bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacodynamics of Moxifloxacin and Levofloxacin at Simulated Epithelial Lining Fluid Drug Concentrations against
Streptococcus pneumoniae
Authors: Florea Naomi R. , Tessier Pamela R. , Zhang Cuilian , Nightingale Charles H. , Nicolau David P.
Published: March 2004

Recent clinical failures associated with levofloxacin treatment for
Streptococcus pneumoniae
infections and growing evidence of frequent mutations in the isolate population have led to increased concerns regarding fluoroquinolone resistance. Our objective was to characterize the efficacies of levofloxacin and moxifloxacin against various genotypes of
S. pneumoniae
after simulated bronchopulmonary exposures. An in vitro model was used to simulate a levofloxacin concentration of 500 mg and a moxifloxacin concentration of 400 mg, which were previously determined to be the concentrations in the epithelial lining fluid of older adults receiving once-daily dosing. The effects of the drugs were tested against six
S. pneumoniae
containing various mutations. Bacterial density and resistance were quantitatively assessed over 48 h. The
S. pneumoniae
isolate with no mutation displayed a 4-log reduction in CFU after treatment with both agents and did not develop resistance. Isolates containing the
parC
or
parE
mutation or both mutations regrew and developed resistance when they were exposed to levofloxacin, despite an unbound area under the concentration-time curve (AUC):MIC ratio of ∼100. When the isolate containing the
parC
and
gyrA
mutations was exposed to levofloxacin, there was a half-log reduction in the number of CFU compared to that for the control, but the isolate subsequently regrew. Likewise, levofloxacin did not kill the isolate containing the
parC
,
gyrA
, and
parE
mutations. Moxifloxacin sustained the killing of all bacterial isolates tested without the development of resistance. Levofloxacin did not sustain bacterial killing and did not prevent the emergence of further resistance in mutants with the
parC
or
parE
mutation or both mutations, even though an unbound AUC:MIC ratio for exposure well above the breakpoint of 30 to 40 established in the literature for
S. pneumoniae
was maintained. Moxifloxacin was effective against all isolates tested, despite the presence of isolates with two- and three-step mutations, for which the MICs were increased.
Efficacy and Pharmacokinetics of Intravenous Nanocapsule Formulations of Halofantrine in
Plasmodium berghei
-Infected Mice
Authors: Mosqueira Vanessa C. F. , Loiseau Philippe M. , Bories Christian , Legrand Philippe , Devissaguet Jean-Philippe , Barratt Gillian
Published: March 2004

The efficacy and pharmacokinetics of a new parenteral formulation of halofantrine were studied in mice infected with
Plasmodium berghei
. The formulation consisted of nanocapsules with an oily core, prepared from either poly(
d,l
-lactide) (PLA) homopolymer or PLA that was surface modified with grafted polyethylene glycol chains. They were compared with a previously described intravenous halofantrine preparation. No toxic effects were observed with halofantrine in form of nanocapsules after intravenous administration for doses of up to 100 mg/kg, whereas the solubilized form in polyethylene glycol-dimethylacetamide was toxic at this dose. The halofantrine-loaded nanocapsules showed activity that was similar to or better than that of the solution in the 4-day test and as a single dose in severely infected mice, with only minimal differences between the two nanocapsule formulations. Halofantrine pharmacokinetics were determined in parallel with parasite development in severely infected mice. Nanocapsules increased the area under the curve for halofantrine in plasma more than sixfold compared with the solution throughout the experimental period of 70 h. Furthermore, nanocapsules induced a significantly faster control of parasite development than the solution in the first 48 h posttreatment. While the parasitemia fell more rapidly with PLA nanocapsules, the effect was more sustained with the surface-modified ones. This is consistent with surface-modified nanocapsules remaining longer in the circulation. These results suggest that nanocapsule formulations could provide a more favorable halofantrine profile in the plasma and reduce the intravenous dose necessary and therefore the toxicity, thus suggesting the use of halofantrine by a parenteral route in severe malaria.
Involvement of Enterobactin Synthesis Pathway in Production of Microcin H47
Authors: Azpiroz María F. , Laviña Magela
Published: March 2004

Microcin H47 (MccH47) is a gene-encoded peptide antibiotic produced by an
Escherichia coli
clinical isolate which is active on strains of gram-negative bacteria. Its uptake by
E. coli
K-12-susceptible cells depends on the presence of any of the outer membrane proteins Cir, Fiu, and FepA, the three catechol receptors of this organism. The nucleotide sequence of a portion of the MccH47 genetic system that had not yet been studied was elucidated. Five open reading frames were identified, three of which corresponded to genes encoding functions related to catechol-type siderophores:
mchA
and
mchS1
are
iroB
and
iroD
homologues, respectively, and
mchS4
was found to promote the production of the catecholate siderophore enterobactin. The possible relationship between enterobactin synthesis and MccH47 production was studied. Enterobactin-deficient strains failed to produce MccH47 when transformed with the antibiotic genetic determinants and upon introduction of the
ent
genetic cluster, the production of both the siderophore and MccH47 was restored. Further studies demonstrated that at least the enterobactin nonribosomal peptide synthase EntF is necessary for MccH47 synthesis. The relationship found between MccH47 and catecholate siderophore production is discussed, and a model outlining MccH47 synthesis is proposed.
Modulation of In Vitro Fungicidal Activity of Human Lactoferrin againstCandida albicansby Extracellular Cation Concentration and Target Cell Metabolic Activity
Authors: Viejo-Díaz Mónica , Andrés María T. , Fierro José F.
Published: March 2004

