bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Characterization and Substrate Preference of a Polycyclic Aromatic Hydrocarbon Dioxygenase from
Cycloclasticus
sp. Strain A5
Authors: Kasai Yuki , Shindo Kazutoshi , Harayama Shigeaki , Misawa Norihiko
Published: November 2003

Cycloclasticus
sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated
phn
genes, although the sequence and order of these
phn
genes were significantly different from the sequence and order of the known PAH-degrading genes. The
phnA1
,
phnA2
,
phnA3
, and
phnA4
genes, which encode the α and β subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The
phnA4A3
gene cluster was located 3.7 kb downstream of the
phnA2
gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the α and β subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved.
Escherichia coli
cells possessing the
phnA1A2A3A4
genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the
E. coli
cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases.
Usefulness of
rpoB
Gene Sequencing for Identification of
Afipia
and
Bosea
Species, Including a Strategy for Choosing Discriminative Partial Sequences
Authors: Khamis Atieh , Colson Philippe , Raoult Didier , Scola Bernard La
Published: November 2003

Bacteria belonging to the genera
Afipia
and
Bosea
are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete
rpoB
sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of
Bosea
and
Afipia
is discussed in light of these results. For strain identification that does not require the complete
rpoB
sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability “hot spots” in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.
New Method Using Sedimentation and Immunomagnetic Separation for Isolation and Enumeration of
Cryptosporidium parvum
Oocysts and
Giardia lamblia
cysts
Authors: Massanet-Nicolau Jaime
Published: November 2003

A new method for the isolation of
Cryptosporidium parvum
oocysts and
Giardia lamblia
cysts from biosolid samples has been developed that utilizes sedimentation and immunomagnetic separation. The method was used to recover stained cysts and oocysts (spike organisms) from primary settled sewage sludge, anaerobically digested sewage sludge, and bovine manure. Recovery efficiencies associated with this method were approximately 40 to 60% and were significantly greater than those associated with similar methods based on sucrose flotation (
P
< 0.001). The recovery efficiency of the sedimentation-based method showed no significant reduction as a result of sample storage for up to 21 days (
P
> 0.05). Recovery efficiencies were determined by spiking samples with prestained cysts and oocysts, allowing them to be differentiated from those naturally present in the biosolid samples. The prestained cysts and oocysts had been fixed in 5% formalin, and the recovery efficiencies associated with this method may be different from recovery efficiencies for fresh cysts or oocysts.
Web-Based Phylogenetic Assignment Tool for Analysis of Terminal Restriction Fragment Length Polymorphism Profiles of Microbial Communities
Authors: Kent Angela D. , Smith Dan J. , Benson Barbara J. , Triplett Eric W.
Published: November 2003

Culture-independent DNA fingerprints are commonly used to assess the diversity of a microbial community. However, relating species composition to community profiles produced by community fingerprint methods is not straightforward. Terminal restriction fragment length polymorphism (T-RFLP) is a community fingerprint method in which phylogenetic assignments may be inferred from the terminal restriction fragment (T-RF) sizes through the use of web-based resources that predict T-RF sizes for known bacteria. The process quickly becomes computationally intensive due to the need to analyze profiles produced by multiple restriction digests and the complexity of profiles generated by natural microbial communities. A web-based tool is described here that rapidly generates phylogenetic assignments from submitted community T-RFLP profiles based on a database of fragments produced by known 16S rRNA gene sequences. Users have the option of submitting a customized database generated from unpublished sequences or from a gene other than the 16S rRNA gene. This phylogenetic assignment tool allows users to employ T-RFLP to simultaneously analyze microbial community diversity and species composition. An analysis of the variability of bacterial species composition throughout the water column in a humic lake was carried out to demonstrate the functionality of the phylogenetic assignment tool. This method was validated by comparing the results generated by this program with results from a 16S rRNA gene clone library.
Diversity and Succession of the Intestinal Bacterial Community of the Maturing Broiler Chicken
Authors: Lu Jiangrang , Idris Umelaalim , Harmon Barry , Hofacre Charles , Maurer John J. , Lee Margie D.
Published: November 2003

The diversity of bacterial floras in the ilea and ceca of chickens that were fed a vegetarian corn-soy broiler diet devoid of feed additives was examined by analysis of 1,230 partial 16S rRNA gene sequences. Nearly 70% of sequences from the ileum were related to those of
Lactobacillus
, with the majority of the rest being related to
Clostridiaceae
(11%),
Streptococcus
(6.5%), and
Enterococcus
(6.5%). In contrast,
Clostridiaceae
-related sequences (65%) were the most abundant group detected in the cecum, with the other most abundant sequences being related to
Fusobacterium
(14%),
Lactobacillus
(8%), and
Bacteroides
(5%). Statistical analysis comparing the compositions of the different 16S rRNA libraries revealed that population succession occurred during some sampling periods. The significant differences among cecal libraries at 3 and 7 days of age, at 14 to 28 days of age, and at 49 days of age indicated that successions occurred from a transient community to one of increasing complexity as the birds aged. Similarly, the ileum had a stable bacterial community structure for birds at 7 to 21 days of age and between 21 to 28 days of age, but there was a very unique community structure at 3 and 49 days of age. It was also revealed that the composition of the ileal and cecal libraries did not significantly differ when the birds were 3 days old, and in fact during the first 14 days of age, the cecal microflora was a subset of the ileal microflora. After this time, the ileum and cecum had significantly different library compositions, suggesting that each region developed its own unique bacterial community as the bird matured.
Long-Term Succession of Structure and Diversity of a Biofilm Formed in a Model Drinking Water Distribution System
Authors: Martiny Adam C. , Jørgensen Thomas M. , Albrechtsen Hans-Jørgen , Arvin Erik , Molin Søren
Published: November 2003

