bluuebunny/crossref_metadata_2025 huggingface dataset:
Mental health and alcohol and other drug training for emergency department workers: One solution to help manage increasing demand
Authors: King Diane L , Kalucy Ross S , De Crespigny Charlotte F , Stuhlmiller Cynthia M , Thomas Lyndall J
Published: April 2004

Objective:  To evaluate a training course for ED staff aiming to improve knowledge and skills in working with mental health and drug/alcohol patients attending EDs.

Methods:  Pre‐ and postcourse questionnaires assessed attitudes and self‐ratings of confidence, knowledge and skills in working with these patients. Follow‐up interviews assessed if new skills or approaches to patient management had been integrated into daily ED practice.

Results:  Little change was observed in the course participants’ attitudes, although reported attitudes were generally appropriate. Self‐ratings of confidence in skills and knowledge showed a significant improvement on all questions following the course. Responses to the follow‐up interviews suggest course information has been retained and integrated into practice, especially in conducting triage and other assessments and taking more time to talk to patients.

Conclusion:  The course has led to staff feeling more confident and competent to help mental health or drug/alcohol patients who attend the ED.
Congenital diaphragmatic hernia masquerading as pneumonia
Authors: Ng Chung‐por , Lo Chi‐biu , Chung Chin‐hung
Published: April 2004

Patients with congenital diaphragmatic hernia usually present in the immediate neonatal period with respiratory distress. However, presentation beyond the neonatal period has sporadically been reported. We report a case of a 4‐year‐old girl who presented with a 1 month history of chronic cough and low‐grade fever, with no response to multiple antibiotics. Initially, both clinical and radiological findings suggested inflammatory chest disease. Fortunately, after performing ultrasound, suspicion was raised and she was later diagnosed to have left congenital diaphragmatic hernia of Bochdalek type. Although rare, this entity should form one of the differential diagnoses of unresolving pneumonia in children.
Postcoital haemoperitoneum: A cause for shock
Authors: Goswami Anu , S Raghavan Subrahmanian , Ghotekar Lekharaj H
Published: April 2004

Postcoital haemoperitoneum rarely occurs without evident vaginal injury. A 21‐year‐old second gravida woman presented to the ED in shock with a history of 8 weeks amenorrhoea and abdominal pain of 20 h duration. The ultrasound examination revealed a live intrauterine pregnancy and fluid in peritoneal cavity. There was a history of coitus 2 hours prior to the onset of pain. At laparotomy, more than 2 L of free blood was found in the peritoneal cavity. A small bleeding peritoneal vessel in pouch of Douglas was identified and ligated.
An unusual knee injury: Isolated tibiofibular dislocation
Authors: Petter Alastair , Davidson Jennifer
Published: April 2004

Isolated proximal tibiofibular dislocation is an extremely rare condition with potentially serious complications if missed. The mechanism of injury, clinical presentation and treatment options are discussed. This injury was more common in previous centuries, when horse riding was the preferred method of transport. The young man we present was involved in a pastime that is rising in popularity and it is therefore likely that this injury will be a more common presentation to EDs.
Relative Ability of Orally AdministeredLactobacillus murinusTo Predominate and Persist in the Porcine Gastrointestinal Tract
Authors: Gardiner Gillian E. , Casey Pat G. , Casey Garrett , Lynch P. Brendan , Lawlor Peadar G. , Hill Colin , Fitzgerald Gerald F. , Stanton Catherine , Ross R. Paul
Published: April 2004

Five porcine-derivedLactobacillusorPediococcusisolates administered to pigs (n= 4), either singly or as a combination at ∼1010CFU per day varied with respect to intestinal survival and persistence. TwoLactobacillus murinusstrains survived best and were excreted at ∼107to 108CFU/g of feces. In contrast,Pediococcus pentosaceusDPC6006 had the lowest fecal count at ∼105CFU/g and was excreted at a significantly lower level than bothL. murinusstrains. FecalL. murinusDPC6003 counts were also significantly higher than bothLactobacillus salivariusDPC6005 andLactobacillus pentosusDPC6004 (∼106CFU/g). TheL. murinusstrains persisted for at least 9 days postadministration in both the feces and the cecum. Animals fed a combination of all five strains excreted ∼107CFU of the administered strains/g, withL. murinuspredominating, as determined by randomly amplified polymorphic DNA PCR. Postadministration, variation was observed between animals fed the strain combination, but in general,L. murinusDPC6002 and DPC6003 andL. pentosusDPC6004 predominated in the feces and the cecum whileP. pentosaceusDPC6006 was detected only in the cecum. Fifteen days after the start of culture administration, mean fecalEnterobacteriaceaecounts were significantly lower in some of the treatment groups. In addition, when mean preadministration counts were compared with those obtained after 21 days of culture administration,Enterobacteriaceaecounts were reduced by ∼87 to 98% in pigs fedL. salivariusDPC6005,P. pentosaceusDPC6006,L. pentosusDPC6004, and the culture mix. In conclusion, the porcine intestinal isolates have potential as probiotic feed additives for pigs, with differences in strain performance highlighting the advantages of using culture combinations.
Simultaneous Degradation of Atrazine and Phenol by
Pseudomonas
sp. Strain ADP: Effects of Toxicity and Adaptation
Authors: Neumann Grit , Teras Riho , Monson Liis , Kivisaar Maia , Schauer Frieder , Heipieper Hermann J.
Published: April 2004

