Biofilm Formation on Stainless Steel by Streptococcus thermophilus UC8547 in Milk Environments Is Mediated by the Proteinase PrtS.

In Streptococcus thermophilus, gene transfer events and loss of ancestral traits over the years contribute to its high level of adaptation to milk environments. Biofilm formation capacity, a phenotype that is lost in the majority of strains, plays a role in persistence in dairy environments, such as milk pasteurization and cheese manufacturing plants. To investigate this property, we have studied S. thermophilus UC8547, a fast-acidifying dairy starter culture selected for its high capacity to form biofilm on stainless steel under environmental conditions resembling the dairy environment. Using a dynamic flow cell apparatus, it was shown that S. thermophilus UC8547 biofilm formation on stainless steel depends on the presence of milk proteins. From this strain, which harbors the prtS gene for the cell wall protease and shows an aggregative phenotype, spontaneous mutants with impaired biofilm capacity can be isolated at high frequency. These mutants lack the PrtS expendable island, as confirmed by comparison of the genome sequence of UC8547Δ3 with that of the parent strain. The prtS island excision occurs between two 26-bp direct repeats located in the two copies of the ISSth1 flanking this genomic island. The central role of PrtS was confirmed by analyzing the derivative strain UC8547Δ16, whose prtS gene was interrupted by an insertional mutation, thereby making it incapable of biofilm formation. PrtS, acting as a binding substance between the milk proteins adhered to stainless steel and S. thermophilus cell envelopes, mediates biofilm formation in dairy environments. This feature provides S. thermophilus with an ecological benefit for its survival and persistence in this environment.IMPORTANCE The increased persistence of S. thermophilus biofilm has consequences in the dairy environment: if, on the one hand, the release of this microorganism from biofilm can promote the fermentation of artisanal cheeses, under industrial conditions it may lead to undesirable contamination of dairy products. The study of the molecular mechanism driving S. thermophilus biofilm formation provides increased knowledge on how an ancestral trait affects relevant phenotypes, such as persistence in the environment and efficiency of growth in milk. This study provides insight into the genetic factors affecting biofilm formation at dairy plants.

Experimental and ab initio studies of the reactive processes in gas phase i-C3H7Br and i-C3H7OH collisions with potassium ions.

Collisions between potassium ions and neutral i-C3H7Br and i-C3H7OH, all in their electronic ground state, have been studied in the 0.10-10.00 eV center of mass (CM) collision energy range, using the radiofrequency-guided ion beam technique. In K(+) + i-C3H7Br collisions KHBr(+) formation was observed and quantified, while the analogous KH2O(+) formation in K(+) + i-C3H7OH was hardly detected. Moreover, formation of the ion-molecule adducts and their decomposition leading to C3H7(+) and either KBr or KOH, respectively, have been observed. For all these processes, absolute cross-sections were measured as a function of the CM collision energy. Ab initio structure calculations at the MP2 level have given information about the potential energy surfaces (PESs) involved. In these, different stationary points have been characterized using the reaction coordinate method, their connectivity being ensured by using the intrinsic-reaction-coordinate method. From the measured excitation function for KHBr(+) formation the corresponding thermal rate constant at 303 K has been calculated. The topology of the calculated PESs allows an interpretation of the main features of the reaction dynamics of both systems, and in particular evidence the important role played by the potential energy wells in controlling the reactivity for the different reaction channels.

Experimental study of the reactive processes in the gas phase K(+) + i-C3H7Cl collisions: a comparison with Li and Na ions.

Reactive collisions between alkali ions (Li(+), Na(+), and K(+)) and halogenated hydrocarbon molecules have been studied recently in our research group. In this paper, we have reported on the K(+) + i-C3H7Cl system in the 0.20-14.00 eV center-of-mass energy range using a radio frequency guided-ion beam apparatus developed in our laboratory. Aiming at increasing our knowledge about this kind of reactions, we compare our latest results for K(+) with those obtained previously for Li(+) and Na(+). While the reaction channels are the same in all three cases, their energy profiles, reactivity, measured reactive cross-section energy dependences, and even their reaction mechanisms, differ widely. By comparing experimentally measured reactive cross-sections as a function of the collision energy with the ab initio calculations for the different potential energy surfaces, a qualitative interpretation of the dynamics of the three reactive systems is presented in the present work.

