bluuebunny/crossref_metadata_2025 huggingface dataset:
Genotypic Analysis of
Mycobacterium tuberculosis
in Two Distinct Populations Using Molecular Beacons: Implications for Rapid Susceptibility Testing
Authors: Piatek Amy S. , Telenti Amalio , Murray Megan R. , El-Hajj Hiyam , Jacobs William R. , Kramer Fred Russell , Alland David
Published: April 2009

Past genotypic studies of
Mycobacterium tuberculosis
may have incorrectly estimated the importance of specific drug resistance mutations due to a number of sampling biases including an overrepresentation of multidrug-resistant (MDR) isolates. An accurate assessment of resistance mutations is crucial for understanding basic resistance mechanisms and designing genotypic drug resistance assays. We developed a rapid closed-tube PCR assay using fluorogenic reporter molecules called molecular beacons to detect reportedly common
M. tuberculosis
mutations associated with resistance to isoniazid and rifampin. The assay was used in a comparative genotypic investigation of two different study populations to determine whether these known mutations account for most cases of clinical drug resistance. We analyzed samples from a reference laboratory in Madrid, Spain, which receives an overrepresentation of MDR isolates similar to prior studies and from a community medical center in New York where almost all of the resistant isolates and an equal number of susceptible controls were available. The ability of the molecular beacon assay to predict resistance to isoniazid and rifampin was also assessed. The overall sensitivity and specificity of the assay for isoniazid resistance were 85 and 100%, respectively, and those for rifampin resistance were 98 and 100%, respectively. Rifampin resistance mutations were detected equally well in isolates from both study populations; however, isoniazid resistance mutations were detected in 94% of the isolates from Madrid but in only 76% of the isolates from New York (
P
= 0.02). In New York, isoniazid resistance mutations were significantly more common in the MDR isolates (94%) than in single-drug-resistant isolates (44%;
P
< 0.001). No association between previously described mutations in the
kasA
gene and isoniazid resistance was found. The first mutations that cause isoniazid resistance may often occur in sequences that have not been commonly associated with isoniazid resistance, possibly in other as yet uncharacterized genes. The molecular beacon assay was simple, rapid, and highly sensitive for the detection of rifampin-resistant
M. tuberculosis
isolates and for the detection of isoniazid resistance in MDR isolates.
Development of a Long-Term Ascending Urinary Tract Infection Mouse Model for Antibiotic Treatment Studies
Authors: Hvidberg Hanne , Struve Carsten , Krogfelt Karen A. , Christensen Nils , Rasmussen Søren N. , Frimodt-Møller Niels
Published: April 2009

A model of ascending unobstructed urinary tract infection (UTI) in mice was developed to study the significance of the antibiotic concentration in urine, serum, and kidney tissue for efficacy of treatment of UTI in general and pyelonephritis in particular. Outbred Ssc-CF1 female mice were used throughout the study, and
Escherichia coli
was used as the pathogen. The virulence of 11 uropathogenic
E. coli
isolates and 1 nonpathogenic laboratory
E. coli
strain was examined. Strain C175-94 achieved the highest counts in the kidneys, and this strain was subsequently used as the infecting organism. The model gave reproducible bladder infections, i.e., bacteria were recovered from 22 of 23 control mice after 3 days, and histological examination of kidney tissue showed that of 14 infected kidneys, 7 (50%) showed major histological changes, whereas 3 of 36 uninfected kidneys showed major histological changes (
P
= 0.018). Once the model was established, the efficacies of different doses of cefuroxime and gentamicin, corresponding to active concentrations in urine only or in urine, serum, and kidney tissue simultaneously, were examined. All cefuroxime doses resulted in significantly lower counts in urine than control treatments, but the dose which produced concentrations of cefuroxime only in urine and not in serum or kidney tissue had no effect on kidney infection. Even low doses of gentamicin (0.05 mg/mouse) resulted in concentrations in renal tissue for prolonged times due to accumulation. All gentamicin doses had a significant effect (compared to the effect of the control treatment) on bacterial counts in urine and kidneys. The antibiotic effect on bacterial counts in bladders was negligible for unknown reasons. Use of the mouse UTI model is feasible for study of the effect of an antibiotic in the urinary system, although the missing antibacterial effect in the bladder needs further evaluation.
In Vitro Susceptibilities of Rapidly Growing Mycobacteria to Telithromycin (HMR 3647) and Seven Other Antimicrobials
Authors: Fernández-Roblas Ricardo , Esteban Jaime , Cabria Froilán , López Juan Carlos , Jiménez Maria Soledad , Soriano Francisco
Published: April 2009

