The present work was carried out to investigate the Antimicrobial Resistance (AMR) of enterococci isolated from raw ewes’ and cows’ milk. The samples were collected from eighteen semi-extensive dairy sheep and cow farms throughout western Sicily. Plate counts, carried out on Rapid Enterococcus Agar commonly used to detect food enterococci, revealed a maximal enterococcal concentration of approximately 4.58 Log Colony Forming Unit (CFU)/mL. Colonies were isolated and differentiated based on genetic analysis by Randomly Amplified Polymorphic DNA (RAPD)-PCR. Thirty-eight different strains were identified. Analysis by a species-specific multiplex PCR assay grouped the strains into three Enterococcus species such as Enterococcus durans, Enterococcus faecalis and Enterococcus faecium. The 38 strains were also investigated for their antimicrobial resistance by a phenotypic approach. All 38 Enterococcus displayed resistance to at least one or more of the antimicrobials tested confirmed that the dairy enterococci could be a vector for the dissemination of antimicrobial resistance. This work showed that enterococci with AMR traits are commonly present in semiextensive dairy sheep and cow farms of western Sicily pointed out the relevance of informing dairy makers and veterinary regarding the antimicrobial use in order to mitigate problems of public health and veterinary medicine.

Background: Lactic Acid Bacteria (LAB) are an important group of microorganisms responsible for the fermentation dairy products. This study was done to identify the dominant lactic acid bacteria in naturally fermented milks from Messinese goat’s breed. Methods: Eighteen individual raw milk samples collected from Messinese goat’s breed were acidified at pH 5.20 and left to spontaneously ferment at 37 °C for 4 days. All samples were analyzed for rod- and coccus-shaped LAB. Also, all presumptive LAB were isolated and differentiated according to their phenotypic properties and genetic polymorphisms and then identified by sequencing the 16S rRNA gene. Data were statistically analyzed using SAS 9.2 software. Results: Levels of coccus- and rod-shaped LAB were in the range of 1.78-7.10 log and 1.00-7.09 log colony forming unit/ml, respectively. The microbiological counts on the two different growth media were significantly (p<0.05) different among the samples. Among 12 identified strains, the most numerous one was Enterococcus hirae (n=4), followed by E. faecium (n=3), while the other species (E. durans, E. faecalis, E. lactis, Lactococcus lactis, and Leuconostoc lactis) included one strain each. Conclusion: The major group identified in this study was mainly represented by members of Enterococcus genus. Although Enterococcus spp. are related to the typicality of some traditional fermented dairy products, this study highlights the need for risk assessment of pathogenic enterococci species for the consumers.

The present work was carried out to investigate the microbiological profile of Sicilian ewes' ricotta cheeses during fifteen years of investigations (2002-2016). The samples were collected between those conferred to the Istituto Zooprofilattico Sperimentale della Sicilia (IZSSi) Adelmo Mirri, Palermo (Italy), by the competent authority during official control, by food business operator in HACCP systems and in research projects. Enterobacteriaceae, Escherichia coli and coagulase-positive staphylococci (CPS) were found only in some samples. Bacillus cereus was detected in c.a. 16% of samples but the level of contaminations did not reach the threshold that leads to significant toxin production. Pathogenic bacteria such as Listeria monocytogenes, Salmonella spp. and Brucella spp. were never detected. Furthermore, a total of 47 of lactic acid bacteria (LAB) strains were identified at species level by sequencing the 16S rRNA gene, resulting in the identification of 10 species belonging to four genera Enterococcus, Lactobacillus, Lactococcus and Leuconostoc, commonly employed as starter and non starter cultures in different traditional cheese. Results of this study highlighted an improvement of the hygienic conditions of the Sicilian ewes' ricotta cheeses during the last ten years of investigation. This observation was confirmed from reduction of undesired microorganisms such as Enterobacteriaceae, E. coli and CPS, used to define the process hygiene criteria. However, in order to improve the final quality of this product are needed further strategy such as the dairy makers training, with the aim to apply a good hygienic practices during the production.

