bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induction and Resuscitation of Viable but Nonculturable
Salmonella enterica
Serovar Typhimurium DT104†
Authors: Gupte A. R. , de Rezende C. L. E. , Joseph S. W. 
Published: November 2003

ABSTRACT

Salmonella enterica
serovar Typhimurium DT104 11601was tested for its ability to maintain viability in minimal, chemically defined solutions. Periodic monitoring of growth and survival in microcosms of different ion concentrations, maintained at various temperatures, showed a gradual decline in culturable organisms (∼235 days) at 5°C. Organisms maintained at a higher temperature (21°C) showed continuous, equivalent CFU per milliliter (∼10
6
) up to 400 days after inoculation. Fluorescence microscopy with Baclight revealed that nonculturable cells were actually viable, while observations with scanning electron microscopy showed that the cells had retained their structural integrity. Temperature upshift (56°C ± 0.5, 15 s) of the nonculturable organisms (5°C) in Trypticase soy broth followed by immediate inoculation onto Trypticase soy agar (TSA) gave evidence of resuscitation. Interestingly,
S. enterica
serovar Typhimurium DT104 from the microcosms at either 5°C (1 to 200 days) or 21°C (1 to 250 days) did not show enhanced growth after intermittent inoculation onto catalase-supplemented TSA. Furthermore, cells from 21°C microcosms exposed to oxidative and osmotic stress showed greater resistance to stresses over increasing times of exposure than did recently grown cells. It is possible that the exceptional survivability and resilience of this particular strain may in part reflect the growing importance of this multidrug-resistant organism, in general, as a cause of intestinal disease in humans. The fact that
S. enterica
serovar Typhimurium DT104 11601 is capable of modifying its physiological characteristics, including entry into and recovery from the viable but nonculturable state, suggests the overall possibility that
S. enterica
serovar Typhimurium DT104 may be able to respond uniquely to various adverse environmental conditions.
Biosynthesis of Yersiniabactin, a Complex Polyketide-Nonribosomal Peptide, Using
Escherichia coli
as a Heterologous Host
Authors: Pfeifer Blaine A. , Wang Clay C. C. , Walsh Christopher T. , Khosla Chaitan 
Published: November 2003

ABSTRACT

The medicinal value associated with complex polyketide and nonribosomal peptide natural products has prompted biosynthetic schemes dependent upon heterologous microbial hosts. Here we report the successful biosynthesis of yersiniabactin (Ybt), a model polyketide-nonribosomal peptide hybrid natural product, using
Escherichia coli
as a heterologous host. After introducing the biochemical pathway for Ybt into
E. coli
, biosynthesis was initially monitored qualitatively by mass spectrometry. Next, production of Ybt was quantified in a high-cell-density fermentation environment with titers reaching 67 ± 21 (mean ± standard deviation) mg/liter and a volumetric productivity of 1.1 ± 0.3 mg/liter-h. This success has implications for basic and applied studies on Ybt biosynthesis and also, more generally, for future production of polyketide, nonribosomal peptide, and mixed polyketide-nonribosomal peptide natural products using
E. coli
.
Diversity and Activity of Methanotrophic Bacteria in Different Upland Soils
Authors: Knief Claudia , Lipski André , Dunfield Peter F. 
Published: November 2003

