bluuebunny/crossref_metadata_2025_split huggingface dataset:
Arginine Catabolism by Sourdough Lactic Acid Bacteria: Purification and Characterization of the Arginine Deiminase Pathway Enzymes from
Lactobacillus sanfranciscensis
CB1
Authors: De Angelis Maria , Mariotti Liberato , Rossi Jone , Servili Maurizio , Fox Patrick F. , Rollán Graciela , Gobbetti Marco 
Published: November 2002

ABSTRACT

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as
Lactobacillus sanfranciscensis
CB1;
Lactobacillus brevis
AM1, AM8, and 10A;
Lactobacillus hilgardii
51B; and
Lactobacillus fructivorans
DD3 and DA106 showed all three enzyme activities.
Lactobacillus plantarum
B14 did not show CK activity.
L. sanfranciscensis
CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37°C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of
Lactobacillus sakei. L. sanfranciscensis
CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in
L. sanfranciscensis
CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by
L. sanfranciscensis
CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by
L. sanfranciscensis
CB1 enhanced cell growth, cell survival during storage at 7°C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.
Growth of Iron(III)-Reducing Bacteria on Clay Minerals as the Sole Electron Acceptor and Comparison of Growth Yields on a Variety of Oxidized Iron Forms
Authors: Kostka Joel E. , Dalton Dava D. , Skelton Hayley , Dollhopf Sherry , Stucki Joseph W. 
Published: November 2002

ABSTRACT

Smectite clay minerals are abundant in soils and sediments worldwide and are typically rich in Fe. While recent investigations have shown that the structural Fe(III) bound in clay minerals is reduced by microorganisms, previous studies have not tested growth with clay minerals as the sole electron acceptor. Here we have demonstrated that a pure culture of
Shewanella oneidensis
strain MR-1 as well as enrichment cultures of Fe(III)-reducing bacteria from rice paddy soil and subsurface sediments are capable of conserving energy for growth with the structural Fe(III) bound in smectite clay as the sole electron acceptor. Pure cultures of
S. oneidensis
were used for more detailed growth rate and yield experiments on various solid- and soluble-phase electron acceptors [smectite, Fe(III) oxyhydroxide FeOOH, Fe(III) citrate, and oxygen] in the same minimal medium. Growth was assessed as direct cell counts or as an increase in cell carbon (measured as particulate organic carbon). Cell counts showed that similar growth of
S. oneidensis
(10
8
cells ml
−1
) occurred with smectitic Fe(III) and on other Fe forms [amorphous Fe(III) oxyhydroxide, and Fe citrate] or oxygen as the electron acceptor. In contrast, cell yields of
S. oneidensis
measured as the increase in cell carbon were similar on all Fe forms tested while yields on oxygen were five times higher, in agreement with thermodynamic predictions. Over a range of particle loadings (0.5 to 4 g liter
−1
), the increase in cell number was highly correlated to the amount of structural Fe in smectite reduced. From phylogenetic analysis of the complete 16S rRNA gene sequences, a predominance of clones retrieved from the clay mineral-reducing enrichment cultures were most closely related to the low-G+C gram-positive members of the
Bacteria
(
Clostridium
and
Desulfitobacterium
) and the δ-
Proteobacteria
(members of the
Geobacteraceae
). Results indicate that growth with smectitic Fe(III) is similar in magnitude to that with Fe(III) oxide minerals and is dependent upon the mineral surface area available. Iron(III) bound in clay minerals should be considered an important electron acceptor supporting the growth of bacteria in soils or sedimentary environments.
Soil Microbial Community Structure across a Thermal Gradient following a Geothermal Heating Event
Authors: Norris Tracy B. , Wraith Jon M. , Castenholz Richard W. , McDermott Timothy R. 
Published: November 2002

ABSTRACT

In this study microbial species diversity was assessed across a landscape in Yellowstone National Park, where an abrupt increase in soil temperature had occurred due to recent geothermal activity. Soil temperatures were measured, and samples were taken across a temperature gradient (35 to 65°C at a 15-cm depth) that spanned geothermally disturbed and unimpacted soils; thermally perturbed soils were visually apparent by the occurrence of dead or dying lodgepole pine trees. Changes in soil microbial diversity across the temperature gradient were qualitatively assessed based on 16S rRNA sequence variation as detected by denaturing gradient gel electrophoresis (DGGE) using both ribosomal DNA (rDNA) and rRNA as PCR templates and primers specific for the
Bacteria
or
Archaea
domain. The impact of the major heating disturbance was apparent in that DGGE profiles from heated soils appeared less complex than those from the unaffected soils. Phylogenetic analysis of a bacterial 16S rDNA PCR clone library from a recently heated soil showed that a majority of the clones belonged to the
Acidobacterium
(51%) and
Planctomyces
(18%) divisions. Agar plate counts of soil suspensions cultured on dilute yeast extract and R2A agar media incubated at 25 or 50°C revealed that thermophile populations were two to three orders of magnitude greater in the recently heated soil. A soil microcosm laboratory experiment simulated the geothermal heating event. As determined by both RNA- and DNA-based PCR coupled with DGGE, changes in community structure (marked change in the DGGE profile) of soils incubated at 50°C occurred within 1 week and appeared to stabilize after 3 weeks. The results of our molecular and culture data suggest that thermophiles or thermotolerant species are randomly distributed in this area within Yellowstone National Park and that localized thermal activity selects for them.
A Novel Fluorescent Protein-Based Biosensor for Gram-Negative Bacteria
Authors: Goh Yan Y. , Ho Bow , Ding Jeak L. 
Published: November 2002