The antimicrobial activities of telithromycin (HMR 3647) and seven other antimicrobials against 94 strains of rapidly growing mycobacteria were determined. Telithromycin at a concentration of 1 μg/ml inhibited
Mycobacterium peregrinum
(100%),
Mycobacterium chelonae
(80%),
Mycobacterium abscessus-Mycobacterium mucogenicum
(44.4%), and
Mycobacterium fortuitum
(2.1%). All or most strains of
M. peregrinum
,
M. fortuitum
, and
M. mucogenicum
were inhibited by 2 μg of quinolones per ml.
Morphological Change in
Pseudomonas aeruginosa
following Antibiotic Treatment of Experimental Infection in Mice and Its Relation to Susceptibility to Phagocytosis and to Release of Endotoxin
Authors: Yokochi Takashi , Narita Kayou , Morikawa Akiko , Takahashi Kazuko , Kato Yutaka , Sugiyama Tuyoshi , Koide Naoki , Kawai Makoto , Fukada Masako , Yoshida Tomoaki
Published: April 2009

The relationship between morphological changes in
Pseudomonas aeruginosa
following antibiotic treatment of experimental infection in mice, susceptibility to phagocytosis, and release of endotoxin was studied. The intraperitoneal administration of
P. aeruginosa
with imipenem or ceftazidime into mice induced morphological changes in the cells 2 h after injection. Round
P. aeruginosa
cells with imipenem treatment became susceptible to phagocytosis by peritoneal cells, whereas long filamentous cells with ceftazidime treatment were hardly phagocytized by peritoneal cells. The morphological changes also affected the plasma endotoxin level in the circulation.
Production of a TEM-24 Plasmid-Mediated Extended-Spectrum β-Lactamase by a Clinical Isolate of
Pseudomonas aeruginosa
Authors: Marchandin Helene , Jean-Pierre Helene , De Champs Christophe , Sirot Danielle , Darbas Helene , Perigault Pierre Francois , Carriere Christian
Published: April 2009

Several
Pseudomonas aeruginosa
strains, including one urinary isolate producing an extended-spectrum β-lactamase TEM-24, were isolated from a long-term-hospitalized woman. Three TEM-24-producing enterobacterial species (
Enterobacter aerogenes
,
Escherichia coli
, and
Proteus mirabilis
) were isolated from the same patient. TEM-24 and the resistance markers for aminoglycosides, chloramphenicol, and sulfonamide were encoded by a 180-kb plasmid transferred by conjugation into
E. coli
HB101.
In Vitro Activities of the New Antifungal Triazole SCH 56592 against Common and Emerging Yeast Pathogens
Authors: Barchiesi Francesco , Arzeni Daniela , Fothergill Annette W. , Di Francesco Luigi Falconi , Caselli Francesca , Rinaldi Michael G. , Scalise Giorgio
Published: April 2009

A broth microdilution method performed in accordance with the National Committee for Clinical Laboratory Standards guidelines was used to compare the in vitro activity of the new antifungal triazole SCH 56592 (SCH) to that of fluconazole (FLC), itraconazole (ITC), and ketoconazole (KETO) against 257 clinical yeast isolates. They included 220 isolates belonging to 12 different species of
Candida
, 15 isolates each of
Cryptococcus neoformans
and
Saccharomyces cerevisiae
, and seven isolates of
Rhodotorula rubra
. The MICs of SCH at which 50% (MIC
50
) and 90% (MIC
90
) of the isolates were inhibited were 0.06 and 2.0 μg/ml, respectively. In general, SCH was considerably more active than FLC (MIC
50
and MIC
90
of 1.0 and 64 μg/ml, respectively) and slightly more active than either ITC (MIC
50
and MIC
90
of 0.25 and 2.0 μg/ml, respectively) and KETO (MIC
50
and MIC
90
of 0.125 and 4.0 μg/ml, respectively). Our in vitro data suggest that SCH has significant potential for clinical development.
Activities of Synthetic Hybrid Peptides against Anaerobic Bacteria: Aspects of Methodology and Stability
Authors: Oh Herin , Hedberg Maria , Wade David , Edlund Charlotta
Published: April 2009

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. One line of investigation is the synthesis of antimicrobial hybrid peptides. The aim of the present investigation was to determine the in vitro activities of 16 cecropin-melittin hybrid peptides (CAMEL analogues) against 60 anaerobic bacterial strains, to compare their activities with those of seven clinically used antimicrobial agents, and to compare different methods for anaerobic susceptibility testing of these peptides. The stability of one of the peptides, temporin B, with different stereoisomeric configurations was investigated in a fecal milieu. The CAMEL analogues showed antimicrobial activity against the anaerobic bacteria, with MICs ranging from 0.125 to 32 μg/ml. The overall activities (the MICs at which 90% of isolates are inhibited) of the CAMEL analogues against anaerobic bacteria were mainly inferior to those of imipenem, clindamycin, and piperacillin but were equal to or superior to those of metronidazole, cefoxitin, ciprofloxacin, and chloramphenicol. The agarose dilution method was found to be an accurate method for the testing of large numbers of bacterial strains. The
d
isomer of temporin B was inactivated more slowly in feces than the
l
isomer. This study shows that the CAMEL analogues are potential agents for the treatment of anaerobic infections.
Induction of a Futile Embden-Meyerhof-Parnas Pathway in
Deinococcus radiodurans
by Mn: Possible Role of the Pentose Phosphate Pathway in Cell Survival
Authors: Zhang Y.-M. , Wong T.-Y. , Chen L.-Y. , Lin C.-S. , Liu J.-K.
Published: April 2009

Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium
Deinococcus radiodurans
. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in
D. radiodurans
. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO
2
. Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of
D. radiodurans
. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance.
Characterization of Two Inducible Phosphate Transport Systems in
Rhizobium tropici
Authors: Botero Lina M. , Al-Niemi Thamir S. , McDermott Timothy R.
Published: April 2009

Rhizobium tropici
forms nitrogen-fixing nodules on the roots of the common bean (
Phaseolus vulgaris
). Like other legume-
Rhizobium
symbioses, the bean-
R. tropici
association is sensitive to the availability of phosphate (P
i
). To better understand phosphorus movement between the bacteroid and the host plant, P
i
transport was characterized in
R. tropici
. We observed two P
i
transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn
5
B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The

K
m

and
V
max
values for the low-affinity system were estimated to be 34 ± 3 μM P
i
and 118 ± 8 nmol of P
i
· min
−1
· mg (dry weight) of cells
−1
, respectively, and the

K
m

and
V
max
values for the high-affinity system were 0.45 ± 0.01 μM P
i
and 86 ± 5 nmol of P
i
· min
−1
· mg (dry weight) of cells
−1
, respectively. Both systems were inducible by P
i
starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide
m
-chlorophenylhydrazone, but P
i
transport through both systems was eliminated by the ATPase inhibitor
N
,
N
′-dicyclohexylcarbodiimide; the P
i
transport rate was correlated with the intracellular ATP concentration. Also, P
i
movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P
i
transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with
kdpE
, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.
Evidence for Microbial Fe(III) Reduction in Anoxic, Mining-Impacted Lake Sediments (Lake Coeur d'Alene, Idaho)
Authors: Cummings David E. , March Anthony W. , Bostick Benjamin , Spring Stefan , Caccavo Frank , Fendorf Scott , Rosenzweig R. Frank
Published: April 2009

Mining-impacted sediments of Lake Coeur d'Alene, Idaho, contain more than 10% metals on a dry weight basis, approximately 80% of which is iron. Since iron (hydr)oxides adsorb toxic, ore-associated elements, such as arsenic, iron (hydr)oxide reduction may in part control the mobility and bioavailability of these elements. Geochemical and microbiological data were collected to examine the ecological role of dissimilatory Fe(III)-reducing bacteria in this habitat. The concentration of mild-acid-extractable Fe(II) increased with sediment depth up to 50 g kg
−1
, suggesting that iron reduction has occurred recently. The maximum concentrations of dissolved Fe(II) in interstitial water (41 mg liter
−1
) occurred 10 to 15 cm beneath the sediment-water interface, suggesting that sulfidogenesis may not be the predominant terminal electron-accepting process in this environment and that dissolved Fe(II) arises from biological reductive dissolution of iron (hydr)oxides. The concentration of sedimentary magnetite (Fe
3
O
4
), a common product of bacterial Fe(III) hydroxide reduction, was as much as 15.5 g kg
−1
. Most-probable-number enrichment cultures revealed that the mean density of Fe(III)-reducing bacteria was 8.3 × 10
5
cells g (dry weight) of sediment
−1
. Two new strains of dissimilatory Fe(III)-reducing bacteria were isolated from surface sediments. Collectively, the results of this study support the hypothesis that dissimilatory reduction of iron has been and continues to be an important biogeochemical process in the environment examined.
Purification and Partial Characterization of a Murein Hydrolase, Millericin B, Produced by
Streptococcus milleri
NMSCC 061
Authors: Beukes M. , Bierbaum G. , Sahl H.-G. , Hastings J. W.
Published: April 2009

Streptococcus milleri
NMSCC 061 was screened for antimicrobial substances and shown to produce a bacteriolytic cell wall hydrolase, termed millericin B. The enzyme was purified to homogeneity by a four-step purification procedure that consisted of ammonium sulfate precipitation followed by gel filtration, ultrafiltration, and ion-exchange chromatography. The yield following ion-exchange chromatography was 6.4%, with a greater-than-2,000-fold increase in specific activity. The molecular weight of the enzyme was 28,924 as determined by electrospray mass spectrometry. The amino acid sequences of both the N terminus of the enzyme (NH
2
SENDFSLAMVSN) and an internal fragment which was generated by cyanogen bromide cleavage (NH
2
SIQTNAPWGL) were determined by automated Edman degradation. Millericin B displayed a broad spectrum of activity against gram-positive bacteria but was not active against
Bacillus subtilis
W23 or
Escherichia coli
ATCC 486 or against the producer strain itself.
N
-Dinitrophenyl derivatization and hydrazine hydrolysis of free amino and free carboxyl groups liberated from peptidoglycan digested with millericin B followed by thin-layer chromatography showed millericin B to be an endopeptidase with multiple activities. It cleaves the stem peptide at the N terminus of glutamic acid as well as the N terminus of the last residue in the interpeptide cross-link of susceptible strains.
Purification and Characterization of 2,6-β-
d
-Fructan 6-Levanbiohydrolase from
Streptomyces exfoliatus
F3-2
Authors: Saito Katsuichi , Kondo Kazuya , Kojima Ichiro , Yokota Atsushi , Tomita Fusao
Published: April 2009

