bluuebunny/crossref_metadata_2025_split huggingface dataset:
Recovery and Detection of
Escherichia coli
O157:H7 in Surface Water, Using Ultrafiltration and Real-Time PCR
Authors: Mull Bonnie , Hill Vincent R. 
Published: April 2009

ABSTRACT

Enterohemorrhagic
Escherichia coli
O157:H7 (EHEC O157:H7) outbreaks have revealed the need for improved analytical techniques for environmental samples. Ultrafiltration (UF) is increasingly recognized as an effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. This study describes the application of hollow-fiber UF as the primary step for concentrating EHEC O157:H7 seeded into 40-liter samples of surface water, followed by an established culture/immunomagnetic-separation (IMS) method and a suite of real-time PCR assays. Three TaqMan assays were used to detect the
stx1, stx2
, and
rfbE
gene targets. The results from this study indicate that approximately 50 EHEC O157:H7 cells can be consistently recovered from a 40-liter surface water sample and detected by culture and real-time PCR. Centrifugation was investigated and shown to be a viable alternative to membrane filtration in the secondary culture/IMS step when water quality limits the volume of water that can be processed by a filter. Using multiple PCR assay sets to detect
rfbE, stx1
, and
stx2
genes allowed for specific detection of EHEC O157:H7 from strains that do not possess all three genes. The reported sample collection and analysis procedure should be a sensitive and effective tool for detecting EHEC O157:H7 in response to outbreaks of disease associated with contaminated water.
Direct Expression of Antimicrobial Peptides in an Intact Form by a Translationally Coupled Two-Cistron Expression System
Authors: Jang Su A , Sung Bong Hyun , Cho Ju Hyun , Kim Sun Chang 
Published: April 2009

ABSTRACT

We describe a novel prokaryotic expression system for the production of cationic antimicrobial peptides (AMPs). The method relies on a translationally coupled two-cistron system, in which the termination codon for the first cistron (which encodes the anionic polypeptide mIFc2, a derivative of human gamma interferon) overlaps with the initiation codon for the second cistron (which encodes a cationic AMP) in the sequence of 5′-TAATG-3′. By forming an insoluble complex with the AMP upon translation, the mIFc2 protein efficiently neutralized the toxicity of the coexpressed cationic AMP and minimized the sensitivity of AMP to proteolytic degradation in a host. The AMPs were retrieved from the insoluble inclusion bodies without any chemical or enzymatic cleavage step by simple cation-exchange chromatography. With our system, ∼100 mg of various AMPs (buforin IIb, parasin I, and pexiganan) were obtained from 1 liter of
Escherichia coli
culture. Our expression system may represent a universal cost-effective solution for the mass production of intact AMPs in their natural forms.
High-Throughput Quantitative Analysis of the Human Intestinal Microbiota with a Phylogenetic Microarray
Authors: Paliy Oleg , Kenche Harshavardhan , Abernathy Frank , Michail Sonia 
Published: April 2009

ABSTRACT

Gut microbiota carry out key functions in health and participate in the pathogenesis of a growing number of diseases. The aim of this study was to develop a custom microarray that is able to identify hundreds of intestinal bacterial species. We used the Entrez nucleotide database to compile a data set of bacterial 16S rRNA gene sequences isolated from human intestinal and fecal samples. Identified sequences were clustered into separate phylospecies groups. Representative sequences from each phylospecies were used to develop a microbiota microarray based on the Affymetrix GeneChip platform. The designed microbiota array contains probes to 775 different bacterial phylospecies. In our validation experiments, the array correctly identified genomic DNA from all 15 bacterial species used. Microbiota array has a detection sensitivity of at least 1 pg of genomic DNA and can detect bacteria present at a 0.00025% level of overall sample. Using the developed microarray, fecal samples from two healthy children and two healthy adults were analyzed for bacterial presence. Between 227 and 232 species were detected in fecal samples from children, whereas 191 to 208 species were found in adult stools. The majority of identified phylospecies belonged to the classes
Clostridia
and
Bacteroidetes
. The microarray revealed putative differences between the gut microbiota of healthy children and adults: fecal samples from adults had more
Clostridia
and less
Bacteroidetes
and
Proteobacteria
than those from children. A number of other putative differences were found at the genus level.
Differential Utilization of Carbon Substrates by Bacteria and Fungi in Tundra Soil
Authors: Rinnan Riikka , Bååth Erland 
Published: April 2009

