bluuebunny/crossref_metadata_2025 huggingface dataset:
Selection and Genetic Characterization of
Streptococcus pneumoniae
Mutants Resistant to the Des-F(6) Quinolone BMS-284756
Authors: Hartman-Neumann Sandra , DenBleyker Kenneth , Pelosi Lenore A. , Lawrence Laura E. , Barrett John F. , Dougherty Thomas J.
Published: July 2002

Existing quinolones are known to target the type II topoisomerases in bacteria. In order to determine which of these targets are of key importance in
Streptococcus pneumoniae
treated with BMS-284756 (T-3811ME), a novel des-F(6) quinolone, resistant mutants were selected in several steps of increasing resistance by plating pneumococci on a series of blood agar plates containing serial twofold-increasing concentrations of drug. After incubation, colonies that arose were selected and passaged twice on antibiotic-containing media at the selection level. Mutants generally showed increases in resistance of four- to eightfold over the prior level of susceptibility. Mutants in the next-higher level of resistance were selected from the previous round of resistant mutants. Subsequently, chromosomal DNA was prepared from parental (R6) pneumococci and from at least three clones from each of four levels of increasing antibiotic resistance. Using PCR primers, 500- to 700-bp amplicons surrounding the quinolone resistance determining regions (QRDR) of
gyrA
,
gyrB
,
parC,
and
parE
genes were prepared from each strain. Internal primers were used to sequence both DNA strands in the regions of approximately 400 bp centered on the QRDR. Mutations identified with increasing levels of resistance included changes in GyrA at Ser-81 and Glu-85 and changes in ParC at Ser-79 and Asp-83. Changes in GyrB and ParE were not observed at the levels of resistance obtained in this selection. The resistance to comparator quinolones (levofloxacin, ciprofloxacin, and moxifloxacin) also increased in four- to eightfold steps with these mutations. The intrinsically greater level of antibacterial activity and thus lower MICs of BMS-284756 observed at all resistance levels in this study may translate to coverage of these resistant pneumococcal strains in the clinic.
TEM-89 β-Lactamase Produced by a
Proteus mirabilis
Clinical Isolate: New Complex Mutant (CMT 3) with Mutations in both TEM-59 (IRT-17) and TEM-3
Authors: Neuwirth Catherine , Madec Stephanie , Siebor Eliane , Pechinot Andre , Duez Jean-Marie , Pruneaux Michele , Fouchereau-Peron Martine , Kazmierczak Antoine , Labia Roger
Published: July 2002

TEM-89 (CMT-3) is the first complex mutant β-lactamase produced by a clinical strain of
Proteus mirabilis
(strain Pm 631). This new enzyme, which has a pI of 6.28, is derived from TEM-3 and has a single amino acid substitution also encountered in TEM-59 (inhibitor-resistant TEM β-lactamase IRT-17): Ser-130 to Gly. TEM-89 hydrolyzed penicillins to the same extent that TEM-3 did but lost almost all hydrolytic activity for cephalosporins and, like TEM-59, was highly resistant to inhibitors.
Novel Scintillation Proximity Assay for Measuring Membrane-Associated Steps of Peptidoglycan Biosynthesis in
Escherichia coli
Authors: Chandrakala B. , Elias Bertha C. , Mehra Upasana , Umapathy N. S. , Dwarakanath P. , Balganesh T. S. , deSousa Sunita M.
Published: July 2002

We have developed a novel, high-throughput scintillation proximity assay to measure the membrane-associated steps (stages 2 and 3) of peptidoglycan synthesis in
Escherichia coli
. At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway.
E. coli
membranes are incubated with the two sugar precursors, UDP–
N
-acetyl muramylpentapeptide and UDP–[
3
H]-
N
-acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinin-coated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.
Diversity of Tn
1546
Elements in Clinical Isolates of Glycopeptide-Resistant Enterococci from Scottish Hospitals
Authors: Brown A. R. , Townsley A. C. , Amyes S. G. B.
Published: July 2002

The Tn
1546
-related elements of 48 Van glycopetide-resistant enterococci were compared. Ten distinct Tn
1546
types were identified with variation primarily due to IS
1542
and IS
1216V
-like insertions. Clonal isolates frequently differed in their Tn
1546
type, indicating instability of Tn
1546
-related elements. A putative hybrid promoter was identified, generated upstream of
vanR
by the insertion of IS
1542
. The presence of this hybrid promoter was associated with constitutive expression of the
van
genes and elevated teicoplanin resistance.
Salmonella Excretion after Cessation of Tosufloxacin Therapy in Acute Nontyphoid Salmonella Enterocolitis
Authors: Ohnishi Kenji , Kimura Kyoko
Published: July 2002

Symptomatic nontyphoid salmonella enterocolitis patients were treated with tosufloxacin (TFLX) at oral doses of 150 mg three times a day for 5 to 7 days, but bacterial relapse without symptoms occurred in 85% of the patients within 15 days after the cessation of therapy. Our study indicates that TFLX may not be an ideal drug against symptomatic salmonella enterocolitis from a bacteriological point of view.
Azasordarins: Susceptibility of Fluconazole-Susceptible and Fluconazole-Resistant Clinical Isolates of
Candida
spp. to GW 471558
Authors: Cuenca-Estrella Manuel , Mellado Emilia , Dı́az-Guerra Teresa M. , Monzón Araceli , Rodrı́guez-Tudela Juan L.
Published: July 2002

