bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutant Prevention Concentrations of Fluoroquinolones for Clinical Isolates of
Streptococcus pneumoniae
Authors: Blondeau Joseph M. , Zhao Xilin , Hansen Glen , Drlica Karl 
Published: July 2002

ABSTRACT

The mutant prevention concentration (MPC) represents a threshold above which the selective proliferation of resistant mutants is expected to occur only rarely. A provisional MPC (MPC
pr
) was defined and measured for five fluoroquinolones with clinical isolates of
Streptococcus pneumoniae
. Based on their potential for restricting the selection of resistant mutants, the five fluoroquinolones, in descending order, were found to be moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of about 90 clinical isolates that lacked a known resistance mutation had a value of MPC
pr
that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. Since MPC
pr
overestimates MPC, these data identify moxifloxacin and gatifloxacin as good candidates for determining whether MPC
pr
can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of resistance.
Resistance to Moxifloxacin in Toxigenic
Clostridium difficile
Isolates Is Associated with Mutations in
gyrA
Authors: Ackermann Grit , Tang Yajarayma J. , Kueper Robert , Heisig Peter , Rodloff Arne C. , Silva Joseph , Cohen Stuart H. 
Published: July 2002

ABSTRACT

Clostridium difficile
is the etiological agent of antibiotic-associated colitis and the most common cause of hospital-acquired infectious diarrhea. Fluoroquinolones such as ciprofloxacin are associated with lower risks of
C. difficile
-associated diarrhea. In this study, we have analyzed 72
C. difficile
isolates obtained from patients with different clinical courses of disease, such as toxic megacolon and relapses; the hospital environment; public places; and horses. They were investigated for their susceptibilities to moxifloxacin (MXF), metronidazole (MEO), and vancomycin (VAN). Mutants highly resistant to fluoroquinolones were selected in vitro by stepwise exposure to increasing concentrations of MXF. The resulting mutants were analyzed for the presence of mutations in the quinolone resistance-determining regions of DNA gyrase (
gyrA
), the production of toxins A and B, and the epidemiological relationship of these isolates. These factors were also investigated using PCR-based methods. All strains tested were susceptible to MEO and VAN. Twenty-six percent of the clinical isolates (19 of 72) were highly resistant to MXF (MIC ≥ 16 μg/ml). Fourteen of these 19 strains contained nucleotide changes resulting in amino acid substitutions at position 83 in the
gyrA
protein. Resistant strains selected in vitro did not contain mutations at that position. These findings indicate that resistance to MXF in a majority of cases may be due to amino acid substitution in the
gyrA
gene.
Antifungal Activity of Eupolauridine and Its Action on DNA Topoisomerases
Authors: Khan Shabana I. , Nimrod Alison C. , Mehrpooya Mohammed , Nitiss John L. , Walker Larry A. , Clark Alice M. 
Published: July 2002

ABSTRACT

The azafluoranthene alkaloid eupolauridine has previously been shown to have in vitro antifungal activity and selective inhibition of fungal topoisomerase I. The present study was undertaken to examine further its selectivity and mode of action. Eupolauridine completely inhibits the DNA relaxation activity of purified fungal topoisomerase I at 50 μg/ml, but it does not stabilize the cleavage complex of either human or fungal topoisomerase I. Cleavage complex stabilization is the mode of action of topoisomerase I targeting drugs of the camptothecin family. Also, unlike camptothecin, eupolauridine does not cause significant cytotoxicity in mammalian cells. To determine if the inhibition of topoisomerase I is the principal mode of antifungal action of eupolauridine,
Saccharomyces cerevisiae
strains with alterations in topoisomerase genes were used in clonogenic assays. The antifungal activity of eupolauridine was not diminished in the absence of topoisomerase I; rather, the cells lacking the enzyme were more sensitive to the drug. Cell-killing activity of eupolauridine was also more pronounced in cells that overexpressed topoisomerase II. In vitro assays with the purified yeast enzyme confirmed that eupolauridine stabilized topoisomerase II covalent complexes. These results indicate that a major target for fungal cell killing by eupolauridine is DNA topoisomerase II rather than topoisomerase I, but does not exclude the possibility that the drug also acts against other targets.
MET17and Hydrogen Sulfide Formation inSaccharomyces cerevisiae
Authors: Spiropoulos Apostolos , Bisson Linda F. 
Published: July 2002