The strain
Pseudomonas
sp. strain ADP is able to degrade atrazine as a sole nitrogen source and therefore needs a single source for both carbon and energy for growth. In addition to the typical C source for
Pseudomonas
, Na
2
-succinate, the strain can also grow with phenol as a carbon source. Phenol is oxidized to catechol by a multicomponent phenol hydroxylase. Catechol is degraded via the
ortho
pathway using catechol 1,2-dioxygenase. It was possible to stimulate the strain in order to degrade very high concentrations of phenol (1,000 mg/liter) and atrazine (150 mg/liter) simultaneously. With cyanuric acid, the major intermediate of atrazine degradation, as an N source, both the growth rate and the phenol degradation rate were similar to those measured with ammonia as an N source. With atrazine as an N source, the growth rate and the phenol degradation rate were reduced to ∼35% of those obtained for cyanuric acid. This presents clear evidence that although the first three enzymes of the atrazine degradation pathway are constitutively present, either these enzymes or the uptake of atrazine is the bottleneck that diminishes the growth rate of
Pseudomonas
sp. strain ADP with atrazine as an N source. Whereas atrazine and cyanuric acid showed no significant toxic effect on the cells, phenol reduces growth and activates or induces typical membrane-adaptive responses known for the genus
Pseudomonas
. Therefore
Pseudomonas
sp. strain ADP is an ideal bacterium for the investigation of the regulatory interactions among several catabolic genes and stress response mechanisms during the simultaneous degradation of toxic phenolic compounds and a xenobiotic N source such as atrazine.
Fum3p, a 2-Ketoglutarate-Dependent Dioxygenase Required for C-5 Hydroxylation of Fumonisins in
Fusarium verticillioides
Authors: Ding Yousong , Bojja Ravi S. , Du Liangcheng
Published: April 2004

Fumonisins are polyketide-derived mycotoxins produced by several agriculturally important
Fusarium
species. The B series fumonisins, FB
1
, FB
2
, FB
3
, and FB
4
, are fumonisins produced by wild-type
Fusarium verticillioides
strains, differing in the number and location of hydroxyl groups attached to the carbon backbone. We characterized the protein encoded by
FUM3
, a gene in the fumonisin biosynthetic gene cluster. The 33-kDa
FUM3
protein (Fum3p) was heterologously expressed and purified from
Saccharomyces cerevisiae.
Yeast cells expressing the Fum3p converted FB
3
to FB
1
, indicating that Fum3p catalyzes the C-5 hydroxylation of fumonisins. This result was verified by assaying the activity of Fum3p purified from yeast cells. The C-5 hydroxylase activity of purified Fum3p required 2-ketoglutarate, Fe
2+
, ascorbic acid, and catalase, all of which are required for 2-ketoglutarate-dependent dioxygenases. The protein also contains two His motifs that are highly conserved in this family of dioxygenases. Thus, Fum3p is a 2-ketoglutarate-dependent dioxygenase required for the addition of the C-5 hydroxyl group of fumonisins.
Occurrence and Phylogenetic Diversity ofSphingomonasStrains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons
Authors: Leys Natalie M. E. J. , Ryngaert Annemie , Bastiaens Leen , Verstraete Willy , Top Eva M. , Springael Dirk
Published: April 2004

Bacterial strains of the genusSphingomonasare often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection ofSphingomonasstrains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting theSphingomonas16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assessSphingomonasdiversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the familySphingomonadaceae. Single-band DGGE profiles were obtained for mostSphingomonasstrains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 104CFU g of soil−1. The analysis ofSphingomonaspopulation structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowestSphingomonasdiversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA geneSphingomonassequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designedSphingomonas-specific PCR-DGGE detection technique successfully analyzed theSphingomonascommunities from polluted soils at the species level and revealed differentSphingomonasmembers not previously detected by culture-dependent detection techniques.
Phenotypic and Genotypic Characteristics and Epidemiological Significance ofctx+Strains ofVibrio choleraeIsolated from Seafood in Malaysia
Authors: Chen Chien-Hsien , Shimada Toshio , Elhadi Nasreldin , Radu Son , Nishibuchi Mitsuaki
Published: April 2004

Of 97 strains ofVibrio choleraeisolated from various seafoods in Malaysia in 1998 and 1999, 20 strains carried thectxgene and produced cholera toxin. Fourteen, one, and five of these toxigenic strains belonged to the O139, O1 Ogawa, and rough serotypes, respectively. The rough strains had therfbgene of the O1 serotype. The toxigenic strains varied in their biochemical characteristics, the amount of cholera toxin produced, their antibiograms, and the presence or absence of the pTLC plasmid sequence. DNA fingerprinting analysis by arbitrarily primed PCR, ribotyping, and a pulsed-field gel electrophoresis method classified the toxigenic strains into 3, 7, and 10 types, respectively. The relatedness of these toxigenic strains to clinical strains isolated in other countries and from international travelers was examined by using a dendrogram constructed from the pulsed-field gel electrophoresis profiles. The results of the examination of the antibiogram and the possession of the toxin-linked cryptic plasmid were consistent with the dendrogram-based relatedness: the O139 strains isolated from Malaysian seafoods could be separated into two groups that appear to have been introduced from the Bengal area independently. The rough strains of Malaysian seafood origin formed one group and belonged to a cluster unique to the Thailand-Malaysia-Laos region, and this group may have persisted in this area for a long period. The single O1 Ogawa strain detected in Malaysian seafood appears to have an origin and route of introduction different from those of the O139 and the rough strains.
Mycobacteria in Water and Loose Deposits of Drinking Water Distribution Systems in Finland
Authors: Torvinen Eila , Suomalainen Sini , Lehtola Markku J. , Miettinen Ilkka T. , Zacheus Outi , Paulin Lars , Katila Marja-Leena , Martikainen Pertti J.
Published: April 2004

