bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptation of
Escherichia coli
O157:H7 to pH Alters Membrane Lipid Composition, Verotoxin Secretion, and Resistance to Simulated Gastric Fluid Acid
Authors: Yuk Hyun-Gyun , Marshall Douglas L.
Published: June 2004

The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37°C) was determined for
Escherichia coli
O157:H7 (HEC, ATCC 43895), an
rpoS
-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic
E. coli
(NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased
cis
-vaccenic acid (C18:1ω7c) in the membrane lipids of all strains. The ratio of
cis
-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10
8
CFU/ml. In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the
rpoS
gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion.
Phylogenetic and Ecological Analysis of Novel Marine Stramenopiles
Authors: Massana Ramon , Castresana Jose , Balagué Vanessa , Guillou Laure , Romari Khadidja , Groisillier Agnès , Valentin Klaus , Pedrós-Alió Carlos
Published: June 2004

Culture-independent molecular analyses of open-sea microorganisms have revealed the existence and apparent abundance of novel eukaryotic lineages, opening new avenues for phylogenetic, evolutionary, and ecological research. Novel marine stramenopiles, identified by 18S ribosomal DNA sequences within the basal part of the stramenopile radiation but unrelated to any previously known group, constituted one of the most important novel lineages in these open-sea samples. Here we carry out a comparative analysis of novel stramenopiles, including new sequences from coastal genetic libraries presented here and sequences from recent reports from the open ocean and marine anoxic sites. Novel stramenopiles were found in all major habitats, generally accounting for a significant proportion of clones in genetic libraries. Phylogenetic analyses indicated the existence of 12 independent clusters. Some of these were restricted to anoxic or deep-sea environments, but the majority were typical components of coastal and open-sea waters. We specifically identified four clusters that were well represented in most marine surface waters (together they accounted for 74% of the novel stramenopile clones) and are the obvious targets for future research. Many sequences were retrieved from geographically distant regions, indicating that some organisms were cosmopolitan. Our study expands our knowledge on the phylogenetic diversity and distribution of novel marine stramenopiles and confirms that they are fundamental members of the marine eukaryotic picoplankton.
Spatial Distribution of Bacterial Communities and Phenanthrene Degradation in the Rhizosphere of
Lolium perenne
L
Authors: Corgié S. C. , Beguiristain T. , Leyval C.
Published: June 2004

Rhizodegradation of organic pollutants, such as polycyclic aromatic hydrocarbons, is based on the effect of root-produced compounds, known as exudates. These exudates constitute an important and constant carbon source that selects microbial populations in the plant rhizosphere, modifying global as well as specific microbial activities. We conducted an experiment in two-compartment devices to show the selection of bacterial communities by root exudates and phenanthrene as a function of distance to roots. Using direct DNA extraction, PCR amplification, and thermal gradient gel electrophoresis screening, bacterial population profiles were analyzed in parallel to bacterial counts and quantification of phenanthrene biodegradation in three layers (0 to 3, 3 to 6, and 6 to 9 mm from root mat) of unplanted-polluted (phenanthrene), planted-polluted, and planted-unpolluted treatments. Bacterial community differed as a function of the distance to roots, in both the presence and the absence of phenanthrene. In the planted and polluted treatment, biodegradation rates showed a strong gradient with higher values near the roots. In the nonplanted treatment, bacterial communities were comparable in the three layers and phenanthrene biodegradation was high. Surprisingly, no biodegradation was detected in the section of planted polluted treatment farthest from the roots, where the bacterial community structure was similar to those of the nonplanted treatment. We conclude that root exudates and phenanthrene induce modifications of bacterial communities in polluted environments and spatially modify the activity of degrading bacteria.
The
Aspergillus parasiticus estA
-Encoded Esterase Converts Versiconal Hemiacetal Acetate to Versiconal and Versiconol Acetate to Versiconol in Aflatoxin Biosynthesis
Authors: Chang Perng-Kuang , Yabe Kimiko , Yu Jiujiang
Published: June 2004

In aflatoxin biosynthesis, the pathway for the conversion of 1-hydroxyversicolorone to versiconal hemiacetal acetate (VHA) to versiconal (VHOH) is part of a metabolic grid. In the grid, the steps from VHA to VHOH and from versiconol acetate (VOAc) to versiconol (VOH) may be catalyzed by the same esterase. Several esterase activities are associated with the conversion of VHA to VHOH, but only one esterase gene (
estA
) is present in the complete aflatoxin gene cluster of
Aspergillus parasiticus
. We deleted the
estA
gene from
A. parasiticus
SRRC 2043, an
O
-methylsterigmatocystin (OMST)-accumulating strain. The
estA-
deleted mutants were pigmented and accumulated mainly VHA and versicolorin A (VA). A small amount of VOAc and other downstream aflatoxin intermediates, including VHOH, versicolorin B, and OMST, also were accumulated. In contrast, a VA-accumulating mutant, NIAH-9, accumulated VA exclusively and neither VHA nor VOAc were produced. Addition of the esterase inhibitor dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the transformation recipient strain RHN1, an
estA
-deleted mutant, or NIAH-9 resulted in the accumulation of only VHA and VOAc. In in vitro enzyme assays, the levels of the esterase activities catalyzing the conversion of VHA to VHOH in the cell extracts of two
estA
-deleted mutants were decreased to approximately 10% of that seen with RHN1. Similar decreases in the esterase activities catalyzing the conversion of VOAc to VOH were also obtained. Thus, the
estA
-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during aflatoxin biosynthesis.
Inactivation of the Flagellin Gene
flaA
in
Magnetospirillum gryphiswaldense
Results in Nonmagnetotactic Mutants Lacking Flagellar Filaments
Authors: Schultheiss Daniel , Kube Michael , Schüler Dirk
Published: June 2004

