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

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

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

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

Layer-by-layer technique to developing functional nanolaminate films with antifungal activity

The layer-by-layer (LbL) deposition method was used to build up alternating layers (five) of different polyelectrolyte solutions (alginate, zein-carvacrol nanocapsules, chitosan and chitosan-carvacrol emulsions) on an aminolysed/charged polyethylene terephthalate (A/C PET) film. These nanolaminated films were characterised by contact angle measurements and through the determination of water vapour (WVTR) and oxygen (O2TR) transmission rates. The effect of active nanolaminated films against the Alternaria sp. and Rhizopus stolonifer was also evaluated. This procedure allowed developing optically transparent nanolaminated films with tuneable water vapour and gas properties and antifungal activity. The water and oxygen transmission rate values for the multilayer films were lower than those previously reported for the neat alginate or chitosan films. The presence of carvacrol and zein nanocapsules significantly decreased the water transmission rate (up to 40 %) of the nanolaminated films. However, the O2TR behaved differently and was only improved (up to 45 %) when carvacrol was encapsulated, i.e. nanolaminated films prepared by alternating alginate with nanocapsules of zein-carvacrol layers showed better oxygen barrier properties than those prepared as an emulsion of chitosan and carvacrol. These films containing zein-carvacrol nanocapsules also showed the highest antifungal activity (30 %), which did not significantly differ from those obtained with the highest amount of carvacrol, probably due to the controlled release of the active agent (carvacrol) from the zein-carvacrol nanocapsules. Thus, this work shows that nanolaminated films prepared with alternating layers of alginate and zein-carvacrol nanocapsules can be considered to improve the shelf-life of foodstuffs.The authors acknowledge financial support from FP7 IP project BECOBIOCAP^. M. J. Fabra is recipients of a Juan de la Cierva contract from the Spanish Ministry of Economy and Competitivity. Maria L. Flores-López thanks Mexican Science and Technology Council (CONACyT, Mexico) for PhD fellowship support (CONACyT Grant Number 215499/310847). The author Miguel A. Cerqueira is a recipient of a fellowship (SFRH/BPD/72753/2010) supported by Fundação para a Ciência e Tecnologia, POPH-QREN and FSE (FCT, Portugal). The authors also thank the FCT Strategic Project of UID/ BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP- 01-0124-FEDER-027462) and the project BBioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes,^ REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2–O Novo Norte), QREN, FEDER. The support of EU Cost Action FA0904 is gratefully acknowledged

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 Greenland Ice Sheet Large Ensemble (GrISLENS): simulating the future of Greenland under climate variability

The Greenland ice sheet has lost ice at an increasing pace over recent decades, driven by a combination of human-caused climate change and internal variability in the climate system. In projections of future ice sheet evolution, internal variability in climate results in uncertainty that cannot be reduced through model improvements due to the intrinsically chaotic nature of the climate system. This study describes the Greenland Ice Sheet Large Ensemble (GrISLENS), the first large-ensemble study of ice sheet evolution under climate variability, which resolves individual outlet glaciers and climate variability calibrated to observations. GrISLENS combines multiple advanced modeling methods, including a stochastic ice sheet model, a coupled atmosphere–ocean model, dynamical surface mass balance downscaling, and statistical techniques for constraining stochastic parameterizations of climate forcing. We quantify the role of internal climate variability in 185-year projections of the Greenland ice sheet under both a high-emission scenario and pre-2000 climate conditions. We find that spread between ensemble members due to internal climate variability represents a substantial fraction of the mean ice sheet change in the first 20–30 years of simulations, which may be important for coastal planning efforts on decadal timescales. This spread between ensemble members decreases to a small fraction of the total ice sheet change past 2050. At the ice sheet scale, uncertainty in ice loss is dominated by the response to surface mass balance variability, while the response to ocean variability is relatively small, though its influence is more important within individual catchments. The GrISLENS ensemble spread is relatively small compared to that of previous studies estimating uncertainty from climate variability in coarse models, which indicates that resolving small-scale features in climate forcing and ice sheet dynamics substantially affects the quantification of internal variability in ice sheet mass change. On longer timescales, human emissions of greenhouse gases and structural and parametric uncertainties in climate and ice sheet models are larger contributors to projection uncertainties. Through our analysis, we identify the need for more robust initialization methods and extension of these large-ensemble methods to the Antarctic ice sheet.</p

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