Drinking water distribution systems were analyzed for viable counts of mycobacteria by sampling water from waterworks and in different parts of the systems. In addition, loose deposits collected during mechanical cleaning of the main pipelines were similarly analyzed. The study covered 16 systems at eight localities in Finland. In an experimental study, mycobacterial colonization of biofilms on polyvinyl chloride tubes in a system was studied. The isolation frequency of mycobacteria increased from 35% at the waterworks to 80% in the system, and the number of mycobacteria in the positive samples increased from 15 to 140 CFU/liter, respectively. Mycobacteria were isolated from all 11 deposits with an accumulation time of tens of years and from all 4 deposits which had accumulated during a 1-year follow-up time. The numbers of mycobacteria were high in both old and young deposits (medians, 1.8 × 10
5
and 3.9 × 10
5
CFU/g [dry weight], respectively). Both water and deposit samples yielded the highest numbers of mycobacteria in the systems using surface water and applying ozonation as an intermediate treatment or posttreatment. The number and growth of mycobacteria in system waters correlated strongly with the concentration of assimilable organic carbon in the water leaving the waterworks. The densities of mycobacteria in the developing biofilms were highest at the distal sites of the systems. Over 90% of the mycobacteria isolated from water and deposits belonged to
Mycobacterium lentiflavum
,
M. tusciae
,
M. gordonae
, and a previously unclassified group of mycobacteria. Our results indicate that drinking water systems may be a source for recently discovered new mycobacterial species.
Protective Effect of Sucrose and Sodium Chloride for
Lactococcus lactis
during Sublethal and Lethal High-Pressure Treatments
Authors: Molina-Höppner Adriana , Doster Wolfgang , Vogel Rudi F. , Gänzle Michael G.
Published: April 2004

The bactericidal effect of hydrostatic pressure is reduced when bacteria are suspended in media with high osmolarity. To elucidate mechanisms responsible for the baroprotective effect of ionic and nonionic solutes,
Lactococcus lactis
was treated with pressures ranging from 200 to 600 MPa in a low-osmolarity buffer or with buffer containing 0.5 M sucrose or 4 M NaCl. Pressure-treated cells were characterized in order to determine viability, the transmembrane difference in pH (ΔpH), and multiple-drug-resistance (MDR) transport activity. Furthermore, pressure effects on the intracellular pH and the fluidity of the membrane were determined during pressure treatment. In the presence of external sucrose and NaCl, high intracellular levels of sucrose and lactose, respectively, were accumulated by
L. lactis
; 4 M NaCl and, to a lesser extent, 0.5 M sucrose provided protection against pressure-induced cell death. The transmembrane ΔpH was reversibly dissipated during pressure treatment in any buffer system. Sucrose but not NaCl prevented the irreversible inactivation of enzymes involved in pH homeostasis and MDR transport activity. In the presence 0.5 M sucrose or 4 M NaCl, the fluidity of the cytoplasmic membrane was maintained even at low temperatures and high pressure. These results indicate that disaccharides protect microorganisms against pressure-induced inactivation of vital cellular components. The protective effect of ionic solutes relies on the intracellular accumulation of compatible solutes as a response to the osmotic stress. Thus, ionic solutes provide only asymmetric protection, and baroprotection with ionic solutes requires higher concentrations of the osmolytes than of disaccharides.
Tri1
in
Fusarium graminearum
Encodes a P450 Oxygenase
Authors: McCormick S. P. , Harris L. J. , Alexander N. J. , Ouellet T. , Saparno A. , Allard S. , Desjardins A. E.
Published: April 2004

Gibberella zeae
(asexual state
Fusarium graminearum
) is a major causal agent of wheat head blight and maize ear rot in North America and is responsible for contamination of grain with deoxynivalenol and related trichothecene mycotoxins. To identify additional trichothecene biosynthetic genes, cDNA libraries were prepared from fungal cultures under trichothecene-inducing conditions in culture and in planta. A gene designated
LH1
that was highly expressed under these conditions exhibited only moderate (59%) similarity to known trichothecene biosynthetic cytochrome P450s. To determine the function of
LH1
, gene disruptants were produced and assessed for trichothecene production. Gene disruptants no longer produced 15-acetyldeoxynivalenol, which is oxygenated at carbon 7 (C-7) and C-8, but rather accumulated calonectrin and 3-deacetylcalonectrin, which are not oxygenated at either C-7 or C-8. These results indicate that gene
LH1
encodes a cytochrome P450 responsible for oxygenation at one or both of these positions. Despite the relatively low level of DNA and amino acid sequence similarity between the two genes,
LH1
from
G. zeae
is the probable homologue of
Tri1
, which encodes a cytochrome P450 required for C-8 oxygenation in
F. sporotrichioides
.
Use of Whole Cells ofPseudomonas aeruginosafor Synthesis of the Antioxidant Hydroxytyrosol via Conversion of Tyrosol
Authors: Allouche N. , Damak M. , Ellouz R. , Sayadi S.
Published: April 2004

