bluuebunny/crossref_metadata_2025 huggingface dataset:
Members of a Readily Enriched β-Proteobacterial Clade Are Common in Surface Waters of a Humic Lake
Authors: Burkert Ulrike , Warnecke Falk , Babenzien Dieter , Zwirnmann Elke , Pernthaler Jakob
Published: November 2003

Humic lakes are systems often characterized by irregular high input of dissolved organic carbon (DOC) from the catchment. We hypothesized that specific bacterial groups which rapidly respond to changes in DOC availability might form large populations in such habitats. Seasonal changes of microbial community composition were studied in two compartments of an artificially divided bog lake with contrasting DOC inputs. These changes were compared to community shifts induced during short-term enrichment experiments. Inocula from the two compartments were diluted 1:10 into water from the more DOC-rich compartment, and inorganic nutrients were added to avoid microbial N and P limitation. The dilutions were incubated for a period of 2 weeks. The microbial assemblages were analyzed by cloning and sequencing of 16S rRNA genes and by fluorescence in situ hybridization with specific oligonucleotide probes. β-Proteobacteria from a cosmopolitan freshwater lineage related to
Polynucleobacter necessarius
(beta II) were rapidly enriched in all treatments. In contrast, members of the class
Actinobacteria
did not respond to the enhanced availability of DOC by an immediate increase in growth rate, and their relative abundances declined during the incubations. In lake water members of the beta II clade seasonally constituted up to 50% of all microbes in the water column. Bacteria from this lineage annually formed a significantly higher fraction of the microbial community in the lake compartment with a higher allochthonous influx than in the other compartment.
Actinobacteria
represented a second numerically important bacterioplankton group, but without clear differences between the compartments. We suggest that the pelagic microbial community of the studied system harbors two major components with fundamentally different growth strategies.
Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of
Escherichia coli
Authors: Overhage Jörg , Steinbüchel Alexander , Priefert Horst
Published: November 2003

The
vaoA
gene from
Penicillium simplicissimum
CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in
Escherichia coli
XL1-Blue under the control of the
lac
promoter, together with the genes
calA
and
calB
, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of
Pseudomonas
sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain
E. coli
XL1-Blue(pSK
vao
mP
calA
m
calB
) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter
−1
. This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter
−1
after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation,
E. coli
XL1-Blue(pSK
vao
mP
calA
m
calB
) was used to produce ferulic acid from eugenol and, subsequently,
E. coli
(pSK
ech
E/H
fcs
) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter
−1
, besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter
−1
. The genes
ehyAB
, encoding eugenol hydroxylase of
Pseudomonas
sp. strain HR199, and
azu
, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in
E. coli
XL1-Blue.
Characterization of aLactococcus lactisStrain That Secretes a Major Epitope of Bovine Beta-Lactoglobulin and Evaluation of Its Immunogenicity in Mice
Authors: Chatel Jean-Marc , Nouaille Sebastien , Adel-Patient Karine , Le Loir Yves , Boe Herman , Gruss Alexandra , Wal Jean-Michel , Langella Philippe
Published: November 2003

Bovine β-lactoglobulin (Blg) is one of the major cow's milk allergens. Peptide 41-60 of Blg (Blg41-60) was described as a murine T-cell determinant and a murine, rat, and human immunoglobulin E (IgE) epitope. The aim of this study was the expression of Blg41-60 as a fusion protein in the food-grade bacteriumLactococcus lactisand the characterization of its immunogenicity in mice. We constructed a recombinant strain ofL. lactiscapable of inducible production and secretion of Blg41-60::Nuc, a fusion protein between Blg41-60 and the mature part of the staphylococcal nuclease (Nuc). The highest production yield of Blg41-60::Nuc (32.5 mg/liter) was reached 4 h after induction. At this time, up to 75% of Blg41-60::Nuc was secreted. When monoclonal antibodies specific for Blg41-60 were used, purified Blg41-60::Nuc and synthetic Blg41-60 exhibited very similar immunoreactivities. Subcutaneous coadministration of purified Blg41-60::Nuc and killed nonrecombinantL. lactisresulted in the induction of specific anti-Blg41-60 IgG2a and IgG1. The IgG1/IgG2a ratio and the lack of specific IgE suggest a Th1-type immune response, i.e., a nonallergic response. Similar administrations of the killed Blg41-60::Nuc-producingL. lactisstrain did not elicit a specific immune response, whereas a transitory mucosal IgA-specific immune response was induced in mice after oral administration of the live Blg41-60::Nuc-producingL. lactisstrain.
Sampling Natural Viral Communities from Soil for Culture-Independent Analyses
Authors: Williamson Kurt E. , Wommack K. Eric , Radosevich Mark
Published: November 2003

