bluuebunny/crossref_metadata_2025 huggingface dataset:
Therapy of Experimental
Pseudomonas
Infections with a Nonreplicating Genetically Modified Phage
Authors: Hagens Steven , Habel André , von Ahsen Uwe , von Gabain Alexander , Bläsi Udo
Published: September 2004

Bacteriophage therapy of bacterial infections has received renewed attention owing to the increasing prevalence of antibiotic-resistant pathogens. A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g., endotoxins of gram-negative bacteria, which mediate the general pathological aspects of septicemia. Here we explored an alternative strategy by using genetically engineered nonreplicating, nonlytic phage to combat an experimental
Pseudomonas aeruginosa
infection. An export protein gene of the
P. aeruginosa
filamentous phage Pf3 was replaced with a restriction endonuclease gene. This rendered the Pf3 variant (Pf3R) nonreplicative and concomitantly prevented the release of the therapeutic agent from the target cell. The Pf3R phage efficiently killed a wild-type host in vitro, while endotoxin release was kept to a minimum. Treatment of
P. aeruginosa
infections of mice with Pf3R or with a replicating lytic phage resulted in comparable survival rates upon challenge with a minimal lethal dose of 3. However, the survival rate after phage therapy with Pf3R was significantly higher than that with the lytic phage upon challenge with a minimal lethal dose of 5. This higher survival rate correlated with a reduced inflammatory response elicited by Pf3R treatment relative to that with the lytic phage. Therefore, this study suggests that the increased survival rate of Pf3R-treated mice could result from reduced endotoxin release. Thus, the use of a nonreplicating modified phage for the delivery of genes encoding proteins toxic to bacterial pathogens may open up a new avenue in antimicrobial therapy.
Intrapulmonary Pharmacokinetics and Pharmacodynamics of Itraconazole and 14-Hydroxyitraconazole at Steady State
Authors: Conte John E. , Golden Jeffrey A. , Kipps Juliana , McIver Marina , Zurlinden Elisabeth
Published: September 2004

We determined the steady-state intrapulmonary pharmacokinetic and pharmacodynamic parameters of orally administered itraconazole (ITRA), 200 mg every 12 h (twice a day [b.i.d.]), on an empty stomach, for a total of 10 doses, in 26 healthy volunteers. Five subgroups each underwent standardized bronchoscopy and bronchoalveolar lavage (BAL) at 4, 8, 12, 16, and 24 h after administration of the last dose. ITRA and its main metabolite, 14-hydroxyitraconazole (OH-IT), were measured in plasma, BAL fluid, and alveolar cells (AC) using high-pressure liquid chromatography. Half-life and area under the concentration-time curves (AUC) in plasma, epithelial lining fluid (ELF), and AC were derived using noncompartmental analysis. ITRA and OH-IT maximum concentrations of drug (Cmax) (mean ± standard deviation) in plasma, ELF, and AC were 2.1 ± 0.8 and 3.3 ± 1.0, 0.5 ± 0.7 and 1.0 ± 0.9, and 5.5 ± 2.9 and 6.6 ± 3.1 μg/ml, respectively. The ITRA and OH-IT AUC for plasma, ELF, and AC were 34.4 and 60.2, 7.4 and 18.9, and 101 and 134 μg · hr/ml. The ratio of theCmaxand the MIC at which 90% of the isolates were inhibited (MIC90), the AUC/MIC90ratio, and the percent dosing interval above MIC90for ITRA and OH-IT concentrations in AC were 1.1 and 3.2, 51 and 67, and 100 and 100%, respectively. Plasma, ELF, and AC concentrations of ITRA and OH-IT declined monoexponentially with half-lives of 23.1 and 37.2, 33.2 and 48.3, and 15.7 and 45.6 h, respectively. An oral dosing regimen of ITRA at 200 mg b.i.d. results in concentrations of ITRA and OH-ITRA in AC that are significantly greater than those in plasma or ELF and intrapulmonary pharmacodynamics that are favorable for the treatment of fungal respiratory infection.
Antimicrobial Activity of Euplotin C, the Sesquiterpene Taxonomic Marker from the Marine Ciliate
Euplotes crassus
Authors: Savoia Dianella , Avanzini Claudio , Allice Tiziano , Callone Emanuela , Guella Graziano , Dini Fernando
Published: September 2004

