bluuebunny/crossref_metadata_2025_split huggingface dataset:
MexXY-OprM Efflux Pump Is Required for Antagonism of Aminoglycosides by Divalent Cations in
Pseudomonas aeruginosa
Authors: Mao Weimin , Warren Mark S. , Lee Angela , Mistry Anita , Lomovskaya Olga 
Published: July 2002

ABSTRACT

Antagonism of aminoglycosides by divalent cations is well documented for
Pseudomonas aeruginosa
and is regarded as one of the problems in aminoglycoside therapy. It is generally considered that divalent cations interfere with uptake of aminoglycosides at both the outer and inner membranes. It has been demonstrated recently that aminoglycosides can be removed from cells of
P. aeruginosa
by the three-component multidrug resistance efflux pump MexXY-OprM. We sought to investigate the interplay between efflux and uptake in resistance to aminoglycosides in
P. aeruginosa
. To do so, we studied the effects of the divalent cations Mg
2+
and Ca
2+
on susceptibility to aminoglycosides in a wild-type strain of
P. aeruginosa
and in mutants either overexpressing or lacking the MexXY-OprM efflux pump. MICs of gentamicin, streptomycin, amikacin, apramycin, netilmicin, and arbekacin were determined in Mueller-Hinton broth in the presence of cations added at concentrations that varied from 0.125 to 8 mM. We found, unexpectedly, that while both Mg
2+
and Ca
2+
antagonized aminoglycosides (up to a 64-fold decrease in susceptibility at 8 mM), antagonism was seen only in the strains of
P. aeruginosa
that contained the functional MexXY-OprM efflux pump. Our results indicate that inhibition of the MexXY-OprM efflux pump should abolish the antagonism of aminoglycosides by divalent cations, regardless of its precise mechanism. This may significantly increase the therapeutic index of aminoglycosides and improve the clinical utility of this important class of antibiotics.
Molecular and Biochemical Characterization of Ambler Class A Extended-Spectrum β-Lactamase CGA-1 from
Chryseobacterium gleum
Authors: Bellais Samuel , Naas Thierry , Nordmann Patrice 
Published: July 2002

ABSTRACT

Antibiotic susceptibility testing by disk diffusion of a
Chryseobacterium gleum
isolate, strain CIP 103039, showed a typical synergy image between clavulanic acid and expanded-spectrum cephalosporins. Shotgun cloning gave a recombinant plasmid in
Escherichia coli
that produced a β-lactamase, CGA-1, with a pI value of 8.9 that conferred resistance to most penicillins (except ureidopenicillins) and narrow-spectrum cephalosporins and an intermediate susceptibility to expanded-spectrum cephalosporins and aztreonam. The CGA-1 amino acid sequence shared only 60% amino acid identity with CME-1 and CME-2 from
Chryseobacterium meningosepticum
, the most closely related β-lactamases. CGA-1 was very likely chromosome encoded. It is a novel member of the PER subgroup of Ambler class A β-lactamases (Bush functional group 2be).
Oral Administration of Cyclopentane Neuraminidase Inhibitors Protects Ferrets against Influenza Virus Infection
Authors: Sweet Clive , Jakeman Kenneth J. , Bush Karen , Wagaman Pamela C. , Mckown Linda A. , Streeter Anthony J. , Desai-Krieger Daksha , Chand Pooran , Babu Yarlagadda S. 
Published: July 2002

ABSTRACT
Several cyclopentane inhibitors of influenza virus neuraminidase that have inhibitory activities in tissue culture similar to those of zanamivir and oseltamivir have recently been described. These new inhibitors have been examined for efficacy against a virulent H3N2 influenza virus when administered orally to infected ferrets. Preliminary studies indicated that oral administration of BCX-1923, BCX-1827, or BCX-1812 (RWJ-270201) at a dose of 5 or 25 mg/kg of body weight was active in ferrets in reducing respiratory and constitutional signs and symptoms, but these antivirals affected virus titers in the upper and lower respiratory tracts only marginally. Of the three compounds, BCX-1812 seemed to be the most efficacious and was examined further at higher doses of 30 and 100 mg/kg. These doses significantly reduced peak virus titers in nasal washes and total virus shedding as measured by areas under the curve. Virus titers in lung homogenates were also reduced compared to those in controls, but the difference was not statistically significant. As was observed with BCX-1812 at lower doses, the nasal inflammatory cellular response, fever, and nasal signs were reduced while ferret activity was not, with activity remaining similar to uninfected animals.
Randomized Controlled Trial of Sequential Intravenous (i.v.) and Oral Moxifloxacin Compared with Sequential i.v. and Oral Co-Amoxiclav with or without Clarithromycin in Patients with Community-Acquired Pneumonia Requiring Initial Parenteral Treatment
Authors: Finch R. , Schürmann D. , Collins O. , Kubin R. , McGivern J. , Bobbaers H. , Izquierdo J. L. , Nikolaides P. , Ogundare F. , Raz R. , Zuck P. , Hoeffken G. 
Published: July 2002