In this study, we examined the long-term development of the overall structural morphology and community composition of a biofilm formed in a model drinking water distribution system with biofilms from 1 day to 3 years old. Visualization and subsequent quantification showed how the biofilm developed from an initial attachment of single cells through the formation of independent microcolonies reaching 30 μm in thickness to a final looser structure with an average thickness of 14.1 μm and covering 76% of the surface. An analysis of the community composition by use of terminal restriction fragment length polymorphisms showed a correlation between the population profile and the age of the sample, separating the samples into young (1 to 94 days) and old (571 to 1,093 days) biofilms, whereas a limited spatial variation in the biofilm was observed. A more detailed analysis with cloning and sequencing of 16S rRNA fragments illustrated how a wide variety of cells recruited from the bulk water initially attached and resulted in a species richness comparable to that in the water phase. This step was followed by the growth of a bacterium which was related to
Nitrospira
, which constituted 78% of the community by day 256, and which resulted in a reduction in the overall richness. After 500 days, the biofilm entered a stable population state, which was characterized by a greater richness of bacteria, including
Nitrospira
,
Planctomyces
,
Acidobacterium
, and
Pseudomonas
. The combination of different techniques illustrated the successional formation of a biofilm during a 3-year period in this model drinking water distribution system.
Analysis, Characterization, and Loci of the
tuf
Genes in
Lactobacillus
and
Bifidobacterium
Species and Their Direct Application for Species Identification
Authors: Ventura Marco , Canchaya Carlos , Meylan Valèrie , Klaenhammer Todd R. , Zink Ralf
Published: November 2003

We analyzed the
tuf
gene, encoding elongation factor Tu, from 33 strains representing 17
Lactobacillus
species and 8
Bifidobacterium
species. The
tuf
sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these
tuf
sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of
tuf
sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as
Lactobacillus casei
group members. These
tuf
gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of
tuf
genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one
tuf
gene in the genomes of lactobacilli and bifidobacteria, but the
tuf
gene was arranged differently in the genomes of these two taxa. Our results revealed that the
tuf
gene in bifidobacteria is flanked by the same gene constellation as the
str
operon, as originally reported for
Escherichia coli
. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the
tuf
gene in four
Lactobacillus
species indicated the same four-gene unit and suggested a novel
tuf
operon specific for the genus
Lactobacillus
.
Influence of Storage on Monodispersed Cells of
Mycobacterium terrae
Used for Quantitative Carrier Test prEN 14563
Authors: Woelk Eva , Goroncy-Bermes Peter , Sand Wolfgang
Published: November 2003

The degree of cell clumping increased with time of storage (1% cell clumps immediately after homogenization and 3 and 6.5% after 48 and 96 h of storage, respectively), and the number of living single cells decreased. Quantitative carrier tests were carried out with these cells using ortho-phthaldialdehyde (OPA) and coco fatty aminoxethylate as biocides. In contrast to OPA, with coco fatty aminoxethylate the reductions obtained with freshly homogenized mycobacteria were significantly higher (
P
= 0.02) than those obtained with mycobacteria kept in the refrigerator for 4 days. Therefore, it is advisable to prepare the test suspension freshly for each test.
FUM9
Is Required for C-5 Hydroxylation of Fumonisins and Complements the Meitotically Defined
Fum3
Locus in
Gibberella moniliformis
Authors: Butchko Robert A. E. , Plattner Ronald D. , Proctor Robert H.
Published: November 2003

Deletion of the
Gibberella moniliformis FUM9
gene resulted in mutants that produce only fumonisins that lack a C-5 hydroxyl group. This phenotype is identical to that of previously described mutants with defective alleles at the meiotically defined
Fum3
locus. Transformation with a wild-type
FUM9
gene into a
Fum3
-defective mutant restored wild-type fumonisin production. These results indicate that the
FUM9
protein catalyzes the C-5 hydroxylation of fumonisins and that
FUM9
and the
Fum3
locus are the same gene.
Update on the Major Clonal Types of Methicillin-Resistant
Staphylococcus aureus
in the Czech Republic
Authors: Melter O. , Aires de Sousa M. , Urbášková P. , Jakubů V. , Žemličková H. , de Lencastre H.
Published: November 2003

