Development and validation of a chemostat gut model to study both planktonic and biofilm modes of growth of Clostridium difficile and human microbiota

Copyright: 2014 Crowther et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.The human gastrointestinal tract harbours a complex microbial community which exist in planktonic and sessile form. The degree to which composition and function of faecal and mucosal microbiota differ remains unclear. We describe the development and characterisation of an in vitro human gut model, which can be used to facilitate the formation and longitudinal analysis of mature mixed species biofilms. This enables the investigation of the role of biofilms in Clostridium difficile infection (CDI). A well established and validated human gut model of simulated CDI was adapted to incorporate glass rods that create a solid-gaseous-liquid interface for biofilm formation. The continuous chemostat model was inoculated with a pooled human faecal emulsion and controlled to mimic colonic conditions in vivo. Planktonic and sessile bacterial populations were enumerated for up to 46 days. Biofilm consistently formed macroscopic structures on all glass rods over extended periods of time, providing a framework to sample and analyse biofilm structures independently. Whilst variation in biofilm biomass is evident between rods, populations of sessile bacterial groups (log10 cfu/g of biofilm) remain relatively consistent between rods at each sampling point. All bacterial groups enumerated within the planktonic communities were also present within biofilm structures. The planktonic mode of growth of C. difficile and gut microbiota closely reflected observations within the original gut model. However, distinct differences were observed in the behaviour of sessile and planktonic C. difficile populations, with C. difficile spores preferentially persisting within biofilm structures. The redesigned biofilm chemostat model has been validated for reproducible and consistent formation of mixed species intestinal biofilms. This model can be utilised for the analysis of sessile mixed species communities longitudinally, potentially providing information of the role of biofilms in CDI.Peer reviewe

SMT19969 as a treatment for Clostridium difficile infection : an assessment of antimicrobial activity using conventional susceptibility testing and an in vitro gut model

© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.OBJECTIVES: We investigated the efficacy of the novel antimicrobial agent SMT19969 in treating simulated Clostridium difficile infection using an in vitro human gut model. METHODS: Concentrations of the predominant cultivable members of the indigenous gut microfloras and C. difficile (total and spore counts) were determined by viable counting. Cytotoxin titres were determined using cell cytotoxicity and expressed as log10 relative units (RU). Clindamycin was used to induce simulated C. difficile PCR ribotype 027 infection. Once high-level cytotoxin titres (≥ 4 RU) were observed, SMT19969 was instilled for 7 days. Two SMT19969 dosing regimens (31.25 and 62.5 mg/L four times daily) were evaluated simultaneously in separate experiments. MICs of SMT19969 were determined against 30 genotypically distinct C. difficile ribotypes. RESULTS: SMT19969 was 7- and 17-fold more active against C. difficile than metronidazole and vancomycin, respectively, against a panel of genotypically distinct isolates (P < 0.05). Both SMT19969 dosing regimens demonstrated little antimicrobial activity against indigenous gut microflora groups except clostridia. SMT19969 inhibited C. difficile growth and repressed C. difficile cytotoxin titres in the gut model. CONCLUSIONS: These data suggest that SMT19969 is a narrow-spectrum and potent antimicrobial agent against C. difficile. Additional studies evaluating SMT19969 in other models of C. difficile infection are warranted, with human studies to place these gut model observations in context.Peer reviewedFinal Published versio

Equilibrium configurations of fluids and their stability in higher dimensions

We study equilibrium shapes, stability and possible bifurcation diagrams of fluids in higher dimensions, held together by either surface tension or self-gravity. We consider the equilibrium shape and stability problem of self-gravitating spheroids, establishing the formalism to generalize the MacLaurin sequence to higher dimensions. We show that such simple models, of interest on their own, also provide accurate descriptions of their general relativistic relatives with event horizons. The examples worked out here hint at some model-independent dynamics, and thus at some universality: smooth objects seem always to be well described by both ``replicas'' (either self-gravity or surface tension). As an example, we exhibit an instability afflicting self-gravitating (Newtonian) fluid cylinders. This instability is the exact analogue, within Newtonian gravity, of the Gregory-Laflamme instability in general relativity. Another example considered is a self-gravitating Newtonian torus made of a homogeneous incompressible fluid. We recover the features of the black ring in general relativity.Comment: 42 pages, 11 Figures, RevTeX4. Accepted for publication in Classical and Quantum Gravity. v2: Minor corrections and references adde