For the first time, a soil bacterium, designatedPseudomonas aeruginosa, was isolated based on its ability to grow on tyrosol as a sole source of carbon and energy. During growth on tyrosol, this strain was capable of promoting the formation of a significant amount of hydroxytyrosol and trace quantities of parahydroxyphenyl acetic acid and 3,4-dihydroxyphenyl acetic acid. The products were confirmed by high-performance liquid chromatography and gas chromatography-mass spectrometry analyses. Using an optimized tyrosol concentration of 2 g liter−1, the maximal hydroxytyrosol yield (80%) was achieved after a 7-h reaction in a growth experiment. To enhance the formation of hydroxytyrosol and prevent its degradation, a resting-cell method usingP. aeruginosawas performed. The growth state of the culture utilized for biomass production, the carbon source on which the biomass was grown, the concentration of the biomass, and the amount of tyrosol that was treated were optimized. The optimal yield of hydroxytyrosol (96%) was obtained after a 7-h reaction using 4 g of tyrosol liter−1and 5 g of cells liter−1pregrown on tyrosol and harvested at the end of the exponential phase. This proposed procedure is an alternative approach to obtain hydroxytyrosol in an environmentally friendly way. In addition, the reaction is easy to perform and can be adapted to a bioreactor for industrial purposes.
Determination of Nitrogen-Fixing Phylotypes in
Lyngbya
sp. and
Microcoleus
chthonoplastes
Cyanobacterial Mats from Guerrero Negro, Baja California, Mexico
Authors: Omoregie Enoma O. , Crumbliss Lori L. , Bebout Brad M. , Zehr Jonathan P.
Published: April 2004

In many environments, biological nitrogen fixation can alleviate nitrogen limitation. The high rates of N
2
fixation often observed in cyanobacterial mats suggest that N
2
fixation may be an important source of N. In this study, organisms expressing
nifH
were identified in a
Lyngbya
sp.- and two
Microcoleus chthonoplastes
-dominated cyanobacterial mats. The pattern of nitrogenase activity was determined for the
Lyngbya
sp. mat and a
Microcoleus chthonoplastes
mat sampled directly in Guerrero Negro, Mexico. Their maximum rates were 23 and 15 μmol of C
2
H
4
m
−2
h
−1
, respectively. The second
Microcoleus
mat, which was maintained in a greenhouse facility, had a maximum rate of 40 μmol of C
2
H
4
m
−2
h
−1
. The overall diel pattern of nitrogenase activity in the three mats was similar, with the highest rates of activity occurring during the dark period. Analysis of
nifH
transcripts by reverse transcription-PCR revealed that several different organisms were expressing
nifH
during the dark period.
nifH
phylotypes recovered from these mats were similar to sequences from the unicellular cyanobacterial genera
Halothece
,
Myxosarcina
, and
Synechocystis,
the filamentous cyanobacterial genera
Plectonema
and
Phormidium
, and several bacterial
nifH
groups. The results of this study indicate that several different organisms, some of which were not previously known to fix nitrogen, are likely to be responsible for the observed dark-period nitrogenase activity in these cyanobacterial mats.
Impact of Consumption of Oligosaccharide-Containing Biscuits on the Fecal Microbiota of Humans
Authors: Tannock Gerald W. , Munro Karen , Bibiloni Rodrigo , Simon Mary A. , Hargreaves Patrick , Gopal Pramod , Harmsen Hermie , Welling Gjalt
Published: April 2004

Human subjects consumed biscuits containing either galacto-oligosaccharides or fructo-oligosaccharides in a double-blinded, crossover study. The impact of supplementing the diet with three biscuits per day on the fecal microbiota was evaluated by selective culture of particular bacterial groups, measurement of β-galactosidase activity, and nucleic acid-based analytical methods (PCR-denaturing gradient gel electrophoresis [PCR-DGGE] and fluorescent in situ hybridization). The composition of the bifidobacterial populations was monitored at the level of species (PCR-DGGE) and strains (pulsed-field gel electrophoresis of DNA digests), and representative cultures were tested quantitatively for their ability to use galacto-oligosaccharides. Technical improvements to DGGE analysis of the microbiota were made by the use of an internal standard that allowed valid comparisons of fragment staining intensities to be made between profiles, the use of S1 nuclease digestion to remove single-stranded DNA to facilitate cloning of DNA sequences cut from gels, and the extraction of RNA to be used as the template in reverse transcription-PCR-DGGE. RNA-DGGE profiles were markedly different (Dice's similarity coefficient, 58.5%) from those generated by DNA-DGGE. Neither the sizes of the bacterial populations nor the DNA-DGGE profiles of the microbiota were altered by the consumption of the biscuits, but the RNA-DGGE profiles were altered by the detection or increased staining intensity of 16S rRNA gene sequences originating from
Bifidobacterium adolescentis
and/or
Colinsella aerofaciens
in the feces of 11 of 15 subjects. β-Galactosidase activity was elevated in the feces of some subjects as a result of biscuit consumption. Subjects differed in the ability of the bifidobacterial strains harbored in their feces to use galacto-oligosaccharides. Our observations suggest that a phylogenetic approach to analysis of the gut ecosystem may not always be optimal and that a more physiological (biochemical) method might be more informative.
Fiber-Degrading Systems of Different Strains of the Genus
Fibrobacter
Authors: Béra-Maillet Christel , Ribot Yves , Forano Evelyne
Published: April 2004

