Detection of hepatitis C virus in an exhumed body identified the origin of a nosocomial transmission that caused multiple fatal diseases.

BACKGROUND: Medico-legal conflicts arise when it is difficult to prove the cause of nosocomial infections. AIM: To report an outbreak of patient-to-patient transmission of hepatitis C virus (HCV) through the repeated use of a multi-dose saline flask during the rinsing of central venous catheters. METHODS: Blood samples were taken from each patient for the comparative analysis of their HCV RNA strains. No samples were available for one patient who died before the investigation started. Despite the known lability of HCV RNA, the body was exhumed four months after burial and postmortem samples were collected. HCV RNA was extracted successfully from liver and spleen samples. Genotyping of all the HCV strains was performed by sequence analysis of the 5'NC untranslated region, the E1 core conserved region and the E1/E2 hypervariable region. FINDINGS: Forensic investigators retraced the route used by two ward nurses, when saline catheter flushes were given to 14 patients with each nurse administering to seven patients. The comparative phylogenetic analysis of all case strains identified the deceased patient as the source of contamination to five patients. CONCLUSIONS: This study highlights the value of sequence analysis as a tool for solving medico-legal conflicts. The High Court of Justice found that a health worker's re-use of a contaminated needle resulted in the nosocomial transmission of HCV.

Fructose-derived advanced glycation end-products drive lipogenesis and skeletal muscle reprogramming via SREBP-1c dysregulation in mice.

Advanced Glycation End-Products (AGEs) have been recently related to the onset of metabolic diseases and related complications. Moreover, recent findings indicate that AGEs can endogenously be formed by high dietary sugars, in particular by fructose which is widely used as added sweetener in foods and drinks. The aim of the present study was to investigate the impact of a high-fructose diet and the causal role of fructose-derived AGEs in mice skeletal muscle morphology and metabolism. C57Bl/6J mice were fed a standard diet (SD) or a 60% fructose diet (HFRT) for 12 weeks. Two subgroups of SD and HFRT mice received the anti-glycative compound pyridoxamine (150 mg/kg/day) in the drinking water. At the end of protocol high levels of AGEs were detected in both plasma and gastrocnemius muscle of HFRT mice associated to impaired expression of AGE-detoxifying AGE-receptor 1. In gastrocnemius, AGEs upregulated the lipogenesis by multiple interference on SREBP-1c through downregulation of the SREBP-inhibiting enzyme SIRT-1 and increased glycation of the SREBP-activating protein SCAP. The AGEs-induced SREBP-1c activation affected the expression of myogenic regulatory factors leading to alterations in fiber type composition, associated with reduced mitochondrial efficiency and muscular strength. Interestingly, pyridoxamine inhibited AGEs generation, thus counteracting all the fructose-induced alterations. The unsuspected involvement of diet-derived AGEs in muscle metabolic derangements and proteins reprogramming opens new perspectives in pathogenic mechanisms of metabolic diseases.

Study of the photochemical transformation of 2-ethylhexyl 4-(dimethylamino)benzoate (OD-PABA) under conditions relevant to surface waters.

We studied the aquatic environmental fate of 2-ethylhexyl 4-(dimethylamino)benzoate (OD-PABA), a widespread sunscreen, to assess its environmental persistence and photoinduced transformation. Direct photolysis is shown to play a key role in phototransformation, and this fast process is expected to be the main attenuation route of OD-PABA in sunlit surface waters. The generation of transformation products (TPs) was followed via HPLC/HRMS. Five (or four) TPs were detected in the samples exposed to UVB (or UVA) radiation, respectively. The main detected TPs of OD-PABA, at least as far as HPLC-HRMS peak areas are concerned, would involve a dealkylation or hydroxylation/oxidation process in both direct photolysis and indirect phototransformation. The latter was simulated by using TiO2-based heterogeneous photocatalysis, involving the formation of nine additional TPs. Most of them resulted from the further degradation of the primary TPs that can also be formed by direct photolysis. Therefore, these secondary TPs might also occur as later transformation intermediates in natural aquatic systems.

Analysis of bacterial community shifts in the gastrointestinal tract of pigs fed diets supplemented with ß-glucan from Laminaria digitata, Laminaria hyperborea and Saccharomyces cerevisiae.