ABSTRACTCommercial isolates ofSaccharomyces cerevisiaediffer in the production of hydrogen sulfide (H2S) during fermentation, which has been attributed to variation in the ability to incorporate reduced sulfur into organic compounds. We transformed two commercial strains (UCD522 and UCD713) with a plasmid overexpressing theMET17gene, which encodes the bifunctionalO-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase), to test the hypothesis that the level of activity of this enzyme limits reduced sulfur incorporation, leading to H2S release. Overexpression ofMET17resulted in a 10- to 70-fold increase in OAS/OAH SHLase activity in UCD522 but had no impact on the level of H2S produced. In contrast, OAS/OAH SHLase activity was not as highly expressed in transformants of UCD713 (0.5- to 10-fold) but resulted in greatly reduced H2S formation. Overexpression of OAS/OAH SHLase activity was greater in UCD713 when grown under low-nitrogen conditions, but the impact on reduction of H2S was greater under high-nitrogen conditions. Thus, there was not a good correlation between the level of enzyme activity and H2S production. We measured cellular levels of cysteine to determine the impact of overexpression of OAS/OAH SHLase activity on sulfur incorporation. While Met17p activity was not correlated with increased cysteine production, conditions that led to elevated cytoplasmic levels of cysteine also reduced H2S formation. Our data do not support the simple hypothesis that variation in OAS/OAH SHLase activity is correlated with H2S production and release.
Inhibition of Luminescence and Virulence in the Black Tiger Prawn (
Penaeus monodon
) Pathogen
Vibrio harveyi
by Intercellular Signal Antagonists
Authors: Manefield Michael , Harris Lachlan , Rice Scott A. , de Nys Rocky , Kjelleberg Staffan 
Published: July 2002

ABSTRACT

Expression of luminescence in the
Penaeus monodon
pathogen
Vibrio harveyi
is regulated by an intercellular quorum sensing mechanism involving the synthesis and detection of two signaling molecules, one of which is
N
-hydroxy butanoyl-
l
-homoserine lactone and the other of which is uncharacterized. Indirect evidence has suggested that virulence, associated with a toxic extracellular protein, and luminescence in
V. harveyi
are coregulated. In this study the effects of an acylated homoserine lactone antagonist produced by the marine alga
Delisea pulchra
on luminescence and toxin production in a virulent strain of
V. harveyi
were analyzed. Luminescence and toxin production were both inhibited by the signal antagonist at concentrations that had no impact on growth. Toxin production was found to be prematurely induced in
V. harveyi
cultures incubated in a 10% conditioned medium. Additionally, a significant reduction in the toxicity of concentrated supernatant extracts from
V. harveyi
cultures incubated in the presence of the signal antagonist, as measured by in vivo toxicity assays in mice and prawns, was observed. These results suggest that intercellular signaling antagonists have potential utility in the control of
V. harveyi
prawn infections.
Activities of Garlic Oil, Garlic Powder, and Their Diallyl Constituents against
Helicobacter pylori
Authors: O'Gara E. A. , Hill D. J. , Maslin D. J. 
Published: July 2002