The in vitro activity of the azasordarin GW 471558 was compared with those of amphotericin B, flucytosine, itraconazole, and ketoconazole against 177 clinical isolates of
Candida
spp. GW 471558 showed potent activity against
Candida albicans
,
Candida glabrata
, and
Candida tropicalis
, even against isolates with decreased susceptibility to azoles.
Candida krusei, Candida parapsilosis, Candida lusitaniae,
and
Candida guilliermondii
are resistant to GW 471558 in vitro (MICs, >128 μg/ml).
Prevalence of Macrolide Resistance Genes in Clinical Isolates of the
Streptococcus anginosus
(“
S. milleri
”) Group
Authors: Jacobs Jan A. , van Baar Gilles J. , London Nancy H. H. J. , Tjhie Jeroen H. T. , Schouls Leo M. , Stobberingh Ellen E.
Published: July 2002

Twenty-two unrelated erythromycin-resistant anginosus group strains (3.2% resistance rate) were assessed for mechanisms of resistance.
Streptococcus anginosus
accounted for 16 of the 22 isolates. Fifteen isolates harbored the
erm
(B) gene. The
erm
(TR) and the
mef
(E) genes were carried by two isolates each. In three isolates, none of these resistance genes was detected by PCR.
Interactions of Quinupristin-Dalfopristin with Eight Other Antibiotics as Measured by Time-Kill Studies with 10 Strains of
Staphylococcus aureus
for Which Quinupristin-Dalfopristin Alone Was Not Bactericidal
Authors: Fuchs Peter C. , Barry Arthur L. , Brown Steven D.
Published: July 2002

Quinupristin-dalfopristin (Q-D) and eight other antimicrobial agents were tested alone and in combination with Q-D in time-kill studies against 10 strains of macrolide-lincosamide-streptogramin B-resistant
Staphylococcus aureus
. Although Q-D is normally a bactericidal drug, it was only bacteriostatic for these isolates. Gentamicin alone was bactericidal against 7 of the 10 strains, and Q-D did not alter that killing effect. However, when vancomycin, cefepime, ceftazidime, imipenem, piperacillin-tazobactam, and ciprofloxacin were bactericidal when tested alone, the killing rates were reduced when combined with Q-D. The clinical significance of this in vitro antagonism is unknown at this time, and more studies are needed.
In Vitro Activities of BMS-284756 against
Chlamydia trachomatis
and Recent Clinical Isolates of
Chlamydia pneumoniae
Authors: Malay Sheila , Roblin Patricia M. , Reznik Tamara , Kutlin Andrei , Hammerschlag Margaret R.
Published: July 2002

The in vitro activities of BMS-284756 (a novel des-fluoroquinolone), levofloxacin, moxifloxacin, and clarithromycin were tested against 5 strains of
Chlamydia trachomatis
and 20 isolates of
Chlamydia pneumoniae
. The MIC at which 90% of the isolates were inhibited and the minimal bactericidal concentration at which 90% of the isolates were killed by BMS-284756 for all isolates of
C. pneumoniae
and
C. trachomatis
was 0.015 μg/ml (range, 0.015 to 0.03 μg/ml). BMS-284756 was the most active quinolone tested.
In Vitro Antibacterial Activities of DQ-113, a Potent Quinolone, against Clinical Isolates
Authors: Tanaka Mayumi , Yamazaki Emi , Chiba Megumi , Yoshihara Kiyomi , Akasaka Takaaki , Takemura Makoto , Sato Kenichi
Published: July 2002

The antibacterial activity of DQ-113, formerly D61-1113, was compared with those of antibacterial agents currently available. MICs at which 90% of the isolates tested are inhibited (MIC
90
s) of DQ-113 against clinical isolates of methicillin-susceptible and -resistant
Staphylococcus aureus
and methicillin-susceptible and -resistant coagulase-negative staphylococci were 0.03, 0.008, 0.03, and 0.06 μg/ml, respectively. Moreover, DQ-113 showed the most potent activity against ofloxacin-resistant and methicillin-resistant
S. aureus
, with a MIC
90
of 0.25μg/ml. DQ-113 inhibited the growth of all strains of
Streptococcus pneumoniae
, including penicillin-resistant strains, and
Streptococcus pyogenes
at 0.06 μg/ml, and DQ-113 was more active than the other quinolones tested against
Enterococcus faecalis
and
Enterococcus faecium
with MIC90s of 0.25 and 2 μg/ml, respectively. Against vancomycin-resistant enterococci, DQ-113 showed the highest activity among the reference compounds, with a MIC range from 0.25 to 2 μg/ml. DQ-113 also showed a potent activity against
Haemophilus influenzae
, including ampicillin-resistant strains (MIC90, 0.015 μg/ml), and
Moraxella catarrhalis
(MIC90, 0.03 μg/ml). The activity of DQ-113 was roughly comparable to that of levofloxacin against all species of
Enterobacteriaceae
. The MICs of DQ-113 against ofloxacin-susceptible
Pseudomonas aeruginosa
ranged from 0.25 to 2 μg/ml, which were four times higher than those of ciprofloxacin. From these results, DQ-113 showed the most potent activity against gram-positive pathogens among antibacterial agents tested.
Effects of Compounds Found in Propolis onStreptococcus mutansGrowth and on Glucosyltransferase Activity
Authors: Koo Hyun , Rosalen Pedro L. , Cury Jaime A. , Park Yong K. , Bowen William H.
Published: July 2002

