bluuebunny/crossref_metadata_2025_split huggingface dataset:
TaqMan PCR for Detection of
Vibrio cholerae
O1, O139, Non-O1, and Non-O139 in Pure Cultures, Raw Oysters, and Synthetic Seawater
Authors: Lyon W. J. 
Published: July 2002

ABSTRACT

Vibrio cholerae
is recognized as a leading human waterborne pathogen. Traditional diagnostic testing for
Vibrio
is not always reliable, because this bacterium can enter a viable but nonculturable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this article, a TaqMan PCR assay is presented for quantitative detection of
V. cholerae
in pure cultures, oysters, and synthetic seawater. Primers and probe were designed from the nonclassical hemolysin (
hly
A) sequence of
V. cholerae
strains. This probe was applied to DNA from 60 bacterial strains comprising 21 genera. The TaqMan PCR assay was positive for all of the strains of
V. cholerae
tested and negative for all other species of
Vibrio
tested. In addition, none of the other genera tested was amplified with the TaqMan primers and probe used in this study. The results of the TaqMan PCR with raw oysters and spiked with
V. cholerae
serotypes O1 and O139 were comparable to those of pure cultures. The sensitivity of the assay was in the range of 6 to 8 CFU g
−1
and 10 CFU ml
−1
in spiked raw oyster and synthetic seawater samples, respectively. The total assay could be completed in 3 h. Quantification of the
Vibrio
cells was linear over at least 6 log units. The TaqMan probe and primer set developed in this study can be used as a rapid screening tool for the presence of
V. cholerae
in oysters and seawater without prior isolation and characterization of the bacteria by traditional microbiological methods.
Persistence of an Occlusion-Negative Recombinant Nucleopolyhedrovirus in
Trichoplusia ni
Indicates High Multiplicity of Cellular Infection
Authors: Bull James C. , Godfray H. C. J. , O'Reilly David R. 
Published: July 2002

ABSTRACT

We use data from the serial passage of co-occluded recombinant
Autographa californica
nuclear polyhedrosis virus (Ac
M
NPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of Ac
M
NPV was constructed with a deletion of its
polyhedrin
gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type Ac
M
NPV and used to infect fifth-instar
Trichoplusia ni
larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 ± 0.3 viral genomes per occlusion body-producing cell.
Citrate Metabolism by
Enterococcus faecalis
FAIR-E 229
Authors: Sarantinopoulos Panagiotis , Kalantzopoulos George , Tsakalidou Effie 
Published: July 2002

ABSTRACT

Citrate metabolism by
Enterococcus faecalis
FAIR-E 229 was studied in various growth media containing citrate either in the presence of glucose or lactose or as the sole carbon source. In skim milk (130 mM lactose, 8 mM citrate), cometabolism of citrate and lactose was observed from the first stages of the growth phase. Lactose was stoichiometrically converted into lactate, while citrate was converted into acetate, formate, and ethanol. When de Man-Rogosa-Sharpe (MRS) broth containing lactose (28 mM) instead of glucose was used,
E. faecalis
FAIR-E 229 catabolized only the carbohydrate. Lactate was the major end product, and small amounts of ethanol were also detected. Increasing concentrations of citrate (10, 40, 70, and 100 mM) added to MRS broth enhanced both the maximum growth rate of
E. faecalis
FAIR-E 229 and glucose catabolism, although citrate itself was not catabolized. Glucose was converted stoichiometrically into lactate, while small amounts of ethanol were produced as well. Finally, when increasing initial concentrations of citrate (10, 40, 70, and 100 mM) were used as the sole carbon sources in MRS broth without glucose, the main end products were acetate and formate. Small amounts of lactate, ethanol, and acetoin were also detected. This work strongly supports the suggestion that enterococcal strains have the metabolic potential to metabolize citrate and therefore to actively contribute to the flavor development of fermented dairy products.
Phylogenetic Analysis of Culturable Dimethyl Sulfide-Producing Bacteria from a
Spartina
-Dominated Salt Marsh and Estuarine Water
Authors: Ansede John H. , Friedman Robert , Yoch Duane C. 
Published: July 2002