ABSTRACTSite-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFPi) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in anEscherichia colihost. The EGFPiproteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide from theE. colilysates, the binding affinities of the EGFPiG10 and G12 to lipid A were established. TheKDvalues of 7.16 × 10−7M for G10 and 8.15 × 10−8M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFPibiosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFPispecifically tagged gram-negative bacteria likeE. coliandPseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment. This dual function of the EGFPiin detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.
Rapid Identification, Differentiation, and Proposed New Taxonomic Classification of
Bifidobacterium lactis
Authors: Ventura Marco , Zink Ralf 
Published: November 2002

ABSTRACT

Identification of
Bifidobacterium lactis
and
Bifidobacterium animalis
is problematic because of phenotypic and genetic homogeneities and has raised the question of whether they belong to one unique taxon. Analysis of the 16S-23S internally transcribed spacer region of
B. lactis
DSM10140
T
,
B. animalis
ATCC 25527
T
, and six potential
B. lactis
strains suggested two distinct clusters. Two specific 16S-23S spacer rRNA gene-targeted primers have been developed for specific detection of
B. animalis
. All of the molecular techniques used (
B. lactis
or
B. animalis
PCR primers, enterobacterial repetitive intergenic consensus PCR) demonstrated that
B. lactis
and
B. animalis
form two main groups and suggest a revision of the strains assigned to
B. animalis
. We propose that
B. lactis
should be separated from
B. animalis
at the subspecies level.
Quantification of Bacteria Adherent to Gastrointestinal Mucosa by Real-Time PCR
Authors: Huijsdens Xander W. , Linskens Ronald K. , Mak Mariëtte , Meuwissen Stephan G. M. , Vandenbroucke-Grauls Christina M. J. E. , Savelkoul Paul H. M. 
Published: November 2002

ABSTRACT

The use of real-time quantitative PCR (5′ nuclease PCR assay) as a tool to study the gastrointestinal microflora that adheres to the colonic mucosa was evaluated. We developed primers and probes based on the 16S ribosomal DNA gene sequences for the detection of
Escherichia coli
and
Bacteroides vulgatus
. DNA was isolated from pure cultures and from gut biopsy specimens and quantified by the 5′ nuclease PCR assay. The assay showed a very high sensitivity: as little as 1 CFU of
E. coli
and 9 CFU of
B. vulgatus
could be detected. The specificities of the primer-probe combinations were evaluated with samples that were spiked with the species most closely related to
E. coli
and
B. vulgatus
and with eight other gut microflora species. Mucosal samples spiked with known amounts of
E. coli
or
B. vulgatus
DNA showed no PCR inhibition. We conclude that the 5′ nuclease PCR assay may be a useful alternative to conventional culture techniques to study the actual in vivo composition of a complex microbial community like the gut microflora.
High Oral Prevalence of
Candida krusei
in Leprosy Patients in Northern Thailand
Authors: Reichart P. A. , Samaranayake L. P. , Samaranayake Y. H. , Grote M. , Pow E. , Cheung B. 
Published: November 2002