The anti-Candidaactivity of the innate defense protein human lactoferrin was investigated. Lactoferrin displayed a clear fungicidal effect againstCandida albicansonly under low-strength conditions. This candidacidal activity was inversely correlated with the extracellular concentration of the monovalent cations and was prevented by Na+and K+(≥30 mM) and by divalent cations (Ca2+and Mg2+at ≥4 mM). A slight cellular release of K+, cytosolic acidification, and a change in the membrane potential were observed inC. albicanscells treated with lactoferrin, suggesting that this protein directly or indirectly interacts with the cytoplasmic membrane. Mitochondrial inhibitors (carbonyl cyanidem-chlorophenylhydrazone, 2,4-dinitrophenol, azide, and antimycin) as well as anaerobic conditions significantly reduced the killing effect of lactoferrin. These results suggest that low-strength conditions and the cellular metabolic state may modulate the candidacidal activity of human lactoferrin.
Mycobacterium tuberculosis
DNA Gyrase: Interaction with Quinolones and Correlation with Antimycobacterial Drug Activity
Authors: Aubry Alexandra , Pan Xiao-Su , Fisher L. Mark , Jarlier Vincent , Cambau Emmanuelle
Published: March 2004

Genome studies suggest that DNA gyrase is the sole type II topoisomerase and likely the unique target of quinolones in
Mycobacterium tuberculosis
. Despite the emerging importance of quinolones in the treatment of mycobacterial disease, the slow growth and high pathogenicity of
M. tuberculosis
have precluded direct purification of its gyrase and detailed analysis of quinolone action. To address these issues, we separately overexpressed the
M. tuberculosis
DNA gyrase GyrA and GyrB subunits as His-tagged proteins in
Escherichia coli
from pET plasmids carrying
gyrA
and
gyrB
genes. The soluble 97-kDa GyrA and 72-kDa GyrB subunits were purified by nickel chelate chromatography and shown to reconstitute an ATP-dependent DNA supercoiling activity. The drug concentration that inhibited DNA supercoiling by 50% (IC
50
) was measured for 22 different quinolones, and values ranged from 2 to 3 μg/ml (sparfloxacin, sitafloxacin, clinafloxacin, and gatifloxacin) to >1,000 μg/ml (pipemidic acid and nalidixic acid). By comparison, MICs measured against
M. tuberculosis
ranged from 0.12 μg/ml (for gatifloxacin) to 128 μg/ml (both pipemidic acid and nalidixic acid) and correlated well with the gyrase IC
50
s (
R
2
= 0.9). Quinolones promoted gyrase-mediated cleavage of plasmid pBR322 DNA due to stabilization of the cleavage complex, which is thought to be the lethal lesion. Surprisingly, the measured concentrations of drug inducing 50% plasmid linearization correlated less well with the MICs (
R
2
= 0.7). These findings suggest that the DNA supercoiling inhibition assay may be a useful screening test in identifying quinolones with promising activity against
M. tuberculosis
. The quinolone structure-activity relationship demonstrated here shows that C-8, the C-7 ring, the C-6 fluorine, and the N-1 cyclopropyl substituents are desirable structural features in targeting
M. tuberculosis
gyrase.
Effect of
rpoB
Mutations Conferring Rifampin Resistance on Fitness of
Mycobacterium tuberculosis
Authors: Mariam Deneke H. , Mengistu Yohannes , Hoffner Sven E. , Andersson Dan I.
Published: March 2004

Rifampin is a major drug used in the treatment of tuberculosis infections, and increasing rifampin resistance represents a worldwide clinical problem. Resistance to rifampin is caused by mutations in the
rpoB
gene, encoding the β-subunit of RNA polymerase. We examined the effect of three different
rpoB
mutations on the fitness of
Mycobacterium tuberculosis
. Rifampin-resistant mutants were isolated from a virulent clinical isolate of
M. tuberculosis
(strain Harlingen) in vitro at a mutation frequency of 2.3 × 10
−8
. Mutations in the
rpoB
gene were identified, and the growth rates of three defined mutants were measured by competition with the susceptible parent strain in laboratory medium and by single cultures in a macrophage cell line and in laboratory medium. All of the mutants showed a decreased growth rate in the three assays. The relative fitness of the mutants varied between 0.29 and 0.96 (that of the susceptible strain was set to 1.0) depending on the specific mutant and assay system. Unexpectedly, the relative fitness ranking of the mutants differed between the different assays. In conclusion, rifampin resistance is associated with a cost that is conditional.
Emerging Plasmid-Mediated Quinolone Resistance Associated with the
qnr
Gene in
Klebsiella pneumoniae
Clinical Isolates in the United States
Authors: Wang Minggui , Sahm Daniel F. , Jacoby George A. , Hooper David C.
Published: March 2004