Propolis, a resinous bee product, has been shown to inhibit the growth of oral microorganisms and the activity of bacterium-derived glucosyltransferases (GTFs). Several compounds, mainly polyphenolics, have been identified in this natural product. The present study evaluated the effects of distinct chemical groups found in propolis on the activity of GTF enzymes in solution and on the surface of saliva-coated hydroxyapatite (sHA) beads. Thirty compounds, including flavonoids, cinnamic acid derivatives, and terpenoids, were tested for the ability to inhibit GTFs B, C, and D fromStreptococcus mutansand GTF fromS. sanguinis(GTF Ss). Flavones and flavonols were potent inhibitors of GTF activity in solution; lesser effects were noted on insolubilized enzymes. Apigenin, a 4′,5,7-trihydroxyflavone, was the most effective inhibitor of GTFs, both in solution (90.5 to 95% inhibition at a concentration of 135 μg/ml) and on the surface of sHA beads (30 to 60% at 135 μg/ml). Antibacterial activity was determined by using MICs, minimum bactericidal concentrations (MBCs), and time-kill studies. Flavanones and some dihydroflavonols, as well as the sesquiterpenett-farnesol, inhibited the growth ofS. mutansandS. sobrinus;tt-farnesol was the most effective antibacterial compound (MICs of 14 to 28 μg/ml and MBCs of 56 to 112 μg/ml).tt-Farnesol (56 to 112 μg/ml) produced a 3-log-fold reduction in the bacterial population after 4 h of incubation. Cinnamic acid derivatives had negligible biological activities. Several of the compounds identified in propolis inhibit GTF activities and bacterial growth. Apigenin is a novel and potent inhibitor of GTF activity, andtt-farnesol was found to be an effective antibacterial agent.
Outbreak of Infections Caused by
Enterobacter cloacae
Producing the Integron-Associated β-Lactamase IBC-1 in a Neonatal Intensive Care Unit of a Greek Hospital
Authors: Kartali Georgia , Tzelepi Eva , Pournaras Spyros , Kontopoulou Constantina , Kontos Fanourios , Sofianou Danai , Maniatis Antonios N. , Tsakris Athanassios
Published: July 2002

Nineteen of 27 ceftazidime-resistant
Enterobacter cloacae
isolates from a neonatal intensive care unit in Thessaloniki, Greece, had genes coding for the novel extended-spectrum β-lactamase IBC-1; 18 of those 19 harbored similar conjugative plasmids and belonged to two distinct genetic lineages. A synergy test with ceftazidime and imipenem enabled us to identify five unrelated
bla
IBC-1
-carrying
E. cloacae
isolates from other wards of the hospital. It seems that this integron-associated gene is capable of dispersing both by clonal spread and by gene dissemination.
High Prevalence of High-Level Ciprofloxacin Resistance in
Neisseria gonorrhoeae
in Tel Aviv, Israel: Correlation with Response to Therapy
Authors: Dan Michael , Poch Francesca , Sheinberg Bracha
Published: July 2002

A survey of the drug susceptibilities of gonococcal isolates from 100 consecutive patients attending clinics in the Tel Aviv area of Israel during the period from February to September 2000 has shown a 61% rate of ciprofloxacin resistance (MICs, ≥1 μg/ml); 51 isolates were highly resistant (MICs, ≥4 μg/ml). Ciprofloxacin-resistant strains were more prevalent among isolates with chromosomally mediated resistance to penicillin and tetracycline. Therapeutic failures with ciprofloxacin correlated with MICs of ≥1 mg/ml (
P
< 0.01). The high rate of ciprofloxacin resistance coinciding with a sharp rise in the incidence of gonorrhea precludes the use of fluoroquinolone drugs for the empirical treatment of gonococcal infections acquired in Israel.
Phylogeny of All Recognized Species of Ammonia Oxidizers Based on Comparative 16S rRNA and
amoA
Sequence Analysis: Implications for Molecular Diversity Surveys
Authors: Purkhold Ulrike , Pommerening-Röser Andreas , Juretschko Stefan , Schmid Markus C. , Koops Hans-Peter , Wagner Michael
Published: July 2002

The current perception of evolutionary relationships and the natural diversity of ammonia-oxidizing bacteria (AOB) is mainly based on comparative sequence analyses of their genes encoding the 16S rRNA and the active site polypeptide of the ammonia monooxygenase (AmoA). However, only partial 16S rRNA sequences are available for many AOB species and most AOB have not yet been analyzed on the
amoA
level. In this study, the 16S rDNA sequence data of 10
Nitrosomonas
species and
Nitrosococcus mobilis
were completed. Furthermore, previously unavailable 16S rRNA sequences were determined for three
Nitrosomonas
sp. isolates and for the gamma-subclass proteobacterium
Nitrosococcus halophilus
. These data were used to revaluate the specificities of published oligonucleotide primers and probes for AOB. In addition, partial
amoA
sequences of 17 AOB, including the above-mentioned 15 AOB, were obtained. Comparative phylogenetic analyses suggested similar but not identical evolutionary relationships of AOB by using 16S rRNA and AmoA as marker molecules, respectively. The presented 16S rRNA and
amoA
and AmoA sequence data from all recognized AOB species significantly extend the currently used molecular classification schemes for AOB and now provide a more robust phylogenetic framework for molecular diversity inventories of AOB. For 16S rRNA-independent evaluation of AOB species-level diversity in environmental samples,
amoA
and AmoA sequence similarity threshold values were determined which can be used to tentatively identify novel species based on cloned
amoA
sequences. Subsequently, 122
amoA
sequences were obtained from 11 nitrifying wastewater treatment plants. Phylogenetic analyses of the molecular isolates showed that in all but two plants only nitrosomonads could be detected. Although several of the obtained
amoA
sequences were only relatively distantly related to known AOB, none of these sequences unequivocally suggested the existence of previously unrecognized species in the wastewater treatment environments examined.
Mechanism of the Incidental Production of a Melanin-Like Pigment during 6-Demethylchlortetracycline Production in
Streptomyces aureofaciens
Authors: Nakano Tetsuo , Miyake Koichiro , Ikeda Masato , Mizukami Toru , Katsumata Ryoichi
Published: July 2002

