bluuebunny/crossref_metadata_2025 huggingface dataset:
Wettability and thermal stability of fluorocarbon films deposited by deep reactive ion etching
Authors: Zhuang Yan Xin , Menon Aric
Published: May 2005

Fluorocarbon films have low surface energy and can be used as antistiction coating for microelectromechanical systems. By using the passivation process in a deep reactive ion etcher, the fluorocarbon films can be deposited and integrated with other processes in the clean room. The properties such as wettability, surface energies, and thermal stability, have been investigated in detail. It has been found that the fluorocarbon films deposited have a static water contact angle of 109° and a surface energy around 14.5mJ∕m2, whereas as-received and as-deposited single silicon, poly silicon, and silicon nitride have a much lower water contact angle and a higher surface energy. The fluorocarbon films keep their good hydrophobicity up to 300°C, and the degradation temperature depends on the thickness of the fluorocarbon films. Decomposition happens at lower temperatures (100–300°C) even though the decomposition rate is quite slow without affecting the contact angle. The decomposition mechanism at low temperatures (less than 300°C) might be different from that at high temperatures. It has been shown that the fluorocarbon film deposited by a deep reactive ion etcher tool provides very high hydrophobicity, low surface energy, good thermal stability, and antiadhesion behavior for use in nanoimprinting lithography.
Characterization of epitaxial germanium grown on (LaxY1−x)2O3∕Si(111) using different surfactants
Authors: Preisler E. J. , Guha S.
Published: May 2005

The uniformity and quality of epitaxial, ultrathin germanium-on-insulator (GOI) layers are studied as a function of Ge regrowth under different surfactant conditions. It is shown that using antimony as a surfactant during the solid phase epitaxial regrowth of the germanium layers provides a higher crystal quality and much flatter surfaces than samples grown without antimony. However, the diffusion of even a small percentage of a monolayer of antimony into the GOI layer may cause a thin n+ “delta-doped” layer to remain on the surface of the wafer, making device fabrication difficult. It is shown that using a surface layer of silicon as a surfactant that is not expected to interact electronically with the Ge (unlike Sb), acceptable surface smoothness may be achieved. Physical mechanisms behind this are discussed.
Effect of O2 flow ratio on the microstructure and stress of room temperature reactively sputtered RuOx thin films
Authors: Shi Junxia , Huang Feng , Weaver Mark L. , Klein Tonya M.
Published: May 2005

RuO x thin films were deposited at room temperature by reactive radio frequency magnetron-sputtering using Ar∕O2 discharges of varying O2 flow ratio (fO2) over the range 10%–50% and were characterized using x-ray diffraction, x-ray reflectivity, x-ray photoelectron spectroscopy, resistivity, and stress-temperature measurements. With the increase of fO2, the film texture changed from (110) to (101). Films deposited with fO2>25% were determined stoichiometric. The residual stresses in as-deposited films were all compressive and increased with addition of O2, except for the film sputtered at fO2=20% which was in biaxial tension. The film deposited at fO2=30% had a low resistivity value of 68μΩcm and near zero stress (<50MPa tensile) after a thermal cycle in air up to 500 °C which is promising for use in microdevices.
Effect of ion entry acceptance conditions on the performance of a quadrupole mass spectrometer operated in upper and lower stability regions
Authors: Turner P. , Taylor S. , Gibson J. R.
Published: May 2005

Computer simulation of ion motion in a quadrupole mass spectrometer has been used to examine the effect of initial ion conditions on performance when operated in the first and third zones of the Mathieu stability diagram. Commercial instruments frequently use round electrodes instead of the better-performing hyperbolic electrodes because the cost of manufacturing is lower. However, adverse features are seen when using round electrodes. Here further insight is provided and a possible method of correction is suggested. For the first time, ion origin for the first stability region for a round electrode quadrupole has been reported.
Effects of substrate temperature on properties of pulsed dc reactively sputtered tantalum oxide films
Authors: Jain Pushkar , Juneja Jasbir S. , Bhagwat Vinay , Rymaszewski Eugene J. , Lu Toh-Ming , Cale Timothy S.
Published: May 2005