Assessment of tetracycline and erythromycin resistance transfer during sausage fermentation by culture-dependent and -independent methods.

The food chain is considered one of the main routes of antibiotic resistance diffusion between animal and human population. The resistance to antimicrobial agents among enterococci could be related to the efficient exchange of transferable genetic elements. In this study a sausage model was used to evaluate the persistence of antibiotic resistant enterococci during meat fermentation and to assess horizontal gene transfer among bacteria involved in meat fermentation. Enterococcus faecalis OG1rf harbouring either pCF10 or pAMß1 plasmid was used as donor strain. The analysis of population dynamics during fermentation confirmed that the human isolate E. faecalis OG1rf was able to colonize the meat ecosystem with similar growth kinetics to that of food origin enterococci and to transfer the mobile genetic elements coding for tetracycline and erythromycin resistances. Transconjugant strains were detected after only two days of fermentation and increased their numbers during ripening even in the absence of selective antibiotic pressure. By means of culture-dependent and -independent molecular techniques, transconjugant strains carrying both tetracycline and erythromycin resistance genes were identified in enterococci, pediococci, lactobacilli and staphylococci groups. Our results suggest that the sausage model provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among food microbiota.

MMP(-2) expression in skeletal muscle after strength training.

The aim of this study was to assess the effects of resistance training on ladders (RTL) on MMP(-2) expression and blood lactate concentration [La-]. 30 male (3 months of age), albino rats were divided into 3 groups: sedentary control (SC, n=10), low resistance exercise training (Low-IntRT, n=10) and high-intensive exercise training (High-IntRT, n=10). Animals of High-IntRT were submitted to a progressively increasing overload in relation to body weight until exhaustion, while the Low-IntRT group performed the same exercise regimen with no external load. The program had a frequency of 3 times per week over 8 weeks. MMP(-2) expression of tibialis anterior muscle and [La-] were measured. While there was a significant increase of MMP(-2) (pro-form) in both groups, only High-IntRT significantly increased MMP(-2) in active-form (p<0.05). Both trained groups exhibited an increase in [La-] when compared to controls, however, the increase in [La-] was significantly higher in the High-IntRT compared to Low-IntRT (p<0.05). Strong correlation was found between MMP(-2) (active form) and [La-] in High-IntRT (r=0.91). RTL in using low and high-intensity exercise can serve as a model to demonstrate different responses of MMP(-2) expression in an animal model. It appears active form expression of MMP(-2) is modulated by exercise intensity.

Olive fertility as affected by cross-pollination and boron.

Self-compatibility of local olive (Olea europaea L.) accessions and of the cultivars "Frantoio" and "Leccino" was investigated in Garda Lake area, northern Italy. Intercompatibility was determined for "Casaliva," "Frantoio," and "Leccino," as well as the effects of foliar Boron applications (0, 262, 525, or 1050 mg.L(-1)) applied about one week before anthesis on fruit set, shotberry set, and on in vitro pollen germination. Following self-pollination, fruit set was significantly lower and the occurrence of shot berries significantly higher than those obtained by open pollination. No significant effect of controlled cross-pollination over self-pollination on fruit set and shotberry set was detectable. B treatments increased significantly fruit set in "Frantoio" and "Casaliva" but not in "Leccino." B sprays had no effect on shotberry set, suggesting that these parthenocarpic fruits did not strongly compete for resources allocation and did not take advantage of increased B tissue levels. Foliar B application enhanced in vitro pollen germination, and the optimal level was higher for pollen germination than for fruit set. Our results highlight the importance of olive cross pollination for obtaining satisfactory fruit set and the beneficial effect of B treatments immediately prior to anthesis, possibly by affecting positively the fertilisation process and subsequent plant source-sink relations linked to fruitlet retention.

