bluuebunny/crossref_metadata_2025 huggingface dataset:
Pyrroloquinoline Quinone-Dependent Dehydrogenases fromKetogulonicigenium vulgareCatalyze the Direct Conversion ofl-Sorbosone tol-Ascorbic Acid
Authors: Miyazaki Taro , Sugisawa Teruhide , Hoshino Tatsuo
Published: February 2006

A novel enzyme,l-sorbosone dehydrogenase 1 (SNDH1), which directly convertsl-sorbosone tol-ascorbic acid (l-AA), was isolated fromKetogulonicigenium vulgareDSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and hemecas the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs producedl-AA, as well as 2-keto-l-gulonic acid (2KGA), froml-sorbosone, suggesting that tautomerization ofl-sorbosone causes the dual conversion by SNDHs. ThesndHgene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase fromAcinetobacter calcoaceticus.The C-terminal one-fifth of the sequence exhibited similarity to ac-type cytochrome with a heme-binding motif. A lysate ofEscherichia colicells expressingsndHexhibited SNDH activity in the presence of PQQ and CaCl2. Gene disruption analysis ofK. vulgareindicated that all of the SNDH proteins are encoded by thesndHgene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells.
In Vivo Functional Assay of a Recombinant Aquaporin inPichia pastoris
Authors: Daniels Mark J. , Wood Malcolm R. , Yeager Mark
Published: February 2006

The water channel protein PvTIP3;1 (α-TIP) is a member of themajorintrinsicprotein (MIP) membrane channel family. We overexpressed this eukaryotic aquaporin in the methylotrophic yeastPichia pastoris, and immunogold labeling of cellular cryosections showed that the protein accumulated in the plasma membrane, as well as vacuolar and other intracellular membranes. We then developed an in vivo functional assay for water channel activity that measures the change in optical absorbance of spheroplasts following an osmotic shock. Spheroplasts of wild-typeP. pastorisdisplayed a linear relationship between absorbance and osmotic shock level. However, spheroplasts ofP. pastorisexpressing PvTIP3;1 showed a break in this linear relationship corresponding to hypo-osmotically induced lysis. It is the difference between control and transformed spheroplasts under conditions of hypo-osmotic shock that forms the basis of our aquaporin activity assay. The aquaporin inhibitor mercury chloride blocked water channel activity but had no effect on wild-type yeast. Osmotically shocked yeast cells were affected only slightly by expression of theEscherichia coliglycerol channel GlpF, which belongs to the MIP family but is a weak water channel. The important role that aquaporins play in human physiology has led to a growing interest in their potential as drug targets for treatment of hypertension and congestive heart failure, as well as other fluid overload states. The simplicity of this assay that is specific for water channel activity should enable rapid screening for compounds that modulate water channel activity.
Colonization of Tomato Root by Pathogenic and Nonpathogenic
Fusarium oxysporum
Strains Inoculated Together and Separately into the Soil
Authors: Olivain Chantal , Humbert Claude , Nahalkova Jarmila , Fatehi Jamshid , L'Haridon Floriane , Alabouvette Claude
Published: February 2006

In soil, fungal colonization of plant roots has been traditionally studied
by indirect methods such as microbial isolation that do not enable
direct observation of infection sites or of interactions between fungal
pathogens and their antagonists. Confocal laser scanning microscopy was
used to visualize the colonization of tomato roots in heat-treated soil
and to observe the interactions between a nonpathogenic strain, Fo47,
and a pathogenic strain, Fol8, inoculated onto tomato roots in soil.
When inoculated separately, both fungi colonized the entire root
surface, with the exception of the apical zone. When both strains were
introduced together, they both colonized the root surface and were
observed at the same locations. When Fo47 was introduced at a higher
concentration than Fol8, it colonized much of the root surface, but
hyphae of Fol8 could still be observed at the same location on the
root. There was no exclusion of the pathogenic strain by the presence
of the nonpathogenic strain. These results are not consistent with the
hypothesis that specific infection sites exist on the root for
Fusarium oxysporum
and instead support the hypothesis that
competition occurs for nutrients rather than for infection
sites.
Induction by
Klebsiella aerogenes
of a Melanin-Like Pigment in
Cryptococcus neoformans
Authors: Frases Susana , Chaskes Stuart , Dadachova Ekaterina , Casadevall Arturo
Published: February 2006

While studying the interaction of
Cryptococcus neoformans
with
Dictyostelium discoideum
, we noticed that yeast colonies in agar with a feeder lawn of
Klebsiella aerogenes
were brown. This finding was intriguing because
C. neoformans
colonies are not pigmented unless they are provided with precursors for melanization. Strains of all
C. neoformans
serotypes produced brown pigment in response to
K. aerogenes
at 22, 30, and 37°C. Pigment production required fungal laccase and was suppressed by high concentrations of glucose. Treatment of brown cells with guanidinium isothiocyanate and hot concentrated HCl yielded particulate material that had the physical and chemical characteristics of melanins. No pigment formation was observed when
C. neoformans
was exposed to live
Escherichia coli
or heat-killed
K. aerogenes
. Analysis of
K. aerogenes
supernatants revealed the presence of dopamine, which can be a substrate for melanin synthesis by
C. neoformans
. Our findings illustrate a remarkable interaction between a pathogenic fungus and a gram-negative bacterium, in which the bacterium produces a substrate that promotes fungal melanization. This observation provides a precedent that could explain the source of a substrate for
C. neoformans
melanization in the environment.
Molecular Assays for DetectingAphanomyces invadansin Ulcerative Mycotic Fish Lesions
Authors: Vandersea Mark W. , Litaker R. Wayne , Yonnish Bryan , Sosa Emilio , Landsberg Jan H. , Pullinger Chris , Moon-Butzin Paula , Green Jason , Morris James A. , Kator Howard , Noga Edward J. , Tester Patricia A.
Published: February 2006