The purpose of our study was the molecular characterization of methicillin-resistant
Staphylococcus aureus
(MRSA) strains isolated in 21 hospitals in the Czech Republic in the period 2000-2002 and comparison with previous results from 1996-1997. Strains were analyzed by pulsed-field gel electrophoresis (PFGE) of
Sma
I digests and ribotyping of
Hin
dIII digests hybridized with a 16S-23S DNA probe. The prevalence of the most clinically important macrolide (
ermA
,
ermB
,
ermC
, and
msrA
) and aminoglycoside (
aph3
′,
ant4
′, and
aac6
′
-aph2
") resistance genes was evaluated as well. Selected isolates representative of each clonal type were analyzed by multilocus sequence typing and by a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome
mec
(SCC
mec
) carried by the bacteria. Our results document the displacement of the Brazilian clone (ST239, SCC
mec
type IIIA, PFGE type B, ribotype H1) by a new clone that we named “Czech clone” (ST239, SCC
mec
type IIIA, PFGE type F, ribotype H6) and the maintenance of the Iberian clone (ST247, SCC
mec
type IA, PFGE type A, ribotype H2) exclusively in one hospital in the Czech Republic. In addition, we found a correlation between the distribution of aminoglycoside resistance genes and MRSA clonal types.
Detection and Identification of
Entamoeba
Species in Stool Samples by a Reverse Line Hybridization Assay
Authors: Verweij Jaco J. , Laeijendecker Daphne , Brienen Eric A. T. , van Lieshout Lisette , Polderman Anton M.
Published: November 2003

Classically, detection of
Entamoeba histolytica
is performed by microscopic examination for characteristic cysts and/or trophozoites in fecal preparations. Differentiation of
E. histolytica
cysts and those of nonpathogenic amoebic species is made on the basis of the appearance and the size of the cysts. However, by classical means objective tools for confirmation and quality control do not exist. Therefore, a reverse line blot hybridization assay was developed to detect a variety of
Entamoeba
species and genetic variants known to infect humans. The assay was performed after amplification with general
Entamoeba
-specific primers. The assay could identify four genetic variants of
Entamoeba polecki
-like cysts as well as
E. histolytica
,
Entamoeba dispar
,
Entamoeba hartmanni
,
Entamoeba moshkovskii
and
Entamoeba coli
and even mixed infections in a range of controls and fecal samples. This technique can be used as an additional standard for diagnosis, epidemiology, and quality control for amoebic infections.
Temporal Shifts in the Dominance of Serotypes of
Shigella dysenteriae
from 1999 to 2002 in Dhaka, Bangladesh
Authors: Talukder Kaisar A. , Islam M. Aminul , Khajanchi Bijay K. , Dutta Dilip K. , Islam Zhahirul , Safa Ashrafus , Alam Khorshed , Hossain A. , Nair G. B. , Sack David A.
Published: November 2003

A total of 358
Shigella dysenteriae
strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrheal Disease Research, Bangladesh, between the years 1999 and 2002 were included in this study.
S. dysenteriae
type 1, the dominant serotype in 1999 (76.4%), declined to 6.5% in 2002. On the other hand,
S. dysenteriae
types 2 to 12 were isolated with increasing frequencies of 19, 67, 73.5, and 87% in 1999, 2000, 2001, and 2002, respectively. Of these, types 2 and 4 were the most dominant serotypes, accounting for more than 18.7 and 28.5% of the total isolates, respectively. There was no isolation of serotypes 5, 7, 8, and 13 during this period. Twenty-eight (7.8%) of the isolates were atypical and agglutinated only with the polyvalent antiserum of
S. dysenteriae
. More than 98% of type 1 strains isolated between 1999 and 2001 were resistant to ampicillin, sulfamethoxazole-trimethoprim, and nalidixic acid. Among other serotypes of
S. dysenteriae
, Nal
r
type 2 strains were isolated in 2001 and 2002. Although heterogeneous plasmid profiles were obtained depending on the presence or absence of a single plasmid, core plasmids were defined for particular serotypes. On the other hand, the same plasmid profile was found to be shared by different serotypes. Interestingly, plasmid patterns of types 2 and 4 were almost identical except that a middle-range plasmid of 70 to 60 MDa was present in type 4 in addition to the core plasmids. All the strains harboring the 140-MDa plasmid were positive for the
ipaH
gene, had Congo red binding abilities, and were positive by the Sereny test, demonstrating their invasive properties.
PCR Assay for Monitoring
Trypanosoma cruzi
Parasitemia in Childhood after Specific Chemotherapy
Authors: Galvão Lúcia M. C. , Chiari Egler , Macedo Andréa M. , Luquetti Alejandro O. , Silva Simonne A. , Andrade Ana Lúcia S. S.
Published: November 2003

Assessment of cure of
Trypanosoma cruzi
infection by antibody seroconversion usually involves several years of follow-up. Parasitological negativity is useless for cure assessment, since even untreated patients mostly show negative results; conversely, positive tests are of great value because they indicate treatment failure. Here, PCR was used to assess the rate of specific chemotherapy failure in a well-characterized Brazilian cohort of
T. cruzi
-seropositive children, who were enrolled in a field trial of benznidazole (Bz) efficacy. Paired blood samples from 111 children were taken at baseline and 36 months after treatment with either Bz (
n
= 58) or a placebo (
n
= 53). DNA extraction and PCR amplification were carried out as previously described, and hybridization was performed with all PCR products. At the end of follow-up, PCR was positive for 39.6% of the patients in the Bz group versus 64.2% in the placebo group (
P
= 0.01). Untreated patients had a 1.6-fold-higher chance of remaining positive by PCR than those in the Bz group (
P
< 0.05). We conclude that PCR is a useful tool for revealing therapeutic failure of
T. cruzi
infection on a short-term basis.
Real-Time PCR Assay for Clinical Management of Human Immunodeficiency Virus-Infected Patients with Visceral Leishmaniasis
Authors: Bossolasco Simona , Gaiera Giovanni , Olchini Davide , Gulletta Maurizio , Martello Leonardo , Bestetti Arabella , Bossi Laura , Germagnoli Luca , Lazzarin Adriano , Uberti-Foppa Caterina , Cinque Paola
Published: November 2003