The secondary metabolite 6-demethylchlortetracycline (6-DCT), which is produced by
Streptomyces aureofaciens
, is used as a precursor of semisynthetic tetracyclines. Strains that produce 6-DCT also produce a melanin-like pigment (MP). The correlation between MP production and 6-DCT production was investigated by using
S. aureofaciens
NRRL 3203. Production of both MP and 6-DCT was repressed by phosphate or ammonium ions, suggesting that syntheses of these compounds are controlled by the same regulators. Ten chlortetracycline-producing recombinants were derived from 6-DCT-producing mutant NRRL 3203 by gene replacement. All of the recombinants produced chlortetracycline but not MP, indicating that MP production is the results of a defect in the 6-methylation step and suggesting that the polyketide nonaketideamide is a common intermediate leading to MP as well as 6-DCT. To further examine the possibility that MP might be synthesized via the 6-DCT-biosynthetic pathway, mutants defective in 6-DCT biosynthesis were derived from a 6-DCT producer. Some of these mutants were able to produce MP, while others, including mutants with mutations in the gene encoding anhydrotetracycline oxygenase, an enzyme catalyzing the penultimate step in the pathway, produced neither 6-DCT nor MP. Production of 6-DCT and production of MP were restored simultaneously by integrative transformation with the corresponding 6-DCT-biosynthetic genes, indicating that some of 6-DCT-biosynthetic enzymes are indispensable for MP production. These findings suggest that a defect in the 6-methylation step results in redirection of carbon flux from a certain intermediate in the 6-DCT-biosynthetic pathway to a shunt pathway and results in MP production.
Expression of the Antifeeding Gene
anfA1
in
Serratia entomophila
Requires RpoS
Authors: Giddens Stephen R. , Tormo Antonio , Mahanty H. Khris
Published: July 2002

The
rpoS
gene of
Serratia entomophila
BC4B was cloned and used to create
rpoS
-mutant strain BC4BRS. Larvae of the New Zealand grass grub
Costelytra zealandica
infected with BC4BRS became amber colored but continued to feed, albeit to a lesser extent than infected larvae. Subsequently, we found that expression of the antifeeding gene
anfA1
in
trans
was substantially reduced in BC4BRS relative to that in the parental strain BC4B. Our data show that a functional
rpoS
gene is vital for full expression of
anfA1
and for development of the antifeeding component of amber disease.
Cloning and Sequencing of the Gene Encoding an Aldehyde Dehydrogenase That Is Induced by Growing
Alteromonas
sp. Strain KE10 in a Low Concentration of Organic Nutrients
Authors: Maeda Toshimichi , Yoshinaga Ikuo , Shiba Tsuneo , Murakami Masatada , Wada Akira , Ishida Yuzaburou
Published: July 2002

The protein composition of
Alteromonas
sp. strain KE10 cultured at two different organic-nutrient concentrations was determined by using two-dimensional polyacrylamide gel electrophoresis. The cellular levels of three proteins, OlgA, -B, and -C, were considerably higher in cells grown in a low concentration of organic nutrient medium (LON medium; 0.2 mg of carbon per liter) than cells grown in a high concentration of organic nutrient medium (HON; 200 mg of C liter
−1
) or cells starved for organic nutrients. In the LON medium, the cellular levels of the Olg proteins were higher at the exponential growth phase than at the stationary growth phase. A sequence of the gene for OlgA revealed that the amino acid sequence had a high degree of similarity to the NAD
+
-dependent aldehyde dehydrogenases of several bacteria. OlgA, expressed in
Escherichia coli
, catalyzed the dehydrogenation of acetaldehyde, propionaldehyde, and butyraldehyde. The aldehyde dehydrogenase activity of KE10 was higher in cells growing exponentially in LON medium than in HON. OlgA may be involved in the growth under low-nutrient conditions. The physiological role of OlgA is discussed here.
Simultaneous Immunofluorescent Detection of Coentrapped Cells in Gel Beads
Authors: Prioult Guenolee , Lacroix Christophe , Turcotte Carl , Fliss Ismaïl
Published: July 2002

An immunofluorescent method involving double color labeling and confocal microscopy was reported to specifically detect lactic acid bacteria and probiotic cells coimmobilized in gels beads. The method described is rapid (4 h) and sensitive and may be useful for studying cell dynamics during mixed-culture starter production using immobilized cells in gel beads. Microscopic observations were perfectly correlated to cell counts obtained using a sandwich enzyme-linked immunosorbent assay.
New Insights into Methyl Bromide Cooxidation by
Nitrosomonas europaea
Obtained by Experimenting with Moderately Low Density Cell Suspensions
Authors: Duddleston Khrystyne N. , Bottomley Peter J. , Porter Angela J. , Arp Daniel J.
Published: July 2002