The effects of substrate heating on the stoichiometry and the electrical properties of pulsed dc reactively sputtered tantalum oxide films over a range of film thickness (0.14to5.4μm) are discussed. The film stoichiometry, and hence the electrical properties, of tantalum oxide films; e.g., breakdown field, leakage current density, dielectric constant, and dielectric loss are compared for two different cases: (a) when no intentional substrate/film cooling is provided, and (b) when the substrate is water cooled during deposition. All other operating conditions are the same, and the film thickness is directly related to deposition time. The tantalum oxide films deposited on the water-cooled substrates are stoichiometric, and exhibit excellent electrical properties over the entire range of film thickness. “Noncooled” tantalum oxide films are stoichiometric up to ∼1μm film thickness, beyond that the deposited oxide is increasingly nonstoichiometric. The presence of partially oxidized Ta in thicker (>∼1μm) noncooled tantalum oxide films causes a lower breakdown field, higher leakage current density, higher apparent dielectric constant, and dielectric loss. The growth of nonstoichiometric tantalum oxide in thicker noncooled films is attributed to decreased surface oxygen concentration due to oxygen recombination and desorption at higher film temperatures (>∼100°C). The quantitative results presented reflect experience with a specific piece of equipment; however, the procedures presented can be used to characterize deposition processes in which film stoichiometry can change.
Low- and high-resistivity silicon substrate characterization using the Al/silicon-rich oxide/Si structure with comparison to the metal oxide semiconductor technique
Authors: Luna-López A. , Aceves-Mijares M. , Malik O. , Glaenzer R.
Published: May 2005

High-resistivity silicon substrates (HRS, NB<1014cm−3) are commonly used, especially in optoelectronic integrated circuits. However, standard metal oxide semiconductor (MOS) characterization methods fail to predict correctly the dopant concentration and lifetime. This is due to the high resistance in series with the MOS capacitor, which causes an erroneous capacitance measurement at high frequency. To overcome this restriction, a different characterization method is proposed, using the electronic transport property of silicon-rich oxide (SRO) films, with aluminum/silicon-rich oxide (Al/SRO/Si) devices and using capacitance-voltage (C-V) and current-voltage (I-V) characteristics, the dopant concentration and lifetime can be estimated with these method. In addition, using low/high-frequency C-V measurements in MOS structure on HRS can be used to determine the dopant concentration. In this work, low-resistivity silicon and HRS substrates are characterized. The results for both type of substrates and for the different methods are compared. It is shown that the results are similar and any of these methods produce reliable results, but the Al/SRO/Si structure has the advantage that the generation lifetime is easily obtained.
Compositional study of silicon oxynitride thin films deposited using electron cyclotron resonance plasma-enhanced chemical vapor deposition technique
Authors: Baumann H. , Sah R. E.
Published: May 2005

We have used backscattering spectrometry and N15(H1,α,γ)C12 nuclear reaction analysis techniques to study in detail the variation in the composition of silicon oxynitride films with deposition parameters. The films were deposited using 2.45GHz electron cyclotron resonance plasma-enhanced chemical vapor deposition (PECVD) technique from mixtures of precursors argon, nitrous oxide, and silane at deposition temperature 90°C. The deposition pressure and nitrous oxide-to-silane gas flow rates ratio have been found to have a pronounced influence on the composition of the films. When the deposition pressure was varied for a given nitrous oxide-to-silane gas flow ratio, the amount of silicon and nitrogen increased with the deposition pressure, while the amount of oxygen decreased. For a given deposition pressure, the amount of incorporated nitrogen and hydrogen decreased while that of oxygen increased with increasing nitrous oxide-to-silane gas flow rates ratio. For nitrous oxide-to-silane gas flow ratio of 5, we obtained films which contained neither chemically bonded nor nonbonded nitrogen atoms as revealed by the results of infrared spectroscopy, backscattering spectrometry, and nuclear reaction analysis. Our results demonstrate the nitrogen-free nearly stoichiometric silicon dioxide films can be prepared from a mixture of precursors argon, nitrous oxide, and silane at low substrate temperature using high-density PECVD technique. This avoids the use of a hazardous and an often forbidden pair of silane and oxygen gases in a plasma reactor.
Characterization of a plasma produced by pulsed arc using an electrostatic double probe
Authors: García L. A. , Pulzara A. O. , Devia A. , Restrepo E.
Published: May 2005

In this work the determination of plasma parameters of a pulsed-arc discharge by using an electrostatic double probe is presented. The system to generate the plasma is composed mainly of a reaction chamber, where the electrodes of different materials (Ti, Zr, Al) were placed in order to identify the variation of the plasma parameters when the cathode material is changed. An automatic system has been implemented for acquiring data starting from the bias of a double electrostatic probe. This system allowed registering complete curves of current-voltage in relatively short times (≈30ms). The electron density ne and electron temperature Te can be extracted from the I-V characteristic curves obtained. The advantage of this technique is its facility to provide the whole quantity of “in situ” information, which can be compared with the theoretical results using numeric methods for the I-V curves simulation. By means of a suitable fit this allowed the determination of the plasma parameters. The values obtained for Te were in the order of 1eV and for ne of about 1013cm−3. A significant variation for ne was not found.
Potential Uptake of
Escherichia coli
O157:H7 from Organic Manure into Crisphead Lettuce
Authors: Johannessen Gro S. , Bengtsson Gunnar B. , Heier Berit T. , Bredholt Sylvia , Wasteson Yngvild , Rørvik Liv Marit
Published: May 2005