The S85 type strain of
Fibrobacter succinogenes
, a major ruminal fibrolytic species, was isolated 49 years ago from a bovine rumen and has been used since then as a model for extensive studies. To assess the validity of this model, we compared the cellulase- and xylanase-degrading activities of several other
F. succinogenes
strains originating from different ruminants, including recently isolated strains, and looked for the presence of 10 glycoside hydrolase genes previously identified in S85. The NR9
F. intestinalis
type strain, representative of the second species of the genus, was also included in this study. DNA-DNA hybridization and 16S rRNA gene sequencing first classified the strains and provided the phylogenetic positions of isolates of both species. Cellulase and xylanase activity analyses revealed similar activity profiles for all
F. succinogenes
strains. However, the F
E
strain, phylogenetically close to S85, presented a poor xylanolytic system and weak specific activities. Furthermore, the HM2 strain, genetically distant from the other
F. succinogenes
isolates, displayed a larger cellulolytic profile on zymograms and higher cellulolytic specific activity.
F. intestinalis
NR9 presented a higher cellulolytic specific activity and a stronger extracellular xylanolytic activity. Almost all glycoside hydrolase genes studied were found in the
F. succinogenes
isolates by PCR, except in the HM2 strain, and few of them were detected in
F. intestinalis
NR9. As expected, the fibrolytic genes of strains of the genus
Fibrobacter
as well as the cellulase and xylanase activities are better conserved in closely related phylogenetic isolates.
Use of PCR-Restriction Fragment Length Polymorphism of
inlA
for Rapid Screening of
Listeria monocytogenes
Strains Deficient in the Ability To Invade Caco-2 Cells
Authors: Rousseaux S. , Olier M. , Lemaître J. P. , Piveteau P. , Guzzo J.
Published: April 2004

A PCR-restriction fragment length polymorphism (RFLP) method was developed in order to screen a large number of strains for impaired adhesion to epithelial cells due to expression of truncated InlA.
inlA
polymorphism was analyzed by PCR-RFLP in order to correlate
inlA
PCR-RFLP profiles and production of truncated InlA. Thirty-seven
Listeria monocytogenes
strains isolated from various sources, including five noninvasive and two invasive reference strains, were screened. Two endonucleases (AluI and Tsp509I) were used, and they generated five composite profiles. Thirteen
L. monocytogenes
isolates were characterized by two specific PCR-RFLP profiles similar to PCR-RFLP profiles of noninvasive reference strains previously described as strains that produce truncated InlA. Ten of the 13 isolates showed low abilities to invade human epithelial Caco-2 cells. However, 4 of the 13 isolates were able to invade Caco-2 cells like reference strains containing complete InlA. Sequencing of
inlA
and Western blot analysis confirmed that truncated InlA was expressed in the 10
L. monocytogenes
strains which were isolated from food. This PCR-RFLP method allowed us to identify 10 new strains expressing a truncated internalin. Based on the results obtained in this study, the PCR-RFLP method seems to be an interesting method for rapidly screening
L. monocytogenes
strains deficient in the ability to invade Caco-2 cells when a sizeable number of strains are studied.
Spatial and Temporal Distribution of Two Diazotrophic Bacteria in the Chesapeake Bay
Authors: Short Steven M. , Jenkins Bethany D. , Zehr Jonathan P.
Published: April 2004

The aim of this study was to initiate autecological studies on uncultivated natural populations of diazotrophic bacteria by examining the distribution of specific diazotrophs in the Chesapeake Bay. By use of quantitative PCR, the abundance of two
nifH
sequences (907h22 and 912h4) was quantified in water samples collected along a transect from the head to the mouth of the Chesapeake Bay during cruises in April and October 2001 and 2002. Standard curves for the quantitative PCR assays demonstrated that the relationship between gene copies and cycle threshold was linear and highly reproducible from 1 to 10
7
gene copies. The maximum number of 907h22 gene copies detected was approximately 140 ml
−1
and the maximum number of 912h4 gene copies detected was approximately 340 ml
−1
. Sequence 912h4 was most abundant at the mouth of the Chesapeake Bay, and in general, its abundance increased with increasing salinity, with the highest abundances observed in April 2002. Overall, the 907h22 phylotype was most abundant at the mid-bay station. Additionally, 907h22 was most abundant in the April samples from the mid-bay and mouth of the Chesapeake Bay. Despite the fact that the Chesapeake Bay is rarely nitrogen limited, our results show that individual nitrogen-fixing bacteria have distinct nonrandom spatial and seasonal distributions in the Chesapeake Bay and are either distributed by specific physical processes or adapted to different environmental niches.
Ethanol-Mediated Variations in Cellular Fatty Acid Composition and Protein Profiles of Two Genotypically Different Strains of
Escherichia coli
O157:H7
Authors: Chiou R. Y.-Y. , Phillips R. D. , Zhao P. , Doyle M. P. , Beuchat L. R.
Published: April 2004