The pathogenic oomyceteAphanomyces invadansis the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detectA. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n= 50) collected in September 2004 were positive forA. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence thatA. invadansis the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusivelyA. invadans.
Characterization of Metabolism in the Fe(III)-Reducing Organism
Geobacter sulfurreducens
by Constraint-Based Modeling
Authors: Mahadevan R. , Bond D. R. , Butler J. E. , Esteve-Nuñez A. , Coppi M. V. , Palsson B. O. , Schilling C. H. , Lovley D. R.
Published: February 2006

Geobacter sulfurreducens
is a well-studied representative of the
Geobacteraceae
, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate
G. sulfurreducens
central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why
Geobacter
species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of
G. sulfurreducens
could synthesize amino acids more efficiently than that of
Escherichia coli
due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of
G. sulfurreducens
central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.
A Quorum-Quenching Approach To Investigate the Conservation of Quorum-Sensing-Regulated Functions within the
Burkholderia cepacia
Complex
Authors: Wopperer Julia , Cardona Silvia T. , Huber Birgit , Jacobi Christoph A. , Valvano Miguel A. , Eberl Leo
Published: February 2006

Taxonomic studies of the past few years have shown that the
Burkholderia cepacia
complex, a heterogeneous group of
B. cepacia
-like organisms, consists of at least nine species.
B. cepacia
complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time,
B. cepacia
complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All
B. cepacia
complex species investigated thus far use quorum-sensing (QS) systems that rely on
N
-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the
Bacillus
sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described
B. cepacia
complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested
B. cepacia
complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and
Caenorhabditis elegans
killing efficiency was regulated by AHL in the large majority of strains investigated.
Functional and Structural Characterization of Thermostabled-Amino Acid Aminotransferases fromGeobacillusspp
Authors: Lee Seung-Goo , Hong Seung-Pyo , Song Jae Jun , Kim Su-Jin , Kwak Mi-Sun , Sung Moon-Hee
Published: February 2006

d-Amino acid aminotransferases (d-AATs) fromGeobacillus toebiiSK1 andGeobacillussp. strain KLS1 were cloned and characterized from a genetic, catalytic, and structural aspect. Although the enzymes were highly thermostable, their catalytic capability was approximately one-third of that of highly activeBacillienzymes, with respective turnover rates of 47 and 55 s−1at 50°C. TheGeobacillusenzymes were unique and shared limited sequence identities of below 45% withd-AATs from mesophilic and thermophilicBacillusspp., except for a hypothetical protein with a 72% identity from theG. kaustophilusgenome. Structural alignments showed that most key residues were conserved in theGeobacillusenzymes, although the conservative residues just before the catalytic lysine were distinctively changed: the 140-LRcD-143 sequence inBacillusd-AATs was 144-EYcY-147 in theGeobacillusd-AATs. When the EYcY sequence from the SK1 enzyme was mutated into LRcD, a 68% increase in catalytic activity was observed, while the binding affinity toward α-ketoglutarate decreased by half. The mutant was very close to the wild-type in thermal stability, indicating that the mutations did not disturb the overall structure of the enzyme. Homology modeling also suggested that the two tyrosine residues in the EYcY sequence from theGeobacillusd-AATs had a π/π interaction that was replaceable with the salt bridge interaction between the arginine and aspartate residues in the LRcD sequence.
Lack of Detrimental Effects of
Bacillus thuringiensis
Cry Toxins on the Insect Predator
Chrysoperla carnea
: a Toxicological, Histopathological, and Biochemical Analysis
Authors: Rodrigo-Simón Ana , de Maagd Ruud A. , Avilla Carlos , Bakker Petra L. , Molthoff Jos , González-Zamora Jose E. , Ferré Juan
Published: February 2006

