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Conclusion: Although older people varied widely in tactile sensitivity, our results show that tactile thresholds increased with age. Objectives: The aim of this study was to evaluate the effects of aging on nasal tactile sensitivity. Methods: A total of 160 healthy patients aged between 50 and 90 years were included. According to their age, patients were divided into groups (A, B, C, D, E, F, G, and H). From the age of 50, each group included subjects with an age range of 5 years (i.e. group A, 50-55 years; group B, 56-60 years, etc.). Each patient's outcome was assessed through the nasal monofilament test: a set of 20 Semmes-Weinstein monofilaments was used to detect nasal sensitivity for both nasal cavities. The sensitivity threshold was recorded as the minimum monofilament size from which patients could detect at least two of three stimuli. Results: In groups D (66-70 years), E (71-75 years), F (76-80 years), G (81-85 years), and H (86-90 years) a significantly (p < 0.05) higher stimulus (171.1 ± 0.34 mg vs 67.7 mg, 167.01 ± 0.31 mg 67.7 mg, 166.54 ± 0.28 mg 67.7 mg, 201.24 ± 0.43 mg 67.7 mg, 165,87 ± 0.27 mg 67.7 mg) was required to trigger a touch response in the monofilament test.

Lactobacillus sakei is widely used as starter in the production process of Italian fermented sausages and its growth and survival are affected by various factors. We studied the differential expression of genome in response to different stresses by the fluorescent differential display (FDD) technique. This study resulted in the development and optimization of an innovative technique, with a high level of reproducibility and quality, which allows the identification of gene expression changes associated with different microbial behaviours under different growth conditions.

Oenococcus oeni strains from traditional Italian red wines of the Basilicata region were investigated on the basis of their physiological and molecular response to different temperatures and ethanol concentrations. All strains were highly resistant to different ethanol concentrations and it has been observed that 7% ethanol was able to stimulate the growth of strains in wine, and 12-13% of ethanol allowed their proliferation. Moreover, strain tolerances to 18 and 42 degrees C were observed. Fingerprinting analysis with fluorescent differential display-PCR and investigation of changes in gene expression during the tolerance process were carried out. The expression gene pattern reflects mechanisms involved in tolerance to environmental conditions. This study establishes and validates a method that enables, with a high reproducibility, different gene expression identification under stress conditions in lactic acid bacteria.

In winemaking Oenococcus (O.) oeni is the most frequent species of lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). Several studies have demonstrated that O. oeni is a quite homogeneous species and strains are difficult to differentiate especially when isolates from the same region are analyzed. In this study, the molecular biodiversity of O. oeni isolated from wines of the same region (Aglianico produced in Basilicata Region, Southern Italy) was evaluated with the aim of designing a molecular approach for discrimination and characterization of the isolates at the strain level. Three molecular techniques were applied: random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), restriction endonucleases analysis-pulsed field gel electrophoresis (REA-PFGE) and differential display PCR (DD-PCR). The results obtained by RAPD-PCR confirmed the difficulty in differentiating isolates. By means of REA-PFGE a higher polymorphism, often related to the origin (winery) of strains, was revealed. However, on analyzing strains isolated from the same winery, only in some cases was more than one REA-PFGE pattern obtained. By analyzing dendrograms constructed on the basis of DD-PCR profiles differentiation of strains isolated from the same winery, in some cases, could be accomplished. The reliability of the DD-PCR in the differentiation of closely related strains suggests that this method could represent an alternative and/or additional tool to other molecular methods, such as REA-PFGE, for fine characterization of oenococcal strains.

Conventional multivariate statistical techniques (hierarchical cluster analysis, linear discriminant analysis) and unsupervised (Kohonen Self Organizing Map) and supervised (Bayesian network) artificial neural networks were compared for as tools for the classification and identification of 352 SDS-PAGE patterns of whole cell proteins of lactic acid bacteria belonging to 22 species of the genera Lactobacillus, Leuconostoc, Enterococcus, Lactococcus and Streptococcus including 47 reference strains. Electrophoretic data were pre-treated using the logistic weighting function described by Piraino et al. [Piraino, P., Ricciardi, A., Lanorte, M. T., Malkhazova, I., Parente, E., 2002. A new procedure for data reduction in electrophoretic fingerprints of whole-cell proteins. Biotechnol. Lett. 24, 1477-1482]. Hierarchical cluster analysis provided a satisfactory classification of the patterns but was unable to discriminate some species (Leuconostoc, Lb. sakei/Lb. curvatus, Lb. acidophilus/Lb. helveticus, Lb. plantarum/Lb. paraplantarum, Lc. lactis/Lc. raffinolactis). A 7 × 7 Kohonen self-organizing map (KSOM), trained with the patterns of the reference strains, provided a satisfactory classification of the patterns and was able to discriminate more species than hierarchical cluster analysis. The map was used in predictive mode to identify unknown strains and provided results which in 85.5% of cases matched the classification obtained by hierarchical cluster analysis. Two supervised tools, linear discriminant analysis and a 23:5:2 Bayesian network were proven to be highly effective in the discrimination of SDS-PAGE patterns of Lc. lactis from those of other species. We conclude that data reduction by logistic weighting coupled to traditional multivariate statistical analysis or artificial neural networks provide an effective tool for the classification and identification of lactic acid bacteria on the basis of SDS-PAGE patterns of whole cell proteins. © 2005 Elsevier B.V. All rights reserved.