An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses. In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy [EFM], and transmission electron microscopy [TEM]) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils. Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain. Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 × 10
8
g of dry soil
−1
), but specific soil-eluant combinations posed significant problems to enumeration by EFM. Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5×10
8
g of dry soil
−1
) that were about five times lower. Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil.
Dynamics of Bacterial and Fungal Communities on Decaying Salt Marsh Grass
Authors: Buchan Alison , Newell Steven Y. , Butler Melissa , Biers Erin J. , Hollibaugh James T. , Moran Mary Ann
Published: November 2003

Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated microbial community. Here we describe the bacterial and fungal assemblages associated with two decomposition stages of
Spartina alterniflora
detritus in a productive southeastern U.S. salt marsh. 16S rRNA genes and 18S-to-28S internal transcribed spacer (ITS) regions were used to target the bacterial and ascomycete fungal communities, respectively, based on DNA sequence analysis of isolates and environmental clones and by using community fingerprinting based on terminal restriction fragment length polymorphism (T-RFLP) analysis. Seven major bacterial taxa (six affiliated with the α-
Proteobacteria
and one with the
Cytophagales
) and four major fungal taxa were identified over five sample dates spanning 13 months. Fungal terminal restriction fragments (T-RFs) were informative at the species level; however, bacterial T-RFs frequently comprised a number of related genera. Amplicon abundances indicated that the salt marsh saprophyte communities have little-to-moderate variability spatially or with decomposition stage, but considerable variability temporally. However, the temporal variability could not be readily explained by either successional shifts or simple relationships with environmental factors. Significant correlations in abundance (both positive and negative) were found among dominant fungal and bacterial taxa that possibly indicate ecological interactions between decomposer organisms. Most associations involved one of four microbial taxa: two groups of bacteria affiliated with the α-
Proteobacteria
and two ascomycete fungi (
Phaeosphaeria spartinicola
and environmental isolate “4clt”).
Plant Transformation by Coinoculation with a Disarmed
Agrobacterium tumefaciens
Strain and an
Escherichia coli
Strain Carrying Mobilizable Transgenes
Authors: Pappas Katherine M. , Winans Stephen C.
Published: November 2003

Transformation of
Nicotiana tabacum
leaf explants was attempted with
Escherichia coli
as a DNA donor either alone or in combination with
Agrobacterium tumefaciens
. We constructed
E. coli
donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an
E. coli
strain expressing the IncN-type conjugation system was coinoculated with a disarmed
A. tumefaciens
strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from
E. coli
to the disarmed
A. tumefaciens
strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct
A. tumefaciens
strains containing binary vectors and could simplify the creation of transgenic plants.
Detection and Identification of
Lactobacillus
Species in Crops of Broilers of Different Ages by Using PCR-Denaturing Gradient Gel Electrophoresis and Amplified Ribosomal DNA Restriction Analysis
Authors: Guan Le Luo , Hagen Karen E. , Tannock Gerald W. , Korver Doug R. , Fasenko Gaylene M. , Allison Gwen E.
Published: November 2003

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10
8
to 10
9
CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the
Lactobacillus acidophilus
group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the
L. acidophilus
group.
Hae
III-ARDRA was used for preliminary identification of isolates in the
L. acidophilus
group and to identify
Lactobacillus reuteri
and
Lactobacillus salivarius. Mse
I-ARDRA generated unique patterns for all species of the
L. acidophilus
group, identifying
Lactobacillus crispatus
,
Lactobacillus johnsonii
, and
Lactobacillus gallinarum
among crop isolates. The results of our study provide comprehensive knowledge of the
Lactobacillus
microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.
Fungal Lipid Accumulation and Development of Mycelial Structures by Two Arbuscular Mycorrhizal Fungi
Authors: van Aarle Ingrid M. , Olsson Pål Axel
Published: November 2003

We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi
Scutellospora calospora
and
Glomus intraradices
when colonizing
Plantago lanceolata
. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1ω5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of
G. intraradices
increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus.
S. calospora
does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for
G. intraradices. G. intraradices
allocated more of its lipids to storage than did
S. calospora
. Thus, within a species, the fatty acid 16:1ω5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1ω5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.
Ecology of Uncultivated
Oscillospira
Species in the Rumen of Cattle, Sheep, and Reindeer as Assessed by Microscopy and Molecular Approaches
Authors: Mackie Roderick I. , Aminov Rustam I. , Hu Wenping , Klieve Athol V. , Ouwerkerk Diane , Sundset Monica A. , Kamagata Yoichi
Published: November 2003

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium
Oscillospira
spp. was studied.
Oscillospira-
specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the
Oscillospira
clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an
Oscillospira-
specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large
Oscillospira
morphotypes, the presence of
Oscillospira
spp. was still detected by
Oscillospira-
specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of
Oscillospira
species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.
Methodology of Protistan Discovery: from rRNA Detection to Quality Scanning Electron Microscope Images
Authors: Stoeck Thorsten , Fowle William H. , Epstein Slava S.
Published: November 2003