Although quinolone resistance commonly results from chromosomal mutation, recent studies indicate that such resistance can also be transferred on plasmids carrying the gene responsible,
qnr
. One hundred ten ciprofloxacin-resistant clinical isolates of
Klebsiella pneumoniae
and
Escherichia coli
from the United States were screened for the
qnr
gene by PCR and Southern hybridization of plasmid DNA. Conjugation experiments were done with azide-resistant
E. coli
J53 as the recipient and selection with azide and sulfonamide, a resistance frequently linked to
qnr
. EcoRI and BamHI digests of
qnr
-hybridizing plasmids were subjected to electrophoresis on agarose gels and probed with
qnr
by Southern hybridization.
qnr
was detected in 8 (11.1%) of 72
K. pneumoniae
strains. These eight positive strains were from six states in the United States.
qnr
was not found in any of the 38
E. coli
strains tested. Quinolone resistance was transferred from seven of the eight probe-positive strains. Transconjugants with
qnr
-hybridizing plasmids had 32-fold increases in ciprofloxacin MICs relative to
E. coli
J53. For all eight strains, the sequence of
qnr
was identical to that originally reported. By size and restriction digests, four plasmids were related to the first-reported plasmid, pMG252, and three were different. Five new
qnr
plasmids encoded FOX-5 β-lactamase, as did pMG252, but two others produced SHV-7 extended-spectrum β-lactamase. Transferable plasmid-mediated quinolone resistance associated with
qnr
is now widely distributed in quinolone-resistant clinical strains of
K. pneumoniae
in the United States. Plasmid-determined quinolone resistance contributes to the increasing quinolone resistance of
K. pneumoniae
isolates and to the linkage previously observed between resistance to quinolones and the latest β-lactam antibiotics.
Relationship between Antiviral Activity and Host Toxicity: Comparison of the Incorporation Efficiencies of 2′,3′-Dideoxy-5-Fluoro-3′-Thiacytidine-Triphosphate Analogs by Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Human Mitochondrial DNA Polymerase
Authors: Feng Joy Y. , Murakami Eisuke , Zorca Suzana M. , Johnson Allison A. , Johnson Kenneth A. , Schinazi Raymond F. , Furman Phillip A. , Anderson Karen S.
Published: March 2004

Emtricitabine [(−)FTC; (−)-β-l-2′-3′-dideoxy-5-fluoro-3′-thiacytidine] is an oxathiolane nucleoside analog recently approved by the Food and Drug Administration for the treatment of human immunodeficiency virus (HIV). Structurally, (−)FTC closely resembles lamivudine [(−)3TC] except that the former is 5-fluorinated on the cytosine ring. In HIV-1 reverse transcriptase (RT) enzymatic assays, the triphosphate of (−)FTC [(−)FTC-TP] was incorporated into both DNA-DNA and DNA-RNA primer-templates nearly 3- and 10-fold more efficiently than (−)3TC-TP. Animal studies and clinical trial studies have demonstrated a favorable safety profile for (−)FTC. However, a detailed study of the incorporation of (−)FTC-TP by human mitochondrial DNA polymerase γ, a host enzyme associated with nucleoside toxicity, is required for complete understanding of the molecular mechanisms of inhibition and toxicity. We studied the incorporation of (−)FTC-TP and its enantiomer (+)FTC-TP into a DNA-DNA primer-template by recombinant human mitochondrial DNA polymerase in a pre-steady-state kinetic analysis. (−)FTC-TP was incorporated 2.9 × 105-, 1.1 × 105-, 1.6 × 103-, 7.9 × 103-, and 100-fold less efficiently than dCTP, ddCTP, (+)3TC-TP, (+)FTC-TP, and (−)3TC-TP, respectively. The rate of removal of (−)FTC-MP from the corresponding chain-terminated 24-mer DNA by polymerase γ's 3′→5′ exonuclease activity was equal to the removal of (+)FTC-MP, 2-fold slower than the removal of (−)3TC-MP and (+)3TC-MP, and 4.6-fold slower than the excision of dCMP. These results demonstrate that there are clear differences between HIV-1 RT and polymerase γ in terms of preferences for substrate structure.
Enhancement of the
mexAB
-
oprM
Efflux Pump Expression by a Quorum-Sensing Autoinducer and Its Cancellation by a Regulator, MexT, of the
mexEF
-
oprN
Efflux Pump Operon in
Pseudomonas aeruginosa
Authors: Maseda Hideaki , Sawada Isao , Saito Kohjiro , Uchiyama Hiroo , Nakae Taiji , Nomura Nobuhiko
Published: March 2004

nfxC
-type cells of
Pseudomonas aeruginosa
that produce the MexEF-OprN efflux pump exhibit resistance to fluoroquinolones and chloramphenicol and hypersusceptibility to most classical β-lactam antibiotics. We investigated the molecular mechanism of how the
nfxC
mutation causes β-lactam hypersusceptibility. The MexAB-OprM extrusion pump transports and confers resistance to β-lactam antibiotics. Interestingly, expression of the
mexAB-oprM
operon reached the highest level during the mid-stationary growth phase in both wild-type and
nfxC
-type mutant strains, suggesting that expression of the
mexAB-oprM
operon may be controlled by cell density-dependent regulation such as quorum sensing. This assumption was verified by demonstrating that exogenous addition of the quorum-sensing autoinducer
N
-butyryl-
l
-homoserine lactone (C4-HSL) enhanced the expression of MexAB-OprM, whereas
N
-(3-oxododecanoyl)-
l
-homoserine lactone had only a slight effect. Furthermore, this C4-HSL-mediated enhancement of
mexAB-oprM
expression was repressed by MexT, a positive regulator of the
mexEF-oprN
operon. It was concluded that β-lactam hypersusceptibility in
nfxC
-type mutant cells is caused by MexT-mediated cancellation of C4-HSL-mediated enhancement of MexAB-OprM expression.
Activities of Artesunate and Primaquine againstAsexual- and Sexual-Stage Parasites in FalciparumMalaria
Authors: Pukrittayakamee Sasithon , Chotivanich Kesinee , Chantra Arun , Clemens Ralf , Looareesuwan Sornchai , White Nicholas J.
Published: March 2004

