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Understanding the actual distribution of different Legionella species in water networks would help prevent outbreaks. Culture investigations followed by serological agglutination tests, with poly/monovalent antisera, still represent the gold standard for isolation and identification of Legionella strains. However, also MALDI-TOF and mip-gene sequencing are currently used. This study was conducted to genetically correlate strains of Legionella non pneumophila (L-np) isolated during environmental surveillance comparing different molecular techniques. Overall, 346 water samples were collected from the water system of four pavilions located in a hospital of the Apulia Region of Italy. Strains isolated from the samples were then identified by serological tests, MALDI-TOF, and mip-gene sequencing. Overall, 24.9% of water samples were positive for Legionella, among which the majority were Legionella pneumophila (Lpn) 1 (52.3%), followed by Lpn2-15 (20.9%), L-np (17.4%), Lpn1 + Lpn2-15 (7.1%), and L-np + Lpn1 (2.3%). Initially, L-np strains were identified as L. bozemanii by monovalent antiserum, while MALDI-TOF and mip-gene sequencing assigned them to L. anisa. More cold water than hot water samples were contaminated by L. anisa (p < 0.001). PFGE, RAPD, Rep-PCR, and SAU-PCR were performed to correlate L. anisa strains. Eleven out of 14 strains identified in all four pavilions showed 100% of similarity upon PFGE analysis. RAPD, Rep-PCR, and SAU-PCR showed greater discriminative power than PFGE. [ABSTRACT FROM AUTHOR]

In this study, 23 samples of traditional wines produced in Southern Italy were subjected to microbiological analyses with the aim to identify and biotype the predominant species of lactic acid bacilli. For this purpose, a multiple approach, consisting in the application of both phenotypic (API 50CHL test) and biomolecular methods (polymerase chain reaction-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing) was used. The results showed that Lactobacillus plantarum was the predominant species, whereas Lb. brevis was detected in lower amount. In detail, out of 80 isolates 58 were ascribable to Lb. plantarum and 22 to Lb. brevis. Randomly amplified polymorphic DNA-polymerase chain reaction was used to highlight intraspecific variability among Lb. plantarum strains. Interestingly, the cluster analysis evidenced a relationship between different biotypes of Lb. plantarum and their origin, in terms of wine variety. Data acquired in this work show the possibility to obtain several malolactic fermentation starter cultures, composed by different Lb. plantarum biotypes, for their proper use in winemaking processes which are distinctive for each wine. [ABSTRACT FROM AUTHOR]

As reported in the European Community regulation, grappa is a spirit beverage made in Italy from marc that has been steam distilled or distilled after the addition of water. Grape marc from red grapes has already undergone alcoholic fermentation with the must and can be distilled immediately. Grape marc from white grapes does not contain ethanol but contains sugars that are fermented by spontaneous anaerobic fermentation during a storage period. The characteristic aroma of grappa consists of a large number of volatile compounds, which arise from various sources, the most important of which is yeast. Very few studies have been undertaken to characterize the natural populations of yeast during the fermentation of grape marc. The goal of this study was to understand how different pHs, temperatures and yeast starter cultures affect the growth and dynamics of yeast species involved in pomace fermentation, which could be the basis for improving the final quality of grappa production. We found that a temperature of 15°C has the greatest effect on improving the quality of the product. Unfortunately, due to the solid state of the grape marc and the impossibility of its mixing, it appears that acidification and the addition of yeast starter cultures during the silage period are not effective. [ABSTRACT FROM AUTHOR]

The aim of this research was the study of indigenous yeasts isolated from spontaneous fermentation of Inzolia grapes, one of the most widespread native white grapes in Sicily (Italy). The use of selective medium for the isolation and the screening for sulphur dioxide tolerance were useful for the first selection among 640 isolates. The yeasts characterized by high SO tolerance were identified at species level by restriction analysis of ITS region; although the majority of isolates were identified as S. cerevisiae, some non- Saccharomyces yeasts were found. Forty-seven selected yeasts, both S. cerevisiae and non- Saccharomyces yeasts, were characterized for genetic and technological diversity. The genetic polymorphism was evaluated by RAPD-PCR analysis, whereas the technological diversity was analyzed by determining the main secondary compounds in the experimental wines obtained by inoculating these yeasts. Both the molecular and metabolic profiles of selected yeasts were able to clearly discriminate S. cerevisiae from non- Saccharomyces yeasts. This research was useful for the constitution of a collection of selected indigenous yeast strains, including S. cerevisiae and non- Saccharomyces species possessing interesting enological traits. This collection represents a source of wild yeasts, among of which it is possible to select indigenous starters able to maintain the specific organoleptic characteristics of Inzolia wine. [ABSTRACT FROM AUTHOR]