Two strains of
Escherichia coli
O157:H7 were grown in tryptic soy broth (TSB, pH 7.1) supplemented with 0, 2.5, 5.0, 7.5, and 10% ethanol at 30°C for up to 54 h. Growth rates in TSB supplemented with 0, 2.5, and 5.0% ethanol decreased with an increase in ethanol concentration. Growth was not observed in TSB supplemented with 7.5 or 10% ethanol. The pH of TSB containing 5.0% ethanol decreased to 5.8 within 12 h and then increased to 7.0 at 54 h. The ethanol content in TSB supplemented with 2.5 or 5.0% ethanol did not change substantially during the first 36 h of incubation but decreased slightly thereafter, indicating utilization or degradation of ethanol by both strains. Glucose was depleted in TSB supplemented with 0, 2.5, or 5.0% ethanol within 12 h. Cells grown under ethanol stress contained a higher amount of fatty acids. With the exceptions of
cis
-oleic acid and nonadecanoic acid, larger amounts of fatty acid were present in stationary-phase cells of the two strains grown in TSB supplemented with 5.0% ethanol for 30 h than in cells grown in TSB without ethanol for 22 h. The
trans
-oleic acid content was 10-fold higher in the cells grown in TSB with 5.0% ethanol than those grown in TSB without ethanol. In contrast,
cis
-oleic acid was not detected in ethanol-stressed cells but was present at concentrations of 0.32 and 0.36 mg/g of cells of the two strains grown in TSB without ethanol. Protein content was higher in ethanol-stressed cells than in nonstressed cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles varied qualitatively as affected by the strain and the presence of ethanol in TSB. An ethanol-mediated protein (28 kDa) was observed in the ethanol-stressed cells but not in control cells. It is concluded that the two test strains of
E. coli
O157:H7 underwent phenotypic modifications in cellular fatty acid composition and protein profiles in response to ethanol stress. The potential for cross protection against subsequent stresses applied in food preservation technologies as a result of these changes is under investigation.
Phenol Hydroxylase and Toluene/
o
-Xylene Monooxygenase from
Pseudomonas stutzeri
OX1: Interplay between Two Enzymes
Authors: Cafaro Valeria , Izzo Viviana , Scognamiglio Roberta , Notomista Eugenio , Capasso Paola , Casbarra Annarita , Pucci Piero , Di Donato Alberto
Published: April 2004

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of
Pseudomonas stutzeri
OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/
o
-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.
Direct Quantification of
Campylobacter jejuni
and
Campylobacter lanienae
in Feces of Cattle by Real-Time Quantitative PCR
Authors: Inglis G. Douglas , Kalischuk Lisa D.
Published: April 2004

Campylobacter
species are fastidious to culture, and the ability to directly quantify biomass in microbiologically complex substrates using real-time quantitative (RTQ) PCR may enhance our understanding of their biology and facilitate the development of efficacious mitigation strategies. This study reports the use of nested RTQ-PCR to directly quantify
Campylobacter jejuni
and
Campylobacter lanienae
in cattle feces. For
C. jejuni
, the single-copy
mapA
gene was selected. For
C. lanienae
, the three-copy 16S rRNA gene was targeted. RTQ-PCR primers were tested alone or they were nested with species-specific primers, and amplification products were detected using the intercalating dye SYBR Green. Nesting did not increase the specificity or sensitivity of
C. jejuni
quantification, and the limit of quantification was 19 to 25 genome copies (≈3 × 10
3
CFU/g of feces). In contrast, nested RTQ-PCR was necessary to confer specificity on
C. lanienae
by targeting the 16S rRNA gene. The limit of quantification was 1.8 genome copies (≈250 CFU/g of feces), and there was no discernible difference between the two
C. lanienae
secondary primer sets evaluated. Detection and quantification of
C. jejuni
in naturally infested cattle feces by RTQ-PCR were comparable to the results of culture-based methods. In contrast, culturing did not detect
C. lanienae
in 6 of 10 fecal samples positive for the bacterium and substantially underestimated cell densities relative to nested RTQ-PCR. The results of this study illustrate that RTQ-PCR can be used to directly quantify campylobacters, including very fastidious species, in a microbiologically and chemically complex substrate. Furthermore, targeting of a multicopy universal gene provided highly sensitive quantification of
C. lanienae
, but nested RTQ-PCR was necessary to confer specificity. This method will facilitate subsequent studies to elucidate the impact of this group of bacteria within the gastrointestinal tracts of livestock and studies of the factors that influence colonization success and shedding.
Evaluation of Five Membrane Filtration Methods for Recovery of
Cryptosporidium
and
Giardia
Isolates from Water Samples
Authors: Wohlsen T. , Bates J. , Gray B. , Katouli M.
Published: April 2004