The
activities of primaquine in combination with quinine or artesunate
against asexual- and sexual-stage parasites were assessed in 176 adult
Thai patients with uncomplicated
Plasmodium falciparum
malaria. Patients were randomized to one of the six following 7-day
oral treatment regimens: (i) quinine alone, (ii) quinine with
tetracycline, (iii) quinine with primaquine at 15 mg/day, (iv) quinine
with primaquine at 30 mg/day, (v) artesunate alone, or (vi) artesunate
with primaquine. Clinical recovery occurred in all patients. There were
no significant differences in fever clearance times, rates of
P.
falciparum
reappearance, or recurrent vivax malaria between the
six treatment groups. Patients treated with artesunate alone or in
combination with primaquine had significantly shorter parasite
clearance times (mean ± standard deviation = 65±
18 versus 79 ± 21 h) and lower gametocyte
carriage rates (40 versus 62.7%) than those treated with quinine
(
P
≤ 0.007). Primaquine did not affect the therapeutic
response (
P
> 0.2). Gametocytemia was detected in 98
patients (56% [22% before treatment and 34%
after treatment]). Artesunate reduced the appearance of
gametocytemia (relative risk [95% confidence
interval] = 0.34 [0.17 to 0.70]), whereas
combinations containing primaquine resulted in shorter gametocyte
clearance times (medians of 66 versus 271 h for quinine
groups and 73 versus 137 h for artesunate groups;
P
≤
0.038). These results suggest that artesunate predominantly
inhibits gametocyte development whereas primaquine accelerates
gametocyte clearance in
P. falciparum
malaria.
Potent Synergistic In Vitro Interaction between Nonantimicrobial Membrane-Active Compounds and Itraconazole against Clinical Isolates of
Aspergillus fumigatus
Resistant to Itraconazole
Authors: Afeltra Javier , Vitale Roxana G. , Mouton Johan W. , Verweij Paul E.
Published: March 2004

To develop new approaches for the treatment of invasive infections caused by
Aspergillus fumigatus
, the in vitro interactions between itraconazole (ITZ) and seven different nonantimicrobial membrane-active compounds—amiodarone (AMD), amiloride, lidocaine, lansoprazole (LAN), nifedipine (NIF), verapamil, and fluphenazine—against seven ITZ-susceptible and seven ITZ-resistant (ITZ-R) strains were evaluated by the checkerboard microdilution method based on National Committee for Clinical Laboratory Standards M-38A guidelines. The nature and the intensity of the interactions were assessed by a nonparametric approach (fractional inhibitory concentration [FIC] index model), a fully parametric response surface approach (Greco model) of the Loewe additivity no-interaction theory, and the nonparametric (Prichard model) and semiparametric response surface approaches of the Bliss independence (BI) no-interaction theory. Statistically significant synergy was found for the combination of ITZ and AMD and the combination of LAN and NIF, although with different intensities against ITZ-R strains. The FIC index values ranged from 1 to 0.02 for ITZ-AMD, 0.53 to 0.04 for ITZ-LAN, and 0.28 to 0.06 for ITZ-NIF. By use of the BI-based model, the strongest synergy was found for the combination of ITZ with AMD, followed by the combination of ITZ and NIF. The parametric models could not be fit adequately because most of the drugs alone did not show any effect and, thus, no sigmoid dose-response. In general, the combination of ITZ with calcium pump blockers displayed in vitro synergistic activity, primarily against ITZ-R strains, and warrants further investigation.
Mutations in a 23S rRNA Gene of
Chlamydia trachomatis
Associated with Resistance to Macrolides
Authors: Misyurina O. Y. , Chipitsyna E. V. , Finashutina Y. P. , Lazarev V. N. , Akopian T. A. , Savicheva A. M. , Govorun V. M.
Published: March 2004

For six clinical isolates of
Chlamydia trachomatis
, in vitro susceptibility to erythromycin, azithromycin, and josamycin has been determined. Four isolates were resistant to all the antibiotics and had the mutations A2058C and T2611C (
Escherichia coli
numbering) in the 23S rRNA gene. All the isolates had mixed populations of bacteria that did and did not carry 23S rRNA gene mutations.
Dissemination ofblaCMY-2amongEscherichia coliIsolates from Food Animals, Retail Ground Meats, and Humans in Southern Taiwan
Authors: Yan Jing-Jou , Hong Ching-Yi , Ko Wen-Chien , Chen Yuan-Jui , Tsai Shu-Huei , Chuang Chin-Luan , Wu Jiunn-Jong
Published: March 2004

Twenty-sixEscherichia coliisolates recovered from food animal feces and retail ground meats and 14 urinaryE. coliisolates from outpatients were shown to carryblaCMY-2. Similar CMY-2-encoding plasmids were found among seven human and three ground-pork isolates. These data indicate the community spread ofblaCMY-2in southern Taiwan.
6,7-Dihydroxyflavone Dramatically Intensifies the Susceptibility of Methicillin-Resistant or -Sensitive
Staphylococcus aureus
to β-Lactams
Authors: Sato Youichi , Shibata Hirofumi , Arakaki Naokatu , Higuti Tomihiko
Published: March 2004