Reactive processes in gas phase Na(+)-iso-C3H7Cl collisions: experimental guided-ion-beam and ab initio studies of the reactions on the ground singlet potential surface of the system up to 12.00 eV.

Reactive processes, taking place when sodium ions collide with neutral iso-C(3)H(7)Cl molecules in the 0.02-12.00 eV range of energies in the center of mass frame, have been studied using an octopole radiofrequency guided-ion-beam apparatus developed in our laboratory. A dehydrohalogenation reaction channel leading to Na(C(3)H(6))(+) formation has been observed up to 1.00 eV while another process producing NaHCl(+) continues up to 4.00 eV. Furthermore, C(3)H(7)(+) formation resulting from decomposition of the reactants, ion-molecule adducts, has also been observed as well as its decomposition into C(2)H(3)(+) on increasing collision energy. Cross-section energy dependences for all these reactions have been obtained in absolute units. The ab initio electronic structure calculations have been done at the MP2 level for the colliding system ground singlet potential surface, giving information on the reactive surface main topological features. From the surface reactants side to the products' one, different potential wells and barriers have been characterized and their connectivity along the reaction evolution has been established using the intrinsic-reaction-coordinate method, thus interpreting the dynamical evolution of the reactants' collision complex to products. Experimental results demonstrate that NaHCl(+) can be produced via different channels. Reaction rate constants at 308.2 K for both dehydrohalogenation reactions have been calculated from measured excitation functions. It has been also confirmed that the reactants adduct decomposition giving C(3)H(7)(+) and NaCl takes place on the same potential surface. A qualitative interpretation of the experimental results in terms of ab initio calculations is also given.

Cross-section energy dependence of the [C6H6-M]+ adduct formation between benzene molecules and alkali ions (M = Li, Na, K).

The association reactions of benzene molecules with alkali ions M(+) (Li(+), Na(+) and K(+)) under single collision conditions have been studied using a radiofrequency-guided-ion-beam apparatus and mass spectrometry characterization of the different adducts. Cross-section energy dependences for [M-C(6)H(6)](+) adduct formation have been measured at collision energies up to 1.20 eV in the center of mass frame. All excitation functions decrease when collision energy increases, showing the expected behaviour for barrierless reactions. From ab initio chemical structure calculations at the MP2(full) level, the formation of the adducts makes evident the alkali ion-benzene non-covalent chemical bond. The cross-section energy dependence and the role of radiative cooling rates and unimolecular decomposition on the stabilization of the energized collision complex are also discussed.

The impact of liver resection on survival for locally advanced intrahepatic cholangiocarcinoma tumors: A propensity score analysis.

BACKGROUND: Aim of work was to investigate the prognostic impact of liver resection (LR) on locally advanced Intrahepatic Cholangiocarcinoma (IC) in comparison to alternative palliative chemotherapy (CTx). METHOD: A retrospective cohort study performed utilizing Surveillance, Epidemiology, and End Results (SEER) database to identify Locally advanced IC patients. Based on the American Joint Committee on Cancer (AJCC) Staging System, locally advanced IC was defined as: Stage III and IVa - 7th edition (7th-ed) or stage III - 8th edition (8th-ed). Study population were sub-classified into: LR group and a propensity score (PS) matched CTx group. RESULTS: In 7th-ed module, the median survival for LR group (n = 154) was 35 months, and the 3-year survival rate was 40.8%. In PS matched CTx group (n = 154); the median survival was 14 months and the 3-year survival rate was 5.5% (P = 0.007). Survival rates were superior for LR group over PS matched CTx group in 8th-ed module as well. Worse prognosis has been reported in LR patients above 65 years old (HR 2.618, P = 001) and in multifocal lesions (HR 1.890, P = 0.025). CONCLUSION: Hepatic resection was associated with a favorable impact on prognosis over chemotherapy for IC stage III and IVa of the 7th edition and for stage IIIb of 8th edition of AJCC staging system. Worse outcome has been observed in LR patients >65 years and with multifocal lesions. Randomized control studies are recommended to confirm the role of surgical resection in the management for advanced cases of IC, and to clarify the related prognostic factors.

