bluuebunny/crossref_metadata_2025 huggingface dataset:
Two-Dimensional Surrogate Contact Modeling for Computationally Efficient Dynamic Simulation of Total Knee Replacements
Authors: Lin Yi-Chung , Haftka Raphael T. , Queipo Nestor V. , Fregly Benjamin J.
Published: March 2009

Computational speed is a major limiting factor for performing design sensitivity and optimization studies of total knee replacements. Much of this limitation arises from extensive geometry calculations required by contact analyses. This study presents a novel surrogate contact modeling approach to address this limitation. The approach involves fitting contact forces from a computationally expensive contact model (e.g., a finite element model) as a function of the relative pose between the contacting bodies. Because contact forces are much more sensitive to displacements in some directions than others, standard surrogate sampling and modeling techniques do not work well, necessitating the development of special techniques for contact problems. We present a computational evaluation and practical application of the approach using dynamic wear simulation of a total knee replacement constrained to planar motion in a Stanmore machine. The sample points needed for surrogate model fitting were generated by an elastic foundation (EF) contact model. For the computational evaluation, we performed nine different dynamic wear simulations with both the surrogate contact model and the EF contact model. In all cases, the surrogate contact model accurately reproduced the contact force, motion, and wear volume results from the EF model, with computation time being reduced from 13minto13s. For the practical application, we performed a series of Monte Carlo analyses to determine the sensitivity of predicted wear volume to Stanmore machine setup issues. Wear volume was highly sensitive to small variations in motion and load inputs, especially femoral flexion angle, but not to small variations in component placements. Computational speed was reduced from an estimated 230hto4h per analysis. Surrogate contact modeling can significantly improve the computational speed of dynamic contact and wear simulations of total knee replacements and is appropriate for use in design sensitivity and optimization studies.
Total Hip Wear Assessment: A Comparison Between Computational and In Vitro Wear Assessment Techniques Using ISO 14242 Loading and Kinematics
Authors: Matsoukas George , Willing Ryan , Kim Il Yong
Published: March 2009

In the present study a direct comparison was made between in vitro total hip wear testing and a computational analysis considering the effects of time and a nonlinear stress-strain relationship for ultrahigh molecular weight polyethylene (UHMWPE) at 37°C. The computational simulation was made correct through calibration to experimental volumetric wear results, and the predicted damage layout on the acetabular liner surface was compared with results estimated from laser scanning of the actual worn specimens. The wear rates for the testing specimens were found to be 17.14±1.23 mg/106 cycles and 19.39±0.79 mg/106 cycles, and the cumulative volumetric wear values after 3×106 cycles were 63.70 mm3 and 64.02 mm3 for specimens 1 and 2, respectively. The value of the calibrated wear coefficient was found to be 5.32(10−10) mm3/N mm for both specimens. The major difference between the computational and experimental wear results was the existence of two damage vectors in the experimental case. The actual location of damage was virtually the same in both cases, and the maximum damage depth of the computational model agreed well with the experiment. The existence of multiple wear vectors may indicate the need for computational approaches to account for multidirectional sliding or strain hardening of UHMWPE. Despite the limitation in terms of describing the overall damage layout, the present computational model shows that simulation can mimic some of the behavior of in vitro wear.
A Numerical Simulation Method to Evaluate the Static and Dynamic Characteristics of Flying Head Sliders on Patterned Disk Surfaces
Authors: Hayashi Takefumi , Yoshida Hideyo , Mitsuya Yasunaga
Published: March 2009

To quantitatively predict the static and dynamic characteristics of a flying head slider on a patterned disk surface, the “mixed averaging method” has been successfully applied to the molecular gas lubrication problem incurred by groove-shaped textures formed over the lubricating surface. First, formulation of the alternative direction implicit method using a “mixed averaged” film thickness was derived and implemented as a numerical simulation system. To analyze several types of surface patterns including their mixture patterns, the mixing ratio for the pressure flow and the shear flow were tabulated and interpolated at every mesh point, and then the spacing variations of typical sliders on a discrete track recording medium were demonstrated.
A Numerical Static Friction Model for Spherical Contacts of Rough Surfaces, Influence of Load, Material, and Roughness
Authors: Chen W. Wayne , Wang Q. Jane
Published: March 2009

The relative motion between two surfaces under a normal load is impeded by friction. Interfacial junctions are formed between surfaces of asperities, and sliding inception occurs when shear tractions in the entire contact area reach the shear strength of the weaker material and junctions are about to be separated. Such a process is known as a static friction mechanism. The numerical contact model of dissimilar materials developed by the authors is extended to evaluate the maximum tangential force (in terms of the static friction coefficient) that can be sustained by a rough surface contact. This model is based on the Boussinesq–Cerruti integral equations, which relate surface tractions to displacements. The materials are assumed to respond elastic perfectly plastically for simplicity, and the localized hardness and shear strength are set as the upper limits of contact pressure and shear traction, respectively. Comparisons of the numerical analysis results with published experimental data provide a validation of this model. Static friction coefficients are predicted for various material pairs in contact first, and then the behaviors of static friction involving rough surfaces are extensively investigated.
A Voronoi Finite Element Study of Fatigue Life Scatter in Rolling Contacts
Authors: Jalalahmadi Behrooz , Sadeghi Farshid
Published: March 2009

