Virulotyping and antimicrobial resistance typing of salmonella enterica serovars relevant to human health in Europe

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

Molecular typing of Salmonella serotypes prevalent in animals in England: assessment of methodology

Salmonella enterica serotypes Derby, Mbandaka, Montevideo, Livingstone, and Senftenberg were among the 10 most prevalent serotypes isolated from farm animals in England and Wales in 1999. These serotypes are of potential zoonotic relevance; however, there is currently no "gold standard" fingerprinting method for them. A collection of isolates representing the former serotypes and serotype Gold Coast were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), and ribotyping. The success of the molecular methods in identifying DNA polymorphisms was different for each serotype. Plasmid profiling was particularly useful for serotype Derby isolates, and it also provided a good level of discrimination for serotype Senftenberg. For most serotypes, we observed a number of nontypeable plasmid-free strains, which represents a limitation of this technique. Fingerprinting of genomic DNA by ribotyping and PFGE produced a significant variation in results, depending on the serotype of the strain. Both PstI/SphI ribotyping and XbaI-PFGE provided a similar degree of strain differentiation for serotype Derby and serotype Senftenberg, only marginally lower than that achieved by plasmid profiling. Ribotyping was less sensitive than PFGE when applied to serotype Mbandaka or serotype Montevideo. Serotype Gold Coast isolates were found to be nontypeable by XbaI-PFGE, and a significant proportion of them were found to be plasmid free. A similar situation applies to a number of serotype Livingstone isolates which were nontypeable by plasmid profiling and/or PFGE. In summary, the serotype of the isolates has a considerable influence in deciding the best typing strategy; a single method cannot be relied upon for discriminating between strains, and a combination of typing methods allows further discrimination.

Life-cycle simulations of shallow frontal waves and the impact of deformation strain

The life-cycle of shallow frontal waves and the impact of deformation strain on their development is investigated using the idealised version of the Met Office non-hydrostatic Unified Model which includes the same physics and dynamics as the operational forecast model. Frontal wave development occurs in two stages; first, a deformation strain is applied to a front and a positive potential vorticity (PV) strip forms, generated by latent heat release in the frontal updraft; second, as the deformation strain is reduced the PV strip breaks up into individual anomalies. The circulations associated with the PV anomalies cause shallow frontal waves to form. The structure of the simulated frontal waves is consistent with the conceptual model of a frontal cyclone. Deeper frontal waves are simulated if the stability of the atmosphere is reduced. Deformation strain rates of different strengths are applied to the PV strip to determine whether a deformation strain threshold exists above which frontal wave development is suppressed. An objective method of frontal wave activity is defined and frontal wave development was found to be suppressed by deformation strain rates $\ge 0.4\times10^{-5}\mbox{s}^{-1}$. This value compares well with observed deformation strain rate thresholds and the analytical solution for the minimum deformation strain rate needed to suppress barotropic frontal wave development. The deformation strain rate threshold is dependent on the strength of the PV strip with strong PV strips able to overcome stronger deformation strain rates (leading to frontal wave development) than weaker PV strips.

The impact of deformation strain on the formation of banded clouds in idealized modeling experiments

Experiments are performed using an idealized version of an operational forecast model to determine the impact on banded frontal clouds of the strength of deformational forcing, low-level baroclinicity, and model representation of convection. Line convection is initiated along the front, and slantwise bands extend from the top of the line-convection elements into the cold air. This banding is attributed primarily to M adjustment. The cross-frontal spreading of the cold pool generated by the line convection leads to further triggering of upright convection in the cold air that feeds into these slantwise bands. Secondary low-level bands form later in the simulations; these are attributed to the release of conditional symmetric instability. Enhanced deformation strain leads to earlier onset of convection and more coherent line convection. A stronger cold pool is generated, but its speed is reduced relative to that seen in experiments with weaker deformational strain, because of inhibition by the strain field. Enhanced low-level baroclinicity leads to the generation of more inertial instability by line convection (for a given capping height of convection), and consequently greater strength of the slantwise circulations formed by M adjustment. These conclusions are based on experiments without a convective-parametrization scheme. Experiments using the standard or a modified scheme for this model demonstrate known problems with the use of this scheme at the awkward 4 km grid length used in these simulations. Copyright © 2008 Royal Meteorological Society

Microscopic Mechanisms of Strain Hardening in Glassy Polymers

The mechanisms underlying the increase in stress for large mechanical strains of a polymer glass, quantified by the strain-hardening modulus, are still poorly understood. In the present paper we aim to elucidate this matter and present new mechanisms. Molecular-dynamics simulations of two polymers with very different strain-hardening moduli (polycarbonate and polystyrene) have been carried out. Nonaffine displacements occur because of steric hindrances and connectivity constraints. We argue that it is not necessary to introduce the concept of entanglements to understand strain hardening, but that hardening is rather coupled with the increase in the rate of nonaffine particle displacements. This rate increases faster for polycarbonate, which has the higher strain-hardening modulus. Also more nonaffine chain stretching is present for polycarbonate. It is shown that the inner distances of such a nonaffinely deformed chain can be well described by the inner distances of the worm-like chain, but with an effective stiffness length (equal to the Kuhn length for an infinite worm-like chain) that increases during deformation. It originates from the finite extensibility of the chain. In this way the increase in nonaffine particle displacement can be understood as resulting from an increase in the effective stiffness length of the perturbed chain during deformation, so that at larger strains a higher rate of plastic events in terms of nonaffine displacement is necessary, causing in turn the observed strain hardening in polymer glasses.