ABSTRACT

Although
Candida albicans
is the most common human yeast pathogen, other
Candida
species such as
C. krusei
are now recognized as emerging agents, especially in patients with human immunodeficiency virus (HIV) disease.
C. krusei
is inherently resistant to the widely used triazole antifungal fluconazole and poses therapeutic problems, especially in systemic candidiasis. In a surveillance study of leprosy patients (with arrested or burnt-out disease) in a leprosarium in northern Thailand, we found a rate of oral carriage of
C. krusei
(36%) significantly (
P
< 0.05) higher than that for a healthy control group (10%). Among the
Candida-
positive patients, 16 of 35 (46%) carried
C. krusei
, while
C. albicans
was the second most common isolate (12 of 35 patients; 34%). The corresponding figures for the control group were 2 of 13 (15%) and 6 of 13 (46%), respectively. Studies of the antifungal resistance of the
C. krusei
isolates from patients indicated that all except one of the isolates were resistant to fluconazole, two isolates were resistant to ketoconazole, and all isolates were sensitive to amphotericin B. Evaluation of their genetic profiles by randomly amplified polymorphic DNA analysis with three different primers and subsequent analysis of the gel profiles by computerized cluster-derived dendrograms revealed that the
C. krusei
isolates from patients belonged to 10 disparate clusters, despite the origin from a single locale. These nascent findings indicate an alarmingly high prevalence of a
Candida
species resistant to a widely used antifungal in a part of the world where HIV disease is endemic.
Evidence that Rodents Are a Reservoir of Hepatitis E Virus for Humans in Nepal
Authors: He Junkun , Innis Bruce L. , Shrestha Mrigendra P. , Clayson Edward T. , Scott Robert M. , Linthicum Kenneth J. , Musser Guy G. , Gigliotti Scott C. , Binn Leonard N. , Kuschner Robert A. , Vaughn David W. 
Published: November 2002

ABSTRACT

Hepatitis E virus (HEV) is an important cause of enterically transmitted hepatitis in developing countries. Sporadic autochthonous cases of hepatitis E have been reported recently in the United States and other industrialized countries. The source of HEV infection in these cases is unknown; zoonotic transmission has been suggested. Antibodies to HEV have been detected in many animals in areas where HEV is endemic and in domestic swine and rats in the United States. There is evidence supporting HEV transmission between swine and humans. Nevertheless, HEV has not been detected in wild rodents. We tested murid rodents and house shrews trapped in Nepal's Kathmandu Valley, where hepatitis E is hyperendemic, for HEV infection. The most commonly trapped species was
Rattus rattus brunneusculus
. Serum samples from 675 animals were tested for immunoglobulin G against HEV by enzyme-linked immunosorbent assay; 78 (12%) were positive, indicating acute or past infection. Antibody prevalence was higher among
R. rattus brunneusculus
and
Bandicota bengalensis
than in
Suncus murinus.
Forty-four specimens from 78 antibody-positive animals had sufficient residual volume for detection of HEV RNA (viremia) by reverse transcription-PCR. PCR amplification detected four animals (9%; three were
R. rattus brunneusculus
and one was
B. bengalensis
) with viremia. Phylogenetic analysis of the four genome sequences (405 bp in the capsid gene) recovered showed that they were identical, most closely related to two human isolates from Nepal (95 and 96% nucleotide homology, respectively), and distinct from HEV sequences isolated elsewhere. These data prove that certain peridomestic rodents acquire HEV in the wild and suggest that cross-species transmission occurs, with rodents serving as a virus reservoir for humans.
Optimization and Evaluation of a PCR Assay for Detecting Toxoplasmic Encephalitis in Patients with AIDS
Authors: Joseph Priya , Calderón Maritza M. , Gilman Robert H. , Quispe Monica L. , Cok Jaime , Ticona Eduardo , Chavez Victor , Jimenez Juan A. , Chang Maria C. , Lopez Martín J. , Evans Carlton A. 
Published: November 2002

ABSTRACT

Toxoplasma gondii
is a common life-threatening opportunistic infection. We used experimental murine
T. gondii
infection to optimize the PCR for diagnostic use, define its sensitivity, and characterize the time course and tissue distribution of experimental toxoplasmosis. PCR conditions were adjusted until the assay reliably detected quantities of DNA derived from less than a single parasite. Forty-two mice were inoculated intraperitoneally with
T. gondii
tachyzoites and sacrificed from 6 to 72 h later. Examination of tissues with PCR and histology revealed progression of infection from blood to lung, heart, liver, and brain, with PCR consistently detecting parasites earlier than microscopy and with no false-positive results. We then evaluated the diagnostic value of this PCR assay in human patients. We studied cerebrospinal fluid and serum samples from 12 patients with AIDS and confirmed toxoplasmic encephalitis (defined as positive mouse inoculation and/or all of the Centers for Disease Control clinical diagnostic criteria), 12 human immunodeficiency virus-infected patients with suspected cerebral toxoplasmosis who had neither CDC diagnostic criteria nor positive mouse inoculation, 26 human immunodeficiency virus-infected patients with other opportunistic infections and no signs of cerebral toxoplasmosis, and 18 immunocompetent patients with neurocysticercosis. Eleven of the 12 patients with confirmed toxoplasmosis had positive PCR results in either blood or cerebrospinal fluid samples (6 of 9 blood samples and 8 of 12 cerebrospinal fluid samples). All samples from control patients were negative. This study demonstrates the high sensitivity, specificity, and clinical utility of PCR in the diagnosis of toxoplasmic encephalitis in a resource-poor setting.
Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids
Authors: Broccolo Francesco , Locatelli Giuseppe , Sarmati Loredana , Piergiovanni Sara , Veglia Fabrizio , Andreoni Massimo , Buttò Stefano , Ensoli Barbara , Lusso Paolo , Malnati Mauro S. 
Published: November 2002

