bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protocol for Specific Isolation of Virulent Strains of
Vibrio vulnificus
Serovar E (Biotype 2) from Environmental Samples
Authors: Sanjuán Eva , Amaro Carmen 
Published: December 2004

ABSTRACT

The eel pathogen
Vibrio vulnificus
biotype 2 comprises at least three serovars, with serovar E being the only one involved in both epizootics of eel vibriosis and sporadic cases of human infections. The virulent strains of this serovar (VSE) have only been recovered from clinical (mainly eel tissue) sources. The main objective of this work was to design and validate a new protocol for VSE-specific isolation from environmental samples. The key element of the new protocol is the broth used for the first step (saline eel serum broth [SEB]), which contains eel serum as a nutritive and selective component. This approach takes advantage of the ability of VSE cells to grow in eel serum and thus to separate themselves from the pool of competitors. The growth yield in SEB after 8 h of incubation was 1,000 times higher for VSE strains than for their putative competitors (including biotype 1 strains of the species). The selective and differential agar
Vibrio vulnificus
medium (VVM) was selected from five selective media for the second step because it gave the highest plating efficiency not only for the VSE group but also for other
V. vulnificus
groups, including biotype 3. The entire protocol was validated by field studies, with alkaline peptone water plus VVM as a control.
V. vulnificus
was isolated by both protocols, but serovar E was only recovered by the new method described here. All selected serovar E isolates were identified as VSE since they were virulent for both eels and iron-overloaded mice and resisted the bactericidal action of eel and iron-overloaded human sera. In conclusion, this new protocol is a suitable method for the isolation of VSE strains from environmental samples and is recommended for epidemiological studies of the pathogenic serovar E.
Synthesis of Novel Lipids in
Saccharomyces cerevisiae
by Heterologous Expression of an Unspecific Bacterial Acyltransferase
Authors: Kalscheuer Rainer , Luftmann Heinrich , Steinbüchel Alexander 
Published: December 2004

ABSTRACT

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium
Acinetobacter calcoaceticus
ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis.
Saccharomyces cerevisiae
accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain
S. cerevisiae
H1246, the disruption of
DGA1
,
LRO1
,
ARE1
, and
ARE2
leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from
A. calcoaceticus
ADP1 in
S. cerevisiae
H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in
Escherichia coli
XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in
S. cerevisiae
H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from
A. calcoaceticus
ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.
Allelic Diversity and Population Structure inOenococcus oenias Determined from Sequence Analysis of Housekeeping Genes
Authors: de las Rivas Blanca , Marcobal Ángela , Muñoz Rosario 
Published: December 2004

ABSTRACTOenococcus oeniis the organism of choice for promoting malolactic fermentation in wine. The population biology ofO. oeniis poorly understood and remains unclear. For a better understanding of the mode of genetic variation within this species, we investigated by using multilocus sequence typing (MLST) with thegyrB,pgm,ddl,recP, andmleAgenes the genetic diversity and genetic relationships among 18O. oenistrains isolated in various years from wines of the United States, France, Germany, Spain, and Italy. These strains have also been characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rRNA gene intergenic spacer region (ISR). Ribotyping grouped the strains into two groups; however, the RFLP analysis of the ISRs showed no differences in the strains analyzed. In contrast, MLST in oenococci had a good discriminatory ability, and we have found a higher genetic diversity than indicated by ribotyping analysis. All sequence types were represented by a single strain, and all the strains could be distinguished from each other because they had unique combinations of alleles. Strains assumed to be identical showed the same sequence type. Phylogenetic analyses indicated a panmictic population structure inO. oeni. Sequences were analyzed for evidence of recombination by split decomposition analysis and analysis of clustered polymorphisms. All results indicated that recombination plays a major role in creating the genetic heterogeneity ofO. oeni. A low standardized index of association value indicated that theO. oenigenes analyzed are close to linkage equilibrium. This study constitutes the first step in the development of an MLST method forO. oeniand the first example of the application of MLST to a nonpathogenic food production bacteria.
Characterization of a Novel Amylolytic Enzyme Encoded by a Gene from a Soil-Derived Metagenomic Library
Authors: Yun Jiae , Kang Seowon , Park Sulhee , Yoon Hyunjin , Kim Myo-Jeong , Heu Sunggi , Ryu Sangyeol 
Published: December 2004