ABSTRACT

Samples from diverse upland soils that oxidize atmospheric methane were characterized with regard to methane oxidation activity and the community composition of methanotrophic bacteria (MB). MB were identified on the basis of the detection and comparative sequence analysis of the
pmoA
gene, which encodes a subunit of particulate methane monooxygenase. MB commonly detected in soils were closely related to
Methylocaldum
spp.,
Methylosinus
spp.,
Methylocystis
spp., or the “forest sequence cluster” (USC α), which has previously been detected in upland soils and is related to
pmoA
sequences of type II MB (
Alphaproteobacteria
). As well, a novel group of sequences distantly related (<75% derived amino acid identity) to those of known type I MB (Gammaproteobacteria) was often detected. This novel “upland soil cluster γ” (USC γ) was significantly more likely to be detected in soils with pH values of greater than 6.0 than in more acidic soils. To identify active MB, four selected soils were incubated with
13
CH
4
at low mixing ratios (<50 ppm of volume), and extracted methylated phospholipid fatty acids (PLFAs) were analyzed by gas chromatography-online combustion isotope ratio mass spectrometry. Incorporation of
13
C into PLFAs characteristic for methanotrophic
Gammaproteobacteria
was observed in all soils in which USC γ sequences were detected, suggesting that the bacteria possessing these sequences were active methanotrophs. A pattern of labeled PLFAs typical for methanotrophic
Alphaproteobacteria
was obtained for a sample in which only USC α sequences were detected. The data indicate that different MB are present and active in different soils that oxidize atmospheric methane.
Application of Real-Time PCR for Quantification of Microcystin Genotypes in a Population of the Toxic CyanobacteriumMicrocystissp
Authors: Kurmayer Rainer , Kutzenberger Thomas 
Published: November 2003

ABSTRACTThe cyanobacteriumMicrocystissp. frequently develops water blooms consisting of organisms with different genotypes that either produce or lack the hepatotoxin microcystin. In order to monitor the development of microcystin (mcy) genotypes during the seasonal cycle of the total population,mcygenotypes were quantified by means of real-time PCR in Lake Wannsee (Berlin, Germany) from June 1999 to October 2000. Standard curves were established by relating cell concentrations to the threshold cycle (the PCR cycle number at which the fluorescence passes a set threshold level) determined by theTaqnuclease assay (TNA) for two gene regions, the intergenic spacer region within the phycocyanin (PC) operon to quantify the total population and themcyBgene, which is indicative of microcystin synthesis. In laboratory batch cultures, the cell numbers inferred from the standard curve by TNA correlated significantly with the microscopically determined cell numbers on a logarithmic scale. The TNA analysis of 10 strains revealed identical amplification efficiencies for both genes. In the field, the proportion ofmcygenotypes made up the smaller part of the PC genotypes, ranging from 1 to 38%. The number ofmcyBgenotypes was one-to-one related to the number of PC genotypes, and parallel relationships between cell numbers estimated via the inverted microscope technique and TNA were found for both genes. It is concluded that the mean proportion of microcystin genotypes is stable from winter to summer and thatMicrocystiscell numbers could be used to infer the mean proportion ofmcygenotypes in Lake Wannsee.
Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis
Authors: Lopez Isabel , Ruiz-Larrea Fernanda , Cocolin Luca , Orr Erica , Phister Trevor , Marshall Megan , VanderGheynst Jean , Mills David A. 
Published: November 2003

ABSTRACTDenaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.
Peptide Ligands That Bind Selectively to Spores of
Bacillus subtilis
and Closely Related Species
Authors: Knurr Jordan , Benedek Orsolya , Heslop Jennifer , Vinson Robert B. , Boydston Jeremy A. , McAndrew Joanne , Kearney John F. , Turnbough Charles L. 
Published: November 2003

