Food-grade titanium dioxide can affect microbiota physiology, adhesion capability, and interbacterial interactions: A study on L. rhamnosus and E. faecium.

Food-grade titanium dioxide (TiO2-FG) is a widespread metal oxide used in the food industries. Recently, the European Food Safety Authority concluded that TiO2-FG cannot be considered safe for consumption due to its genotoxicity; however, its effect on the gut microbiota has not yet been completely unraveled. We studied the effects of TiO2-FG (0.125 mg/mL) on Lactobacillus rhamnosus GG (LGG) and Enterococcus faecium NCIMB10415 (Ent), in particular some physiological and phenotypic traits (growth kinetics, bile salts, and ampicillin resistance) and their interactions with the host (auto-aggregation, biofilm formation, and adhesion on Caco-2/TC7 monolayers) and other gut microorganisms (antimicrobial activity towards pathogens). The results obtained revealed that TiO2-FG alters both LGG and Ent growth and lowers bile resistance (62 and 34.5%, respectively) and adhesion on Caco-2/TC7 monolayers (34.8 and 14.16%, respectively). The other outcomes were strictly species-specific: Ent showed a lower ampicillin sensitivity (14.48%) and auto-aggregation (38.1%), while LGG showed a reduced biofilm formation (37%) and antimicrobial activity towards Staphylococcus aureus (35.73%). Overall, these results suggest an adverse effect of TiO2-FG on both the endogenous and exogenously administered probiotics, contributing to the argument against using TiO2-FG as a food additive.

Putative probiotics decrease cell viability and enhance chemotherapy effectiveness in human cancer cells: role of butyrate and secreted proteins.

Recent advances have highlighted probiotic role in preventing colorectal cancer, by promoting differentiation, inhibiting proliferation, and inducing apoptosis in colonocytes. Here, three ascertained probiotics (L. rhamnosus GG ATCC 53103, L. reuterii DSM 17938 and L. johnsonii LC1) and four food-isolated putative probiotics (L. plantarum S2, L. plantarum O2, L. pentosus S3, L. rhamnosus 14E4) were investigated for their ability to adhere to HT29 cancer cells and to inhibit their and the chemoresistant counterpart (HT29-dx cells) proliferation. Three putative probiotics (S2, S3 and 14E4) were able to decrease viability of both sensitive and chemo-resistant HT-29 cells. Supposing this effect related to secreted metabolites (namely short chain fatty acids (SCFA), exopolysaccharides (EPS) and extracellular proteins) we tested the efficacy of extracellular extracts and butyrate with or without the chemotherapeutic agent doxorubicin (DOXO) (10 µM, 4 h). Increased production of mitochondrial reactive oxygen species (ROS) in HT29 and HT29-dx cells was observed. Moreover, cell exposure to DOXO (10 µM, 24 h) and extracellular extracts (48 h) reduced cell viability. Comparative phenotypic and secretome analyses on the effective/non effective strains, revealed quantitative/qualitative differences in EPS content and protein profiles, suggesting that P40, phage-tail-like and capsid-like proteins may be also involved. These results suggest that food-isolated bacteria releasing bioactive compounds (butyrate, EPS and peculiar proteins) may control cancer cell proliferation and improve their response to chemotherapy.

Enterococcus faecium NCIMB10415 responds to norepinephrine by altering protein profiles and phenotypic characters.