We examined the rates and sustainability of methyl bromide (MeBr) oxidation in moderately low density cell suspensions (∼6 × 10
7
cells ml
−1
) of the NH
3
-oxidizing bacterium
Nitrosomonas europaea
. In the presence of 10 mM NH
4
+
and 0.44, 0.22, and 0.11 mM MeBr, the initial rates of MeBr oxidation were sustained for 12, 12, and 24 h, respectively, despite the fact that only 10% of the NH
4
+
, 18% of the NH
4
+
, and 35% of the NH
4
+
, respectively, were consumed. Although the duration of active MeBr oxidation generally decreased as the MeBr concentration increased, similar amounts of MeBr were oxidized with a large number of the NH
4
+
-MeBr combinations examined (10 to 20 μmol mg [dry weight] of cells
−1
). Approximately 90% of the NH
3
-dependent O
2
uptake activity and the NO
2
−
-producing activity were lost after
N. europaea
was exposed to 0.44 mM MeBr for 24 h. After MeBr was removed and the cells were resuspended in fresh growth medium, NO
2
−
production increased exponentially, and 48 to 60 h was required to reach the level of activity observed initially in control cells that were not exposed to MeBr. It is not clear what percentage of the cells were capable of cell division after MeBr oxidation because NO
2
−
accumulated more slowly in the exposed cells than in the unexposed cells despite the fact that the latter were diluted 10-fold to create inocula which exhibited equal initial activities. The decreases in NO
2
−
-producing and MeBr-oxidizing activities could not be attributed directly to NH
4
+
or NH
3
limitation, to a decrease in the pH, to the composition of the incubation medium, or to toxic effects caused by accumulation of the end products of oxidation (NO
2
−
and formaldehyde) in the medium. Additional cooxidation-related studies of
N. europaea
are needed to identify the mechanism(s) responsible for the MeBr-induced loss of cell activity and/or viability, to determine what percentages of cells damaged by cooxidative activities are culturable, and to determine if cooxidative activity interferes with the regulation of NH
3
-oxidizing activity.
Rhizosphere Soil Aggregation and Plant Growth Promotion of Sunflowers by an Exopolysaccharide-Producing
Rhizobium
sp. Strain Isolated from Sunflower Roots
Authors: Alami Younes , Achouak Wafa , Marol Christine , Heulin Thierry
Published: July 2002

Root-adhering soil (RAS) forms the immediate environment where plants take up water and nutrients for their growth. We report the effect of an exopolysaccharide (EPS)-producing rhizobacterium (strain YAS34) on the physical properties of sunflower (
Helianthus annuus
L.) RAS, associated with plant growth promotion, under both water stress and normal water supply conditions. Strain YAS34 was isolated as a major EPS-producing bacterium from the rhizoplane of sunflowers grown in a French dystric cambisol. Strain YAS34 was assigned to the
Rhizobium
genus by 16S ribosomal DNA gene sequencing. Inoculation of sunflower seeds and soil with strain YAS34 caused a significant increase in RAS per root dry mass (dm) (up to 100%) and a significant increase in soil macropore volume (12 to 60 μm in diameter). The effect of inoculation on sunflower shoot dm (up to +50%) and root dm (up to +70%) was significant under both normal and water stress conditions. Inoculation with strain YAS34 modified soil structure around the root system, counteracting the negative effect of water deficit on growth. Using [
15
N]nitrate, we showed that inoculation made the use of fertilizer more effective by increasing nitrogen uptake by sunflower plantlets.
Relationship of Exo-β-
d
-Galactofuranosidase Kinetic Parameters to the Number of Phosphodiesters in
Penicillium fellutanum
Peptidophosphogalactomannan: Enzyme Purification and Kinetics of Glycopeptide and Galactofuran Chain Hydrolysis
Authors: Tuekam Brigitte A. , Park Yong-Il , Unkefer Clifford J. , Gander John E.
Published: July 2002

Extracellular
Penicillium fellutanum
exo-β-
d
-galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP
2
GM
ii
and pP
25
GM
ii
(containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1-
O
-methyl-β-
d
-galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP
2
GM
ii
or pP
25
GM
ii
. The
k
cat
/

K
m

value for pP
25
GM
ii
is 1.7 × 10
3
M
−1
s
−1
, that for 1-
O
-methyl-β-
d
-galactofuranoside is 1.1 × 10
4
M
−1
s
−1
, that for pP
2
GM
ii
is 1.7 × 10
4
M
−1
s
−1
, and those for 5-
O
-β-
d
-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 × 10
5
and 4.1 × 10
5
M
−1
s
−1
, respectively. Variability in the
k
cat
/

K
m

values is due primarily to differences in

K
m

values; the
k
−1
/
k
1
ratio likely provides the most influence on

K
m
. k

cat
increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pP
x
GM
ii
added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml
−1
h
−1
. No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pP
x
GM
ii
and of related polymers is proportional to the
k
cat
/

K
m

value of each polymer. The kinetic data show that the
k
cat
/

K
m

value increases as the number of phosphodiesters of pP
x
GM
ii
decreases, also resulting in an increase in the activity of exo-β-
d
-galactofuranosidase.
Identification of a Replication Protein and Repeats Essential for DNA Replication of the Temperate Lactococcal Bacteriophage TP901-1
Authors: Østergaard Solvej , Brøndsted Lone , Vogensen Finn K.
Published: July 2002

DNA replication of the temperate lactococcal bacteriophage TP901-1 was shown to involve the gene product encoded by
orf13
and the repeats located within the gene. Sequence analysis of 1,500 bp of the early transcribed region of the phage genome revealed a single-stranded DNA binding protein analogue (ORF12) and the putative replication protein (ORF13). The putative origin of replication was identified as series of repeats within
orf13
and was shown to confer a TP901-1 resistance phenotype when present in
trans
. Site-specific mutations were introduced into the replication protein and into the repeats. The mutations were introduced into the TP901-1 prophage by homologous recombination by using a vector with a temperature-sensitive replicon. Subsequent analysis of induced phages showed that the protein encoded by
orf13
and the repeats within
orf13
were essential for phage TP901-1 amplification. In addition, analyses of internal phage DNA replication showed that the ORF13 protein and the repeats are essential for phage TP901-1 DNA replication in vivo. These results show that
orf13
encodes a replication protein and that the repeats within the gene are the origin of replication.
Cloning of Genomic DNA of
Lactococcus lactis
That Restores Phage Sensitivity to an Unusual Bacteriophage sk1-Resistant Mutant
Authors: Kraus Jennifer , Geller Bruce L.
Published: July 2002