ABSTRACT

Little is known about the contribution of bacteria and fungi to decomposition of different carbon compounds in arctic soils, which are an important carbon store and possibly vulnerable to climate warming. Soil samples from a subarctic tundra heath were incubated with
13
C-labeled glucose, acetic acid, glycine, starch, and vanillin, and the incorporation of
13
C into different phospholipid fatty acids (PLFA; indicative of growth) and neutral lipid fatty acids (NLFA; indicative of fungal storage) was measured after 1 and 7 days. The use of
13
C-labeled substrates allowed the addition of substrates at concentrations low enough not to affect the total amount of PLFA. The label of glucose and acetic acid was rapidly incorporated into the PLFA in a pattern largely corresponding to the fatty acid concentration profile, while glycine and especially starch were mainly taken up by bacteria and not fungi, showing that different groups of the microbial community were responsible for substrate utilization. The
13
C-incorporation from the complex substrates (starch and vanillin) increased over time. There was significant allocation of
13
C into the fungal NLFA, except for starch. For glucose, acetic acid, and glycine, the allocation decreased over time, indicating use of the storage products, whereas for vanillin incorporation into fungal NLFA increased during the incubation. In addition to providing information on functioning of the microbial communities in an arctic soil, our study showed that the combination of PLFA and NLFA analyses yields additional information on the dynamics of substrate degradation.
Maternally and Naturally Acquired Antibodies to Shiga Toxins in a Cohort of Calves Shedding Shiga-ToxigenicEscherichia coli
Authors: Fröhlich Julia , Baljer Georg , Menge Christian 
Published: April 2009

ABSTRACTCalves become infected with Shiga toxin-producingEscherichia coli(STEC) early in life, which frequently results in long-term shedding of the zoonotic pathogen. Little is known about the animals' immunological status at the time of infection. We assessed the quantity and dynamics of maternal and acquired antibodies to Shiga toxins (Stx1 and Stx2), the principal STEC virulence factors, in a cohort of 27 calves. Fecal and serum samples were taken repeatedly from birth until the 24th week of age. Sera, milk, and colostrums of dams were also assessed. STEC shedding was confirmed by detection ofstxin fecal cultures. Stx1- and Stx2-specific antibodies were quantified by Vero cell neutralization assay and further analyzed by immunoblotting. By the eighth week of age, 13 and 15 calves had at least onestx1-type and at least onestx2-type positive culture, respectively. Eleven calves had first positive cultures only past that age. Sera and colostrums of all dams and postcolostral sera of all newborn calves contained Stx1-specific antibodies. Calf serum titers decreased rapidly within the first 6 weeks of age. Only five calves showed Stx1-specific seroconversion. Maternal and acquired Stx1-specific antibodies were mainly directed against the StxA1 subunit. Sparse Stx2-specific titers were detectable in sera and colostrums of three dams and in postcolostral sera of their calves. None of the calves developed Stx2-specific seroconversion. The results indicate that under natural conditions of exposure, first STEC infections frequently coincide with an absence of maternal and acquired Stx-specific antibodies in the animals' sera.
Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable
Legionella pneumophila
Cells in Environmental Water Samples
Authors: Delgado-Viscogliosi Pilar , Solignac Lydie , Delattre Jean-Marie 
Published: April 2009

ABSTRACT

PCR-based methods have been developed to rapidly screen for
Legionella pneumophila
in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for
L. pneumophila
that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable
L. pneumophila
cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the
L. pneumophila
PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of
L. pneumophila
cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating
L. pneumophila
bacteria in water and, in comparison with conventional qPCR techniques used to monitor
Legionella
, has the advantage of selectively amplifying only viable cells.
Matrix Metalloproteinases as Drug Targets in Infections Caused by Gram-Negative Bacteria and in Septic Shock
Authors: Vanlaere Ineke , Libert Claude 
Published: April 2009

