Microbial Succession in Spontaneously Fermented Grape Must Before, During and After Stuck Fermentation

The microbial succession in spontaneously fermenting Riesling must was investigated from the beginning(pressing) until the end (sulphuring) of the fermentation in two harvest years (2008 and 2009) at a Mosellewinery (Germany). In both years, the fermentation was interrupted by a stuck period. The length of thestuck period varied considerably (20 weeks in 2008 and one week in 2009). Different yeasts (Candida,Debaryomyces, Pichia, Hanseniaspora, Saccharomyces, Metschnikowia, Cryptococcus, Filobasidium andRhodotorula) and bacteria (Gluconobacter, Asaia, Acetobacter, Oenococcus, Lactobacillus, Bacillus andPaenibacillus) were isolated successively by plating. The main fermenting organism was Saccharomycesuvarum. Specific primers were developed for S. uvarum, H. uvarum and C. boidinii, followed by thedetermination of the total cell counts with qPCR. The initial glucose concentration differed between thetwo years and was 116 g/L in 2008 and 85.4 g/L in 2009. Also, the fructose concentrations were differentin both years (114 g/L in 2008 and 77.8 g/L in 2009). The stuck period appeared when the glucose/fructoseratio was 0.34 and 0.12 respectively. The microbiota changed during the stuck period

Bactericidal action of carvacrol towards the food pathogen Bacillus cereus : a case study of a novel approach to mild food preservation

A new trend in food preservation is the use of mild preservation systems, instead of more severe techniques such as heating, freezing or addition of chemical preservatives. Carvacrol, a phenolic compound present in the essential oil fraction of oreganum and thyme, is known for its antimicrobial activity since ancient times. This thesis describes a study of the antimicrobial activity of carvacrol towards the foodborne pathogen B. cereus . Carvacrol shows a dose-related inhibition of growth of B. cereus . Concentrations of 0.75 mM and higher inhibit growth completely at 8°C. Below 0.75 mM, carvacrol extends the lag-phase and reduces the specific growth rate as well as the final population density. Exposure to 0.75-3 mM carvacrol decreases the number of viable cells of B. cereus exponentially. Spores are approximately two fold more resistant towards carvacrol than vegetative cells.The incubation and exposure temperature have a significant influence on the sensitivity of B. cereus to carvacrol. An increase of the growth temperature from 8°C to 30°C decreases the fluidity of the membrane of vegetative cells and as a consequence, B. cereus becomes less sensitive to carvacrol. The change in membrane fluidity is probably the result of a higher percentage of lower melting lipids in the membranes at 8°C (chemical process) as an adaptation to lower temperature. Cells need to maintain an adequate proportion of the liquid-crystalline lipid in the membrane, as this is the ideal physical state of the membrane. On the other hand, an increase of the exposure temperature from 8 to 30°C, reduces the viability again. This can be explained by an increase of the membrane fluidity at a higher temperature as a result of melting of the lipids (physical process). At a higher membrane fluidity, relatively more carvacrol can dissolve in the membrane and the cells will be exposed to relatively higher concentrations than at a lower membrane fluidity.Not only the temperature plays a role in the activity of carvacrol, also pH is an important factor. The sensitivity of B. cereus to carvacrol is reduced at pH 7, compared to other pH-values between pH 4.5 and 8.5.Carvacrol interacts with the cytoplasmic membrane by changing its permeability for cations such as K +and H +. Consequently, the dissipation of the membrane potential (Δψ) andΔpH leads to inhibition of essential processes in the cell, such as ATP synthesis, and finally to cell death. At carvacrol concentrations as low as 0.15 mM,Δψis completely dissipated, however the viable count of B. cereus is not affected.Vegetative cells of B. cereus can adapt to carvacrol when the compound is present at concentrations below the MIC-value. Compared to non-adapted cells, lower concentrations of carvacrol are needed to obtain the same reduction in viable count of adapted cells. Adapted cells were found to have a lower membrane fluidity, caused by a change in the fatty acid composition and head group composition of the phospholipids in the cytoplasmic membrane. Adaptation to 0.4 mM carvacrol increases the phase transition temperature of the lipid bilayer (T m ) from 20.5°C to 28.3°C. Addition of carvacrol to cell suspensions of adapted B. cereus cells decreases T m again to 19.5°C, approximately the same value as was found for non-adapted cells in the absence of carvacrol.Incubation of cooked rice in the presence of different carvacrol concentrations results in a dose-related reduction of the viable count of B. cereus . Concentrations of 0.15 mg/g and above, reduce the viable count, leading to full suppression of growth at 0.38 mg/g. The influence of carvacrol on the viable count is dependent on the initial inoculum size. Although carvacrol is an effective inhibitor of growth of B. cereus in rice, it could affect the flavour and taste of the product at concentrations where full suppression of growth is observed. However, strong synergistic activity is observed when carvacrol is combined with the biosynthetic precursor cymene or the flavour enhancer soya sauce. This makes it possible to use lower carvacrol concentrations and consequently a smaller influence on the sensoric properties of the rice is expected.Besides its influence on the viability of vegetative cells, carvacrol also shows inhibition of diarrhoeal toxin production by B. cereus at concentrations below the MIC-value. Addition of 0.06 mg/ml carvacrol to the growth medium, inhibits the toxin to 21% of the control (no carvacrol added). The inhibition correlates with the reduction of the viable count of B. cereus in the presence of carvacrol. At the same time, the total amount of cells did not change. In mushroom soup, also an inhibition of the toxin production was observed, however, the viable count did not change. This effect on the toxin production is most probably caused by a lack of sufficient metabolic energy, since carvacrol affects ATP synthesis. The cell will use its low levels of ATP to maintain its viability, rather than using it for toxin production or excretion. It could also be possible that the decreased toxin synthesis in BHI was the result of the lower amount of viable cells. The inhibition of toxin production at carvacrol concentrations which do permit growth of B. cereus , reduces the risk of food intoxication by this pathogen.In conclusion, carvacrol may play an important role in future as a natural antimicrobial compound. However, its application will most probably be in combination with other natural antimicrobial systems.</p