Magnetotactic bacteria synthesize magnetosomes, which cause them to orient and migrate along magnetic field lines. The analysis of magnetotaxis and magnetosome biomineralization at the molecular level has been hindered by the unavailability of genetic methods, namely the lack of a means to introduce directed gene-specific mutations. Here we report a method for knockout mutagenesis by homologous recombination in
Magnetospirillum gryphiswaldense
. Multiple flagellin genes, which are unlinked in the genome, were identified in
M. gryphiswaldense
. The targeted disruption of the flagellin gene
flaA
was shown to eliminate flagella formation, motility, and magnetotaxis. The techniques described in this paper will make it possible to take full advantage of the forthcoming genome sequences of
M. gryphiswaldense
and other magnetotactic bacteria.
Comparison of Total Culturable Virus Assay and Multiplex Integrated Cell Culture-PCR for Reliability of Waterborne Virus Detection
Authors: Lee Hwa Kyung , Jeong Yong Seok
Published: June 2004

The total culturable virus assay (TCVA) and an integrated cell culture-PCR (ICC-PCR) were compared in parallel to evaluate their detection reliability. Source, finished, and tap water samples from three drinking water treatment plant systems were analyzed by TCVA, and every cell culture dish was subsequently examined by reverse transcription (RT) multiplex PCR using enterovirus- and adenovirus-specific primers. Twenty-seven of 180 (15%) inoculated dishes exhibited cytopathic effects (CPE). Virus concentrations for source water ranged from 3.3 to 21.0 most probable numbers of infectious units (MPN) per 100 liters. No finished or tap water samples were positive. On the other hand, 38 (21%) of the dishes were positive in multiplex ICC-PCR. Virus concentrations ranged from 4.5 to 10.2 MPN/100 liters for source water and 0 to 0.9 MPN/100 liters for finished and tap water. In spite of its superior sensitivity, the ICC-PCR assay resulted in lower virus concentration values than the TCVA for two of the source water sites. Retest of the CPE-positive dishes using reovirus-specific RT-PCR revealed that 24 of the 27 (89%) dishes were also positive for reoviruses. These observations suggested that the detection reliability of ICC-PCR is restricted by the primer sets that are integrated in the reaction mixture. The observation of an uneven distribution of PCR-positive culture dishes in a given sample raises an additional caution that simple extrapolation of the ICC-PCR result from the analysis of a limited fraction of collected samples should be avoided to minimize possible over- and underestimation of the amount of virus.
Comparison of Coliforms and Coliphages as Tools for Assessment of Viral Contamination in River Water
Authors: Skraber S. , Gassilloud B. , Gantzer C.
Published: June 2004

The aim of the study was to evaluate the presence of pathogenic viruses in the Moselle River and to compare the usefulness of thermotolerant coliforms and somatic coliphages as tools for river water quality assessment in terms of viral contamination. Thermotolerant coliforms and somatic coliphages were enumerated by standardized methods in 170 samples of river water drawn from five sampling sites along the Moselle River (eastern France). BGM cell culture and integrated cell culture-reverse transcription-PCR DNA enzyme immunoassay were used to determine the presence of pathogenic viral genome (
Enterovirus
and
Norovirus
genogroup II [GGII]) and infectious
Enterovirus
spp. in 90 1-liter samples. No infectious
Enterovirus
spp. were isolated, but
Enterovirus
and
Norovirus
GGII genomes were detected in 38% of the samples.
Norovirus
GGII genome was mostly detected in winter, whereas
Enterovirus
genome was mostly detected in summer and fall. Somatic coliphages appeared to be less sensitive to higher river water temperature than thermotolerant coliforms. Furthermore, the number of river water samples positive for pathogenic viral genome increased with increasing concentration of somatic coliphages, whereas coliform concentration was unrelated to viral genome contamination. Consequently somatic coliphages, which are less sensitive to environmental factors than thermotolerant coliforms in river water, would provide a promising tool for assessment of river water quality in terms of fecal and viral pollution.
Isolation, Characterization, and Identification of Bacterial Contaminants in Semifinal Gelatin Extracts
Authors: De Clerck E. , Vanhoutte T. , Hebb T. , Geerinck J. , Devos J. , De Vos P.
Published: June 2004

Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol.25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA genesequencing, species-specificgyrBPCR, andgyrAandrpoBsequencing and were tested for gelatinase activity. The majority of isolates belonged to members ofBacillusor related endospore-forming genera. Representative strains were identified asBacillus cereus,Bacillus coagulans,Bacillus fumarioli,Bacillus amyloliquefaciens,Bacillus licheniformis,Bacillus pumilus,Bacillus sonorensis,Bacillus subtilis,Bacillus gelatini,Bacillus thermoamylovorans,Anoxybacillus contaminans,Anoxybacillus flavithermus,Brevibacillus agri,Brevibacillus borstelensis, andGeobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications.
Identification of Differential Gene Expression in Bacteria Associated with Coral Black Band Disease by Using RNA-Arbitrarily Primed PCR
Authors: Frias-Lopez Jorge , Bonheyo George T. , Fouke Bruce W.
Published: June 2004