To investigate the potential transfer of
Escherichia coli
O157:H7 from contaminated manure to fresh produce, lettuce seedlings were transplanted into soil fertilized with bovine manure which had been inoculated with approximately 10
4
CFU g
−1
E. coli
O157:H7. The lettuce was grown for approximately 50 days in beds in climate-controlled rooms in a greenhouse. As the bacterium was not detected in the edible parts of the lettuce, the outer leaves of the lettuce, or the lettuce roots at harvest it was concluded that transmission of
E. coli
O157:H7 from contaminated soil to lettuce did not occur. The pathogen persisted in the soil for at least 8 weeks after fertilizing but was not detected after 12 weeks. Indigenous
E. coli
was detected only sporadically on the lettuce at harvest, and enterococci were not detected at all. The numbers of enterococci declined more rapidly than those of
E. coli
in the soil.
Pseudomonas fluorescens
, which inhibited growth of
E. coli
O157:H7 in vitro, was isolated from the rhizosphere.
Relationship between Bacterioplankton Richness, Respiration, and Production in the Southern North Sea
Authors: Reinthaler Thomas , Winter Christian , Herndl Gerhard J.
Published: May 2005

We investigated the relationship between bacterioplankton production (BP), respiration (BR), and community composition measured by terminal restriction fragment length polymorphism in the southern North Sea over a seasonal cycle. Major changes in bacterioplankton richness were apparent from April to December. While cell-specific BP decreased highly significantly with increasing bacterioplankton richness, cell-specific BR was found to be variable along the richness gradient, suggesting that bacterioplankton respiration is rather independent from shifts in the bacterial community composition. As a consequence, the bacterial growth efficiency [BGE = BP/(BP + BR)] was negatively related to bacterioplankton richness, explaining ∼43% of the variation in BGE. Our results indicate that despite the observed shifts in the community composition, the main function of the bacterioplankton, the remineralization of dissolved organic carbon to CO
2
, is rather stable.
Metabolic Network Analysis of
Streptomyces tenebrarius
, a
Streptomyces
Species with an Active Entner-Doudoroff Pathway
Authors: Borodina Irina , Schöller Charlotte , Eliasson Anna , Nielsen Jens
Published: May 2005

Streptomyces tenebrarius
is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When
S. tenebrarius
is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin.
13
C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in
Streptomyces
. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.
Contribution of
Archaea
to Total Prokaryotic Production in the Deep Atlantic Ocean
Authors: Herndl Gerhard J. , Reinthaler Thomas , Teira Eva , van Aken Hendrik , Veth Cornelius , Pernthaler Annelie , Pernthaler Jakob
Published: May 2005

Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic
Archaea
(
Crenarchaeota
and
Euryarchaeota
) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While
Euryarchaeota
were found to be more dominant in nearsurface waters,
Crenarchaeota
were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of
Euryarchaeota
and
Crenarchaeaota
taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of
Crenarchaeota
taking up inorganic carbon increased with depth, while
Euryarchaeota
taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.
Reduced Proteolysis of Secreted Gelatin and Yps1-Mediated α-Factor Leader Processing in a
Pichia pastoris kex2
Disruptant
Authors: Werten Marc W. T. , de Wolf Frits A.
Published: May 2005

Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned
Pichia pastoris
homologs of the
Saccharomyces cerevisiae
basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the
P. pastoris KEX2
gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The
S. cerevisiae
α-factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the
YPS1
gene had no effect on gelatin degradation or processing of the α-factor propeptide. When both the
KEX2
and
YPS1
genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that
P. pastoris kex2
and
yps1
single-gene disruptants can be used for the α-factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.
Construction, Analysis, and β-Glucanase Screening of a Bacterial Artificial Chromosome Library from the Large-Bowel Microbiota of Mice
Authors: Walter Jens , Mangold Marco , Tannock Gerald W.
Published: May 2005

A metagenomic (community genomic) library consisting of 5,760 bacterial
artificial chromosome clones was prepared in
Escherichia coli
DH10B from DNA extracted from the large-bowel microbiota of BALB/c
mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp
(range, 8 to 150 kbp) in size. A functional screen of the library forβ
-glucanase activity was conducted using lichenin agar plates
and Congo red solution. Three clones with β-glucanase activity
were detected. The inserts of these three clones were sequenced and
annotated. Open reading frames (ORF) that encoded putative proteins
with identity to glucanolytic enzymes (lichenases and laminarinases)
were detected by reference to databases. Other putative genes were
detected, some of which might have a role in environmental sensing,
nutrient acquisition, or coaggregation. The insert DNA from two clones
probably originated from uncultivated bacteria because the ORF had low
sequence identity with database entries, but the genes associated with
the remaining clone resembled sequences reported in
Bacteroides
species.
Utilization of the Plant Hormone Indole-3-Acetic Acid for Growth by
Pseudomonas putida
Strain 1290
Authors: Leveau Johan H. J. , Lindow Steven E.
Published: May 2005