ABSTRACT

Chronic
Helicobacter pylori
disease is reduced with
Allium
vegetable intake. This study was designed to assess the in vivo anti-
H. pylori
potential of a variety of garlic substances. The garlic materials all showed substantial but widely differing anti-
H. pylori
effects against all strains and isolates tested. The MICs (range, 8 to 32 μg/ml) and minimum bactericidal concentrations (MBCs) (range, 16 to 32 μg/ml) of undiluted garlic oil (GO) were smaller than those of garlic powder (GP) (MIC range, 250 to 500 μg/ml; MBC range, 250 to 500 μg/ml) but greater than the MIC of allicin (4.0 μg/ml) (Table 2) present in GP. Allicin (MIC, 6 μg/ml; MBC, 6 μg/ml) was more potent than diallyl disulfide (MIC range, 100 to 200 μg/ml; MBC range, 100 to 200 μg/ml), its corresponding sulfide, but of a strength similar to that of diallyl tetrasulfide (MIC range, 3 to 6 μg/ml; MBC range, 3 to 6 μg/ml). Antimicrobial activity of the diallyl sulfides increased with the number of sulfur atoms. Time course viability studies and microscopy showed dose-dependent anti-
H. pylori
effects with undiluted GO, GP, allicin, and diallyl trisulfide after a lag phase of ca. 1 to 2 h. Substantial in vitro anti-
H. pylori
effects of pure GO and GP and their diallyl sulfur components exist, suggesting their potential for in vivo clinical use against
H. pylori
infections.
Detection of
Ralstonia solanacearum
Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay
Authors: Weller S. A. , Elphinstone J. G. , Smith N. C. , Boonham N. , Stead D. E. 
Published: July 2002

ABSTRACT

A fluorogenic (TaqMan) PCR assay was developed to detect
Ralstonia solanacearum
strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of
R. solanacearum
, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of
Taq
DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42
R. solanacearum
and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of
R. solanacearum
to ≥10
2
cells ml
−1
was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of
R. solanacearum
.
Unlabeled Helper Oligonucleotides Increase the In Situ Accessibility to 16S rRNA of Fluorescently Labeled Oligonucleotide Probes
Authors: Fuchs Bernhard M. , Glöckner Frank Oliver , Wulf Jörg , Amann Rudolf 
Published: July 2002

ABSTRACT

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in
Escherichia coli
DSM 30083
T
. The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for
E. coli
. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.
Relative Contributions of Bacteria, Protozoa, and Fungi to In Vitro Degradation of Orchard Grass Cell Walls and Their Interactions
Authors: Lee S. S. , Ha J. K. , Cheng K.-J. 
Published: July 2002

ABSTRACT
To assess the relative contributions of microbial groups (bacteria, protozoa, and fungi) in rumen fluids to the overall process of plant cell wall digestion in the rumen, representatives of these groups were selected by physical and chemical treatments of whole rumen fluid and used to construct an artificial rumen ecosystem. Physical treatments involved homogenization, centrifugation, filtration, and heat sterilization. Chemical treatments involved the addition of antibiotics and various chemicals to rumen fluid. To evaluate the potential activity and relative contribution to degradation of cell walls by specific microbial groups, the following fractions were prepared: a positive system (whole ruminal fluid), a bacterial (B) system, a protozoal (P) system, a fungal (F) system, and a negative system (cell-free rumen fluid). To assess the interactions between specific microbial fractions, mixed cultures (B+P, B+F, and P+F systems) were also assigned. Patterns of degradation due to the various treatments resulted in three distinct groups of data based on the degradation rate of cell wall material and on cell wall-degrading enzyme activities. The order of degradation was as follows: positive and F systems > B system > negative and P systems. Therefore, fungal activity was responsible for most of the cell wall degradation. Cell wall degradation by the anaerobic bacterial fraction was significantly less than by the fungal fraction, and the protozoal fraction failed to grow under the conditions used. In general, in the mixed culture systems the coculture systems demonstrated a decrease in cellulolysis compared with that of the monoculture systems. When one microbial fraction was associated with another microbial fraction, two types of results were obtained. The protozoal fraction inhibited cellulolysis of cell wall material by both the bacterial and the fungal fractions, while in the coculture between the bacterial fraction and the fungal fraction a synergistic interaction was detected.
Distribution of Oxytetracycline Resistance Plasmids between Aeromonads in Hospital and Aquaculture Environments: Implication of Tn
1721
in Dissemination of the Tetracycline Resistance Determinant Tet A
Authors: Rhodes Glenn , Huys Geert , Swings Jean , Mcgann Patrick , Hiney Maura , Smith Peter , Pickup Roger W. 
Published: July 2002