Each year, thousands of new protistan 18S rRNA sequences are detected in environmental samples. Many of these sequences are molecular signatures of new protistan species, classes, and/or kingdoms that have never been seen before. The main goal of this study was to enable visualization of these novel organisms and to conduct quality ultrastructural examination. We achieved this goal by modifying standard procedures for cell fixation, fluorescence in situ hybridization, and scanning electron microscopy (SEM) and by making these methodologies work in concert. As a result, the same individual cell can now be detected by 18S rRNA-targeted fluorochrome-labeled probes and then viewed by SEM to reveal its diagnostic morphological characteristics. The method was successfully tested on a wide range of protists (alveolates, stramenopiles, kinetoplastids, and cryptomonads). The new methodology thus opens a way for fine microscopy studies of many organisms previously known exclusively by their 18S rRNA sequences.
Development and Application of a
dapB
-Based In Vivo Expression Technology System To Study Colonization of Rice by the Endophytic Nitrogen-Fixing Bacterium
Pseudomonas stutzeri
A15
Authors: Rediers Hans , Bonnecarrère Victoria , Rainey Paul B. , Hamonts Kelly , Vanderleyden Jos , De Mot René
Published: November 2003

Pseudomonas stutzeri
A15 is a nitrogen-fixing bacterium isolated from paddy rice. Strain A15 is able to colonize and infect rice roots. This strain may provide rice plants with fixed nitrogen and hence promote plant growth. In this article, we describe the use of
dapB
-based in vivo expression technology to identify
P. stutzeri
A15 genes that are specifically induced during colonization and infection (
cii
). We focused on the identification of
P. stutzeri
A15 genes that are switched on during rice root colonization and are switched off during free-living growth on synthetic medium. Several transcriptional fusions induced in the rice rhizosphere were isolated. Some of the corresponding genes are involved in the stress response, chemotaxis, metabolism, and global regulation, while others encode putative proteins with unknown functions or without significant homology to known proteins.
Agrobacterium Bioassay Strain for Ultrasensitive Detection of
N
-Acylhomoserine Lactone-Type Quorum-Sensing Molecules: Detection of Autoinducers in
Mesorhizobium huakuii
Authors: Zhu Jun , Chai Yunrong , Zhong Zengtao , Li Shunpeng , Winans Stephen C.
Published: November 2003

An ultrasensitive bioassay system for the detection of
N
-acylhomoserine lactones (AHLs) was constructed in
Agrobacterium tumefaciens
by using the T7 expression system to overproduce the AHL receptor TraR. This strain detected many diverse AHLs, some at extremely low concentrations. We used this strain to detect for the first time AHLs made by
Mesorhizobium huakuii
, which symbiotically fixes nitrogen in association with the legume
Astragalus sinicus
, a source of green manure throughout eastern Asia.
Site-Specific Manifestations of Invasive Group A Streptococcal Disease: Type Distribution and Corresponding Patterns of Virulence Determinants
Authors: Vlaminckx Bart J. M. , Mascini Ellen M. , Schellekens Joop , Schouls Leo M. , Paauw Armand , Fluit Ad C. , Novak Rodger , Verhoef Jan , Schmitz Franz Josef
Published: November 2003

As part of a national surveillance program on invasive group A streptococci (GAS), isolates that caused specific manifestations of invasive GAS disease in The Netherlands were collected between 1992 and 1996. These site-specific GAS infections involved meningitis, arthritis, necrotizing fasciitis, and puerperal sepsis. An evaluation was performed to determine whether GAS virulence factors correlate with these different disease manifestations. PCRs were developed to detect 9 genes encoding exotoxins and 12 genes encoding fibronectin binding proteins. The genetic backgrounds of all isolates were determined by M genotyping and pulsed-field gel electrophoresis (PFGE) analysis. The predominant M types included M1, M2, M3, M4, M6, M9, M12, and M28. Most M types were associated with all manifestations of GAS disease. However, M2 was found exclusively in patients with puerperal sepsis, M6 predominated in patients with meningitis, and M12 predominated in patients with GAS arthritis. While characteristic gene profiles were detected in most M types, the resolution of detection of different gene profiles within M genotypes was enhanced by PFGE analysis, which clearly demonstrated the existence of some clonal lineages among invasive GAS isolates in The Netherlands. M1 isolates comprised a single clone carrying highly mitogenic toxin genes (speA,smeZ) and were associated with toxic shock-like syndrome. Toxin profiles were highly conserved among the most virulent strains, such as M1 and M3.
Comparison of Real-Time PCR and Culture for Detection of
Porphyromonas gingivalis
in Subgingival Plaque Samples
Authors: Boutaga Khalil , van Winkelhoff Arie Jan , Vandenbroucke-Grauls Christina M. J. E. , Savelkoul Paul H. M.
Published: November 2003

