bluuebunny/crossref_metadata_2025_split huggingface dataset:
Direct Real-Time PCR Quantification of
Campylobacter jejuni
in Chicken Fecal and Cecal Samples by Integrated Cell Concentration and DNA Purification
Authors: Rudi Knut , Høidal Hilde Kristin , Katla Tone , Johansen Birgit Klungseth , Nordal John , Jakobsen Kjetill S. 
Published: February 2004

ABSTRACT

Campylobacter jejuni
is a major cause of diarrheal disease and food-borne gastroenteritis. The main reservoir of
C. jejuni
in poultry is the cecum, with an estimated content of 6 to 8 log
10
CFU/g. If a flock is infected with
C. jejuni
, the majority of the birds in that flock will harbor the bacterium. Diagnostics at the flock level could thus be an important control point. The aim of the work presented here was to develop a complete quantitative PCR-based detection assay for
C. jejuni
obtained directly from cecal contents and fecal samples. We applied an approach in which the same paramagnetic beads were used both for cell isolation and for DNA purification. This integrated approach enabled both fully automated and quantitative sample preparation and a DNA extraction method. We developed a complete quantitative diagnostic assay through the combination of the sample preparation approach and real-time 5′-nuclease PCR. The assay was evaluated both by spiking the samples with
C. jejuni
and through the detection of
C. jejuni
in naturally colonized chickens. Detection limits between 2 and 25 CFU per PCR and a quantitative range of >4 log
10
were obtained for spiked fecal and cecal samples. Thirty-one different poultry flocks were screened for naturally colonized chickens. A total of 262 (204 fecal and 58 cecal) samples were analyzed. Nineteen of the flocks were
Campylobacter
positive, whereas 12 were negative. Two of the flocks contained
Campylobacter
species other than
C. jejuni
. There was a large difference in the
C. jejuni
content, ranging from 4 to 8 log
10
CFU/g of fecal or cecal material, for the different flocks tested. Some issues that have not yet promoted much attention are the prequantitative differences in the ability of
C. jejuni
to colonize poultry and the importance of these differences for causing human disease through food contamination. Understanding the colonization kinetics in poultry is therefore of great importance for controlling human infections by this bacterium.
Characterization and Development of Two Reporter Gene Systems for
Clostridium acetobutylicum
Authors: Feustel Lothar , Nakotte Stephan , Dürre Peter 
Published: February 2004

ABSTRACT

The use of
lacZ
from
Thermoanaerobacterium thermosulfurigenes
(encoding β-galactosidase) and
lucB
from
Photinus pyralis
(encoding luciferase) as reporter genes in
Clostridium acetobutylicum
was analyzed with promoters of genes required for solventogenesis and acidogenesis. Both systems proved to be well suited and allowed the detection of differences in promoter strength at least up to 100-fold. The luciferase assay could be performed much faster and comes close to online measurement. Resequencing of
lacZ
revealed a sequence error in the original database entry, which resulted in β-galactosidase with an additional 31 amino acids. Cutting off part of the gene encoding this C terminus resulted in decreased enzyme activity. The
lacZ
reporter data showed that
bdhA
(encoding butanol dehydrogenase A) is expressed during the early growth phase, followed by
sol
(encoding butyraldehyde/butanol dehydrogenase E and coenzyme A transferase) and
bdhB
(encoding butanol dehydrogenase B) expression.
adc
(encoding acetoacetate decarboxylase) was also induced early. There is about a 100-fold difference in expression between
adc
and
bdhB
(higher) and
bdhA
and the
sol
operon (lower). The
lucB
reporter activity could be increased 10-fold by the addition of ATP to the assay. Washing of the cells proved to be important in order to prevent a red shift of bioluminescence in an acidic environment (for reliable data).
lucB
reporter measurements confirmed the expression pattern of the
sol
and
ptb-buk
(encoding phosphotransbutyrylase and butyrate kinase) operons as determined by the
lacZ
reporter and showed that the expression level from the
ptb
promoter is 59-fold higher than that from the
sol
operon promoter.
New Perspective on Uncultured Bacterial Phylogenetic Division OP11
Authors: Harris J. Kirk , Kelley Scott T. , Pace Norman R. 
Published: February 2004