The effect of Cry proteins of
Bacillus thuringiensis
on the green lacewing (
Chrysoperla carnea
) was studied by using a holistic approach which consisted of independent, complementary experimental strategies. Tritrophic experiments were performed, in which lacewing larvae were fed
Helicoverpa armigera
larvae reared on Cry1Ac, Cry1Ab, or Cry2Ab toxins. In complementary experiments, a predetermined amount of purified Cry1Ac was directly fed to lacewing larvae. In both experiments no effects on prey utilization or fitness parameters were found. Since binding to the midgut is an indispensable step for toxicity of Cry proteins to known target insects, we hypothesized that specific binding of the Cry1A proteins should be found if the proteins were toxic to the green lacewing. In control experiments, Cry1Ac was detected bound to the midgut epithelium of intoxicated
H. armigera
larvae, and cell damage was observed. However, no binding or histopathological effects of the toxin were found in tissue sections of lacewing larvae. Similarly, Cry1Ab or Cry1Ac bound in a specific manner to brush border membrane vesicles from
Spodoptera exigua
but not to similar fractions from green lacewing larvae. The in vivo and in vitro binding results strongly suggest that the lacewing larval midgut lacks specific receptors for Cry1Ab or Cry1Ac. These results agree with those obtained in bioassays, and we concluded that the Cry toxins tested, even at concentrations higher than those expected in real-life situations, do not have a detrimental effect on the green lacewing when they are ingested either directly or through the prey.
Temperature-Regulated Formation of Mycelial Mat-Like Biofilms by
Legionella pneumophila
Authors: Piao Zhenyu , Sze Chun Chau , Barysheva Oksana , Iida Ken-ichiro , Yoshida Shin-ichi
Published: February 2006

Fifty strains representing 38 species of the genus
Legionella
were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25°C, 37°C, and 42°C. Strains of
Legionella pneumophila
, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of
L. pneumophila
biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37°C or 42°C than at 25°C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25°C than at 37°C or 42°C. On glass surfaces, the biofilms were formed faster but attached less stably at 37°C or 42°C than at 25°C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37°C or 42°C were mycelial mat like and were composed of filamentous cells, while at 25°C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a
recA
null mutant of
L. pneumophila
was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37°C. The possibility of this biofilm to represent a novel strategy by
L. pneumophila
to compete for proliferation among the environmental microbiota is discussed.
Dynamic Transition of a Methanogenic Population in Response to the Concentration of Volatile Fatty Acids in a Thermophilic Anaerobic Digester
Authors: Hori Tomoyuki , Haruta Shin , Ueno Yoshiyuki , Ishii Masaharu , Igarashi Yasuo
Published: February 2006

In this study, the microbial community succession in a thermophilic methanogenic bioreactor under deteriorative and stable conditions that were induced by acidification and neutralization, respectively, was investigated using PCR-mediated single-strand conformation polymorphism (SSCP) based on the 16S rRNA gene, quantitative PCR, and fluorescence in situ hybridization (FISH). The SSCP analysis indicated that the archaeal community structure was closely correlated with the volatile fatty acid (VFA) concentration, while the bacterial population was impacted by pH. The archaeal community consisted mainly of two species of hydrogenotrophic methanogen (i.e., a
Methanoculleus
sp. and a
Methanothermobacter
sp.) and one species of aceticlastic methanogen (i.e., a
Methanosarcina
sp.). The quantitative PCR of the 16S rRNA gene from each methanogen revealed that the
Methanoculleus
sp. predominated among the methanogens during operation under stable conditions in the absence of VFAs. Accumulation of VFAs induced a dynamic transition of hydrogenotrophic methanogens, and in particular, a drastic change (i.e., an approximately 10,000-fold increase) in the amount of the 16S rRNA gene from the
Methanothermobacter
sp. The predominance of the one species of hydrogenotrophic methanogen was replaced by that of the other in response to the VFA concentration, suggesting that the dissolved hydrogen concentration played a decisive role in the predominance. The hydrogenotrophic methanogens existed close to bacteria in aggregates, and a transition of the associated bacteria was also observed by FISH analyses. The degradation of acetate accumulated during operation under deteriorative conditions was concomitant with the selective proliferation of the
Methanosarcina
sp., indicating effective acetate degradation by the aceticlastic methanogen. The simple methanogenic population in the thermophilic anaerobic digester significantly responded to the environmental conditions, especially to the concentration of VFAs.
Two Subpopulations of
Listeria monocytogenes
Occur at Subinhibitory Concentrations of Leucocin 4010 and Nisin
Authors: Hornbæk Tina , Brockhoff Per B. , Siegumfeldt Henrik , Budde Birgitte Bjørn
Published: February 2006

In situ analyses of single
Listeria monocytogenes
cells at subinhibitory concentrations of leucocin 4010 and nisin revealed two subpopulations when measured by fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester. One subpopulation consisted of cells with a dissipated pH gradient (ΔpH), and the other consisted of cells that maintained ΔpH. The proportion of cells belonging to each subpopulation was estimated, and the concentrations of bacteriocins required to dissipate ΔpH for 90% of the cell population (ED
90
) was predicted. ED
90
increased after the addition of sodium chloride (1 to 3% [wt/vol]) to the bacteriocin solutions, while ED
90
decreased by the addition of sodium nitrite (60 and 100 ppm). Other meat additives, including sodium phosphate, sodium lactate, sodium citrate, and sodium acetate slightly increased ED
90
. The inhibitory effect of sodium chloride on the antilisterial activity of leucocin 4010 and nisin was confirmed on the surfaces of meat sausages. This study highlights the important practical implications of applying subinhibitory concentrations of bacteriocins, which results in unaffected target cells. In situ analyses by FRIM in combination with modeling of single-cell data can be applied to ensure that sufficient concentrations of bacteriocins are used in food preservation.
Txp40, a Ubiquitous Insecticidal Toxin Protein from
Xenorhabdus
and
Photorhabdus
Bacteria
Authors: Brown S. E. , Cao A. T. , Dobson P. , Hines E. R. , Akhurst R. J. , East P. D.
Published: February 2006

