bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods
Authors: Jean Julie , D'Souza Doris H. , Jaykus Lee-Ann 
Published: November 2004

ABSTRACT

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10
−1
reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10
0
to 10
2
reverse transcription-PCR-detectable units (or PFU)/9 cm
2
in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.
Heavy-Metal Stress and Developmental Patterns of Arbuscular Mycorrhizal Fungi
Authors: Pawlowska Teresa E. , Charvat Iris 
Published: November 2004

ABSTRACT

The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species,
Glomus etunicatum
and
Glomus intraradices
, had different levels of sensitivity to metal stress.
G. etunicatum
was more sensitive to all three metals than was
G. intraradices
. A unique response of increased presymbiotic hyphal extension occurred in
G. intraradices
exposed to Cd and Pb. Presymbiotic hyphae of
G. intraradices
formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In
G. intraradices
grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by
G. intraradices
spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.
Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a
Sphingomonas
Strain That Degrades Various Polycyclic Aromatic Hydrocarbons
Authors: Demanèche Sandrine , Meyer Christine , Micoud Julien , Louwagie Mathilde , Willison John C. , Jouanneau Yves 
Published: November 2004

ABSTRACT

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism
Sphingomonas
sp. strain CHY-1 were investigated. [
14
C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in
Novosphingobium aromaticivorans
F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (
phnB
). The second locus contained five genes encoding an extradiol dioxygenase (
phnC
), a ferredoxin (
phnA3
), another oxygenase component (PhnII), and an isomerase (
phnD
). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (
phnA3
) and a reductase (
phnA4
). Disruption of the
phnA1
a
gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in
Escherichia coli
functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.
DNA Macroarray Profiling of
Lactococcus lactis
subsp.
lactis
IL1403 Gene Expression during Environmental Stresses
Authors: Xie Yi , Chou Lan-szu , Cutler Adele , Weimer Bart 
Published: November 2004

ABSTRACT

This report describes the use of an oligonucleotide macroarray to profile the expression of 375 genes in
Lactococcus lactis
subsp.
lactis
IL1403 during heat, acid, and osmotic stress. A set of known stress-associated genes in IL1403 was used as the internal control on the array. Every stress response was accurately detected using the macroarray, compared to data from previous reports. As a group, the expression patterns of the investigated metabolic genes were significantly altered by heat, acid, and osmotic stresses. Specifically, 13 to 18% of the investigated genes were differentially expressed in each of the environmental stress treatments. Interestingly, the methionine biosynthesis pathway genes (
metA-metB1
and
metB2-cysK
) were induced during heat shock, but methionine utilization genes, such as
metK
, were induced during acid stress. These data provide a possible explanation for the differences between acid tolerance mechanisms of
L. lactis
strains IL1403 and MG1363 reported previously. Several groups of transcriptional responses were common among the stress treatments, such as repression of peptide transporter genes, including the
opt
operon (also known as
dpp
) and
dtpT
. Reduction of peptide transport due to environmental stress will have important implications in the cheese ripening process. Although stress responses in lactococci were extensively studied during the last decade, additional information about this bacterium was gained from the use of this metabolic array.
Seasonal Shedding of MultipleCryptosporidiumGenotypes in California Ground Squirrels (Spermophilus beecheyi)
Authors: Atwill Edward R. , Phillips Ralph , Pereira Maria Das Graças C. , Li Xunde , McCowan Brenda 
Published: November 2004

