bluuebunny/crossref_metadata_2025 huggingface dataset:
Quinupristin-Dalfopristin Combined with β-Lactams for Treatment of Experimental Endocarditis Due to
Staphylococcus aureus
Constitutively Resistant to Macrolide-Lincosamide-Streptogramin B Antibiotics
Authors: Vouillamoz J. , Entenza J. M. , Féger C. , Glauser M. P. , Moreillon P.
Published: July 2002

Quinupristin-dalfopristin (Q-D) is an injectable streptogramin active against most gram-positive pathogens, including methicillin-resistant
Staphylococcus aureus
(MRSA). In experimental endocarditis, however, Q-D was less efficacious against MRSA isolates constitutively resistant to macrolide-lincosamide-streptogram B (C-MLS
B
) than against MLS
B
-susceptible isolates. To circumvent this problem, we used the checkerboard method to screen drug combinations that would increase the efficacy of Q-D against such bacteria. β-Lactams consistently exhibited additive or synergistic activity with Q-D. Glycopeptides, quinolones, and aminoglycosides were indifferent. No drugs were antagonistic. The positive Q-D–β-lactam interaction was independent of MLS
B
or β-lactam resistance. Moreover, addition of Q-D at one-fourth the MIC to flucloxacillin-containing plates decreased the flucloxacillin MIC for MRSA from 500 to 1,000 mg/liter to 30 to 60 mg/liter. Yet, Q-D–β-lactam combinations were not synergistic in bactericidal tests. Rats with aortic vegetations were infected with two C-MLS
B
-resistant MRSA isolates (isolates AW7 and P8) and were treated for 3 or 5 days with drug dosages simulating the following treatments in humans: (i) Q-D at 7 mg/kg two times a day (b.i.d.) (a relatively low dosage purposely used to help detect positive drug interactions), (ii) cefamandole at constant levels in serum of 30 mg/liter, (iii) cefepime at 2 g b.i.d., (iv) Q-D combined with either cefamandole or cefepime. Any of the drugs used alone resulted in treatment failure. In contrast, Q-D plus either cefamandole or cefepime significantly decreased valve infection compared to the levels of infection for both untreated controls and those that received monotherapy (
P
< 0.05). Importantly, Q-D prevented the growth of highly β-lactam-resistant MRSA in vivo. The mechanism of this beneficial drug interaction is unknown. However, Q-D–β-lactam combinations might be useful for the treatment of complicated infections caused by multiple organisms, including MRSA.
β-Lactamases in
Shigella flexneri
Isolates from Hong Kong and Shanghai and a Novel OXA-1-Like β-Lactamase, OXA-30
Authors: Siu L. K. , Lo J. Y. C. , Yuen K. Y. , Chau P. Y. , Ng M. H. , Ho P. L.
Published: July 2002

Ninety-one ampicillin-resistant
Shigella flexneri
strains from Hong Kong and Shanghai were studied for production of β-lactamases. TEM-1-like and OXA-1-like enzymes were identified in 21 and 79% of the strains, respectively, by isoelectric focusing (IEF). No difference in the pattern of β-lactamase production was found between strains from Hong Kong and Shanghai. Four ribotypes were detected. Over 88% of OXA-producing strains had the same ribotype. All TEM-1-like strains harbored a plasmid which hybridized positively with the
bla
TEM
probe. Total DNA from OXA-1-like strains failed to hybridize or only hybridized weakly with an OXA probe. The OXA resistance was not transferable. OXA-1-like enzymes exhibited substrate and inhibition profiles similar to that of OXA-1 and were shown to have a pI of 7.3 by further IEF using a narrow-range ampholine gel. The gene encoding the OXA-1-like enzyme from one isolate (CH-07) was cloned, sequenced, and found to differ from
bla
OXA-1
at codon 131 (AGA→GGA; Arg to Gly), resulting in the novel designation OXA-30. The predominance of OXA-type enzymes in ampicillin-resistant
S. flexneri
suggests host preference for specific β-lactamases.
Relationships of the Area under the Curve/MIC Ratio to Different Integral Endpoints of the Antimicrobial Effect: Gemifloxacin Pharmacodynamics in an In Vitro Dynamic Model
Authors: Firsov Alexander A. , Lubenko Irene Y. , Portnoy Yury A. , Zinner Stephen H. , Vostrov Sergey N.
Published: July 2002