ABSTRACT

Organisms belonging to the OP11 candidate phylogenetic division of
Bacteria
have been detected only in rRNA-based sequence surveys of environmental samples. Preliminary studies indicated that such organisms represented by the sequences are abundant and widespread in nature and highly diverse phylogenetically. In order to document more thoroughly the phylogenetic breadth and environmental distribution of this diverse group of organisms, we conducted further molecular analyses on environmental DNAs. Using PCR techniques and primers directed toward each of the five described subdivisions of OP11, we surveyed 17 environmental DNAs and analyzed rRNA gene sequences in 27 clonal libraries from 14 environments. Ninety-nine new and unique sequences were determined completely, and approximately 200 additional clones were subjected to partial sequencing. Extensive phylogenetic comparisons of the new sequences to those representing other bacterial divisions further resolved the phylogeny of the bacterial candidate division OP11 and identified two new candidate bacterial divisions, OP11-derived 1 (OD1) and Sulphur River 1 (SR1). The widespread environmental distribution of representatives of the bacterial divisions OD1, OP11, and SR1 suggests potentially conspicuous biogeochemical roles for these organisms in their respective environments. The information on environmental distribution offers clues for attempts to culture landmark representatives of these novel bacterial divisions, and the sequences are specific molecular signatures that provide for their identification in other contexts.
Characterization of Bioluminescent Derivatives of Assimilable Organic Carbon Test Bacteria
Authors: Haddix Pryce L. , Shaw Nancy J. , LeChevallier Mark W. 
Published: February 2004

ABSTRACT

The assimilable organic carbon (AOC) test is a standardized measure of the bacterial growth potential of treated water. We describe the design and initial development of an AOC assay that uses bioluminescent derivatives of AOC test bacteria. Our assay is based on the observation that bioluminescence peaks at full cell yield just prior to the onset of the stationary phase during growth in a water sample.
Pseudomonas fluorescens
P-17 and
Spirillum
sp. strain NOX bacteria were mutagenized with
luxCDABE
operon fusion and inducible transposons and were selected on minimal medium. Independent mutants were screened for high luminescence activity and predicted AOC assay sensitivity. All mutants tested were able to grow in tap water under AOC assay conditions. Strains P-17 I5 (with
p
-aminosalicylate inducer) and NOX I3 were chosen for use in the bioluminescence AOC test. Peak bioluminescence and plate count AOC were linearly related for both test bacteria, though data suggest that the P-17 bioluminescence assay requires more consistent luminescence monitoring. Bioluminescence results were obtained 2 or 3 days postinoculation, compared with 5 days for the ATP luminescence AOC assay and 8 days for the plate count assay. Plate count AOC assay results for nonmutant and bioluminescent bacteria from 36 water samples showed insignificant differences, indicating that the luminescent bacteria retained a full range of AOC measurement capability. This bioluminescence method is amenable to automation with a microplate format with programmable reagent injection.
Chromate-Reducing Properties of Soluble Flavoproteins from
Pseudomonas putida
and
Escherichia coli
Authors: Ackerley D. F. , Gonzalez C. F. , Park C. H. , Blake R. , Keyhan M. , Matin A. 
Published: February 2004

ABSTRACT

Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins—ChrR (from
Pseudomonas putida
) and YieF (from
Escherichia coli
)—were examined. Both are dimers and reduce chromate efficiently to Cr(III) (
k
cat
/

K
m

= ∼2 × 10
4
M
−1
· s
−1
). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.
Electrotransformation of
Clostridium thermocellum
Authors: Tyurin Michael V. , Desai Sunil G. , Lynd Lee R. 
Published: February 2004

ABSTRACT

Electrotransformation of several strains of
Clostridium thermocellum
was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of
C. thermocellum
with subsequent PCR specific to the
mls
gene on the plasmid, as well as by retransformation of
Escherichia coli
. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 ± 0.5) × 10
5
transformants per μg of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the
mls
gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 ± 1.8) × 10
4
transformants per μg of plasmid DNA for strain ATCC 27405 and ∼1 × 10
3
transformants per μg of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was ∼50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in
C. thermocellum
that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.
Multi-Virulence-Locus Sequence Typing ofListeria monocytogenes
Authors: Zhang Wei , Jayarao Bhushan M. , Knabel Stephen J. 
Published: February 2004

ABSTRACTA multi-virulence-locus sequence typing (MVLST) scheme was developed for subtypingListeria monocytogenes, and the results obtained using this scheme were compared to those of pulsed-field gel electrophoresis (PFGE) and the published results of other typing methods, including ribotyping (RT) and multilocus sequence typing (MLST). A set of 28 strains (eight different serotypes and three known genetic lineages) ofL. monocytogeneswas selected from a strain collection (n> 1,000 strains) to represent the genetic diversity of this species. Internal fragments (ca. 418 to 469 bp) of three virulence genes (prfA,inlB, andinlC) and three virulence-associated genes (dal,lisR, andclpP) were sequenced and analyzed. Multiple DNA sequence alignment identified 10 (prfA), 19 (inlB), 13 (dal), 10 (lisR), 17 (inlC), and 16 (clpP) allelic types and a total of 28 unique sequence types. Comparison of MVLST with automatedEcoRI-RT and PFGE withApaI enzymatic digestion showed that MVLST was able to differentiate strains that were indistinguishable by RT (13 ribotypes; discrimination index = 0.921) or PFGE (22 profiles; discrimination index = 0.970). Comparison of MVLST with housekeeping-gene-based MLST analysis showed that MVLST provided higher discriminatory power for serotype 1/2a and 4b strains than MLST. Cluster analysis based on the intragenic sequences of the selected virulence genes indicated a strain phylogeny closely related to serotypes and genetic lineages. In conclusion, MVLST may improve the discriminatory power of MLST and provide a convenient tool for studying the local epidemiology ofL. monocytogenes.
Diazotrophic Community Structure and Function in Two Successional Stages of Biological Soil Crusts from the Colorado Plateau and Chihuahuan Desert
Authors: Yeager Chris M. , Kornosky Jennifer L. , Housman David C. , Grote Edmund E. , Belnap Jayne , Kuske Cheryl R. 
Published: February 2004