Microlevel material failure has been recognized as one of the main modes of failure for rolling contact fatigue (RCF) of bearing. Therefore, microlevel features of materials will be of significant importance to RCF investigation. At the microlevel, materials consist of randomly shaped and sized grains, which cannot be properly analyzed using the classical and commercially available finite element method. Hence, in this investigation, a Voronoi finite element method (VFEM) was developed to simulate the microstructure of bearing materials. The VFEM was then used to investigate the effects of microstructure randomness on rolling contact fatigue. Here two different types of randomness are considered: (i) randomness in the microstructure due to random shapes and sizes of the material grains, and (ii) the randomness in the material properties considering a normally (Gaussian) distributed elastic modulus. In this investigation, in order to determine the fatigue life, the model proposed by Raje et al. (“A Numerical Model for Life Scatter in Rolling Element Bearings,” ASME J. Tribol., 130, pp. 011011-1–011011-10), which is based on the Lundberg–Palmgren theory (“Dynamic Capacity of Rolling Bearings,” Acta Polytech. Scand., Mech. Eng. Ser., 1(3), pp. 7–53), is used. This model relates fatigue life to a critical stress quantity and its corresponding depth, but instead of explicitly assuming a Weibull distribution of fatigue lives, the life distribution is obtained as an outcome of numerical simulations. We consider the maximum range of orthogonal shear stress and the maximum shear stress as the critical stress quantities. Forty domains are considered to study the effects of microstructure on the fatigue life of bearings. It is observed that the Weibull slope calculated for the obtained fatigue lives is in good agreement with previous experimental studies and analytical results. Introduction of inhomogeneous elastic modulus and initial flaws within the material domain increases the average critical stresses and decreases the Weibull slope.
Experimental Analysis of Model-Based Roentgen Stereophotogrammetric Analysis (MBRSA) on Four Typical Prosthesis Components
Authors: Seehaus Frank , Emmerich Judith , Kaptein Bart L. , Windhagen Henning , Hurschler Christof
Published: March 2009

Classical marker-based roentgen stereophotogrammetric analysis (RSA) is an accurate method of measuring in vivo implant migration. A disadvantage of the method is the necessity of placing tantalum markers on the implant, which constitutes additional manufacturing and certification effort. Model-based RSA (MBRSA) is a method by which pose-estimation of geometric surface-models of the implant is used to detect implant migration. The placement of prosthesis markers is thus no longer necessary. The accuracy of the pose-estimation algorithms used depends on the geometry of the prosthesis as well as the accuracy of the surface models used. The goal of this study was thus to evaluate the experimental accuracy and precision of the MBRSA method for four different, but typical prosthesis geometries, that are commonly implanted. Is there a relationship existing between the accuracy of MBRSA and prosthesis geometries? Four different prosthesis geometries were investigated: one femoral and one tibial total knee arthroplasty (TKA) component and two different femoral stem total hip arthroplasty (THA) components. An experimental phantom model was used to simulate two different implant migration protocols, whereby the implant was moved relative to the surrounding bone (relative prosthesis-bone motion (RM)), or, similar to the double-repeated measures performed to assess accuracy clinically, both the prosthesis and the surrounding bone model (zero relative prosthesis-bone motion (ZRM)) were moved. Motions were performed about three translational and three rotational axes, respectively. The maximum 95% confidence interval (CI) for MBRSA of all four prosthesis investigated was better than −0.034 to 0.107 mm for in-plane and −0.217 to 0.069 mm for out-of-plane translation, and from −0.038 deg to 0.162 deg for in-plane and from −1.316 deg to 0.071 deg for out-of-plane rotation, with no clear differences between the ZRM and RM protocols observed. Accuracy in translation was similar between TKA and THA components, whereas rotational accuracy about the long axis of the hip stem THA components was worse than the TKA components. The data suggest that accuracy and precision of MBRSA seem to be equivalent to the classical marker-based RSA method, at least for the nonsymmetric implant geometries investigated in this study. The model-based method thus allows the accurate measurement of implant migration without requiring prosthesis markers, and thus presents new opportunities for measuring implant migration where financial or geometric considerations of marker placement have thus far been prohibitive factors.
On the Application of Stress Triaxiality Formula for Plane Strain Fracture Testing
Authors: Bai Yuanli , Teng Xiaoqing , Wierzbicki Tomasz
Published: March 2009