This study aimed to describe the effects of refrigerated storage up to 180days on microbiological, chemical, physical, and sensory characteristics of a PDO ewes’ raw milk stretched cheese. To this aim, a total of 224 cheeses were manufactured in four consecutive production weeks, and series of 32 of them were examined before packaging and after 15, 30, 60, 90, 120, and 180d of storage at 4±2°C in the dark, respectively. Lactic acid bacteria cocci displayed the highest levels (7.8 Log CFU*g−1) during early storage and decreased progressively over time (7.4 Log CFU*g−1), while the opposite trend was observed for lactic acid bacteria rods (from 6.5 to 7.3 Log CFU*g−1). TMC and enterocci significantly increased during the storage. Chemical parameters showed a natural increase of proteolytic index during storage, an increase of pH (from 5.44 to 5.92), salt (from 2.08 to 2.40% of DM) and a decrease of aw (from 0.984 to 0.971). Storage modified the color of the cheeses, provoked a slight browning, while a* value (red–green) and b* value (yellow–blue) increased until 30days and then remained unchanged. Cheese fatty acids composition didn’t show particular trend during the storage, while several panel test parameters changed. Cheeses after 180days of storage showed higher solubility, greater odor of butter and less odor of milk than fresh cheeses, that determined an high overall satisfaction of the panelists at the end of storage.

ABSTRACT The main hypothesis of this work was that Sicilian forestry resources are suitable for the production of equipment to be used in cheese making and indigenous milk lactic acid bacteria (LAB) are able to develop stable biofilms providing starter and nonstarter cultures necessary for curd fermentation and cheese ripening, respectively. Hence, the present work was carried out with deproteinized whey to evaluate LAB biofilm formation on different woods derived from tree species grown in Sicily. Microbiological and scanning electron microscopy analyses showed minimal differences in microbial levels and compositions for the neoformed biofilms. The specific investigation of Salmonella spp., Listeria monocytogenes, Escherichia coli, coagulase-positive staphylococci (CPS), and sulfite-reducing anaerobes did not generate any colony for all vats before and after bacterial adhesion. LAB populations dominated all vat surfaces. The highest levels (7.63 log CFU/cm2) were registered for thermophilic cocci. Different colonies were characterized physiologically, biochemically, and genetically (at strain and species levels). Six species within the genera Enterococcus, Lactobacillus, Lactococcus, and Streptococcus were identified. The species most frequently present were Lactobacillus fermentum and Lactococcus lactis. LAB found on the surfaces of the wooden vats in this study showed interesting characteristics important for dairy manufacture. To thoroughly investigate the safety of the wooden vat, a test of artificial contamination on new Calabrian chestnut (control wood) vats was carried out. The results showed that LAB represent efficient barriers to the adhesion of the main dairy pathogens, probably due to their acidity and bacteriocin generation. IMPORTANCE This study highlights the importance of using wooden vats for traditional cheese production and provides evidence for the valorization of the Sicilian forest wood resources via the production of dairy equipment.

Casein genes are highly polymorphic and the high degree of variability has qualitative and quantitative effects on milk composition thereby affecting chemical, physical and technological properties of goat milk. The aim of this work was to evaluate the genetic polymorphisms of the alpha s2-casein (CSN1S2) gene in the endangered Girgentana dairy goat breed in order to assess the genotypes distribution, as it is known genotype influences technological and nutritional milk properties. The study was performed on 207 sample of Girgentana goat breed, analysed with different PCR protocols. The most frequent alleles was A (0.722), followed by F (0.225), C (0.051) and E (0.002) while B, D and 0 alleles were not found. Genotypes detected were AA (0.512), AF (0.338), AC (0.082), FF (0.043), CF (0.020) and EF (0.005). Our results suggested that Girgentana goat breed could be used for the production of milk with high fat and protein content and with optimal technological ability, suitable for cheese making.

Polymorphisms of genes encoding key factors for the control and activation of inflammatory response and coagulation cascade regulation may play a role in genetic susceptibility to acute myocardial infarction (AMI). This study sought to analyze the effect of TNF -308G/A and pro-thrombin (FII) 20210G/A polymorphisms on the laboratory parameters of young patients affected by AMI. Results indicated that TNF -308A positive genotype frequencies were increased in these patients and that a genetically determined higher production of TNF-α is associated in young subjects to a more severe cardiac damage as depicted by higher levels of troponin, Creatine kinase-MB Isoenzyme (mCK-MB) and a significant increased plasma fibrinogen levels. Similar and probably additive effects on might have a genetically determined increased production of pro-thrombin even if no significant differences in genotype frequencies of pro-thrombin (FII) 20210G/A polymorphisms were observed in this study. All together these results, indicating the relationship among genetically determined TNFα and FII production and increased levels of tissue damage markers of AMI, suggest that a complex genetic background, might be involved in susceptibility to AMI in young men influencing the extension and severity of the disease.