Most integral endpoints of the antimicrobial effect are determined over an arbitrarily chosen time period, such as the dosing interval (τ), regardless of the actual effect duration. Unlike the τ-related endpoints, the intensity of the antimicrobial effect (
I
E
) does consider its duration—from time zero to the time when bacterial counts on the regrowth curve achieve the same maximal numbers as in the absence of the antimicrobial. To examine the possible impact of this fundamental difference on the relationships of the antimicrobial effect to the ratio of the area under the concentration-time curve (AUC) to the MIC, a clinical isolate of
Staphylococcus aureus
was exposed to simulated gemifloxacin pharmacokinetics over a 40-fold range of AUC/MIC ratios, from 11 to 466 h. In each run,
I
E
and four τ-related endpoints, including the area under the time-kill curve (AUBC), the area above the curve (AAC), the area between the control growth and time-kill curves (ABBC), and the ABBC related to the area under the control growth curve (AUGC), were calculated for τ = 24 h. Unlike the
I
E
, which displayed pseudolinear relationships with the AUC/MIC ratio; each τ-related endpoint showed a distinct saturation at potentially therapeutic AUC/MIC ratios (116 to 466 h) when the antimicrobial effect persisted longer than τ. This saturation results from the underestimation of the true effect and may be eliminated if ABBC, AAC, and AUBC (but not AUGC) are modified and determined in the same manner as the
I
E
to consider the actual effect duration. These data suggest a marginal value of the τ-related endpoints as indices of the total antimicrobial effect. Since all of them respond to AUC/MIC ratio changes less than the
I
E
, the latter is preferable in comparative pharmacodynamic studies.
Antimicrobial Susceptibilities of Unique
Stenotrophomonas maltophilia
Clinical Strains
Authors: Valdezate Sylvia , Vindel Ana , Loza Elena , Baquero Fernando , Cantón Rafael
Published: July 2002

Susceptibility to 41 antimicrobials was studied with 99
Stenotrophomonas maltophilia
strains, and different pulsed-field gel electrophoresis profiles were identified among 130 prospectively collected isolates. Moxalactam, doxycycline, minocycline, and clinafloxacin displayed the highest activity (≥98% susceptibility). Ticarcillin resistance (75%) was reverted by clavulanate in 25% of strains. Trimethoprim-sulfamethoxazole resistance was 26.2% (≥4 [trimethoprim]/76 [sulfamethoxazole] μg/ml) and dropped to 11.1% when an 8/152-μg/ml breakpoint was applied based on its bimodal MIC distribution. Resistance was lower when unique strains were considered, because clonal organisms contribute to resistance.
Activation of Multiple Antibiotic Resistance in Uropathogenic
Escherichia coli
Strains by Aryloxoalcanoic Acid Compounds
Authors: Balagué Claudia , Véscovi Eleonora Garcı́a
Published: July 2002

Clofibric and ethacrynic acids are prototypical pharmacological agents administered in the treatment of hypertrigliceridemia and as a diuretic agent, respectively. They share with 2,4-dichlorophenoxyacetic acid (the widely used herbicide known as 2,4-D) a chlorinated phenoxy structural moiety. These aryloxoalcanoic agents (AOAs) are mainly excreted by the renal route as unaltered or conjugated active compounds. The relatedness of these agents at the structural level and their potential effect on therapeutically treated or occupationally exposed individuals who are simultaneously undergoing a bacterial urinary tract infection led us to analyze their action on uropathogenic, clinically isolated
Escherichia coli
strains. We found that exposure to these compounds increases the bacterial resistance to an ample variety of antibiotics in clinical isolates of both uropathogenic and nonpathogenic
E. coli
strains. We demonstrate that the AOAs induce an alteration of the bacterial outer membrane permeability properties by the repression of the major porin OmpF in a
micF
-dependent process. Furthermore, we establish that the antibiotic resistance phenotype is primarily due to the induction of the MarRAB regulatory system by the AOAs, while other regulatory pathways that also converge into
micF
modulation (OmpR/EnvZ, SoxRS, and Lrp) remained unaltered. The fact that AOAs give rise to uropathogenic strains with a diminished susceptibility to antimicrobials highlights the impact of frequently underestimated or ignored collateral effects of chemical agents.
Susceptibilities of
Mycoplasma hominis, M. pneumoniae
, and
Ureaplasma urealyticum
to GAR-936, Dalfopristin, Dirithromycin, Evernimicin, Gatifloxacin, Linezolid, Moxifloxacin, Quinupristin-Dalfopristin, and Telithromycin Compared to Their Susceptibilities to Reference Macrolides, Tetracyclines, and Quinolones
Authors: Kenny George E. , Cartwright Frank D.
Published: July 2002