RNA-arbitrarily primed PCR techniques have been applied for the first time to identify differential gene expression in black band disease (BBD), a virulent coral infection that affects reef ecosystems worldwide. The gene activity for the BBD mat on infected surfaces of the brain coral
Diploria strigosa
was compared with that for portions of the BBD mat that were removed from the coral and suspended nearby in the seawater column. The results obtained indicate that three genes (DD 95-2, DD 95-4, and DD 99-9) were up-regulated in the BBD bacterial mat on the coral surface compared to the transcript base levels observed in the BBD mat suspended in seawater. Clone DD 95-4 has homology with known amino acid ABC transporter systems in bacteria, while clone DD 99-9 exhibits homology with chlorophyll A apoprotein A1 in cyanobacteria. This protein is essential in the final conformation of photosystem I P700. DD 95-2, the only gene that was fully repressed in the BBD mat samples suspended in seawater, exhibited homology with the AraC-type DNA binding domain-containing proteins. These transcriptional activators coordinate the expression of genes essential for virulence in many species of gram-negative bacteria.
Transformation of
Leuconostoc carnosum
4010 and Evidence for Natural Competence of the Organism
Authors: Helmark Søren , Hansen Mette E. , Jelle Birthe , Sørensen Kim I. , Jensen Peter R.
Published: June 2004

Plasmid transformation in
Leuconostoc carnosum
4010 was analyzed. A successful transformation protocol for
L. carnosum
was established by modifying an existing protocol for
Lactococcus lactis
. Several parameters, including the number of generations that the cells had grown at the time of harvest, glycine concentration, the time of incubation for phenotypic expression, and the electrical field strength, were investigated and proved to have influence on the transformation frequency. Electrocompetence was found to be transient and to peak in the early exponential growth phase. Optimized conditions resulted in transformation frequencies of up to 6.7 × 10
5
transformants per microgram of plasmid DNA. A total of five plasmids in
L. carnosum
were successfully introduced and maintained. Interestingly, we discovered that DNA uptake was of a frequency of 3 × 10
−6
to 19 × 10
−6
transformants per CFU in the absence of an applied electrical field. We concluded that
L. carnosum
is naturally competent.
Isolation and Phylogenetic Analysis of Novel Viruses Infecting the Phytoplankton
Phaeocystis globosa
(Prymnesiophyceae)
Authors: Brussaard C. P. D. , Short S. M. , Frederickson C. M. , Suttle C. A.
Published: June 2004

Viruses infecting the harmful bloom-causing alga
Phaeocystis globosa
(Prymnesiophyceae) were readily isolated from Dutch coastal waters (southern North Sea) in 2000 and 2001. Our data show a large increase in the abundance of putative
P. globosa
viruses during blooms of
P. globosa
, suggesting that viruses are an important source of mortality for this alga. In order to examine genetic relatedness among viruses infecting
P. globosa
and other phytoplankton, DNA polymerase gene (
pol
) fragments were amplified and the inferred amino acid sequences were phylogenetically analyzed. The results demonstrated that viruses infecting
P. globosa
formed a closely related monophyletic group within the family
Phycodnaviridae
, with at least 96.9% similarity to each other. The sequences grouped most closely with others from viruses that infect the prymnesiophyte algae
Chrysochromulina brevifilum
and
Chrysochromulina strobilus
. Whether the
P. globosa
viruses belong to the genus
Prymnesiovirus
or form a separate group needs further study. Our data suggest that, like their phytoplankton hosts, the
Chrysochromulina
and
Phaeocystis
viruses share a common ancestor and that these prymnesioviruses and their algal host have coevolved.
Use of
Sinorhizobium meliloti
as an Indicator for Specific Detection of Long-Chain
N
-Acyl Homoserine Lactones
Authors: Llamas Inmaculada , Keshavan Neela , González Juan E.
Published: June 2004

Population-density-dependent gene expression in gram-negative bacteria involves the production of signal molecules characterized as
N
-acyl homoserine lactones (AHLs). The synthesis of AHLs by numerous microorganisms has been identified by using biosensor strains based on the
Agrobacterium tumefaciens
and
Chromobacterium violaceum
quorum-sensing systems. The symbiotic nitrogen-fixing bacterium
Sinorhizobium meliloti
is rapidly becoming a model organism for the study of quorum sensing. This organism harbors at least three different quorum-sensing systems (Sin, Mel, and Tra), which play a role in its symbiotic relationship with its host plant, alfalfa. The Sin system is distinguished among them for the production of long-chain AHLs, including C
18
-HL, the longest AHL reported so far. In this work, we show that construction of a
sinI
::
lacZ
transcriptional fusion results in a strain that detects long-chain AHLs with exquisite sensitivity. Overexpression of the SinR regulator protein from a vector promoter increases its sensitivity without loss of specificity. We also show that the resulting indicator strain can recognize long-chain AHLs produced by unrelated bacteria such as
Paracoccus denitrificans
and
Rhodobacter capsulatus
. This
S. meliloti
indicator strain should serve as a tool for the specific detection of long-chain AHLs in new systems.
Protein Expression by
Streptococcus mutans
during Initial Stage of Biofilm Formation
Authors: Welin J. , Wilkins J. C. , Beighton D. , Svensäter G.
Published: June 2004

Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents. In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface. In this study we have investigated the changes induced in the protein synthesis by contact of
Streptococcus mutans
with a surface. Log-phase planktonic cells of
S. mutans
were allowed to adhere to a glass slide for 2 h in the presence of a
14
C-amino acid mixture. Nonadhered cells were washed away, and the adhered cells were removed by sonication. The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis. Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished ≥1.3-fold in the biofilm cells. Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis. Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting. The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface.
Monitoring gp43 Antigenemia in Paracoccidioidomycosis Patients during Therapy
Authors: Marques da Silva Silvia Helena , Queiroz-Telles Flávio , Colombo Arnaldo Lopes , Blotta Maria Heloisa Souza Lima , Lopes José Daniel , Pires de Camargo Zoilo
Published: June 2004

Paracoccidioidomycosis (PCM) is a systemic fungal disease that is particularly important among individuals living and working in rural areas of endemicity in Latin America. Detection of anti-
Paracoccidioides brasiliensis
antibodies is of limited value due to false-negative results. Detection of
P. brasiliensis
-gp43 circulating antigen is a practical approach for a specific diagnosis of the disease. In a previous study we described an inhibition enzyme-linked immunosorbent assay able to detect the 43-kDa
P. brasiliensis
antigen in sera of 100% of patients with the acute form of PCM and in 95.31 and 100% of patients with the chronic multifocal and unifocal forms of PCM. To investigate its potential application for the follow-up of PCM patients during treatment, antigen levels were monitored at regular intervals for up 8 to 12 months in serum samples from 23 patients. The results showed that treatment with itraconazole resulted in decreasing levels of circulating gp43 that were correlated with the reduction of anti-gp43 antibodies. It was also observed that by the end of 12 months of treatment gp43 levels were <5 μg/ml in all patients.
SHV-12-Like Extended-Spectrum-β-Lactamase-Producing Strains of
Salmonella enterica
Serotypes Babelsberg and Enteritidis Isolated in France among Infants Adopted from Mali
Authors: Weill François-Xavier , Demartin Marie , Tandé Didier , Espié Emmanuelle , Rakotoarivony Ignace , Grimont Patrick A. D.
Published: June 2004

From December 2002 to June 2003, 14 cultures of
Salmonella enterica
serotype Babelsberg and 6 cultures of serotype Enteritidis, isolated in France from internationally adopted children, were identified at the French National Reference Center for
Salmonella
. All serotype Babelsberg isolates were related, as determined by pulsed-field gel electrophoresis, and all serotype Enteritidis strains displayed the same phage type. All serotype Enteritidis and seven serotype Babelsberg isolates produced an SHV-12-like extended-spectrum β-lactamase as determined by sequencing of PCR products and by isoelectrofocusing. Some serotype Enteritidis isolates exhibited additional antimicrobial resistance (aminoglycosides, tetracycline, chloramphenicol, sulfonamides, and trimethoprim). Our investigation indicated that these
Salmonella
isolates were certainly acquired in the same orphanage in Bamako, Mali, before the children were adopted by French families. An inappropriate use of ceftriaxone was probably the cause of the emergence of such strains. There is an urgent need to determine the origin of the contamination and to introduce adequate antibiotic protocols into this orphanage to prevent further transmission and dissemination. Screening for infections and follow-up, adapted to the origin of the internationally adopted children, should be recommended.
Molecular and Morphological Identification ofColletotrichumSpecies of Clinical Interest
Authors: Cano Josep , Guarro Josep , Gené Josepa
Published: June 2004

Colletotrichumspecies have caused human infections in recent years. Because of the difficulties in recognizing them in vitro, we have designed a quick and unambiguous molecular test, based on the amplification of a specific fragment of the internal transcribed spacer 1 region, to distinguish anyColletotrichumisolate from other fungi, including the common pathogenic species. Analysis of the sequences of the ribosomal DNA (rDNA) fragment showed sufficient variability to clearly separate the five species ofColletotrichumthat are of clinical interest, i.e.,Colletotrichum coccodes,C. crassipes,C. dematium,C. gloeosporioides, andC. graminicola.Sequencing of the D1-D2 region of the large-subunit rDNA gene also supported these results. Additionally, we reviewed the most suitable morphological characteristics for the in vitro identification of these increasingly important opportunistic fungi.
Transposition Rates of
Mycobacterium tuberculosis
IS
6110
Restriction Fragment Length Polymorphism Patterns
Authors: Eilers Paul H. C. , van Soolingen Dick , Thi Ngoc Lan Nguyen , Warren Rob M. , Borgdorff Martien W.
Published: June 2004