An Open-Source Storage Solution for Cryo-Electron Microscopy Samples

Cryo-electron microscopy (cryo-EM) enables the study of biological structures in situ in great detail and to solve protein structures at Ångstrom level resolution. Due to recent advances in instrumentation and data processing, the field of cryo-EM is a rapidly growing. Access to facilities and national centers that house the state-of-the-art microscopes is limited due to the ever-rising demand, resulting in long wait times between sample preparation and data acquisition. To improve sample storage, we have developed a cryo-storage system with an efficient, high storage capacity that enables sample storage in a highly organized manner. This system is simple to use, cost-effective and easily adaptable for any type of grid storage box and dewar and any size cryo-EM laboratory.Microbial Biotechnolog

The Amsterdam wrist rules: The multicenter prospective derivation and external validation of a clinical decision rule for the use of radiography in acute wrist trauma

Background: Although only 39 % of patients with wrist trauma have sustained a fracture, the majority of patients is routinely referred for radiography. The purpose of this study was to derive and externally validate a clinical decision rule that selects patients with acute wrist trauma in the Emergency Department (ED) for radiography. Methods: This multicenter prospective study consisted of three components: (1) derivation of a clinical prediction model for detecting wrist fractures in patients following wrist trauma; (2) external validation of this model; and (3) design of a clinical decision rule. The study was conducted in the EDs of five Dutch hospitals: one academic hospital (derivation cohort) and four regional hospitals (external validation cohort). We included all adult patients with acute wrist trauma. The main outcome was fracture of the wrist (distal radius, distal ulna or carpal bones) diagnosed on conventional X-rays. Results: A total of 882 patients were analyzed; 487 in the derivation cohort and 395 in the validation cohort. We derived a clinical prediction model with eight variables: age; sex, swelling of the wrist; swelling of the anatomical snuffbox, visible deformation; distal radius tender to palpation; pain on radial deviation and painful axial compression of the thumb. The Area Under the Curve at external validation of this model was 0.81 (95 % CI: 0.77-0.85). The sensitivity and specificity of the Amsterdam Wrist Rules (AWR) in the external validation cohort were 98 % (95 % CI: 95-99 %) and 21 % (95 % CI: 15 %-28). The negative predictive value was 90 % (95 % CI: 81-99 %). Conclusions: The Amsterdam Wrist Rules is a clinical prediction rule with a high sensitivity and negative predictive value for fractures of the wrist. Although external validation showed low specificity and 100 % sensitivity could not be achieved, the Amsterdam Wrist Rules can provide physicians in the Emergency Department with a useful screening tool to select patients with acute wrist trauma for radiography. The upcoming implementation study will further reveal the impact of the Amsterdam Wrist Rules on the anticipated reduction of X-rays requested, missed fractures, Emergency Department waiting times and health care costs. Trial registration: This study was registered in the Dutch Trial Registry, reference number NTR2544 on October 1st, 2010