Theoretical and experimental studies have shown that stress triaxiality is the key parameter controlling the magnitude of the fracture strain. Smooth and notched round bar specimens are mostly often used to quantify the effect of stress triaxiality on ductile fracture strain. There is a mounting evidence (Bai and Wierzbicki, 2008, “A New Model of Metal Plasticity and Fracture With Pressure and Lode Dependence,” Int. J. Plast., 24(6), pp. 1071–1096) that, in addition to the stress triaxiality, the normalized third deviatoric stress invariant (equivalent to the Lode angle parameter) should also be included in characterization of ductile fracture. The calibration using round notched bars covers only a small range of possible stress states. Plane strain fracture tests provide additional important data. Following Bridgman’s stress analysis inside the necking of a plane strain specimen, a closed-form solution is derived for the stress triaxiality inside the notch of a flat-grooved plane strain specimen. The newly derived formula is verified by finite element simulations. The range of stress triaxiality in round notched bars and flat-grooved specimens is similar, but the values of the Lode angle parameter are different. These two groups of tests are therefore very useful in constructing a general 3D fracture locus. The results of experiments and numerical simulations on 1045 and DH36 steels have proved the applicability of the closed-form solution and have demonstrated the effect of the Lode angle parameter on the fracture locus.
Basic Electrochemical Thermodynamic Studies of Fuel Cells and Fuel Cell Hybrids
Authors: Williams M. , Horita T. , Yamagi K. , Sakai N. , Yokokawa H.
Published: March 2009

It is important to understand the maximum possible thermal efficiency a device is capable of obtaining and then what of this it actually achieves. In this paper it is shown that the thermal efficiency is a product of the voltage efficiency and the maximum possible thermal efficiency. One can mathematically demonstrate that for any elemental direct anodic oxidation reaction for a simple hybrid system, any fuel cell, and any operating temperature, any pressure, the maximum reversible work is equal to the free energy of reaction at the standard state. This is useful in defining an intrinsic fuel cell exergetic efficiency. An equation for thermal efficiency as a product of exergetic efficiency and maximum possible thermal efficiency is developed and presented for loosely integrated fuel cell turbine hybrids. From these simple studies alone one would conclude that the efficiency potential of fuel cells is expanded through simple fuel cell turbine hybrids.
Nonconventional Fuels for High-Temperature Fuel Cells: Status and Issues
Authors: Cigolotti V. , McPhail S. , Moreno A.
Published: March 2009

The pressing environmental and political necessities of modern international society call for a suitable array of contingency solutions to the energy question. One valid alternative to fossil fuels, for example, is the use of alternative or nonconventional fuels, derived from waste or biomass. Combining these resources with fuel cell applications would provide a significant contribution to environmentally friendly and efficient energy use. Through a comprehensive literature survey and the collection of practical case studies and operational experience, an assessment of the potential for coupling with high-temperature fuel cells of three technologies of alternative fuel production—landfill, anaerobic digestion, and gasification—has been attempted. Though landfill is the easiest technology, anaerobic digestion produces superior quality gas and has the benefit of yielding extra fertilizer, in the form of digestate. Gasification is the most demanding of the technologies but is very flexible in its feedstock. Furthermore, using steam as a gasifying agent produces high quality syngas. However, the main issue with all three technologies is the removal of contaminants, in particular, sulfur. The application of high-temperature gas cleanup is demonstrated to bring considerable advantages on system level when gasification of nonconventional fuels is considered. Ultimately, the reforming step is a key aspect for optimal cost-effective integration of these alternative systems. The review provided establishes the key characteristics of alternative fuel conversion by landfill, anaerobic digestion, and gasification, and exposes the major points of attention for their subsequent application in high-temperature fuel cells. Indications of the measures required and the developments in the field of basic research and system integration are given to provide clear paths of activity, which should bring about the wide-scale implementation of a truly promising application of fuel cell systems.
Modeling Carbon Monoxide Direct Oxidation in Solid Oxide Fuel Cells
Authors: Andreassi Luca , Toro Claudia , Ubertini Stefano
Published: March 2009

In the present study, the results of the numerical implementation of a mathematical model of a planar anode-supported SOFC are reported. In particular, model results are validated and discussed when the fuel is a mixture of hydrogen and carbon monoxide, focusing on the importance of simulating direct oxidation of carbon monoxide. The mathematical model is solved in a 3D environment and the key issue is the validation comparing with experimental data, which is made in different operating conditions to establish the reliability of the presented model. The results show the importance of simulating direct oxidation of carbon monoxide and its effect on the fuel cell performance.
Discrimination of Viable and Dead Fecal
Bacteroidales
Bacteria by Quantitative PCR with Propidium Monoazide
Authors: Bae Sungwoo , Wuertz Stefan
Published: March 2009

Propidium monoazide (PMA) was optimized to discriminate between viable and dead
Bacteroides fragilis
cells and extracellular DNA at different concentrations of solids using quantitative PCR. Conditions of 100 μM PMA and a 10-min light exposure also excluded DNA from heat-treated cells of nonculturable
Bacteroidales
in human feces and wastewater influent and effluent.
Pyrosequencing of the Chaperonin-60 Universal Target as a Tool for Determining Microbial Community Composition
Authors: Schellenberg John , Links Matthew G. , Hill Janet E. , Dumonceaux Tim J. , Peters Geoffrey A. , Tyler Shaun , Ball T. Blake , Severini Alberto , Plummer Francis A.
Published: March 2009

We compared dideoxy sequencing of cloned chaperonin-60 universal target (
cpn60
UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using
cpn60
universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the
cpn60
data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of
cpn60
UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.
Antimicrobial Properties of a Novel Silver-Silica Nanocomposite Material
Authors: Egger Salome , Lehmann Rainer P. , Height Murray J. , Loessner Martin J. , Schuppler Markus
Published: March 2009