We have demonstrated that 6,7-dihydroxyflavone by itself has only a weak antibacterial effect on methicillin-resistant
Staphylococcus aureus
(MRSA) but that at concentrations less than MIC it synergistically elevates the susceptibility of clinically isolated MRSA and methicillin-sensitive
S. aureus
strains to β-lactam antibiotics from 8- to 32,800-fold.
Increasing Prevalence of Methicillin-Resistant
Staphylococcus aureus
Causing Nosocomial Infections at a University Hospital in Taiwan from 1986 to 2001
Authors: Hsueh Po-Ren , Teng Lee-Jene , Chen Wen-Hwei , Pan Huei-Ju , Chen Mei-Lin , Chang Shan-Chwen , Luh Kwen-Tay , Lin Fang-Yue
Published: March 2004

A rapid emergence of nosocomial methicillin-resistant
Staphylococcus aureus
(MRSA) infection (from 26.3% in 1986 to 77% in 2001) was found. The susceptibility of 200 nonduplicate blood isolates of MRSA and 100 MRSA isolates causing refractory bacteremia to 22 antimicrobial agents disclosed that glycopeptides, quinupristin-dalfopristin, and linezolid remained the most active agents.
Clonal Relatedness of Erythromycin-Resistant
Streptococcus pyogenes
Isolates in Germany
Authors: Reinert Ralf R. , Lütticken Rudolf , Sutcliffe Joyce A. , Tait-Kamradt Amelia , Cil Murat Y. , Schorn Holger M. , Bryskier André , Al-Lahham Adnan
Published: March 2004

In a nationwide study in Germany, a total of 381
Streptococcus pyogenes
were collected. Erythromycin A-resistant strains were characterized for the underlying resistance genotype, showing 55.6% had the efflux type
mef
(A), 31.5% had
erm
(A), and 13.0% had
erm
(B). A total of 23 different multilocus sequence types were observed.
Reduction in Glycopeptide Resistance in Vancomycin-Resistant Enterococci as a Result of
vanA
Cluster Rearrangements
Authors: Lee Wee Gyo , Huh Ji Young , Cho Sung Ran , Lim Young Ae
Published: March 2004

The molecular characterization of five clinical isolates of
vanA
-containing vancomycin-resistant enterococci with altered resistance to glycopeptides was examined. One strain represented an IS
1216V
insertion accompanied by partial deletion of the reading frame of
vanX
following a transposition event. The other four strains represented IS
1216V
within the
vanX
-
vanY
intergenic region associated with deletion of
vanY
or
vanZ
.
Caspofungin Resistance inCandida albicans: Correlating Clinical Outcome with Laboratory Susceptibility Testing of Three Isogenic Isolates Serially Obtained from a Patient with Progressive Candida Esophagitis
Authors: Hernandez Steve , López-Ribot José L. , Najvar Laura K. , McCarthy Dora I. , Bocanegra Rosie , Graybill John R.
Published: March 2004

A patient with azole-refractory thrush-esophagitis responded initially to caspofungin, but the treatment eventually failed. In a murine model, caspofungin was effective against two early isolates for which the MICs of caspofungin were low, but it was less effective against a late isolate for which the MIC of caspofungin was greater. We concluded that there is a correlation between in vivo failure and rising in vitro caspofungin MICs.
Molecular Analysis of Metallo-β-Lactamase Gene
bla
SPM-1
-Surrounding Sequences from Disseminated
Pseudomonas aeruginosa
Isolates in Recife, Brazil
Authors: Poirel Laurent , Magalhaes Marcelo , Lopes Miguel , Nordmann Patrice
Published: March 2004

The spread of clonally related carbapenem-resistant
Pseudomonas aeruginosa
producing the metallo-β-lactamase SPM-1 was found in Recife, Brazil. Upstream of
bla
SPM-1
, a novel common region (CR4) was identified, comprising an open reading frame,
orf495
, whose product shares significant identity with putative recombinases, such as Orf513. CR4 may be responsible for mobilization and expression of
bla
SPM-1
.
Macrolide Resistance Determinants of Invasive and Noninvasive Group B Streptococci in a Turkish Hospital
Authors: Acikgoz Ziya Cibali , Almayanlar Ebru , Gamberzade Sohret , Gocer Safiye
Published: March 2004

Macrolide resistance in 156 consecutive group B streptococcal isolates was investigated. Thirty-five isolates (22.4%) had inducible (80%) or constitutive (20%) erythromycin resistance. The genes responsible were
erm
(B),
erm
(A) subclass
erm
(TR), and
erm
(B) plus
erm
(TR) in 62.9, 2.9, and 8.6% of isolates, respectively. Nine isolates (25.7%) harbored neither
mef
nor detectable
erm
genes.
Field Evaluation of a Novel Colorimetric Method—Double-Site Enzyme-Linked Lactate Dehydrogenase Immunodetection Assay—To Determine Drug Susceptibilities of
Plasmodium falciparum
Clinical Isolates from Northwestern Thailand
Authors: Brockman Alan , Singlam Sittaporn , Phiaphun Lucy , Looareesuwan Sornchai , White Nicholas J. , Nosten François
Published: March 2004

A double-site enzyme-linked lactate dehydrogenase enzyme immunodetection assay was tested against field isolates of
Plasmodium falciparum
for assessing in vitro drug susceptibilities to a wide range of antimalarial drugs. Its sensitivity allowed the use of parasite densities as low as 200 parasites/μl of blood. Being a nonisotopic, colorimetric assay, it lies within the capabilities of a modest laboratory at the district level.
Repression of Tax Expression Is Associated both with Resistance of Human T-Cell Leukemia Virus Type 1-Infected T Cells to Killing by Tax-Specific Cytotoxic T Lymphocytes and with Impaired Tumorigenicity in a Rat Model
Authors: Nomura Machiko , Ohashi Takashi , Nishikawa Keiko , Nishitsuji Hironori , Kurihara Kiyoshi , Hasegawa Atsuhiko , Furuta Rika A. , Fujisawa Jun-ichi , Tanaka Yuetsu , Hanabuchi Shino , Harashima Nanae , Masuda Takao , Kannagi Mari
Published: March 2004