ABSTRACT

It has been estimated that less than 1% of the microorganisms in nature can be cultivated by conventional techniques. Thus, the classical approach of isolating enzymes from pure cultures allows the analysis of only a subset of the total naturally occurring microbiota in environmental samples enriched in microorganisms. To isolate useful microbial enzymes from uncultured soil microorganisms, a metagenome was isolated from soil samples, and a metagenomic library was constructed by using the pUC19 vector. The library was screened for amylase activity, and one clone from among approximately 30,000 recombinant
Escherichia coli
clones showed amylase activity. Sequencing of the clone revealed a novel amylolytic enzyme expressed from a novel gene. The putative amylase gene (
amyM
) was overexpressed and purified for characterization. Optimal conditions for the enzyme activity of the AmyM protein were 42°C and pH 9.0; Ca
2+
stabilized the activity. The amylase hydrolyzed soluble starch and cyclodextrins to produce high levels of maltose and hydrolyzed pullulan to panose. The enzyme showed a high transglycosylation activity, making α-(1, 4) linkages exclusively. The hydrolysis and transglycosylation properties of AmyM suggest that it has novel characteristics and can be regarded as an intermediate type of maltogenic amylase, α-amylase, and 4-α-glucanotransferase.
Molecular and Genetic Characterization of Propionicin F, a Bacteriocin fromPropionibacterium freudenreichii
Authors: Brede Dag Anders , Faye Therese , Johnsborg Ola , Ødegård Inger , Nes Ingolf F. , Holo Helge 
Published: December 2004

ABSTRACTThis work describes the purification and characterization of propionicin F, the first bacteriocin isolated fromPropionibacterium freudenreichii. The bacteriocin has a bactericidal activity and is only active against strains ofP. freudenreichii. Propionicin F appears to be formed through a processing pathway new to bacteriocins. The mass of the purified bacteriocin was determined by mass spectrometry, and the N-terminal amino acid sequence was determined by Edman degradation. Sequencing ofpcfA, the bacteriocin structural gene, revealed that propionicin F corresponds to a 43-amino-acid peptide in the central part of a 255-amino-acid open reading frame, suggesting that mature propionicin F is excised from the probacteriocin by N- and C-terminal proteolytic modifications. DNA sequencing and Northern blot hybridizations revealed thatpcfAis cotranscribed with genes encoding a putative proline peptidase and a protein from the radicalS-adenosylmethionine family. A gene encoding an ABC transporter was also identified in close proximity to the bacteriocin structural gene. The potential role of these genes in propionicin F maturation and secretion is discussed.
In Vivo
31
P Nuclear Magnetic Resonance Investigation of Tellurite Toxicity in
Escherichia coli
Authors: Lohmeier-Vogel Elke M. , Ung Shiela , Turner Raymond J. 
Published: December 2004

ABSTRACT

Here we compare the physiological state of
Escherichia coli
exposed to tellurite or selenite by using the noninvasive technique of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. We studied glucose-fed
Escherichia coli
HB101 cells containing either a normal pUC8 plasmid with no tellurite resistance determinants present or the pTWT100 plasmid which contains the resistance determinants
tehAB
. No differences could be observed in intracellular ATP levels, the presence or absence of a transmembrane pH gradient, or the levels of phosphorylated glycolytic intermediates when resistant cells were studied by
31
P NMR in the presence or absence of tellurite. In the sensitive strain, we observed that the transmembrane pH gradient was dissipated and intracellular ATP levels were rapidly depleted upon exposure to tellurite. Only the level of phosphorylated glycolytic intermediates remained the same as observed with resistant cells. Upon exposure to selenite, no differences could be observed by
31
P NMR between resistant and sensitive strains, suggesting that the routes for selenite and tellurite reduction within the cells differ significantly, since only tellurite is able to collapse the transmembrane pH gradient and lower ATP levels in sensitive cells. The presence of the resistance determinant
tehAB
, by an as yet unidentified detoxification event, protects the cells from uncoupling by tellurite.
Milk Contamination and Resistance to Processing Conditions Determine the Fate of
Lactococcus lactis
Bacteriophages in Dairies
Authors: Madera Carmen , Monjardín Cristina , Suárez Juan E. 
Published: December 2004