This study was designed to evaluate the effects of algal and yeast ß-glucans on the porcine gastrointestinal microbiota, specifically the community of Lactobacillus, Bifidobacterium and coliforms. A total of 48 pigs were fed four diets over a 28-day period to determine the effect that each had on these communities. The control diet consisted of wheat and soya bean meal. The remaining three diets contained wheat and soya bean meal supplemented with ß-glucan at 250 g/tonne from Laminaria digitata, Laminaria hyperborea or Saccharomyces cerevisiae. Faecal samples were collected from animals before feeding each diet and after the feeding period. The animals were slaughtered the following day and samples were collected from the stomach, ileum, caecum, proximal colon and distal colon. Alterations in Lactobacillus in the gastrointestinal tract (GIT) were analysed using denaturing gradient gel electrophoresis (DGGE) profiles generated by group-specific 16S rRNA gene PCR amplicons. Plate count analysis was also performed to quantify total coliforms. DGGE profiles indicated that all ß-glucan diets provoked the emergence of a richer community of Lactobacillus. The richest community of lactobacilli emerged after feeding L. digitata (LD ß-glucan). Plate count analysis revealed that the L. hyperborea (LH ß-glucan) diet had a statistically significant effect on the coliform counts in the proximal colon in comparison with the control diet. ß-glucan from L. digitata and S. cerevisiae also generally reduced coliforms but to a lesser extent. Nevertheless, the ß-glucan diets did not significantly reduce levels of Lactobacillus or Bifidobacterium. DGGE analysis of GIT samples indicated that the three ß-glucan diets generally promoted the establishment of a more varied range of Lactobacillus species in the caecum, proximal and distal colon. The LH ß-glucan had the most profound reducing effect on coliform counts when compared with the control diet and diets supplemented with L. digitata and S. cerevisiae ß-glucans.

In vitro inhibition of Clostridium difficile and Clostridium perfringens by commercial probiotic strains.

Probiotics have gained importance in human and veterinary medicine to prevent and control clostridial enteric disease. Limited information is available on the ability of different probiotic bacteria used in food products to inhibit Clostridium difficile and Clostridium perfringens. The objective of this study was to examine the in vitro inhibitory effects of selected commercial bacterial strains on pathogenic clostridia and their growth characteristics under simulated gastrointestinal conditions. The inhibitory effects of 17 commercial strains of Lactobacillus (n = 16) and Bifidobacterium (n = 1) on the reference strains of C. difficile and C. perfringens were assessed by an agar well diffusion assay and by a broth culture inhibition assay using cell-free supernatant harvested at different growth phases, with and without pH neutralization. To study growth characteristics, probiotic strains were cultivated in different acid and bile environments, and growth in the modified media was compared to growth in standard medium. In the agar well diffusion assay, supernatant obtained from two probiotic strains inhibited the growth of both reference and clinical strains of C. perfringens. This effect as seen when supernatant was assessed with and without pH neutralization. Supernatants obtained from 10 probiotic strains inhibited C. difficile only when supernatant was added without pH neutralization. In the broth culture inhibition assay, growth of C. perfringens and C. difficile was inhibited by supernatant without pH neutralization from 5 and 10 probiotic strains, respectively. All potential probiotic strains were able to grow at pH 4.0 and in the presence of 0.15% and 0.3% bile but none were able to grow or survive at pH 2.0. Altogether five probiotic strains [Lactobacillus plantarum (n = 2), Lactobacillus rhamnosus (n = 2), Bifidobacterium animalis lactis (n = 1)] were shown to inhibit all strains of C. difficile and C. perfringens. The inhibitory effect was probiotic strain-specific. Two strains showed a pH-independent inhibitory effect likely due to production of either antibiotics or bacteriocins inhibiting C. perfringens only. These strains have favourable growth characteristics for use as probiotics and their efficacy as prophylactic or therapeutic measures against clostridial enteric disease should be further evaluated by clinical trials in animals.

Identification of the unknown transformation products derived from lincomycin using LC-HRMS technique.

In this paper, a comprehensive study of the fate of an antibiotic, lincomycin, in the aquatic environment is presented. High-resolution mass spectrometry was employed to assess the evolution of the process over time. Formation of intermediate compounds was followed by high performance liquid chromatography-high resolution mass spectrometry (LC-HRMS); accurate mass-to-charge ratios of parent ions were reported with inaccuracy below 1 mmu, which guarantee the correct assignment of their molecular formula in all cases, while their MS(2) and MS(3) spectra showed several structural-diagnostic ions that allowed to characterize the different transformation products (TPs) and to discriminate the isobaric species. The simulation of phototransformation occurring in the aquatic environment and the identification of biotic and abiotic TPs of the pharmaceutical compound were carried out in different experimental conditions: dark experiments, homogeneous photolysis and heterogeneous photocatalysis using titanium dioxide, in order to recreate conditions similar to those found in the environment. Twenty-one main species were identified afterwards lincomycin transformation. Several isomeric species were formed and characterized by analyzing MS and MS(n) spectra and by comparison with parent molecule fragmentation pathways. The major transformation process for lincomycin is hydroxylation either at N-alkyl side chain or at the pyrrolidine moiety. In addition, oxidation/reduction, demethylation or cleavage of pyranose ring occurs. Based on this information and additional assessment of profiles over time of formation/disappearance of each species, it was possible to recognize the transformation pathways followed by the drug.