We have isolated from plant surfaces several bacteria with the ability to catabolize indole-3-acetic acid (IAA). One of them, isolate 1290, was able to utilize IAA as a sole source of carbon, nitrogen, and energy. The strain was identified by its 16S rRNA sequence as
Pseudomonas putida
. Activity of the enzyme catechol 1,2-dioxygenase was induced during growth on IAA, suggesting that catechol is an intermediate of the IAA catabolic pathway. This was in agreement with the observation that the oxygen uptake by IAA-grown
P. putida
1290 cells was elevated in response to the addition of catechol. The inability of a
catR
mutant of
P. putida
1290 to grow at the expense of IAA also suggests a central role for catechol as an intermediate in IAA metabolism. Besides being able to destroy IAA, strain 1290 was also capable of producing IAA in media supplemented with tryptophan. In root elongation assays,
P. putida
strain 1290 completely abolished the inhibitory effect of exogenous IAA on the elongation of radish roots. In fact, coinoculation of roots with
P. putida
1290 and 1 mM concentration of IAA had a positive effect on root development. In coinoculation experiments on radish roots, strain 1290 was only partially able to alleviate the inhibitory effect of bacteria that in culture overproduce IAA. Our findings imply a biological role for strain 1290 as a sink or recycler of IAA in its association with plants and plant-associated bacteria.
Application of Cation-Coated Filter Method to Detection of Noroviruses, Enteroviruses, Adenoviruses, and Torque Teno Viruses in the Tamagawa River in Japan
Authors: Haramoto Eiji , Katayama Hiroyuki , Oguma Kumiko , Ohgaki Shinichiro
Published: May 2005

The occurrence of human enteric viruses in surface water in the Tamagawa River, Japan, was surveyed for 1 year, from April 2003 to March 2004. Sixty-four samples were collected from six sites along the river, and 500 ml of the sample was concentrated using the cation-coated filter method, which was developed in our previous study. This method showed recovery yields of 56% ± 32% (
n
= 37) for surface water samples inoculated with polioviruses. More than one kind of tested virus was detected in 43 (67%) of 64 samples by TaqMan PCR. Noroviruses and adenoviruses were detected in a high positive ratio; 34 (53%), 28 (44%), and 29 (45%) of 64 samples were positive for norovirus genotype 1 and genotype 2 and adenoviruses, respectively. The mean concentrations of norovirus genotype 1 or genotype 2 determined by real-time PCR were 0.087 and 0.61 genome/ml, respectively, showing much higher values in winter (0.21 genome/ml for genotype 1 and 2.3 genomes/ml for genotype 2). Enteroviruses were detected by both direct PCR (6 of 64 samples; 9%) and cell culture PCR (2 of 64 samples; 3%). Torque teno viruses, emerging hepatitis viruses, were also isolated in three samples (5%). The concentration of total coliforms and the presence of F-specific phages showed a high correlation with the presence of viruses, which suggested that the simultaneous use of total coliforms and F-specific phages as indicators of surface water may work to monitor viral contamination.
Processive Endoglucanase Active in Crystalline Cellulose Hydrolysis by the Brown Rot Basidiomycete
Gloeophyllum trabeum
Authors: Cohen Roni , Suzuki Melissa R. , Hammel Kenneth E.
Published: May 2005

Brown rot basidiomycetes have long been thought to lack the processive cellulases that release soluble sugars from crystalline cellulose. On the other hand, these fungi remove all of the cellulose, both crystalline and amorphous, from wood when they degrade it. To resolve this discrepancy, we grew
Gloeophyllum trabeum
on microcrystalline cellulose (Avicel) and purified the major glycosylhydrolases it produced. The most abundant extracellular enzymes in these cultures were a 42-kDa endoglucanase (Cel5A), a 39-kDa xylanase (Xyn10A), and a 28-kDa endoglucanase (Cel12A). Cel5A had significant Avicelase activity—4.5 nmol glucose equivalents released/min/mg protein. It is a processive endoglucanase, because it hydrolyzed Avicel to cellobiose as the major product while introducing only a small proportion of reducing sugars into the remaining, insoluble substrate. Therefore, since
G. trabeum
is already known to produce a β-glucosidase, it is now clear that this brown rot fungus produces enzymes capable of yielding assimilable glucose from crystalline cellulose.
Phenotypic and Genotypic Selection of Microbiota Surviving under Dental Restorations
Authors: Paddick J. S. , Brailsford S. R. , Kidd E. A. M. , Beighton D.
Published: May 2005