Xenorhabdus
and
Photorhabdus
are gram-negative bacteria that produce a range of proteins that are toxic to insects. We recently identified a novel 42-kDa protein from
Xenorhabdus nematophila
that was lethal to the larvae of insects such as
Galleria mellonella
and
Helicoverpa armigera
when it was injected at doses of 30 to 40 ng/g larvae. In the present work, the toxin gene
txp40
was identified in another 59 strains of
Xenorhabdus
and
Photorhabdus
, indicating that it is both highly conserved and widespread among these bacteria. Recombinant toxin protein was shown to be active against a variety of insect species by direct injection into the larvae of the lepidopteran species
G. mellonella
,
H. armigera
, and
Plodia interpunctella
and the dipteran species
Lucilia cuprina
. The protein exhibited significant cytotoxicity against two dipteran cell lines and two lepidopteran cell lines but not against a mammalian cell line. Histological data from
H. armigera
larvae into which the toxin was injected suggested that the primary site of action of the toxin is the midgut, although some damage to the fat body was also observed.
Analysis of Expressed Sequence Tag Data and Gene Expression Profiles Involved in Conidial Germination of
Fusarium oxysporum
Authors: Deng Ye , Dong Haitao , Jin Qingchao , Dai Cheng'en , Fang Yongqi , Liang Shen , Wang Ke , Shao Jing , Lou Yichun , Shi Wenqin , Vakalounakis Demetrios J. , Li Debao
Published: February 2006

We obtained 3,372 tentative unique transcripts (TUTs) from a cDNA library of
Fusarium oxysporum
. A cDNA array with 3,158 TUTs was produced to analyze gene expression profiles in conidial germination. It seems that
ras
and other signaling genes, e.g.,
ccg
, cooperatively initiate conidial germination in
Fusarium
by increasing protein synthesis.
mRNA-Based Parallel Detection of Active Methanotroph Populations by Use of a Diagnostic Microarray
Authors: Bodrossy Levente , Stralis-Pavese Nancy , Konrad-Köszler Marianne , Weilharter Alexandra , Reichenauer Thomas G. , Schöfer David , Sessitsch Angela
Published: February 2006

A method was developed for the mRNA-based application of microbial diagnostic microarrays to detect active microbial populations. DNA- and mRNA-based analyses of environmental samples were compared and confirmed via quantitative PCR. Results indicated that mRNA-based microarray analyses may provide additional information on the composition and functioning of microbial communities.
Characterization of Bacterial Communities Associated with Deep-Sea Corals on Gulf of Alaska Seamounts
Authors: Penn Kevin , Wu Dongying , Eisen Jonathan A. , Ward Naomi
Published: February 2006

Although microbes associated with shallow-water corals have been reported, deepwater coral microbes are poorly characterized. A cultivation-independent analysis of Alaskan seamount octocoral microflora showed that
Proteobacteria
(classes
Alphaproteobacteria
and
Gammaproteobacteria
),
Firmicutes
,
Bacteroidetes
, and
Acidobacteria
dominate and vary in abundance. More sampling is needed to understand the basis and significance of this variation.
Improvement of Oligonucleotide Probe Design Criteria for Functional Gene Microarrays in Environmental Applications
Authors: Liebich Jost , Schadt Christopher W. , Chong Song C. , He Zhili , Rhee Sung-Keun , Zhou Jizhong
Published: February 2006

To optimize oligonucleotide probe design criteria, PCR products with different similarities to probes were hybridized to a functional gene microarray designed to detect homologous genes from different organisms. In contrast to more restrictive probe designs based on a single criterion, simultaneous consideration of the percent similarity (≤90%), the length of identical sequence stretches (≤20 bases), and the binding free energy (≥−35 kcal mol
−1
) was found to be predictive of probe specificity.
Measurement of Respiration Rates ofMethylobacterium extorquensAM1 Cultures by Use of a Phosphorescence-Based Sensor
Authors: Strovas Tim J. , Dragavon Joe M. , Hankins Tyler J. , Callis James B. , Burgess Lloyd W. , Lidstrom Mary E.
Published: February 2006

Respiration rates of bacterial cultures can be a powerful tool in gauging the effects of genetic manipulation and environmental changes affecting overall metabolism. We present an optical method for measuring respiration rates using a robust phosphorescence lifetime-based sensor and off-the-shelf technology. This method was tested with the facultative methylotrophMethylobacterium extorquensAM1 to demonstrate subtle mutant phenotypes.
Caenorhabditis elegans
Is a Model Host for
Listeria monocytogenes
Authors: Thomsen Line E. , Slutz Sandra S. , Tan Man-Wah , Ingmer Hanne
Published: February 2006