Volumes of polytopes in spaces of constant curvature

We overview the volume calculations for polyhedra in Euclidean, spherical and hyperbolic spaces. We prove the Sforza formula for the volume of an arbitrary tetrahedron in $H^3$ and $S^3$. We also present some results, which provide a solution for Seidel problem on the volume of non-Euclidean tetrahedron. Finally, we consider a convex hyperbolic quadrilateral inscribed in a circle, horocycle or one branch of equidistant curve. This is a natural hyperbolic analog of the cyclic quadrilateral in the Euclidean plane. We find a few versions of the Brahmagupta formula for the area of such quadrilateral. We also present a formula for the area of a hyperbolic trapezoid.Comment: 22 pages, 9 figures, 58 reference

Trigonometry of spacetimes: a new self-dual approach to a curvature/signature (in)dependent trigonometry

A new method to obtain trigonometry for the real spaces of constant curvature and metric of any (even degenerate) signature is presented. The method encapsulates trigonometry for all these spaces into a single basic trigonometric group equation. This brings to its logical end the idea of an absolute trigonometry, and provides equations which hold true for the nine two-dimensional spaces of constant curvature and any signature. This family of spaces includes both relativistic and non-relativistic homogeneous spacetimes; therefore a complete discussion of trigonometry in the six de Sitter, minkowskian, Newton--Hooke and galilean spacetimes follow as particular instances of the general approach. Any equation previously known for the three classical riemannian spaces also has a version for the remaining six spacetimes; in most cases these equations are new. Distinctive traits of the method are universality and self-duality: every equation is meaningful for the nine spaces at once, and displays explicitly invariance under a duality transformation relating the nine spaces. The derivation of the single basic trigonometric equation at group level, its translation to a set of equations (cosine, sine and dual cosine laws) and the natural apparition of angular and lateral excesses, area and coarea are explicitly discussed in detail. The exposition also aims to introduce the main ideas of this direct group theoretical way to trigonometry, and may well provide a path to systematically study trigonometry for any homogeneous symmetric space.Comment: 51 pages, LaTe

An analysis of type F2 software measurement standards for profile surface texture parameters

This paper reports on an in-depth analysis of ISO 5436 part 2 type F2 reference software for the calculation of profile surface texture parameters that has been performed on the input, implementation and output results of the reference software developed by the National Physical Laboratory (NPL), the National Institute of Standards and Technology (NIST) and Physikalisch-Technische Bundesanstalt (PTB). Surface texture parameters have been calculated for a selection of 17 test data files obtained from the type F1 reference data sets on offer from NPL and NIST. The surface texture parameter calculation results show some disagreements between the software methods of the National Metrology Institutes. These disagreements have been investigated further, and some potential explanations are given

Genetic Evaluation of Hip Score in UK Labrador Retrievers

Hip dysplasia is an important and complex genetic disease in dogs with both genetic and environmental influences. Since the osteoarthritis that develops is irreversible the only way to improve welfare, through reducing the prevalence, is through genetic selection. This study aimed to evaluate the progress of selection against hip dysplasia, to quantify potential improvements in the response to selection via use of genetic information and increases in selection intensity, and to prepare for public provision of estimated breeding values (EBV) for hip dysplasia in the UK. Data consisted of 25,243 single records of hip scores of Labrador Retrievers between one and four years old, from radiographs evaluated between 2000 and 2007 as part of the British Veterinary Association (BVA) hip score scheme. A natural logarithm transformation was applied to improve normality and linear mixed models were evaluated using ASREML. Genetic correlations between left and right scores, and total hip scores at one, two and three years of age were found to be close to one, endorsing analysis of total hip score in dogs aged one to three as an appropriate approach. A heritability of 0.35±0.016 and small but significant litter effect (0.07±0.009) were estimated. The observed trends in both mean hip score and mean EBV over year of birth indicate that a small genetic improvement has been taking place, approximately equivalent to avoiding those dogs with the worst 15% of scores. Deterministic analysis supported by simulations showed that a 19% greater response could be achieved using EBV compared to phenotype through increases in accuracy alone. This study establishes that consistent but slow genetic improvement in the hip score of UK Labrador Retrievers has been achieved over the previous decade, and demonstrates that progress may be easily enhanced through the use of EBVs and more intense selection