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.
Human T-Cell Lymphotropic Virus Type 1 Open Reading Frame II-Encoded p30
II
Is Required for In Vivo Replication: Evidence of In Vivo Reversion
Authors: Silverman Lee R. , Phipps Andrew J. , Montgomery Andrew , Ratner Lee , Lairmore Michael D.
Published: March 2004

Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia/lymphoma and exhibits high genetic stability in vivo. HTLV-1 contains four open reading frames (ORFs) in its pX region. ORF II encodes two proteins, p30
II
and p13
II
, both of which are incompletely characterized. p30
II
localizes to the nucleus or nucleolus and has distant homology to the transcription factors Oct-1, Pit-1, and POU-M1. In vitro studies have demonstrated that at low concentrations, p30
II
differentially regulates cellular and viral promoters through an interaction with CREB binding protein/p300. To determine the in vivo significance of p30
II
, we inoculated rabbits with cell lines expressing either a wild-type clone of HTLV-1 (ACH.1) or a clone containing a mutation in ORF II, which eliminated wild-type p30
II
expression (ACH.30.1). ACH.1-inoculated rabbits maintained higher HTLV-1-specific antibody titers than ACH.30.1-inoculated rabbits, and all ACH.1-inoculated rabbits were seropositive for HTLV-1, whereas only two of six ACH.30.1-inoculated rabbits were seropositive. Provirus could be consistently PCR amplified from peripheral blood mononuclear cell (PBMC) DNA in all ACH.1-inoculated rabbits but in only three of six ACH.30.1-inoculated rabbits. Quantitative competitive PCR indicated higher PBMC proviral loads in ACH.1-inoculated rabbits. Interestingly, sequencing of ORF II from PBMC of provirus-positive ACH.30.1-inoculated rabbits revealed a reversion to wild-type sequence with evidence of early coexistence of mutant and wild-type sequence. Our data provide evidence that HTLV-1 must maintain its key accessory genes to survive in vivo and that in vivo pressures select for maintenance of wild-type ORF II gene products during the early course of infection.
Interplay between Alpha/Beta and Gamma Interferons with B, T, and Natural Killer Cells in the Defense against Herpes Simplex Virus Type 1
Authors: Vollstedt Sabine , Arnold Susi , Schwerdel Cornelia , Franchini Marco , Alber Gottfried , Di Santo James P. , Ackermann Mathias , Suter Mark
Published: March 2004

The essential components of the immune system that control primary and chronic infection with herpes simplex virus type 1 (HSV-1) in mice were investigated. Infection within the first few days can be controlled by alpha/beta interferon (IFN-α/β) alone without significant contribution of B, T, or NK cells. IFN-α/β and IFN-γ cooperate in the elimination of virus in the absence of these lymphocytes. In contrast, B, T, or NK cells appear to be required to control persistent infection with HSV-1. These results suggest that distinct and essential immune elements are recruited in a time-dependent fashion to control acute and persistent HSV-1 infection.
Defective RNA Replication and Late Gene Expression in Temperature-Sensitive Influenza Viruses Expressing Deleted Forms of the NS1 Protein
Authors: Falcón Ana M. , Marión Rosa M. , Zürcher Thomas , Gómez Paulino , Portela Agustín , Nieto Amelia , Ortín Juan
Published: March 2004

Influenza A virus mutants expressing C-terminally deleted forms of the NS1 protein (NS1-81 and NS1-110) were generated by plasmid rescue. These viruses were temperature sensitive and showed a small plaque size at the permissive temperature. The accumulation of virion RNA in mutant virus-infected cells was reduced at the restrictive temperature, while the accumulation of cRNA or mRNA was not affected, indicating that the NS1 protein is involved in the control of transcription versus replication processes in the infection. The synthesis and accumulation of late virus proteins were reduced in NS1-81 mutant-infected cells at the permissive temperature and were essentially abolished for both viruses at the restrictive temperature, while synthesis and accumulation of nucleoprotein (NP) were unaffected. Probably as a consequence, the nucleocytoplasmic export of virus NP was strongly inhibited at the restrictive temperature. These results indicate that the NS1 protein is essential for nuclear and cytoplasmic steps during the virus cycle.
Poly(A)- and Primer-Independent RNA Polymerase of
Norovirus
Authors: Fukushi Shuetsu , Kojima Shigeyuki , Takai Reiko , Hoshino Fuminori B. , Oka Tomoichiro , Takeda Naokazu , Katayama Kazuhiko , Kageyama Tsutomu
Published: March 2004