Here we report that
Caenorhabditis elegans
nematodes fed
Listeria monocytogenes
die over the course of several days, as a consequence of an accumulation of bacteria in the worm intestine. Mutant strains previously shown to be important for virulence in mammalian models were also found to be attenuated in their virulence in
C. elegans
. However, ActA, which is required for actin-based intracellular motility, appears to be dispensable during infection of
C. elegans
, indicating that
L. monocytogenes
remains extracellular in
C. elegans
.
Freezing Induces Biased Results in the Molecular Detection of
Flavobacterium columnare
Authors: Suomalainen Lotta-Riina , Reunanen Hilkka , Ijäs Ritva , Valtonen E. Tellervo , Tiirola Marja
Published: February 2006

Specific PCR detection and electron microscopy of
Flavobacterium columnare
revealed the risk of false-negative results in molecular detection of this fish pathogen. Freezing and thawing destroyed the cells so that DNA was for the most part undetectable by PCR. The detection of bacteria was also weakened after prolonged enrichment cultivation of samples from infected fish.
Isolation ofRickettsia felisin the Mosquito Cell Line C6/36
Authors: Horta Maurício C. , Labruna Marcelo B. , Durigon Edison L. , Schumaker Teresinha T. S.
Published: February 2006

We report the isolation and establishment ofRickettsia felisin the C6/36 cell line. Rickettsial growth was intense, always with 90 to 100% of cells being infected after few weeks. The rickettsial isolate was confirmed by testing infected cells by PCR and sequencing fragments of three majorRickettsiagenes (gltA,ompB, and the 17-kDa protein gene).
Activities of Micafungin against 315 Invasive Clinical Isolates of Fluconazole-Resistant
Candida
spp
Authors: Messer S. A. , Diekema D. J. , Boyken L. , Tendolkar S. , Hollis R. J. , Pfaller M. A.
Published: February 2006

Micafungin is a new echinocandin exhibiting broad-spectrum activity against
Candida
spp. The activity of the echinocandins against
Candida
species known to express intrinsic or acquired resistance to fluconazole is of interest. We determined the MICs of micafungin and caspofungin against 315 invasive clinical (bloodstream and other sterile-site) isolates of fluconazole-resistant
Candida
species obtained from geographically diverse medical centers between 2001 and 2004. MICs were determined using broth microdilution according to the CLSI reference method M27-A2. RPMI 1640 was used as the test medium, and we used the MIC endpoint of prominent growth reduction at 24 h. Among the 315 fluconazole-resistant
Candida
isolates, 146 (46%) were
C. krusei
, 110 (35%) were
C. glabrata
, 41 (13%) were
C. albicans
, and 18 (6%) were less frequently isolated species. Micafungin had good in vitro activity against all fluconazole-resistant
Candida
spp. tested; the MICs at which 50% (MIC
50
) and 90% (MIC
90
) of isolates were inhibited were 0.03 μg/ml and 0.06 μg/ml, respectively. All the fluconazole-resistant
Candida
spp. were inhibited at a micafungin MIC that was ≤1 μg/ml. Among the most common fluconazole-resistant
Candida
spp. tested in the collection,
C. glabrata
exhibited the lowest micafungin MICs (MIC
90
, ≤0.015 μg/ml), followed by
C. albicans
(MIC
90
, 0.03 μg/ml) and
C. krusei
(MIC
90
, 0.06 μg/ml). The new echinocandin micafungin has excellent in vitro activity against 315 invasive clinical isolates of fluconazole-resistant
Candida
, which represents the largest collection to date of fluconazole-resistant
Candida
isolates tested against micafungin. Micafungin may prove useful in the treatment of infections due to azole-resistant
Candida
.
Evaluation of the New GenoType Mycobacterium Assay for Identification of Mycobacterial Species
Authors: Russo Cristina , Tortoli Enrico , Menichella Donato
Published: February 2006

The new commercial assay GenoType Mycobacterium, intended for the identification of mycobacteria, was evaluated with a panel of 197 strains belonging to 86 different taxa. The system, which relies on solid-phase reverse hybridization, is composed of two different kits: GenoType CM for the identification of more frequently detected mycobacterial species and GenoType AS for less common species. The sensitivity and specificity were 97.9% and 92.4% for the CM kit and 99.3% and 99.4% for the AS kit. Our results show that the system may represent a useful tool to substantially enlarge the number of mycobacteria that can be reliably identified in clinical laboratories.
Comparison of Two Commercially Available DNA Line Probe Assays for Detection of Multidrug-Resistant
Mycobacterium tuberculosis
Authors: Mäkinen Johanna , Marttila Harri J. , Marjamäki Merja , Viljanen Matti K. , Soini Hanna
Published: February 2006