To determine the rate at which IS
6110
restriction fragment length polymorphism (RFLP) patterns in
Mycobacterium tuberculosis
change over time, we applied a smooth nonparametric survival model to several data sets, including data from previous publications on the rate of change. The results strongly suggest a simple parametric model, with an instantaneous change at time zero and essentially a zero rate of change thereafter. Our interpretation of the results is that at the time of collection of the first isolate, more than one strain is present. We speculate that the selection of mutant strains is most likely during rapid growth, revival of the dormant bacteria, and/or adaptation to a new host. The parameter most accurately describing changing RFLP patterns is the proportion of isolates with band changes, rather than the half-life or the rate of change.
Molecular Evidence of Infections withBabesia gibsoniParasites in Japan and Evaluation of the Diagnostic Potential of a Loop-Mediated Isothermal Amplification Method
Authors: Ikadai Hiromi , Tanaka Hiroko , Shibahara Nona , Matsuu Aya , Uechi Masami , Itoh Naoyuki , Oshiro Sugao , Kudo Noboru , Igarashi Ikuo , Oyamada Takashi
Published: June 2004

Detection and analysis ofBabesia gibsoniinfection were performed with whole-blood samples collected between July 2002 and July 2003 from 945 and 137 dogs from the Aomori and Okinawa Prefectures of Japan, respectively, by PCR and loop-mediated isothermal amplification (LAMP). On the basis of the criterion for positivity by PCR, 3.9% (37 of 945) and 10.9% (15 of 137) of the dogs hadB. gibsoniDNA. All 37 positive animals from Aomori Prefecture were male Tosa dogs (Japanese mastiff). The 15 dogs from Okinawa Prefecture with positive PCR assay results were of various breeds, ages, and sexes. The 18S ribosomal DNA (18S rDNA) sequences from all samples showed 100% homology to each other and to publishedB. gibsonisequences. The limits of detection ofB. gibsoniparasitemia by the PCR and LAMP methods with an 18S rDNA-based primer set were 0.0005% each. A comparison of the PCR and LAMP methods with microscopic examination for the detection ofB. gibsoniinfections in blood samples from 945 field dogs in Aomori Prefecture and 137 field dogs in Okinawa Prefecture showed that 37 and 15 dogs, respectively, were positive by the PCR and LAMP methods and that 16 and 12 dogs, respectively, were positive by light microscopic examination. All samples found to be positive by microscopic examination were also positive by the PCR and LAMP methods. The results of the PCR and LAMP methods agreed for samples with positive results by either method. Moreover, nonspecific reactions were not observed by the LAMP method. These results suggest that the LAMP method provides a useful tool for the detection ofB. gibsoniinfections in dogs.
Distinct Diversity of the
cag
Pathogenicity Island among
Helicobacter pylori
Strains in Japan
Authors: Azuma Takeshi , Yamakawa Akiyo , Yamazaki Shiho , Ohtani Masahiro , Ito Yoshiyuki , Muramatsu Atsushi , Suto Hiroyuki , Yamazaki Yukinao , Keida Yoshihide , Higashi Hideaki , Hatakeyama Masanori
Published: June 2004

The severity of
Helicobacter pylori
-related disease is correlated with the presence of a
cag
pathogenicity island (PAI). Genetic diversity within the
cag
PAI may have a modifying effect on the pathogenic potential of the infecting strain. We analyzed the complete
cag
PAI sequences of 11 representative Japanese strains according to their
vacA
genotypes and clinical effects and examined the relationship between the diversity of the
cag
PAI and clinical features. The
cag
PAI genes were divided into two major groups, a Western and a Japanese group, by phylogenetic analysis based on the entire
cag
PAI sequences. The predominant Japanese strains formed a Japanese cluster which was different from the cluster formed by Western strains. The diversity of the
cag
PAI was associated with the
vacA
and
cagA
genotypes. All strains with the s1c
vacA
genotype were in the Japanese cluster. In addition, all strains with the East Asian-type
cagA
genotype were also in the Japanese cluster. Patients infected with the Japanese-cluster strain had high-grade gastric mucosal atrophy. These results suggest that a distinct diversity of the
cag
PAI of
H. pylori
is present among Japanese strains and that this diversity may be involved in the development of atrophic gastritis and may increase the risk for gastric cancer.
Simple, Rapid Latex Agglutination Test for Serotyping of Pneumococci (Pneumotest-Latex)
Authors: Slotved H.-C. , Kaltoft M. , Skovsted I. C. , Kerrn M. B. , Espersen F.
Published: June 2004

The “gold standard” for epidemiological typing ofStreptococcus pneumoniae(pneumococcus) is the capsular reaction test (Neufeld test) with antisera against the 90 pneumococcal polysaccharide capsules, i.e., serotyping. The method is labor intensive and requires a certain level of experience to be performed satisfactory, and thus it has been restricted for use in specialized reference or research laboratories. Surveillance of the serotype distribution of pneumococci that cause infections is important to secure an optimal composition of pneumococcal vaccines and to monitor antibiotic resistance in pneumococci. At Statens Serum Institut, a simple latex agglutination test for serotyping of pneumococci has been developed. The Pneumotest-Latex kit consists of 14 different pooled pneumococcus antisera (pools A to I and pools P to T) applied to latex particles. In a blind test of 352 isolates (with all 90 serotypes represented), 336 (95.5%) were typed or grouped correctly by the Pneumotest-Latex; in addition, 2 (7%) of 30 strains regarded as nontypeable or rough strains were serotyped, and the serotypes of these two isolates were confirmed by the capsular reaction test with type-specific antisera. The Pneumotest-Latex seems to be a sensitive method for serotyping or grouping of the majority of pneumococcal strains. By use of this ready-to-perform latex agglutination kit (Pneumotest-Latex), serotyping of pneumococci can gain more ground as a tool in prevention of pneumococcal diseases.
Sequence-Based Identification of Aerobic Actinomycetes
Authors: Patel Jean Baldus , Wallace Richard J. , Brown-Elliott Barbara A. , Taylor Tony , Imperatrice Carol , Leonard Deborah G. B. , Wilson Rebecca W. , Mann Linda , Jost Kenneth C. , Nachamkin Irving
Published: June 2004