ABSTRACT

As part of an effort to develop detectors for selected species of bacterial spores, we screened phage display peptide libraries for 7- and 12-mer peptides that bind tightly to spores of
Bacillus subtilis
. All of the peptides isolated contained the sequence Asn-His-Phe-Leu at the amino terminus and exhibited clear preferences for other amino acids, especially Pro, at positions 5 to 7. We demonstrated that the sequence Asn-His-Phe-Leu-Pro (but not Asn-His-Phe-Leu) was sufficient for tight spore binding. We observed equal 7-mer peptide binding to spores of
B. subtilis
and its most closely related species,
Bacillus amyloliquefaciens
, and slightly weaker binding to spores of the closely related species
Bacillus globigii
. These three species comprise one branch on the
Bacillus
phylogenetic tree. We did not detect peptide binding to spores of several
Bacillus
species located on adjacent and nearby branches of the phylogenetic tree nor to vegetative cells of
B. subtilis
. The sequence Asn-His-Phe-Leu-Pro was used to identify
B. subtilis
proteins that may employ this peptide for docking to the outer surface of the forespore during spore coat assembly and/or maturation. One such protein, SpsC, appears to be involved in the synthesis of polysaccharide on the spore coat. SpsC contains the Asn-His-Phe-Leu-Pro sequence at positions 6 to 10, and the first five residues of SpsC apparently must be removed to allow spore binding. Finally, we discuss the use of peptide ligands for bacterial detection and the use of short peptide sequences for targeting proteins during spore formation.
Protistan Grazing Analysis by Flow Cytometry Using Prey Labeled by In Vivo Expression of Fluorescent Proteins
Authors: Fu Yutao , O'Kelly Charles , Sieracki Michael , Distel Daniel L. 
Published: November 2003

ABSTRACT

Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate
Paraphysomonas imperforata
on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species,
Escherichia coli
,
Enterobacter aerogenes
, and
Pseudomonas putida. Micromonas pusilla
, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of
P. imperforata
was influenced by prey type, size, and condition. In competitive feeding trials,
P. imperforata
consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for
P. putida
, the largest prey species examined; smaller cells of
P. putida
were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.
Isolation and Identification of
Photobacterium phosphoreum
from an Unexpected Niche: Migrating Salmon
Authors: Budsberg K. J. , Wimpee C. F. , Braddock J. F. 
Published: November 2003

ABSTRACT

Six luminous bacteria were isolated from migrating salmon in the Yukon River, Alaska. All isolates were identified as
Photobacterium phosphoreum
. Previous studies suggest that
P. phosphoreum
is an exclusively marine bacterium, while our Alaskan isolates are from salmon which migrated up to 1,228 km from the marine environment.
Rectoanal Mucosal Swab Culture Is More Sensitive Than Fecal Culture and Distinguishes
Escherichia coli
O157:H7-Colonized Cattle and Those Transiently Shedding the Same Organism
Authors: Rice Daniel H. , Sheng Haiqing Q. , Wynia Stacey A. , Hovde Carolyn J. 
Published: November 2003

ABSTRACT

Enrichment and direct (nonenrichment) rectoanal mucosal swab (RAMS) culture techniques were developed and compared to traditional fecal culture for the detection of
Escherichia coli
O157:H7 in experimentally infected and naturally infected cattle. Holstein steers (
n
= 16) orally dosed with
E. coli
O157:H7 were sampled after bacterial colonization starting 15 days postinoculation. Enrichment RAMS cultures (70.31% positive) were more sensitive than enrichment fecal cultures with 10 g of feces (46.88% positive) at detecting
E. coli
O157:H7 (
P
< 0.01). Holstein bull calves (
n
= 15) were experimentally exposed to
E. coli
O157:H7 by penning them with
E. coli
O157:H7-positive calves. Prior to bacterial colonization (1 to 14 days postexposure), enriched fecal cultures were more sensitive at detecting
E. coli
O157:H7 than enriched RAMS cultures (
P
< 0.01). However, after colonization (40 or more days postexposure), the opposite was true and RAMS culture was more sensitive than fecal culture (
P
< 0.05). Among naturally infected heifers, enriched RAMS or fecal cultures were equally sensitive (
P
= 0.5), but direct RAMS cultures were more sensitive than either direct or enriched fecal cultures at detecting
E. coli
O157:H7 (
P
< 0.01), with 25 of 144, 4 of 144, and 10 of 108 samples, respectively, being culture positive. For both experimentally and naturally infected cattle, RAMS culture predicted the duration of infection. Cattle transiently shedding
E. coli
O157:H7 for <1 week were positive by fecal culture only and not by RAMS culture, whereas colonized animals (which were culture positive for an average of 26 days) were positive early on by RAMS culture. RAMS culture more directly measured the relationship between cattle and
E. coli
O157:H7 infection than fecal culture.
Novel Enzyme-Linked Minisequence Assay for Genotypic Analysis of Human Immunodeficiency Virus Type 1 Drug Resistance
Authors: Sugiura Wataru , Shimada Kazunori , Matsuda Masakazu , Chiba Tomoko , Myint Lay , Okano Aiko , Yamada Kaneo 
Published: November 2003