Replication of positive-strand caliciviruses is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of
Norovirus
(NV), a member of the family
Caliciviridae
, we used a recombinant baculovirus system to express an enzymatically active RdRp protein from the 3D region of the NV genome and defined conditions for optimum enzymatic activity. Using an RNA template from the NV 3′ genomic region, we observed similar levels of enzymatic activity in assays with and without a poly(A) tail. RdRp activity was not significantly affected by the addition of an RNA primer to the reaction mixture. Thus, the NV RdRp exhibited primer- and poly(A)-independent RNA polymerase activity. While the RdRp inhibitor phosphonoacetic acid inhibited NV RdRp activity, another gliotoxin did not. The active recombinant NV RdRp will be of benefit to studies of NV replication and will facilitate the development of specific inhibitors of NV proliferation.
Membrane Cofactor Protein Is a Receptor for Adenoviruses Associated with Epidemic Keratoconjunctivitis
Authors: Wu Eugene , Trauger Sunia A. , Pache Lars , Mullen Tina-Marie , Von Seggern Daniel J. , Siuzdak Gary , Nemerow Glen R.
Published: March 2004

Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.
Highly Effective Control of an AIDS Virus Challenge in Macaques by Using Vesicular Stomatitis Virus and Modified Vaccinia Virus Ankara Vaccine Vectors in a Single-Boost Protocol
Authors: Ramsburg Elizabeth , Rose Nina F. , Marx Preston A. , Mefford Megan , Nixon Douglas F. , Moretto Walter J. , Montefiori David , Earl Patricia , Moss Bernard , Rose John K.
Published: March 2004

Previous studies have shown that vaccination and boosting of rhesus macaques with attenuated vesicular stomatitis virus (VSV) vectors encoding Env and Gag proteins of simian immunodeficiency virus-human immunodeficiency virus (SHIV) hybrid viruses protect rhesus macaques from AIDS after challenge with the highly pathogenic SHIV 89.6P (23). In the present study, we compared the effectiveness of a single prime-boost protocol consisting of VSV vectors expressing SHIV Env, Gag, and Pol proteins to that of a protocol consisting of a VSV vector prime followed with a single boost with modified vaccinia virus Ankara (MVA) expressing the same SHIV proteins. After challenge with SHIV 89.6P, MVA-boosted animals controlled peak challenge viral loads to less than 2 × 10
6
copies/ml (a level significantly lower than that seen with VSV-boosted animals and lower than those reported for other vaccine studies employing the same challenge). MVA-boosted animals have shown excellent preservation of CD4
+
T cells, while two of four VSV-boosted animals have shown significant loss of CD4
+
T cells. The improved protection in MVA-boosted animals correlates with trends toward stronger prechallenge CD8
+
-T-cell responses to SHIV antigens and stronger postchallenge SHIV-neutralizing antibody production.
Recombinant Modified Vaccinia Virus Ankara Expressing a Soluble Form of Glycoprotein B Causes Durable Immunity and Neutralizing Antibodies against Multiple Strains of Human Cytomegalovirus
Authors: Wang Zhongde , La Rosa Corinna , Maas Rebecca , Ly Heang , Brewer John , Mekhoubad Shahram , Daftarian Pirouz , Longmate Jeff , Britt William J. , Diamond Don J.
Published: March 2004

Human cytomegalovirus (CMV) is a viral pathogen that infects both genders, who remain asymptomatic unless they receive immunosuppressive drugs or acquire infections that cause reactivation of latent virus. CMV infection also causes serious birth defects following primary maternal infection during gestation. A safe and effective vaccine to limit disease in this population continues to be elusive. A well-studied antigen is glycoprotein B (gB), which is the principal target of neutralizing antibodies (NAb) towards CMV in humans and has been implicated as the viral partner in the receptor-mediated infection by CMV in a variety of cell types. Antibody-mediated virus neutralization has been proposed as a mechanism by which host immunity could modify primary infection. Towards this goal, an attenuated poxvirus, modified vaccinia virus Ankara (MVA), has been constructed to express soluble CMV gB (gB680-MVA) to induce CMV NAb. Very high levels of gB-specific CMV NAb were produced after two doses of the viral vaccine. NAb were durable within a twofold range for up to 6 months. Neutralization titers developed in immunized mice are equivalent to titers found clinically after natural infection. This viral vaccine, expressing gB derived from CMV strain AD169, induced antibodies that neutralized CMV strains of three different genotypes. Remarkably, preexisting MVA and vaccinia virus (poxvirus) immunity did not interfere with subsequent immunizations of gB680-MVA. The safety characteristics of MVA, combined with the robust immune response to CMV gB, suggest that this approach could be rapidly translated into the clinic.
Spread of Hepatitis B Viruses In Vitro Requires Extracellular Progeny and May Be Codetermined by Polarized Egress
Authors: Funk A. , Hohenberg H. , Mhamdi M. , Will H. , Sirma H.
Published: March 2004

Viruses can spread by different mechanisms: via intracellular particles through cell junctions to neighboring cells or via secreted virions to adjacent or remote cells. The observation of clusters of hepadnavirus-infected cells both in vivo and in primary hepatocytes neither proves the first mechanism nor excludes the second. In order to test which mechanism, if not both, is used by hepatitis B viruses in order to spread, we used primary duck hepatocytes and duck hepatitis B virus (DHBV) as an infection model. If extracellular progeny virus alone determines spreading, neutralizing antisera or drugs blocking virus binding to hepatocytes should abolish secondary infection. In order to test this, we used DHBV envelope-specific neutralizing antisera, as well as suramin, a known inhibitor of infection. Both reagents strongly reduced hepatocellular attachment of viral particles and almost completely abolished primary infection, whereas an ongoing intracellular infection was not affected as long as no progeny virus was released. In contrast, incubation of infected primary hepatocytes with these reagents during release of progeny virus completely prevented secondary infection. Moreover, the combination of electron and immunofluorescence microscopy analyses revealed the residence of viral particles in cytoplasmic vesicles preferentially located near the basolateral membrane of infected hepatocytes. Taken together, these data strongly suggest that hepatitis B viruses mainly spread by secreted, extracellular progeny and point to polarized egress of viral particles into intercellular compartments, which restricts their diffusion and favors transmission of virus to adjacent cells.
Epstein-Barr Virus Nuclear Antigen Leader Protein Induces Expression of Thymus- and Activation-Regulated Chemokine in B Cells
Authors: Kanamori Mikiko , Watanabe Shinya , Honma Reiko , Kuroda Masayuki , Imai Shosuke , Takada Kenzo , Yamamoto Naoki , Nishiyama Yukihiro , Kawaguchi Yasushi
Published: March 2004