ABSTRACT

The objective of this study was to characterize the community structure and activity of N
2
-fixing microorganisms in mature and poorly developed biological soil crusts from both the Colorado Plateau and Chihuahuan Desert. Nitrogenase activity was approximately 10 and 2.5 times higher in mature crusts than in poorly developed crusts at the Colorado Plateau site and Chihuahuan Desert site, respectively. Analysis of
nifH
sequences by clone sequencing and the terminal restriction fragment length polymorphism technique indicated that the crust diazotrophic community was 80 to 90% heterocystous cyanobacteria most closely related to
Nostoc
spp. and that the composition of N
2
-fixing species did not vary significantly between the poorly developed and mature crusts at either site. In contrast, the abundance of
nifH
sequences was approximately 7.5 times greater (per microgram of total DNA) in mature crusts than in poorly developed crusts at a given site as measured by quantitative PCR. 16S rRNA gene clone sequencing and microscopic analysis of the cyanobacterial community within both crust types demonstrated a transition from a
Microcoleus vaginatus
-dominated, poorly developed crust to mature crusts harboring a greater percentage of
Nostoc
and
Scytonema
spp. We hypothesize that ecological factors, such as soil instability and water stress, may constrain the growth of N
2
-fixing microorganisms at our study sites and that the transition to a mature, nitrogen-producing crust initially requires bioengineering of the surface microenvironment by
Microcoleus vaginatus
.
Occurrence, Structure, and Mobility of Tn
1546
-Like Elements in Environmental Isolates of Vancomycin-Resistant Enterococci
Authors: Guardabassi L. , Dalsgaard A. 
Published: February 2004

ABSTRACT

The occurrence, structure, and mobility of Tn
1546-
like elements were studied in environmental vancomycin-resistant enterococci (VRE) isolated from municipal sewage, activated sludge, pharmaceutical waste derived from antibiotic production, seawater, blue mussels, and soil. Of 200 presumptive VRE isolates tested, 71 (35%) harbored

vanA

. Pulsed-field gel electrophoresis analysis allowed the detection of 26 subtypes, which were identified as
Enterococcus faecium
(
n
= 13),
E. casseliflavus
(
n
= 6),
E. mundtii
(
n
= 3),
E. faecalis
(
n
= 3), and
E. durans
(
n
= 1) by phenotypic tests and 16S ribosomal DNA sequencing. Long PCR-restriction fragment length polymorphism (L-PCR-RFLP) analysis of Tn
1546
-like elements and PCR analysis of internal regions revealed the presence of seven groups among the 29 strains studied. The most common group (group 1) corresponded to the structure of Tn
1546
in the prototype strain
E. faecium
BM4147. Two novel L-PCR-RFLP patterns (groups 3 and 4) were found for
E. casseliflavus
strains. Indistinguishable Tn
1546-
like elements occurred in VRE strains belonging to different species or originating from different sources. Interspecies plasmid-mediated transfer of vancomycin resistance to
E. faecium
BM4105 was demonstrated for
E. faecalis
,
E. mundtii
, and
E. durans
. This study indicates that VRE, including species other than
E. faecium
and
E. faecalis
, are widespread in nature and in environments that are not exposed to vancomycin selection and not heavily contaminated with feces, such as seawater, blue mussels, and nonagricultural soil. Tn
1546
-like elements can readily transfer between enterococci of different species and ecological origins, therefore raising questions about the origin of these transposable elements and their possible transfer between environmental and clinical settings.
Detection and Differentiation of In Vitro-Spiked Bacteria by Real-Time PCR and Melting-Curve Analysis
Authors: Klaschik S. , Lehmann L. E. , Raadts A. , Book M. , Gebel J. , Hoeft A. , Stuber F. 
Published: February 2004