The susceptibilities of
Mycoplasma hominis
,
Mycoplasma pneumoniae
, and
Ureaplasma urealyticum
to eight new antimicrobial agents were determined by agar dilution.
M. pneumoniae
was susceptible to the new glycylcycline GAR-936 at 0.12 μg/ml and evernimicin at 4 μg/ml, but it was resistant to linezolid. It was most susceptible to dirithromycin, quinupristin-dalfopristin, telithromycin, reference macrolides, and josamycin.
M. hominis
was susceptible to linezolid, evernimicin, and GAR-936. It was resistant to macrolides and the ketolide telithromycin but susceptible to quinupristin-dalfopristin and josamycin.
U. urealyticum
was susceptible to evernimicin (8 to 16 μg/ml) and resistant to linezolid. It was less susceptible to GAR-936 (4.0 μg/ml) than to tetracycline (0.5 μg/ml). Telithromycin and quinupristin-dalfopristin were the most active agents against ureaplasmas (0.06 μg/ml). The new quinolone gatifloxacin was active against
M. pneumoniae
and
M. hominis
at 0.12 to 0.25 μg/ml and active against ureaplasmas at 1.0 μg/ml. The MICs of macrolides were markedly affected by pH, with an 8- to 32-fold increase in the susceptibility of
M. pneumoniae
as the pH increased from 6.9 to 7.8. A similar increase in susceptibility with increasing pH was also observed with ureaplasmas. Tetracyclines showed a fourfold increase of activity as the pH decreased 1 U, whereas GAR-936 showed a fourfold decrease in activity with a decrease in pH.
DNA Sequence Analysis of
rdxA
and
frxA
from 12 Pairs of Metronidazole-Sensitive and -Resistant Clinical
Helicobacter pylori
Isolates
Authors: Kwon D. H. , Hulten K. , Kato M. , Kim J. J. , Lee M. , El-Zaatari F. A. K. , Osato M. S. , Graham D. Y.
Published: July 2002

We previously reported that inactivation of
rdxA
and/or
frxA
converted
Helicobacter pylori
from metronidazole sensitive to metronidazole resistant. To examine the individual roles of
rdxA
and
frxA
in the development of metronidazole resistance in
H. pylori
, we examined the status of
rdxA
and
frxA
from 12 pairs of metronidazole-sensitive and -resistant
H. pylori
isolates obtained following unsuccessful therapy containing metronidazole. Arbitrary primed fingerprinting analyses revealed that the genotypes of 11 sensitive and resistant pairs of strains were essentially identical. Amino acid sequence identities of RdxA and FrxA from the 14 metronidazole-sensitive isolates ranged from 92 to 98% and 95 to 98%, respectively, compared to that of
H. pylori
J99 (MIC, 1 μg/ml). All strains with high-level metronidazole resistance (MICs, 128 μg/ml) contained premature truncation of both RdxA and FrxA caused by nonsense and/or frameshift mutations. Strains with intermediate resistance to metronidazole (MICs, 32 to 64 μg/ml) contained a single premature truncation and/or altered RdxA and FrxA caused by nonsense, frameshift, and unique missense mutations. The low-level metronidazole-resistant strains (MICs, 8 μg/ml) contained unique missense mutations in FrxA but no specific changes in RdxA. The results demonstrate that alterations in both the
rdxA
and
frxA
genes are required for moderate and high-level metronidazole resistance and that metronidazole resistance that develops during anti-
H. pylori
therapy containing metronidazole is most likely to involve a single sensitive strain infection rather than a coinfection with a metronidazole-resistant strain.
In Vitro Antifungal Activities of the New Triazole UR-9825 against Clinically Important Filamentous Fungi
Authors: Capilla Javier , Ortoneda Montserrat , Pastor Francisco Javier , Guarro Josep
Published: July 2002

We used a modified reference microdilution method (the M-38P method) to evaluate the in vitro activities of the new triazole UR-9825 in comparison with those of amphotericin B against 77 strains of opportunistic filamentous fungi. UR-9825 was clearly more active than amphotericin B against all fungi except
Fusarium solani
and
Scytalidium
spp. Notably, UR-9825 had low MICs for
Aspergillus fumigatus
and
Paecilomyces lilacinus
(MICs at which 90% of isolates are inhibited, 0.125 μg/ml for both species).
Type II Topoisomerase Quinolone Resistance-Determining Regions of
Aeromonas caviae
, A. hydrophila, and
A. sobria
Complexes and Mutations Associated with Quinolone Resistance
Authors: Goñi-Urriza Marisol , Arpin Corinne , Capdepuy Michèle , Dubois Véronique , Caumette Pierre , Quentin Claudine
Published: July 2002

Most
Aeromonas
strains isolated from two European rivers were previously found to be resistant to nalidixic acid. In order to elucidate the mechanism of this resistance, 20 strains of
Aeromonas caviae
(
n
= 10),
A. hydrophila
(
n
= 5), and
A. sobria
(
n
= 5) complexes, including 3 reference strains and 17 environmental isolates, were investigated. Fragments of the
gyrA
,
gyrB
,
parC
, and
parE
genes encompassing the quinolone resistance-determining regions (QRDRs) were amplified by PCR and sequenced. Results obtained for the six sensitive strains showed that the GyrA, GyrB, ParC, and ParE QRDR fragments of
Aeromonas
spp. were highly conserved (≥96.1% identity), despite some genetic polymorphism; they were most closely related to those of
Vibrio
spp.,
Pseudomonas
spp., and members of the family
Enterobacteriaceae
(72.4 to 97.1% homology). All 14 environmental resistant strains carried a point mutation in the GyrA QRDR at codon 83, leading to the substitution Ser-83→Ile (10 strains) or Ser-83→Arg. In addition, seven strains harbored a mutation in the ParC QRDR either at position 80 (five strains), generating a Ser-80→Ile (three strains) or Ser-80→Arg change, or at position 84, yielding a Glu-84→Lys modification. No amino acid alterations were discovered in the GyrB and ParE QRDRs. Double
gyrA-parC
missense mutations were associated with higher levels of quinolone resistance compared with the levels associated with single
gyrA
mutations. The most resistant strains probably had an additional mechanism(s) of resistance, such as decreased accumulation of the drugs. Our data suggest that, in mesophilic
Aeromonas
spp., as in other gram-negative bacteria, gyrase and topoisomerase IV are the primary and secondary targets for quinolones, respectively.
Efficacy of Antifungal Therapy in a Nonneutropenic Murine Model of Zygomycosis
Authors: Dannaoui Eric , Mouton Johan W. , Meis Jacques F. G. M. , Verweij Paul E.
Published: July 2002