SUMMARYThe mammalian immune system is optimized to cope effectively with the constant threat of pathogens. However, when the immune system overreacts, sepsis, severe sepsis, or septic shock can develop. Despite extensive research, these conditions remain the leading cause of death in intensive care units. The matrix metalloproteinases (MMPs) constitute a family of proteases that are expressed in developmental, physiological, and pathological processes and also in response to infections. Studies using MMP inhibitors and MMP knockout mice indicate that MMPs play essential roles in infection and in the host defense against infection. This review provides a brief introduction to some basic concepts of infections caused by gram-negative bacteria and reviews reports describing MMP expression and inhibition, as well as studies with MMP-deficient mice in models of infection caused by gram-negative bacteria and of septic shock. We discuss whether MMPs should be considered novel drug targets in infection and septic shock.
Efflux-Mediated Antifungal Drug Resistance
Authors: Cannon Richard D. , Lamping Erwin , Holmes Ann R. , Niimi Kyoko , Baret Philippe V. , Keniya Mikhail V. , Tanabe Koichi , Niimi Masakazu , Goffeau Andre , Monk Brian C. 
Published: April 2009

SUMMARYFungi cause serious infections in the immunocompromised and debilitated, and the incidence of invasive mycoses has increased significantly over the last 3 decades. Slow diagnosis and the relatively few classes of antifungal drugs result in high attributable mortality for systemic fungal infections. Azole antifungals are commonly used for fungal infections, but azole resistance can be a problem for some patient groups. High-level, clinically significant azole resistance usually involves overexpression of plasma membrane efflux pumps belonging to the ATP-binding cassette (ABC) or the major facilitator superfamily class of transporters. The heterologous expression of efflux pumps in model systems, such Saccharomyces cerevisiae, has enabled the functional analysis of efflux pumps from a variety of fungi. Phylogenetic analysis of the ABC pleiotropic drug resistance family has provided a new view of the evolution of this important class of efflux pumps. There are several ways in which the clinical significance of efflux-mediated antifungal drug resistance can be mitigated. Alternative antifungal drugs, such as the echinocandins, that are not efflux pump substrates provide one option. Potential therapeutic approaches that could overcome azole resistance include targeting efflux pump transcriptional regulators and fungal stress response pathways, blockade of energy supply, and direct inhibition of efflux pumps.
Emergence of the Severe Syndrome and Mortality Associated with Dengue and Dengue-Like Illness: Historical Records (1890 to 1950) and Their Compatibility with Current Hypotheses on the Shift of Disease Manifestation
Authors: Kuno Goro 
Published: April 2009

SUMMARYOutbreaks of the severe dengue syndrome, dengue hemorrhagic fever (DHF), emerged beginning in the 1950s, marking a dramatic change in the dengue syndrome. While intense investigations in multiple directions have been conducted for many years to elucidate the intrinsic mechanisms conducive to the development of DHF, no consensus has yet emerged. Meanwhile, relatively little attention has been paid to the occurrence of severe dengue and death prior to the 1950s. This comprehensive review was designed to evaluate outbreak records in the early dengue history to better understand the epidemiologic background and other factors that existed before the emergence of DHF outbreaks. By applying a set of stringent criteria to remove unreliable data as much as possible and by interpreting the results conservatively, a short list of etiologically more reliable outbreaks with high mortality was obtained. The results show that severe dengue syndrome, clinically very much compatible with DHF, occurred far more frequently in multiple locations than it had been assumed before; that the magnitudes of mortality in several outbreaks were not negligible; and that the epidemiologic background features shared among these outbreaks in the early period were, with the exceptions of more limited demographic changes, generally similar to the post-1950 conditions.
Growth at High pH and Sodium and Potassium Tolerance in Media above the Cytoplasmic pH Depend on ENA ATPases in
Ustilago maydis
Authors: Benito Begoña , Garciadeblás Blanca , Pérez-Martín José , Rodríguez-Navarro Alonso 
Published: April 2009