ABSTRACT

Dimethylsulfoniopropionate (DMSP), an abundant osmoprotectant found in marine algae and salt marsh cordgrass, can be metabolized to dimethyl sulfide (DMS) and acrylate by microbes having the enzyme DMSP lyase. A suite of DMS-producing bacteria isolated from a salt marsh and adjacent estuarine water on DMSP agar plates differed markedly from the pelagic strains currently in culture. While many of the salt marsh and estuarine isolates produced DMS and methanethiol from methionine and dimethyl sulfoxide, none appeared to be capable of producing both methanethiol and DMS from DMSP. DMSP, and its degradation products acrylate and β-hydroxypropionate but not methyl-3-mecaptopropionate or 3-mercaptopropionate, served as a carbon source for the growth of all the α- and β- but only some of the γ-proteobacterium isolates. Phylogenetic analysis of 16S rRNA gene sequences showed that all of the isolates were in the group
Proteobacteria
, with most of them belonging to the α and γ subclasses. Only one isolate was identified as a β-proteobacterium, and it had >98% 16S rRNA sequence homology with a terrestrial species of
Alcaligenes faecalis
. Although bacterial population analysis based on culturability has its limitations, bacteria from the α and γ subclasses of the
Proteobacteria
were the dominant DMS producers isolated from salt marsh sediments and estuaries, with the γ subclass representing 80% of the isolates. The α-proteobacterium isolates were all in the
Roseobacter
subgroup, while many of the γ-proteobacteria were closely related to the pseudomonads; others were phylogenetically related to
Marinomonas, Psychrobacter
, or
Vibrio
species. These data suggest that DMSP cleavage to DMS and acrylate is a characteristic widely distributed among different phylotypes in the salt marsh-estuarine ecosystem.
Acetate Oxidation Coupled to Fe(III) Reduction in Hyperthermophilic Microorganisms
Authors: Tor Jason M. , Kashefi Kazem , Lovley Derek R. 
Published: July 2002

ABSTRACT

No hyperthermophilic microorganisms have previously been shown to anaerobically oxidize acetate, the key extracellular intermediate in the anaerobic oxidation of organic matter. Here we report that two hyperthermophiles,
Ferroglobus placidus
and “
Geoglobus ahangari
,” grow at 85°C by oxidizing acetate to carbon dioxide, with Fe(III) serving as the electron acceptor. These results demonstrate that acetate could potentially be metabolized within the hot microbial ecosystems in which hyperthermophiles predominate, rather than diffusing to cooler environments prior to degradation as has been previously proposed.
Comparison of Different Approaches To Quantify
Staphylococcus aureus
Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese
Authors: Hein Ingeborg , Lehner Angelika , Rieck Petra , Klein Kurt , Brandl Ernst , Wagner Martin 
Published: July 2002

ABSTRACT

Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of
Staphylococcus aureus
cells by targeting the thermonuclease (
nuc
) gene. Purified DNA extracts from pure cultures of
S. aureus
were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60
nuc
gene copies/μl) than using a fluorigenic TaqMan probe (6
nuc
gene copies/μl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify
S. aureus
cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 × 10
2
to 6.4 × 10
2
copies of the
nuc
gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and
nuc
gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of
S. aureus
in cheese by performing RTQ-PCR.
Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells
Authors: Lim Eelin L. , Tomita Aoy V. , Thilly William G. , Polz Martin F. 
Published: July 2002

ABSTRACT

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate
Cafeteria roenbergensis
as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated
C. roenbergensis
cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 10
4
cells ml
−1
. The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells ml
−1
by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples.
Nitrous Oxide Formation in the Colne Estuary, England: the Central Role of Nitrite
Authors: Dong Liang F. , Nedwell David B. , Underwood Graham J. C. , Thornton Daniel C. O. , Rusmana Iman 
Published: July 2002