Three isolates of zygomycetes belonging to three different genera (Rhizopus microsporus,Absidia corymbifera, andApophysomyces elegans) were used to produce a disseminated infection in nonimmunocompromised mice. The therapeutic efficacy of amphotericin B, given intraperitoneally at doses ranging from 0.5 to 4.5 mg/kg of body weight/day, oral itraconazole at 100 mg/kg/day, and oral terbinafine at 150 mg/kg/day was evaluated in this model. The markers of antifungal efficacy were the median survival time, the mortality rate, and the percentage of infected organs. Organ culture was performed along with microscopic direct examinations of tissues to assess the presence of an active infection. An acute and lethal infection was obtained in untreated mice challenged with each of the three strains. The data obtained for direct examinations and qualitative cultures indicate that, due to the nonseptate nature of the hyphae, each technique gives different information and should be used together with the others. Against all three strains, amphotericin B yielded a 90 to 100% survival rate. Itraconazole was inactive againstR. microsporusbut significantly reduced mortality in mice infected withA. corymbiferaorA. elegans.Terbinafine had no beneficial effects againstR. microsporusandA. corymbiferadespite documented absorption of the drug. Overall, only limited correlations were observed between MICs determined in vitro and in vivo efficacy of the drugs. The efficacy of itraconazole in these models of zygomycosis suggests that this drug, as well as the new azole compounds presently under development, warrants close evaluation.
Characterization of Reutericyclin Produced by
Lactobacillus reuteri
LTH2584
Authors: Gänzle Michael G. , Höltzel Alexandra , Walter Jens , Jung Günther , Hammes Walter P.
Published: July 2002

Lactobacillus reuteri
LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including
Lactobacillus
spp.,
Bacillus subtilis
,
B. cereus
,
Enterococcus faecalis
,
Staphylococcus aureus
, and
Listeria innocua
. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of
Escherichia coli
was inhibited. Reutericyclin exhibited a bactericidal mode of action against
Lactobacillus sanfranciscensis
,
Staphylococcus aureus
, and
B. subtilis
and triggered the lysis of cells of
L. sanfranciscensis
in a dose-dependent manner. Germination of spores of
B. subtilis
was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of
L. reuteri
LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C
8
chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. Höltzel, M. G. Gänzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766–2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of
L. reuteri
LTH2584 corresponded to that of purified as well as synthetic reutericyclin.
Bacterial Origin and Community Composition in the Barley Phytosphere as a Function of Habitat and Presowing Conditions
Authors: Normander Bo , Prosser Jim I.
Published: July 2002

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (
Hordeum vulgare
) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.
Improved Secretion of Native Human Insulin-Like Growth Factor 1 from
gas1
Mutant
Saccharomyces cerevisiae
Cells
Authors: Vai Marina , Brambilla Luca , Orlandi Ivan , Rota Nicola , Ranzi Bianca Maria , Alberghina Lilia , Porro Danilo
Published: July 2002

We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The
hIGF-1
gene was fused to the mating factor α prepro- leader sequence under the control of the constitutive
ACT1
promoter. We found that the inactivation of the
GAS1
gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.
Endosymbiotic Microbiota of the Bamboo PseudococcidAntonina crawii(Insecta, Homoptera)
Authors: Fukatsu Takema , Nikoh Naruo
Published: July 2002