An investigation of the dynamics of intramammary infections acquired during the dry period on European dairy farms

The dry period is acknowledged as playing a key role in mastitis epidemiology and yet surprisingly few studies have explored dry period infection dynamics in detail. The aim of this study was to investigate the dynamics of intramammary infection across a cohort of dairy herds in Europe. Five hundred and twenty-two cows were recruited from 12 farms in 6 European countries. All cows received antibiotic dry cow therapy but teat sealants were not used. All quarters of all cows were sampled for bacteriology at drying off and in the week immediately postcalving. Two ipsilateral quarters were also sampled for bacteriology in each cow 2 and 6 wk after drying off. Cows were body condition scored and teats assessed for cleanliness at all sampling time points and for the presence of a keratin plug during the dry period. Other cow-level parameters such as historic somatic cell counts and milk yields before drying off were collated from farm records. Univariable and multivariable analyses were undertaken to investigate the etiology, prevalence, and dynamics of infection during the dry period and associated influential factors. In summary, environmental mastitis pathogens predominated. Although gram-positive major pathogens were typically well controlled and did not increase in prevalence across the dry period, gram-negative pathogens generally increased in prevalence. There was an increase in the number of quarters that yielded no growth across the dry period, although this was driven by minor rather than major mastitis pathogen control. Other than the presence of a gram-positive or gram-negative pathogen 6 wk after drying off, the measured parameters were not influential when considering their effect on the presence of pathogens postcalving. Analysis also suggested that the early and mid dry period may be more important with respect to the timing of acquisition of infection than previously thought. We observed substantial variation in the etiology and prevalence of different pathogens on different farms with, in all cases, at least one of the 12 herds experiencing the opposite of the others with respect to increases and decreases in pathogen prevalence. Overall, this study confirms the importance of the dry period in mastitis epidemiology but highlights the importance of assessing and understanding infection dynamics on individual units. The lack of influence of the cow and quarter factors measured in this study suggests that herd and management factors may be more influential

Mixed linear model approach adapted for genome-wide association studies.

5 5 t e c h n i c a l r e p o r t s Mixed linear model (MLM) methods have proven useful in controlling for population structure and relatedness within genome-wide association studies. However, MLM-based methods can be computationally challenging for large datasets. We report a compression approach, called &apos;compressed MLM&apos;, that decreases the effective sample size of such datasets by clustering individuals into groups. We also present a complementary approach, &apos;population parameters previously determined&apos; (P3D), that eliminates the need to re-compute variance components. We applied these two methods both independently and combined in selected genetic association datasets from human, dog and maize. The joint implementation of these two methods markedly reduced computing time and either maintained or improved statistical power. We used simulations to demonstrate the usefulness in controlling for substructure in genetic association datasets for a range of species and genetic architectures. We have made these methods available within an implementation of the software program TASSEL. Although genome-wide association studies (GWAS) have the potential to pinpoint genetic polymorphisms underlying human diseases and agriculturally important traits, false discoveries are a major concern 1 and can be partially attributed to spurious associations caused by population structure and unequal relatedness among individuals in a given cohort. Population stratification was initially addressed using general linear model (GLM)-based methods such as structured association 2 , genomic control 3 and family-based tests of association 4 . The introduction of MLM approaches has more recently been demonstrated as an improved method to simultaneously account for population structure and unequal relatedness among individuals 5 . In the MLM-based methods, population structure 2,6 is fit as a fixed effect, whereas kinship among individuals is incorporated as the variance-covariance structure of the random effect for the individuals. Regardless of the applied statistical method, GWAS require large sample sizes to achieve sufficient statistical power 7 , especially in order to detect the small effect polymorphisms that underlie most complex traits 8 . For the MLM approach, datasets with these large sample sizes create a heavy computational burden because the computing time for solving a MLM increases with the cube of the number of individuals fit as a random effect. The earliest effort to reduce the size of the random effect in an MLM can be traced back to the sire model approach used in animal breeding 9-12 , which replaces an individual&apos;s genetic effect with that of its sire. Consequently, the sire-model approach requires pedigrees, which are not always available, and which in particular are often not available in plant studies. Even with available pedigrees, the use of a marker-based kinship is preferred because of its higher accurac