ABSTRACTTwelve percent of 853 California ground squirrels (Spermophilus beecheyi) from six different geographic locations in Kern County, Calif., were found to be shedding on average 44,482 oocysts g of feces−1. The mean annual environmental loading rate ofCryptosporidiumoocysts was 57,882 oocysts squirrel−1day−1, with seasonal patterns of fecal shedding ranging from <10,000 oocysts squirrel−1day−1in fall, winter, and spring to levels of 2 × 105oocysts squirrel−1day−1in summer. Juveniles were about twice as likely as adult squirrels to be infected and shed higher concentrations of oocysts than adults did, with particularly high levels of infection and shedding being found among juvenile male squirrels. Based on DNA sequencing of a portion of the 18S small-subunit rRNA gene, there existed three genotypes ofCryptosporidiumspecies in these populations of squirrels (Sbey03a, Sbey03b, and Sbey03c; accession numbersAY462231toAY462233, respectively). These unique DNA sequences were most closely related (96 to 97% homology) to porcineC. parvum(AF115377) andC. wrairi(AF115378). Inoculating BALB/c neonatal mice with up to 10,000 Sbey03b or Sbey03c fresh oocysts from different infected hosts did not produce detectable levels of infection, suggesting that this common genotype shed by California ground squirrels is not infectious for mice and may constitute a new species ofCryptosporidium.
Changes in Bacterioplankton Composition under Different Phytoplankton Regimens
Authors: Pinhassi Jarone , Sala Maria Montserrat , Havskum Harry , Peters Francesc , Guadayol Òscar , Malits Andrea , Marrasé Cèlia 
Published: November 2004

ABSTRACT

The results of empirical studies have revealed links between phytoplankton and bacterioplankton, such as the frequent correlation between chlorophyll
a
and bulk bacterial abundance and production. Nevertheless, little is known about possible links at the level of specific taxonomic groups. To investigate this issue, seawater microcosm experiments were performed in the northwestern Mediterranean Sea. Turbulence was used as a noninvasive means to induce phytoplankton blooms dominated by different algae. Microcosms exposed to turbulence became dominated by diatoms, while small phytoflagellates gained importance under still conditions. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments showed that changes in phytoplankton community composition were followed by shifts in bacterioplankton community composition, both as changes in the presence or absence of distinct bacterial phylotypes and as differences in the relative abundance of ubiquitous phylotypes. Sequencing of DGGE bands showed that four
Roseobacter
phylotypes were present in all microcosms. The microcosms with a higher proportion of phytoflagellates were characterized by four phylotypes of the
Bacteroidetes
phylum: two affiliated with the family
Cryomorphaceae
and two with the family
Flavobacteriaceae
. Two other
Flavobacteriaceae
phylotypes were characteristic of the diatom-dominated microcosms, together with one
Alphaproteobacteria
phylotype (
Roseobacter
) and one
Gammaproteobacteria
phylotype (
Methylophaga
). Phylogenetic analyses of published
Bacteroidetes
16S rRNA gene sequences confirmed that members of the
Flavobacteriaceae
are remarkably responsive to phytoplankton blooms, indicating these bacteria could be particularly important in the processing of organic matter during such events. Our data suggest that quantitative and qualitative differences in phytoplankton species composition may lead to pronounced differences in bacterioplankton species composition.
Intracellular Proliferation of
Legionella pneumophila
in
Hartmannella vermiformis
in Aquatic Biofilms Grown on Plasticized Polyvinyl Chloride
Authors: Kuiper Melanie W. , Wullings Bart A. , Akkermans Antoon D. L. , Beumer Rijkelt R. , van der Kooij Dick 
Published: November 2004

ABSTRACT

The need for protozoa for the proliferation of
Legionella pneumophila
in aquatic habitats is still not fully understood and is even questioned by some investigators. This study shows the in vivo growth of
L. pneumophila
in protozoa in aquatic biofilms developing at high concentrations on plasticized polyvinyl chloride in a batch system with autoclaved tap water. The inoculum, a mixed microbial community including indigenous
L. pneumophila
originating from a tap water system, was added in an unfiltered as well as filtered (cellulose nitrate, 3.0-μm pore size) state. Both the attached and suspended biomasses were examined for their total amounts of ATP, for culturable
L. pneumophila
, and for their concentrations of protozoa.
L. pneumophila
grew to high numbers (6.3 log CFU/cm
2
) only in flasks with an unfiltered inoculum. Filtration obviously removed the growth-supporting factor, but it did not affect biofilm formation, as determined by measuring ATP. Cultivation, direct counting, and 18S ribosomal DNA-targeted PCR with subsequent sequencing revealed the presence of
Hartmannella vermiformis
in all flasks in which
L. pneumophila
multiplied and also when cycloheximide had been added. Fluorescent in situ hybridization clearly demonstrated the intracellular growth of
L. pneumophila
in trophozoites of
H. vermiformis
, with 25.9% ± 10.5% of the trophozoites containing
L. pneumophila
on day 10 and >90% containing
L. pneumophila
on day 14. Calculations confirmed that intracellular growth was most likely the only way for
L. pneumophila
to proliferate within the biofilm. Higher biofilm concentrations, measured as amounts of ATP, gave higher
L. pneumophila
concentrations, and therefore the growth of
L. pneumophila
within engineered water systems can be limited by controlling biofilm formation.
Influence of an Oyster Reef on Development of the Microbial Heterotrophic Community of an Estuarine Biofilm
Authors: Nocker Andreas , Lepo Joe E. , Snyder Richard A. 
Published: November 2004