We investigated the utility of 500-bp 16S rRNA gene sequencing for identifying clinically significant species of aerobic actinomycetes. A total of 28 reference strains and 71 clinical isolates that included members of the generaStreptomyces,Gordonia, andTsukamurellaand 10 taxa ofNocardiawere studied. Methods of nonsequencing analyses included growth and biochemical analysis, PCR-restriction enzyme analysis of the 439-bp Telenti fragment of the 65hspgene, susceptibility testing, and, for selected isolates, high-performance liquid chromatography. Many of the isolates were included in prior taxonomic studies. Sequencing ofNocardiaspecies revealed that members of the group were generally most closely related to the American Type Culture Collection (ATCC) type strains. However, the sequences ofNocardia transvalensis,N. otitidiscaviarum, andN. novaisolates were highly variable; and it is likely that each of these species contains multiple species. We propose that these three species be designated complexes until they are more taxonomically defined. The sequences of several taxa did not match any recognized species. Among other aerobic actinomycetes, each group most closely resembled the associated reference strain, but with some divergence. The study demonstrates the ability of partial 16S rRNA gene sequencing to identify members of the aerobic actinomycetes, but the study also shows that a high degree of sequence divergence exists within many species and that many taxa within theNocardiaspp. are unnamed at present. A major unresolved issue is the type strain ofN. asteroides, as the present one (ATCC 19247), chosen before the availability of molecular analysis, does not represent any of the common taxa associated with clinical nocardiosis.
Multiplex PCR for Distinguishing the Most Common Phase-1 Flagellar Antigens of
Salmonella
spp
Authors: Herrera-León Silvia , McQuiston John R. , Usera Miguel A. , Fields Patricia I. , Garaizar Javier , Echeita M. Aurora
Published: June 2004

Most
Salmonella
serotypes alternatively express either phase-1 or phase-2 flagellar antigens, encoded by the
fliC
and
fljB
genes, respectively. Flagellar phase reversal for the identification of both flagellar antigens is not necessary at the genetic level. Variable internal regions of the
fliC
genes encoding the H:i, H:r, H:l,v, H:e,h, H:z
10
, H:b, and H:d antigens have been sequenced; and the specific sites for each antigen in selected
Salmonella
serotypes have been determined. These results, together with flagellar G-complex variable internal sequences obtained by the Foodborne and Diarrheal Diseases Branch at the Centers for Disease Control and Prevention in Atlanta, Ga., have been used to design a multiplex PCR to identify the G-complex antigens as well as the H:i, H:r, H:l,v, H:e,h, Hz
10
, H:b, and H:d first-phase antigens. These antigens are part of the most common
Salmonella
serotypes possessing first-phase flagellar antigens.
Salmonella enterica
serotype Enteritidis is identified by adding a specific primer pair published previously (P. G. Agron, R. L. Walker, H. Kinde, S. J. Sawyer, D. C. Hayes, J. Wollard, and G. L. Andersen, Appl. Environ. Microbiol. 67:4984-4991, 2001). This multiplex PCR includes 13 primers. A total of 161
Salmonella
strains associated with 72 different serotypes were tested. Each strain generated one first-phase-specific antigen fragment ranging from 100 to 500 bp;
Salmonella
serotype Enteritidis, however, generated two amplicons of 500 bp that corresponded to the G complex and a 333-bp serotype-specific amplicon, respectively. Twenty-three strains representing 19 serotypes with flagellar genes different from those targeted in this work did not generate any fragments. The method is quick, specific, and reproducible and is independent of the phase expressed by the bacteria when they are tested.
Etiology of Sporadic Cases of Pediatric Acute Gastroenteritis in Asturias, Spain, and Genotyping and Characterization of Norovirus Strains Involved
Authors: Boga José Antonio , Melón Santiago , Nicieza Inés , de Diego Isabel , Villar Mercedes , Parra Francisco , de Oña María
Published: June 2004

From November 2000 to October 2001, a reverse transcription-PCR using primers directed to the norovirus RNA polymerase coding region was included in a viral and bacterial routine screening to diagnose sporadic cases of acute gastroenteritis among children in Asturias, Spain. The role of noroviruses (8.6% of the positively diagnosed cases) as the cause of sporadic pediatric gastroenteritis was evaluated with respect to the detection rates of other gastroenteritis-associated viruses and bacteria. The results indicated that noroviruses were less common than rotaviruses (36.9%),
Campylobacter
spp. (28.8%), and
Salmonella
spp. (18.4%) but more frequent than astroviruses (4.3%), adenoviruses (3.8%), and
Yersinia
spp. (2.2%). Mixed infections involving noroviruses were rarely observed (0.5%). The presence of a norovirus-associated pediatric gastroenteritis peak in summer, as well as the complete absence of norovirus-associated cases in colder months, challenges the view that norovirus infections exclusively have wintertime seasonality. On the other hand, phylogenetic analysis of the amplified fragments showed that the norovirus strains responsible were closely related. A further study using the full-length capsid region showed that these strains could be included into genogroup II, Bristol/Lorsdale cluster, and were closely related to the 1995 and 1996 U.S. subset of strains associated with outbreaks recorded worldwide between 1995 and 1996.
Comparison of Blood Smear, Antigen Detection, and Nested-PCR Methods for Screening Refugees from Regions Where Malaria Is Endemic after a Malaria Outbreak in Quebec, Canada
Authors: Ndao Momar , Bandyayera Etienne , Kokoskin Evelyne , Gyorkos Theresa W. , MacLean J. Dick , Ward Brian J.
Published: June 2004