Nanotechnology enables development and production of novel silver-based composite materials. We used in vitro tests to demonstrate the antimicrobial activity of a silver-silica nanocomposite compared to the activities of conventional materials, such as silver nitrate and silver zeolite. A silver-silica-containing polystyrene material was manufactured and shown to possess strong antimicrobial properties.
Quantitative Approach to Determining the Contribution of Viable-but-Nonculturable Subpopulations to Malolactic Fermentation Processes
Authors: Quirós Covadonga , Herrero Mónica , García Luis A. , Díaz Mario
Published: March 2009

Different sizes of viable-but-nonculturable cell subpopulations of a lactic acid bacterium strain were induced by adding increasing amounts of SO2. The experimental data obtained here were fitted to a segregated kinetic model developed previously. This procedure allowed us to determine in quantitative terms the contribution of this physiological state to malolactic fermentation.
Percolation and Survival of
Escherichia coli
O157:H7 and
Salmonella enterica
Serovar Typhimurium in Soil Amended with Contaminated Dairy Manure or Slurry
Authors: Semenov Alexander V. , van Overbeek Leo , van Bruggen Ariena H. C.
Published: March 2009

The effect of cattle manure and slurry application on percolation and survival of
Escherichia coli
O157:H7 and
Salmonella enterica
serovar Typhimurium was investigated for different soil depths after the addition of water. Four treatments were chosen for the first set of experiments: (i) addition of inoculated farmyard manure on the soil surface, (ii) mixing of inoculated farmyard manure with the top 10 cm of soil, (iii) addition of inoculated slurry on the soil surface, and (iv) injection of inoculated slurry into the top 10 cm of the soil. Homogeneity of water distribution in the soil profile was confirmed by a nondestructive nuclear magnetic resonance method. Survival data were fitted to a modified logistic model, and estimated survival times were compared. In the second set of experiments, pathogen-inoculated farmyard manure or slurry was applied to soil columns with 1-month-old lettuce plants. More pathogen cells percolated to greater depths after slurry than after manure application. Survival of
E. coli
O157:H7 was significantly longer in soil with slurry than in that with manure, while survival of
Salmonella
serovar Typhimurium was equally high with manure and slurry. The densities of the pathogens were not different in the rhizosphere compared to the bulk soil with manure, while the densities were higher by 0.88 ± 0.11 and 0.71 ± 0.23 log CFU per g (dry weight), respectively, in the rhizosphere than in bulk soil after slurry application. Our results suggest that surface application of manure may decrease the risk of contamination of groundwater and lettuce roots compared to injection of slurry.
Detection of Adenoviruses and Rotaviruses in Drinking Water Sources Used In Rural Areas of Benin, West Africa
Authors: Verheyen Jens , Timmen-Wego Monika , Laudien Rainer , Boussaad Ibrahim , Sen Sibel , Koc Aynur , Uesbeck Alexandra , Mazou Farouk , Pfister Herbert
Published: March 2009

Diseases associated with viruses also found in environmental samples cause major health problems in developing countries. Little is known about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. We established a method to analyze 10 liters of water from drinking water sources in a rural area of Benin for the presence of adenoviruses and rotaviruses. Overall, 541 samples from 287 drinking water sources were tested. A total of 12.9% of the sources were positive for adenoviruses and 2.1% of the sources were positive for rotaviruses at least once. Due to the temporary nature of viral contamination in drinking water sources, the probability of virus detection increased with the number of samples taken at one test site over time. No seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Overall, 3 of 15 surface water samples (20%) and 35 of 247 wells (14.2%) but also 2 of 25 pumps (8%) tested positive for adenoviruses or rotaviruses. The presence of latrines within a radius of 50 m in the vicinity of pumps or wells was identified as being a risk factor for virus detection. In summary, viral contamination was correlated with the presence of latrines in the vicinity of drinking water sources, indicating the importance of appropriate decision support systems in these socioeconomic prospering regions.
Reduction of Tumor Necrosis Factor Alpha-Inducing Capacity of RecombinantLactobacillus caseivia Expression ofSalmonellaOmpC
Authors: Kajikawa A. , Igimi S.
Published: March 2009

The insertion of a heterologous gene into commensal bacteria is a common technique to develop a delivery agent for vaccination and therapies, but the pleiotropic effects of genetic modifications need to be investigated before its use in practical applications. Although supplemental properties provided by the expression of heterologous antigens have been reported, the negative or side effects on the immune-modulating properties caused by recombination are barely understood. In the present study, we fortuitously found that the secretion of tumor necrosis factor alpha (TNF-α) from murine macrophages was reduced by recombinantLactobacillus caseiexpressingSalmonellaOmpC compared to the stimulation of TNF-α secretion by nonexpressingL. casei. This reduction could not be attributed to OmpC as a purified protein. The main component of the OmpC-expressing strain included in the attenuation of TNF-α release seemed to be the cell wall, which exhibited higher sensitivity againstN-acetylmuramidase than that of nonexpressing strains. These results suggest that the recombinant strain expressing a specific heterologous antigen might be digested rapidly in macrophages and lose immune-stimulating capability at an early time point.
Effects of Experimental Choices and Analysis Noise on Surveys of the “Rare Biosphere”
Authors: Hamp Timothy J. , Jones W. Joe , Fodor Anthony A.
Published: March 2009