The long-term established symbiosis between gut microbiota and humans is based upon a dynamic equilibrium that, if unbalanced, could lead to the development of diseases. Despite the huge amount of data concerning the microbiota-gut-brain-axis, little information is available on what happens at the molecular level in bacteria, when exposed to human signals. In the present study, the physiological effects exerted by norepinephrine (NE), a human hormone present in significant amounts in the host gut, were analyzed using the commensal/probiotic strain Enterococcus faecium NCIMB10415 as a target. The aim was to compare the protein profiles of treated and untreated bacteria and relating these proteome patterns to some phenotypic modifications important for bacteria-host interaction. Actually, to date, only pathogens have been considered. Combining a gel-free/label-free proteomic analysis with the evaluation of bile salts resistance, biofilm formation and autoaggregation ability (as well as with the bacterial growth kinetics), allowed to detect changes induced by NE treatment on all the tested probiotic properties. Furthermore, exposure to the bioactive molecule increased the abundance of proteins related to stress response and to host-microbe interaction, such as moonlight proteins involved in adhesion and immune stimulation. The results of this investigation demonstrated that, not only pathogens, but also commensal gut bacteria are affected by host-derived hormones, underlining the importance of a correct cross-signalling in the maintenance of gut homeostasis. SIGNIFICANCE: The crucial role played by the human gut microbiota in ensuring host homeostasis and health is definitively ascertained as suggested by the holobiome concept. The present research was intended to shed light on the endocrinological perturbations possibly affecting microbiota. The microbial model used in this study belongs to Enterococcus faecium species, whose controversial role as gut commensal and opportunistic pathogen in the gut ecosystem is well recognized. The results obtained in the present investigation clearly demonstrate that E. faecium NCIMB10415 can sense and respond to norepinephrine, a human hormone abundant at the gut level, by changing protein profiles and physiology, inducing changes that could favor survival and colonization of the host tissues. To our knowledge, this is the first proteomic report concerning the impact of a human hormone on a commensal/probiotic bacterium, since previous research has focused on exploring the effects of neuroendocrine molecules on growth and virulence of pathogenic species.

Influence of ethanol, malate and arginine on histamine production of Lactobacillus hilgardii isolated from an Italian red wine.

Wine, like other fermented foods, may contain biogenic amines produced by lactic acid bacteria via amino acids decarboxylation. The most relevant amines from the toxicological standpoint are histamine and tyramine. The complexity of fermented substrates makes it difficult to suggest a priori how variables can modulate amine production. Lactobacillus hilgardii ISE 5211 was isolated from an Italian red wine. Besides producing lactate from malate, this strain is also able to convert arginine to ornithine and histidine to histamine. In the present investigation we studied the influence of malate, arginine and ethanol on histamine accumulation by L. hilgardii ISE 5211. Ethanol concentrations above 13% inhibit both histamine accumulation and bacterial growth; concentrations below 9% affect neither growth nor histamine production. However, an ethanol concentration of 11% allows a low but continuous accumulation of histamine to occur. Arginine also delays histamine accumulation, while malate appears to have no effect on histidine-histamine conversion.

Correction: Selenium effects on the metabolism of a Se-metabolizing Lactobacillus reuteri: analysis of envelope-enriched and extracellular proteomes.

Correction for 'Selenium effects on the metabolism of a Se-metabolizing Lactobacillus reuteri: analysis of envelope-enriched and extracellular proteomes' by E. Mangiapane et al., Mol. BioSyst., 2014, 10, 1272-1280.

Ten years of subproteome investigations in lactic acid bacteria: A key for food starter and probiotic typing.

The definition of safety and efficacy of food-employed bacteria as well as probiotic strains is a continuous, often unattended, challenge. Proteomic techniques such as 2DE, DIGE and LC/LC-MS/MS are suitable and powerful tools to reveal new aspects (positive and negative) of "known" and "unknown" strains that can be employed in food making and as nutraceutical supplements for human health. Unfortunately, these techniques are not used as extensively as it should be wise. The present report describes the most significant results obtained by our research group in 10years of study on subproteomes in bacteria, chiefly lactic acid bacteria. Production of desired and undesired metabolites, differences between strains belonging to same species but isolated from different ecological niches, the effect of cryoprotectants on survival to lyophilization as well as the adhesive capability of strains, were elucidated by analysis of cytosolic, membrane-enriched, surface and extracellular proteomes. The present review opens a window on a yet largely underexplored field and highlights the huge potential of subproteome investigations for more rational choice of microbial strains as food starters, probiotics and for production of nutraceuticals. These analyses will hopefully contribute to manufacturing safer and healthier food and food supplements in the near future. This article is part of a Special Issue entitled: HUPO 2014.