Manufacture of Fior di Latte cheese by incorporation of probiotic lactobacilli.

This work aimed to select heat-resistant probiotic lactobacilli to be added to Fior di Latte (high-moisture cow milk Mozzarella) cheese. First, 18 probiotic strains belonging to Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus reuteri were screened. Resistance to heating (65 or 55°C for 10 min) varied markedly between strains. Adaptation at 42°C for 10 min increased the heat resistance at 55°C for 10 min of all probiotic lactobacilli. Heat-adapted L. delbrueckii ssp. bulgaricus SP5 (decimal reduction time at 55°C of 227.4 min) and L. paracasei BGP1 (decimal reduction time at 55°C of 40.8 min) showed the highest survival under heat conditions that mimicked the stretching of the curd and were used for the manufacture of Fior di Latte cheese. Two technology options were chosen: chemical (addition of lactic acid to milk) or biological (Streptococcus thermophilus as starter culture) acidification with or without addition of probiotics. As determined by random amplified polymorphic DNA-PCR and 16S rRNA gene analyses, the cell density of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 in chemically or biologically acidified Fior di Latte cheese was approximately 8.0 log(10)cfu/g. Microbiological, compositional, biochemical, and sensory analyses (panel test by 30 untrained judges) showed that the use of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 enhanced flavor formation and shelf-life of Fior di Latte cheeses.

The use of Lactobacillus brevis PS1 to in vitro inhibit the outgrowth of Fusarium culmorum and other common Fusarium species found on barley.

A total of 129 lactic acid bacteria (LAB) were screened for antifungal activity against common Fusarium spp. isolated from brewing barley. Four out of the five most inhibiting isolates were identified as Lactobacillus brevis, whereas one belonged to Weissella cibaria. L. brevis PS1, the isolate showing the largest inhibition spectrum, was selected and the influence of its freeze-dried cell-free supernatant (cfsP) on germination of macroconidia as well as mycelia growth was investigated using Fusarium culmorum as target organism.Addition of cfsP into the growth medium at concentrations > or = 2% altered the growth morphology of F.culmorum, whereas at concentrations > 5% the outgrowth of germ tubes from macroconidia was delayed and distorted. The presence of 10% cfsP completely inhibited the outgrowth of F. culmorum macroconidia. The activity of the compounds produced by L. brevis PS1 was higher at low pH values, i.e. pH < 5. Heating and/or proteolytic treatment reduced the inhibitory activity of cfsP, indicating that L. brevis produces organic acids and proteinaceous compounds which are active against Fusarium spp.

The use of sourdough fermented by antifungal LAB to reduce the amount of calcium propionate in bread.

Addition of sourdough is a common practice in the bakery industry to improve, among other quality parameters, the shelf life of bread. In this study, sourdough fermented by antifungal Lactobacillus plantarum strains was investigated for the ability to inhibit growth of common bread spoilage fungi. In both in vitro and sourdough wheat bread system, the antifungal sourdoughs significantly affected the outgrowth of Aspergillus niger, Fusarium culmorum, or Penicillium expansum spores, however on wheat bread outgrowth of Penicillium roqueforti spores was not affected. In an attempt to reduce the amounts of chemical additives in bread, the antifungal sourdoughs were used in combination with calcium propionate (CAP) and possible synergistic effects were evaluated. Presence of 3000 ppm CAP in the bread did not affect the outgrowth of P. roqueforti, whereas outgrowth of the other fungi was retarded. A strong synergistic effect was observed when CAP and antifungal sourdoughs were combined into the bread formulation, and outgrowth of P. roqueforti was affected. The use of reduced CAP amount (1000 ppm) showed significant inhibition only when antifungal sourdough was added. Remarkably, the increase in shelf life achieved was higher than that obtained using 3000 ppm of CAP alone. In conclusion, the results of this study clearly show that the addition of antifungal sourdough has the potential to reduce the levels of chemical additives needed in the bakery industry to ensure the microbiological safety of bread.