The effects of sealing infected carious dentine below dental restorations on the phenotypic and genotypic diversity of the surviving microbiota was investigated. It was hypothesized that the microbiota would be subject to nutrient limitation or nutrient simplification, as it would no longer have access to dietary components or salivary secretion for growth. The available nutrients would be limited primarily to serum proteins passing from the pulp through the patent dentinal tubules to the infected dentine. Ten lesions were treated, and infected dentine was sealed below dental restorations for approximately 5 months. Duplicate standardized samples of infected dentine were taken at baseline and after the removal of the restorations. The baseline microbiota were composed primarily of
Lactobacillus
spp.,
Streptococcus mutans
,
Streptococcus parasanguinis
,
Actinomyces israelii
, and
Actinomyces gerencseriae
. None of these taxa were isolated among the microbiota of the dentine samples taken after 5 months, which consisted of only
Actinomyces naeslundii
,
Streptococcus oralis
,
Streptococcus intermedius
, and
Streptococcus mitis.
The microbiota of the final sample exhibited a significantly (
P
< 0.001) increased ability to produce glycosidic enzymes (sialidase, β-
N
-acetylglucosaminidase, and β-galactosidase), which liberate sugars from glycoproteins. The genotypic diversity of
S. oralis
and
A. naeslundii
was significantly (
P
= 0.002 and
P
= 0.001, respectively) reduced in the final samples. There was significantly (
P
< 0.001) greater genotypic diversity within these taxa between the pairs of dentine samples taken at baseline than was found in the 5-month samples, indicating that the dentine was more homogenous than it was at baseline. We propose that during the interval between placement of the restorations and their removal, the available nutrient, primarily serum proteins, or the relative simplicity and homogeneity of the nutrient supply significantly affected the surviving microbiota. The surviving microbiota was less complex, based on compositional, phenotypic, and genotypic analyses, than that isolated from carious lesions which were also exposed to salivary secretions and pH perturbations.
Efficacy of Two Peroxygen-Based Disinfectants for Inactivation of
Cryptosporidium parvum
Oocysts
Authors: Quilez Joaquin , Sanchez-Acedo Caridad , Avendaño Catalina , del Cacho Emilio , Lopez-Bernad Fernando
Published: May 2005

Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate
Cryptosporidium parvum
oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4′,6′-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of
C. parvum
oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting
C. parvum
oocyst viability). These findings indicate the potential efficacy of both disinfectants for
C. parvum
oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.
Application of a pH-Sensitive Fluoroprobe (C-SNARF-4) for pH Microenvironment Analysis in
Pseudomonas aeruginosa
Biofilms
Authors: Hunter Ryan C. , Beveridge Terry J.
Published: May 2005

An important feature of microbial biofilms is the development of four-dimensional physical and chemical gradients in space and time. There is need for novel approaches to probe these so-called microenvironments to determine their effect on biofilm-specific processes. In this study, we describe the use of seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) for pH microenvironment analysis in
Pseudomonas aeruginosa
biofilms. C-SNARF-4 is a fluorescent ratiometric probe that allows pH quantification independent of probe concentration and/or laser intensity. By confocal scanning laser microscopy, C-SNARF-4 revealed pH heterogeneity throughout the biofilm in both the
x
,
y
and
x
,
z
planes, with values ranging from pH 5.6 (within the biofilm) to pH 7.0 (bulk fluid). pH values were typically remarkably different than those just a few micrometers away. Although this probe has been successfully used in a number of eukaryotic systems, problems have been reported which describe spectral emission changes as a result of macromolecular interactions with the fluorophore. To assess how the biofilm environment may influence fluorescent properties of the dye, fluorescence of C-SNARF-4 was quantified via spectrofluorometry while the probe was suspended in various concentrations of representative biofilm matrix components (i.e., proteins, polysaccharides, and bacterial cells) and growth medium. Surprisingly, our data demonstrate that few changes in emission spectra occur as a result of matrix interactions below pH 7. These studies suggest that C-SNARF-4 can be used as a reliable indicator of pH microenvironments, which may help elucidate their influence on the medical and geobiological roles of natural biofilms.
tir
- and
stx
-Positive
Escherichia coli
in Stream Waters in a Metropolitan Area
Authors: Higgins James A. , Belt Kenneth T. , Karns Jeffrey S. , Russell-Anelli Jonathan , Shelton Daniel R.
Published: May 2005