ABSTRACT

Oxytetracycline-resistant (OT
r
) mesophilic aeromonads were recovered from untreated hospital effluent (72 isolates) and from fish farm hatchery tanks (91 isolates) at sites within the English Lake District, Cumbria, England. The transfer of OT
r
plasmids from these isolates was investigated. Using
Escherichia coli
J53-1 as a recipient, 11 isolates from the hospital site and 6 isolates from the fish farm site transferred OT
r
plasmids (designated pFBAOT1 to 17). Original isolates were identified using fatty acid methyl ester and fluorescent amplified fragment length polymorphism comparisons as either
Aeromonas hydrophila
HG3 (eight isolates),
A. veronii
b.v. sobria HG8 (six isolates), and
A. caviae
HGB5 (one isolate). One isolate remained unidentified, and one could not be assigned a taxonomic designation beyond the genus level. Plasmids pFBAOT1 to -17 were screened for the presence of the tetracycline resistance determinants Tet A to E and Tet G. Only determinant Tet A (10 plasmids) was detected in these plasmids, with 7
tet
gene determinants remaining unclassified. In all cases, Tet A was located on a 5.5-kb
Eco
RI restriction fragment. Hybridization with
inc-rep
probes N, P, Q, W, and U showed pFBAOT3, -4, -5, -6, -7, -9, and -11, from the hospital environment, to be IncU plasmids. Further, restriction fragment length polymorphism (RFLP) analyses and DNA probing demonstrated that pFBAOT plasmids were closely related to IncU OT
r
plasmids pASOT, pASOT2, pASOT3, pRAS1 (originally isolated from
A. salmonicida
strains from fish farms in Scotland and Norway, respectively), and pIE420 (isolated from a German hospital
E. coli
strain). In addition, DNA analyses demonstrated that plasmids pRAS1 and pIE420 had identical RFLP profiles and that all fragments hybridized to each other. The presence of tetracycline resistance transposon Tn
1721
in its entirety or in a truncated form in these plasmids was demonstrated. These results provided direct evidence that related tetracycline resistance-encoding plasmids have disseminated between different
Aeromonas
species and
E. coli
and between the human and aquaculture environments in distinct geographical locations. Collectively, these findings provide evidence to support the hypothesis that the aquaculture and human compartments of the environment behave as a single interactive compartment.
Toluene-Degrading Bacteria Are Chemotactic towards the Environmental Pollutants Benzene, Toluene, and Trichloroethylene
Authors: Parales Rebecca E. , Ditty Jayna L. , Harwood Caroline S. 
Published: July 2002

ABSTRACT

The bioremediation of polluted groundwater and toxic waste sites requires that bacteria come into close physical contact with pollutants. This can be accomplished by chemotaxis. Five motile strains of bacteria that use five different pathways to degrade toluene were tested for their ability to detect and swim towards this pollutant. Three of the five strains (
Pseudomonas putida
F1,
Ralstonia pickettii
PKO1, and
Burkholderia cepacia
G4) were attracted to toluene. In each case, the response was dependent on induction by growth with toluene.
Pseudomonas mendocina
KR1 and
P. putida
PaW15 did not show a convincing response. The chemotactic responses of
P. putida
F1 to a variety of toxic aromatic hydrocarbons and chlorinated aliphatic compounds were examined. Compounds that are growth substrates for
P. putida
F1, including benzene and ethylbenzene, were chemoattractants.
P. putida
F1 was also attracted to trichloroethylene (TCE), which is not a growth substrate but is dechlorinated and detoxified by
P. putida
F1. Mutant strains of
P. putida
F1 that do not oxidize toluene were attracted to toluene, indicating that toluene itself and not a metabolite was the compound detected. The two-component response regulator pair TodS and TodT, which control expression of the toluene degradation genes in
P. putida
F1, were required for the response. This demonstration that soil bacteria can sense and swim towards the toxic compounds toluene, benzene, TCE, and related chemicals suggests that the introduction of chemotactic bacteria into selected polluted sites may accelerate bioremediation processes.
Growth of
Pseudomonas mendocina
on Fe(III) (Hydr)Oxides
Authors: Hersman L. E. , Forsythe J. H. , Ticknor L. O. , Maurice P. A. 
Published: July 2002