ABSTRACTNitrate and nitrite concentrations in the water and nitrous oxide and nitrite fluxes across the sediment-water interface were measured monthly in the River Colne estuary, England, from December 1996 to March 1998. Water column concentrations of N2O in the Colne were supersaturated with respect to air, indicating that the estuary was a source of N2O for the atmosphere. At the freshwater end of the estuary, nitrous oxide effluxes from the sediment were closely correlated with the nitrite concentrations in the overlying water and with the nitrite influx into the sediment. Increases in N2O production from sediments were about 10 times greater with the addition of nitrite than with the addition of nitrate. Rates of denitrification were stimulated to a larger extent by enhanced nitrite than by nitrate concentrations. At 550 μM nitrite or nitrate (the highest concentration used), the rates of denitrification were 600 μmol N · m−2· h−1with nitrite but only 180 μmol N · m−2· h−1with nitrate. The ratios of rates of nitrous oxide production and denitrification (N2O/N2× 100) were significantly higher with the addition of nitrite (7 to 13% of denitrification) than with nitrate (2 to 4% of denitrification). The results suggested that in addition to anaerobic bacteria, which possess the complete denitrification pathway for N2formation in the estuarine sediments, there may be two other groups of bacteria: nitrite denitrifiers, which reduce nitrite to N2via N2O, and obligate nitrite-denitrifying bacteria, which reduce nitrite to N2O as the end product. Consideration of free-energy changes during N2O formation led to the conclusion that N2O formation using nitrite as the electron acceptor is favored in the Colne estuary and may be a critical factor regulating the formation of N2O in high-nutrient-load estuaries.
Bacillus stearothermophilus
Neopullulanase Selective Hydrolysis of Amylose to Maltose in the Presence of Amylopectin
Authors: Kamasaka Hiroshi , Sugimoto Kazuhisa , Takata Hiroki , Nishimura Takahisa , Kuriki Takashi 
Published: July 2002

ABSTRACT

The specificity of
Bacillus stearothermophilus
TRS40 neopullulanase toward amylose and amylopectin was analyzed. Although this neopullulanase completely hydrolyzed amylose to produce maltose as the main product, it scarcely hydrolyzed amylopectin. The molecular mass of amylopectin was decreased by only one order of magnitude, from approximately 10
8
to 10
7
Da. Furthermore, this neopullulanase selectively hydrolyzed amylose when starch was used as a substrate. This phenomenon, efficient hydrolysis of amylose but not amylopectin, was also observed with cyclomaltodextrinase from alkaliphilic
Bacillus
sp. strain A2-5a and maltogenic amylase from
Bacillus licheniformis
ATCC 27811. These three enzymes hydrolyzed cyclomaltodextrins and amylose much faster than pullulan. Other amylolytic enzymes, such as bacterial saccharifying α-amylase, bacterial liquefying α-amylase, β-amylase, and neopullulanase from
Bacillus megaterium
, did not exhibit this distinct substrate specificity at all, i.e., the preference of amylose to amylopectin.
Taxis Response of Various Denitrifying Bacteria to Nitrate and Nitrite
Authors: Lee Dong Yun , Ramos Adela , Macomber Lee , Shapleigh James P. 
Published: July 2002