We evaluated the efficiency of five membrane filters for recovery of
Cryptosporidium parvum
oocysts and
Giardia lamblia
cysts. These filters included the Pall Life Sciences Envirochek (EC) standard filtration and Envirochek high-volume (EC-HV) membrane filters, the Millipore flatbed membrane filter, the Sartorius flatbed membrane filter (SMF), and the Filta-Max (FM) depth filter. Distilled and surface water samples were spiked with 10 oocysts and 10 cysts/liter. We also evaluated the recovery efficiency of the EC and EC-HV filters after a 5-s backwash postfiltration. The backwashing was not applied to the other filtration methods because of the design of the filters. Oocysts and cysts were visualized by using a fluorescent monoclonal antibody staining technique. For distilled water, the highest percent recovery for both the oocysts and cysts was obtained with the FM depth filter. However, when a 5-s backwash was applied, the EC-HV membrane filter (EC-HV-R) was superior to other filters for recovery of both oocysts (
n
= 53 ± 15.4 per 10 liters) and cysts (
n
= 59 ± 11.5 per 10 liters). This was followed by results of the FM depth filter (oocysts, 28.2 ± 8,
P
= 0.015; cysts, 49.8 ± 12.2,
P
= 0.4260), and SMF (oocysts, 16.2 ± 2.8,
P
= 0.0079; cysts, 35.2 ± 3,
P
= 0.0079). Similar results were obtained with surface water samples.
Giardia
cysts were recovered at higher rates than were
Cryptosporidium
oocysts with all five filters, regardless of backwashing. Although the time differences for completion of filtration process were not significantly different among the procedures, the EC-HV filtration with 5-s backwash was less labor demanding.
Subtilosin Production by Two
Bacillus subtilis
Subspecies and Variance of the
sbo-alb
Cluster
Authors: Stein Torsten , Düsterhus Stefanie , Stroh Anke , Entian Karl-Dieter
Published: April 2004

Eight different
Bacillus subtilis
strains and
Bacillus atrophaeus
were found to produce the bacteriocin subtilosin A. On the basis of the subtilosin gene (
sbo
) sequences two distinct classes of
B. subtilis
strains were distinguished, and they fell into the two
B. subtilis
subspecies (
B. subtilis
subsp.
subtilis
and
B. subtilis
subsp.
spizizenii
). The entire sequence of the subtilosin gene cluster of a
B. subtilis
subsp.
spizizenii
strain,
B. subtilis
ATCC 6633, was determined. This sequence exhibited a high level of homology to the sequence of the
sbo-alb
gene locus of
B. subtilis
168. By using primer extension analysis the transcriptional start sites of
sbo
in
B. subtilis
strains ATCC 6633 and 168 were found to be 47 and 45 bp upstream of the
sbo
start codon, respectively. Our results provide insight into the incipient evolutionary divergence of the two
B. subtilis
subspecies.
Analysis of Gene Expression in
Escherichia coli
in Response to Changes of Growth-Limiting Nutrient in Chemostat Cultures
Authors: Hua Qiang , Yang Chen , Oshima Taku , Mori Hirotada , Shimizu Kazuyuki
Published: April 2004

Studies of steady-state metabolic fluxes in
Escherichia coli
grown in nutrient-limited chemostat cultures suggest remarkable flux alterations in response to changes of growth-limiting nutrient in the medium (Hua et al., J. Bacteriol.
185:
7053-7067, 2003). To elucidate the physiological adaptation of cells to the nutrient condition through the flux change and understand the molecular mechanisms underlying the change in the flux, information on gene expression is of great importance. DNA microarray analysis was performed to investigate the global transcriptional responses of steady-state cells grown in chemostat cultures with limited glucose or ammonia while other environmental conditions and the growth rate were kept constant. In slow-growing cells (specific growth rate of 0.10 h
−1
), 9.8% of a total of 4,071 genes investigated, especially those involved in amino acid metabolism, central carbon and energy metabolism, transport system and cell envelope, were observed to be differentially expressed between the two nutrient-limited cultures. One important characteristic of
E. coli
grown under nutrient limitation was its capacity to scavenge carbon or nitrogen from the medium through elevating the expression of the corresponding transport and assimilation genes. The number of differentially expressed genes in faster-growing cells (specific growth rate of 0.55 h
−1
), however, decreased to below half of that in slow-growing cells, which could be explained by diverse transcriptional responses to the growth rate under different nutrient limitations. Independent of the growth rate, 92 genes were identified as being differentially expressed. Genes tightly related to the culture conditions were highlighted, some of which may be used to characterize nutrient-limited growth.
Deletion and Complementation of the Mating Type (
MAT
) Locus of the Wheat Head Blight Pathogen
Gibberella zeae
Authors: Desjardins A. E. , Brown D. W. , Yun S.-H. , Proctor R. H. , Lee T. , Plattner R. D. , Lu S.-W. , Turgeon B. G.
Published: April 2004

Gibberella zeae
, a self-fertile, haploid filamentous ascomycete, causes serious epidemics of wheat (
Triticum aestivum
) head blight worldwide and contaminates grain with trichothecene mycotoxins. Anecdotal evidence dating back to the late 19th century indicates that
G. zeae
ascospores (sexual spores) are a more important inoculum source than are macroconidia (asexual spores), although the fungus can produce both during wheat head blight epidemics. To develop fungal strains to test this hypothesis, the entire mating type (
MAT1
) locus was deleted from a self-fertile (
MAT1
-
1
/
MAT1
-
2
), virulent, trichothecene-producing wild-type strain of
G. zeae
. The resulting
MAT
deletion (
mat1
-
1
/
mat1
-
2
) strains were unable to produce perithecia or ascospores and appeared to be unable to mate with the fertile strain from which they were derived. Complementation of a
MAT
deletion strain by transformation with a copy of the entire
MAT
locus resulted in recovery of production of perithecia and ascospores.
MAT
deletion strains and
MAT
-complemented strains retained the ability to produce macroconidia that could cause head blight, as assessed by direct injection into wheat heads in greenhouse tests. Availability of
MAT
-null and
MAT
-complemented strains provides a means to determine the importance of ascospores in the biology of
G. zeae
and perhaps to identify novel approaches to control wheat head blight.
Oligotrophic Bacterioplankton with a Novel Single-Cell Life Strategy
Authors: Simu Karin , Hagström Åke
Published: April 2004