Streptomyces exfoliatus
F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from
S. exfoliatus
F3-2 was identified as a novel 2,6-β-
d
-fructan 6-levanbiohydrolase (EC
3.2.1.64
).
Detection and Characterization of Plasmid pJP4 Transfer to Indigenous Soil Bacteria
Authors: Newby D. T. , Josephson K. L. , Pepper I. L.
Published: April 2009

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism,
Escherichia coli
ATCC 15224, by plate mating with
Ralstonia eutropha
JMP134. Genes on this plasmid encode mercury resistance and partial 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The
E. coli
donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10
7
and 10
8
transconjugants g of dry soil
−1
for samples supplemented with 500 and 1,000 μg of 2,4-D g of dry soil
−1
, respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus
Burkholderia
or the genus
Pseudomonas
. No mercury-resistant, 2,4-D-degrading microorganisms containing large plasmids or the
tfdB
gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the
tfdB
gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.
Production and Comparison of Peptide Siderophores from Strains of Distantly Related Pathovars of
Pseudomonas syringae
and
Pseudomonas viridiflava
LMG 2352
Authors: Bultreys Alain , Gheysen Isabelle
Published: April 2009

The production of peptide siderophores and the variation in siderophore production among strains of
Pseudomonas syringae
and
Pseudomonas viridiflava
were investigated. An antibiose test was used to select a free amino acid-containing agar medium favorable for production of fluorescent siderophores by two
P. syringae
strains. A culture technique in which both liquid and solid asparagine-containing culture media were used proved to be reproducible and highly effective for inducing production of siderophores in a liquid medium by the fluorescent
Pseudomonas
strains investigated. Using asparagine as a carbon source appeared to favor siderophore production, and relatively high levels of siderophores were produced when certain amino acids were used as the sole carbon and energy sources. Purified chelated siderophores of strains of
P. syringae
pv. syringae,
P. syringae
pv. aptata,
P. syringae
pv. morsprunorum,
P. syringae
pv. tomato, and
P. viridiflava
had the same amino acid composition and spectral characteristics and were indiscriminately used by these strains. In addition, nonfluorescent strains of
P. syringae
pv. aptata and
P. syringae
pv. morsprunorum were able to use the siderophores in biological tests. Our results confirmed the proximity of
P. syringae
and
P. viridiflava
; siderotyping between pathovars of
P. syringae
was not possible. We found that the spectral characteristics of the chelated peptide siderophores were different from the spectral characteristics of typical pyoverdins. Our results are discussed in relation to the ecology of the organisms and the conditions encountered on plant surfaces.
Endo-Xylogalacturonan Hydrolase, a Novel Pectinolytic Enzyme
Authors: van der Vlugt-Bergmans C. J. B. , Meeuwsen P. J. A. , Voragen A. G. J. , van Ooyen A. J. J.
Published: April 2009

We screened an
Aspergillus tubingensis
expression library constructed in the yeast
Kluyveromyces lactis
for xylogalacturonan-hydrolyzing activity in microwell plates by using a bicinchoninic acid assay. This assay detects reducing carbohydrate groups when they are released from a carbohydrate by enzymatic activity. Two
K. lactis
recombinants exhibiting xylogalacturonan-hydrolyzing activity were found among the 3,400 colonies tested. The cDNA insert of these recombinants encoded a 406-amino-acid protein, designated XghA, which was encoded by a single-copy gene,
xghA
. A multiple-sequence alignment revealed that XghA was similar to both polygalacturonases (PGs) and rhamnogalacturonases. A detailed examination of conserved regions in the sequences of these enzymes revealed that XghA resembled PGs more. High-performance liquid chromatography and matrix-assisted laser desorption ionization–time of flight mass spectrometry of the products of degradation of xylogalacturonan and saponified modified hairy regions of apple pectin by XghA demonstrated that this enzyme uses an endo type of mechanism. XghA activity appeared to be specific for a xylose-substituted galacturonic acid backbone.
Antimicrobial Actions of Degraded and Native Chitosan against Spoilage Organisms in Laboratory Media and Foods
Authors: Rhoades J. , Roller S.
Published: April 2009