Porphyromonas gingivalis
is a major pathogen in destructive periodontal disease in humans. Detection and quantification of this microorganism are relevant for diagnosis and treatment planning. The prevalence and quantity of
P. gingivalis
in subgingival plaque samples of periodontitis patients were determined by anaerobic culture and real-time PCR amplification of the 16S small-subunit rRNA gene. The PCR was performed with primers and a fluorescently labeled probe specific for the
P. gingivalis
16S rRNA gene. By the real-time PCR assay, as few as 1 CFU of
P. gingivalis
could be detected. Subgingival plaque samples from 259 adult patients with severe periodontitis were analyzed.
P. gingivalis
was detected in 111 (43%) of the 259 subgingival plaque samples by culture and in 138 (53%) samples by PCR. The sensitivity, specificity, and positive and negative predictive values of the real-time PCR were 100, 94, 94, and 100%, respectively. We conclude that real-time PCR confirms the results of quantitative culture of
P. gingivalis
and offers significant advantages with respect to the rapidity and sensitivity of detection of
P. gingivalis
in subgingival plaque samples.
Molecular Epidemiology of
Proteus mirabilis
Infections of the Catheterized Urinary Tract
Authors: Sabbuba N. A. , Mahenthiralingam E. , Stickler D. J.
Published: November 2003

Proteus mirabilis
compromises the care of many patients undergoing long-term indwelling bladder catheterization. It forms crystalline bacterial biofilms in catheters which block the flow of urine, causing either incontinence due to leakage or painful distention of the bladder due to urinary retention. If it is not dealt with, catheter blockage can lead to pyelonephritis and septicemia. We have examined the epidemiology of catheter-associated
P. mirabilis
infections by use of pulsed-field gel electrophoresis (PFGE) of
Not
I restriction enzyme digests of bacterial DNA. This technique was shown to be more discriminatory than the classical phenotypic Dienes typing technique. We demonstrated that each of 42 isolates from diverse environmental sources and 10 of 12 isolates from blood, wound swabs, and mid-stream urine samples of hospitalized patients had distinct genotypes. Examination of a set of 55 isolates of
P. mirabilis
, each from a different clinical or environmental source, identified 49 distinct genotypes and 43 Dienes types. The index of discrimination was 0.993 for the PFGE method and 0.988 for the Dienes method. Applying the PFGE method to isolates from catheter-associated urinary tract infections confirmed that the strains present in the crystalline catheter biofilms were identical to those isolated from the same patient's urine. An analysis of samples taken during a prospective study of infections in catheterized nursing home patients revealed that a single genotype of
P. mirabilis
can persist in the urinary tract despite many changes of catheter, periods of noncatheterization, and antibiotic therapy.
Geographic Distribution and Clonal Diversity of
Streptococcus pneumoniae
Serotype 1 Isolates
Authors: Brueggemann Angela B. , Spratt Brian G.
Published: November 2003

Serotype 1 pneumococci are a major cause of serious disease and have been associated with outbreaks but are rarely carried. The high attack rate and lack of coverage of this serotype by the heptavalent conjugate vaccine prompted the characterization of a geographically diverse collection of 166 serotype 1 isolates from recent cases of invasive disease. The isolates were resolved by multilocus sequence typing into 16 clones, which clustered into three major lineages with very different geographic distributions. Lineage A isolates were exclusively from Europe and North America, lineage B isolates were predominantly from Africa and Israel, and lineage C isolates were mainly from Chile. There was no clear association between the presence of individual clones within a country and the prevalence of serotype 1 disease.
Characterization of Isolates of Methicillin-Resistant
Staphylococcus aureus
from Hong Kong by Phage Typing, Pulsed-Field Gel Electrophoresis, and Fluorescent Amplified-Fragment Length Polymorphism Analysis
Authors: Ip M. , Lyon D. J. , Chio F. , Enright M. C. , Cheng A. F.
Published: November 2003

The genetic relatedness of 127 methicillin-resistant
Staphylococcus aureus
(MRSA) isolates, belonging to five major types as identified by pulsed-field gel electrophoresis (PFGE) and antibiotic resistance profiles, was examined further using phage typing and fluorescent amplified fragment length polymorphism (FAFLP). The MRSA isolates were recovered from patients at the Prince of Wales Hospital (PWH), Hong Kong, over a 13-year period, 1988 to 2000. These strains were also compared with representatives of the well-described MRSA international clones and with epidemic MRSA strains (eMRSA) 1 to 16 from the United Kingdom. Phage typing distinguished two major “clones” at this hospital: all of the phage type 1 (PT1) isolates belonged to PFGE types A, C, D, and E, while most of the PT2 isolates were associated with PFGE type B, which exhibited a unique antibiotic resistance profile. MRSA isolates belonging to PFGE subtype A2 were indistinguishable from the British eMRSA-1, while isolates of PFGE type B were closely related to eMRSA-9 by PFGE. Based on FAFLP, all five predominant PFGE types at the PWH belonged to one group and fell into the same cluster as eMRSA-1, -4, -7, -9, and -11 isolates. Multilocus sequence typing and staphylococcal cassette chromosome
mec
typing classified representatives of our MRSA isolates as members of the same clone (ST239-MRSA-III). Thus, the predominant MRSA isolates frin the PWH in the last decade are closely related to early United Kingdom eMRSA clones 1, 4, and 11 and are members of a lineage that includes the Brazilian MRSA clone.
Enzyme-Linked Immunosorbent Assay Employing a Recombinant Antigen for Detection of Protective Antibody against Swine Erysipelas
Authors: Imada Yumiko , Mori Yasuyuki , Daizoh Masaji , Kudoh Kazuma , Sakano Tetsuya
Published: November 2003