ABSTRACT

We introduce a consensus real-time PCR protocol for the detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and plasma. This prototype detection system enables an exact Gram stain classification and, in particular, screening for specific species of 17 intensive care unit-relevant bacteria by means of fluorescence hybridization probes and melting-curve analysis in a one-run experiment. One strain of every species was tested at a final density of 10
6
CFU/ml. All bacteria examined except
Staphylococcus aureus
and
Staphylococcus epidermidis
could be differentiated successfully;
S. aureus
and
S. epidermidis
could only be classified as “
Staphylococcus
species.” The hands-on time for preparation of the DNA, performance of the PCR, and evaluation of the PCR results was less than 4 h. Nevertheless, this prototype detection system requires more clinical validation.
Antigen Capture Competitive Enzyme-Linked Immunosorbent Assays Using Baculovirus-Expressed Antigens for Diagnosis of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus
Authors: Mecham J. O. , Wilson W. C. 
Published: February 2004

ABSTRACTBluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are orbiviruses that infect both livestock and wild ruminants. Antigenic cross-reactivity between BTV and EHDV often results in serologic misdiagnosis. Competitive enzyme-linked immunosorbent assays (c-ELISAs) show increased sensitivity and specificity for the identification of these viral diseases; however, the preparation of cell culture-derived viral antigen for these tests is laborious and variable from batch to batch, and the resulting antigen may be infectious. To overcome these problems, the genes coding for a structural protein, VP7, of BTV and EHDV were cloned into baculovirus and the recombinant proteins were expressed in Sf9 cultured insect cells. Recombinant viral proteins released into the baculovirus-infected Sf9 cell culture supernatant were used in antigen capture c-ELISAs (Ag Cap c-ELISA) tests that specifically detected antibody in the serum of cattle experimentally infected with BTV and EHDV. The diagnostic utility of the Ag Cap c-ELISA was demonstrated by comparison with a commercial c-ELISA. The Ag Cap c-ELISA offers the advantages of using an easily produced, easily standardized, noninfectious antigen that does not require further purification or concentration.
DNA Differential Diagnosis of Taeniasis and Cysticercosis by Multiplex PCR
Authors: Yamasaki Hiroshi , Allan James C. , Sato Marcello Otake , Nakao Minoru , Sako Yasuhito , Nakaya Kazuhiro , Qiu Dongchuan , Mamuti Wulamu , Craig Philip S. , Ito Akira 
Published: February 2004

ABSTRACT

Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome
c
oxidase subunit 1 gene yielded evident differential products unique for
Taenia saginata
and
Taenia asiatica
and for American/African and Asian genotypes of
Taenia solium
with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9
T. solium
carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a
T. solium
carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to
T. solium
carriers, 827-bp markers were detected by multiplex PCR in all
T. saginata
carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections.
Multicenter Evaluation of the Performance Characteristics of the Bayer VERSANT HCV RNA 3.0 Assay (bDNA)
Authors: Elbeik Tarek , Surtihadi Johan , Destree Mark , Gorlin Jed , Holodniy Mark , Jortani Saeed A. , Kuramoto Ken , Ng Valerie , Valdes Roland , Valsamakis Alexandra , Terrault Norah A. 
Published: February 2004

ABSTRACTIn this multicenter evaluation, the VERSANT HCV RNA 3.0 Assay (bDNA) (Bayer Diagnostics, Tarrytown, N.Y.) was shown to have excellent reproducibility, linearity, and analytical sensitivity across specimen collection matrices (serum, EDTA, ACD-A), and hepatitis C virus (HCV) genotypes 1 to 6. The VERSANT HCV bDNA Assay has a reportable range of 615 to 7,690,000 (7.69 × 106) IU/ml. The total coefficient of variation (CV) ranged from 32.4% at 615 IU/ml to 17% at 6.8 × 106IU/ml. The assay was linear across the reportable range. Analytical specificity of 98.8% was determined by testing 999 specimens from volunteer blood donors. Evaluation of HCV genotypes using RNA transcripts of representative clones of 1a, 1b, 2a, 2b, 2c, 3a, 4a, 5a, and 6a and patient specimens showed that the largest difference between genotype 1, upon which the assay is standardized, and non-1 genotypes was within 1.5-fold. Testing of potentially interfering endogenous substances and exogenous substances and conditions found no interference in HCV-positive or HCV-negative specimens except for unconjugated bilirubin at concentrations of ≥20 mg/dl and protein at concentrations of ≥9 g/dl. Biological variability was estimated from 29 clinically stable individuals not on HCV therapy who were tested weekly over an 8-week period. The combined estimate of total (biologic plus assay) variability was 0.15 log10standard deviation (CV, 36.1%), a fold change of 2.6. Thus, the observed fold change between any two consecutive HCV RNA measures is expected to be less than 2.6-fold (equivalent to 0.41 log10IU/ml) 95% of the time in clinically stable individuals.