Purification, biochemical properties and substrate specificity of a catechol 1,2-dioxygenase from a phenol degrading Acinetobacter radioresistens.

A catechol 1,2-dioxygenase (C1,2O) has been purified to homogeneity from Acinetobacter radioresistens grown on phenol as the sole carbon and energy source. The C1,2O appears to be a homodimer, with a molecular mass of 78,000 Da. At relatively high ionic strengths (0.5 M Na2SO4) subunit dissociation occurs and the monomeric unit (38,700 Da) is shown to be active. This phenomenon has never been observed before in dioxygenases. The purified C1,2O contains 0.96 iron(III) ions per unit and spectroscopic measurements suggest the presence of one high-spin iron(III) ion in an environment characteristic of intradiol cleaving enzymes. The NH2-terminal amino acid sequence has been determined and compared to the primary structures of intradiol rings cleaving dioxygenases from other Acinetobacter strains revealing 45% homology with the benzoate-grown A. calcoaceticus ADP-1 and an identity of only one of the 20 amino acids sequenced for the phenol-grown A. calcoaceticus NCIB 8250.

Improved resistance to transition metals of a cobalt-substituted alcohol dehydrogenase 1 from Saccharomyces cerevisiae.

Cobalt-substituted alcohol dehydrogenase 1 was purified from a yeast culture of Saccharomyces cerevisiae. Its reactivity towards different transition metals was tested and compared with the native zinc enzyme. The cobalt enzyme displayed a catalytic efficiency 100-fold higher than that of the zinc enzyme. Copper, nickel and cadmium exerted a mixed-type inhibition, with a scale of inhibition efficiency: Cu(2+)>Ni(2+)>Cd(2+). In general, a higher resistance of the modified protein to the inhibitory action of transition metals was observed, with two orders of magnitude for copper I(50). The presence of nickel in the complexes enzyme-coenzyme-inhibitor-substrate resulted in a decrease of the ampholytic nature of the catalytic site. On the contrary, cadmium and copper exerted an enhancement of this parameter. Electrostatic or other types of interactions may be involved in conferring a good resistance in the basic pH range, making cobalt enzyme very suitable for biotechnological processes.

Catalytic and spectroscopic characterisation of a copper-substituted alcohol dehydrogenase from yeast.

Yeast alcohol dehydrogenase (Y-ADH) is widely studied for its biotechnological importance and various attempts to improve its catalytic properties have been made. In this paper, a catalytically active metal-substituted Y-ADH was prepared in vitro by substituting one zinc atom with copper. EPR and Raman spectroscopy suggest that copper maintains the same co-ordination geometry as zinc in native Y-ADH. The active Cu-ADH shows lower substrate affinity and lower specific activity (SA) than native ADH, but greater than a previously obtained Co-ADH. Furthermore, Cu-ADH maintains its catalytic efficiency in a wider pH range than native enzyme.

Extraction, purification and characterization of ADH1 from the budding yeast Kluyveromyces marxianus.

The enzyme ADH1 has been extracted and purified from the budding yeast Kluyveromyces marxianus, and its enzymatic activity has been compared, with the ADH1 extracted and purified in the same way from the well known yeast Saccharomyces cerevisiae. K. marxianus ADH1 has an optimal temperature higher than the S. cerevisiae enzyme (45-50 degrees vs 35 degrees C), a better stability to pH variations in the oxidative reaction (pH optimum 7.5), a lower Michaelis constant for acetaldehyde, and a good catalytic activity both for fermentative and oxidative reactions. In fact, while in Saccharomyces the constants ratio (velocity constant fermentation/velocity constant oxidation) is about 20,000, in Kluyveromyces the same ratio is only 15. Even if these two Genera are quite related (they belong to the same subfamily) it seems that their ADH1s possess different catalytic properties.