When planning a survey of 16S rRNA genes from a complex environment, investigators face many choices including which primers to use and how to taxonomically classify sequences. In this study, we explored how these choices affected a survey of microbial diversity in a sample taken from the aerobic basin of the activated sludge of a North Carolina wastewater treatment plant. We performed pyrosequencing reactions on PCR products generated from primers targeting the V1-V2, V6, and V6-V7 variable regions of the 16S rRNA gene. We compared these sequences to 16S rRNA gene sequences found in a whole-genome shotgun pyrosequencing run performed on the same sample. We found that sequences generated from primers targeting the V1-V2 variable region had the best match to the whole-genome shotgun reaction across a range of taxonomic classifications from phylum to family. Pronounced differences between primer sets, however, occurred in the “rare biosphere” involving taxa that we observed in fewer than 11 sequences. We also examined the results of analysis strategies comparing a classification scheme using a nearest-neighbor approach to directly classifying sequences with a naïve Bayesian algorithm. Again, we observed pronounced differences between these analysis schemes in infrequently observed taxa. We conclude that if a study is meant to probe the rare biosphere, both the experimental conditions and analysis choices will have a profound impact on the observed results.
Characterization of a Polycyclic Aromatic Hydrocarbon Degradation Gene Cluster in a Phenanthrene-Degrading
Acidovorax
Strain
Authors: Singleton David R. , Guzmán Ramirez Liza , Aitken Michael D.
Published: March 2009

Acidovorax
sp. strain NA3 was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil that had been treated in a bioreactor and enriched with phenanthrene. The 16S rRNA gene of the isolate possessed 99.8 to 99.9% similarity to the dominant sequences recovered during a previous stable-isotope probing experiment with [U-
13
C]phenanthrene on the same soil (D. R. Singleton, S. N. Powell, R. Sangaiah, A. Gold, L. M. Ball, and M. D. Aitken, Appl. Environ. Microbiol. 71:1202-1209, 2005). The strain grew on phenanthrene as a sole carbon and energy source and could mineralize
14
C from a number of partially labeled PAHs, including naphthalene, phenanthrene, chrysene, benz[
a
]anthracene, and benzo[
a
]pyrene, but not pyrene or fluoranthene. Southern hybridizations of a genomic fosmid library with a fragment of the large subunit of the ring-hydroxylating dioxygenase gene from a naphthalene-degrading
Pseudomonas
strain detected the presence of PAH degradation genes subsequently determined to be highly similar in both nucleotide sequence and gene organization to an uncharacterized
Alcaligenes faecalis
gene cluster. The genes were localized to the chromosome of strain NA3. To test for gene induction by selected compounds, RNA was extracted from amended cultures and reverse transcribed, and cDNA associated with the enzymes involved in the first three steps of phenanthrene degradation was quantified by quantitative real-time PCR. Expression of each of the genes was induced most strongly by phenanthene and to a lesser extent by naphthalene, but other tested PAHs and PAH metabolites had negligible effects on gene transcript levels.
Characterization of a
Dehalobacter
Coculture That Dechlorinates 1,2-Dichloroethane to Ethene and Identification of the Putative Reductive Dehalogenase Gene
Authors: Grostern Ariel , Edwards Elizabeth A.
Published: March 2009

Dehalobacter
and “
Dehalococcoides
” spp. were previously shown to be involved in the biotransformation of 1,1,2-trichloroethane (1,1,2-TCA) and 1,2-dichloroethane (1,2-DCA) to ethene in a mixed anaerobic enrichment culture. Here we report the further enrichment and characterization of a
Dehalobacter
sp. from this mixed culture in coculture with an
Acetobacterium
sp. Through a series of serial transfers and dilutions with acetate, H
2
, and 1,2-DCA, a stable coculture of
Acetobacterium
and
Dehalobacter
spp. was obtained, where
Dehalobacter
grew during dechlorination. The isolated
Acetobacterium
strain did not dechlorinate 1,2-DCA. Quantitative PCR with specific primers showed that
Dehalobacter
cells did not grow in the absence of a chlorinated electron acceptor and that the growth yield with 1,2-DCA was 6.9 (±0.7) × 10
7
16S rRNA gene copies/μmol 1,2-DCA degraded. PCR with degenerate primers targeting reductive dehalogenase genes detected three distinct
Dehalobacter
/
Desulfitobacterium
-type sequences in the mixed-parent culture, but only one of these was present in the 1,2-DCA-H
2
coculture. Reverse transcriptase PCR revealed the transcription of this dehalogenase gene specifically during the dechlorination of 1,2-DCA. The 1,2-DCA-H
2
coculture could dechlorinate 1,2-DCA but not 1,1,2-TCA, nor could it dechlorinate chlorinated ethenes. As a collective, the genus
Dehalobacter
has been show to dechlorinate many diverse compounds, but individual species seem to each have a narrow substrate range.
Relatedness of
Escherichia coli
Strains with Different Susceptibility Phenotypes Isolated from Swine Feces during Ampicillin Treatment
Authors: Bibbal D. , Dupouy V. , Prère M. F. , Toutain P. L. , Bousquet-Mélou A.
Published: March 2009