Diarrheagenic
Escherichia coli
, which may include the enteropathogenic
E. coli
and the enterohemorrhagic
E. coli
, are a significant cause of diarrheal disease among infants and children in both developing and developed areas. Disease outbreaks related to freshwater exposure have been documented, but the presence of these organisms in the urban aquatic environment is not well characterized. From April 2002 through April 2004 we conducted weekly surveys of streams in the metropolitan Baltimore, Md., area for the prevalence of potentially pathogenic
E. coli
by using PCR assays targeting the
tir
and
stx
1
and
stx
2
genes. Coliforms testing positive for the presence of the
tir
gene were cultured from 653 of 1,218 samples (53%), with a greater prevalence associated with urban, polluted streams than in suburban and forested watershed streams. Polluted urban streams were also more likely to test positive for the presence of one of the
stx
genes. Sequence analysis of the
tir
amplicon, as well as the entire
tir
gene from three isolates, indicated that the pathogenic
E. coli
present in the stream waters has a high degree of sequence homology with the
E. coli
O157:H7 serotype. Our data indicate that pathogenic
E. coli
are continually deposited into a variety of stream habitats and suggest that this organism may be a permanent member of the gastrointestinal microflora of humans and animals in the metropolitan Baltimore area.
Suppressive Subtractive Hybridization of and Differences in Gene Expression Content of Calcifying and Noncalcifying Cultures ofEmiliania huxleyiStrain 1516
Authors: Nguyen Binh , Bowers Robert M. , Wahlund Thomas M. , Read Betsy A.
Published: May 2005

The marine coccolithophoridEmiliania huxleyiis a cosmopolitan alga intensely studied in relation to global carbon cycling, biogeochemistry, marine ecology, and biomineralization processes. The biomineralization capabilities of coccolithophorids have attracted the attention of scientists interested in exploiting this ability for the development of materials science and biomedical and biotechnological applications. Although it has been well documented that biomineralization inE. huxleyiis promoted by growth under phosphate-limited conditions, the genes and proteins that govern the processes of calcification and coccolithogenesis remain unknown. Suppressive subtractive hybridization (SSH) libraries were constructed from cultures grown in phosphate-limited and phosphate-replete media as tester and driver populations for reciprocal SSH procedures. Positive clones from each of the two libraries were randomly selected, and dot blotting was performed for the analysis of expression patterns. A total of 513 clones from the phosphate-replete library and 423 clones from the phosphate-limited library were sequenced, assembled, and compared to sequences in GenBank using BLASTX. Of the 103 differentially expressed gene fragments from the phosphate-replete library, 34% showed significant homology to other known proteins, while only 23% of the 65 differentially expressed gene fragments from the phosphate-limited library showed homology to other proteins. To further assess mRNA expression, real-time RT-PCR analysis was employed and expression profiles were generated over a 14-day time course for three clones from the phosphate-replete library and five clones from the phosphate-limited library. The fragments isolated provide the basis for future cloning of full-length genes and functional analysis.
Methane-Oxidizing Bacteria in a California Upland Grassland Soil: Diversity and Response to Simulated Global Change
Authors: Horz Hans-Peter , Rich Virginia , Avrahami Sharon , Bohannan Brendan J. M.
Published: May 2005

We investigated the diversity of methane-oxidizing bacteria (i.e., methanotrophs) in an annual upland grassland in northern California, using comparative sequence analysis of the
pmoA
gene. In addition to identifying type II methanotrophs commonly found in soils, we discovered three novel
pmoA
lineages for which no cultivated members have been previously reported. These novel
pmoA
clades clustered together either with clone sequences related to “RA 14” or “WB5FH-A,” which both represent clusters of environmentally retrieved sequences of putative atmospheric methane oxidizers. Conservation of amino acid residues and rates of nonsynonymous versus synonymous nucleotide substitution in these novel lineages suggests that the
pmoA
genes in these clades code for functionally active methane monooxygenases. The novel clades responded to simulated global changes differently than the type II methanotrophs. We observed that the relative abundance of type II methanotrophs declined in response to increased precipitation and increased atmospheric temperature, with a significant antagonistic interaction between these factors such that the effect of both together was less than that expected from their individual effects. Two of the novel clades were not observed to respond significantly to these environmental changes, while one of the novel clades had an opposite response, increasing in relative abundance in response to increased precipitation and atmospheric temperature, with a significant antagonistic interaction between these factors.
Production of Autoinducer 2 in
Salmonella enterica
Serovar Thompson Contributes to Its Fitness in Chickens but Not on Cilantro Leaf Surfaces
Authors: Brandl M. T. , Miller W. G. , Bates A. H. , Mandrell R. E.
Published: May 2005