The specificities and sensitivities of five recombinant proteins of the surface protective antigen (SpaA) of
Erysipelothrix rhusiopathiae
were examined by indirect enzyme-linked immunosorbent assay (ELISA) with the aim of developing a reliable serological test for the detection of protective antibody against
E. rhusiopathiae
. Fully mature protein and the N-terminal 416 amino acids (SpaA416) showed sufficient antigenicities, and further examination was done with SpaA416 because of its higher yield. The antibody titers of pigs experimentally immunized with commercial live vaccine and two types of inactivated vaccines clearly increased after immunization, and all pigs were completely protected against challenge with virulent strains. On the other hand, the antibody titers of nonimmunized control pigs remained very low until they were challenged, and all showed severe symptoms or subsequently died. Interference with the production of antibody against live vaccine by maternal antibody or porcine respiratory and reproductive syndrome virus infection 1 week after vaccination was also clearly detected. Because the ELISA titer correlated well with the protection results, the specificity and sensitivity of the ELISA were further evaluated with sera collected from pigs reared on 1 farm on which animals had acute septicemia, 2 farms on which the animals were infected or free from infection, and 10 farms on which the animals were vaccinated with live vaccine, among others. The ELISA titers clearly revealed the conditions of the herds. These results indicate that the SpaA416 ELISA is an effective method not only for evaluating pigs for the presence of protective antibody levels resulting from vaccination or maternal antibody but also for detecting antibody produced by natural infection. This test has important potential for the effective control of swine erysipelas.
High Diversity ofankASequences ofAnaplasma phagocytophilumamongIxodes ricinusTicks in Germany
Authors: von Loewenich Friederike D. , Baumgarten Birgit U. , Schröppel Klaus , Geißdörfer Walter , Röllinghoff Martin , Bogdan Christian
Published: November 2003

In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes ofAnaplasma phagocytophilumstrains infecting GermanIxodes ricinusticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022I. ricinusticks were investigated for infection withA. phagocytophilumby nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA andgroESLgenes were sequenced. The complete coding sequence of theankAgene could be determined in 24 samples. The 16S rRNA andgroESLgene sequences were as much as 100% identical to known sequences. FifteenankAsequences were ≥99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, GermanI. ricinusticks most likely harborA. phagocytophilumstrains that can cause disease in humans. Nine additional sequences were clearly different from knownankAsequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use theankAgene for further phylogenetic analyses ofA. phagocytophilumand to investigate the biology and pathogenicity of strains that differ in theankAgene.
Characterization of the Natural Population of
Bartonella henselae
by Multilocus Sequence Typing
Authors: Iredell J. , Blanckenberg D. , Arvand M. , Grauling S. , Feil E. J. , Birtles R. J.
Published: November 2003

Investigations of the population genetics of
Bartonella henselae
have demonstrated a high level of diversity among strains, and the delineation of isolates into one of two subtypes, type I (Houston) and type II (Marseille), represented by specific 16S ribosomal DNA (rDNA) sequences, has long been considered the most significant genotypic division within the species. This belief is challenged by recent work suggesting a role for horizontal gene exchange in generating intraspecies diversity. We attempted to resolve this issue and extend exploration of the population structure of
B. henselae
by using multilocus sequence typing (MLST) to examine the distribution of polymorphisms within nine different genes in a sample of 37 human and feline isolates. MLST distinguished seven sequence types (STs) that resolved into three distinct lineages, suggesting a clonal population structure for the species, and support for these divisions was obtained by macrorestriction analysis using pulsed-field gel electrophoresis. The distribution of STs among isolates recovered from human infections was not random, and such isolates were significantly more often associated with one particular ST, lending further support to the suggestion that specific genotypes contribute disproportionately to the disease burden in humans. All but one isolate lay on lineages that bore the representative strain of either the Houston or Marseille subtype. However, the distribution of the two 16S rDNA alleles among the isolates was not entirely congruent with their lineage allocations, indicating that this is not a sensitive marker of the clonal divisions within the species. The inheritances of several of the genes studied could not be reconciled with one another, providing further evidence of horizontal gene transfer among
B. henselae
strains and suggesting that recombination has a role in shaping the genetic character of bartonellae.
Prevalence, Molecular Epidemiology, and Clinical Significance of Heterogeneous Glycopeptide-Intermediate
Staphylococcus aureus
in Liver Transplant Recipients
Authors: Bert Frédéric , Clarissou Juliette , Durand François , Delefosse Didier , Chauvet Chantal , Lefebvre Patricia , Lambert Nicole , Branger Catherine
Published: November 2003