To evaluate the usefulness of a real-time PCR for
Leishmania
DNA in the diagnosis and follow-up of patients with human immunodeficiency virus type 1 (HIV-1) and
Leishmania
coinfection,
Leishmania
DNA levels were measured in whole peripheral blood from 25 HIV-infected patients with clinical features suggestive of visceral leishmaniasis.
Leishmania
DNA was detected in 10 of 25 patients with microscopically confirmed visceral leishmaniasis and in none of those without this disease. Following treatment with liposomal amphotericin B, a clinical response was observed in 9 of 10 patients, in association with significantly decreased parasite loads. Seven patients relapsed clinically a median of 110 days after the end of treatment, in association with substantial increases in
Leishmania
DNA levels.
Leishmania
DNA levels correlated with the clinical course of visceral leishmaniasis, and their measurement at diagnosis and during and after treatment seems to be useful in the clinical management of HIV-infected patients with this disease.
Evaluation and Field Validation of PCR Tests for Detection of
Actinobacillus pleuropneumoniae
in Subclinically Infected Pigs
Authors: Fittipaldi Nahuel , Broes André , Harel Josée , Kobisch Marylène , Gottschalk Marcelo
Published: November 2003

Eight PCR tests were evaluated for their abilities to detect
Actinobacillus pleuropneumoniae
in swine tonsils. At first they were compared regarding their specificities by using
A. pleuropneumoniae
and related bacterial species and their analytical sensitivities by using tonsils experimentally infected in vitro. PCRs were carried out both directly with tonsil homogenates (direct PCR) and after culture of the sample (after-culture PCR). Most tests demonstrated good specificities; however, some tests gave false-positive results with some non-
A. pleuropneumoniae
species. High degrees of variation in the analytical sensitivities among the tests were observed for the direct PCRs (10
9
to 10
2
CFU/g of tonsil), whereas those of most of the after-culture PCRs were similar (10
2
CFU/g of tonsil). In a second phase, the effects of sample storage time and storage conditions were evaluated by using tonsils from experimentally infected animals. Storage at −20°C allowed the detection of the organism for at least 4 months. Finally, the
omlA
PCR test described by Savoye et al. (C. Savoye et al., Vet. Microbiol. 73:337-347, 2000) and the commercially available Adiavet App PCR test were further validated with field samples. Their effectiveness was compared to those of standard and immunomagnetic separation-based methods of bacterial isolation. In addition, a comparison of tonsil biopsy specimens (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted.
A. pleuropneumoniae
was neither isolated nor detected by PCR from a herd serologically negative for
A. pleuropneumoniae
. PCR was more sensitive than the standard isolation method with whole tonsils from three infected herds. After-culture PCR offered the highest degree of sensitivity (93 and 83% for the
omlA
and Adiavet App PCRs, respectively). The PCR detection rate was higher with whole tonsils than with tonsil biopsy specimens. Good agreement (κ = 0.65) was found between the presence of
A. pleuropneumoniae
in tonsils and the individual serological status of the animals.
Experience with the MicroSeq D2 Large-Subunit Ribosomal DNA Sequencing Kit for Identification of Commonly Encountered, Clinically Important Yeast Species
Authors: Hall Leslie , Wohlfiel Sherri , Roberts Glenn D.
Published: November 2003

Experience with a MicroSeq D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing kit for identification of yeast species commonly encountered in the mycology laboratory at Mayo Clinic is described here. A total of 131 isolates of yeasts recovered from clinical specimens were included in the study. Phenotypic methods used for initial identification included germ tube formation, urease production, microscopic morphological features on cornmeal agar, and an API 20C AUX system; all isolates were sequenced using a MicroSeq D2 LSU rDNA sequencing kit. Nucleic acid sequencing identified 93.9% of the isolates to the correct genus and species. A total of 100 of the isolates (representing 19 species of
Candida
) were sequenced, and 98% gave results concordant with identifications made by the API 20C AUX system; distance scores ranged from 0 to 1.88%, with an average value of 0.23%.
Candida dubliniensis
was not included in the MicroSeq database and was identified as
Candida albicans
. A total of 32 isolates representing 9 other genera (including
Cryptococcus
,
Filobasidium
,
Kloeckera
,
Malassezia
,
Pichia
,
Sporidiobolus
,
Rhodotorula
,
Zygosaccharomyces
, and
Trichosporon
) were included, and 81.3% showed concordant results when phenotypic and sequencing results were compared. Most discrepancies were attributed to the lack of inclusion of the species in the MicroSeq or API 20C AUX database. The MicroSeq D2 LSU rDNA sequencing kit appears to be accurate and useful for the identification of yeasts that might be seen in a clinical laboratory.
Use of O-Antigen Gene Cluster-Specific PCRs for the Identification and O-Genotyping of
Yersinia pseudotuberculosis
and
Yersinia pestis
Authors: Bogdanovich Tatiana , Carniel Elisabeth , Fukushima Hiroshi , Skurnik Mikael
Published: November 2003