ABSTRACT

The objective of the present trial was to compare the efficacy, safety, and tolerability of moxifloxacin (400 mg) given intravenously (i.v.) once daily followed by oral moxifloxacin (400 mg) for 7 to 14 days with the efficacy, safety, and tolerability of co-amoxiclav (1.2 g) administered by i.v. infusion three times a day followed by oral co-amoxiclav (625 mg) three times a day, with or without clarithromycin (500 mg) twice daily (i.v. or orally), for 7 to 14 days in adult patients with community-acquired pneumonia requiring initial parenteral therapy. A total of 628 patients were enrolled and assessed by evaluation of their clinical and bacteriological responses 5 to 7 days and 21 to 28 days after administration of the last dose of study medication. Although the trial was designed, on the basis of predefined outcomes, to demonstrate the equivalence of the two regimens, the results showed statistically significant higher clinical success rates (for moxifloxacin, 93.4%, and for comparator regimen, 85.4%; difference [Δ], 8.05%; 95% confidence interval [CI], 2.91 to 13.19%;
P
= 0.004) and bacteriological success rates (for moxifloxacin, 93.7%, and for comparator regimen, 81.7%; Δ, 12.06%; 95% CI, 1.21 to 22.91%) for patients treated with moxifloxacin. This superiority was seen irrespective of the severity of the pneumonia and whether or not the combination therapy included a macrolide. The time to resolution of fever was also statistically significantly faster for patients who received moxifloxacin (median time, 2 versus 3 days), and the duration of hospital admission was approximately 1 day less for patients who received moxifloxacin. The treatment was converted to oral therapy immediately after the initial mandatory 3-day period of i.v. administration for a larger proportion of patients in the moxifloxacin group than patients in the comparator group (151 [50.2%] versus 57 [17.8%] patients). There were fewer deaths (9 [3.0%] versus 17 [5.3%]) and fewer serious adverse events (38 [12.6%] versus 53 [16.5%]) in the moxifloxacin group than in the comparator group. The rates of drug-related adverse events were comparable in both groups (38.9% in each treatment group). The overall incidence of laboratory abnormalities was similar in both groups. Thus, it is concluded that monotherapy with moxifloxacin is superior to that with a standard combination regimen of a β-lactam and a β-lactamase inhibitor, co-amoxiclav, with or without a macrolide, clarithromycin, in the treatment of patients with community-acquired pneumonia admitted to a hospital.
mRNA Detection by Reverse Transcription-PCR for Monitoring Viability over Time in an
Enterococcus faecalis
Viable but Nonculturable Population Maintained in a Laboratory Microcosm
Authors: del Mar Lleò Maria , Pierobon Sabrina , Tafi Maria Carla , Signoretto Caterina , Canepari Pietro 
Published: July 2002

ABSTRACT

The viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria when they are exposed to hostile environmental conditions. It has been shown that VBNC forms of bacteria are no longer capable of growing on conventional bacteriological media but conserve pathogenic factors and/or genes. It is thus necessary to develop methods capable of detecting nonculturable bacteria and of establishing their viability when the microbiological quality of environments is monitored. In this study we demonstrated that a gene was expressed during the VBNC state in a low-nutrient-concentration microcosm through detection of
Enterococcus faecalis pbp5
mRNA by reverse transcription-PCR over a 3-month period. The presence of mRNA correlated with metabolic activity and resuscitation capability, indicating the viability of the VBNC cells.
Molecular Characterization and Identification of Biocontrol Isolates of
Trichoderma
spp
Authors: Hermosa M. R. , Grondona I. , Iturriaga E. A. , Diaz-Minguez J. M. , Castro C. , Monte E. , Garcia-Acha I. 
Published: July 2002

ABSTRACT

The most common biological control agents (BCAs) of the genus
Trichoderma
have been reported to be strains of
Trichoderma virens
,
T. harzianum
, and
T. viride
. Since
Trichoderma
BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as “
Trichoderma harzianum
” Rifai and one biocontrol strain recognized as
T. viride
, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups:
T. harzianum-T. inhamatum
complex,
T. longibrachiatum
,
T. asperellum
, and
T. atroviride-T. koningii
complex. ITS2 sequences were also useful for locating the biocontrol strains in
T. atroviride
within the complex
T. atroviride-T. koningii
. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of
T. harzianum
, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi
Phoma betae
,
Rosellinia necatrix
,
Botrytis cinerea
, and
Fusarium oxysporum
f. sp.
dianthi
in three different media.
Reductive Dehalogenation of Trichloroacetic Acid by
Trichlorobacter thiogenes
gen. nov., sp. nov
Authors: De Wever Helene , Cole James R. , Fettig Michael R. , Hogan Deborah A. , Tiedje James M. 
Published: July 2002