ABSTRACT

The taxis response of
Rhodobacter sphaeroides
2.4.1 and 2.4.3,
Rhodopseudomonas palustris
, and
Agrobacterium tumefaciens
to nitrate and nitrite was evaluated by observing the macroscopic behavior of cells suspended in soft agar and incubated under various conditions.
R. sphaeroides
2.4.3, which is capable of both nitrate and nitrite reduction, showed a taxis response to both nitrate and nitrite.
R. sphaeroides
2.4.1, which contains nitrate reductase but not nitrite reductase, did not show a taxis response towards either nitrogen oxide. Insertional inactivation of the nitrite reductase structural gene or its transcriptional regulator, NnrR, in strain 2.4.3 caused a loss of a taxis response towards both nitrate and nitrite. An isolate of 2.4.1 carrying a copy of the nitrite reductase gene from 2.4.3 showed a taxis response to both nitrogen oxides. The taxis response of 2.4.3 was observed under anaerobic conditions, suggesting that the taxis response was due to nitrate and nitrite respiration, not to inhibition of oxygen respiration by respiration of nitrogen oxides. Strain 2.4.3 showed a taxis response to nitrate and nitrite under photosynthetic and aerobic conditions. Changing the carbon source in the culture medium caused an unexpected subtle shift in the taxis response of 2.4.3 to nitrite. A taxis response to nitrogen oxides was also observed in
R. palustris
and
A. tumefaciens. R. palustris
exhibited a taxis response to nitrite but not to nitrate, while
A. tumefaciens
exhibited a response to both compounds.
Cytoskeletal Alterations in Lipopolysaccharide-Induced Bovine Vascular Endothelial Cell Injury and Its Prevention by Sodium Arsenite
Authors: Chakravortty Dipshikha , Koide Naoki , Kato Yutaka , Sugiyama Tsuyoshi , Kawai Makoto , Fukada Masako , Yoshida Tomoaki , Yokochi Takashi 
Published: July 2002

ABSTRACT
Morphological changes, especially cytoskeletal alterations, in lipopolysaccharide (LPS)-induced vascular endothelial cell injury were studied by using LPS-susceptible bovine aortic endothelial cells (BAEC). BAEC in cultures with LPS showed cell rounding, shrinking, and intercellular gap formation. In those cells, LPS caused the disorganization of actin, tubulin, and vimentin. LPS also induced a reduction in the F-actin pool and an elevation in the G-actin pool. Cytoskeletal disorganization affected transendothelial permeability across the endothelial monolayer. Pretreatment of BAEC with sodium arsenite (SA) prevented alterations in LPS-induced BAEC injury. However, posttreatment with SA had no protective effect on them. SA upregulated the expression of heat shock protein in the presence of LPS. The role of SA in prevention of LPS-induced BAEC injury is discussed.
Use of Synthetic Cardiolipin and Lecithin in the Antigen Used by the Venereal Disease Research Laboratory Test for Serodiagnosis of Syphilis
Authors: Castro Arnold R. , Morrill William E. , Shaw Walter A. , Gale David C. , Park Mahin M. , Peregrino-Ferreira Luiz A. , Bazzo Maria L. , Pope Victoria 
Published: July 2002

ABSTRACT

The Venereal Disease Research Laboratory (VDRL) test is a microflocculation test for syphilis that uses an antigen containing cardiolipin, lecithin, and cholesterol. For more than 50 years, the preparation of natural cardiolipin and lecithin for this test has been based on the Pangborn method which involves isolating and purifying these components from beef hearts. This process is tedious and time-consuming and results in a variable purity range. In our studies, we found that a VDRL antigen using synthetic tetramyristoyl cardiolipin and synthetic 1-palmitoyl-2-oleoyl-
sn
-glycero-3-phosphocholine (lecithin) was as specific in detecting syphilis as a VDRL antigen made with natural components. In 85% of the cases, we obtained an endpoint titer of 1/2 or 1 dilution more than a titer obtained with a VDRL antigen made with natural components. The use of these pure synthetic compounds, with a purity of 99%, would offer advantages in the standardization and stability of the VDRL antigen. Because this antigen is the basic ingredient in the preparation of nontreponemal reagents such as the rapid plasma reagin, toluidine red unheated serum test, and the unheated serum reagin, the use of this synthetic VDRL antigen should also increase the reactivity of these reagents.
Evaluation of Antibodies against a Rubella Virus Neutralizing Domain for Determination of Immune Status
Authors: Cordoba Patricia , Lanoel Alejandra , Grutadauria Sergio , Zapata Marta 
Published: July 2002