An unusual, spontaneous, phage sk1-resistant mutant (RMSK1/1) of
Lactococcus lactis
C2 apparently blocks phage DNA entry into the host. Although no visible plaques formed on RMSK1/1, this host propagated phage at a reduced efficiency. This was evident from center-of-infection experiments, which showed that 21% of infected RMSK1/1 formed plaques when plated on its phage-sensitive parental strain, C2. Moreover, viable cell counts 0 and 4 h after infection were not significantly different from those of an uninfected culture. Further characterization showed that phage adsorption was normal, but burst size was reduced fivefold and the latent period was increased from 28.5 to 36 min. RMSK1/1 was resistant to other, but not all, similar phages. Phage sensitivity was restored to RMSK1/1 by transformation with a cloned DNA fragment from a genomic library of a phage-sensitive strain. Characterization of the DNA that restored phage sensitivity revealed an open reading frame with similarity to sequences encoding lysozymes (β-1,4-
N
-acetylmuramidase) and lysins from various bacteria, a fungus, and phages of
Lactobacillus
and
Streptococcus
and also revealed DNA homologous to noncoding sequences of temperate phage of
L. lactis
, DNA similar to a region of phage sk1, a gene with similarity to tRNA genes, a prophage attachment site, and open reading frames with similarities to
sun
and to sequences encoding phosphoprotein phosphatases and protein kinases. Mutational analyses of the cloned DNA showed that the region of homology with lactococcal temperate phage was responsible for restoring the phage-sensitive phenotype. The region of homology with DNA of lactococcal temperate phage was similar to DNA from a previously characterized lactococcal phage that suppresses an abortive infection mechanism of phage resistance. The region of homology with lactococcal temperate phage was deleted from a phage-sensitive strain, but the strain was not phage resistant. The results suggest that the cloned DNA with homology to lactococcal temperate phage was not mutated in the phage-resistant strain. The cloned DNA apparently suppressed the mechanism of resistance, and it may do so by mimicking a region of phage DNA that interacts with components of the resistance mechanism.
Secretion of Active Recombinant Human Tissue Plasminogen Activator Derivatives in
Escherichia coli
Authors: Manosroi Jiradej , Tayapiwatana Chatchai , Götz Friedrich , Werner Rolf G. , Manosroi Aranya
Published: July 2002

The DNA fragment coding for kringle 2 plus serine protease domains (
K2S
) of tissue plasminogen activator (tPA) was inserted into a phagemid vector, pComb3HSS. In the recombinant vector, pComb3H-
K2S
, the
K2S
gene was fused to
gpIII
of ΦM13 and linked to the OmpA signal sequence. The resulting gene,
rK2S-gpIII,
was inducibly expressed in
Escherichia coli
XL-1 Blue. The protein was presented on the phage particle. To stop the expression of
gpIII,
a stop codon between
K2S
and the
gpIII
gene was inserted by site-directed mutagenesis. This mutated vector, MpComb3H-
K2S
, was transformed in XL-1 Blue. After induction with IPTG (isopropyl-β-
d
-thiogalactopyranoside), rK2S was found both in the periplasm as an inactive form of approximately 32% and in the culture supernatant as an active form of approximately 68%. The secreted form of rK2S was partially purified by ammonium sulfate (55%) precipitation. The periplasmic form was isolated from whole cells by chloroform extraction. The fibrin binding site of kringle 2 was demonstrated in all expressed versions (phage-bound, periplasmic, and secreted forms) using the monoclonal anti-kringle 2 antibody (16/B). Only the secreted form of rK2S revealed a fibrinogen-dependent amidolytic activity with the specific activity of 236 IU/μg. No amidolytic activity of rK2S was observed in either the periplasmic or the phage-bound form. The secretion of rK2S as an active enzyme offers a novel approach for the production of the active-domain deletion mutant tPA, rK2S, without any requirements for bacterial compartment preparation and in vitro refolding processes. This finding is an important technological advance in the development of large-scale, bacterium-based tPA production systems.
Increased Enterocyte Production in Gnotobiotic Rats Mono-Associated with
Lactobacillus rhamnosus
GG
Authors: Banasaz M. , Norin E. , Holma R. , Midtvedt T.
Published: July 2002

There is increasing scientific and commercial interest in using beneficial microorganisms (i.e., probiotics) to enhance intestinal health. Of the numerous microbial strains examined,
Lactobacillus rhamnosus
GG has been most extensively studied. Daily intake of
L. rhamnosus
GG shortens the course of rotavirus infection by mechanisms that have not been fully elucidated. Comparative studies with germfree and conventional rats have shown that the microbial status of an animal influences the intestinal cell kinetics and morphology. The present study was undertaken to study whether establishment of
L. rhamnosus
GG as a mono-associate in germfree rats influences intestinal cell kinetics and morphology.
L. rhamnosus
GG was easily established in germfree rats. After 3 days of mono-association, the rate of mitoses in the upper part of the small intestine (jejunum 1) increased as much as 14 and 22% compared to the rates in germfree and conventional counterparts, respectively. The most striking alteration in morphology was an increase in the number of cells in the villi. We hypothesis that the compartmentalized effects of
L. rhamnosus
GG may represent a reparative event for the mucosa.
Variability and Immunogenicity of Human Immunodeficiency Virus Type 1 p24 Gene Quasispecies
Authors: Iroegbu Johanna , Birk Markus , Lazdina Una , Sönnerborg Anders , Sällberg Matti
Published: July 2002

