Multiplex PCR to detect bacteriophages from natural whey cultures of buffalo milk and characterisation of two phages active against Lactococcus lactis, фApr-1 and фApr-2

This work investigated bacteriophage induced starter failures in artisanal buffalo Mozzarella production plants in Southern Italy. Two hundred and ten samples of whey starter cultures were screened for bacteriophage infection. Multiplex polymerase chain reaction (PCR) revealed phage infection in 28.56% of samples, all showing acidification problems during cheese making. Based on DNA sequences, bacteriophages for Lactococcus lactis (L. lactis), Lactobacillus delbruekii (L. delbruekii) and Streptococcus thermophilus (S. thermophilus) were detected. Two phages active against L.  lactis, фApr-1 and фApr-2, were isolated and characterised. The genomes, approximately 31.4 kb and 31 kb for фApr-1 and фApr-2 respectively, consisted of double-stranded linear DNA with pac-type system. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed one major structural protein of approximately 32.5 kDa and several minor proteins. This is the first report of phage isolation in buffalo milk and of the use of multiplex PCR to screen and study the diversity of phages against Lactic Acid Bacteria (LAB) strains in artisanal Water Buffalo Mozzarella starters

Gastroenteritis Outbreak at Holiday Resort, Central Italy

Gastroenteritis Outbreak at Holiday Resort, Central Ital

Proprieta nutraceutiche e nutrizionali del dolcificante Stevia Rebaudiana Bertoni. Evidenze di efficacia nel trattamento del diabete mellito di tipo 2.

“PROPRIETA’ NUTRACEUTICHE E NUTRIZIONALI DEL DOLCIFICANTE STEVIA REBAUDIANA BERTONI. EVIDENZE DI EFFICACIA NEL TRATTAMENTO DEL DIABETE MELLITO DI TIPO 2.” Il diabete mellito è una patologia in continuo aumento nei paesi industrializzati, caratterizzata da iperglicemia e da alterazioni metaboliche conseguenti a difetti della secrezione o dell’azione dell’insulina oppure ad entrambi i meccanismi. L’attuale classificazione del diabete mellito include 4 classi cliniche: diabete mellito di tipo 1, diabete mellito di tipo 2, diabete mellito gestazionale e altre forme di diabete mellito dovuto ad altre cause, come per esempio i difetti genetici nella funzione delle cellule β, malattie del pancreas esocrino e forme indotte dai farmaci. Il diabete mellito di tipo 1 è caratterizzato dalla distruzione delle cellule β del pancreas con conseguente deficienza di insulina. Esiste una predisposizione genetica, anche se altri fattori sono stati considerati come cause scatenanti o promuoventi, tra cui l’azione di virus, sostanze tossiche e fattori ambientali. Il diabete mellito di tipo 2 è la forma di diabete più frequente. Anche qui la predisposizione genetica ha un ruolo essenziale, tuttavia la malattia può essere aggravata da fattori esogeni come la dieta e la sedentarietà. I criteri per la diagnosi di diabete mellito comprendono: - Riscontro di almeno due valori di glicemia a digiuno ≥ 126 mg/dl (assenza di apporto calorico da almeno 8 ore). Oppure: - Sintomi dell’iperglicemia + una glicemia casuale a random ≥ 200 mg/dl. Oppure: - Glicemia a 2 ore dal pasto di 200 mg/dl durante un test per la tolleranza glucidica orale. La mia tesi si è focalizzata sul diabete mellito di tipo 2 e sulle implicazioni di una sostituzione del saccarosio, nella dieta, con l’ edulcorante Stevia rebaudiana Bertoni. La Stevia è un piccolo arbusto ramificato della famiglia delle Asteraceae, nativa della regione di Amambay nel Nord-Est del Paraguay. E’ stata classificata botanicamente nel 1899 da Moisés Santiago Bertoni. Il principio dolce fu isolato nel 1909 e solo nel 1931 l’estratto è stato purificato per produrre lo stevioside. Lo stevioside, circa 300 volte più dolce del saccarosio, è stato descritto come un glicoside comprendente tre molecole di glucosio attaccate ad un aglicone, lo steviolo. Durante gli anni ’70, sono stati isolati altri composti come il rebaudioside A, con un potere dolcificante anche superiore a quello dello stevioside. Gli studi farmacologici finora condotti con i glicosidi dello steviolo hanno dimostrato la loro sicurezza e assenza di tossicità. Hanno dimostrato, inoltre, che questi sono scarsamente assorbiti dopo l’esposizione orale, e che necessitano di essere idrolizzati dalla microflora del colon in steviolo. Lo steviolo, nel fegato, subisce la coniugazione con l’acido glucuronico per formare glucuronide steviolo, che è escreto per via urinaria. I glicosidi steviolici vengono largamente impiegati dall’industria alimentare come dolcificante di bevande e cibi, rientrando nella categoria degli additivi alimentari con la sigla E 960. JECFA e FDA hanno stabilito una dose accettabile di assunzione quotidiana di steviolo puro (≥ 95%) come additivo alimentare di 0-4 mg/kg di peso corporeo, equivalenti a 12 mg/kg di peso corporeo per il rebaudioside A. Nel 2013 sono stati studiati gli effetti dell’estratto ottenuto dalle foglie di Stevia su ratti resi sperimentalmente diabetici per trattamento con streptozotocina. Alla fine dello studio sono stati osservati bassi livelli di glucosio nel sangue e una piccola riduzione del peso del rene nei ratti alimentati con l’estratto di Stevia. Altri studi sono stati condotti su pazienti con diabete mellito di tipo 2, ed è stato confermato l’effetto ipoglicemizzante della Stevia, con abbassamento dei livelli di glucosio e di insulina plasmatici. Si può concludere affermando che la Stevia è un dolcificante sicuro e non tossico, può essere utilizzato nella dietoterapia dei pazienti diabetici e il suo uso a lungo termine può risultare vantaggioso nella prevenzione delle malattie cardiovascolari nei pazienti diabetici