[Frequency of in vitro sensitivity to netilmicin and ceftizoxime on selected highly resistant flora in surgical intensive therapy]. / Studio delle frequenze di sensibilità "in vitro" a netilmicina e ceftizoxima su una flora altamente resistente selezionata in terapia intensiva chirurgica.

The in vitro sensitivity of 132 gram positive and gram negative bacterial strains to Netilmycin and Ceftizoxime was assessed in order to update the statistics on the a priori efficacy of the antibacterial drugs. The flora studied were carefully selected as an extreme case of resistance to the standard antibiotics. Netilmycin was found to be effective against Staphylococci, Pseudomonas and Enterobacter, Ceftizoxime against Serratia, Acinetobacter, Proteus and Klebsiella. Both drugs were equally effective against E. coli and Citrobacter.

[Postoperative monitoring of patients operated on for digestive tract neoplasms using tumor markers. Preliminary results]. / Monitoraggio postoperatorio di pazienti operati per neoplasia digestiva mediante marcatori tumorali. Risultati preliminari.

The report analyses the comparative efficacy of three different tumour markers (CEA, GICA and TPA) in providing indications for the postoperative monitoring of patients treated for cancer of the digestive tract. Assessment of effectiveness of chemotherapy produced different results according to the site of the tumour (stomach, colon or pancreas). In the case of cancers of the pancreatic head, the influence of jaundice on the markers was also evaluated, to discover which marker provided most information on the tumour investigated.

[Hospital infections in an emergency surgery ward]. / Infezioni ospedaliere in un reparto di chirurgia d'urgenza.

A study was conducted on hospital infections in an emergency surgery ward. The survey lasted one year and confirmed the higher incidence of infections after contaminated and dirty operations. Rates of infection were also high after clean operations due to the large number of subjects at risk among the group examined. The most commonly encountered micro-organisms in the infections of surgical wounds were, in order, E. coli, Str. faecalis and Staph. aureus, the latter being only minimally responsive to the standard antibiotics. Several proposals are advanced for prophylaxis in the attempt to halt the circulation of strains with a multiple resistance to antibiotics.

[Use of a single daily dose of tobramycin sulfate for the prevention of infections caused by the positioning of fixed drains in body cavities]. / Uso della tobramicina solfato a dose unica giornaliera nella prevenzione delle infezioni da posizionamento di drenaggi a dimora in cavità corporee.

The clinical and bacterial efficiency of tobramycin sulphate administered once a day (150 mg/day) in the prevention of pleural cavity and bladder infections was evaluated by considering 30 surgical patients fitted with thoracostomic tubes or bladder catheters. Pleural cavity sterility was maintained in 74% of cases and bladder sterility was maintained or obtained in 100% of cases. These results confirm the success of the treatment.

[Variations in serum kininase II activity in acute jaundice]. / Variazioni dell'attività sierica kininasica II negli itteri acuti.

An assay of Kininase II activity in the serum of 92 jaundice patients revealed a significant difference between the group with viral hepatitis (268.48 +/- 70.93 U/ml), that with biliary obstructions (133.05 +/- 37.64 U/ml) and with cirrhosis (173.76 +/- 79.56 U/ml). The increase encountered in patients with medical jaundice correlates well with total bilirubinaemia but not with cytolysis enzymes. This suggests failed demolition of ACE by the hepatocyte during cellular stress.

[Infusion and transfusion therapy in polytraumatized patients]. / Terapia infusionale e trasfusionale nei politraumatizzati.

Considerations of a physiopathological nature are expressed with regard to haemorrhagic multiple-injury patients in the light of the experience acquired in first-aid practice at the University of Turin department of emergency surgery. While treatment of the basic injury is the key to therapy, sufficient infusion management and careful monitoring of coagulation parameters before and during transfusion may be of fundamental importance in survival. By the same token, not all blood products available can be used indiscriminately. A treatment and monitoring protocol for the reinstatement of volaemia and corpuscle mass, and for the correction of coagulopathy due to loss, consumption and wash-out is proposed.