The aim of this study was to examine the dynamics of the development of resistance in fecal
Escherichia coli
populations during treatment with ampicillin for 7 days in pigs. Before treatment, only 6% of the isolates were ampicillin resistant, whereas more than 90% of the isolates were resistant after days 4 and 7 of treatment. Ampicillin-resistant
E. coli
isolates were mainly multiresistant, and 53% of the isolates from the treated pigs had one phenotype that included resistance to six antibiotics (ampicillin, chloramphenicol, sulfonamides, tetracycline, trimethoprim, and streptomycin) at day 7. Determination of the frequency of the four phylogenetic groups showed that there was a shift in the
E. coli
population in ampicillin-treated pigs; before treatment 75% of the isolates belonged to phylogroup B1, whereas at day 7 85% of the isolates belonged to phylogroup A. Pulsed-field gel electrophoresis (PFGE) typing revealed that ampicillin treatment selected ampicillin-resistant isolates with genotypes which were present before treatment. Comparison of antimicrobial phenotypes and PFGE genotypes showed that resistance traits were disseminated by vertical transmission through defined strains. One PFGE genotype, associated with the six-antibiotic-resistant phenotype and including a specific combination of resistance determinants, was predominant among the ampicillin-resistant strains before treatment and during treatment. These data indicate that ampicillin administration selected various ampicillin-resistant isolates that were present in the digestive tract before any treatment and that
E. coli
isolates belonging to one specific PFGE genotype encoding resistance to six antibiotics became the predominant strains as soon as ampicillin was present in the digestive tract.
AguR Is Required for Induction of the
Streptococcus mutans
Agmatine Deiminase System by Low pH and Agmatine
Authors: Liu Yaling , Zeng Lin , Burne Robert A.
Published: March 2009

Acidic conditions and the presence of exogenous agmatine are required to achieve maximal expression of the agmatine deiminase system (AgDS) of
Streptococcus mutans
. Here we demonstrate that the transcriptional activator of the AgDS, AguR, is required for the responses to agmatine and to low pH. Linker scanning mutagenesis was used to create a panel of mutated
aguR
genes that were utilized to complement an
aguR
deletion mutant of
S. mutans
. The level of production of the mutant proteins was shown to be comparable to that of the wild-type AguR protein. Mutations in the predicted DNA binding domain of AguR eliminated activation of the
agu
operon. Insertions into the region connecting the DNA binding domain to the predicted extracellular and transmembrane domains were well tolerated. In contrast, a variety of mutants were isolated that had a diminished capacity to respond to low pH but retained the ability to activate AgDS gene expression in response to agmatine, and vice versa. Also, a number of mutants were unable to respond to either agmatine or low pH. AguD, which is a predicted agmatine-putrescine antiporter, was found to be a negative regulator of AgDS gene expression in the absence of exogenous agmatine but was not required for low-pH induction of the AgDS genes. This study reveals that the control of AgDS gene expression by both agmatine and low pH is coordinated through the AguR protein and begins to identify domains of the protein involved in sensing and signaling.
UV Light Inactivation of Bacterial Biothreat Agents
Authors: Rose L. J. , O'Connell H.
Published: March 2009

Seven species of bacterial biothreat agents were tested for susceptibility to UV light (254 nm). All gram-negative organisms tested required <12 mJ/cm
2
for a 4-log
10
reduction in viability (inactivation). Tailing off of the
B. anthracis
spore inactivation curves began close to the 2-log
10
inactivation point, with a fluence of approximately 40 mJ/cm
2
, and 3-log
10
inactivation still was not achieved with a fluence of 120 mJ/cm
2
.
Generation of Single-Copy Transposon Insertions in
Clostridium perfringens
by Electroporation of Phage Mu DNA Transposition Complexes
Authors: Lanckriet A. , Timbermont L. , Happonen L. J. , Pajunen M. I. , Pasmans F. , Haesebrouck F. , Ducatelle R. , Savilahti H. , Van Immerseel F.
Published: March 2009

Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in
Clostridium perfringens
has been restricted to the use of Tn
916
-based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in
C. perfringens
, which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of
C. perfringens
.
Streptomyces
Development in Colonies and Soils
Authors: Manteca Angel , Sanchez Jesus
Published: March 2009

Streptomyces
development was analyzed under conditions resembling those in soil. The mycelial growth rate was much lower than that in standard laboratory cultures, and the life span of the previously named first compartmentalized mycelium was remarkably increased.
Cellobiose Dehydrogenase from the Ligninolytic Basidiomycete
Ceriporiopsis subvermispora
Authors: Harreither Wolfgang , Sygmund Christoph , Dünhofen Evelyn , Vicuña Rafael , Haltrich Dietmar , Ludwig Roland
Published: March 2009