Strains of the marine ciliate protist
Euplotes crassus
produce exclusive terpenoids called euplotins that play an ecological role. Among these derivatives, euplotin C is the main of four secondary metabolites isolated from cultures of this protozoon and represents the sesquiterpene taxonomic marker from
E. crassus
. Because different terpenoid metabolites of plant origin showed a certain antimicrobial activity, we assessed the compound euplotin C, purified by high-pressure liquid chromatography and solubilized in two solubility enhancers, against the protozoa
Leishmania major
and
Leishmani infantum
, the fungus
Candida albicans
, and nine strains of gram-positive and gram-negative microorganisms. An activity of euplotin C against
Leishmania
promastigotes was demonstrated (50% lethal doses were 4.6 or 8.1 μg/ml depending on the agent used to solubilize the compound), while the effect was less evident on
Candida
and nearly absent on bacteria. A nonsignificant cytotoxicity (50% lethal dose, >200 μg/ml) against the J774 cell line was observed. A leishmanicidal activity was also shown by the living, euplotin-producing cells of
E. crassus
cultured together with promastigotes; this activity increased with time from 10 min to 6 h of incubation. This study provides an initial rationale for the evaluation of euplotin C and other similar natural products as alternative or possibly synergistic compounds for current antiprotozoon chemotherapeutics.
In Vitro Comparison of Topical Microbicides for Prevention of Human Immunodeficiency Virus Type 1 Transmission
Authors: Dezzutti Charlene S. , James V. Nicole , Ramos Artur , Sullivan Sharon T. , Siddig Aladin , Bush Timothy J. , Grohskopf Lisa A. , Paxton Lynn , Subbarao Shambavi , Hart Clyde E.
Published: September 2004

A standardized protocol was used to compare cellular toxicities and anti-human immunodeficiency virus type 1 (HIV-1) activities of candidate microbicides formulated for human use. The microbicides evaluated were cellulose acetate phthalate (CAP), Carraguard, K-Y plus nonoxynol-9 (KY-N9), PRO 2000 (0.5 and 4%), SPL7013 (5%), UC781 (0.1 and 1%), and Vena Gel, along with their accompanying placebos. Products were evaluated for toxicity on cervical and colorectal epithelial cell lines, peripheral blood mononuclear cells (PBMCs), and macrophages (MΦ) by using an ATP release assay, and they were tested for their effect on transepithelial resistance (TER) of polarized epithelial monolayers. Anti-HIV-1 activity was evaluated in assays for transfer of infectious HIV-1 from epithelial cells to activated PBMCs and for PBMC and MΦ infection. CAP, Carraguard, PRO 2000, SPL7013, and UC781 along with their placebos were 20- to 50-fold less toxic than KY-N9 and Vena Gel. None of the nontoxic product concentrations disrupted the TER. Transfer of HIV-1
Ba-L
from epithelial cells to PBMCs and PBMC and MΦ infection with laboratory-adapted HIV-1
Ba-L
and HIV-1
LAI
isolates were inhibited by all products except Carraguard, KY-N9, and Vena Gel. KY-N9, Vena Gel, and Carraguard were not effective in blocking PBMC infection with primary HIV-1
A
, HIV-1
C
, and HIV-1
CRF01-AE
isolates. The concordance of these toxicity results with those previously reported indicates that our protocol may be useful for predicting toxicity in vivo. Moreover, our systematic anti-HIV-1 testing provides a rational basis for making better informed decisions about which products to consider for clinical trials.
Caspofungin Uptake Is Mediated by a High-Affinity Transporter in
Candida albicans
Authors: Paderu Padmaja , Park Steven , Perlin David S.
Published: September 2004