Instability of surface-temperature filaments in strain and shear

The effects of uniform straining and shearing on the stability of a surface quasi-geostrophic temperature filament are investigated. Straining is shown to stabilize perturbations for wide filaments but only for a finite time until the filament thins to a critical width, after which some perturbations can grow. No filament can be stabilized in practice, since there are perturbations that can grow large for any strain rate. The optimally growing perturbations, defined as solutions that reach a certain threshold amplitude first, are found numerically for a wide range of parameter values. The radii of the vortices formed through nonlinear roll-up are found to be proportional to θ/s, where θ is the temperature anomaly of the filament and s the strain rate, and are not dependent on the initial size of the filament. Shearing is shown to reduce the normal-mode growth rates, but it cannot stabilize them completely when there are temperature discontinuities in the basic state; smooth filaments can be stabilized completely by shearing and a simple scaling argument provides the shear rate required. Copyright © 2010 Royal Meteorological Society

Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 degrees C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed. (c) 2008 Elsevier Inc. All rights reserved.

Thiamine is synthesized by a salvage pathway in Rhizobium leguminosarum bv. viciae strain 3841

In the absence of added thiamine, Rhizobium leguminosarum bv. viciae strain 3841 does not grow in liquid medium and forms only "pin" colonies on agar plates, which contrasts with the good growth of Sinorhizobium meliloti 1021, Mesorhizobium loti 303099, and Rhizobium etli CFN42. These last three organisms have thiCOGE genes, which are essential for de novo thiamine synthesis. While R. leguminosarum bv. viciae 3841 lacks thiCOGE, it does have thiMED. Mutation of thiM prevented formation of pin colonies on agar plates lacking added thiamine, suggesting thiamine intermediates are normally present. The putative functions of ThiM, ThiE, and ThiD are 4-methyl-5-(beta-hydroxyethyl) thiazole (THZ) kinase, thiamine phosphate pyrophosphorylase, and 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) kinase, respectively. This suggests that a salvage pathway operates in R. leguminosarum, and addition of HMP and THZ enabled growth at the same rate as that enabled by thiamine in strain 3841 but elicited no growth in the thiM mutant (RU2459). There is a putative thi box sequence immediately upstream of the thiM, and a gfp-mut3.1 fusion to it revealed the presence of a promoter that is strongly repressed by thiamine. Using fluorescent microscopy and quantitative reverse transcription-PCR, it was shown that thiM is expressed in the rhizosphere of vetch and pea plants, indicating limitation for thiamine. Pea plants infected by RU2459 were not impaired in nodulation or nitrogen fixation. However, colonization of the pea rhizosphere by the thiM mutant was impaired relative to that of the wild type. Overall, the results show that a thiamine salvage pathway operates to enable growth of Rhizobium leguminosarum in the rhizosphere, allowing its survival when thiamine is limiting.

Effect of time of day and rabbit strain on patterns of aortic wall permeability

Lipid deposits occur more frequently downstream of branch points than upstream in immature rabbit and human aortas but the opposite pattern is seen in mature vessels. These distributions correlate spatially with age-related patterns of aortic permeability, observed in rabbits, and may be determined by them. The mature but not the immature pattern of permeability is dependent on endogenous nitric oxide synthesis. Although the transport patterns have hitherto seemed robust, recent studies have given the upstream pattern in some mature rabbits but the downstream pattern in others. Here we show that transport in mature rabbits is significantly skewed to the downstream pattern in the afternoon compared with the morning (P < 0.05), and switches from a downstream to an upstream pattern at around 21 months in rabbits of the Murex strain, but at twice this age in Highgate rabbits (P < 0.001). The effect of time of day was not explained by changes in nitric oxide production, assessed from plasma levels of nitrate and nitrate, nor did it correlate with conduit artery tone, assessed from the shape of the peripheral pulse wave. The effect of strain could not be explained by variation in nitric oxide production nor by differences in wall structure. The effects of time of day and rabbit strain on permeability patterns explain recent discrepancies, provide a useful tool for investigating underlying mechanisms and may have implications for human disease.

Damage rate is a predictor of fatigue life and creep strain rate in fatigue of tensile human cortical bone samples

We present results on the growth of damage in 29 fatigue tests of human femoral cortical bone from four individuals, aged 53–79. In these tests we examine the interdependency of stress, cycles to failure, rate of creep strain, and rate of modulus loss. The behavior of creep rates has been reported recently for the same donors as an effect of stress and cycles (Cotton, J. R., Zioupos, P., Winwood, K., and Taylor, M., 2003, "Analysis of Creep Strain During Tensile Fatigue of Cortical Bone," J. Biomech. 36, pp. 943–949). In the present paper we first examine how the evolution of damage (drop in modulus per cycle) is associated with the stress level or the "normalized stress" level (stress divided by specimen modulus), and results show the rate of modulus loss fits better as a function of normalized stress. However, we find here that even better correlations can be established between either the cycles to failure or creep rates versus rates of damage than any of these three measures versus normalized stress. The data indicate that damage rates can be excellent predictors of fatigue life and creep strain rates in tensile fatigue of human cortical bone for use in practical problems and computer simulations.