ABSTRACT
Milk contamination by phages, the susceptibility of the phages to pasteurization, and the high levels of resistance to phage infection of starter strains condition the evolution dynamics of phage populations in dairy environments. Approximately 10% (83 of 900) of raw milk samples contained phages of the quasi-species c2 (72%), 936 (24%), and P335 (4%). However, 936 phages were isolated from 20 of 24 (85%) whey samples, while c2 was detected in only one (4%) of these samples. This switch may have been due to the higher susceptibility of c2 to pasteurization (936-like phages were found to be approximately 35 times more resistant than c2 strains to treatment of contaminated milk in a plate heat exchanger at 72°C for 15 s). The restriction patterns of 936-like phages isolated from milk and whey were different, indicating that survival to pasteurization does not result in direct contamination of the dairy environment. The main alternative source of phages (commercial bacterial starters) does not appear to significantly contribute to phage contamination. Twenty-four strains isolated from nine starter formulations were generally resistant to phage infection, and very small progeny were generated upon induction of the lytic cycle of resident prophages. Thus, we postulate that a continuous supply of contaminated milk, followed by pasteurization, creates a factory environment rich in diverse 936 phage strains. This equilibrium would be broken if a particular starter strain turned out to be susceptible to infection by one of these 936-like phages, which, as a consequence, became prevalent.
Integral Strategy for Evaluation of Fecal Indicator Performance in Bird-Influenced Saline Inland Waters
Authors: Kirschner Alexander K. T. , Zechmeister Thomas C. , Kavka Gerhard G. , Beiwl Christian , Herzig Alois , Mach Robert L. , Farnleitner Andreas H. 
Published: December 2004

ABSTRACT

Wild birds are an important nonpoint source of fecal contamination of surface waters, but their contribution to fecal pollution is mostly difficult to estimate. Thus, to evaluate the relation between feces production and input of fecal indicator bacteria (FIB) into aquatic environments by wild waterfowl, we introduced a new holistic approach for evaluating the performance of FIB in six shallow saline habitats. For this, we monitored bird abundance, fecal pellet production, and the abundance of FIB concomitantly with a set of environmental variables over a 9-month period. For estimating fecal pellet production, a new protocol of fecal pellet counting was introduced, which was called fecal taxation (FTX). We could show that, over the whole range of investigated habitats, bird abundance, FTX values, and FIB abundance were highly significantly correlated and could demonstrate the good applicability of the FTX as a meaningful surrogate parameter for recent bird abundances and fecal contamination by birds in shallow aquatic ecosystems. Presumptive enterococci (ENT) were an excellent surrogate parameter of recent fecal contamination in these saline environments for samples collected at biweekly to monthly sampling intervals while presumptive
Escherichia coli
and fecal coliforms (FC) were often undetectable. Significant negative correlations with salinity indicated that
E. coli
and FC survival was hampered by osmotic stress. Statistical analyses further revealed that fecal pollution-associated parameters represented one system component independent from other environmental variables and that, besides feces production, rainfall, total suspended solids (direct), and trophy (indirect) had significant positive effects on ENT concentrations. Our holistic approach of linking bird abundance, feces production, and FIB detection with environmental variables may serve as a powerful model for application to other aquatic ecosystems.
Detection of Pathogenic
Vibrio
spp. in Shellfish by Using Multiplex PCR and DNA Microarrays
Authors: Panicker Gitika , Call Douglas R. , Krug Melissa J. , Bej Asim K. 
Published: December 2004

ABSTRACT

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with
vvh
and
viuB
for
Vibrio vulnificus
, with
ompU
,
toxR
,
tcpI
, and
hlyA
for
V. cholerae
, and with
tlh
,
tdh
,
trh
, and open reading frame 8 for
V. parahaemolyticus
helped to ensure that total and pathogenic strains, including subtypes of the three
Vibrio
spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10
2
to 10
3
CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of
V. vulnificus
(100%) and
V. parahaemolyticus
(83%). However,
V. cholerae
was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.
Construction and Characterization of a Highly Efficient
Francisella
Shuttle Plasmid
Authors: Maier Tamara M. , Havig Andrea , Casey Monika , Nano Francis E. , Frank Dara W. , Zahrt Thomas C. 
Published: December 2004