This work aimed to evaluate the antibiotic susceptibility of 63 Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese, of Lactobacillus GG and of the type strain L. rhamnosus DSM 20021. Antimicrobial susceptibility was determined by the disc diffusion method on 41 antibiotics. Inhibition zone diameter was carefully measured and the results (the mean of four determinations) were expressed in terms of resistance or susceptibility. All the strains isolated from cheese showed resistance to six antibiotics (cefixime, vancomycin, neomycin, enoxacin, pefloxacin and sulphamethoxazole plus trimethoprim). The strain DSM 20021T was resistant to nine antibiotics (the previous six plus cephalexin, bacitracin and lincomycin), while the commercial strain L. GG showed resistance to eighteen antibiotics. A high strain-specific resistance to different antibiotics was ascertained in Lactobacillus rhamnosus isolated from cheese. The results obtained in this study confirm that antibiotic resistance is a very important feature in the selection of potentially probiotic lactic acid bacteria., Sensibilité aux antibiotiques de souches de Lactobacillus rhamnosus isolées du fromage Parmigiano Reggiano. Le but de cette étude était l'évaluation de la sensibilité aux antibiotiques de 63 souches de Lactobacillus rhamnosus isolées du fromage Parmigiano Reggiano, de la souche Lactobacillus GG et de la souche type de L. rhamnosus DSM 20021. La sensibilité ou la résistance à 41 antibiotiques de ces 65 souches de L. rhamnosus a été déterminée avec le test de diffusion de disque en gélose. Les diamètres des zones d'inhibition, mesurés avec précision, ont permis d'individualiser pour chaque antibiotique testé les souches résistantes et celles sensibles. Toutes les souches isolées du fromage se sont avérées résistantes à six antibiotiques (céfixime, vancomycine, néomycine, enoxacine, péfloxacine et triméthoprime + sulfaméthoxazole). La souche type DSM 20021T était résistante à neuf antibiotiques (les six précédents plus cefalexine, bacitracine et lincomycine), tandis que la souche commerciale L. GG a montré une résistance à 18 antibiotiques. Une résistance souche-spécifique à différents antibiotiques a été montrée pour les souches de Lactobacillus rhamnosus isolées du fromage. Les résultats obtenus dans cette étude ont confirmé que la résistance aux antibiotiques est une caractéristique très importante pour sélectionner les bactéries lactiques potentiellement probiotiques.

Countries of the Mediterranean area are characterized by production of artisanal cheeses, obtained from goat, sheep, cow and buffalo raw milk. The numbers and species of yeasts in the different cheeses are variable, but some species are more frequently detected than others. Kluyveromyces marxianus, K. lactis with their anamorph, Candida kefir, Debaryomyces hansenii and C. famata, C. colliculosa and C. catenulata are dominant species in several cheeses. However, Saccharomyces cerevisiae is often detected in pasta filata cheeses, such as Water Buffalo Mozzarella (WBM) or Cacio Cavallo Podolico. Recently, a comprehensive study of yeasts isolated from Mozzarella cheese produced in Basilicata (Southern Italy) has been carried out. The study has focused on lactose and/or galactose fermenting species (Kluyveromyces and Saccharomyces) to evaluate their role on the functional and sensory properties of the product. End products in milk were evaluated and the biodiversity in terms of production of sulphur dioxide, higher alcohols, ethyl acetate, and acetaldehyde was studied. In particular, S. cerevisiae strains from Water Buffalo Mozzarella cheese, compared to strains isolated from different habitats, such as wine, exhibited considerable difference in the production of some volatile compounds. The diversity observed could be related to the particular microhabitat of S. cerevisiae occurring in whey cheese of water buffalo milk.

We have demonstrated that Rhizobium leguminosarum strain LPR1105 contains a heat stable and a heat labile glutamine synthetase (EC 6.3.1.2) activity similar to those described for other Rhizobiaceae. Most of the activity is heat stable when this strain is grown on glutamine as sole nitrogen source, but most is heat labile when grown on nitrate. Using a gene bank of R. leguminosarum DNA we have isolated two clones, which code for heat stable (p7D9) and heat labile (p4F7) glutamine synthetase activity, by complementing the glutamine auxotrophy of Klebsiella pneumoniae glnA mutants. Cross-hybridization of p7D9 with a fragment of the glnA gene of K. pneumoniae was observed, but no cross-hybridization between p7D9 and p4F7 was found. Since these two regions hybridize to genomic DNA of R. leguminosarum they are probably the structural genes for GSI and GSII, and the availability of these genes will make it possible to test this hypothesis. Clone p4F7 complements an ntrC+ but not an ntrC K. pneumoniae glnA mutant, suggesting that the ntrC gene is required for the complementation of the glutamine auxotrophy by this plasmid.

To investigate the physical organization of ribosomal RNA genes of two bobbed ( bb ) loci carried by the Dp(1; f)122 free duplication, a wild type and a deleted one derived from it, genomic DNAs from and adult females were analyzed by restriction enzyme digestions. We found that in the bb mutant there was a loss of uninterrupted genes, while genes interrupted by type I and type II insertions remained apparently unchanged. This is an indication that at least in this wild type bb + locus, carried by the 122 free duplication, the different repeating units are not distributed randomly. In fact, after digestion of the rDNA carried by the bb + duplication with the enzyme Bam HI that cuts only in type I insertions, we have obtained long uncleaved fragments of DNA containing uninterrupted genes.