We characterized the intracellular symbiotic microbiota of the bamboo pseudococcidAntonina crawiiby performing a molecular phylogenetic analysis in combination with in situ hybridization. Almost the entire length of the bacterial 16S rRNA gene was amplified and cloned fromA. crawiiwhole DNA. Restriction fragment length polymorphism analysis revealed that the clones obtained included three distinct types of sequences. Nucleotide sequences of the three types were determined and subjected to a molecular phylogenetic analysis. The first sequence was a member of the γ subdivision of the divisionProteobacteria(γ-Proteobacteria) to which no sequences in the database were closely related, although the sequences of endosymbionts of other homopterans, such as psyllids and aphids, were distantly related. The second sequence was a β-Proteobacteriasequence and formed a monophyletic group with the sequences of endosymbionts from other pseudococcids. The third sequence exhibited a high level of similarity to sequences ofSpiroplasmaspp. from ladybird beetles and a tick. Localization of the endosymbionts was determined by using tissue sections ofA. crawiiand in situ hybridization with specific oligonucleotide probes. The γ- and β-Proteobacteriasymbionts were packed in the cytoplasm of the same mycetocytes (or bacteriocytes) and formed a large mycetome (or bacteriome) in the abdomen. The spiroplasma symbionts were also present intracellularly in various tissues at a low density. We observed that the anterior poles of developing eggs in the ovaries were infected by the γ- and β-Proteobacteriasymbionts in a systematic way, which ensured vertical transmission. Five representative pseudococcids were examined by performing diagnostic PCR experiments with specific primers; the β-Proteobacteriasymbiont was detected in all five pseudococcids, the γ-Proteobacteriasymbiont was found in three, and the spiroplasma symbiont was detected only inA. crawii.
Prevention of Yeast Spoilage in Feed and Food by the Yeast Mycocin HMK
Authors: Lowes K. F. , Shearman C. A. , Payne J. , MacKenzie D. , Archer D. B. , Merry R. J. , Gasson M. J.
Published: July 2002

The yeastWilliopsis mrakiiproduces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin inAspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with nativeW. mrakiimycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application ofA. nigermycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts,A. nigermycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.
Biochemical and Molecular Characterization of a Laccase from
Marasmius quercophilus
Authors: Dedeyan Boghos , Klonowska Agnieszka , Tagger Simone , Tron Thierry , Iacazio Gilles , Gil Gérard , Le Petit Jean
Published: July 2002

The basidiomycete
Marasmius quercophilus
is commonly found during autumn on the decaying litter of the evergreen oak (
Quercus ilex
L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the “blue copper oxidase” family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75°C, and the

K
m

was 7.1 μM. The structural gene,
lac1
, was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.
Nickel Availability and
hupSL
Activation by Heterologous Regulators Limit Symbiotic Expression of the
Rhizobium leguminosarum
bv. Viciae Hydrogenase System in Hup
−
Rhizobia
Authors: Brito Belén , Monza Jorge , Imperial Juan , Ruiz-Argüeso Tomás , Palacios Jose Manuel
Published: July 2002

A limited number of
Rhizobium
and
Bradyrhizobium
strains possess a hydrogen uptake (Hup) system that recycles the hydrogen released from the nitrogen fixation process in legume nodules. To extend this ability to rhizobia that nodulate agronomically important crops, we investigated factors that affect the expression of a cosmid-borne Hup system from
Rhizobium leguminosarum
bv. viciae UPM791 in
R. leguminosarum
bv. viciae,
Rhizobium etli
,
Mesorhizobium loti
, and
Sinorhizobium meliloti
Hup
−
strains. After cosmid pAL618 carrying the entire
hup
system of strain UPM791 was introduced, all recipient strains acquired the ability to oxidize H
2
in symbioses with their hosts, although the levels of hydrogenase activity were found to be strain and species dependent. The levels of hydrogenase activity were correlated with the levels of nickel-dependent processing of the hydrogenase structural polypeptides and with transcription of structural genes. Expression of the NifA-dependent
hupSL
promoter varied depending on the genetic background, while the
hyp
operon, which is controlled by the FnrN transcriptional regulator, was expressed at similar levels in all recipient strains. With the exception of the
R. etli
-bean symbiosis, the availability of nickel to bacteroids strongly affected hydrogenase processing and activity in the systems tested. Our results indicate that efficient transcriptional activation by heterologous regulators and processing of the hydrogenase as a function of the availability of nickel to the bacteroid are relevant factors that affect hydrogenase expression in heterologous rhizobia.
Superoxide Dismutase Activity in
Pseudomonas putida
Affects Utilization of Sugars and Growth on Root Surfaces
Authors: Kim Young Cheol , Miller Charles D. , Anderson Anne J.
Published: July 2002

To investigate the role of superoxide dismutases (SOD) in root colonization and oxidative stress, mutants of
Pseudomonas putida
lacking manganese-superoxide dismutase (MnSOD) (
sodA
), iron-superoxide dismutase (FeSOD) (
sodB
), or both were generated. The
sodA sodB
mutant did not grow on components washed from bean root surfaces or glucose in minimal medium. The
sodB
and
sodA sodB
mutants were more sensitive than wild type to oxidative stress generated within the cell by paraquat treatment. In single inoculation of SOD mutants on bean, only the
sodA sodB
double mutant was impaired in growth on root surfaces. In mixed inoculations with wild type, populations of the
sodA
mutant were equal to those of the wild type, but levels of the
sodB
mutant and, to a great extent, the
sodA sodB
mutant, were reduced. Confocal microscopy of young bean roots inoculated with green fluorescent protein-tagged cells showed that wild type and SOD single mutants colonized well predominantly at the root tip but that the
sodA sodB
double mutant grew poorly at the tip. Our results indicate that FeSOD in
P. putida
is more important than MnSOD in aerobic metabolism and oxidative stress. Inhibition of key metabolic enzymes by increased levels of superoxide anion may cause the impaired growth of SOD mutants in vitro and in planta.
Influence of Cadmium and Mercury on Activities of Ligninolytic Enzymes and Degradation of Polycyclic Aromatic Hydrocarbons by
Pleurotus ostreatus
in Soil
Authors: Baldrian Petr , in der Wiesche Carsten , Gabriel Jiří , Nerud František , Zadražil František
Published: July 2002