Analysis of creep strain during tensile fatigue of cortical bone

During fatigue tests of cortical bone specimens, at the unload portion of the cycle (zero stress) non-zero strains occur and progressively accumulate as the test progresses. This non-zero strain is hypothesised to be mostly, if not entirely, describable as creep. This work examines the rate of accumulation of this strain and quantifies its stress dependency. A published relationship determined from creep tests of cortical bone (Journal of Biomechanics 21 (1988) 623) is combined with knowledge of the stress history during fatigue testing to derive an expression for the amount of creep strain in fatigue tests. Fatigue tests on 31 bone samples from four individuals showed strong correlations between creep strain rate and both stress and “normalised stress” (σ/E) during tensile fatigue testing (0–T). Combined results were good (r2=0.78) and differences between the various individuals, in particular, vanished when effects were examined against normalised stress values. Constants of the regression showed equivalence to constants derived in creep tests. The universality of the results, with respect to four different individuals of both sexes, shows great promise for use in computational models of fatigue in bone structures.

Synthesis of prebiotic galactooligosaccharides using whole cells of a novel strain, Bifidobacterium bifidum NCIMB 41171

A novel strain of Bifidobacterium bifidum NCIMB 41171, isolated from a faecal sample from a healthy human volunteer and able to express beta-galactosidase activity, was used in synthesis reactions for the production of galactooligosaccharide from lactose. The beta-galactosidase activity of whole bifidobacterial cells showed an optimum activity at pH 6.8-7.0 and 40 degrees C. The transgalactosylation activity of the B. bifidum cells from 50% (w/w) lactose resulted in a galactooligosaccharide mixture (20% w/w) comprising (w/w): 25% disaccharides, 35% trisaccharides, 25% tetrasaccharides and 15% pentasaccharides. Using different initial lactose concentrations, the conversion rate to galactooligosaccharides was maximum (35%) when 55% (w/w) lactose was used. In fermentation experiments, B. bifidum showed an increased preference towards the produced galactooligosaccharide mixture, displaying higher growth rate and short-chain fatty acid production when compared with commercially available oligosaccharides.

Volatile compounds produced by the probiotic strain Lactobacillus plantarum NCIMB 8826 in cereal-based substrates

The production of volatile compounds by the probiotic strain, Lactobacillus plantarum NCIMB 8826, in cereal-based media (oat, wheat. barley and malt) was investigated. Sixty compounds, including fatty acids and their esters, amides, alcohols, aldehydes, aromatic hydrocarbons, furans, ketones, peroxides and pyrans, were identified. L. plantarum significantly changed the aroma profile of the four cereal broths. and each substrate showed a specific volatiles profile. Oat and barley media were the substrates more influenced by the fermentation process. The most abundant volatiles detected in oat, wheat, barley and malt were oleic acid, linoleic acid. acetic acid, and 5-hydroxymethylfurfural, respectively. Analysis of these products confirmed the heterofermentative pathway of L plantarum. Maillard compounds were not detected during sterilisation and fermentation. This study is the first to report the volatile composition of probiotic drinks produce with non-supplemented cereal-based media and the results obtained could contribute to the development of new non-dairy probiotic formulations. (C) 2009 Elsevier Ltd. All rights reserved.

An in vitro study of the probiotic potential of a bile-salt-hydrolyzing Lactobacillus fermentum strain, and determination of its cholesterol-lowering properties

This study evaluated the use of a bile-salt-hydrolyzing Lactobacillus fermentum strain as a probiotic with potential hypocholesterolemic properties. The effect of L. fermentum on representative microbial populations and overall metabolic activity of the human intestinal microbiota was investigated using a three-stage continuous culture system. Also, the use of galactooligosaccharides as a prebiotic to enhance growth and/or activity of the Lactobacillus strain was evaluated. Administration of L. fermentum resulted in a decrease in the overall bifidobacterial population (ca. 1 log unit). In the in vitro system, no significant changes were observed in the total bacterial, Lactobacillus, Bacteroides, and clostridial populations through L. fermentum supplementation. Acetate production decreased by 9 to 27%, while the propionate and butyrate concentrations increased considerably (50 to 90% and 52 to 157%, respectively). A general, although lesser, increase in the production of lactate was observed with the administration of the L. fermentum strain. Supplementation of the prebiotic to the culture medium did not cause statistically significant changes in either the numbers or the activity of the microbiota, although an increase in the butyrate production was seen (29 to 39%). Results from this in vitro study suggest that L.Fermentum KC5b is a candidate probiotic which may affect cholesterol metabolism. The short-chain fatty acid concentrations, specifically the molar proportion of propionate and/or bile salt deconjugation, are probably the major mechanism involved in the purported cholesterol-lowering properties of this strain.