Molecular analyses of a large population of isolates, previously identified as group B or genetic group A4 of the Colletotrichum acutatum species complex, mainly of Italian origin from olive, but also from other hosts collected since 1992, confirmed a well-resolved phylogenetic lineage with distinctive phenotypic characters which can be recognized as a separate species. Based on RAPD genomic fingerprinting, ITS and β-tubulin DNA sequences, this species was clearly distinct from C. acutatum sensu stricto, C. fioriniae and C. simmondsii as well as from the genetic groups A1, A6, A7 and A8, all previously referred to as C. acutatum sensu lato. Group A4 is widespread in Europe, being responsible for olive anthracnose epidemics in some Mediterranean countries, including Greece, Italy, Montenegro, Portugal and Spain; moreover, it causes anthracnose diseases on a wide range of other hosts including about 20 different genera of woody and herbaceous plants, ornamentals and fruit trees. This new anamorphic taxon is described as Colletotrichum clavatum sp. nov. [ABSTRACT FROM AUTHOR]

The aim of this study was to characterize dairy Lactobacillus plantarum-group strains on the basis of their phenotypic and genotypic profiles in order to increase the knowledge on the ecology and biodiversity within this wild Lactobacillus group. In addition, their in vitro potential probiotic properties were evaluated with a view to identifying potential interesting applications. Among the strains, both physiological and biochemical characteristics differed noticeably, indicating biodiverse phenotypic differences. Genotyping experiments using randomly amplified polymorphic DNA (RAPD)-PCR with primer M13 also showed a remarkable heterogeneity among the strains and allowed the strains to be grouped into the species L. plantarum and L. pentosus/L. paraplantarum. With regard to probiotic functional characteristics, the L. plantarum strains 31C and 143C and the L. plantarum strains 64FS and 61FS, isolated from Caciotta and Fiore Sardo cheeses, respectively, survived simulated gastrointestinal conditions and were considered to be acid and bile tolerant. The majority of the strains exhibited antagonistic activity towards Escherichia coli ATCC 43895 and L. sakei DSM 20017, but only one of these strains was found to produce a bacteriocin-like compound. The results of this study suggest the presence of both phenotypic and genotypic variation within the L. plantarum group isolated from the two different Italian cheeses. Further investigation and development as potential probiotic strains is required. [ABSTRACT FROM AUTHOR]

Eleven sourdoughs from Molise region (Southern-Italy) were subjected to microbiological analyses in order to select predominant lactobacilli species to be utilised as starter culture for bread production. A multiple approach was used, consisting of the growth in different culture media, DGGE analysis, 16S rRNA gene sequencing and RAPD-PCR typing. Forty-three lactobacilli were identified and four different species, facultatively or obligately heterofermentative lactobacilli, were found. Lactobacillus plantarum and Lactobacillus brevis represented the prevailing lactobacilli, while Lactobacillus casei and Lactobacillus paracasei ssp. paracasei were detected only in few samples. The use of different media demonstrated that there is no efficient medium for the study of sourdoughs and the cultivation in different substrates remains the best tool to obtain a picture of lactic acid bacteria population. DGGE and 16S rRNA gene sequencing allowed to obtain a reliable identification of strains, while RAPD-PCR resulted a suitable method for typing lactobacilli at strain level. [ABSTRACT FROM AUTHOR]

Abstract: The occurrence of yeast microflora in raw goat''s milk collected from 62 dairy farms located in different areas of Sardinia was evaluated. Candida zeylanoides was the most frequently occurring species followed by different Basidiomycetous species. In the strains isolated some biochemical characteristics of technological interest were investigated and a predominance of lipolytic yeast species was found. We employed a simple method of DNA extraction that in a minimal time and with low-cost provided a high quality of DNA for RAPD analysis of 32 isolates of C. zeylanoides. The primers M13 and CDU were used and at 40% of similarity, two distinct clusters were observed. The presence of C. krissii species was supposed but further molecular studies are needed to exclude the presence of an as-yet-undescribed species. [Copyright &y& Elsevier]