The white-rot fungus
Pleurotus ostreatus
was able to degrade the polycyclic aromatic hydrocarbons (PAHs) benzo[
a
]anthracene, chrysene, benzo[
b
]fluoranthene, benzo[
k
]fluoranthene, benzo[
a
]pyrene, dibenzo[
a
,
h
]anthracene, and benzo[
ghi
]perylene in nonsterile soil both in the presence and in the absence of cadmium and mercury. During 15 weeks of incubation, recovery of individual compounds was 16 to 69% in soil without additional metal. While soil microflora contributed mostly to degradation of pyrene (82%) and benzo[
a
]anthracene (41%), the fungus enhanced the disappearance of less-soluble polycyclic aromatic compounds containing five or six aromatic rings. Although the heavy metals in the soil affected the activity of ligninolytic enzymes produced by the fungus (laccase and Mn-dependent peroxidase), no decrease in PAH degradation was found in soil containing Cd or Hg at 10 to 100 ppm. In the presence of cadmium at 500 ppm in soil, degradation of PAHs by soil microflora was not affected whereas the contribution of fungus was negligible, probably due to the absence of Mn-dependent peroxidase activity. In the presence of Hg at 50 to 100 ppm or Cd at 100 to 500 ppm, the extent of soil colonization by the fungus was limited.
Expression of
nifH
Genes in Natural Microbial Assemblages in Lake George, New York, Detected by Reverse Transcriptase PCR
Authors: Zani Sabino , Mellon Mark T. , Collier Jackie L. , Zehr Jonathan P.
Published: July 2002

A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-μm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a
nifH
gene, including representatives of unicellular and filamentous cyanobacteria, the α- and γ-subdivisions of the division
Proteobacteria
(α- and γ-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing
nifH
transcripts, clustered with the unicellular and filamentous cyanobacteria, α-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (
nifH
-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.
Purification and Biochemical Characterization of Mutacin I from the Group I Strain of
Streptococcus mutans
, CH43, and Genetic Analysis of Mutacin I Biosynthesis Genes
Authors: Qi Fengxia , Chen Ping , Caufield Page W.
Published: July 2002

Previously, we reported isolation and characterization of mutacin III and genetic analysis of mutacin III biosynthesis genes from the group III strain of
Streptococcus mutans
, UA787 (F. Qi, P. Chen, and P. W. Caufield, Appl. Environ. Microbiol. 65:3880–3887, 1999). During the same process of isolating the mutacin III structural gene, we also cloned the structural gene for mutacin I. In this report, we present purification and biochemical characterization of mutacin I from the group I strain CH43 and compare mutacin I and mutacin III biosynthesis genes. The mutacin I biosynthesis gene locus consists of 14 genes in the order
mutR
, -
A
, -
A
′, -
B
, -
C
, -
D
, -
P
, -
T
, -
F
, -
E
, -
G
,
orfX
,
orfY
,
orfZ. mutA
is the structural gene for mutacin I, while
mutA
′ is not required for mutacin I activity. DNA and protein sequence analysis revealed that mutacins I and III are homologous to each other, possibly arising from a common ancestor. The mature mutacin I is 24 amino acids in size and has a molecular mass of 2,364 Da. Ethanethiol modification and peptide sequencing of mutacin I revealed that it contains six dehydrated serines, four of which are probably involved with thioether bridge formation. Comparison of the primary sequence of mutacin I with that of mutacin III and epidermin suggests that mutacin I likely has the same bridging pattern as epidermin.
Iron Stress in Open-Ocean Cyanobacteria (
Synechococcus
,
Trichodesmium
, and
Crocosphaera
spp.): Identification of the IdiA Protein
Authors: Webb E. A. , Moffett J. W. , Waterbury J. B.
Published: July 2002