ABSTRACT
We constructed a novel tool for genotypic analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance by using an enzyme-linked minisequence assay (ELMA). ELMA is a combination of hybridization and a 1-base extension reaction, and we designed the assay to detect five mutations conferring nucleoside analogue resistance (M41L, D67N, K70R, T215Y, and M184V) and six mutations conferring protease inhibitor resistance (D30N, M46I, G48V, V82A, I84V, and L90M). At all detection points, ELMA demonstrated high sensitivity and specificity, sufficient for clinical use. Compared to that obtained by direct sequencing, the genotypic information obtained by ELMA is limited to the targeted loci for which it was designed. However, ELMA proves advantageous in several respects. The assay does not require expensive equipment, such as an autosequencer, and can be performed in regular clinical diagnostic laboratories. Therefore ELMA can be a candidate for a drug resistance monitoring assay to be introduced in developing countries. In addition, ELMA demonstrated higher sensitivity in the detection of minor resistant populations. We successfully detected a minor virus population (10%) by the assay. The high sensitivity and specificity of the assay recommend it as a first screening assay for drug resistance surveillance.
Metronidazole-Resistant
Helicobacter pylori
Is More Prevalent in Patients with Nonulcer Dyspepsia than in Peptic Ulcer Patients in a Multiethnic Asian Population
Authors: Lui Sook-Yin , Yeoh Khay-Guan , Ho Bow 
Published: November 2003

ABSTRACT

The trend of increasing prevalence of antibiotic resistance among
Helicobacter pylori
strains has been suggested as a cause of the failure of treatment of
H. pylori
infections. In this study, 120 of 211 antral biopsy specimens from patients with dyspeptic symptoms were found to harbor
H. pylori.
The isolates from the 120 specimens were tested by the agar dilution method, and 38 (31.7%) were found to be metronidazole resistant. Among the 211 subjects, 81 of 115 (70.4%) patients with peptic ulcer (PU) were infected with
H. pylori
, whereas 39 of 96 (40.6%) patients with nonulcer dyspepsia (NUD) were infected with
H. pylori
. Interestingly, significantly more NUD patients than PU patients harbored metronidazole-resistant
H. pylori
(22 of 39 [56.4%] and 16 of 81 [19.8%], respectively;
P
< 0.001). A similar pattern was also observed among NUD patients of different ethnicities but not between male and female patients (23 of 78 [29.5%] and 15 of 42 [35.7%], respectively;
P
= 0.54). In the posttreatment follow-up, five of six patients who had positive urea breath test results, indicating treatment failure, were NUD patients. Of these, four harbored metronidazole-resistant
H. pylori
strains. This further illustrates the relevance of metronidazole-resistant
H. pylori
in NUD patients. The significantly higher percentage of metronidazole-resistant
H. pylori
isolates in NUD patients may be attributed to the protection offered by the mucus layer of the nonulcerated stomach to the bacteria that reside below it, resulting in organism exposure to sublethal concentrations of metronidazole and leading to the induction of metronidazole resistance. The results demonstrate that the
H. pylori
isolates colonizing NUD patients are more likely to be resistant to metronidazole. It will therefore be useful to reevaluate the use of metronidazole in the treatment of NUD patients infected with
H. pylori
.
Distribution of Intimin Subtypes amongEscherichia coliIsolates from Ruminant and Human Sources
Authors: Ramachandran Vidiya , Brett Kim , Hornitzky Michael A. , Dowton Mark , Bettelheim Karl A. , Walker Mark J. , Djordjevic Steven P. 
Published: November 2003