Despite the conserved nature of the human immunodeficiency virus type 1 (HIV-1)gaggene, multiple quasispecies of the p24 gene coexist in HIV-1-infected patients. We cloned and sequenced 31 p24 genes from four HIV-1-infected patients. The intrapatient homology between the p24 genes ranged from 97.1 to 99.1%, whereas the interpatient homology ranged from 91.5 to 93.8%, suggesting a host-specific evolution. Synonymous and nonsynonymous nucleotide changes were evenly distributed in the p24 gene, with 27 and 28%, respectively, located within host human leukocyte antigen class I recognition sites. This would suggest only a minor influence from the host cytotoxic T-cell response on the evolution of the p24 gene. The importance of minor variations within p24 was analyzed by designing DNA-based immunogens from two distinct p24 quasispecies genes simultaneously derived from one patient. In plasmid-immunizedH-2b,H-2d, andH-2khaplotype mice, a clear influence from the host major histocompatibility complex was noted on the immune responses, fully consistent with those noted when a recombinant p24 protein is used as the immunogen. The two p24 DNA immunogens did not differ in their immunogenicity, indicating that the limited genetic variability (<1%) had little influence on the immune responses.
Neutralization Profiles of Sera from Human Immunodeficiency Virus (HIV)-Infected Individuals: Relationship to HIV Viral Load and CD4 Cell Count
Authors: Nokta Mostafa , Turk Patricia , Loesch Kimberly , Pollard Richard B.
Published: July 2002

The relationship of the neutralizing activity (NA) profile of sera from human immunodeficiency virus (HIV)-infected individuals to the HIV viral load and the absolute CD4 count was examined. The NA of 24 serum samples against autologous isolates (AI) and HIV type 1 strain MN was examined. Three NA patterns were recognized. Nine sera neutralized both AI and MN (+/+), six sera neutralized MN but not AI (−/+), and nine sera failed to neutralize both AI and MN (−/−). The identification of the three neutralization patterns (+/+, −/+, and −/−) indicated that resistance to neutralization was progressive. A reciprocal relationship between the viral burden of the patients and the NA profiles was observed. The nine subjects with a −/− NA profile had a plasma viral load of ≥5 × 10
4
copies/ml and a cellular viral burden of ≥1,122 infectious units per million viable cells, which were significantly different from those of the other groups (
P
< 0.02). These patterns were independent of the phenotypic characteristics of the virus. Longitudinally, subjects with a −/− profile at baseline gained their HIV-specific NA by 24 weeks of antiretroviral therapy when this was associated with a ≥1-log
10
decline in the plasma HIV viral load. The sera from week 24 from some patients were able to neutralize both the 24-week and the baseline dominant virus isolates. A change in CD4 cell count of 50 or more in either direction predicted a −/− or +/+ profile. The verification of the autologous NA profile might be important in selecting patients who may benefit from immune-based therapies involving neutralizing monoclonal antibodies.
Identification and Strain Differentiation of
Vibrio cholerae
by Using Polyclonal Antibodies against Outer Membrane Proteins
Authors: Martı́nez-Govea A. , Ambrosio J. , Gutiérrez-Cogco L. , Flisser A.
Published: July 2002

Cholera is caused only by O1 and O139
Vibrio cholerae
strains. For diagnosis, 3 working days are needed for bacterial isolation from human feces and for biochemical characterization. Here we describe the purification of bacterial outer membrane proteins (OMP) from
V. cholerae
O1 Ogawa, O1 Inaba, and O139 strains, as well as the production of specific antisera and their use for fecal
Vibrio
antigen detection. Anti-OMP antisera showed very high reactivity and specificity by enzyme-linked immunosorbent assay (ELISA) and dot-ELISA. An inmunodiagnostic assay for
V. cholerae
detection was developed; this assay avoids preenrichment and costly equipment and can be used for epidemiological surveillance and clinical diagnosis of cases, considering that prompt and specific identification of bacteria is mandatory in cholera.
Serum Antibody Response to Polysaccharides in Children with Recurrent Respiratory Tract Infections
Authors: López-Yap Alberto , Abdelnour Arturo , Lomonte Bruno , Porras Oscar
Published: July 2002

We evaluated children (15-months old and older) with recurrent upper respiratory tract infections and normal levels of immunoglobulins in serum for specific polysaccharide immunodeficiency using an enzyme-linked immunosorbent assay method. Results showed that of 12 patients vaccinated with Act-HIB vaccine, one did not develop specific antibodies to
Haemophilus influenzae
type b, demonstrating that such immunodeficiency is present in Costa Rican children.
Chemokine Receptor CCR5 Δ32 Genetic Analysis Using Multiple Specimen Types and the NucliSens Basic Kit
Authors: Tetali Suryakumari , Lee Eun Mi , Kaplan Mark H. , Romano Joseph W. , Ginocchio Christine C.
Published: July 2002