The uptake of the echinocandin drug caspofungin acetate in
Candida albicans
was evaluated at drug levels at or near the MIC for the organism. Maximal uptake was achieved in 10 min and was energy independent. A saturable transport system, consistent with a facilitated-diffusion carrier, was observed with the unlabeled drug competing with the labeled drug for uptake and efflux. More than 90% of the transported drug was observed in a single kinetic compartment that was available for efflux, indicating that the drug was free in the cytoplasm following uptake. Efflux was also energy independent but was sensitive to the presence of a fully loaded carrier on both faces of the bilayer. Overall, the data presented are consistent with the presence of a high-affinity facilitated-diffusion transporter that mediates caspofungin uptake and could be a potential source of transport-related reduced susceptibility.
[
18
F]Ciprofloxacin, a New Positron Emission Tomography Tracer for Noninvasive Assessment of the Tissue Distribution and Pharmacokinetics of Ciprofloxacin in Humans
Authors: Brunner Martin , Langer Oliver , Dobrozemsky Georg , Müller Ulrich , Zeitlinger Markus , Mitterhauser Markus , Wadsak Wolfgang , Dudczak Robert , Kletter Kurt , Müller Markus
Published: September 2004

The biodistribution and pharmacokinetics of the fluorine-18-labeled fluoroquinolone antibiotic [
18
F]ciprofloxacin in tissue were studied noninvasively in humans by means of positron emission tomography (PET). Special attention was paid to characterizing the distribution of [
18
F]ciprofloxacin to select target tissues. Healthy volunteers (
n
= 12) were orally pretreated for 5 days with therapeutic doses of unlabeled ciprofloxacin. On day 6, subjects received a tracer dose (mean injected amount, 700 ± 55 MBq, which contained about 0.6 mg of unlabeled ciprofloxacin) of [
18
F]ciprofloxacin as an intravenous bolus. Thereafter, PET imaging and venous blood sampling were initiated. Time-radioactivity curves were measured for liver, kidney, lung, heart, spleen, skeletal muscle, and brain tissues for up to 6 h after radiotracer administration. The first application of [
18
F]ciprofloxacin in humans has demonstrated the safety and utility of the newly developed radiotracer for pharmacokinetic PET imaging of the tissue ciprofloxacin distribution. Two different tissue compartments of radiotracer distribution could be identified. The first compartment including the kidney, heart, and spleen, from which the radiotracer was washed out relatively quickly (half-lives [
t
1/2
s], 68, 57, and 106 min, respectively). The second compartment comprised liver, muscle, and lung tissue, which displayed prolonged radiotracer retention (
t
1/2
, >130 min). The highest concentrations of radioactivity were measured in the liver and kidney, the main organs of excretion (standardized uptake values [SUVs], 4.9 ± 1.0 and 9.9 ± 4.4, respectively). The brain radioactivity concentrations were very low (<1 kBq · g
−1
) and could therefore not be quantified. Transformation of SUVs into absolute concentrations (in micrograms per milliliter) allowed us to relate the concentrations at the target site to the susceptibilities of bacterial pathogens. In this way, the frequent use of ciprofloxacin for the treatment of a variety of infections could be corroborated.
Mannose-Specific Plant Lectins from the
Amaryllidaceae
Family Qualify as Efficient Microbicides for Prevention of Human Immunodeficiency Virus Infection
Authors: Balzarini Jan , Hatse Sigrid , Vermeire Kurt , Princen Katrien , Aquaro Stefano , Perno Carlo-Federico , De Clercq Erik , Egberink Herman , Vanden Mooter Guy , Peumans Willy , Van Damme Els , Schols Dominique
Published: September 2004