ABSTRACTThe intimin geneeae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenicEscherichia coli(EPEC) and some Shiga toxin-producingE. coli(STEC) strains from all other pathotypes of diarrheagenicE. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (α1, α2, β, γ, κ, ε, η, ι, λ, θ, and ζ) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int-μ, Int-ν, and Int-ξ. The PCR-RFLP assay was used to screen 213eae-positiveE. coliisolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 werestx-negative (stx−) andehxA-positive (ehxA+) isolates, and 42 werestx−andehxA-negative isolates. Int-β, the most commonly identifiedeaesubtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-ζ (39 of 213 [18.3%] isolates; 11 serotypes), Int-θ (25 of 213 [11.7%] isolates; 15 serotypes), Int-γ (19 of 213 [8.9%] isolates; 9 serotypes), and Int-ε (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes α1, α2, κ, λ, ξ, μ, ν, and ι were infrequently identified; and Int-η was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (α, β-ξ, γ, κ, ε-η-ν, ι-μ, λ, θ, and ζ). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containingE. colistrains.
Quantifying Adenovirus-Neutralizing Antibodies by Luciferase Transgene Detection: Addressing Preexisting Immunity to Vaccine and Gene Therapy Vectors
Authors: Sprangers Mieke C. , Lakhai Wandena , Koudstaal Wouter , Verhoeven Marielle , Koel Björn F. , Vogels Ronald , Goudsmit Jaap , Havenga Menzo J. E. , Kostense Stefan 
Published: November 2003

ABSTRACT
The presence of various levels of anti-adenovirus serotype 5 (Ad5)-neutralizing antibodies in humans is thought to contribute to the inconsistent clinical results obtained so far in diverse gene transfer and vaccination studies and might preclude universal dosing with recombinant Ad5. Prescreening of individuals eligible for Ad5 or alternative serotype treatment and subsequently tailoring the vector dose might aid in ensuring the consistency of clinical parameters. For this purpose, a qualified Ad neutralization assay is required. Here we have tested the different protocols used to date to determine anti-Ad neutralizing activity. Based on simplicity, speed, high throughput, sensitivity, and robustness, we propose a qualified assay in which Ad neutralization is monitored by luciferase reporter gene expression.
Genetic Diversity among
Borrelia
Strains Determined by Single-Strand Conformation Polymorphism Analysis of the
ospC
Gene and Its Association with Invasiveness
Authors: Lagal Vanessa , Postic Danièle , Ruzic-Sabljic Eva , Baranton Guy 
Published: November 2003

ABSTRACT

Lyme borreliosis (LB) is a tick-borne spirochetal infection caused by three
Borrelia
species:
Borrelia afzelii
,
B. garinii
, and
B. burgdorferi
sensu stricto. LB evolves in two stages: a skin lesion called erythema migrans and later, different disseminated forms (articular, neurological, cardiac…). Previous research based on analysis of
ospC
sequences allowed the definition of 58 groups (divergence of <2% within a group and >8% between groups). Only 10 of these groups include all of the strains isolated from disseminated forms that are considered invasive. The aim of this study was to determine whether or not invasive strains belong to restricted
ospC
groups by testing human clinical strains isolated from disseminated forms. To screen for
ospC
genetic diversity, we used single-strand conformation polymorphism (SSCP) analysis. Previously known
ospC
sequences from 44 different strains were first tested, revealing that each
ospC
group had a characteristic SSCP pattern. Therefore, we studied 80 disseminated-form isolates whose
ospC
sequences were unknown. Of these, 28 (35%) belonged to previously known invasive groups. Moreover, new invasive groups were identified: six of
B. afzelii
, seven of
B. garinii
, and one of
B. burgdorferi
sensu stricto. This study confirmed that invasive strains are not distributed among all 69
ospC
groups but belong to only 24 groups. This suggests that OspC may be involved in the invasiveness of
B. burgdorferi
.