ABSTRACT

Potassium and Na
+
effluxes across the plasma membrane are crucial processes for the ionic homeostasis of cells. In fungal cells, these effluxes are mediated by cation/H
+
antiporters and ENA ATPases. We have cloned and studied the functions of the two ENA ATPases of
Ustilago maydis
,
U. maydis
Ena1 (UmEna1) and UmEna2. UmEna1 is a typical K
+
or Na
+
efflux ATPase whose function is indispensable for growth at pH 9.0 and for even modest Na
+
or K
+
tolerances above pH 8.0. UmEna1 locates to the plasma membrane and has the characteristics of the low-Na
+
/K
+
-discrimination ENA ATPases. However, it still protects
U. maydis
cells in high-Na
+
media because Na
+
showed a low cytoplasmic toxicity. The UmEna2 ATPase is phylogenetically distant from UmEna1 and is located mainly at the endoplasmic reticulum. The function of UmEna2 is not clear, but we found that it shares several similarities with
Neurospora crassa
ENA2, which suggests that endomembrane ENA ATPases may exist in many fungi. The expression of
ena1
and
ena2
transcripts in
U. maydis
was enhanced at high pH and at high K
+
and Na
+
concentrations. We discuss that there are two modes of Na
+
tolerance in fungi: the high-Na
+
-content mode, involving ENA ATPases with low Na
+
/K
+
discrimination, as described here for
U. maydis
, and the low-Na
+
-content mode, involving Na
+
-specific ENA ATPases, as in
Neurospora crassa
.
Formation of Hirano Bodies after Inducible Expression of a Modified Form of an Actin-Cross-Linking Protein
Authors: Reyes Juan F. , Stone Karen , Ramos Jeanie , Maselli Andrew 
Published: April 2009

ABSTRACT

Hirano bodies are cytoplasmic inclusions composed mainly of actin and actin-associated proteins. The formation of Hirano bodies during various neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis, has been reported. Although the underlying molecular mechanisms that lead to the formation of these inclusions in the brain are not known, expression of the C-terminal fragment (CT) (amino acids 124 to 295) from the endogenous 34-kDa actin-binding protein of
Dictyostelium discoideum
leads to the formation of actin inclusions in vivo. In the current study, we report the development of an inducible expression system to study the early phases of Hirano body formation using an inducible promoter system (
rnrB
). By fusing the CT to a green fluorescent protein (CT-GFP), we monitored protein expression and localization by fluorescence microscopy, flow cytometry, and Western blot analysis. We observed an increase in the number and size of inclusions formed following induction of the CT-GFP vector system. Time-lapse microscopy studies revealed that the CT-GFP foci associated with the cell cortex and fused to form a single large aggregate. Transmission electron microscopy further demonstrates that these inclusions have a highly ordered ultrastructure, a pathological hallmark of Hirano bodies observed in postmortem brain samples from patients with various neurodegenerative disorders. Collectively, this system provides a method to visualize and characterize the events that surround early actin inclusion formation in a eukaryotic model.
Matrix Metalloproteinase 9 Plays a Key Role in Lyme Arthritis but Not in Dissemination ofBorrelia burgdorferi
Authors: Heilpern Andrew J. , Wertheim Warren , He Jia , Perides George , Bronson Roderick T. , Hu Linden T. 
Published: April 2009

ABSTRACTBorrelia burgdorferi, the causative agent of Lyme arthritis, does not produce any exported proteases capable of degrading extracellular matrix despite the fact that it is able to disseminate from a skin insertion site to infect multiple organs. Prior studies have shown thatB. burgdorferiinduces the host protease, matrix metalloproteinase 9 (MMP-9), and suggested that the induction of MMP-9 may allow the organism to disseminate and produce local tissue destruction. We examined the role of MMP-9 in dissemination ofB. burgdorferiand pathogenesis of Lyme arthritis. In a MMP-9−/−mouse model, MMP-9 was not required for the dissemination of the spirochete to distant sites. However, MMP-9−/−exhibited significantly decreased arthritis compared to wild-type mice. The decrease in arthritis was not due to an inability to control infection since the spirochete numbers in the joints were identical. Levels of inflammatory chemokines and cytokines were also similar in MMP-9−/−and wild-type mice. We examined whether decreased inflammation in MMP-9−/−mice may be the result of decreased production of neoattractants by MMP-9-dependent cleavage of collagen. MMP-9 cleavage of type I collagen results in increased monocyte chemoattraction. MMP-9 plays an important role in regulating inflammation in Lyme arthritis, potentially through the cleavage of type I collagen.
Simultaneous Inactivation of Sigma Factors B and D Interferes with Light Acclimation of the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Pollari Maija , Ruotsalainen Virpi , Rantamäki Susanne , Tyystjärvi Esa , Tyystjärvi Taina 
Published: April 2009