The plant lectins derived from
Galanthus nivalis
(Snowdrop) (GNA) and
Hippeastrum
hybrid (Amaryllis) (HHA) selectively inhibited a wide variety of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains and clinical (CXCR4- and CCR5-using) isolates in different cell types. They also efficiently inhibited infection of T lymphocytes by a variety of mutant virus strains. GNA and HHA markedly prevented syncytium formation between persistently infected HUT-78/HIV cells and uninfected T lymphocytes. The plant lectins did not measurably affect the antiviral activity of other clinically approved anti-HIV drugs used in the clinic when combined with these drugs. Short exposure of the lectins to cell-free virus particles or persistently HIV-infected HUT-78 cells markedly decreased HIV infectivity and increased the protective (microbicidal) activity of the plant lectins. Flow cytometric analysis and monoclonal antibody binding studies and a PCR-based assay revealed that GNA and HHA do not interfere with CD4, CXCR4, CCR5, and DC-SIGN and do not specifically bind with the membrane of uninfected cells. Instead, GNA and HHA likely interrupt the virus entry process by interfering with the virus envelope glycoprotein. HHA and GNA are odorless, colorless, and tasteless, and they are not cytotoxic, antimetabolically active, or mitogenic to human primary T lymphocytes at concentrations that exceed their antivirally active concentrations by 2 to 3 orders of magnitude. GNA and HHA proved stable at high temperature (50°C) and low pH (5.0) for prolonged time periods and can be easily formulated in gel preparations for microbicidal use; they did not agglutinate human erythrocytes and were not toxic to mice when administered intravenously.
Genomic Approach to Identification of Mutations Affecting Caspofungin Susceptibility in
Saccharomyces cerevisiae
Authors: Markovich Sarit , Yekutiel Aya , Shalit Itamar , Shadkchan Yona , Osherov Nir
Published: September 2004

The antifungal agent caspofungin (CAS) specifically interferes with glucan synthesis and cell wall formation. To further study the cellular processes affected by CAS, we analyzed a
Saccharomyces cerevisiae
mutant collection (4,787 individual knockout mutations) to identify new genes affecting susceptibility to the drug. This collection was screened for increased CAS sensitivity (CAS-IS) or increased CAS resistance (CAS-IR). MICs were determined by the broth microdilution method. Disruption of 20 genes led to CAS-IS (four- to eightfold reductions in the MIC). Eleven of the 20 genes are involved in cell wall and membrane function, notably in the protein kinase C (PKC) integrity pathway (
MID2
,
FKS1
,
SMI1
, and
BCK1
), chitin and mannan biosynthesis (
CHS3
,
CHS4
,
CHS7
, and
MNN10
), and ergosterol biosynthesis (
ERG5
and
ERG6
). Four of the 20 genes (
TPO1
,
VPS65
,
VPS25
, and
CHC1
) are involved in vacuole and transport functions, 3 of the 20 genes (
CCR4
,
POP2
, and
NPL3
) are involved in the control of transcription, and 2 of the 20 genes are of unknown function. Disruption of nine additional genes led to CAS-IR (a fourfold increase of MIC). Five of these nine genes (
SLG1
,
ERG3
,
VRP1
,
CSG2
, and
CKA2
) are involved in cell wall function and signal transduction, and two of the nine genes (
VPS67
and
SAC2
) are involved in vacuole function. To assess the specificity of susceptibility to CAS, the MICs of amphotericin B, fluconazole, flucytosine, and calcofluor for the strains were tested. Seven of 20 CAS-IS strains (with disruption of
FKS1
,
SMI1
,
BCK1
,
CHS4
,
ERG5
,
TPO1
, and
ILM1
) and 1 of 9 CAS-IR strains (with disruption of
SLG1
) demonstrated selective susceptibility to CAS. To further explore the importance of PKC in CAS susceptibility, the activity of the PKC inhibitor staurosporine in combination with CAS was tested against eight
Aspergillus
clinical isolates by the microdilution assay. Synergistic or synergistic-to-additive activities were found against all eight isolates by use of both MIC and minimum effective concentration endpoints.
Fusidic Acid-Resistant Mutants of
Salmonella enterica
Serovar Typhimurium Have Low Levels of Heme and a Reduced Rate of Respiration and Are Sensitive to Oxidative Stress
Authors: Macvanin Mirjana , Ballagi Andras , Hughes Diarmaid
Published: September 2004