Guided-ion-beam and ab initio study of the Li+, K+, and Rb+ association reactions with gas-phase butanone and cyclohexanone in their ground electronic states.

The association reactions between Li(+), K(+), and Rb(+) (M) and butanone and cyclohexanone molecules under single collision conditions have been studied using a radiofrequency-guided ion-beam apparatus, characterizing the adducts by mass spectrometry. The excitation function for the [M-(molecule)](+) adducts (in arbitrary units) has been obtained at low collision energies in the 0.10 eV up to a few eV range in the center of mass frame. The measured relative cross sections decrease when collision energy increases, showing the expected energy dependence for adduct formation. The energetics and structure of the different adducts have been calculated ab initio at the MP2(full) level, showing that the M(+)-molecule interaction takes place through the carbonyl oxygen atom, as an example of a nontypical covalent chemical bond. The cross-section energy dependence and the role of radiative cooling rates allowing the stabilization of the collision complexes are also discussed.

An experimental guided-ion-beam and ab initio study of the ion-molecule gas-phase reactions between Li+ ions and iso-C3H7Cl in their ground electronic state.

Reactive collisions between Li(+) ions and i-C(3)H(7)Cl molecules have been studied in the 0.20-12.00 eV center-of-mass energy range using an octopole radio frequency guided-ion beam apparatus recently developed in our laboratory. At low collision energies, dehydrohalogenation reactions giving rise to Li(C(3)H(6))(+) and Li(HCl)(+) are the main reaction channels, while at higher ones C(3)H(7)(+) and C(2)H(3)(+) become dominant, all their reactive cross sections having been measured as a function of the collision energy. To obtain information about the potential energy surfaces (PESs) on which the reactive processes take place, ab initio calculations at the MP2 level have been performed. For dehydrohalogenations, the reactive ground singlet PES shows ion-molecule adduct formation in both the reactant and product sides of the surface. Following the minimum energy path connecting both minima, an unstable intermediate and the corresponding barriers, both lying below the reactant's energy, have been characterized. The entrance channel ion-molecule adduct is also involved in the formation of C(3)H(7)(+), which then generates C(2)H(3)(+) via an CH(4) unimolecular elimination. A qualitative interpretation of the experimental results based on ab initio calculations is also included.

Endoscopic treatment of bile duct complications after orthotopic liver transplantation.

AIM: To assess the indications and results of endoscopic retrograde cholangio-pancreatography (ERCP) in patients who have undergone ortotopic liver transplantation (OLT). METHODS: We reviewed data from 42 consecutive patients who underwent ERCP for biliary complications after OLT over an 8-year period, in particular recording indications and success of the treatment after a mean of 17 months follow-up. RESULTS: Cholangiograms performed in 33/42 patients (79%) displayed anastomotic strictures in 17 patients (52%), bile duct stones in 8 (24%), both bile duct stones and an anastomotic stricture in 2 (6%), papillary stenosis in 1 (3%), and anastomotic biliary leakage in 1 (3%). In contrast, the contrastogram was normal in four patients (12%). Stone extraction was completed in 9/10 patients (90%) with a mean of 1.2 sessions, while stricture dilation was achieved in 12/19 patients (63%) after a mean of 1.7 sessions, by stent positioning (n = 7), balloon dilation (n = 4), or Soehendra dilator (n = 1). Both biliary leakage and papillary stenosis were cured by ERCP. Only one procedure-related complication -- severe pancreatitis (2.4%) -- was observed and no mortality. CONCLUSION: ERCP is a safe and effective mode of management of bile duct complications after OLT. It should be attempted before a surgical approach. Better results are obtained for treatment of biliary stones than of anastomotic strictures.

A new experimental model of isolated perfused pig liver to support acute hepatic failure.

Liver transplantation still represents the only effective treatment for patients with liver failure, but many patients die while awaiting transplantation, even though many attempts have been made to increase the organ procurement rates and to partially support hepatic function in recent years. Our aim was to design an "open" ex vivo perfused liver model to simplify liver support using an isolated porcine liver perfused with arterial and portal blood from the recipient pig, while monitoring the metabolic capacity of the supporting graft. It was possible to perfuse the isolated liver for 6 hours as a bridging procedure with satisfactory hemodynamic homeostasis controlled by software biofeedbacks.