We investigated the prevalence, molecular epidemiology, and clinical significance of heterogeneous glycopeptide-intermediate
Staphylococcus aureus
(hGISA) isolates in 48 liver transplant recipients infected or colonized with methicillin-resistant
S. aureus
over a 5-year period. Strains were screened for hGISA on Mueller-Hinton agar containing 5 mg of teicoplanin per liter. Heterogeneous glycopeptide resistance was confirmed by the E-test method with a dense inoculum and a simplified method of population analysis. hGISA strains were found in 13 (27%) of the 48 patients studied. Eleven of the 13 strains shared a common multiresistant phenotype with homogeneous methicillin resistance and gentamicin resistance, and they were closely related according to the results of pulsed-field gel electrophoresis. Only 2 of the 13 patients infected or colonized with hGISA strains had previously received glycopeptide therapy. Most patients were successfully treated with vancomycin, but one patient who failed to respond to vancomycin subsequently died. These results suggest that the high prevalence of hGISA among our patients was due to the clonal spread of a multiresistant strain.
Comparison between Indirect Immunofluorescence Assay and Shell Vial Culture for Detection of Mumps Virus from Clinical Samples
Authors: Reina Jordi , Ballesteros Francisca , Ruiz de Gopegui Enrique , Munar Maria , Mari Margarita
Published: November 2003

We report a prospective comparison of the efficacies of an indirect immunofluorescence assay (IFA) and shell vial culture (SVC) of throat swab and urine samples from patients with mumps. Throat swab samples were used for the IFA; the urine samples and throat swabs were inoculated into vials of Vero cells. We studied 62 patients by using 62 throat swabs and 50 urine samples (50 patients with both samples). Sixty (96.7%) throat samples were positive in the SVC, and 61 (98.3%) were positive in the IFA. For the 50 patients from whom both samples were available, the IFA was positive in 50 (100%) cases, the urine sample was positive in 49 (98%) cases, and the throat swab was positive in 48 (96%) cases (
P
> 0.05). This comparison of throat swabs and urine samples has shown that the two clinical samples are similar in efficacy.
Genetic Characterization of Newly Reintroduced Dengue Virus Type 3 in Martinique (French West Indies)
Authors: Peyrefitte Christophe N. , Couissinier-Paris Patricia , Mercier-Perennec Véronique , Bessaud Maël , Martial Jenny , Kenane Nadia , Durand Jean-Paul A. , Tolou Hugues J.
Published: November 2003

Dengue type 3 viruses were isolated from patients in Martinique between 1999 and 2002. This serotype had not been detected on the island in the last 20 years. Genomic sequence determination and analysis showed great stability of the virus during the period studied.
Molecular Variation in the Variable-Length PCR Target and 120-Kilodalton Antigen Genes ofEhrlichia chaffeensisfrom White-Tailed Deer (Odocoileus virginianus)
Authors: Yabsley Michael J. , Little Susan E. , Sims Ethan J. , Dugan Vivien G. , Stallknecht David E. , Davidson William R.
Published: November 2003

Genes encoding two surface-expressed antigens ofEhrlichia chaffeensis, the variable-length PCR target (VLPT) and the 120-kDa antigen, which contain variable numbers of tandem repeats, were characterized forE. chaffeensisfrom white-tailed deer (Odocoileus virginianus). Both genes from infected deer contained numbers of repeats similar to those reported in genes from humans and ticks, although a new variant of the 120-kDa antigen gene containing five repeat units and coinfection with multiple VLPT and 120-kDa antigen gene genetic types were detected. Sequence analysis of both genes revealed more nucleotide variation than previously reported forE. chaffeensisfrom infected humans or ticks. This is the most extensive study ofE. chaffeensisVLPT and 120-kDa antigen gene genetic variation to date and is the first to examine genetic variation inE. chaffeensisfrom a nonhuman vertebrate host.
Performance of the Focus and Kalon Enzyme-Linked Immunosorbent Assays for Antibodies to Herpes Simplex Virus Type 2 Glycoprotein G in Culture-Documented Cases of Genital Herpes
Authors: Morrow Rhoda Ashley , Friedrich David , Krantz Elizabeth
Published: November 2003

Glycoprotein G-based herpes simplex virus type 2 (HSV-2) enzyme-linked immunosorbent assays from Focus and Kalon were performed with specimens from 118 patients with culture-documented genital herpes episodes, and their results were compared. Sensitivity was 52% by Kalon and 86% by Focus for first HSV-2 episodes and 100% (for each of the two tests) for recurrent HSV-2. Median times to seroconversion were 120 days by the Kalon assay, 21 days by the Focus assay, and 68 days by Western blotting assay. Values for specificity were 100% (Kalon) and 93% (Focus).
Evaluation of Genotyping Large Numbers of
Escherichia coli
Isolates by Enterobacterial Repetitive Intergenic Consensus-PCR
Authors: Meacham Kiersten J. , Zhang Lixin , Foxman Betsy , Bauer Richard J. , Marrs Carl F.
Published: November 2003