The objective of this study was to determine whether chitosan (poly-β-1,4-glucosamine) and hydrolysates of chitosan can be used as novel preservatives in foods. Chitosan was hydrolyzed by using oxidative-reductive degradation, crude papaya latex, and lysozyme. Mild hydrolysis of chitosan resulted in improved microbial inactivation in saline and greater inhibition of growth of several spoilage yeasts in laboratory media, but highly degraded products of chitosan exhibited no antimicrobial activity. In pasteurized apple-elderflower juice stored at 7°C, addition of 0.3 g of chitosan per liter eliminated yeasts entirely for the duration of the experiment (13 days), while the total counts and the lactic acid bacterial counts increased at a slower rate than they increased in the control. Addition of 0.3 or 1.0 g of chitosan per kg had no effect on the microbial flora of houmous, a chickpea dip; in the presence of 5.0 g of chitosan per kg, bacterial growth but not yeast growth was substantially reduced compared with growth in control dip stored at 7°C for 6 days. Improved antimicrobial potency of chitosan hydrolysates like that observed in the saline and laboratory medium experiments was not observed in juice and dip experiments. We concluded that native chitosan has potential for use as a preservative in certain types of food but that the increase in antimicrobial activity that occurs following partial hydrolysis is too small to justify the extra processing involved.
A New Chemolithoautotrophic Arsenite-Oxidizing Bacterium Isolated from a Gold Mine: Phylogenetic, Physiological, and Preliminary Biochemical Studies
Authors: Santini Joanne M. , Sly Lindsay I. , Schnagl Roger D. , Macy Joan M.
Published: April 2009

A previously unknown chemolithoautotrophic arsenite-oxidizing bacterium has been isolated from a gold mine in the Northern Territory of Australia. The organism, designated NT-26, was found to be a gram-negative motile rod with two subterminal flagella. In a minimal medium containing only arsenite as the electron donor (5 mM), oxygen as the electron acceptor, and carbon dioxide-bicarbonate as the carbon source, the doubling time for chemolithoautotrophic growth was 7.6 h. Arsenite oxidation was found to be catalyzed by a periplasmic arsenite oxidase (optimum pH, 5.5). Based upon 16S rDNA phylogenetic sequence analysis, NT-26 belongs to the
Agrobacterium/Rhizobium
branch of the α-
Proteobacteria
and may represent a new species. This recently discovered organism is the most rapidly growing chemolithoautotrophic arsenite oxidizer known.
Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress
Authors: Amor Kaouther Ben , Breeuwer Pieter , Verbaarschot Patrick , Rombouts Frank M. , Akkermans Antoon D. L. , De Vos Willem M. , Abee Tjakko
Published: April 2009

Using a flow cytometry-based approach, we assessed the viability of
Bifidobacterium lactis
DSM 10140 and
Bifidobacterium adolescentis
DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC
4
(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.
Utilization of Ganglioside-Degrading
Paenibacillus
sp. Strain TS12 for Production of Glucosylceramide
Authors: Sumida Tomomi , Sueyoshi Noriyuki , Ito Makoto
Published: April 2009

Gangliosides, sialic acid-containing glycosphingolipids, are membrane constituents of vertebrates and are known to have important roles in cellular differentiation, adhesion, and recognition. We report here the isolation of a bacterium capable of degrading gangliotetraose-series gangliosides and a new method for the production of glucosylceramide with this bacterium. GM1a ganglioside was found to be sequentially degraded by
Paenibacillus
sp. strain TS12, which was isolated from soil, as follows: GM1a → asialo GM1 → asialo GM2 → lactosylceramide → glucosylceramide. TS12 was found to produce a series of ganglioside-degrading enzymes, such as sialidases, β-galactosidases, and β-hexosaminidases. TS12 also produced β-glucosidases, but glucosylceramide was somewhat resistant to the bacterial enzyme under the conditions used. Taking advantage of the specificity, we developed a new method for the production of glucosylceramide using TS12 as a biocatalyst. The method involves the conversion of crude bovine brain gangliosides to glucosylceramide by coculture with TS12 and purification of the product by chromatography with Wakogel C-300 HG.
The Murein Hydrolase of the Bacteriophage φ3626 Dual Lysis System Is Active against All Tested
Clostridium perfringens
Strains
Authors: Zimmer Markus , Vukov Natǎsa , Scherer Siegfried , Loessner Martin J.
Published: April 2009