Cytokines act as pleiotropic polypeptides able to regulate inflammatory/immune responses and to provide important signals in physiological and pathological processes. Several cytokines (Th1, Th2, and Th17) seem to be involved in the pathophysiology of Behçet’s disease, a chronic immune-mediated disease characterized by oral and genital lesions and ocular inflammation. Its individual susceptibility seems to be modulated by genetic variants in genes codifying these cytokines. Th1 and Th17 seem to be involved in the disease’s active phases, and Th2 seems to affect the development or severity of the disease; however, contrasting data are reported. In this study, some genetic variants of the Th1/Th2 cytokine genes were investigated in Sicilian patients and age- and gender-matched controls. Three very significant associations with Behçet’s disease were detected, and combined genotypes associated with increased disease risk were identified. Results obtained point to the key role of Th1/Th2 cytokine genetic variants in disease susceptibility.

Background: The genetic basis of complex diseases like ischemic stroke probably consists of several predisposing risk factors, such as genes involved in inflammation and thrombotic pathways. On this basis the aim of our study was to evaluate the role of SNPs (single nucleotide polymorphisms) of some pro-inflammatory/anti-inflammatory and coagulation/fibrinolytic genes in patients with acute ischemic stroke.Methods: The study population consisted of 144 consecutive Caucasian adult patients who were hospitalized in the Internal Medicine Department at the University of Palermo between November 2006 and January 2008, and who met inclusion criteria. The cases were patients admitted with a diagnosis of acute ischemic stroke, and age-matched (+/- 3 years) control subjects: patients admitted to our Internal Medicine Department for any cause other than acute cardiovascular and cerebrovascular events and for routine checkup examinations.Methods: Molecular analysis of alleles at the 308 nucleotide (-308G/A) of TNF-alpha gene, -1082/-819 haplotypes of IL-10 gene, IL-1RN exon 2 VNR polymorphism, alleles at the 174 nucleotide (-174G/C) of IL-6 gene, PAI-1675 5G/4G polymorphism, alleles at the 7351 nucleotide (-7351C/T) of tPA gene was undertaken in both patient groups.Results: We analyzed 96 subjects with acute ischemic stroke and 48 control subjects. We observed a significantly higher frequency of IL-10 1082 AA genotype in stroke patients with a significant risk trend. We also reported a higher frequency in stroke subjects with a significant risk trend of the TPA 7351-CT genotype and of IL-1RN-VNTR 86 bp 2/2 genotype. Moreover, we observed a significant relationship with TOAST subtype only with regard to CC TPA genotype and 1/1 IL-1 VNTR 86 bp and lacunar strokes.Conclusions: Ischemic stroke is a common multifactor disease, which is affected by a number of genetic mutations and environmental factors. Our findings showing a relationship between pro-inflammatory/anti-inflammatory and thrombotic/fibrinolytic genes SNPs and ischemic stroke may contribute to delineate a possible stroke risk profile in subjects with cerebrovascular risk factors. (C) 2012 Elsevier Ltd. All rights reserved.

Infection risk, sepsis and mortality after severe burn are primarily determined by patient age, burn size, and depth. Whether genetic differences contribute to otherwise unexpected variability in outcomes is unknown. We sought to determine whether there was an association between IL-6, IL-10 and IL-17 polymorphisms with cytokine production and development of sepsis.We evaluated 71 patients with burns >= 15% TBSA and 109 healthy subjects. The genotypes of IL-6 (-174C/G), IL-10 (-819C/T and -1082A/G) and IL-17 (7488T/C) polymorphisms were identified applying polymerase chain reaction protocols. The cytokine levels in serum were determined with enzyme-linked immunoabsorbent assays.Our results demonstrated no significant differences in the genotype frequencies studied between burn patients and healthy subjects. No significant associations were found among IL-6 and IL-17F genotypes and the related cytokine serum levels. Only IL-10 promoter -1082GG genotype was related to an increased IL-10 production in burned patients. In addition, septic subjects bearing -1082G/G genotype have shown the highest and non-septic bearing -1082A/* genotypes the lowest IL-10 serum levels. All together these data seem to indicate that genetically determined individual difference in IL-10 production might influence the susceptibility to septic complications in burned patients and suggest that these markers might be useful in burned patient management. (C) 2011 Elsevier Ltd and ISBI. All rights reserved.