[Exogenous infections and environmental flora. Criteria for the characterization of bacteria and parameters influencing their spread]. / Infezioni esogene e flora ambientale. Alcuni criteri per caratterizzare i batteri ed i parametri che ne influenzano l'espansione.

An assessment was made of environmental micro-organisms isolated in the University of Turin Intensive Surgical Care Unit and Emergency Surgery Department to determine the possible exogenous origin of post-operative infections. The enzymatic characters and antibiotypes were determined for some species, and subsequently compared with similar characters of the "in vivo" isolated flora. A monthly disinfection method based on comparison among three antiseptic agents (chlorexidine, formaldehyde and formotetronium) considerably reduced the flora. The data obtained, while showing that most of the infections observed were endogenous, also provided indications on the way in which the spread of certain Gram-negative germs, often involved in post-operative and post-traumatic infections, could be contained.

[Clinical use of a minicomputer in the evaluation of chemico-clinical parameters in the diagnosis of hepato-biliary-pancreatic diseases of surgical significance. 3 methods compared]. / L'uso clinico di un minicomputer per la valutazione di alcuni parametri chimico-clinici nella diagnostica delle malattie epato-bilio-pancreatiche di interesse chirurgico. Tre metodi a confronto.

Bayes theorem; multivariance analysis, and the discriminating function of Mahalanobis applied to sixteen laboratory examinations employed in the diagnosis of diseases of the extrahepatic bile ducts and pancreas gave the following results: 1) the weight of each test was first measured in general terms, and then with regard to different levels of differentiation, with a classification in decreasing order in each case; 2) little redundancy was noted, and few examinations were of relatively negligible importance; furthermore, combinations of several tests did not lead to positive results; 3) reclassification according to the formula of Bayes and by means of the discriminating function proved sound supports for clinical decision, since they provided 70% and 80% of exact classification respectively; 4) the importance of the minicomputer in the construction of experimental diagnostic models was substantiated.

[Diagnostic and prognostic value of the carcinoembryonic antigen in colonic cancer]. / Valore diagnostico e prognostico del CEA nel carcinoma del colon.

A group of 50 patients operated for carcinoma of the colon in June 1980-December 1981 with a follow-up period of min. 2 months-max. 15 months is presented. C.E.A. measurements were taken in all patients pre and post-operatively and then every 2 months in an attempt to establish its diagnostic and prognostic value. In immediate diagnostic and prognostic terms, pre-operative C.E.A. appears to be insignificant. However both pre-operative C.E.A. and C.E.A. at a distance of 6 months were found to be reliable for long term (max. 15 months) prognosis.

Selenium effects on the metabolism of a Se-metabolizing Lactobacillus reuteri: analysis of envelope-enriched and extracellular proteomes.

Selenium (Se) has received great attention in the last few years, as it is considered to be essential for human health (prevention of viral infections, heart diseases and ageing-related diseases). Se deficiency can be counteracted by the administration of selenium-enriched probiotics that are able to convert inorganic selenium into less toxic and more bio-available organic forms. This study was performed on Lactobacillus reuteri Lb2 BM DSM 16143, a probiotic LAB previously demonstrated to be able to fix Se into selenocysteines. The aim was to assess Se influence on its metabolism, by a 2-DE proteomic approach, on two different cellular districts: envelope-enriched and extracellular proteomes. While in the envelope-enriched fraction 15 differentially expressed proteins were identified, in the extracellular proteome no quantitative difference was detected. However, at a molecular level, we observed the insertion of Se into selenocysteine, exclusively under the stimulated conditions. The obtained results confirmed the possibility to use L. reuteri Lb2 BM DSM 16143 as a carrier of organic Se that can be easily released in the gut becoming available for the human host.