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in cultures of the selective delignifier
Ceriporiopsis subvermispora
when grown on a cellulose- and yeast extract-based liquid medium. CDH amounted to up to 2.5% of total extracellular protein during latter phases of the cultivation and thus suggested an important function for the fungus under the given conditions. The enzyme was purified 44-fold to apparent homogeneity. It was found to be present in two glycoforms of 98 kDa and 87 kDa with carbohydrate contents of 16 and 4%, respectively. The isoelectric point of both glycoforms is around 3.0, differing by 0.1 units, which is the most acidic value so far reported for a CDH. By using degenerated primers of known CDH sequences, one
cdh
gene was found in the genomic DNA, cloned, and sequenced. Alignment of the 774-amino-acid protein sequence revealed a high similarity to CDH from other white rot fungi. One notable difference was found in the longer interdomain peptide linker, which might affect the interdomain electron transfer at higher temperatures. The preferred substrate of
C. subvermispora
CDH is cellobiose, while glucose conversion is strongly discriminated by a 155,000-fold-lower catalytic efficiency. This is a typical feature of a basidiomycete CDH, as are the acidic pH optima for all tested electron acceptors in the range from 2.5 to 4.5.
Cloning and Characterization of the Biosynthetic Gene Cluster for Tomaymycin, an SJG-136 Monomeric Analog
Authors: Li Wei , Chou ShenChieh , Khullar Ankush , Gerratana Barbara
Published: March 2009

Tomaymycin produced by
Streptomyces achromogenes
is a naturally produced pyrrolobenzodiazepine (PBD). The biosynthetic gene cluster for tomaymycin was identified and sequenced. The gene cluster analysis reveals a novel biosynthetic pathway for the anthranilate moiety of PBDs. Gene replacement and chemical complementation studies were used to confirm the proposed biosynthetic pathway.
Biosynthesis of Sibiromycin, a Potent Antitumor Antibiotic
Authors: Li Wei , Khullar Ankush , Chou ShenChieh , Sacramo Ashley , Gerratana Barbara
Published: March 2009

Pyrrolobenzodiazepines, a class of natural products produced by actinomycetes, are sequence selective DNA alkylating compounds with significant antitumor properties. Among the pyrrolo[1,4]benzodiazepines (PBDs) sibiromycin, one of two identified glycosylated PBDs, displays the highest affinity for DNA and the most potent antitumor properties. Despite the promising antitumor properties clinical trials of sibiromycin were precluded by the cardiotoxicity effect in animals attributed to the presence of the C-9 hydroxyl group. As a first step toward the development of sibiromycin analogs, we have cloned and localized the sibiromycin gene cluster to a 32.7-kb contiguous DNA region. Cluster boundaries tentatively assigned by comparative genomics were verified by gene replacement experiments. The sibiromycin gene cluster consisting of 26 open reading frames reveals a “modular” strategy in which the synthesis of the anthranilic and dihydropyrrole moieties is completed before assembly by the nonribosomal peptide synthetase enzymes. In addition, the gene cluster identified includes open reading frames encoding enzymes involved in sibirosamine biosynthesis, as well as regulatory and resistance proteins. Gene replacement and chemical complementation studies are reported to support the proposed biosynthetic pathway.
Lateral Transfer of Genes for Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) Degradation
Authors: Andeer Peter F. , Stahl David A. , Bruce Neil C. , Strand Stuart E.
Published: March 2009

Recent studies demonstrated that degradation of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by species of
Rhodococcus
,
Gordonia
, and
Williamsia
is mediated by a novel cytochrome P450 with a fused flavodoxin reductase domain (XplA) in conjunction with a flavodoxin reductase (XplB). Pulse field gel analysis was used to localize
xplA
to extrachromosomal elements in a
Rhodococcus
sp. and distantly related
Microbacterium
sp. strain MA1. Comparison of
Rhodococcus rhodochrous
11Y and
Microbacterium
plasmid sequences in the vicinity of
xplB
and
xplA
showed near identity (6,710 of 6,721 bp). Sequencing of the associated 52.2-kb region of the
Microbacterium
plasmid pMA1 revealed flanking insertion sequence elements and additional genes implicated in RDX uptake and degradation.
Development and Application of a Novel Peptide Nucleic Acid Probe for the Specific Detection of
Cronobacter
Genomospecies (
Enterobacter sakazakii
) in Powdered Infant Formula
Authors: Almeida C. , Azevedo N. F. , Iversen C. , Fanning S. , Keevil C. W. , Vieira M. J.
Published: March 2009

Here, we report a fluorescence in situ hybridization (FISH) method for rapid detection of
Cronobacter
strains in powdered infant formula (PIF) using a novel peptide nucleic acid (PNA) probe. Laboratory tests with several
Enterobacteriaceae
species showed that the specificity and sensitivity of the method were 100%. FISH using PNA could detect as few as 1 CFU per 10 g of
Cronobacter
in PIF after an 8-h enrichment step, even in a mixed population containing bacterial contaminants.
Transfer of Antibiotic Resistance Marker Genes between Lactic Acid Bacteria in Model Rumen and Plant Environments
Authors: Toomey Niamh , Monaghan Áine , Fanning Séamus , Bolton Declan
Published: March 2009