We analyzed a large collection of
Escherichia coli
isolates typed by enterobacterial repetitive intergenic consensus (ERIC)-PCR with BioNumerics gel analysis software. However, the interexperimental variation of ERIC-PCR caused the computer software to classify repeated isolates as different. ERIC-PCR should not be used as the sole determinant of genetic similarity when typing large numbers of bacterial isolates via computer software.
Frequency of Binary Toxin Genes among
Clostridium difficile
Strains That Do Not Produce Large Clostridial Toxins
Authors: Geric Barbara , Johnson Stuart , Gerding Dale N. , Grabnar Miklavz , Rupnik Maja
Published: November 2003

Pathogenic strains of
Clostridium difficile
commonly produce two large clostridial toxins (LCTs), A and B, virulence factors responsible for
C. difficile
disease. Some strains have been reported to produce an additional toxin, a binary toxin designated CDT. Binary toxin has cytotoxic effects on cells in culture, but its role in human disease is not yet defined. In this study we examined the frequency of binary toxin genes (
cdtB
and
cdtA
) among
C. difficile
isolates that do not produce LCTs (A
−
B
−
) from a large United States-based collection organized by restriction endonuclease analysis (REA) typing. Of 58 strains tested, 9 (15.5%) were
cdtB
and
cdtA
positive, including 4 of 46 (8.7%) non-LCT-producing REA groups, with an estimated prevalence of at least 2% of all non-LCT-producing isolates within the collection. Five of the binary toxin-positive strains belonged to toxinotype XI, which does not produce LCTs but has minor parts of the LCT coding region or pathogenicity locus (PaLoc). We describe two new binary toxin-positive variants, one without any remnant of the LCT genes. This previously unknown variation was found in three isolates that were unrelated by REA typing. LCT-negative, binary toxin-positive strains were isolated from symptomatic and asymptomatic patients and from the hospital environment.
Restriction Enzyme Analysis of Ribosomal DNA Shows that
Candida inconspicua
Clinical Isolates Can Be Misidentified as
Candida norvegensis
with Traditional Diagnostic Procedures
Authors: Majoros L. , Kardos G. , Belák Á. , Maráz A. , Asztalos L. , Csánky E. , Barta Z. , Szabó B.
Published: November 2003

We identified 29 yeast isolates from 22 patients using the API ID32C panel. Twenty-eight of these isolates were
Candida norvegensis
and one was
C. inconspicua
. Although
C. norvegensis
is considered a pseudohypha-producing species, only one isolate produced pseudohyphae. Restriction enzyme analysis of PCR-amplified ribosomal DNA with four different enzymes proved that all isolates were
C. inconspicua
.
Molecular Surveillance of
Cryptosporidium
spp. in Raw Wastewater in Milwaukee: Implications for Understanding Outbreak Occurrence and Transmission Dynamics
Authors: Zhou Ling , Singh Ajaib , Jiang Jianlin , Xiao Lihua
Published: November 2003

Six
Cryptosporidium
spp. were found in 50 of 179 Milwaukee wastewater samples collected weekly over a year. Of the eight subtypes of
Cryptosporidium hominis
and
Cryptosporidium parvum
present, allele Ib was found in 14 of 16 samples, and its sequence was identical to that of the subtype in human samples from the 1993 Milwaukee outbreak of cryptosporidiosis.
Collaborative Consensus for Optimized Multilocus Sequence Typing of
Candida albicans
Authors: Bougnoux M.-E. , Tavanti A. , Bouchier C. , Gow N. A. R. , Magnier A. , Davidson A. D. , Maiden M. C. J. , d'Enfert C. , Odds F. C.
Published: November 2003

A panel of 86 different
Candida albicans
isolates was subjected to multilocus sequence typing (MLST) in two laboratories to obtain sequence data for 10 published housekeeping gene fragments. Analysis of data for all possible combinations of five, six, seven, eight, and nine of the fragments showed that a set comprising the fragments
AAT1a
,
ACC1
,
ADP1
,
MPIb
,
SYA1
,
VPS13
, and
ZWF1b
was the smallest that yielded 86 unique diploid sequence types for the 86 isolates. This set is recommended for future MLST with
C. albicans
.
Prevalence of Group A Streptococcal Carriers in Asymptomatic Children and Clonal Relatedness among Isolates in Malatya, Turkey
Authors: Durmaz Riza , Durmaz Bengul , Bayraktar Mehmet , Ozerol Ibrahim Halil , Kalcioglu Mahmut Tayyar , Aktas Elif , Cizmeci Zeynep
Published: November 2003

In our study, the prevalence of nasopharyngeal
Streptococcus pyogenes
was 130 (14.3%) of 909 healthy children. Isolates were found to be susceptible to all antibiotics tested. Pulsed-field gel electrophoresis and arbitrarily primed PCR revealed that 34 (32.4%) of the 105 isolates and 41 (40.6%) of the 101 isolates typed, respectively, were clonally indistinguishable.
Infections in International Pregnancy Study: Performance of the Optical Immunoassay Test for Detection of Group B Streptococcus
Authors: Thinkhamrop Jadsada , Limpongsanurak Sompop , Festin Mario R. , Daly Sean , Schuchat Anne , Lumbiganon Pisake , Zell Elizabeth , Chipato Tsungai , Win Aye Aye , Perilla Mindy J. , Tolosa Jorge E. , Whitney Cynthia G.
Published: November 2003