ABSTRACT

Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 10
6
viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 μg) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of <15 and 23%, respectively. Furthermore, the use of the calibrator improves the accuracy of quantitation and decreases the intersample variability (CV, 9 and 6%, respectively). The sensitivity and specificity of the assay were tested with a series of clinical specimens obtained from patients affected by various HHV-8-related diseases, as well as from a wide number of controls. In conclusion, our calibrated real-time PCR assay provides a reliable high-throughput method for quantitation of HHV-8 DNA in clinical and laboratory specimens.
Characterization of Tn
1546
in Vancomycin-Resistant
Enterococcus faecium
Isolated from Canine Urinary Tract Infections: Evidence of Gene Exchange between Human and Animal Enterococci
Authors: Simjee S. , White D. G. , McDermott P. F. , Wagner D. D. , Zervos M. J. , Donabedian S. M. , English L. L. , Hayes J. R. , Walker R. D. 
Published: November 2002

ABSTRACT

Thirty-five enterococcal isolates were recovered from dogs diagnosed with urinary tract infections at the Michigan State University Veterinary Teaching Hospital over a 2-year period (1996 to 1998). Isolated species included
Enterococcus faecium
(
n
= 13),
Enterococcus faecalis
(
n
= 7),
Enterococcus gallinarum
(
n
= 11), and
Enterococcus casseliflavus
(
n
= 4). Antimicrobial susceptibility testing revealed several different resistance phenotypes, with the majority of the enterococcal isolates exhibiting resistance to three or more antibiotics. One
E. faecium
isolate, CVM1869, displayed high-level resistance to vancomycin (MIC > 32 μg/ml) and gentamicin (MIC > 2,048 μg/ml). Molecular analysis of this isolate revealed the presence of Tn
1546
(
vanA
), responsible for high-level vancomycin resistance, and Tn
5281
carrying
aac6′-aph2"
, conferring high-level aminoglycoside resistance. Pulsed-field gel electrophoresis analysis revealed that CVM1869 was a canine
E. faecium
clone that had acquired Tn
1546
, perhaps from a human vancomycin-resistant
E. faecium
. Transposons Tn
5281
and Tn
1546
were located on two different conjugative plasmids. Sequence analysis revealed that in Tn
1546
, ORF1 had an 889-bp deletion and an IS
1216V
insertion at the 5′ end and an IS
1251
insertion between
vanS
and
vanH
. To date, this particular form of Tn
1546
has only been described in human clinical vancomycin-resistant enterococcus isolates unique to the United States. Additionally, this is the first report of a vancomycin-resistant
E. faecium
isolated from a companion animal in the United States.
Novel Single-Tube Agar-Based Test System for Motility Enhancement and Immunocapture of
Escherichia coli
O157:H7 by H7 Flagellar Antigen-Specific Antibodies
Authors: Murinda Shelton E. , Nguyen Lien T. , Ivey Susan J. , Almeida Raul A. , Oliver Stephen P. 
Published: November 2002

ABSTRACT

This paper describes a novel single-tube agar-based technique for motility enhancement and immunoimmobilization of
Escherichia coli
O157:H7. Motility indole ornithine medium and agar (0.4%, wt/vol) media containing either nutrient broth, tryptone broth, or tryptic soy broth (TSBA) were evaluated for their abilities to enhance bacterial motility. Twenty-six
E. coli
strains, including 19 O157:H7 strains, 1 O157:H
−
strain, and 6 generic
E. coli
strains, were evaluated. Test bacteria were stab inoculated in the center of the agar column, and tubes were incubated at 37°C for 18 to 96 h. Nineteen to 24 of the 26 test strains (73.1 to 92.3%) were motile in the different media. TSBA medium performed best and was employed in subsequent studies of motility enhancement and H7 flagellar immunocapture. H7 flagellar antiserum (30 and 60 μl) mixed with TSBA was placed as a band (1 ml) in the middle of an agar column separating the top (3-ml) and bottom (3-ml) agar layers. The top agar layer was inoculated with the test bacterial strains. The tubes were incubated at 37°C for 12 to 18 h and for 18 to 96 h. The specificity and sensitivity of the H7 flagellar immunocapture tests were 75 and 100%, respectively. The procedure described is simple and sensitive and could be adapted easily for routine use in laboratories that do not have sophisticated equipment and resources for confirming the presence of H7 flagellar antigens. Accurate and rapid identification of H7 flagellar antigen is critical for the complete characterization of
E. coli
O157:H7, owing to the immense clinical, public health, and economic significance of this food-borne pathogen.