Hepatitis C virus-related cirrhosis as a significant mortality factor in intention-to-treat analysis in liver transplantation.

INTRODUCTION: Liver transplantation represents the gold standard for the treatment of chronic liver disease. The whole transplantation process was assessed using an intention-to-treat analysis and considering patients from the time of their inclusion on the list and throughout lengthy follow-up. MATERIALS AND METHODS: From January 1, 1999 to June 1, 2004, 373 adults joined the waiting list for liver transplantation at our institution. The main variables analyzed were: age, gender, etiology, Model for End-stage Liver Disease score, Child-Pugh class, United Network for Organ Sharing (UNOS) status. Global survival was evaluated using intention-to-treat analysis from the time of patient inclusion in the list to the end of their late follow-up. RESULTS: The median waiting time was 20 months (range 0.1 to 70.2). By univariate analysis, the variables significantly influencing survival when patients joined the waiting list were: encephalopathy; ascites, poor nutritional status, Child-Pugh class C, UNOS 2, hepatitis C virus (HCV) and bilirubin > 2 mg/dL. By multivariate analysis, only HCV-related cirrhosis emerged as having an independent prognostic value. By intention-to-treat analysis, the 5-year survival rate was 67% and 79% for HCV-positive and HCV-negative patients, respectively (P = .0003). CONCLUSIONS: HCV-related cirrhosis is an independent prognostic factor for survival according to an intention-to-treat analysis. Different inclusion criteria or treatments while on the waiting list and after transplantation need to be considered in the future for HCV-positive patients.

Prognostic impact of model for end-stage liver disease score in patients undergoing liver transplantation with suboptimal livers.

BACKGROUND/AIMS: The aim of this retrospective study is to analyze the prognostic impact of Model for End-Stage Liver Disease (MELD) score in patients undergoing liver transplantation (OLT) with suboptimal livers. METHODS: Between January 2002 and January 2006, 160 adult patients with liver cirrhosis received a whole liver for primary OLT at our institution including 81 with a suboptimal liver (SOL group) versus 79 with an optimal liver (group OL). The definition of suboptimal liver was: one major criterion (age >60 years, steatosis >20%) or at least two minor criteria: sodium >155 mEq/L, Intensive Care Unit stay >7 days, dopamine >10 microg/kg/min, abnormal liver tests, and relevant hemodynamic instability. RESULTS: Baseline recipients characteristics were comparable in the two study groups. The SOL group had a significantly greater number of early graft deaths (<30 days) than the OL group, while the 3-year Kaplan-Meier patient survivals were similar. Using logistic regression, MELD score was significantly related to patient death only in the SOL group (P = .01), and the receiver operator characteristics curve method identified 17 as the best MELD cutoff with the 3-year survival of 93% versus 85% for MELD < or =7 versus >17, respectively (P > 05). In comparison, it was 94% and 72% in the SOL group (P < .05). Similarly, MELD >17 was significantly associated with early graft death rates only in the SOL group. CONCLUSION: This study advised surgeons to not use suboptimal livers for patients with advanced MELD scores, thus supporting a donor-recipient matching policy.

Combined liver and kidney transplantation: analysis of Padova experience.

BACKGROUND/AIM: The main indications for combined liver and kidney transplantation (CLKT) are as follows: (1) cirrhosis with renal damage dependent or not upon liver disease, (2) renal failure with dialysis and concomitant liver end-stage disease, (3) congenital diseases, and (4) enzymatic liver deficiency with concomitant renal failure. The aim of this study was to evaluate our results with CLKT both in adult and pediatric patients. METHODS: From September 1995 to September 2006, 15 CLKT (2.8%) among 541 liver transplantations included 4 pediatric patients (27%). The main indications for CLKT were hepatitis C virus (HCV) and polycystic diseases in adult patients, and primary hyperoxaluria in pediatric patients. RESULTS: The double transplantation was performed from the same donor in all cases. All adult patients received whole liver grafts, whereas 3 split transplants and 1 whole liver graft were transplanted in the pediatric patients. Median liver and kidney cold ischemia times were 468 and 675 minutes, respectively. After a median follow-up of 36 months (range, 1-125), the overall survival rate was 80%. Five-year patient and graft survival rates were 100% for adult CLKT, whereas they were 50% for pediatric patients. We observed only 2 cases (18%) of delayed renal function, requiring temporary hemodialysis with progressive graft improvement. There was only 1 case of kidney retransplantation due to early graft nonfunction in a pediatric patient. CONCLUSION: Although CLKT is related to major surgical risks, results after transplantation are satisfactory with an evident immunological advantage.