A clinical decision rule for the use of plain radiography in children after acute wrist injury: development and external validation of the Amsterdam Pediatric Wrist Rules

Background: In most hospitals, children with acute wrist trauma are routinely referred for radiography. Objective: To develop and validate a clinical decision rule to decide whether radiography in children with wrist trauma is required. Materials and methods: We prospectively developed and validated a clinical decision rule in two study populations. All children who presented in the emergency department of four hospitals with pain following wrist trauma were included and evaluated for 18 clinical variables. The outcome was a wrist fracture diagnosed by plain radiography. Results: Included in the study were 787 children. The prediction model consisted of six variables: age, swelling of the distal radius, visible deformation, distal radius tender to palpation, anatomical snuffbox tender to palpation, and painful or abnormal supination. The model showed an area under the receiver operator characteristics curve of 0.79 (95% CI: 0.76-0.83). The sensitivity and specificity were 95.9% and 37.3%, respectively. The use of this model would have resulted in a 22% absolute reduction of radiographic examinations. In a validation study, 7/170 fractures (4.1%, 95% CI: 1.7-8.3%) would have been missed using the decision model. Conclusion: The decision model may be a valuable tool to decide whether radiography in children after wrist trauma is required

<Book Reviews> R. W. Jones and P. B. Kenen (eds.), Handbook of International Economics, Volume 1

textabstractBackground Abdominal aortic aneurysm (AAA) repair has been performed by various surgical specialties for many years. Endovascular aneurysm repair (EVAR) may be a disruptive technology, having an impact on which specialties care for patients with AAA. Therefore, we examined the proportion of AAA repairs performed by various specialties over time in the United States and evaluated the impact of the introduction of EVAR. Methods The Nationwide Inpatient Sample (2001-2009) was queried for intact and ruptured AAA and for open repair and EVAR. Specific procedures were used to identify vascular surgeons (VSs), cardiac surgeons (CSs), and general surgeons (GSs) as well as interventional cardiologists and interventional radiologists for states that reported unique treating physician identifiers. Annual procedure volumes were subsequently calculated for each specialty. Results We identified 108,587 EVARs and 85,080 open AAA repairs (3011 EVARs and 12,811 open repairs for ruptured AAA). VSs performed an increasing proportion of AAA repairs during the study period (52% in 2001 to 66% in 2009; P <.001). GSs and CSs performed fewer repairs during the same period (25% to 17% [P <.001] and 19% to 13% [P <.001], respectively). EVAR was increasingly used for intact (33% to 78% of annual cases; P <.001) as well as ruptured AAA repair (5% to 28%; P <.001). The proportion of intact open repairs performed by VSs increased from 52% to 65% (P <.001), whereas for EVAR, the proportion went from 60% to 67% (P <.001). The proportion performed by VSs increased for ruptured open repairs from 37% to 53% (P <.001) and for ruptured EVARs from 28% to 73% (P <.001). Compared with treatment by VSs, treatment by a CS (0.55 [0.53-0.56]) and GS (0.66 [0.64-0.68]) was associated with a decreased likelihood of undergoing endovascular rather than open AAA repair. Conclusions VSs are performing an increasing majority of AAA repairs, in large part driven by the increased utilization of EVAR for both intact and ruptured AAA repair. However, GSs and CSs still perform AAA repair. Further studies should examine the implications of these national trends on the outcome of AAA repair

Brucella periplasmic protein EipB is a molecular determinant of cell envelope integrity and virulence

Microbial Biotechnolog

Teichoic acids anchor distinct cell wall lamellae in an apically growing bacterium

The bacterial cell wall is a multicomponent structure that provides structural support and protection. In monoderm species, the cell wall is made up predominantly of peptidoglycan, teichoic acids and capsular glycans. Filamentous monoderm Actinobacteria incorporate new cell-wall material at their tips. Here we use cryo-electron tomography to reveal the architecture of the actinobacterial cell wall ofStreptomyces coelicolor. Our data shows a density difference between the apex and subapical regions. Removal of teichoic acids results in a patchy cell wall and distinct lamellae. Knock-down oftagOexpression using CRISPR-dCas9 interference leads to growth retardation, presumably because build-in of teichoic acids had become rate-limiting. Absence of extracellular glycans produced by MatAB and CslA proteins results in a thinner wall lacking lamellae and patches. We propose that theStreptomycescell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.Eveline Ultee et al. reveal the architecture of the actinobacterial cell wall of a polar growing bacteriumStreptomyces coelicolor, using cryo-electron tomography. This study suggests that theStreptomycescell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.Microbial Biotechnolog