ABSTRACT

In cyanobacteria, gene expression is regulated mainly at the level of transcription initiation, which is mediated by the RNA polymerase holoenzyme. The RNA polymerase core is catalytically active, while the σ factor recognizes promoter sequences. Group 2 σ factors are similar to the principal σ factor but are nonessential. Group 2 σ factors SigB and SigD are structurally the most similar σ factors in
Synechocystis
sp. strain PCC 6803. Under standard growth conditions, simultaneous inactivation of
sigB
and
sigD
genes did not affect the growth, but the photosynthesis and growth of the Δ
sigBD
strain were slower than in the control strain at double light intensity. Light-saturated electron transfer rates and the fluorescence and thermoluminescence measurements showed that photosynthetic light reactions are fully functional in the Δ
sigBD
strain, but absorption and 77 K emission spectra measurements suggest that the light-harvesting system of the Δ
sigBD
strain does not acclimate normally to higher light intensity. Furthermore, the Δ
sigBD
strain is more sensitive to photoinhibition under bright light because impaired upregulation of
psbA
genes leads to insufficient PSII repair.
GerO, a Putative Na
+
/H
+
-K
+
Antiporter, Is Essential for Normal Germination of Spores of the Pathogenic Bacterium
Clostridium perfringens
Authors: Paredes-Sabja Daniel , Setlow Peter , Sarker Mahfuzur R. 
Published: April 2009

ABSTRACT

The genome of the pathogen
Clostridium perfringens
encodes two proteins, GerO and GerQ, homologous to monovalent cation transporters suggested to have roles in the germination of spores of some
Bacillus
species. GerO and GerQ were able to transport monovalent cations (K
+
and/or Na
+
) in
Escherichia coli
, and
gerO
and
gerQ
were expressed only in the mother cell compartment during
C. perfringens
sporulation.
C. perfringens
spores lacking GerO were defective in germination with a rich medium, KCl,
l
-asparagine, and a 1:1 chelate of Ca
2+
and dipicolinic acid (DPA), but not with dodecylamine, and the defect was prior to DPA release in germination. All defects in
gerO
spores were complemented by ectopic expression of wild-type
gerO
. Loss of GerQ had much smaller effects on spore germination, and these effects were most evident in spores also lacking GerO. A modeled structure of GerO was similar to that of the
E. coli
Na
+
/H
+
antiporter NhaA, and GerO, but not GerQ contained two adjacent Asp residues thought to be important in the function of this group of cation transporters. Replacement of these adjacent Asp residues in GerO with Asn reduced the protein's ability to complement the germination defect in
gerO
spores but not the ability to restore cation transport to
E. coli
cells defective in K
+
uptake. Together, these data suggest that monovalent cation transporters play some role in
C. perfringens
spore germination. However, it is not clear whether this role is directly in germination or perhaps in spore formation.
Inhibition of Selenium Metabolism in the Oral Pathogen
Treponema denticola
Authors: Jackson-Rosario Sarah , Self William T. 
Published: April 2009

ABSTRACT

In this report we provide evidence that the antimicrobial action of stannous salts and a gold drug, auranofin, against
Treponema denticola
is mediated through inhibition of the metabolism of selenium for synthesis of selenoproteins.
RemA (YlzA) and RemB (YaaB) Regulate Extracellular Matrix Operon Expression and Biofilm Formation inBacillus subtilis
Authors: Winkelman Jared T. , Blair Kris M. , Kearns Daniel B. 
Published: April 2009

ABSTRACTBiofilms are multicellular aggregates stabilized by an extracellular matrix. InBacillus subtilis, the biofilm matrix is composed of an extracellular polysaccharide and the secreted protein TasA. Expression of both of the matrix components is repressed by the DNA-binding master regulator, SinR. Here we identify two small protein regulators of the extracellular matrix: RemA (formerly YlzA) and RemB (formerly YaaB). Mutation of RemA or RemB impairs pellicle formation, complex colony architecture, and motility inhibition in asinRmutant background. Both proteins are required for the activation of the matrix biosynthesis operons and appear to act in parallel to SinR and two other known biofilm regulators, AbrB and DegU.