Liver Retransplantation for Hepatic Abscess Due to Hepatic Artery Thrombosis: A Case Report.

INTRODUCTION: Hepatic artery thrombosis (HAT) is a well-recognized complication of liver transplantation (LT). HAT is an important risk factor for infectious, in particular hepatic abscess, which can cause graft loss and increasing morbidity and mortality. CASE REPORT: We present a case report of complicated LT in a 52-year-old Caucasian man with primary sclerosing cholangitis. In 2007 the patient was included on the waiting list in Padua for LT. In 2012 the patient underwent percutaneous transhepatic biliary drainage for bile duct stricture, complicated with acute pancreatitis. A diagnostic laparoscopy was performed with choledochotomy and Kehr's T tube drainage. On February 14, 2012, the patient underwent LT with arterial reconstruction and choledochojejunostomy. The postoperative course was complicated with HAT, multiple liver abscesses, and sepsis associated with bacteremia due to Enterococcus faecium despite massive intravenous antibiotic therapy and percutaneous drainages. On November 28, 2012, the patient underwent retransplantation. Four years after transplantation the patient is still in good general condition. CONCLUSION: Hepatic abscess formation secondary to HAT following LT is a major complication associated with important morbidity and mortality. In selected cases retransplantation should be considered as our case demonstrates.

Relationships among ensiling, nutritional, fermentative, microbiological traits and contamination in corn silages addressed with partial least squares regression.

The objective of this work was to reduce the predictor dimensionality and to develop a model able to forecast contamination in corn silages. A survey on 33 dairy farms was performed, and samples from core, lateral, and apical parts of the feed-out face of corn silage bunkers were analyzed for chemical, biological (digestible and indigestible NDF), fermentative (pH, ammonia nitrogen, lactic acid, VFA, and ethanol), and microbiological (yeasts and molds) traits. Corn silage samples were analyzed for cell and spore counts by adoption of a molecular DNA-based method. A partial least squares (PLS) regression with a leave-one-out cross-validation method was used to reduce the dimensionality of the original predictors ( = 30) by projecting the independent variables into latent constructs. In a first step of the model development, the importance of independent variables in predicting contamination was assessed by plotting factor loadings of both dependent and independent variables on the first 2 components and by verifying for each predictor the variable influence on projection values adopting the Wold's criterion as well as the entity of standardized regression coefficients. Three ensiling characteristics (bunker type, presence of lateral wrap plastic film, and penetration resistance as a measurement of the ensiled mass density), a chemical trait (DM), 9 characterizations of the fermentative profile (pH, ammonia nitrogen, acetic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, ethanol, and lactic acid), and 2 microbiological traits (yeasts and molds) were retained as important terms in the PLS model. Three reduced-variable PLS (rPLS) regressions-the first based on ensiling, chemical, fermentative, and microbiological retained important variables (rPLSecfm); the second based on chemical, fermentative, and microbiological retained important traits (rPLScfm); and the last based on only chemical and fermentative retained important variables (rPLScf)-were performed. The model that best fit the measurements was rPLSecfm. The rPLScfm and rPLScf models had similar regression performances but higher mean square errors of prediction than rPLSecfm. However, all tested models seemed adequate to rank corn silages for low, medium, and high risks of contamination. To avoid the visit on farm by trained people required to measure penetration resistance, the use of the rPLScf model is suggested as a useful tool to assess the risk of in corn silage.