Two commercially available DNA line probe assays, Genotype MTBDR and INNO-LiPA Rif. TB, were evaluated for their abilities to detect resistance to isoniazid (INH) and rifampin (RIF) in 52
Mycobacterium tuberculosis
isolates. The test results were compared to those obtained by phenotypic drug susceptibility testing and sequencing. Compared to the results of phenotypic drug susceptibility testing, the Genotype MTBDR test results were concordant for INH for 47 of the 52 (90.4%) isolates, and both the Genotype MTBDR and the INNO-LiPA Rif. TB test results were concordant for RIF for 51 of the 52 (98.1%) isolates. The Genotype MTBDR test results correlated with the sequencing results for 48 of the 52 (92.3%) isolates and the INNO-LiPA Rif. TB results for 50 of the 52 (96.2%) isolates. Both assays are useful for the rapid screening of
M. tuberculosis
isolates obtained from patients suspected of having multidrug-resistant tuberculosis, but the GenoType MTBDR assay has the advantage of being able to detect resistance to both INH and RIF simultaneously.
Influence of Anti-FloR Antibody on Florfenicol Accumulation in Florfenicol-Resistant
Escherichia coli
and Enzyme-Linked Immunosorbent Assay for Detection of Florfenicol-Resistant
E. coli
Isolates
Authors: Wu Beibei , Xia Chun , Du Xiangdang , Cao Xingyuan , Shen Jianzhong
Published: February 2006

To detect florfenicol-resistant
Escherichia coli
isolates by enzyme-linked immunosorbent assay (ELISA), anti-FloR1 antibodies were produced in mice using a recombinant glutathione
S
-transferase (GST)-FloR1 protein, which was expressed in a prokaryote expression system, as the antigen. The specificity of the murine anti-GST-FloR1 antibody and its influence on florfenicol accumulation in florfenicol-resistant isolates were investigated using Western blotting and high-performance liquid chromatography, respectively. Western blotting using the anti-FloR1 antibody showed specific binding of the antibody to the florfenicol-resistant FloR protein. Preincubation of florfenicol-resistant strains with the antibody significantly increased the intracellular accumulation of florfenicol and enhanced the bacterial susceptibility to florfenicol, suggesting that antibody binding to the FloR protein inhibited the activity of the efflux protein conferred by the
floR
gene. Analyses of florfenicol-resistant and -sensitive isolates by ELISA using the anti-FloR1 antibody showed good correlation between FloR protein expression and the
floR
genotype. The anti-FloR1 antibody-based ELISA is a useful tool for the detection of florfenicol-resistant bacteria harboring the
floR
gene.
Neisseria bacilliformis
sp. nov. Isolated from Human Infections
Authors: Han Xiang Y. , Hong Tao , Falsen Enevold
Published: February 2006

Most
Neisseria
species are gram-negative cocci or diplococci; currently,
N. elongata
is the only species of human origin with a bacillary morphology. Here, we report isolation and characterization of eight strains of another bacillary
Neisseria
species from human infections. The organisms caused or contributed to either oral cavity-related or respiratory tract infections, and two strains were isolated from blood cultures. The 16S rRNA gene sequences of these organisms, being homogenous or nearly so (99.4 to 100% identity), matched at <96% known
Neisseria
species and formed a distinct group within the genus. Analysis of the cellular fatty acids showed broad similarity with a few
Neisseria
species. The organisms were gram negative and measured 0.6 μm by 1.3 to 3.0 μm. They grew well on chocolate agar and on sheep blood agar but did not grow on modified Thayer-Martin agar. They were positive for oxidase and negative for indole production. There was no acid production from dextrose, lactose, maltose, or sucrose. The tests for catalase reaction, nitrate reduction, and tributilin varied with the strains. These results suggest that these organisms represent a novel species within the genus
Neisseria
, for which the name
Neisseria bacilliformis
sp. nov. is proposed. The type strain is MDA2833 = ATCC BAA-1200
T
= CCUG50858
T
. Distinction between
N. bacilliformis
and
N. elongata
can be made confidently by 16S rRNA gene sequencing or cellular fatty acid profiling but may be difficult by morphology or routine biochemical tests.
Genotyping Measles Virus by Real-Time Amplification Refractory Mutation System PCR Represents a Rapid Approach for Measles Outbreak Investigations
Authors: Waku-Kouomou Diane , Alla Amal , Blanquier Bariza , Jeantet Damien , Caidi Hayat , Rguig Ahmed , Freymuth François , Wild Fabian T.
Published: February 2006

Real-time PCR has been developed to genotype measles virus (MV) isolates. MV strains circulating in epidemics in Gabon in 1984, Cameroon in 2001, Morocco in 2003, and France in 2004 were investigated. We developed a real-time amplification refractory mutation system PCR (RT-AMRS PCR) using SYBR green fluorescent dye. Six pairs of primers for RT-ARMS PCR were designed to specifically amplify genotypes A, B2, B3.1, B3.2, C2, and D7. Genotypes could be differentiated by melting curve analysis. All strains were also confirmed by direct sequencing. Using the result obtained by direct sequencing and phylogenetic analysis as the reference, the accuracy of MV by RT-ARMS PCR and melting curve analysis was 97%. However, the latter method is more rapid and sensitive than the former method. This method could be a useful tool for molecular epidemiological studies of MV, providing an efficient alternative for large-scale studies.
Multilocus Microsatellite Typing as a New Tool for Discrimination of
Leishmania infantum
MON-1 Strains
Authors: Ochsenreither Sebastian , Kuhls Katrin , Schaar Matthias , Presber Wolfgang , Schönian Gabriele
Published: February 2006