ABSTRACT

We characterized microbial biofilm communities developed over two very closely located but distinct benthic habitats in the Pensacola Bay estuary using two complementary cultivation-independent molecular techniques. Biofilms were grown for 7 days on glass slides held in racks 10 to 15 cm over an oyster reef and an adjacent muddy sand bottom. Total biomass and optical densities of dried biofilms showed dramatic differences for oyster reef versus non-oyster reef biofilms. This study assessed whether the observed spatial variation was reflected in the heterotrophic prokaryotic species composition. Genomic biofilm DNA from both locations was isolated and served as a template to amplify 16S rRNA genes with universal eubacterial primers. Fluorescently labeled PCR products were analyzed by terminal restriction fragment length polymorphism, creating a genetic fingerprint of the composition of the microbial communities. Unlabeled PCR products were cloned in order to construct a clone library of 16S rRNA genes. Amplified ribosomal DNA restriction analysis was used to screen and define ribotypes. Partial sequences from unique ribotypes were compared with existing database entries to identify species and to construct phylogenetic trees representative of community structures. A pronounced difference in species richness and evenness was observed at the two sites. The biofilm community structure from the oyster reef setting had greater evenness and species richness than the one from the muddy sand bottom. The vast majority of the bacteria in the oyster reef biofilm were related to members of the γ- and δ-subdivisions of
Proteobacteria
, the
Cytophaga-Flavobacterium -Bacteroides
cluster, and the phyla
Planctomyces
and
Holophaga-Acidobacterium
. The same groups were also present in the biofilm harvested at the muddy sand bottom, with the difference that nearly half of the community consisted of representatives of the
Planctomyces
phylum. Total species richness was estimated to be 417 for the oyster reef and 60 for the muddy sand bottom, with 10.5% of the total unique species identified being shared between habitats. The results suggest dramatic differences in habitat-specific microbial diversity that have implications for overall microbial diversity within estuaries.
Adhesion of
Enterococcus faecalis
in the Nonculturable State to Plankton Is the Main Mechanism Responsible for Persistence of This Bacterium in both Lake and Seawater
Authors: Signoretto Caterina , Burlacchini Gloria , Lleò Maria del Mar , Pruzzo Carla , Zampini Massimiliano , Pane Luigi , Franzini Giorgio , Canepari Pietro 
Published: November 2004

ABSTRACT
The presence of enterococci in lake and seawater in an 18-month survey comparing molecular (PCR and quantitative PCR) and culture methods was evaluated, as well as the possibility that zooplankton could act as reservoirs for enterococci. Samples of both water and zooplankton were collected monthly from a Lake Garda site and an Adriatic Sea site. In lake water, the positive samples numbered 13 of 54 (24%) by culture and 32 of 54 (59%) when PCR was applied. In seawater, they numbered 0 of 51 by culture and 18 of 51 (35%) by PCR. Enterococci were found either totally bound to plankton or totally in water, depending on the presence or absence of plankton, respectively. These results clearly indicate that the PCR assay is a powerful tool for detecting fecal indicators and pathogens in the environment, thus providing a much more sensitive method than culture.
Use of Transposon-Transposase Complexes To Create Stable Insertion Mutant Strains of
Francisella tularensis
LVS
Authors: Kawula Thomas H. , Hall Joshua D. , Fuller James R. , Craven Robin R. 
Published: November 2004