Food-borne illness caused by
Salmonella enterica
has been linked traditionally to poultry products but is associated increasingly with fresh fruits and vegetables. We have investigated the role of the production of autoinducer 2 (AI-2) in the ability of
S. enterica
serovar Thompson to colonize the chicken intestine and the cilantro phyllosphere. A mutant of
S. enterica
serovar Thompson that is defective in AI-2 production was constructed by insertional mutagenesis of
luxS
. The population size of the
S. enterica
serovar Thompson parental strain was significantly higher than that of its LuxS
−
mutant in the intestine, spleen, and droppings of chicks 12 days after their oral inoculation with the strains in a ratio of 1:1. In contrast, no significant difference in the population dynamics of the parental and LuxS
−
strain was observed after their inoculation singly or in mixtures onto cilantro plants. Digital image analysis revealed that 54% of
S. enterica
serovar Thompson cells were present in large aggregates on cilantro leaves but that the frequency distributions of the size of aggregates formed by the parental strain and the LuxS
−
mutant were not significantly different. Carbon utilization profiles indicated that the AI-2-producing strain utilized a variety of amino and organic acids more efficiently than its LuxS
−
mutant but that most sugars were utilized similarly in both strains. Thus, inherent differences in the nutrients available to
S. enterica
in the phyllosphere and in the chicken intestine may underlie the differential contribution of AI-2 synthesis to the fitness of
S. enterica
in these environments.
Polychlorinated Biphenyl Rhizoremediation by
Pseudomonas fluorescens
F113 Derivatives, Using a
Sinorhizobium meliloti nod
System To Drive
bph
Gene Expression
Authors: Villacieros Marta , Whelan Clare , Mackova Martina , Molgaard Jesper , Sánchez-Contreras María , Lloret Javier , Aguirre de Cárcer Daniel , Oruezábal Roke I. , Bolaños Luis , Macek Thomas , Karlson Ulrich , Dowling David N. , Martín Marta , Rivilla Rafael
Published: May 2005

Rhizoremediation of organic chemicals requires high-level expression of biodegradation genes in bacterial strains that are excellent rhizosphere colonizers.
Pseudomonas fluorescens
F113 is a biocontrol strain that was shown to be an excellent colonizer of numerous plant rhizospheres, including alfalfa. Although a derivative of F113 expressing polychlorinated biphenyl (PCB) biodegradation genes (F113pcb) has been reported previously, this strain shows a low level of
bph
gene expression, limiting its rhizoremediation potential. Here, a high-level expression system was designed from rhizobial
nod
gene regulatory relays. Nod promoters were tested in strain F113 by using β-galactosidase transcriptional fusions. This analysis showed that nodbox 4 from
Sinorhizobium meliloti
has a high level of expression in F113 that is dependent on an intact
nodD1
gene. A transcriptional fusion of a nodbox cassette containing the
nodD1
gene and nodbox 4 fused to a
gfp
gene was expressed in the alfalfa rhizosphere. The
bph
operon from
Burkholderia
sp. strain LB400 was cloned under the control of the nodbox cassette and was inserted as a single copy into the genome of F113, generating strain F113L::1180. This new genetically modified strain has a high level of BphC activity and grows on biphenyl as a sole carbon and energy source at a growth rate that is more than three times higher than that of F113pcb. Degradation of PCBs 3, 4, 5, 17, and 25 was also much faster in F113L::1180 than in F113pcb. Finally, the modified strain cometabolized PCB congeners present in Delor103 better than strain LB400, the donor of the
bph
genes used.
Changes in Nitrogen-Fixing and Ammonia-Oxidizing Bacterial Communities in Soil of a Mixed Conifer Forest after Wildfire
Authors: Yeager Chris M. , Northup Diana E. , Grow Christy C. , Barns Susan M. , Kuske Cheryl R.
Published: May 2005

This study was undertaken to examine the effects of forest fire on two important groups of N-cycling bacteria in soil, the nitrogen-fixing and ammonia-oxidizing bacteria. Sequence and terminal restriction fragment length polymorphism (T-RFLP) analysis of
nifH
and
amoA
PCR amplicons was performed on DNA samples from unburned, moderately burned, and severely burned soils of a mixed conifer forest. PCR results indicated that the soil biomass and proportion of nitrogen-fixing and ammonia-oxidizing species was less in soil from the fire-impacted sites than from the unburned sites. The number of dominant
nifH
sequence types was greater in fire-impacted soils, and
nifH
sequences that were most closely related to those from the spore-forming taxa
Clostridium
and
Paenibacillus
were more abundant in the burned soils. In T-RFLP patterns of the ammonia-oxidizing community, terminal restriction fragments (TRFs) representing
amoA
cluster 1, 2, or 4
Nitrosospira
spp. were dominant (80 to 90%) in unburned soils, while TRFs representing
amoA
cluster 3A
Nitrosospira
spp. dominated (65 to 95%) in fire-impacted soils. The dominance of
amoA
cluster 3A
Nitrosospira
spp. sequence types was positively correlated with soil pH (5.6 to 7.5) and NH
3
-N levels (0.002 to 0.976 ppm), both of which were higher in burned soils. The decreased microbial biomass and shift in nitrogen-fixing and ammonia-oxidizing communities were still evident in fire-impacted soils collected 14 months after the fire.
The Gene
yggE
Functions in Restoring Physiological Defects of
Escherichia coli
Cultivated under Oxidative Stress Conditions
Authors: Kim SunYoung , Nishioka Motomu , Hayashi Shuhei , Honda Hiroyuki , Kobayashi Takeshi , Taya Masahito
Published: May 2005