The
Leishmania donovani
complex, which consists of
L. donovani
,
L. infantum
-
L. chagasi
, and
L. archibaldi
, is responsible for visceral manifestations of leishmaniasis. Multilocus enzyme electrophoresis is the standard method for the characterization and identification of strains of
Leishmania.
For
L. infantum
, the predominance of zymodeme MON-1 significantly reduces the discriminative power of this approach. In the present study, we developed 17 independent polymorphic microsatellite markers for the typing of strains of
L. infantum
, with the main emphasis on zymodeme MON-1. The discriminative powers of 11 markers selected from among these markers were tested by using a panel of 63 isolates of the
L. donovani
complex. Unique multilocus genotypes were observed for the strains analyzed, with only three exceptions. Model-based and distance-based analyses of the data set showed comparable results. It was possible to discriminate between
L. donovani
sensu stricto, a non-MON-1 group of
L. infantum
isolates, and a MON-1 group of
L. infantum
isolates. Within MON-1, three clusters with geographical correlations became apparent. The frequency of heterozygosity in the alleles analyzed varied extremely between the different groups of isolates. The main clusters described are not consistent with species definitions based on isoenzyme analysis but confirm the results of former PCR-based investigations.
Bead-Based Multiplex Genotyping of Human Papillomaviruses
Authors: Schmitt Markus , Bravo I. G. , Snijders Peter J. F. , Gissmann Lutz , Pawlita Michael , Waterboer Tim
Published: February 2006

Typing of human papillomaviruses (HPV) by DNA hybridization procedures, such as reverse line blot (RLB) assay, is sensitive and well validated. However, the application of these assays to high-throughput analyses is limited. Here, we describe the development of multiplex human papillomavirus genotyping (MPG), a quantitative and sensitive high-throughput procedure for the identification of multiple high- and low-risk genital HPV genotypes in a single reaction. MPG is based on the amplification of HPV DNA by a general primer PCR (GP5+/6+) and the subsequent detection of the products with type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads (Luminex suspension array technology). Up to 100 different HPV types can be detected simultaneously with MPG, and the method is fast and labor saving. We detected all 22 HPV types examined with high specificity and reproducibility (the median interplate coefficient of variation was below 10%). Detection limits for the different HPV types varied between 100 and 800 pg of PCR products. We compared the performance of MPG to an established RLB assay on GP5+/6+-PCR products derived from 94 clinical samples. The evaluation showed an excellent agreement (kappa = 0.922) but also indicated a higher sensitivity of MPG. In conclusion, MPG appears to be highly suitable for large-scale epidemiological studies and vaccination trials as well as for routine diagnostic purposes.
Two New Colorimetric Methods for Early Detection of Vancomycin and Oxacillin Resistance in
Staphylococcus aureus
Authors: Coban Ahmet Y. , Bozdogan Bülent , Cihan Cigdem Cekic , Cetinkaya Ebru , Bilgin Kemal , Darka Ozge , Akgunes Alper , Durupinar Belma , Appelbaum Peter C.
Published: February 2006

We developed two colorimetric methods for the detection of vancomycin- and oxacillin-resistant
Staphylococcus aureus
in ≤6 h: (i) a nitrate reductase assay and (ii) a resazurin microplate method. MICs agreed with results obtained by CLSI methods for oxacillin. However, detection of vancomycin resistance required a larger inoculum. These methods may be recommended for the detection of vancomycin- and oxacillin-resistant
S. aureus.
Use of Different Stains for Microscopic Evaluation of Corneal Scrapings for Diagnosis of Microsporidial Keratitis
Authors: Joseph Joveeta , Murthy Somasheila , Garg Prashant , Sharma Savitri
Published: February 2006

Retrospective evaluation of potassium hydroxide plus calcofluor white (KOH+CFW), Gram, Giemsa, and modified Ziehl-Neelsen (1% H
2
SO
4
, cold) stains for the detection of microsporidia in corneal scrapings from 30 patients showed KOH+CFW and acid-fast stains to be most efficient (29/30 [96.7%] and 28/30 [93.3%], respectively) in the diagnosis of microsporidial keratitis.
Use of Cefoxitin-Based Selective Broth for Improved Detection of Methicillin-Resistant
Staphylococcus aureus
Authors: Fang Hong , Hedin Göran
Published: February 2006

A cefoxitin-based selective broth was evaluated for its efficiency in detecting methicillin-resistant
Staphylococcus aureus
(MRSA) by the use of laboratory reference strains, clinical isolates of different clones, and clinical samples. The cefoxitin-based broth was proved to be more sensitive and rapid for the detection of MRSA strains, especially heterogeneously resistant strains, than the oxacillin-based broth.
Proximal Region of the Gene Encoding Cytadherence-Related Protein Permits Molecular Typing of
Mycoplasma genitalium
Clinical Strains by PCR-Restriction Fragment Length Polymorphism
Authors: Musatovova Oxana , Herrera Caleb , Baseman Joel B.
Published: February 2006

Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified proximal region of the gene encoding cytadherence accessory protein P110 (MG192) revealed DNA sequence divergences among 54
Mycoplasma genitalium
clinical strains isolated from the genitourinary tracts of women attending a sexually transmitted disease-related health clinic, plus one from the respiratory tract and one from synovial fluid. Seven of 56 (12.5%) strains exhibited RFLPs following digestion of the proximal region with restriction endonuclease MboI or RsaI, or both. No sequence variability was detected in the distal portion of the gene.
Unique NS5b Hepatitis C Virus Gene Sequence Consensus Database Is Essential for Standardization of Genotype Determinations in Multicenter Epidemiological Studies
Authors: Laperche Syria , Saune Karine , Dény Paul , Duverlie Gilles , Alain Sophie , Chaix Marie-Laure , Gaudy Catherine , Lunel Françoise , Pawlotsky Jean-Michel , Payan Christopher , Pozzetto Bruno , Tamalet Catherine , Thibault Vincent , Vallet Sophie , Bouchardeau Françoise , Izopet Jacques , Lefrère Jean-Jacques
Published: February 2006

A multicenter study of NS5b hepatitis C virus (HCV) genotype determination involving 12 laboratories demonstrates that any laboratory with expertise in sequencing techniques would be able to provide a reliable HCV genotype for clinical and epidemiological purposes as long as they are provided a consensus reference sequence database.
Timely Diagnosis of Disseminated Toxoplasmosis by Sputum Examination
Authors: Laibe Sophy , Ranque Stéphane , Curtillet Catherine , Faraut Françoise , Dumon Henri , Franck Jacqueline
Published: February 2006

The diagnosis of disseminated toxoplasmosis in a 14-year-old allogeneic bone marrow recipient with graft-versus-host disease was determined by the detection of
Toxoplasma gondii
tachyzoites in sputum smears. Sputum analysis is a valuable alternative in the clinical assessment of pulmonary toxoplasmosis, especially when conventional invasive techniques are not practicable.
Capnocytophaga canimorsus
Mycotic Abdominal Aortic Aneurysm: Why the Mailman Is Afraid of Dogs
Authors: Tierney David M. , Strauss Leigh P. , Sanchez Jason L.
Published: February 2006

We describe the first case report of a mycotic abdominal aortic aneurysm secondary to
Capnocytophaga canimorsus
in an immunocompetent 65-year-old man. The exposure occurred after his dog scratched his arm and subsequently licked the wound. A review of
C. canimorsus
, a rarely pathogenic but potentially devastating bacterium, is also presented.
Direct Detection of
Cardiobacterium hominis
in Serum from a Patient with Infective Endocarditis by Broad-Range Bacterial PCR
Authors: Gatselis N. , Malli E. , Papadamou G. , Petinaki E. , Dalekos G. N.
Published: February 2006

Bacterial DNA was detected directly in the serum of a patient with endocarditis by broad-range 16S rRNA PCR followed by sequencing and analysis of the results by the BLAST search. Using these methods,
Cardiobacterium hominis
was identified in 2 days from the date of serum collection. The microorganism was also isolated and identified using conventional methods (bacterial culture and biochemical tests) 17 days from the date of sample collection. This is the first report showing the direct detection of
C. hominis
in a patient's serum using molecular-based methods, emphasizing their potential usefulness as additional and rapid diagnostic tools for the detection and identification of fastidious bacteria.
ATVs: motorized toys or vehicles for children?
Authors: Yanchar N L , Kennedy R , Russell C
Published: February 2006

Objectives:To compare the nature of injuries from all-terrain vehicles (ATVs) to those from bicycling, dirtbikes/motocross, and motor vehicle crashes.

Design:Data on injuries from the mechanisms outlined above were obtained through CHIRPP (the Canadian Hospitals Injury Reporting and Prevention Program) and hospital records.

Setting:A Canadian tertiary pediatric center.

Subjects:Cases presenting to the emergency department over a 10 year period.

Main outcome measures:Comparison between demographics, mechanisms and natures of injuries sustained, disposition from the emergency department, and lengths of hospital stay.

Results:Contrary to bicycling, ATV related injuries occurred among older ages and appeared to result less often from loss of control. Severe injuries resulting in deep soft tissue trauma and fracture/dislocations were 1.7 and 1.5 times, respectively, more frequent among ATV trauma than bicycling (p<0.01). In addition, ATV related injuries were located more frequently in the trunkal, hip, lower extremity, and spinal regions. Conversely, ATV related trauma bore significant similarities regarding body part and nature of the injury to both motor vehicle crash (MVC) and dirtbike related injuries. Akin to dirtbike and MVC related trauma, ATV related injuries more frequently required admission to the ward or intensive care unit compared to bicycling injuries (30.8%v9.6%, p<0.0001), and used a proportionally larger amount of hospital resources with respect to overall in-hospital and intensive care unit days.

Conclusions:Although ATVs may be considered recreational for children, their associated injury patterns, severity, and costs to the healthcare system more closely resemble those from motorized vehicles and are more significant than bicycling. Strict policy to reflect this must be developed and acknowledged by the public, industry, and legislative bodies.