ABSTRACT

The protective immune responses against rubella virus (RV) are related to its neutralizing epitopes, an issue that is important to consider when assessing the immune status of patients with remote infection. In the present paper, we compare the antibodies detected by a synthetic-peptide-based enzyme immunoassay (EIA) with antibodies detected by the traditional technique of hemagglutination inhibition (HIA) in patients with remote RV infection. The synthetic peptide used as an antigen (SP15) represents a neutralizing epitope that corresponds to amino acids 208 to 239 of the E1 glycoprotein. The SP15-EIA was developed, all variables that affected the assay were standardized, and the test was validated using reference sera. Serum samples (
n
= 129) from patients with remote RV infection were tested by HIA and SP15-EIA. Discrepant sera were assayed by MEIA (IMX/Abbot). The comparison between HIA and SP15-EIA, taking HIA as the standard methodology for determining immune status, showed that SP15-EIA is very specific and sensitive for detecting protecting antibodies (specificity, 100%; sensitivity, 98.20%). This study demonstrates that antibodies against the neutralizing domain represented by SP15 would be important in the memory response after natural infection and may be a good tool in the determination of the true immune status of patients with remote infection with regard to RV.
Role of Genomic Typing in Taxonomy, Evolutionary Genetics, and Microbial Epidemiology
Authors: van Belkum Alex , Struelens Marc , de Visser Arjan , Verbrugh Henri , Tibayrenc Michel 
Published: July 2002

SUMMARY
Currently, genetic typing of microorganisms is widely used in several major fields of microbiological research. Taxonomy, research aimed at elucidation of evolutionary dynamics or phylogenetic relationships, population genetics of microorganisms, and microbial epidemiology all rely on genetic typing data for discrimination between genotypes. Apart from being an essential component of these fundamental sciences, microbial typing clearly affects several areas of applied microbiogical research. The epidemiological investigation of outbreaks of infectious diseases and the measurement of genetic diversity in relation to relevant biological properties such as pathogenicity, drug resistance, and biodegradation capacities are obvious examples. The diversity among nucleic acid molecules provides the basic information for all fields described above. However, researchers in various disciplines tend to use different vocabularies, a wide variety of different experimental methods to monitor genetic variation, and sometimes widely differing modes of data processing and interpretation. The aim of the present review is to summarize the technological and fundamental concepts used in microbial taxonomy, evolutionary genetics, and epidemiology. Information on the nomenclature used in the different fields of research is provided, descriptions of the diverse genetic typing procedures are presented, and examples of both conceptual and technological research developments for Escherichia coli are included. Recommendations for unification of the different fields through standardization of laboratory techniques are made.
Phagosome Acidification Has Opposite Effects on Intracellular Survival ofBordetella pertussisandB. bronchiseptica
Authors: Schneider Boris , Gross Roy , Haas Albert 
Published: July 2002

ABSTRACTBordetella pertussisis readily killed after uptake by professional phagocytes, whereas its close relativeBordetella bronchisepticais not and can persist intracellularly for days. Phagocytosis of members of either species by a mouse macrophage cell line results in transport of the bacteria to a phagosomal compartment positive for the lysosome-associated membrane protein 1, the protease cathepsin D, and the late endosomal vacuolar proton-pumping ATPase but negative for the early endosome antigen 1 and the early endosomal transferrin receptor. In addition, we demonstrate thatBordetella-containing phagosomes rapidly acidify to pH 4.5 to 5.0. Taken together, these data demonstrate thatBordetella-containing phagosomes rapidly mature to an acidic late endosomal/lysosomal compartment. Following up on this observation, we determined thatB. pertussisdoes not survive in bacterial growth media adjusted to a pH of 4.5, whereas this pH has only minor effects on the growth ofB. bronchiseptica. Raising the intracellular pH in infected macrophages by the addition of bafilomycin A1, ammonium chloride, or monensin increases the survival of acid-sensitiveB. pertussisbut, surprisingly, decreases that of acid-tolerantB. bronchiseptica. In summary, we hypothesize that the differential survival ofB. pertussisandB. bronchisepticain macrophages is, at least in part, due to the differences in their acid tolerance.