ABSTRACT

Francisella tularensis
is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of
F. tularensis
has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the
F. tularensis
live vaccine strain (LVS). A Tn
5
-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into
F. tularensis
LVS by electroporation. An insertion frequency of 2.6 × 10
−8
± 0.87 × 10
−8
per cell was consistently achieved using this method. There are 178 described Tn
5
consensus target sites distributed throughout the
F. tularensis
genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn
5
target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn
5
-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in
F. tularensis
.
In Vitro Laser Ablation of Natural Marine Biofilms
Authors: Nandakumar Kanavillil , Obika Hideki , Utsumi Akihiro , Ooie Toshihiko , Yano Tetsuo 
Published: November 2004

ABSTRACT

We studied the efficiency of pulsed low-power laser irradiation of 532 nm from an Nd:YAG (neodymium-doped yttrium-aluminum-garnet) laser to remove marine biofilm developed on titanium and glass coupons. Natural biofilms with thicknesses of 79.4 ± 27.8 μm (titanium) and 107.4 ± 28.5 μm (glass) were completely disrupted by 30 s of laser irradiation (fluence, 0.1 J/cm
2
). Laser irradiation significantly reduced the number of diatoms and bacteria in the biofilm (paired
t
test;
P
< 0.05). The removal was better on titanium than on glass coupons.
Combined Phospholipid Biomarker-16S rRNA Gene Denaturing Gradient Gel Electrophoresis Analysis of Bacterial Diversity and Physiological Status in an Intertidal Microbial Mat
Authors: Villanueva Laura , Navarrete Antoni , Urmeneta Jordi , White David C. , Guerrero Ricardo 
Published: November 2004

ABSTRACT
A combined lipid biomarker-16S rRNA gene denaturing gradient gel electrophoresis analysis was used to monitor changes in the physiological status, biomass, and microbial composition of a microbial mat. In the morning hours, an increase in the biomass of layers containing a high density of phototrophs and a decrease in the growth rate in the deep layers were observed. The combined approach also revealed differences in major groups of microorganisms, including green nonsulfur, gram-positive, and heterotrophic bacteria.
Diversity of Toxic Shock Syndrome Toxin 1-Positive
Staphylococcus aureus
Isolates
Authors: Warner John E. , Onderdonk Andrew B. 
Published: November 2004

ABSTRACT

Staphylococcus aureus
isolates from women with nasal, anal, or vaginal colonization were evaluated for population diversity by pulsed-field gel electrophoresis. Cluster analysis of restriction patterns revealed diversity indices of 0.89 and 0.99 for toxic shock syndrome toxin 1-positive and -negative isolates, respectively. Toxin-producing strains were isolated more frequently from the nares than from other sites.
Selection of Single-Chain Antibodies against the VP8* Subunit of Rotavirus VP4 Outer Capsid Protein and Their Expression inLactobacillus casei
Authors: Monedero Vicente , Rodríguez-Díaz Jesús , Viana Rosa , Buesa Javier , Pérez-Martínez Gaspar 
Published: November 2004

ABSTRACTSingle-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression inLactobacillus caseiof one of the scFv were constructed.L. caseiwas able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies.
A METHOD FOR THE TREATMENT OF A SLOPING SEA BOTTOM IN THE PARABOLIC APPROXIMATION
Authors: PAPADAKIS J. S. , PELLONI B. 
Published: November 2004

The impedance boundary condition for the parabolic approximation is derived in the case of a sea bottom profile sloping at a constant angle, as a non-local boundary condition imposed exactly at the interface. This condition is integrated into the IFD code for the numerical computation of the pressure field and implemented to test its accuracy in some benchmark cases, for which the backscattered field is negligible. It is shown that by avoiding the sloping interface, the results obtained are closer to the benchmark results given by normal mode codes solving the full Helmholtz equation, such as the 2-way COUPLE code, than those of the standard IFD or other 1-way codes, at least for problems that do not have significant backscattering effects.