Clostridium perfringens
commonly occurs in food and feed, can produce an enterotoxin frequently implicated in food-borne disease, and has a substantial negative impact on the poultry industry. As a step towards new approaches for control of this organism, we investigated the cell wall lysis system of
C. perfringens
bacteriophage φ3626, whose dual lysis gene cassette consists of a holin gene and an endolysin gene. Hol3626 has two membrane-spanning domains (MSDs) and is a group II holin. A positively charged beta turn between the two MSDs suggests that both the amino terminus and the carboxy terminus of Hol3626 might be located outside the cell membrane, a very unusual holin topology. Holin function was experimentally demonstrated by using the ability of the holin to complement a deletion of the heterologous phage λ
S
holin in λΔSthf. The endolysin gene
ply3626
was cloned in
Escherichia coli
. However, protein synthesis occurred only when bacteria were supplemented with rare tRNA
Arg
and tRNA
Ile
genes. Formation of inclusion bodies could be avoided by drastically lowering the expression level. Amino-terminal modification by a six-histidine tag did not affect enzyme activity and enabled purification by metal chelate affinity chromatography. Ply3626 has an N-terminal amidase domain and a unique C-terminal portion, which might be responsible for the specific lytic range of the enzyme. All 48 tested strains of
C. perfringens
were sensitive to the murein hydrolase, whereas other clostridia and bacteria belonging to other genera were generally not affected. This highly specific activity towards
C. perfringens
might be useful for novel biocontrol measures in food, feed, and complex microbial communities.
Development of 16S rRNA-Gene-Targeted Group-Specific Primers for the Detection and Identification of Predominant Bacteria in Human Feces
Authors: Matsuki Takahiro , Watanabe Koichi , Fujimoto Junji , Miyamoto Yukiko , Takada Toshihiko , Matsumoto Kazumasa , Oyaizu Hiroshi , Tanaka Ryuichiro
Published: April 2009

For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the
Bacteroides fragilis
group
, Bifidobacterium,
the
Clostridium coccoides
group, and
Prevotella
were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the
B. fragilis
group, 22 isolates of
Bifidobacterium
, 65 isolates of the
C. coccoides
group, and 17 isolates of
Prevotella
, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of
Collinsella
, 24 isolates of the
Clostridium leptum
subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of
Bifidobacterium
is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol.
68:
2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.
Characterization of SixLeuconostoc fallaxBacteriophages Isolated from an Industrial Sauerkraut Fermentation
Authors: Barrangou Rodolphe , Yoon Sung-Sik , Breidt, Frederick , Fleming Henry P. , Klaenhammer Todd R.
Published: April 2009

Six bacteriophages active againstLeuconostoc fallaxstrains were isolated from industrial sauerkraut fermentation brines. These phages were characterized as to host range, morphology, structural proteins, and genome fingerprint. They were exclusively lytic against the speciesL. fallaxand had different host ranges among the strains of this species tested. Morphologically, three of the phages were assigned to the familySiphoviridae, and the three others were assigned to the familyMyoviridae. Major capsid proteins detected by electrophoresis were distinct for each of the two morphotypes. Restriction fragment length polymorphism analysis and randomly amplified polymorphic DNA fingerprinting showed that all six phages were genetically distinct. These results revealed for the first time the existence of bacteriophages that are active againstL. fallaxand confirmed the presence and diversity of bacteriophages in a sauerkraut fermentation. Since a variety ofL. fallaxstrains have been shown to be present in sauerkraut fermentation, bacteriophages active againstL. fallaxare likely to contribute to the microbial ecology of sauerkraut fermentation and could be responsible for some of the variability observed in this type of fermentation.
Molecular Characterization of Cadmium Resistance inStreptococcus thermophilusStrain 4134: an Example of Lateral Gene Transfer
Authors: Schirawski Jan , Hagens Werner , Fitzgerald Gerald F. , van Sinderen Douwe
Published: April 2009

Two genes (cadCStandcadASt[subscriptStrepresentsStreptococcus thermophilus]), located on the chromosome ofS. thermophilus4134, were shown to constitute a cadmium/zinc resistance cassette. The genes seem to be organized in an operon, and their transcription is cadmium dependent in vivo. The proposed product of thecadAopen reading frame (CadASt) is highly similar to P-type cadmium efflux ATPases, whereas the predicted protein encoded bycadCSt(CadCSt) shows high similarity to ArsR-type regulatory proteins. The observed homologies and G+C content of this cassette and surrounding regions suggest that this DNA was derived fromLactococcus lactisand may have been introduced relatively recently into theS. thermophilus4134 genome by a lateral gene transfer event. The complete cassette confers cadmium and zinc resistance to bothS. thermophilusandL. lactis, but expression ofcadAStalone is sufficient to give resistance. By using electrophoretic mobility shift assays it was shown that the CadCStprotein is a DNA binding protein that binds specifically to its own promoter region, possibly to two copies of an inverted repeat, and that this CadCSt-DNA interaction is lost in the presence of cadmium. UsinglacZfusion constructs it was shown that the cadmium-dependent expression of CadAStis mediated by the negative regulator CadCSt. A model for the regulation of the expression of cadmium resistance inS. thermophilusis discussed.
Isolation and Molecular Analysis of the Gene Cluster for the Arginine Deiminase System from
Streptococcus gordonii
DL1
Authors: Dong Yiqian , Chen Yi-Ywan M. , Snyder Jennifer A. , Burne R. A.
Published: April 2009