Yersinia pestis
is a very recently evolved clone of
Yersinia pseudotuberculosis
serotype O:1b. This close relationship causes potential difficulties in DNA-based diagnostic methods. Analysis of the O-antigen gene clusters in these two organisms identified two regions that were used to specifically identify
Y. pestis
-
Y. pseudotuberculosis
as a group or
Y. pestis
alone. Both PCR assays were found to be 100% specific when tested on a large collection of
Yersinia
species and other
Enterobacteriaceae
. Furthermore, advantage was taken of the different setups of the O-antigen gene clusters of the 21 known
Y. pseudotuberculosis
serotypes to develop a multiplex PCR assay to replace the conventional serotyping method of
Y. pseudotuberculosis
by O-genotyping. The multiplex PCR assay contained nine sets of specific PCRs in a single tube and when used on
Y. pseudotuberculosis
reference strains allowed the distinction of 14 individual serotypes and two duplex serotypes (O:4a-O:8 and O:12-O:13). Serotype O:7, O:9, and O:10 strains required additional PCRs for O-genotyping. Once applied to
Y. pseudotuberculosis
strains of various origins, a very good correlation between classical serotypes and O-genotypes was observed, although some discrepancies were found. O-genotyping also proved useful to correct misidentification of some strains and to type
Y. pseudotuberculosis
isolates that had lost the expression of the O-antigen. The PCR-based O-genotyping can easily be applied in conventional laboratories, without the need for tedious preparation of a large set of specific antisera.
Duplex Real-Time SYBR Green PCR Assays for Detection of 17 Species of Food- or Waterborne Pathogens in Stools
Authors: Fukushima Hiroshi , Tsunomori Yoshie , Seki Ryotaro
Published: November 2003

A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using the QIAamp DNA Stool Mini kit was evaluated with regard to detection of 8 of 17 species of food- or waterborne pathogens in five stool specimens in 2 h or less. The protocol used the same LC-PCR with 20 pairs of specific primers. The products formed were identified based on a melting point temperature (

T
m

) curve analysis. The 17 species of food- or waterborne pathogens examined were enteroinvasive
Escherichia coli
, enteropathogenic
E. coli
, enterohemorrhagic
E. coli
, enterotoxigenic
E. coli
, enteroaggregative
E. coli
,
Salmonella
spp.,
Shigella
spp.,
Yersinia enterocolitica
,
Yersinia pseudotuberculosis
,
Campylobacter jejuni
,
Vibrio cholerae
,
Vibrio parahaemolyticus
,
Vibrio vulnificus
,
Aeromonas
spp.,
Staphylococcus aureus
,
Clostridium perfringens
, and
Bacillus cereus.
No interference with the LC-PCR assay was observed when stool specimens were artificially inoculated with each bacterial species. The detection levels were approximately 10
5
food- or waterborne pathogenic bacteria per g of stool. The protocol for processing stool specimens for less than 10
4
food- or waterborne pathogenic bacteria per g of stool requires an overnight enrichment step to achieve adequate sensitivity. However, the rapid amplification and reliable detection of specific genes of greater than 10
5
food- or waterborne pathogenic bacteria per g in samples should facilitate the diagnosis and management of food- or waterborne outbreaks.
Comparison of DNA Amplification, mRNA Amplification, and DNA Hybridization Techniques for Detection of Cytomegalovirus in Bone Marrow Transplant Recipients
Authors: Diaz-Mitoma Francisco , Leger Chantal , Miller Helen , Giulivi Antonio , Frost Rita , Shaw Laura , Huebsch Lothar
Published: November 2003

A total of 676 specimens from 63 recipients of bone marrow allografts were tested for cytomegalovirus (CMV) by the following assays: CMV pp67 NucliSens (NS), AMPLICOR CMV MONITOR (RA), and the Digene CMV DNA test (DG). In a consensus analysis, the sensitivities and specificities were 60 and 99% (NS), 96 and 98% (RA), and 90 and 76% (DG), respectively; for detection of symptomatic CMV infection, they were 60 and 97% (NS), 65 and 97% (RA), and 95 and 77% (DG), respectively. In multivariate analysis, the major risk factor for symptomatic CMV infection was an increase in the viral load in the DG assay; in univariate analyses, maximum viral loads in both DG and RA assays and a rising viral load in the RA assay were also significant. The earliest detection of CMV replication was provided by the RA assay (mean, 39 days posttransplantation), followed by the DG assay (mean, 48 days) and the NS assay (mean, 58 days).
Detection of
Gallibacterium
spp. in Chickens by Fluorescent 16S rRNA In Situ Hybridization
Authors: Bojesen Anders Miki , Christensen Henrik , Nielsen Ole Lerberg , Olsen John Elmerdahl , Bisgaard Magne
Published: November 2003