ABSTRACT

A bacterium able to grow via reductive dechlorination of trichloroacetate was isolated from anaerobic soil enrichments. The isolate, designated strain K1, is a member of the δ proteobacteria and is related to other known sulfur and ferric iron reducers. In anaerobic mineral media supplemented with acetate and trichloroacetate, its doubling time was 6 h. Alternative electron donor and acceptors were acetoin and sulfur or fumarate, respectively. Trichloroacetate dehalogenation activity was constitutively present, and the dechlorination product was dichloroacetate and chloride. Trichloroacetate conversion seemed to be coupled to a novel sulfur-sulfide redox cycle, which shuttled electrons from acetate oxidation to trichloroacetate reduction. In view of its unique physiological characteristics, the name
Trichlorobacter thiogenes
is suggested for strain K1.
Additional Evidence that Juvenile Oyster Disease Is Caused by a Member of the
Roseobacter
Group and Colonization of Nonaffected Animals by
Stappia stellulata
-Like Strains
Authors: Boettcher Katherine J. , Barber Bruce J. , Singer John T. 
Published: July 2002

ABSTRACT

Juvenile oyster disease (JOD) causes significant annual mortalities of hatchery-produced Eastern oysters,
Crassostrea virginica
, cultured in the Northeast. We have reported that a novel species of the α-proteobacteria
Roseobacter
group (designated CVSP) was numerically dominant in JOD-affected animals sampled during the 1997 epizootic on the Damariscotta River, Maine. In this study we report the isolation of CVSP bacteria from JOD-affected oysters during three separate epizootics in 1998. These bacteria were not detected in nonaffected oysters at the enzootic site, nor in animals raised at a JOD-free site. Animals raised at the JOD enzootic site that were unaffected by JOD were stably and persistently colonized by
Stappia stellulata
-like strains. These isolates (designated M1) inhibited the growth of CVSP bacteria in a disk-diffusion assay and thus may have prevented colonization of these animals by CVSP bacteria in situ. Laboratory-maintained
C. virginica
injected with CVSP bacteria experienced statistically significant elevated mortalities compared to controls, and CVSP bacteria were recovered from these animals during the mortality events. Together, these results provide additional evidence that CVSP bacteria are the etiological agent of JOD. Further, there are no other descriptions of specific marine α-proteobacteria that have been successfully cultivated from a defined animal host. Thus, this system presents an opportunity to investigate both bacterial and host factors involved in the establishment of such associations and the role of the invertebrate host in the ecology of these marine α-proteobacteria.
Conversion of Milled Pine Wood by Manganese Peroxidase from
Phlebia radiata
Authors: Hofrichter Martin , Lundell Taina , Hatakka Annele 
Published: July 2002

ABSTRACT

Purified manganese peroxidase (MnP) from the white-rot basidiomycete
Phlebia radiata
was found to convert in vitro milled pine wood (MPW) suspended in an aqueous reaction solution containing Tween 20, Mn
2+
, Mn-chelating organic acid (malonate), and a hydrogen peroxide-generating system (glucose-glucose oxidase). The enzymatic attack resulted in the polymerization of lower-molecular-mass, soluble wood components and in the partial depolymerization of the insoluble bulk of pine wood, as demonstrated by high-performance size exclusion chromatography (HPSEC). The surfactant Tween 80 containing unsaturated fatty acid redsidues promoted the disintegration of bulk MPW. HPSEC showed that the depolymerization yielded preferentially lignocellulose fragments with a predominant molecular mass of ca. 0.5 kDa. MnP from
P. radiata
(MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37°C with vigorous stirring, and 65 and 35% of the activity applied was retained in Tween 20 and Tween 80 reaction mixtures, respectively. In the course of reactions, major part of the Mn-chelator malonate was decomposed (85 to 87%), resulting in an increase of pH from 4.4 to >6.5. An aromatic nonphenolic lignin structure (β-O-4 dimer), which is normally not attacked by MnP, was oxidizible in the presence of pine wood meal. This finding indicates that certain wood components may promote the degradative activities of MnP in a way similar to that promoted by Tween 80, unsaturated fatty acids, or thiols.
Synthesis of Polyhydroxyalkanoate in the Peroxisome of
Saccharomyces cerevisiae
by Using Intermediates of Fatty Acid β-Oxidation
Authors: Poirier Yves , Erard Nadine , Petétot Jean MacDonald-Comber 
Published: July 2002