We evaluated the Strep B optical immunoassay (OIA; ThermoBiostar, Inc.) for detecting light and heavy group B streptococcus colonization in 1,306 pregnant women. The women were examined at 20 to 32 weeks gestation and were from six countries. Compared to culture, the sensitivity and specificity of OIA were 13.3 and 98.4%, respectively, for light colonization and 41.5 and 97.7%, respectively, for heavy colonization.
Disseminated Trichosporonosis Caused by
Trichosporon loubieri
Authors: Marty Francisco M. , Barouch Dan H. , Coakley Eoin P. , Baden Lindsey R.
Published: November 2003

Trichosporonosis is an emerging invasive fungal infection in immunosuppressed patients; a case of disseminated infection caused by
Trichosporon loubieri
presented confirms its role as a human pathogen.
Capnocytophaga
sp. Isolated from a Cat with Chronic Sinusitis and Rhinitis
Authors: Frey Erin , Pressler Barrak , Guy James , Pitulle Christian , Breitschwerdt Edward
Published: November 2003

A
Capnocytophaga
sp. was inadvertently isolated from a cat with chronic sinusitis and rhinitis when cytopathic effects were observed in Crandall-Reese feline kidney cells that had been inoculated with oropharyngeal swab samples. Although
Capnocytophaga
spp. are of considerable zoonotic importance, their clinical relevance for dogs or cats has not been established. However, failure to do so may be attributed to the infrequent use of specialized isolation techniques that are required to grow
Capnocytophaga
spp. To our knowledge, successful isolation of these organisms from a cat with nasopharyngeal disease has not been reported.
Implantable Defibrillator Pocket Infection and Bacteremia Caused by
Nocardia nova
Complex Isolate
Authors: Boell Keith , Gotoff Robert , Foltzer Michael , Storm Randle , Bourbeau Paul P.
Published: November 2003

Nocardia
spp. are common environmental organisms that, to our knowledge, have never been implicated as causing an implantable defibrillator or pacemaker infection. We describe a 70-year-old male with a recent implantable cardiac defibrillator revision and subsequent device infection and bacteremia caused by a
Nocardia nova
complex isolate.
NEW DUAL RELATIONS BETWEEN QUANTUM FIELD THEORY AND STRING REGIMES IN CURVED BACKGROUNDS
Authors: RAMON MEDRANO M. , SANCHEZ N. G.
Published: November 2003

A ℛ "dual" transform is introduced which relates Quantum Field Theory and String regimes, both in a curved background with D-non-compact dimensions. This operation maps the characteristic length of one regime into the other (and, as a consequence, mass domains as well). The ℛ-transform is not an assumed or a priori imposed symmetry but is revealed by the QFT and String dynamics in curved backgrounds. The Hawking–Gibbons temperature and the string maximal or critical temperature are ℛ-mapped one into the other. If back reaction of quantum matter is included, Quantum Field Theory and String phases appear, and ℛ-relations between them manifest as well. These ℛ-transformations are explicitly shown in two relevant examples: Black Hole and de Sitter spacetimes.
ON PROBLEMS OF THE LAGRANGIAN QUANTIZATION OF W3-GRAVITY
Authors: GEYER B. , GITMAN D. M. , LAVROV P. M. , MOSHIN P. YU.
Published: November 2003

We consider the two-dimensional model of W3-gravity within Lagrangian quantization methods for general gauge theories. We use the Batalin–Vilkovisky formalism to study the arbitrariness in the realization of the gauge algebra. We obtain a one-parametric nonanalytic extension of the gauge algebra, and a corresponding solution of the classical master equation, related via an anticanonical transformation to a solution corresponding to an analytic realization. We investigate the possibility of closed solutions of the classical master equation in the Sp (2)-covariant formalism and show that such solutions do not exist in the approximation up to the third order in ghost and auxiliary fields.
COVARIANT WIGNER FUNCTION APPROACH TO THE BOLTZMANN EQUATION FOR MIXING FERMIONS IN CURVED SPACE–TIME
Authors: YAMAMOTO KAZUHIRO
Published: November 2003

Based on the covariant Wigner function approach we derive the quantum Boltzmann equation for fermions with flavor mixing in general curved space–time. This work gives a rigorous theoretical framework to investigate the flavor oscillation phenomena taking the gravitational effect into account. It is shown that the Boltzmann equation of the lowest order of the expansion with respect to ℏ reproduces the previous result which was derived in the relativistic limit on the Minkowski background space–time. It is demonstrated that the familiar formula for the vacuum neutrino oscillation can be obtained by solving the Boltzmann equation. Higher order effects of the ℏ-expansion are also briefly discussed.