The egg consumption of the average household in Italy

A survey was conducted over a one-year period by means of telephone interviews with 7 991 Italian households to establish the domestic consumption of eggs, the distribution by source of supply, seasonal variations and storage and preparation methods used. Eggs are mainly purchased from large retailers (53%), followed by small retailers (25.2%), direct purchase from producers (16%), and local or itinerant markets (5.8%). It was found that 69.9% of households buy packaged eggs; 92% of households store them in the refrigerator, although this percentage varies considerably, according to the type of presentation (packaged or loose) and the number of eggs bought. Italian households mainly eat eggs cooked (48.9%), followed by partly cooked (35.0%) and raw (16.1%)

Assessment of hygienic quality of some types of cheese sampled from retail outlets

The authors evaluated the prevalence of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella spp. and Staphylococcus enterotoxin, in 2 132 samples selected from six types of cheese on the basis of recorded consumption in Italy in 2004. In L. monocytogenes-positive samples the precise level of contamination was determined. To define the physical-chemical characteristics of the selected natural cheeses, the pH values, water activity and sodium chloride content were determined. The results suggest that blue and soft cheeses (Brie, Camembert, Gorgonzola and Taleggio) are more likely to be contaminated with L. monocytogenes. The mean prevalence of L. monocytogenes in the six types of cheese was 2.4% (from 0.2% in Asiago and Crescenza to 6.5% in Taleggio), with contamination levels of up to 460 MPN/g. No presence of Salmonella spp. and E. coli O157 was found in any sample. Staphylococcus enterotoxin was found in 0.6% of the samples examined. Physical and chemical parameter values confirmed that all types of cheese are considered capable of supporting the growth of L. monocytogenes. The study confirmed the need to apply effective control at production and sales levels to reduce the probability of contamination by L. monocytogenes. This micro-organism can attain high levels of contamination in food products, such as cheeses that have a long shelf-life when associated with difficulties of maintaining appropriate storage temperatures in both sales points and in the home

Caratterizzazione di ceppi di Staphylococcus aureus isolati da alimenti destinati all’uomo

È stata condotta un’indagine per valutare la contaminazione da Staphylococcus aureus di alcune categorie di alimenti di origine animale. Dei 350 prodotti esaminati il 14,0% è risultato contaminato da S. aureus, con prevalenze variabili a seconda del gruppo di alimenti considerato: 19,3% per i preparati di carne fresca; 13,3% formaggi freschi; 3,6% per i prodotti di pasticceria e 7,7% per le preparazioni gastronomiche. I ceppi di S. aureus isolati sono stati sottoposti a conferma dell’identificazione della regione16S rDNA e successivamente sottoposti al test di agglutinazione inversa al lattice (SET-RPLA, Oxoid) per individuare quelli enterotossigeni. I risultati sono stati confrontati con i dati ottenuti sottoponendo gli stessi ceppi alla ricerca dei geni codificanti le enterotossine (SE) sea, seb, sec, sed, see, seg, seh, sei e la Toxic Shock Syndrome Toxin-1 (TSST-1). Al test RPLA (8/49) il 16,3% dei ceppi sono risultati produttori di enterotossine mentre alla PCR (24/49) il 48,97% sono risultati portatori di uno o più geni per la produzione di SE e, quindi, potenzialmente enterotossigeni

Characterisation of Staphylococcus aureus strains isolated from food for human consumption