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in transformation of primary B lymphocytes to continuously proliferating lymphoblastoid cell lines (LCLs). To identify cellular genes in B cells whose expression is regulated by EBNA-LP, we performed microarray expression profiling on an EBV-negative human B-cell line, BJAB cells, that were transduced by a retroviral vector expressing the EBV EBNA-LP (BJAB-LP cells) and on BJAB cells that were transduced with a control vector (BJAB-vec cells). Microarray analysis led to the identification of a cellular gene encoding the CC chemokine TARC as a novel target gene that was induced by EBNA-LP. The levels of TARC mRNA expression and TARC secretion were significantly up-regulated in BJAB-LP compared with BJAB-vec cells. Induction of TARC was also observed when a subline of BJAB cells was converted by a recombinant EBV. Among the EBV-infected B-cell lines with the latency III phenotype that were tested, the LCLs especially secreted significantly high levels of TARC. The level of TARC secretion appeared to correlate with the level of full-length EBNA-LP expression. These results indicate that EBV infection induces TARC expression in B cells and that EBNA-LP is one of the viral gene products responsible for the induction.
Purified Recombinant Bluetongue Virus VP1 Exhibits RNA Replicase Activity
Authors: Boyce Mark , Wehrfritz Josa , Noad Rob , Roy Polly
Published: March 2004

The polymerase protein of all known double-stranded RNA (dsRNA) viruses is located within a complex subviral core particle that is responsible for transcription of the viral genome. For members of the family
Reoviridae
, this particle allows messenger sense RNA synthesis while sequestering the viral genome away from cellular dsRNA surveillance systems during infection of eukaryotic cells. The core particle of bluetongue virus (BTV) consists of the major structural proteins VP3 and VP7 and the minor enzymatic proteins VP1 (polymerase), VP4 (capping enzyme), and VP6 (helicase). In this report we have characterized fully processive dsRNA synthesis by VP1 from a viral plus-strand RNA template in the absence of the other proteins of the BTV core. This replicase activity consists of de novo initiation of synthesis, followed by elongation of the minus strand. Purified VP1 exhibits little sequence specificity for BTV plus-strand template, suggesting that the choice of viral over nonviral RNA template comes from its association with other proteins within the viral core.
tRNA-Like Structure Regulates Translation of Brome Mosaic Virus RNA
Authors: Barends Sharief , Rudinger-Thirion Joëlle , Florentz Catherine , Giegé Richard , Pleij Cornelis W. A. , Kraal Barend
Published: March 2004

For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3′ poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS
TYMV
of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3′ TLS
BMV
(about 200 nucleotides) with determinants for tyrosylation. We discovered TLS
BMV
-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS
BMV
tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS
BMV
in
trans
. Intriguingly, a subdomain of the TLS
BMV
could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS
BMV
during the BMV infection cycle.
Induction of Autoantibodies to CCR5 in Macaques and Subsequent Effects upon Challenge with an R5-Tropic Simian/Human Immunodeficiency Virus
Authors: Chackerian Bryce , Briglio Lindsey , Albert Paul S. , Lowy Douglas R. , Schiller John T.
Published: March 2004

Antibodies against CCR5, the major coreceptor for human immunodeficiency virus type 1 (HIV-1), may have antiviral potential as viral fusion inhibitors. In this study, we generated a virus-like particle (VLP)-based vaccine that effectively breaks B-cell tolerance and elicits autoantibodies against CCR5 in pig-tailed macaques. Initial studies in mice identified a polypeptide comprising the N-terminal domain of pig-tailed macaque CCR5 fused to streptavidin that, when conjugated at high density to bovine papillomavirus major capsid protein L1 VLPs, induced high-titer immunoglobulin G (IgG) that bound to a macaque CCR5-expressing cell line in vitro. In macaques, CCR5 peptide-conjugated VLP preparations induced high-avidity anti-CCR5 IgG autoantibody responses, and all five immunized macaques generated IgG that could block infection of CCR5-tropic simian/human immunodeficiency virus SHIV
SF162P3
in vitro. Although the anti-CCR5 IgG titers declined with time, autoantibody levels were boosted upon revaccination. Vaccinated macaques remained healthy for a period of over 3 years after the initial immunization, and no decline in the number of CCR5-expressing T cells was detected. To test the prophylactic efficacy of CCR5 autoantibodies, immunized macaques were challenged with SHIV
SF162P3
. Although the plasma-associated virus in half of six control macaques declined to undetectable levels, viral loads were lower, declined more rapidly, and eventually became undetectable in all five macaques in which CCR5 autoantibodies had been elicited. In addition, in the four vaccinated macaques with higher autoantibody titers, viral loads and time to control of viremia were significantly decreased relative to controls, indicating the possibility that CCR5 autoantibodies contributed to the control of viral replication.