strains from different wines was evaluated by numerical analysis of (i) pulsed-field gel electrophoresis (PFGE) patterns with endonuclease ApaI and (ii) randomly amplified polymorphic DNA (RAPD)-PCR fingerprints with four oligonucleotide primers. Sixty-two percent of the strains could be distinguished by PFGE, whereas most strains were identified by distinct RAPD-PCR profiles and associated according to the geographical origin. Because of its rapidity and reliability, RAPD-PCR appeared to be a suitable method for typing and monitoring O. oeni strains in winemaking.

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

Genus- and species-specific PCR-based detection of dairy propionibacteria in environmental samples by using primers targeted to the genes encoding 16S rRNA.

PCR assays with primers targeted to the genes encoding 16S rRNA were developed for detection of dairy propionibacteria. Propionibacterium thoenii specific oligonucleotide PT3 was selected after partial resequencing. Tests allowed the detection of less than 10 cells per reaction from milk and cheese and 10(2) cells per reaction from forage and soil.

Design and evaluation of malolactic enzyme gene targeted primers for rapid identification and detection of Oenococcus oeni in wine.

Rapid identification and detection of Oenococcus oeni was achieved by species-specific PCR. Two primers flanking a 1025 bp region of the O. oeni gene encoding the malolactic enzyme were designed. The expected DNA amplificate was obtained only when purified DNA from O. oeni was used. The identity of PCR product was confirmed by nested PCR and restriction analysis. Within 8 h, 10(3) cfu ml-1 of oenococci were detected in fermenting grape must containing 10(7) yeast cells, whereas the detection limit in wine was 10(4) cfu ml-1. The rapidity and reliability of the PCR procedure established suggests that the method may be profitably applied in winery laboratories for quality control.

Identification and clustering of dairy propionibacteria by RAPD-PCR and CGE-REA methods.

A total of 67 classical propionic acid bacteria (PAB) strains, 10 of which were from type culture collections and 57 from milk, typical Italian cheeses, acid whey and feed flour of different regions, were analysed by Randomly Amplified Polymorphic DNA (RAPD-PCR) and by Conventional Gel Electrophoresis Restriction Endonuclease Analysis (CGE-REA). The genotypic traits achieved using RAPD-PCR with three primers (OPL-01, OPL-02 and OPL-05) and SmaI CGE-REA patterns were compared by numerical analysis and allowed a clear distinction of four clusters corresponding to the currently described species of classical propionibacteria according to type and reference strains positions. No discrepancies exist in species recognition between the two methods; 36 isolates were identified as Propionibacterium freudenreichii, 15 as P. jensenii, four as P. acidipropionici and two as P. thoenii. Many differences, however, were observed in intraspecific clustering. Numerical comparison of RAPD-PCR profiles appeared to be a suitable method for highlighting the presence of particular phenotypic characters, while intraspecific differentiation obtained by CGE-REA analysis allowed association of strains at high similarity levels on the basis of their geographical origin.