An investigation was conducted to evaluate Staphylococcus aureus contamination in various types of food of animal origin. Of the 350 samples examined, 14.0% were found to be contaminated with S. aureus. Prevalence rates varied according to type, namely: 19.3% for fresh meat products, 13.3% for fresh cheeses, 3.6% for bakery products and 7.7% for deli products. The isolated S. aureus strains then underwent 16S rDNA polymerase chain reaction (PCR) followed by reverse latex agglutination tests to identify enterotoxigenic strains. The results were compared with data obtained by subjecting the same strains to tests for the genes coding for the S. aureus enterotoxins (SEs) sea, seb, sec, sed, see, seg, seh, sei and toxic shock syndrome toxin—1 (TSST—1). Reversed passive latex agglutination (RPLA) testing revealed that 16.3% of strains (8/49) produced enterotoxins, while on PCR, 48.97% (24/49) were found to carry one or more genes for the production of SEs, and were therefore potentially enterotoxigenic

Definition of a standard protocol to determine the growth potential of Listeria monocytogenes and Yersinia enterocolitica in pork sausage produced in Abruzzo Region, Italy

Pork meat products consumed raw or after a short period of fermentation can be considered at risk for food safety. Sausages (fresh sausage made from pork meat) are produced in several Italian regions, with variation in ingredients. In some Italian Regions, including Abruzzo, these products are frequently consumed raw or undercooked, after a variable period of fermentation. The European Community food regulation promotes the use of <em>challenge tests</em> to determine safety levels. This study is aimed to ensure safety of Abruzzo’s sausages, compared with growth potential (δ) of <em>Listeria monocytogenes</em> and <em>Yersinia enterocolitica</em>, and also aims to define an experimental standard protocol document to carry out challenge tests. Guidelines classify ready-to-eat foods in categories that are able to support (δ&gt;0.5 log₁₀ ufc/g) and not support (δ≤0.5 log₁₀ ufc/g) the growth of <em>Listeria monocytogenes</em>. The products were manufactured according to traditional recipes and were contaminated in laboratory. Results from the experiment yielded information useful to assess the ability of these products to support the growth of pathogenic microorganisms. The batches of sausages were stored at 8, 12, 18 and 20°C to get statistical evaluation. The results showed that, despite the conditioning of the storage temperature and the level of water activity, both organisms remain in the product in concentrations similar to those leading or being able to increase its charge. In particular, the period of greatest consumption of this product (7/8 days of preparation) corresponds to the period of greatest growth of pathogenic microorganisms studied, except for those stored at a temperature of 8°C, which are safer for the consumer

Detection of Vibrio splendidus and related species in Chamelea gallina sampled in the Adriatic along the Abruzzi coastline

Vibrio species are an important and widespread component of marine microbial communities. Some Vibrio strains are potentially pathogenic to marine vertebrates and invertebrates. The aim of this study was to identify vibrios, in particular Vibrio splendidus and related species, isolated from clams (Chamelea gallina) collected along the coasts of the Abruzzi region from May to October 2007. The isolates obtained were phenotyped and classified as belonging to the genus Vibrio. The strains underwent biochemical testing in accordance with Alsina’s scheme for V. splendidus identification. Molecular analysis of the 16S-23S intergenic space region and recA gene was used to identify V. splendidus and related species. All the samples examined were found to contain halophylic Vibrio species, with V. alginolyticus, V. splendidus-related species and V. mediterranei most commonly found. A polymerase chain reaction of the 16S-23S intergenic space region and sequencing of the recA gene from isolates confirmed that phenotyping of Vibrio species is not sufficient to distinguish between different species. Differentiation of the highly related species among V. splendidus-related clusters remains an important issue. In this regard, our data suggests sequencing the recA genes was far more discriminatory than sequencing 16S rDNA for this purpose

Characterisation of Listeria monocytogenes strains isolated from soft and semi-soft cheeses sampled in a region of Italy

A total of 47 Listeria monocytogenes strains isolated in a survey of cheeses sampled from retail outlets were characterised. Five cheeses (Gorgonzola, Taleggio, Asiago, Crescenza and Brie) were chosen from the most popular soft and semi-soft cheeses consumed in Italy and most commonly contaminated with L. monocytogenes. The serotype and antibiotic resistance pattern were determined for each strain and their macrorestriction profile was analysed with pulsed-field gel electrophoresis (PFGE). The main serotypes detected were 1/2a (76.6%) and 1/2c (21.3%). Serotype 1/2b was found in only one sample. A total of 97.9% of strains were resistant to oxacillin (OX), 80.9% to lincomycin (L) and 78.7% to clindamycin (CC). Of these strains, 17% were found to be resistant to two antibiotics (OX-CC or OX-L) while 70.2% were resistant to three antibiotics (OX-CC-L). No strains were susceptible to all the compounds tested. A combined analysis of the macrorestriction profiles AscI and ApaI identified eleven pulsotypes divided into three clusters. Two pulsotypes predominated, accounting for 57.4% and 21.3% of the isolated strains. Analysis of the PFGE profiles did not reveal any correlation between pulsotype and type of cheese, producer or retail outlet. A temporal analysis revealed that one pulsotype was persistent throughout the study period, with the exception of August and September, in which time a different pulsotype was detected. This variability suggests the influence of factors affecting the dynamics of the contamination of these products. Large-scale studies could help clarify this phenomenon