Abstract: The composition of yeast microflora in artisanal “Pecorino di Filiano” cheese, a typical product of the Basilicata region of Southern Italy, was studied during ripening. The isolates were identified by restriction analysis of the 18S rDNA amplified region with the combined use of Hinf I and Cfo I enzymes. The majority of the isolates were identified as Debaryomyces hansenii, whereas two yeasts were identified as Kluyveromyces lactis and one as Dekkera anomala. To evaluate natural biodiversity, D. hansenii “Pecorino di Filiano” isolates were submitted to genetic and technological characterization. RAPD-PCR analysis with P80 (5CGCGTGCCCA3) primer revealed significant polymorphism among D. hansenii isolates. About 30% of the isolates showed single molecular profiles, whereas the other D. hansenii yeasts were separated into three main patterns, differing for both the ripening time and the isolation source. Furthermore, the yeasts showed significant variability in their, “proteolytic activity”. This work demonstrated the high predominance of D. hansenii among the yeast population of “Pecorino di Filiano” cheese, probably in consequence of the traditional salting process, which was selected for this salt tolerant species. This preliminary study allowed us to isolate autochthonous D. hansenii yeasts potentially useful as starters for the production of this artisanal cheese. [Copyright &y& Elsevier]

Due to the natural fragmentation of its habitats, Aphanius fasciatus, a Cyprininodontid living in coastal brackish-water habitats, is distributed discontinuously with considerable isolation of its populations, and characterized by a high degree of osteological and genetic differentiation among them. The genetic relationships in four different Italian populations were studied using the control region (D-loop) mitochondrial DNA sequence and the polymorphisms generated by RAPD-PCR. The results revealed a well-defined genetic structuring, that was also confirmed by the high Gst and Fst values, and a close relationship between the populations of Ganzirri and Lesina, as well as the isolation of the other two Sicilian populations. Moreover, with respect to the mitochondrial DNA analyses, the total genetic variance was higher among populations, but lower in the case of the RAPD polymorphisms. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, 68 enterococcal isolates collected from dairy products in north–west Italy were characterised by randomly amplified polymorphic DNA-PCR (RAPD-PCR) and by their technologically relevant biochemical properties. After the comparison of RAPD-PCR profiles with the profiles of the enterococcal type strains, isolates were assigned to the species Enterococcus faecalis (35 isolates), E. faecium (27 isolates) and E. durans (6 isolates). Phenotypic identification based on carbohydrate fermentation profile galleries gave different results from RAPD-PCR in 17.6% of the cases (10 E. faecium and 2 E. durans isolates). With respect to biochemical activities, the majority of the strains displayed weak acidification activity in milk. High values for redox activity were detected for 80% of E. faecalis isolates, while E. faecium provided the most lipolytic species. Proteolytic activity was generally low, except for 10 E. faecalis isolates that displayed values tyrosine of milk. None of the isolates carried vanA or vanB (vancomycin resistance) genetic determinants. [Copyright &y& Elsevier]

Abstract: In the Friuli Venezia Giulia region, in the North East of Italy, a traditional fermented sausage is produced without the use of microbial starters. It is characterized at the end of the ripening period by accentuated acidity, slight sourness and elastic, semi-hard consistency. In this study, three fermentations, carried out in different seasons (winter, spring and summer) were followed analyzing the microbiological, physicochemical and sensory aspects of this product. The sausages were characterized by an important microbial activity of lactic acid bacteria and micro/staphylococci that resulted in a product with a final pH of about 5.6–5.7. An interesting aspect was the high number of fecal enterococci that can play an important role in the definition of the organoleptic profile of the final product. No Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus were ever isolated from the raw materials or the fermented sausages during the maturation, underlining the safety of this product. The final water activity of the product was 0.91–0.92. One hundred and fifty lactic acid bacteria were isolated and identified by molecular methods to understand which species were more predominant in the product. Lactobacillus curvatus and Lactobacillus sakei were the most numerous (54 and 64 strains isolated, respectively) and they were the only species common to all three fermentations. A cluster analysis of the profiles obtained from these strains after RAPD-PCR highlighted a population distribution that was fermentation-specific. [Copyright &y& Elsevier]