The arginine deiminase (AD) system (ADS) is one of two major ammonia-generating pathways in the oral cavity that play important roles in oral biofilm pH homeostasis and oral biofilm ecology. To initiate a study of the
Streptococcus gordonii
ADS, the ADS gene cluster was isolated from subgenomic DNA libraries of
S. gordonii
DL1 by using an
arcB
-specific probe. Nucleotide sequence analysis revealed six open reading frames (ORFs) that were arranged contiguously; the first five ORFs were transcribed in the same direction, as an apparent operon, and the sixth was transcribed in the opposite direction. The ORFs were found to share significant homologies and to correspond closely in molecular mass to previously characterized
arc
genes; thus, they were designated
arcA
(AD)
, arcB
(ornithine carbamyltransferase)
, arcC
(carbamate kinase)
, arcD
(arginine-ornithine antiporter)
, arcT
(dipeptidase), and
arcR
(regulator). A putative σ
70
promoter (
ParcA
[TTGTGT-N
19
-TAGAAT]) was mapped 5′ to
arcA
by primer extension, and the expression of
ParcA
was shown to be inducible by arginine and repressible by glucose, in agreement with AD specific activities measured in the wild-type strain. To investigate the function of ArcR in the differential expression of the
arc
operon,
arcR
was insertionally inactivated by a KM resistance marker flanked by T4 transcription/translation termination signals, and the expression of
ParcA
was monitored by primer extension in the wild-type and ArcR-deficient strains. Lower levels of
arcA
expression, as well as lower levels of AD activity, were consistently observed in the ArcR-deficient strain compared to wild-type cells, regardless of the growth conditions. Thus, ArcR is a transcriptional activator that is required for induction and optimal expression of the
S. gordonii
ADS gene cluster.
Effect of Soil Ammonium Concentration on N
2
O Release and on the Community Structure of Ammonia Oxidizers and Denitrifiers
Authors: Avrahami Sharon , Conrad Ralf , Braker Gesche
Published: April 2009

The effect of ammonium addition (6.5, 58, and 395 μg of NH
4
+
-N g [dry weight] of soil
−1
) on soil microbial communities was explored. For medium and high ammonium concentrations, increased N
2
O release rates and a shift toward a higher contribution of nitrification to N
2
O release occurred after incubation for 5 days at 4°C. Communities of ammonia oxidizers were assayed after 4 weeks of incubation by denaturant gradient gel electrophoresis (DGGE) of the
amoA
gene coding for the small subunit of ammonia monooxygenase. The DGGE fingerprints were invariably the same whether the soil was untreated or incubated with low, medium, or high ammonium concentrations. Phylogenetic analysis of cloned PCR products from excised DGGE bands detected
amoA
sequences which probably belonged to
Nitrosospira
16S rRNA clusters 3 and 4. Additional clones clustered with
Nitrosospira
sp. strains Ka3 and Ka4 and within an
amoA
cluster from unknown species. A
Nitrosomonas-
like
amoA
gene was detected in only one clone. In agreement with the
amoA
results, community profiles of total bacteria analyzed by terminal restriction fragment length polymorphism (T-RFLP) showed only minor differences. However, a community shift occurred for denitrifier populations based on T-RFLP analysis of
nirK
genes encoding copper-containing nitrite reductase with incubation at medium and high ammonia concentrations. Major terminal restriction fragments observed in environmental samples were further described by correspondence to cloned
nirK
genes from the same soil. Phylogenetic analysis grouped these clones into clusters of soil
nirK
genes. However, some clones were also closely related to genes from known denitrifiers. The shift in the denitrifier community was probably the consequence of the increased supply of oxidized nitrogen through nitrification. Nitrification activity increased upon addition of ammonium, but the community structure of ammonium oxidizers did not change.
Differential Enantioselective Transformation of Atropisomeric Polychlorinated Biphenyls by Multiple Bacterial Strains with Different Inducing Compounds
Authors: Singer Andrew C. , Wong Charles S. , Crowley David E.
Published: April 2009

Five polychlorinated biphenyl (PCB)-degrading bacteria were tested for the ability to differentiate between the enantiomers of four atropisomeric PCB congeners (2,2′,3,6-tetra-CB; 2,2′,3,3′,6-penta-CB; 2,2′,3,4′,6-penta-CB; and 2,2′,3,5′,6-penta-CB) after growth in the presence of tryptone-soytone, biphenyl, carvone, or cymene. Enantioselectivity was shown to vary with respect to strain, congener, and cosubstrate.
Diversity of Diazotrophic Unicellular Cyanobacteria in the Tropical North Atlantic Ocean
Authors: Falcón Luisa I. , Cipriano Frank , Chistoserdov Andrei Y. , Carpenter Edward J.
Published: April 2009

We present data on the genetic diversity and phylogenetic affinities of N
2
-fixing unicellular cyanobacteria in the plankton of the tropical North Atlantic Ocean. Our dinitrogenase gene (
nifH
) sequences grouped together with a group of cyanobacteria from the subtropical North Pacific; another subtropical North Pacific group was only distantly related. Most of the 16S ribosomal DNA sequences from our tropical North Atlantic samples were closely allied with sequences from a symbiont of the diatom
Climacodium frauenfeldianum
. These findings suggest a complex pattern of evolutionary and ecological divergence among unicellular cyanobacteria within and between ocean basins.
Anti-Alpha-Hemolysin Monoclonal Antibodies Mediate Protection againstStaphylococcus aureusPneumonia
Authors: Ragle Brook E. , Bubeck Wardenburg Juliane
Published: April 2009