A large fraction of the marine bacterioplankton community is unable to form colonies on agar surfaces, which so far no experimental evidence can explain. Here we describe a previously undescribed growth behavior of three non-colony-forming oligotrophic bacterioplankton, including a SAR11 cluster representative, the world's most abundant organism. We found that these bacteria exhibit a behavior that promotes growth and dispersal instead of colony formation. Although these bacteria do not form colonies on agar, it was possible to monitor growth on the surface of seawater agar slides containing a fluorescent stain, 4′,6′-diamidino-2-phenylindole (DAPI). Agar slides were prepared by pouring a solution containing 0.7% agar and 0.5 μg of DAPI per ml in seawater onto glass slides. Prompt dispersal of newly divided cells explained the inability to form colonies since immobilized cells (cells immersed in agar) formed microcolonies. The behavior observed suggests a life strategy intended to optimize access of individual cells to substrates. Thus, the inability to form colonies or biofilms appears to be part of a K-selected population strategy in which oligotrophic bacteria explore dissolved organic matter in seawater as single cells.
Identification of Genes Associated with Morphology in
Aspergillus niger
by Using Suppression Subtractive Hybridization
Authors: Dai Ziyu , Mao Xingxue , Magnuson Jon K. , Lasure Linda L.
Published: April 2004

The morphology of citric acid production strains of
Aspergillus niger
is sensitive to a variety of factors, including the concentration of manganese (Mn
2+
). Upon increasing the Mn
2+
concentration in
A. niger
(ATCC 11414) cultures to 14 ppb or higher, the morphology switches from pelleted to filamentous, accompanied by a rapid decline in citric acid production. The molecular mechanisms through which Mn
2+
exerts effects on morphology and citric acid production in
A. niger
cultures have not been well defined, but our use of suppression subtractive hybridization has identified 22 genes responsive to Mn
2+
. Fifteen genes were differentially expressed when
A. niger
was grown in media containing 1,000 ppb of Mn
2+
(filamentous form), and seven genes were expressed in 10 ppb of Mn
2+
(pelleted form). Of the 15 filament-associated genes, seven are novel and eight share 47 to 100% identity with genes from other organisms. Five of the pellet-associated genes are novel, and the other two genes encode a pepsin-type protease and polyubiquitin. All 10 genes with deduced functions are either involved in amino acid metabolism-protein catabolism or cell regulatory processes. Northern blot analysis showed that the transcripts of all 22 genes were rapidly enhanced or suppressed by Mn
2+
. Steady-state mRNA levels of six selected filament-associated genes remained high during 5 days of culture in a filamentous state and remained low under pelleted growth conditions. The opposite behavior was observed for four selected pellet-associated genes. The full-length cDNA of the filament-associated clone,
Brsa-25
, was isolated. Antisense expression of
Brsa-25
permitted pelleted growth and increased citrate production at concentrations of Mn
2+
that were higher than the parent strain could tolerate. These results suggest the involvement of the newly isolated genes in the regulation of
A. niger
morphology.
Targeted Isolation of a Designated Region of the
Bacillus subtilis
Genome by Recombinational Transfer
Authors: Tomita Satoshi , Tsuge Kenji , Kikuchi Yo , Itaya Mitsuhiro
Published: April 2004

A method for positional cloning of the
Bacillus subtilis
genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes
ppsABCDE
encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for
ppsABCDE
was constructed using a
B. subtilis
low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a
ppsABCDE
deletion mutant.
Vibrio fischeriσ54Controls Motility, Biofilm Formation, Luminescence, and Colonization
Authors: Wolfe Alan J. , Millikan Deborah S. , Campbell Joy M. , Visick Karen L.
Published: April 2004

In this study, we demonstrated that the putativeVibrio fischeri rpoNgene, which encodes σ54, controls flagellar biogenesis, biofilm development, and bioluminescence. We also show thatrpoNplays a requisite role initiating the symbiotic association ofV. fischeriwith juveniles of the squidEuprymna scolopes.
Sulfide Ameliorates Metal Toxicity for Deep-Sea Hydrothermal Vent Archaea
Authors: Edgcomb Virginia P. , Molyneaux Stephen J. , Saito Mak A. , Lloyd Karen , Böer Simone , Wirsen Carl O. , Atkins Michael S. , Teske Andreas
Published: April 2004

The chemical stress factors for microbial life at deep-sea hydrothermal vents include high concentrations of heavy metals and sulfide. Three hyperthermophilic vent archaea, the sulfur-reducing heterotrophs
Thermococcus fumicolans
and
Pyrococcus
strain GB-D and the chemolithoautotrophic methanogen
Methanocaldococcus jannaschii
, were tested for survival tolerance to heavy metals (Zn, Co, and Cu) and sulfide. The sulfide addition consistently ameliorated the high toxicity of free metal cations by the formation of dissolved metal-sulfide complexes as well as solid precipitates. Thus, chemical speciation of heavy metals with sulfide allows hydrothermal vent archaea to tolerate otherwise toxic metal concentrations in their natural environment.