bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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

ABSTRACTIn 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

ABSTRACT

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.
Measuring parent attributes and supervision behaviors relevant to child injury risk: examining the usefulness of questionnaire measures
Authors: Morrongiello B A , House K 
Published: April 2004

Objective:This study aimed to identify self report questionnaire measures of parent attributes and behaviors that have relevance for understanding injury risk among children 2–5 years of age, and test a new Parent Supervision Attributes Profile Questionnaire (PSAPQ) that was developed to measure aspects of protectiveness and parent supervision.Methods:Naturalistic observations were conducted of parents’ supervision of children on playgrounds, with questionnaires subsequently completed by the parent to measure parent education, family income, parent personality attributes, attributes relevant to parent supervision, and beliefs about parents’ control over the child’s health status. These measures were then related to children’s risk taking and injury history.Results:Visual supervision, auditory supervision, and physical proximity were highly intercorrelated, indicating that parents employed all types of behaviors in service of supervision, rather than relying predominantly on one type of supervisory behavior. Physical proximity was the only aspect of supervision behavior that served a protective function and related to children’s risk taking behaviors: parents who remained close to their children had children who engaged in less risk taking. On questionnaires, parents who reported more conscientiousness, protectiveness, worry about safety, vigilance in supervision, confidence in their ability to keep their child safe, and belief in control over their child’s health had children who showed less risk taking and/or experienced fewer injuries. The new PSAPQ measure was associated with specific aspects of supervision as well as children’s risk taking and injury history.Conclusions:This study reveals several parent attributes and behaviors with relevance for child injury risk that can be measured via self report questionnaires, including the new PSAPQ.
A quantification of preventable unintentional childhood injury mortality in the United States
Authors: Philippakis A , Hemenway D , Alexe D M , Dessypris N , Spyridopoulos T , Petridou E 
Published: April 2004

Objective:To calculate the preventable fraction of unintentional childhood injury deaths in the United States.Design:Ecological study of cause specific unintentional childhood injury mortality rates across the 50 states (and the District of Columbia) of the United States (US) over the 10 year period 1989–98.Methods:The internet accessible database from the National Center for Injury Prevention and Control was used in order to estimate unintentional childhood (0–14 years) injury mortality rates by external cause and time trends over the study period for each of the US states and for the four major geographical regions of the country. In the principal analysis, a calculation was made of the fraction and absolute number of unintentional childhood injury deaths that could have been prevented annually if the mortality rate in the region with the lowest rate also existed in the remaining three. In another scenario, the lowest external cause specific unintentional childhood injury mortality rates from the 50 US states and the District of Columbia were summed to provide the “ideal” lowest conceivable unintentional childhood injury mortality rate from all causes. Ecological correlations between unintentional childhood injury mortality rates from specified external causes, median income, and percent of the population with a college degree were made.Main outcome measures:Unintentional childhood injury mortality rates by cause.Results:Unintentional childhood injury mortality rate declined by 3.5% per year in the country as a whole. If every region of the US had experienced the same injury rate as the Northeast, then one third of all unintentional childhood injuries would not have occurred.More optimistic scenarios indicate that up to two thirds of all unintentional childhood injury deaths could be prevented. Across states, unintentional childhood injury mortality is strongly inversely related to median income.Conclusions:About one third of all unintentional childhood injury deaths in the US are preventable with the means and resources available in the Northeastern states. Among the relevant characteristics in the Northeast region, in comparison with other US regions, are the higher education level of parents, the lower gun ownership, the higher population density that implies shorter distances traveled by cars, a better developed emergency medical system, and the existence of several injury prevention programs.
Incomplete and biased perpetrator coding among hospitalized assaults for women in the United States
Authors: Weiss H B , Ismailov R M , Lawrence B A , Miller T R 
Published: April 2004

Background:Since 1997, hospital discharge data have included external cause of injury (E codes) for designating perpetrator relationship in assaults. For intentional injuries, guidelines require using two E codes; one for the injury mechanism and another (E967.n) identifying perpetrator relationship. Completeness and characteristics of the use of these codes have not been studied on a multistate level among states with complete E coding.Methods:Hospital discharge data for 1997 were solicited from states with good E coding completeness. Data were received from 19 states (51.9% of women in the United States, ages 15–49). For assaulted women, a regression model was constructed to identify factors associated with perpetrator code assignment using age, payment source, pregnancy status, race, and severity as covariates.Results:Among 137 887 injured hospitalized women age 15–49, there were 7402 assaults (5.4%). Among all assaults to women, perpetrator coding was poor (8.8%). Among those that were perpetrator coded, 83.7% were spouse/partner related. Age was positively associated with probability of having a perpetrator code (p<0.001). Those paid by a private source were 42.9% more likely to have a perpetrator code (p = 0.007). Pregnant women were seven times more likely to have a perpetrator code (p<0.001). Non-white women were 66.8% less likely to have a perpetrator code (p<0.001) than white women.Conclusions:The poor use of perpetrator codes in hospital discharge data minimizes their usefulness for surveillance of serious injury from intimate partner violence. An implication of this research is the need to understand the gaps and strengthen the completeness of perpetrator documentation and coding. The findings suggest caution when interpreting the results from existing hospital discharge data based intimate partner violence surveillance systems.
Motor vehicle crash deaths related to police pursuits in the United States
Authors: Rivara F P , Mack C D 
Published: April 2004

Objective:To determine the number and characteristics of motor vehicle crash deaths related to police pursuits in the United States.Methods:Analysis of the Fatality Analysis Reporting System and the Crashworthiness Data System of the National Highway Traffic Safety Administration for the years 1994–2002.Results:There were 2654 fatal crashes involving 3965 vehicles and 3146 fatalities during the nine year study period. Of these, 1088 were to people not in the fleeing vehicle. These crashes often occurred at high speed, in the night, on local roads. Most of the pursued drivers had prior motor vehicle related convictions.Conclusions:Many deaths related to police pursuits are to innocent victims. Given that most of the pursued drivers had prior convictions, alternative means of detaining them should be explored.