ABSTRACT

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads.
Saccharomyces cerevisiae
was transformed with the
Pseudomonas aeruginosa
PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the
Brassica napus
isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant
S
.
cerevisiae
growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that
S
.
cerevisiae
expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the β-oxidation of fatty acids present in the media.
S
.
cerevisiae
can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.
Portable System for Microbial Sample Preparation and Oligonucleotide Microarray Analysis
Authors: Bavykin Sergei G. , Akowski James P. , Zakhariev Vladimir M. , Barsky Viktor E. , Perov Alexander N. , Mirzabekov Andrei D. 
Published: July 2002

ABSTRACT

We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate
Escherichia coli, Bacillus subtilis, Bacillus thuringiensis
, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.
Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from
Lactobacillus sakei
Authors: Sanz Yolanda , Toldrá Fidel 
Published: July 2002

ABSTRACT

An X-prolyl-dipeptidyl peptidase has been purified from
Lactobacillus sakei
by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu
2+
, Hg
2+
, and Zn
2+
). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates.

K
m

s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-
p
-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.
Best Viral Elution Method Available for Quantification of Enteroviruses in Sludge by Both Cell Culture and Reverse Transcription-PCR
Authors: Monpoeho S. , Maul A. , Mignotte-Cadiergues B. , Schwartzbrod L. , Billaudel S. , Ferré V. 
Published: July 2002

ABSTRACT
The aim of this study was to select one or several virus extraction techniques that enable simultaneous detection of enterovirus genomes and infectious particles in different types of urban sludge. Eight techniques were compared by using 16 different liquid and solid sludge samples. The numbers of infectious enteroviruses in cell cultures were determined by using the most-probable-number method. The enterovirus genome was quantified by a single-tube reverse transcription-PCR using TaqMan technology. The results were statistically analyzed by Friedman's test, a nonparametric test for analysis of randomized block data using only ranks in terms of extraction technique efficiency. Two techniques seemed to yield higher viral titers as determined by simultaneous detection by cell culture and PCR. The first involved a 10% beef extract solution at pH 9 and sonication; the second involved a 0.3 M NaCl–7% beef extract solution at pH 7.5 followed by Freon treatment. In solid sludge, no significant differences were observed among the eight techniques tested. Both of the best techniques can be used for simultaneous detection of infectious enterovirus particles and genomes in any type of urban sludge.
Rapid Evolution of a Sexual Reproduction Gene in Centric Diatoms of the Genus
Thalassiosira
Authors: Armbrust E. Virginia , Galindo H. M. 
Published: July 2002

ABSTRACT

Sexual reproduction is commonly assumed to occur in the vast majority of diatoms due to the intimate association of this process with cell size control. Surprisingly, however, little is known about the impact of sexual events on diatom population dynamics. The
Sig1
gene is strongly upregulated during sexual reproduction in the centric diatom
Thalassiosira weissflogii
and has been hypothesized to encode a protein involved in gamete recognition. In the present study, degenerate PCR primers were designed and used to amplify a portion of
Sig1
from three closely related species in the cosmopolitan genus
Thalassiosira,
Thalassiosira oceanica
,
Thalassiosira guillardii
, and
Thalassiosira pseudonana
. Identification of
Sig1
in these three additional species facilitated development of this gene as a molecular marker for diatom sexual events. Examination of the new sequences indicated that multiple copies of
Sig1
are probably present in the genome. Moreover, compared to the housekeeping gene
β
-tubulin
, the
Sig1
genes of isolates of
T. weissflogii
collected from different regions of the Atlantic and Pacific oceans displayed high levels of divergence. The
Sig1
genes of the four closely related
Thalassiosira
species also displayed high levels of sequence divergence compared to the levels observed with a second gene,
Fcp
, probably explaining why
Sig1
could not be amplified from more distantly related species. The high levels of sequence divergence both within and between species suggest that
Sig1
is rapidly evolving in a manner reminiscent of the manner observed in other genes that encode gamete recognition proteins. A simple model is presented for
Sig1
evolution and the implications of such a rapidly evolving sexual reproduction gene for diatom speciation and population dynamics.
Rapid Confirmation of
Clostridium perfringens
by Using Chromogenic and Fluorogenic Substrates
Authors: Adcock Philip W. , Saint Christopher P. 
Published: July 2002

ABSTRACT

The use of 4-methylumbelliferyl phosphate (MUP) and
ortho
-nitrophenyl-β-
d
-galactopyranoside (ONPG) for the identification of
Clostridium perfringens
was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for
C. perfringens
confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life.