Gallibacterium
has recently been included as a new genus of the family
Pasteurellaceae
Pohl 1981, which encompasses bacteria previously reported as
Pasteurella anatis
, “
Actinobacillus salpingitidis
,” and avian
Pasteurella haemolytica
-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus
Gallibacterium
. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families
Pasteurellaceae
,
Enterobacteriaceae
, and
Flavobacteriaceae
, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of
Gallibacterium
. Additionally, determination of the spatial distribution and the host cell affiliation of
Gallibacterium
at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of
Gallibacterium
infections in chickens.
Bloodstream Isolates of
Enterococcus faecium
Enriched with the Enterococcal Surface Protein Gene,
esp
, Show Increased Adhesion to Eukaryotic Cells
Authors: Lund Bodil , Edlund Charlotta
Published: November 2003

Infection-derived
Enterococcus faecium
strains enriched with
esp
had increased ability to adhere to Caco-2 cells (
P
< 0.05) and were less genetically diverse than
esp
-negative isolates.
esp
-negative
E. faecium
fecal isolates from healthy individuals adhered significantly better than
esp
-negative infection isolates (
P
< 0.05), indicating additional factors of importance to adhesion.
Detection and Sequence Analysis of Danish and Swedish Strains of Mink Astrovirus
Authors: Mittelholzer Christian , Englund Lena , Hedlund Kjell-Olof , Dietz Hans-Henrik , Svensson Lennart
Published: November 2003

The sequences of mink astroviruses collected from 11 farms in Denmark and Sweden were analyzed and found to be homologous with one another but different from those of other astroviruses. A species-specific reverse transcriptase-PCR for mink astrovirus was established and shown to be suitable for the analysis of clinical samples.
Comparison of Hepatitis C Virus Genotyping by 5′ Noncoding Region- and Core-Based Reverse Transcriptase PCR Assay with Sequencing and Use of the Assay for Determining Subtype Distribution in India
Authors: Lole Kavita S. , Jha Jyotsna A. , Shrotri Sandhya P. , Tandon Badri N. , Prasad V. G. Mohan , Arankalle Vidya A.
Published: November 2003

Phylogenetic analysis of the sequences of the 5′ noncoding regions (5′NCR) of 149 samples from hepatitis C virus (HCV) RNA-positive chronic carriers representing northern, southern, eastern, and western India showed that type 3 and type 1 are the predominant genotypes circulating in India, with an overall prevalence of 53.69 and 38.25%, respectively. Type 4 viruses (6.04%) were seen only in southern India. Sequence analysis of the core region of 51 of the above isolates enabled us to classify them further into subtypes as 1b (number of isolates [
n
] = 10), 1a (
n
= 6), 3a (
n
= 9), 3g (
n
= 14), 3f (
n
= 1), and 4d (
n
= 3). Three new subtypes were identified for the first time and designated as 3i (
n
= 5), 3j (
n
= 2), and 6l (
n
= 1). Sequencing the 5′NCR could differentiate HCV types, whereas classification at the level of subtype was possible with sequence analysis of the core region.
Isolation and Characterization of Dually Reactive Strains of Group A Rotavirus from Hospitalized Children
Authors: Ranshing Sujata S. , Kelkar Shobhana D.
Published: November 2003

Seven rotavirus strains dually reactive to serotype G1- and G2-specific monoclonal antibodies (MAbs) from hospitalized children with rotavirus diarrhea were culture adapted. Six strains were neutralized with G1 antiserum to a higher titer than that of G2, and one was neutralized with G1 and G2 antisera to equal titers. Of these, four strains were also neutralized with G6 antiserum. Five strains with short RNA pattern could not be serotyped, and the remaining two strains with long RNA pattern were serotyped as G1 strains. In addition, two strains showing dual reactivity to G2 and G4 MAbs and one G2-like strain from a nontypeable specimen were isolated. The dual reactivity of the isolates could not be attributed to mixed infections.
Efficient Extraction of Viral DNA and Viral RNA by the Chemagic Viral DNA/RNA Kit Allows Sensitive Detection of Cytomegalovirus, Hepatitis B Virus, and Hepatitis G Virus by PCR
Authors: Kleines Michael , Schellenberg Kirsten , Ritter Klaus
Published: November 2003

The Chemagic Viral DNA/RNA kit was evaluated for extraction of cytomegalovirus (CMV), hepatitis B virus (HBV), and hepatitis G virus (HGV) by using the QIAamp DNA Blood Mini kit and the QIAamp Viral RNA Mini kit as reference protocols. The extraction efficiencies of the different kits for CMV DNA and HBV DNA were not distinguishable, but the extraction efficiency for HGV RNA was better with the Chemagen protocol. All clinical specimens tested HBV DNA- or HGV RNA-positive after QIAGEN protocols for extraction were confirmed by using the Chemagen protocol. The Chemagen kit failed to confirm one of 75 CMV DNA-positive specimens. Thus, a new competitive extraction method using a technology with a high potential for automation is available.
Invasive Mycotic Infections Caused by
Chaetomium perlucidum
, a New Agent of Cerebral Phaeohyphomycosis
Authors: Barron M. A. , Sutton D. A. , Veve R. , Guarro J. , Rinaldi M. , Thompson E. , Cagnoni P. J. , Moultney K. , Madinger N. E.
Published: November 2003