ABSTRACT

Although iron (Fe) is an essential element for almost all living organisms, little is known regarding its acquisition from the insoluble Fe(III) (hydr)oxides in aerobic environments. In this study a strict aerobe,
Pseudomonas mendocina
, was grown in batch culture with hematite, goethite, or ferrihydrite as a source of Fe.
P. mendocina
obtained Fe from these minerals in the following order: goethite > hematite > ferrihydrite. Furthermore, Fe release from each of the minerals appears to have occurred in excess, as evidenced by the growth of
P. mendocina
in the medium above that of the insoluble Fe(III) (hydr)oxide aggregates, and this release was independent of the mineral's surface area. These results demonstrate that an aerobic microorganism was able to obtain Fe for growth from several insoluble Fe minerals and did so with various growth rates.
Variation in Susceptibility to
Bacillus thuringiensis
Toxins among Unselected Strains of
Plutella xylostella
Authors: González-Cabrera J. , Herrero S. , Sayyed A. H. , Escriche B. , Liu Y. B. , Meyer S. K. , Wright D. J. , Tabashnik B. E. , Ferré J. 
Published: July 2002

ABSTRACT

So far, the only insect that has evolved resistance in the field to
Bacillus thuringiensis
toxins is the diamondback moth (
Plutella xylostella
). Documentation and analysis of resistant strains rely on comparisons with laboratory strains that have not been exposed to
B. thuringiensis
toxins. Previously published reports show considerable variation among laboratories in responses of unselected laboratory strains to
B. thuringiensis
toxins. Because different laboratories have used different unselected strains, such variation could be caused by differences in bioassay methods among laboratories, genetic differences among unselected strains, or both. Here we tested three unselected strains against five
B. thuringiensis
toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, and Cry1Da) using two bioassay methods. Tests of the LAB-V strain from The Netherlands in different laboratories using different bioassay methods yielded only minor differences in results. In contrast, side-by-side comparisons revealed major genetic differences in susceptibility between strains. Compared with the LAB-V strain, the ROTH strain from England was 17- to 170-fold more susceptible to Cry1Aa and Cry1Ac, respectively, whereas the LAB-PS strain from Hawaii was 8-fold more susceptible to Cry1Ab and 13-fold more susceptible to Cry1Da and did not differ significantly from the LAB-V strain in response to Cry1Aa, Cry1Ac, or Cry1Ca. The relative potencies of toxins were similar among LAB-V, ROTH, and LAB-PS, with Cry1Ab and Cry1Ac being most toxic and Cry1Da being least toxic. Therefore, before choosing a standard reference strain upon which to base comparisons, it is highly advisable to perform an analysis of variation in susceptibility among field and laboratory populations.
Role of Intermolecular Disulfide Bonds of the Organic Solvent-Stable PST-01 Protease in Its Organic Solvent Stability
Authors: Ogino Hiroyasu , Uchiho Takeshi , Yokoo Jyunko , Kobayashi Reina , Ichise Rikiya , Ishikawa Haruo 
Published: July 2002

ABSTRACT

The PST-01 protease is secreted by the organic solvent-tolerant microorganism
Pseudomonas aeruginosa
PST-01 and is stable in the presence of various organic solvents. Therefore, the PST-01 strain and the PST-01 protease are very useful for fermentation and reactions in the presence of organic solvents, respectively. The organic solvent-stable PST-01 protease has two disulfide bonds (between Cys-30 and Cys-58 and between Cys-270 and Cys-297) in its molecule. Mutant PST-01 proteases in which one or both of the disulfide bonds were deleted were constructed by site-directed mutagenesis, and the effect of the disulfide bonds on the activity and the various stabilities was investigated. The disulfide bond between Cys-270 and Cys-297 in the PST-01 protease was found to be essential for its activity. The disulfide bond between Cys-30 and Cys-58 played an important role in the organic solvent stability of the PST-01 protease.