The aim of our study was to evaluate the possibility of using multiplex analysis of the cytokine profile as a marker for successful aging by comparing cytokine plasmatic levels of a group of Sicilian nonagenarians with those of young controls. We analyzed a panel of 17 cytokines, comprehensive of haematopoietic factors T helper 1 (Th1), Th2, inflammation regulatory cytokines, and chemokines. The assay was carried out using the Luminex system. Interleukin (IL)-6 levels (p = 0.01) were increased in nonagenarians, whereas no modifications of other proinflammatory cytokines and chemokines were observed. Interferon-gamma (IFN-γ) and IL-2 levels are unmodified, suggesting a substantial maintenance of relevant T cell functions. In addition, a significant increase of IL-12 serum levels in nonagenarians versus young controls that might be related to the increase of natural killer (NK) cell functions characterizing aging processes was observed. The analysis of Th2 cytokines show an increase of IL-13 and a reduction of IL-4 levels mirroring the maintenance of some effector's mechanisms of the immunoresponse in advanced ages. Our results suggest that the multiplex analysis of cytokine levels might be useful in defining a successful aging profile.

The demographic and social changes of the past decades have determined improvements in public health and longevity. So, the number of centenarians is increasing as a worldwide phenomenon. Scientists have focused their attention on centenarians as optimal model to address the biological mechanisms of "successful and unsuccessful ageing". They are equipped to reach the extreme limits of human life span and, most importantly, to show relatively good health, being able to perform their routine daily life and to escape fatal age-related diseases, such as cardiovascular diseases and cancer. Thus, particular attention has been centered on their genetic background and immune system. In this review, we report our data gathered for over 10 years in Sicilian centenarians. Based on results obtained, we suggest longevity as the result of an optimal performance of immune system and an over-expression of anti-inflammatory sequence variants of immune/inflammatory genes. However, as well known, genetic, epigenetic, stochastic and environmental factors seem to have a crucial role in ageing and longevity. Epigenetics is associated with ageing, as demonstrated in many studies. In particular, ageing is associated with a global loss of methylation state. Thus, the aim of future studies will be to analyze the weight of epigenetic changes in ageing and longevity.

Background: Today the treatment with botulinum toxin (BTX) is the most used molecular surgery of strabismus and other eye-movement disorders, as alternative to traditional surgery.However the temporary effect of BTX treatment requires the research of alternative therapies.To this purpose the in vitro effects of three type II RIPs from plants (lanceolin, stenodactylin and ricin)and of the skeletal musclespecific immunotoxin saporin-mAb73 were evaluated on muscle cells.Methods: The RIPs and the immunotoxin were tested for their cytotoxicity in three cell lines: L6E9 (myoblasts), TE671 and RD/18 (rhabdomyosarcoma), both undifferentiated and differentiated.The aspecific toxicity was evaluated on conjunctival IOBA-NHC cell line.Protein synthesis inhibition, viability and apoptotic changes were assayed.Results: All substances showed a strong cytotoxic effect on protein synthesis and viability, with IC50 and LC50 ranging from 0.1 nM to 0.01 pM.Lanceolin and stenodactylin were 1-2 logs more toxic than ricin and 2-3 logs more toxic than the immunotoxin.Myoblasts were particularly susceptible to stenodactylin (IC50<0.01pM).All RIP-treated cells showed typical morphological apoptotic changes and no signs of necrosis.In further experiments miming in vivo treatment, no toxic effects were reported on conjunctival IOBA-NHC cell line.Conclusions: The strong cytotoxicity observed for stenodactylin at very low dose could be compatible with loco-regional treatments in strabismus and eye-movement disorders.It could be possible to modulate the effect on muscle fibers and to obtain a complete ablation of myoblasts, gaining more durable effects as compared to BTX treatment.Moreover, the absence of necrosis should avoid flogistic side effects.

Background: It is well known that exercise has beneficial effects on health. Although intense exercise is experienced by the body as a condition of stress,a well designed training has long term beneficial effects on the organism of an athlete. Less it is known about the effects that the genetic background might have on training adaptation and on the consequent modification of laboratory parameters. Methods: In our study we evaluated the blood chemistry parameters of a group of 41 athletes compared with a group of 45 amateur athletes, to assess whether the training has effects on their variation. In addition we typed our subjects for polymorphisms -308 A/G of the Tumor Necrosis Factor-α (TNFα) and -1082 A/G of Interleukin-10 (IL10), Results: After statistical analysis, performed with Mann-Whitney Test, we can observe a statistically significant (p value< 0,05) increase of basophils, eosinophils, monocytes, and total bilirubin and decreased levels of neutrophils, glucose, electrolytes and AST in professionals than amateurs. These parameters were not modified by the genetic background. Actually the training modification observed were independent by the presence of pro-inflammatory (carrier allele A of -1082 A/G of IL10) or anti-inflammatory alleles (subjects A negative for -308 A/G of TNF) Conclusion: The genetic polymorphisms analyzed do not influence changes in laboratory parameters values induced by professional training.