Cyanobacteria are prominent constituents of the marine biosphere that account for a significant percentage of oceanic primary productivity. In an effort to resolve how open-ocean cyanobacteria persist in regions where the Fe concentration is thought to be limiting their productivity, we performed a number of Fe stress experiments on axenic cultures of marine
Synechococcus
spp.
, Crocosphaera
sp.
,
and
Trichodesmium
sp. Through this work, we determined that all of these marine cyanobacteria mount adaptive responses to Fe stress, which resulted in the induction and/or repression of several proteins. We have identified one of the Fe stress-induced proteins as an IdiA homologue. Genomic observations and laboratory data presented herein from open-ocean
Synechococcus
spp. are consistent with IdiA having a role in cellular Fe scavenging. Our data indicate that IdiA may make an excellent marker for Fe stress in open-ocean cyanobacterial field populations. By determining how these microorganisms respond to Fe stress, we will gain insight into how and when this important trace element can limit their growth in situ
.
This knowledge will greatly increase our understanding of how marine Fe cycling impacts oceanic processes, such as carbon and nitrogen fixation.
Study of Genetic Diversity of Eukaryotic Picoplankton in Different Oceanic Regions by Small-Subunit rRNA Gene Cloning and Sequencing
Authors: Dı́ez Beatriz , Pedrós-Alió Carlos , Massana Ramon
Published: July 2002

Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad
Paraphysomonas
, cercomonads, and fungi. There were two relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.
The Viable But Nonculturable State of
Ralstonia solanacearum
May Be Involved in Long-Term Survival and Plant Infection
Authors: Grey Brian E. , Steck Todd R.
Published: July 2002

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium
Ralstonia solanacearum
was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of
R. solanacearum
cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of
R. solanacearum
entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.
Direct Incorporation of Glucosamine and
N
-Acetylglucosamine into Exopolymers by
Gluconacetobacter xylinus
(=
Acetobacter xylinum
) ATCC 10245: Production of Chitosan-Cellulose and Chitin-Cellulose Exopolymers
Authors: Lee Jin W. , Deng Fang , Yeomans Walter G. , Allen Alfred L. , Gross Richard A. , Kaplan David L.
Published: July 2002

Gluconacetobacter xylinus
(=
Acetobacter xylinum
) ATCC 10245 incorporated 2-amino-2-deoxy-
d
-glucose (glucosamine) and 2-acetamido-2-deoxy-
d
-glucose (
N
-acetylglucosamine), but not 3-
O
-methyl-
d
-glucose or 2-deoxy-
d
-glucose into exopolymers. Incorporation was confirmed by gas chromatography with and without mass spectrometry, Fourier transform infrared, and
1
H nuclear magnetic resonance. The average molar percentage of glucosamine and
N
-acetylglucosamine in the exopolymers was about 18%.
Are the Enzyme Immunoassays for Antibodies to Pneumococcal Capsular Polysaccharides Serotype Specific?
Authors: Soininen Anu , van den Dobbelsteen Germie , Oomen Lukas , Käyhty Helena
Published: July 2002

The specificity of antibody binding to pneumococcal capsular polysaccharides (Pnc PSs) measured by enzyme immunoassay (EIA) was studied by inhibition of antibody binding by homologous and heterologous PSs. We found extensive cross-reactivity of antibody binding to type 6B, 19F, and 23F PSs but not to type 14 PS, even after treatment with cell wall PS (CPS). The cross-reactive antibody was highly prevalent in sera of infants and adults with naturally acquired antibody, but not in sera of infants and adults immunized with pneumococcal vaccines. However, a type 11A antibody response was seen after vaccination with heterologous PSs. Monoclonal antibodies prepared against a type 6B PS-tetanus toxoid conjugate recognized also other than the specific type of PS in the EIA, implying the possible existence of a cross-reactive epitope. Remarkable differences in specificity among type 6B PS preparations from different manufacturers were found. Moreover, different lots of type 11A PS from the same manufacturer showed differences in specificity. The results suggest that some Pnc PS preparations may contain cross-reactive epitopes or impurities, other than CPS, that are common to many types of Pnc PS. The specificity of antibodies, especially in sera from nonimmunized subjects, measured by EIA can be questioned.
Lyme Borreliosis in Rhesus Macaques: Effects of Corticosteroids on Spirochetal Load and Isotype Switching of Anti-Borrelia burgdorferiAntibody
Authors: Pachner Andrew R. , Amemiya Kei , Bartlett Melanie , Schaefer Henry , Reddy Kiran , Zhang Wei-Fen
Published: July 2002

ExperimentalBorrelia burgdorferiinfection of rhesus monkeys is an excellent model of Lyme disease and closely parallels the infection in humans. Little is known about the interaction of host immunity with the spirochete in patients with chronic infection. We hypothesized that rapid development of anti-B. burgdorferiantibody in immunocompetent nonhuman primates (NHPs) is the major determinant of the reduction of the spirochetal load in Lyme borreliosis. This hypothesis was tested by measurement of the spirochetal load by PCR in association with characterization of the anti-B. burgdorferihumoral immune response in immunocompetent NHPs versus that in corticosteroid-treated NHPs. Although anti-B. burgdorferiimmunoglobulin G (IgG) antibody was effectively inhibited in dexamethasone (Dex)-treated NHPs, anti-B. burgdorferiIgM antibody levels continued to rise after the first month and reached levels in excess of IgM levels in immunocompetent NHPs. This vigorous production of anti-B. burgdorferiIgM antibodies was also studied in vitro by measurement of antibody produced byB. burgdorferi-stimulated peripheral blood mononuclear cells. Despite these high IgM antispirochetal antibodies in Dex-treated NHPs, spirochetal loads were much higher in these animals. These data indicate that Dex treatment results in interference with isotype switching in this model and provide evidence that anti-B. burgdorferiIgG antibody is much more effective than IgM antibody in decreasing the spirochetal load in infected animals.
Maturational Changes in Peripheral Lymphocyte Subsets Pertinent to Monitoring Human Immunodeficiency Virus-Infected Chinese Pediatric Patients
Authors: Kam Kai Man , Leung Wai Lin , Wong Ka Hing , Lee Shui Shan , Hung Mi Yim , Kwok Mei Yee
Published: July 2002