Three wild-type dairy isolates of lactic acid bacteria (LAB) and one
Lactococcus lactis
control strain were analyzed for their ability to transfer antibiotic resistance determinants (plasmid or transposon located) to two LAB recipients using both in vitro methods and in vivo models. In vitro transfer experiments were carried out with the donors and recipients using the filter mating method. In vivo mating examined transfer in two natural environments, a rumen model and an alfalfa sprout model. All transconjugants were confirmed by Etest, PCR, pulsed-field gel electrophoresis, and Southern blotting. The in vitro filter mating method demonstrated high transfer frequencies between all LAB pairs, ranging from 1.8 × 10
−5
to 2.2 × 10
−2
transconjugants per recipient. Transconjugants were detected in the rumen model for all mating pairs tested; however, the frequencies of transfer were low and inconsistent over 48 h (ranging from 1.0 × 10
−9
to 8.0 × 10
−6
transconjugants per recipient). The plant model provided an environment that appeared to promote comparatively higher transfer frequencies between all LAB pairs tested over the 9-day period (transfer frequencies ranged from 4.7 × 10
−4
to 3.9 × 10
−1
transconjugants per recipient). In our test models, dairy cultures of LAB can act as a source of mobile genetic elements encoding antibiotic resistance that can spread to other LAB. This observation could have food safety and public health implications.
PCR-Based Method Using Propidium Monoazide To Distinguish Viable from Nonviable
Bacillus subtilis
Spores
Authors: Rawsthorne H. , Dock C. N. , Jaykus L. A.
Published: March 2009

This paper describes a molecular-based method which is able to discriminate between viable and inactivated
Bacillus subtilis
spores by utilizing the DNA-intercalating dye propidium monoazide. The approach should be valuable in our attempt to employ molecular methods to streamline the evaluation of process validation using bacterial endospores.
Eukaryotic
nirK
Genes Encoding Copper-Containing Nitrite Reductase: Originating from the Protomitochondrion?
Authors: Kim Sang-Wan , Fushinobu Shinya , Zhou Shengmin , Wakagi Takayoshi , Shoun Hirofumi
Published: March 2009

Although denitrification or nitrate respiration has been found among a few eukaryotes, its phylogenetic relationship with the bacterial system remains unclear because orthologous genes involved in the bacterial denitrification system were not identified in these eukaryotes. In this study, we isolated a gene from the denitrifying fungus
Fusarium oxysporum
that is homologous to the bacterial
nirK
gene responsible for encoding copper-containing nitrite reductase (NirK). Characterization of the gene and its recombinant protein showed that the fungal
nirK
gene is the first eukaryotic ortholog of the bacterial counterpart involved in denitrification. Additionally, recent genome analyses have revealed the occurrence of
nirK
homologs in many fungi and protozoa, although the denitrifying activity of these eukaryotes has never been examined. These eukaryotic homolog genes, together with the fungal
nirK
gene of
F
.
oxysporum
, are grouped in the same branch of the phylogenetic tree as the
nirK
genes of bacteria, archaea, and eukaryotes, implying that eukaryotic
nirK
and its homologs evolved from a single ancestor (possibly the protomitochondrion). These results show that the fungal denitrifying system has the same origin as its bacterial counterpart.
Comparative Genomic Hybridization Analysis of Two Predominant Nordic Group I (Proteolytic)Clostridium botulinumType B Clusters
Authors: Lindström Miia , Hinderink Katja , Somervuo Panu , Kiviniemi Katri , Nevas Mari , Chen Ying , Auvinen Petri , Carter Andrew T. , Mason David R. , Peck Michael W. , Korkeala Hannu
Published: March 2009

Comparative genomic hybridization analysis of 32 Nordic group IClostridium botulinumtype B strains isolated from various sources revealed two homogeneous clusters, clusters BI and BII. The type B strains differed from reference strain ATCC 3502 by 413 coding sequence (CDS) probes, sharing 88% of all the ATCC 3502 genes represented on the microarray. The two Nordic type B clusters differed from each other by their response to 145 CDS probes related mainly to transport and binding, adaptive mechanisms, fatty acid biosynthesis, the cell membranes, bacteriophages, and transposon-related elements. The most prominent differences between the two clusters were related to resistance to toxic compounds frequently found in the environment, such as arsenic and cadmium, reflecting different adaptive responses in the evolution of the two clusters. Other relatively variable CDS groups were related to surface structures and the gram-positive cell wall, suggesting that the two clusters possess different antigenic properties. All the type B strains carried CDSs putatively related to capsule formation, which may play a role in adaptation to different environmental and clinical niches. Sequencing showed that representative strains of the two type B clusters both carried subtype B2 neurotoxin genes. As many of the type B strains studied have been isolated from foods or associated with botulism, it is expected that the two group IC. botulinumtype B clusters present a public health hazard in Nordic countries. Knowing the genetic and physiological markers of these clusters will assist in targeting control measures against these pathogens.