We report the first two cases of invasive human mycoses caused by the phaeoid ascomycete,
Chaetomium perlucidum
, and review the English literature regarding invasive
Chaetomium
infections. Fatal disseminated disease involving the brain, heart, lungs, and spleen is described in an acute myelogenous leukemia patient. A second patient with a history of asthma and chronic bronchiectasis experiencing right-middle-lobe syndrome grew
C. perlucidum
from lung tissue. This study adds
C. perlucidum
to the list of other known neurotropic
Chaetomium
species,
C. atrobrunneum
and
C. strumarium
, and also documents this organism's ability to disseminate beyond the central nervous system.
Presumed Endocarditis Caused by BRO β-Lactamase-Producing
Moraxella lacunata
in an Infant with Fallot's Tetrad
Authors: Nagano Noriyuki , Sato Junichi , Cordevant Christophe , Nagano Yukiko , Taguchi Fumiaki , Inoue Matsuhisa
Published: November 2003

A case of presumed endocarditis caused by
Moraxella lacunata
in a 15-month-old male infant with Fallot's tetrad is described. This infection may have occurred as the result of transmission of this organism between the father and his son. This is the first report of BRO β-lactamase-producing
M. lacunata
causing presumed endocarditis.
Allergic Fungal Sinusitis Associated with
Trichoderma longibrachiatum
Authors: Tang Patrick , Mohan Subhash , Sigler Lynne , Witterick Ian , Summerbell Richard , Campbell Iivi , Mazzulli Tony
Published: November 2003

We describe allergic fungal sinusitis caused by
Trichoderma longibrachiatum
in a patient with a history of atopy and asthma. A Gram stain of a sinus biopsy specimen was initially thought to contain yeast cells, but when
Trichoderma
was recovered in culture, these cells were subsequently recognized as chlamydospores. The patient was successfully managed with a combination of sinus lavage, oral corticosteroids, itraconazole, and allergen immunotherapy. This case also points out that careful scrutiny of direct smears is required to ensure that fungal structures are not misinterpreted.
Outbreak of
Saccharomyces cerevisiae
Subtype
boulardii
Fungemia in Patients Neighboring Those Treated with a Probiotic Preparation of the Organism
Authors: Cassone Marco , Serra Pietro , Mondello Francesca , Girolamo Antonietta , Scafetti Sandro , Pistella Eleonora , Venditti Mario
Published: November 2003

We report an outbreak of
Saccharomyces cerevisiae
subtype
boulardii
fungemia among three intensive care unit roommates of patients receiving lyophilized preparations of this fungus. The fungemia was probably due to central venous catheter contamination and resolved after fluconazole treatment. The need for stringent application of proper hygiene when using a probiotic preparation of this organism is emphasized.
QUANTUM POTENTIAL APPLIED IN THE COUPLING OF CLASSICAL GRAVITY AND QUANTUM MECHANICS
Authors: LAN DANG MONG
Published: November 2003

It is shown that the quantum potential may be used in the form of an equation of state together with the equation of hydrostatic equilibrium to obtain quantum-gravitational bodies, which are polytropes of index 2 in the Newtonian case and geons of Planck mass and radius in relativistic case.
A GEOMETRIC INTERPRETATION OF THE SELBERG INTEGRAL
Authors: YOSHIDA MASAAKI
Published: November 2003

A geometric interpretation of the reciprocity relation of the Selberg integral is given. Combinatorial topological properties of the point-configuration spaces are used. It has an application to the conformal field theory.
TIME AND TACHYON
Authors: SEN ASHOKE
Published: November 2003

Recent analysis suggests that the classical dynamics of a tachyon on an unstable D-brane is described by a scalar Born–Infeld type action with a runaway potential. The classical configurations in this theory at late time are in one to one correspondence with the configuration of a system of noninteracting (incoherent), nonrotating dust. We discuss some aspects of canonical quantization of this field theory coupled to gravity, and explore, following an earlier work on this subject, the possibility of using the scalar field (tachyon) as the definition of time in quantum cosmology. At late "time" we can identify a subsector in which the scalar field decouples from gravity and we recover the usual Wheeler–de Witt equation of quantum gravity.
CONSISTENT INTERACTIONS IN A THREE-DIMENSIONAL THEORY WITH TENSOR GAUGE FIELDS OF DEGREES TWO AND THREE
Authors: SALIU SOLANGE-ODILE
Published: November 2003

All consistent interactions in a three-dimensional theory with tensor gauge fields of degrees two and three are obtained by means of the deformation of the solution to the master equation combined with cohomological techniques. The local BRST cohomology of this model allows the deformation of the Lagrangian action, accompanying gauge symmetries and gauge algebra. The relationship with the Chern–Simons theory is discussed.
BITS AND PIECES IN LOGARITHMIC CONFORMAL FIELD THEORY
Authors: FLOHR MICHAEL A. I.
Published: November 2003

These are notes of my lectures held at the first School & Workshop on Logarithmic Conformal Field Theory and its Applications, September 2001 in Tehran, Iran.