Increased complexity of the species composition of lactic acid bacteria in human feces revealed by alternative incubation condition.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with primers specific for lactic acid bacteria (LAB) was applied to investigate various media and incubation conditions to recover LAB from human feces. Samples were plated on selective and nonselective media and incubated under standard condition (37 degrees C, anaerobiosis) for fecal LAB as well as alternative condition (30 degrees C, 2% O2). PCR-DGGE analyses of resuspended bacterial biomass (RBB) obtained from agar plates revealed that the species composition of the recovered LAB was affected more strongly by the incubation condition than by the used medium. It was observed that food-associated LAB, such as Lactobacillus sakei and Leuconostoc mesenteroides, hitherto not described as intestinal inhabitants, are more easily selected when the alternative incubation condition is used. Identification of randomly picked colonies grown under the alternative condition showed that L. sakei is one of the predominant food-associated LAB species, reaching counts of up to 106 CFU/g feces. Comparison of the results of bacteriological culture with those obtained by PCR-DGGE analysis of the RBB showed that investigation of RBB is a fast and reliable method to gain insight into the species composition of culturable LAB in feces.

Mild bleeding diathesis in a boy with combined severe haemophilia B (C(10400)-->T) and heterozygous factor V Leiden.

Haemophilia B patients with factor IX (FIX) activity < 1% are usually characterized by severe bleeding episodes early in life. We report a case of sporadic severe haemophilia B, clinically characterized by mild bleeding diathesis. The presence of anamnestic thrombophlebitis in the patient's mother prompted us to investigate a possible associated hypercoagulable condition. Resistance to activated protein C due to factor V R506Q mutation was present in the mother and in the propositus, in the homozygous and heterozygous form, respectively. Molecular analysis of the FIX gene led to the identification of a nonsense mutation resulting in a stop codon at position 50, previously described and usually responsible for a severe pattern of haemophilia B. The implications of this unusual association are discussed.

A dysfunctional factor X (factor X San Giovanni Rotondo) present at homozygous and double heterozygous level: identification of a novel microdeletion (delC556) and missense mutation (Lys(408)-->Asn) in the factor X gene. A study of an Italian family.

Low levels of factor X (F.X) were detected in a 4-year-old boy who experienced acute lymphoblastic leukemia and bleeding manifestations. Laboratory data suggested the presence of a dysfunctional F.X molecule. Two novel F.X gene mutations were identified in the proband that was double heterozygous for both: a microdeletion (delC556) in exon VI resulting in a frameshift leading to a termination codon at position 226. This deletion was found in six family members with reduced F.X antigen and activity levels. A second mutation characterised by a G(1344)-->C transversion in exon VIII was detected in the proband resulting in a Lys(408)-->Asn substitution. This latter mutation was present in several asymptomatic family members from the paternal and the maternal side. The proband's sister was homozygous for the Lys(408)-->Asn substitution and exhibited low F.X activity with a normal antigen level. The naturally occurring F.X Lys(408)-->Asn (F.X(K408N)) variant was isolated from plasma of either homozygous or double heterozygous individuals. NH(2)-terminal sequencing of the heavy chain of F.X(K408N) failed to show any sequence abnormality in patients who were also carriers of the delC556, suggesting that this latter lesion accounted for the lack of F.X synthesis. Purified F.X Lys(408)-->Asn had an identical behaviour to normal F.X as judged by SDS-PAGE and immunoblotting. Clotting assay using purified F.X(K408N) and F.X-deficient plasma resulted in a laboratory phenotype similar to that observed in a homozygous subject for F.X Lys(408)-->Asn substitution. This is the first characterisation of a naturally occurring F.X variant with a mutation at the COOH-terminal end of the molecule.

Aspartic protease inhibitors. An integrated approach for the design andsynthesis of diaminodiol-based peptidomimetics.

Aspartic proteases play key roles in a variety of pathologies, including acquired immunodeficiency syndrome. Peptidomimetic inhibitors can act as drugs to combat these pathologies. We have developed an integrated methodology for preparing human immunodeficiency virus (HIV)-1 aspartic protease diaminodiol inhibitors, based on a computational method that predicts the potential inhibitory activity of the designed structures in terms of calculated enzyme-inhibitor complexation energies. This is combined with a versatile synthetic strategy that couples a high degree of stereochemical control in the central diaminodiol module with complete flexibility in the choice of side chains in the core and in flanking residues. A series of 23 tetrameric, pentameric and hexameric inhibitors, with a wide range of calculated relative complexation energies (-47.2 to +117 kJ.mol-1) and predicted hydrophobicities (logPo/w = 1.8-8.4) was thus assembled from readily available amino acids and carboxylic acids. The IC50 values for these compounds ranged from 3.2 nM to 90 microM, allowing study of correlations between structure and activity, and individuation of factors other than calculated complexation energies that determine the inhibition potency. Multivariable regression analysis revealed the importance of side-chain bulkiness and rigidity at the P2, P2' positions, suggesting possible improvements for the prediction process used to select candidate structures.