Staphylococcus aureuspneumonia is one of the most common invasive diseases caused by this human pathogen.S. aureusalpha-hemolysin, a pore-forming cytotoxin, is an essential virulence factor in the pathogenesis of pneumonia. Vaccine-based targeting of this toxin provides protection against lethal staphylococcal pneumonia in a murine model system, suggesting that a monoclonal antibody-based therapy may likewise prove to be efficacious for prevention and treatment of this disease. We report the generation of two distinct anti-alpha-hemolysin monoclonal antibodies that antagonize toxin activity, preventing human lung cell injury in vitro and protecting experimental animals against lethalS. aureuspneumonia. Each of these two monoclonal antibodies recognized an epitope within the first 50 amino acid residues of the mature toxin and blocked the formation of a stable alpha-hemolysin oligomer on the target cell surface. Active immunization with the first 50 amino acids of the toxin also conferred protection againstS. aureuspneumonia. Together, these data reveal passive and active immunization strategies for prevention or therapy of staphylococcal pneumonia and highlight the potential role that a critical epitope may play in defining human susceptibility to this deadly disease.
The foundations of London Bridge
Authors: Nash J. K. T. L.
Published: April 2009

A bridge across the river Thames has existed at London for 1000 years and possibly more. The early bridges were built on timber piles but these were subject to constant erosion. In 1176 Peter of Colechurch built a masonry bridge on piers which rested on starlings and this lasted through various vicissitudes for over 600 years. Constant maintenance of the foundations was required and in 1831 this was eventually replaced by Rennie's famous bridge which lasted for 140 years. This bridge was founded on a timber grillage which rested on timber piles. Immediately after it was completed it was carefully examined by three independent engineers, relative levels were taken and the same points on the piers were re-levelled after 30 years and again after 130 years. Due to high applied stresses, continuing secondary compression of the clay took place and the resulting differential settlement eventually led to the bridge being replaced. Rennie's bridge was rebuilt in Arizona in 1973.
Fourth Canadian Geotechnical Colloquium: Strength and slope stability in Canadian soft clay deposits
Authors: Lefebvre Guy
Published: April 2009

It is generally admitted that the long term stability of clay slopes should be analysed in terms of effective stresses, which necessitates the knowledge of the pore pressure conditions and of the relationship between the shear strength and the effective stresses. This paper deals with the determination of shear strength for stability analysis of clay slopes in Eastern Canada. First the various concepts pertinent to slope stability in soft clay are examined and a certain justification of the use of the post-peak or large strain strength is demonstrated. In a second part, 14 case records of natural slopes or permanent excavation slopes are presented and analysed in terms of the post-peak strength. Finally, values of post-peak strength determined in the laboratory for more than 30 sites are presented and it is attempted to derive correlations by which the shear strength parameters could be readily approximated from other properties such as the apparent preconsolidation pressure or the vane strength.
An empirical evaluation of pressuremeter test data
Authors: Arnold M.
Published: April 2009

In situ pressuremeter tests have in recent years come into wide use in the determination of the parameters to be used for foundation design. Although theoretically correct analytical expressions have been developed for the radial expansion pressure versus radial strain relationship, these are generally cumbersome and difficult to use and in practice a graphical procedure is commonly used to derive the maximum undrained shear stress and the undrained modulus value from the test data.To overcome the disadvantages that this graphical procedure involves an empirically-derived analytical relationship based on Kondner's hyperbolic stress–strain relationship is presented here, by means of which all the derived parameters can be obtained simply and without recourse to graphical construction.
THE EFFECTS OF UNCERTAINTY ON THE VOLUNTARY PRIVATE PROVISION OF IMPURE PUBLIC GOODS
Authors: KIM ILTAE
Published: April 2009

This paper examines the effects of uncertainty on an individual's own contribution to the provision of the collective good using an impure public good model. Two types of uncertainty analyzing free-riding behavior are evaluated: (i) uncertainty surrounding the contributions of others to the public characteristic and (ii) uncertainty surrounding the response of others to an individual's own contribution. We extend previous studies by examining both the compensated and uncompensated effects of increases in such risks on the provision of the collective good. We also establish the conditions that are sufficient to determine both compensated and the total, uncompensated effects of an increase in risk on the voluntary provision of the collective good.
VALUATION OF EUROPEAN INSTALLMENT PUT OPTION: VARIATIONAL INEQUALITY APPROACH
Authors: YANG ZHOU , YI FAHUAI
Published: April 2009

In this paper, we consider a parabolic variational inequality arising from the valuation of European installment put option. We prove the existence and uniqueness of the solution to the problem. Moreover, we obtain C∞ regularity and the bounds of the free boundary. Eventually, we show its numerical result by the binomial method.