ABSTRACT

Francisella tularensis
is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in
Francisella
spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both
Francisella
and
Escherichia coli
. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 × 10
7
CFU μg of DNA
−1
in
F. tularensis
LVS,
Francisella novicida
U112, and
E. coli
DH5α. Furthermore, this plasmid was stably maintained in
F. tularensis
LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly,
F. tularensis
LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in
F. tularensis
LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a
Francisella
shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of
Francisella
in macrophages. This new tool should significantly enhance genetic manipulation and characterization of
F. tularensis
and other
Francisella
biotypes.
Genotypes of
Cryptosporidium
Species Infecting Fur-Bearing Mammals Differ from Those of Species Infecting Humans
Authors: Zhou Ling , Fayer Ronald , Trout James M. , Ryan Una M. , Schaefer Frank W. , Xiao Lihua 
Published: December 2004

ABSTRACT

Of 471 specimens examined from foxes, raccoons, muskrats, otters, and beavers living in wetlands adjacent to the Chesapeake Bay, 36 were positive for five types of
Cryptosporidium
, including the
C. canis
dog and fox genotypes,
Cryptosporidium
muskrat genotypes I and II, and
Cryptosporidium
skunk genotype. Thus, fur-bearing mammals in watersheds excreted host-adapted
Cryptosporidium
oocysts that are not known to be of significant public health importance.
The battering of informed consent
Authors: Kottow M 
Published: December 2004

Autonomy has been hailed as the foremost principle of bioethics, and yet patients’ decisions and research subjects’ voluntary participation are being subjected to frequent restrictions. It has been argued that patient care is best served by a limited form of paternalism because the doctor is better qualified to take critical decisions than the patient, who is distracted by illness. The revival of paternalism is unwarranted on two grounds: firstly, because prejudging that the sick are not fully autonomous is a biased and unsubstantial view; secondly, because the technical knowledge of healthcare professionals does not include the ethical qualifications and prerogative to decide for others.Clinical research settings are even more prone to erode subjects’ autonomy than clinical settings because of the tendency and temptation to resort to such practices as shading the truth when consent to participation is sought, or waiving consent altogether when research is done in emergency settings.Instead of supporting such dubious practices with unconvincing arguments, it would seem to be the task of bioethics to insist on reinforcing autonomy.
Willingness to donate: an interview study before liver transplantation
Authors: Walter M , Papachristou C , Danzer G , Klapp B F , Frommer J 
Published: December 2004

Objectives:The introduction of the living donation in organ transplantation introduces important new psychological conflicts and ethical questions in the transplantation process. Operation related risks, as well as dependencies in the family structure, generate considerable pressure on potential donors. The aim of the study was to reconstruct the determinants of willingness to donate before transplantation.Methods:Evaluation of 20 taped and transcribed interviews oriented to current approaches in qualitative interview research. The approach used is based on grounded theory, qualitative content analysis, and the concept of the ideal type.Results:Before surgery, “openly motivated” donors push for an operation, leaving no room for ambivalence in the evaluation process. They idealise the relationship with the recipient, and link their donation with the individual—partly in subconscious expectations and wishes. In contrast, “openly ambivalent” donors formulate their anxieties and express arguments against donation.Conclusions:Statements that claim ambivalence towards donation or utterance of arguments against donation indicate earlier coercion. Before transplantation, potential donors should have the opportunity to discuss their emotional situation to help their decision making process.
FORCE GENERATION DUE TO FLUCTUATIONS OF MEDIA AND BOUNDARIES
Authors: GOLESTANIAN RAMIN 
Published: December 2004

In a fluctuating medium, whether of quantum, thermal, or non-thermal origins, an interaction is induced between external objects that modify the fluctuations. These interactions can appear in a vast variety of systems, leading to a plethora of interesting phenomena. Notable examples of these include:1. like-charge attraction in the presence of multivalent counterions;2. Ludwig–Soret effect in charged colloids;3. mass renormalization of moving defects in a phononic background and moving metallic objects in electromagnetic quantum vacuum;4. dissipation due to motion-induced radiation.Another related class of problems corresponds to stirring the media by dynamic deformations of the embedded bodies and benefiting from the back-reaction of the stirred media for force generation, such as force generation in swimming. The fluctuation-induced forces are statistical in nature, and this could make their measurements very difficult, because the actual value of the force might deviate most of the time from the predicted average value.