These notes cover only selected parts of the by now quite extensive knowledge on logarithmic conformal field theories. In particular, I discuss the proper generalization of null vectors towards the logarithmic case, and how these can be used to compute correlation functions. My other main topic is modular invariance, where I discuss the problem of the generalization of characters in the case of indecomposable representations, a proposal for a Verlinde formula for fusion rules and identities relating the partition functions of logarithmic conformal field theories to such of well known ordinary conformal field theories.

The two main topics are complemented by some remarks on ghost systems, the Haldane-Rezayi fractional quantum Hall state, and the relation of these two to the logarithmic c=-2 theory.
IMAGING SURFACE STRUCTURES BY DIRECT PHASING
Authors: MARKS L. D. , PLASS R. , DORSET D.
Published: November 2003

We report success in applying direct phasing methods to produce images of surface structures at the atomic scale from intensity data collected using transmission electron diffraction.
ELECTRON DIFFRACTION AND PHOTOEMISSION STUDIES OF CLEAN AND WATER-COVERED Si(5,5,12) AND Si(112) SURFACES
Authors: RANKE W. , XING Y. R.
Published: November 2003

The structure of silicon surfaces in the orientation range (113)–(5,5,12)–(337)–(112) has been investigated using high resolution LEED and photoemission both on a spherical and on flat samples. We find that Si(5,5,12) [5.3° from (113) and 0.7° from (337)] is the only stable orientation between (113) and (111) and confirm the result of Baski et al. [Science269, 1556 (1995)] that it has a 2 × 1 superstructure with a very large unit cell of 7.68 × 53.5 Å2. Adsorption measurements of water on Si(5,5,12) yield a mobile precursor kinetics with two kinds of regions saturating at 0.25 and 0.15 ML which are related to adsorption on different sites. Using these results, a modified structure model is proposed. Surfaces between (113) and (5,5,12) separate into facets of these two orientations; between (5,5,12) and (112), they separate into (5,5,12) and (111) facets. (337) facets in this range may be considered as defective (5,5,12) facets.
NiPC/Si(111)(7 × 7) STUDIED WITH XPS, STM AND TAPPING MODE AIR AFM
Authors: OTTAVIANO L. , di Nardo S. , LOZZI L. , PASSACANTANDO M. , PICOZZI P. , SANTUCCI S.
Published: November 2003

We evaporated a few angstroms of nickel phthalocyanine (NiPC) in ultrahigh vacuum (UHV) on clean single crystal substrates of Si (111)(7×7) and studied in situ the structural and electronic properties of the interface with X-ray photoelectron spectroscopy (XPS) and scanning tunneling microscopy (STM). The mesoscopic morphology of the samples has also been studied in air with a tapping mode atomic force microscope (TM-AFM). XPS measurements with variation of the NiPC thickness suggest planar adsorption of the first layer of admolecules; in particular, we found evidences for stronger chemisorption at the outer benzene rings of the PC molecule. UHV STM measurements confirm the XPS results; despite the lack of intermolecular resolution we show an image suggesting chemisorption commensurate with the substrate lattice and planar stacking consistent with the intermolecular stacking in the known crystalline phases of metal phthalocyanines. TM-AFM shows a growth mode in terms of flat islands of 20–35 Å typical height and a few hundreds of nm width. The potential of imaging with TM-AFM elastic sample properties of soft materials deposited on hard substrates is addressed.
Circulating levels of inflammatory cytokines (IL-1β and TNF-α), resistance to activated protein C, thrombin and fibrin generation in uncomplicated pregnancies
Authors: Arkel Yale , Paidas Michael , Lockwood Charles , Ku De-Hui
Published: November 2003

We studied 33 women during normal uneventful pregnancies and with no history of previous adverse pregnancy events for markers of activated coagulation and thrombin activity including prothrombin fragment 1.2(PF1.2), thrombin- antithrombin (TAT), and soluble fibrin polymer (SFP). In addition, we measured potential thrombin generation through the addition of thromboplastin to patient plasma in the presence of a throm-bin-specific chromogenic substrate determined serially over a period of time - Endogenous Thrombin Potential assay (ETP). This assay was performed with plasma treated and untreated with activated protein C (APC). The fibrinolytic system was assessed by measurement of thrombin activatable fibrinolysis inhibitor (TAFI). These findings were correlated with the levels of pro-inflammatory cytokines, interleukine-1β and tumor necrosis factor-α. Our data supports previous reports that indicate that resistance to activated protein C and coagulation activation markers are commonly increased in the later 2/3rds of pregnancy. There are no differences in thrombin generation potential, as determined by the ETP assay without the addition of APC, in the three trimesters. However, the thrombin reserve (TR), the ETP result without APC divided by the ETP result with the addition of APC, is increased above the reference range in the 2nd and 3rd trimesters. Patients with increased TR and resistance to APC had increased levels of TNF-α. Increased proinflammatory cytokines are reportedly associated with changes in the APC system with a decrease in the ability to generate APC. A sub-group of pregnancies with APC resistance had increased levels of TNF-α and may be important in the risk for adverse pregnancy outcomes.