The importation of malaria into a region where it is not endemic raises many concerns, including the timely delivery of appropriate care, safety of the blood supply, and the risk of autochthonous transmission. There is presently no consensus on the best way to screen mobile populations for malaria. Between August 2000 and March 2001, 535 refugees arrived in Quebec, Canada, from Tanzanian camps. Within 4 weeks of resettlement of the first group of 224, the McGill University Centre for Tropical Diseases noted an outbreak of malaria across the province (15 cases over a 3-week period). This group (group 1) was traced and screened for malaria between 3 and 4 months after arrival in Canada. Subsequent groups of 106 and 205 refugees were screened immediately upon arrival in Canada (group 2) and immediately prior to their departure from refugee camps (group 3), respectively. A single EDTA-blood sample was obtained from 521 refugees for testing by thick and thin blood smears (groups 1 and 2), antigen detection (ICT Malaria Pf and OptiMAL; group 1 only), and nested PCR (all groups). Overall, 98 of 521 refugees were found to be infected (18.8%). The vast majority of infections (81 of 98) were caused by
Plasmodium falciparum
alone. Using PCR as the “gold standard,” both microscopy (sensitivity, 50%; specificity, 100%) and antigen detection (ICT sensitivity, 37.5%; ICT specificity, 100%; OptiMAL sensitivity, 29.1%; OptiMAL specificity, 95.6%) performed poorly. None of the PCR-positive subjects were symptomatic at the time of testing, and only two had recently had symptoms compatible with malaria (with or without diagnosis and treatment). Active surveillance of migrants from regions of intense malaria transmission can reduce the risk of morbidity in the migrant population and mitigate against transmission to the host population. Our data demonstrate that PCR is, by far, the most powerful tool for such surveillance.
DNA Sequence Analysis of the PorB Protein of Nonserotypeable Serogroup C ET-15 Meningococci Suggests a Potential Mutational Hot Spot on Their Serotype Antigens
Authors: Law Dennis K. S. , Henderson Averil M. , Tsang Raymond S. W.
Published: June 2004

The nucleotide sequences of the PorB proteins from 28 nonserotypeable serogroup C ET-15 meningococci recovered from invasive meningococcal disease cases were determined. PCR amplification of the
porB
genes responsible for encoding the serotype antigen was used for DNA sequence determination and identification of the nature of the serotype antigen. DNA sequencing revealed that three strains were of serotype 2a, and of the remaining 25 strains, 20 were found to have an identical single point mutation in the region of the VR3 gene, which encodes surface-exposed loop VI, where the serotype 2a epitope resides. This nonsynonymous mutation was confirmed by synthetic peptide immunochemical analysis to confer new serospecificity to these serotype 2a mutants. This finding of a potential novel mutational hot spot on the PorB proteins of meningococci may have implications for pathogenesis and vaccine development.
High-Efficiency Detection of Severe Acute Respiratory Syndrome Virus Genetic Material
Authors: Weidmann Manfred , Zanotto Paolo M. D. A. , Weber Friedemann , Spiegel Martin , Brodt Hans Rheinhard , Hufert Frank T.
Published: June 2004

A Taqman amplicon targeting the nucleocapsid gene of severe acute respiratory syndrome coronavirus (SARS-CoV) is 5 log
10
times more sensitive for SARS-CoV target RNA extracted from infected cells and 2.79 log
10
times more sensitive for RNA extracted from patient material of the index case in Frankfurt than an amplicon targeting the polymerase gene.
Incidence of Constitutive and Inducible Clindamycin Resistance in
Staphylococcus aureus
and Coagulase-Negative Staphylococci in a Community and a Tertiary Care Hospital
Authors: Schreckenberger Paul C. , Ilendo Elizabeth , Ristow Kathryn L.
Published: June 2004

The incidences of inducible clindamycin resistance at two hospitals (an inner-city hospital and a suburban community hospital) were 7 and 12% for methicillin-resistant
Staphylococcus aureus
, 20 and 19% for methicillin-susceptible
S
.
aureus
, and 14 and 35% for coagulase-negative staphylococci, respectively. Given the variability of inducible resistance to clindamycin found in our two hospitals, we conclude that susceptibility testing of staphylococci should include the disk diffusion induction test (D-test).
Evaluation of the Methicillin-Resistant
Staphylococcus aureus
(MRSA)-Screen Latex Agglutination Test for Detection of MRSA of Animal Origin
Authors: Lee John H. , Jeong Jae-Myung , Park Yong-Ho , Choi Sung-Sun , Kim Yong-Hwan , Chae Joon-Seok , Moon Jin-San , Park Hyun , Kim Shin , Eo Seong-Kug
Published: June 2004