DNA microarray analysis showed that
yfiD
,
yggB
, and
yggE
genes were up-regulated when superoxide dismutase (SOD)-deficient
Escherichia coli
IM303 (I4) was cultivated under the oxidative stress generated by photoexcited TiO
2
, and pYFD, pYGB, and pYGE were constructed by inserting the respective genes into a pUC 19 vector. The content of reactive oxygen species (ROS) in IM303 (I4) cells carrying pYGE was reduced to 31% of ROS content in the control cells with pUC 19. In the culture of wild-type strain,
E. coli
MM294, in the medium with paraquat (10 μmol/l), maximum specific growth rate of the cells with pYGE was about five times higher than that of the control cells, with a decreased ROS content in the former cells. The introduction of pYGE also suppressed the occurrence of the cells with altered amino acid requirement in the culture of MM294 cells with paraquat.
Molecular Analysis of Human, Porcine, and Poultry
Enterococcus faecium
Isolates and Their
erm
(B) Genes
Authors: De Leener E. , Martel A. , De Graef E. M. , Top J. , Butaye P. , Haesebrouck F. , Willems R. , Decostere A.
Published: May 2005

Fifty-nine
erm
(B)-positive
Enterococcus faecium
strains isolated from pigs, broilers, and humans were typed using multilocus sequence typing (MLST), and the coding sequence of the
erm
(B) gene was determined. Identical
erm
(B) gene sequences were detected in genetically unrelated isolates. Furthermore, genetically indistinguishable strains were found to contain different
erm
(B) alleles. This may suggest that horizontal exchange of the
erm
(B) gene between animal and human
E. faecium
strains or the existence of a common reservoir of
erm
(B) genes might be more important than direct transmission of resistant strains.
Workload Due to
Aspergillus fumigatus
and Significance of the Organism in the Microbiology Laboratory of a General Hospital
Authors: Bouza E. , Guinea J. , Peláez T. , Pérez-Molina J. , Alcalá L. , Muñoz P.
Published: May 2005

The increase in the immunocompromised population and the incidence of invasive aspergillosis (IA) are leading to an overinterpretation of the potential clinical significance of many isolates of
Aspergillus fumigatus
. Our work prospectively assesses the workload of the isolation of
A. fumigatus
and its clinical significance in the microbiology laboratory of a large teaching hospital. During a 3-year period, all patients from whom
A. fumigatus
was isolated were prospectively monitored and classified as having IA or “nonsignificant” disease. A point score based on the prediction of five easily obtained laboratory and clinical parameters was applied. We found 404
A. fumigatus
isolates in 260 patients (1/1,000 microbiology laboratory samples; 2.1 patients/10,000 admissions). A total of 90 isolates (22.3%) were from patients with IA. Of the 260 patients, 31 (12%) had invasive disease (IA), and the remaining 229 had “nonsignificant” disease. A score based on points for five parameters was applied to our population. It was constructed as follows: “sample obtained by invasive procedures” (1 point), “presence of two or more positive samples from the same patient” (1 point), “leukemia” (2 points), “neutropenia” (5 points), and “corticosteroid treatment” (2 points). Patients with a score of 0 had only a 2.5% probability of IA. Those with a score of 1 or 2 had an increased probability of 10.3%. The probabilities rose to 40% and 70%, respectively, for patients with a score of 3 or 4 or a score of ≥5. A simple score based on five easily available parameters may be of help to microbiologists and clinicians to predict the risk of IA.
Genotypic Study of
Pneumocystis jirovecii
in Human Immunodeficiency Virus-Positive Patients in Thailand
Authors: Siripattanapipong Suradej , Worapong Jeerapun , Mungthin Mathirut , Leelayoova Saovanee , Tan-ariya Peerapan
Published: May 2005

Pneumocystis jirovecii
is one of the common opportunistic infections in human immunodeficiency virus (HIV)-infected patients in Thailand. Information regarding genotypic and epidemiological of this organism in Thai patients is not available. We analyzed the genotypes of 28
P. jirovecii
-positive specimens from bronchoalveolar lavage and sputum samples from HIV-infected Thai patients based on nucleotide variations of the internal transcribed spacer regions 1 and 2 of the rRNA gene. Thirteen genotypes were the same as previously reported outside Thailand. Ten genotypes, which included Bp, Er, Eq, Ic, Ir, Ip, Rc, Rp, Qb, and Qq, were new. Ir and Rp were unique and dominant types observed in HIV-infected Thai patients. Thirteen specimens (46.4%) were infected with a single type of
P. jirovecii
, and fifteen (53.6%) were mixed infections. These differences may be used as genotypic markers for studying the epidemiology and transmission of
P. jirovecii
in the Thai population.