Resistance to HIV-1 infection and delayed disease progression have been associated with a 32-bp deletion (Δ32) in the gene encoding the CCR5 chemokine receptor. In the present study we describe the modification of a nucleic acid sequence-based amplification (NASBA)-based CCR5 genotyping assay for a NucliSens Basic Kit (Organon Teknika, Durham, N.C.) format using a new target-specific sandwich oligonucleotide detection methodology. The new method permitted the use of generic electrochemiluminescent probes supplied in the NucliSens Basic Kit, whereas the original NASBA method required expensive target-specific ruthenium detection probes. The Basic Kit CCR5 Δ32 genotypic analysis was in 100% concordance with both the original NASBA assay and DNA PCR results. This study also evaluated the use of multiple specimen types, including peripheral blood mononuclear cells (PBMC), whole blood, dried blood spots, buccal scrapings, and plasma, for CCR5 genotype analysis. The sensitivities of the three assays were comparable when PBMC or whole blood was the specimen source. In contrast, when dried blood spots, buccal scrapings, or plasma was used as the sample source, the sensitivity of DNA PCR was 80.95, 42.8, or 0%, respectively, compared to 100% sensitivity obtained with the original NASBA and Basic Kit NASBA assays. Our study indicates that the NucliSens Basic Kit NASBA assay is very sensitive and specific for CCR5 Δ32 genotyping using multiple sample types.
Antibodies against
Streptococcus agalactiae
Proteins c
α
and R4 in Sera from Pregnant Women from Norway and Zimbabwe
Authors: Moyo Sylvester R. , Maeland Johan A. , Mudzori James
Published: July 2002

Group B streptococci (GBS) express strain-variable and surface-localized proteins, which are important serotype markers and targets of protective antibodies. These include the c
α
and R4 proteins, one or the other of which is expressed by approximately 75% of clinical GBS isolates. These proteins have been considered vaccine candidates. In this study, the c
α
and R4 proteins were extracted by trypsin digestion of GBS and purified by sequential precipitation with trichloroacetic acid and ammonium sulfate followed by gel filtration chromatography. The proteins were used as antigens in an indirect enzyme-linked immunosorbent assay (ELISA) to measure the levels of c
α
- and R4-reactive antibodies in sera from pregnant women from Norway (
n
= 100) and from Zimbabwe (
n
= 124). Antibody levels in the Norwegian group of women were significantly higher than in the Zimbabwean group, and a higher proportion of the Norwegian women contained appreciable levels of antibodies against both proteins. The antibodies traversed the placental barrier. With individual sera, a significant correlation between the anti-c
α
and anti-R4 antibody levels was observed and each of the two protein antigens effectively competed for human serum antibodies both against itself and against the other antigen. Inhibition ELISA results demonstrated specificity for each of the proteins of immune antibodies raised in rabbits. These results demonstrate that (i) the majority of women of childbearing age have antibodies against c
α
and R4, (ii) the levels of these antibodies differ among pregnant women in different parts of the world, and (iii) the normal human serum antibodies may target a common c
α
and R4 protein site, whereas immune antibodies targeted a different site(s) specific for each protein.
Schistosomiasis in the People's Republic of China: Prospects and Challenges for the 21st Century
Authors: Ross Allen G. P. , Sleigh Adrian C. , Li Yuesheng , Davis George M. , Williams Gail M. , Jiang Zheng , Feng Zheng , McManus Donald P.
Published: July 2002

Schistosomiasis japonica is a serious communicable disease and a major disease risk for more than 30 million people living in the tropical and subtropical zones of China. Infection remains a major public health concern despite 45 years of intensive control efforts. It is estimated that 865,000 people and 100,250 bovines are today infected in the provinces where the disease is endemic, and its transmission continues. Unlike the other schistosome species known to infect humans, the oriental schistosome, Schistosoma japonicum, is a true zoonotic organism, with a range of mammalian reservoirs, making control efforts extremely difficult. Clinical features of schistosomiasis range from fever, headache, and lethargy to severe fibro-obstructive pathology leading to portal hypertension, ascites, and hepatosplenomegaly, which can cause premature death. Infected children are stunted and have cognitive defects impairing memory and learning ability. Current control programs are heavily based on community chemotherapy with a single dose of the drug praziquantel, but vaccines (for use in bovines and humans) in combination with other control strategies are needed to make elimination of the disease possible. In this article, we provide an overview of the biology, epidemiology, clinical features, and prospects for control of oriental schistosomiasis in the People's Republic of China.
Role of Lipopolysaccharide Phase Variation in Susceptibility of
Haemophilus influenzae
to Bactericidal Immunoglobulin M Antibodies in Rabbit Sera
Authors: Erwin Alice L. , Brewah Yambasu A. , Couchenour Debra A. , Barren Philip R. , Burke Stephen J. , Choi Gil H. , Lathigra Raju , Hanson Mark S. , Weiser Jeffrey N.
Published: July 2002

The effect of phase variation of lipopolysaccharide (LPS) structure on the susceptibility of
Haemophilus influenzae
to complement-dependent killing by normal human sera and normal rat sera has been described previously. The phase-variable structure phosphorylcholine (ChoP) confers susceptibility to human serum, since ChoP on the bacterial cell surface binds to serum C-reactive protein and activates complement. In contrast, expression of galα1,4gal, a second phase-variable epitope that is also found on human glycoconjugates, confers resistance to human serum. We studied the role of phase variation of these structures in the susceptibilities of
H. influenzae
KW20 (Rd) and a clinical isolate of nontypeable
H. influenzae
to killing by rabbit sera, which often possess naturally acquired complement-dependent bactericidal activity for unencapsulated
H. influenzae
. Expression of ChoP increased the resistance of strain KW20 to killing by bactericidal rabbit sera. In contrast, the serum resistance of a clinical isolate, H233, was unaffected by ChoP expression but was reduced by galα1,4gal expression. The rabbit sera with bactericidal activity (but not the nonbactericidal sera) all contained immunoglobulin M (IgM) antibodies able to bind to the surface of
H. influenzae
bacteria, as detected by flow cytometry, and contained IgM antibodies to LPS purified from strain KW20. Preincubation of sera with LPS reduced their bactericidal activity. Bactericidal activity was recovered quantitatively in an IgM-enriched fraction of sera. It is concluded that naturally occuring bactericidal activity for unencapsulated
H. influenzae
is largely due to IgM antibodies directed against phase-variable structures of the LPS.