Methicillin (oxacillin)-resistant staphylococci (MRS) have emerged as major clinical and epidemiological pathogens, and there have been frequent reports of MRS infections in the veterinary field. The MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) was compared with an oxacillin agar screen test, MIC determination, and
mecA
PCR assay, the “gold standard.” In an analysis of 15
mecA
-positive and 48
mecA
-negative
S
.
aureus
animal isolates, as well as 9
mecA
-positive and 147
mecA
-negative, coagulase-negative staphylococcal animal isolates, the latex agglutination test surpassed the widely used oxacillin agar screen method and MIC determination, with a sensitivity and a specificity of 100%. The MRSA-Screen test is a reliable and rapid method of detecting MRS in the veterinary field.
Utility of Reverse Transcriptase PCR for Rapid Diagnosis of Influenza A Virus Infection and Detection of Amantadine-Resistant Influenza A Virus Isolates
Authors: Pachucki Constance T. , Khurshid M. Aijaz , Nawrocki John
Published: June 2004

A reverse transcriptase PCR was developed to detect 50 or 5,000 RNA copies of influenza A virus per ml in throat swab specimens. The assay was more sensitive than the Directigen Flu A test. The technique was also used to detect amantadine-resistant isolates.
Sequencing and Sequence Analysis of VP7 and NSP5 Genes Reveal Emergence of a New Genotype of Bovine Group B Rotaviruses in India
Authors: Barman P. , Ghosh S. , Das S. , Varghese V. , Chaudhuri S. , Sarkar S. , Krishnan T. , Bhattacharya S. K. , Chakrabarti A. , Kobayashi N. , Naik T. N.
Published: June 2004

Three bovine group B rotavirus strains were detected from diarrheic calves during a surveillance study of rotaviral diarrhea in West Bengal, India. The sequence analysis of VP7 and NSP5 genes of these strains demonstrates a high degree of sequence variation from other group B rotavirus strains, indicating the emergence of a new genotype.
Outbreak of Infection with Hepatitis A Virus (HAV) Associated with a Foodhandler and Confirmed by Sequence Analysis Reveals a New HAV Genotype IB Variant
Authors: Chironna Maria , Lopalco Pierluigi , Prato Rosa , Germinario Cinzia , Barbuti Salvatore , Quarto Michele
Published: June 2004

An outbreak of infection with hepatitis A virus associated with a foodhandler and involving 26 subjects occurred in Southern Italy. Sequence analysis of the VP3-VP1 and VP1-P2A junctions confirmed that the outbreak was due to a point source and allowed the identification of a new genotype IB variant. This report confirms the usefulness of sequence-based molecular fingerprinting during outbreaks.
Evaluation of a New Particle Gel Immunoassay for Determination of Antibodies against
Treponema pallidum
Authors: Schmidt Bruno L.
Published: June 2004

A new particle gel immunoassay (DiaMed AG, Cressier sur Morat, Switzerland) with three recombinant
Treponema pallidum
antigens was evaluated with serum samples from patients with syphilis (
n
= 124) and patients without syphilis (
n
= 490). It proved to be a simple, rapid (20 min), and useful test with sensitivity, specificity, and positive and negative predictive values of 91.9, 99.8, 99.2, and 98%, respectively.
First U.S. Report of Subcutaneous Phaeohyphomycosis Caused byVeronaea botryosain a Heart Transplant Recipient and Review of the Literature
Authors: Sutton D. A. , Rinaldi M. G. , Kielhofner M.
Published: June 2004

Veronaea botryosais a rare agent of human phaeohyphomycosis. We describe the first case of subcutaneous disease occurring in the United States, alert clinicians to the second report of a transplant-associated mycosis in a heart transplant recipient, extend the previously defined area of endemicity, and review the literature.
Subtype B Human Immunodeficiency Virus (HIV) Type 1 Mutant That Escapes Detection in a Fourth-Generation Immunoassay for HIV Infection
Authors: Gaudy Catherine , Moreau Alain , Brunet Sylvie , Descamps Jean-Michel , Deleplanque Pascale , Brand Denys , Barin Francis
Published: June 2004

We report a case of human immunodeficiency virus (HIV) type 1 infection not detected by a highly sensitive combined antigen-antibody assay. The virus was a subtype B strain harboring a unique sequence within the immunodominant epitope of the transmembrane glycoprotein. Immunochemical analysis indicated that this sequence was probably responsible for the failure to detect HIV antibodies.
Recent Advances in the Functionalizations of the Upper Rims of Thiacalix[4]arenes. A Review
Authors: Parola Stephane , Desroches Cedric
Published: June 2004

The chemistry of the thiacalixarenes is very recent compared to that of similar calixarenes. Several papers have recently reported interesting progress in the chemistry of thiacalixarenes. Until recently most of the reactions concerned the substitutions of the lower rims while only very few were dealing with the upper rims. Indeed it was shown that the macrocycles often behave in a very different way compared to the similar calixarenes, and the well-known reactions such as nitration, bromination or formylation could not be directly transposed to the thiacalixarenes. However many important intermediates for upper-rim substitution were recently reported and are resumed in this paper together with further functionalizations of the upper rims and potential developments in the chemistry and applications of thiacalixarenes. A review with 40 references.