On the basis of results of testing of 212 peripheral blood samples from ethnic Chinese individuals in five age groups, ranging from birth to adulthood, by standardized flow cytometry techniques, we studied the maturational processes that are pertinent to monitoring the human immunodeficiency virus (HIV)-infected Chinese pediatric population. While the numbers of peripheral total white cells and percent lymphocytes declined from birth to adulthood, the percent CD3+T lymphocytes was steady among all age groups studied. The numbers of CD3+CD4+(T-helper) cells decreased markedly after the first year of life, followed by a slower decline afterward and then a slight increase before adulthood. The trend for CD3+CD8+(T-suppressor) cells, however, was an increase among individuals of all age ranges. The numbers of CD19+CD3−(B cells) increased only during the first year of life and then declined steadily, while natural killer (NK) cells showed the opposite pattern. Comparison of the results with those of studies done with a Caucasian population showed that both peripheral T-helper and T-suppressor cell numbers were low after the first year of life in the Chinese pediatric population in comparison with those in a Caucasian pediatric population. Lower B-cell counts and higher NK-cell counts were seen after the first year of life in the Chinese population than in the Caucasian population. It is important that for each HIV-infected population normative ranges of the lymphocyte subset be established to monitor HIV-infected pediatric patients.
Highly Active Antiretroviral Therapy in Human Immunodeficiency Virus Type 1-Infected Children: Analysis of Cellular Immune Responses
Authors: Blazevic Vesna , Jankelevich Shirley , Steinberg Seth M. , Jacobsen Freda , Yarchoan Robert , Shearer Gene M.
Published: July 2002

The present study analyzes the effect of highly active antiretroviral therapy (HAART) on restoration of cellular immunity in human immunodeficiency virus (HIV)-infected children over a 24-week period following initiation of HAART with ritonavir, nevirapine, and stavudine. The immunological parameters evaluated at four time points (at enrollment and at 4, 12, and 24 weeks of therapy) included cytokine production by monocytes as well as T-cell proliferation in response to mitogen, alloantigen, and recall antigens including HIV type 1 envelope peptides. Circulating levels of interleukin-16 (IL-16) were measured, in addition to CD4+T-cell counts, plasma HIV RNA levels, and the delayed-type hypersensitivity (DTH) response. At enrollment the children exhibited defects in several immune parameters measured. Therapy increased CD4+T-cell counts and decreased viral loads significantly. By contrast, the only immunological parameter that was significantly increased was IL-12 p70 production by monocytes; the DTH response toCandida albicansalso showed a strong increase in patients becoming positive. In conclusion, these results demonstrate that HAART in HIV-infected children affects the dynamics of HIV replication and the CD4+T-cell count over 24 weeks, similar to the pattern seen in HIV-infected adults. Furthermore, these data indicate improvement in antigen-presenting cell immunological function in HIV-infected children induced by HAART.
Monoclonal Antibodies to Immunodominant Epitope ofTropheryma whipplei
Authors: Liang Zhongxing , La Scola Bernard , Raoult Didier
Published: July 2002

Recent isolation ofTropheryma whipplei(formerlyTrophyrema whippelii), the agent of Whipple’s disease, from the cardiac valve of a patient with Whipple’s disease endocarditis now allows the detection of reactive epitopes that could be used in a serological assay. In order to propose an enzyme-linked immunosorbent assay (ELISA) that uses recombinantT. whippleiantigen, we first determined by Western blotting of human, mouse, and rabbit antisera that the common immunodominant epitope is an 84-kDa protein. We then produced 13 monoclonal antibodies (MAbs) againstT. whipplei, 12 of which recognize this immunodominant epitope. These MAbs did not react with phylogenetically closely related bacteria or bacteria previously shown to be cross-reactive withT. whipplei, but they did react with two other strains ofT. whippleiisolated, one from an ocular sample and the other from a duodenal biopsy specimen. By confocal microscopy, the MAbs allowed detection ofT. whippleiwithin infected fibroblasts. The identification of the 84-kDa antigen with our MAbs will make it possible to develop a diagnostic antigen for use in a diagnostic ELISA for Whipple’s disease.