Mutations in the translation elongation factor G (EF-G) make
Salmonella enterica
serovar Typhimurium resistant to the antibiotic fusidic acid. Fus
r
mutants are hypersensitive to oxidative stress and rapidly lose viability in the presence of hydrogen peroxide. We show that this phenotype is associated with reduced activity of two catalase enzymes, HPI (a bifunctional catalase-hydroperoxidase) and HPII (a monofunctional catalase). These catalases require the iron-binding cofactor heme for their activity. Fus
r
mutants have a reduced rate of transcription of
hemA
, a gene whose product catalyzes the first committed step in heme biosynthesis. Hypersensitivity of Fus
r
mutants to hydrogen peroxide is abolished by the presence of δ-aminolevulinic acid, the precursor of heme synthesis, in the growth media and by the addition of glutamate or glutamine, amino acids required for the first step in heme biosynthesis. Fluorescence measurements show that the level of heme in a Fus
r
mutant is significantly lower than it is in the wild type. Heme is also an essential cofactor of cytochromes in the electron transport chain of respiration. We found that the rate of respiration is reduced significantly in Fus
r
mutants. Sequestration of divalent iron in the growth media decreases the sensitivity of Fus
r
mutants to oxidative stress. Taken together, these results suggest that Fus
r
mutants are hypersensitive to oxidative stress because their low levels of heme reduce both catalase activity and respiration capacity. The sensitivity of Fus
r
mutants to oxidative stress could be associated with loss of viability due to iron-mediated DNA damage in the presence of hydrogen peroxide. We argue that understanding the specific nature of antibiotic resistance fitness costs in different environments may be a generally useful approach in identifying physiological processes that could serve as novel targets for antimicrobial agents.
Novel Concentration-Killing Curve Method for Estimation of Bactericidal Potency of Antibiotics in an In Vitro Dynamic Model
Authors: Liu Y. Q. , Zhang Y. Z. , Gao P. J.
Published: September 2004

The bactericidal pharmacodynamics of antibiotics againstEscherichia coliwere analyzed by a concentration-killing curve (CKC) approach, and the novel parameters median bactericidal concentration (BC50) and bactericidal intensity (r) for bactericidal potency were proposed. By using the agar plate method, about 500E. colicells were inoculated onto Luria-Bertani plates containing a series of antibiotic concentrations, and after 24 h of incubation at 37°C, all the viable colonies were enumerated. This resulted in a sigmoidal CKC that could be perfectly fitted (R2> 0.9) with the functionN=N0/[1 + er(x− BC50)], whereNis number of colonies surviving on each plate with anxseries of concentrations of an antibiotic, andN0represents the meaningful inoculum size. Construction of the CKC method was based on the bactericidal effect of each antibiotic against the bacterial strain versus the concentration in two dimensions and may be a more valid, accurate, and reproducible method for estimating the bactericidal effect than the endpoint minimum bactericidal concentration (MBC) method. Mathematically, the CKC approach was point symmetrical toward its inflexion (BC50,N0/2); thus, 2BC50could replace MBC. The parameter BC1can be defined as BC50+ [ln(N0− 1)/r], which is the drug concentration at which only one colony survived and which is the least critical value of MBC in the CKC. The variater, which determined the tangent slope on inflexion whenN0was limited, could estimate the bactericidal intensity of an antibiotic. This verified that the CKC approach may be useful in studies with other classes of antibiotics and has considerable value as a tool for the accurate and proper administration of antibiotics.
VanD-Type Vancomycin-Resistant
Enterococcus faecium
and
Enterococcus faecalis
Authors: Depardieu Florence , Kolbert Mathias , Pruul Hendrik , Bell Jan , Courvalin Patrice
Published: September 2004

Enterococcus faecium
clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 μg/ml, respectively) and to low levels of teicoplanin (4 μg/ml), and
Enterococcus faecalis
clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 μg/ml) and susceptible to teicoplanin (0.25 μg/ml), were studied. They were constitutively resistant by synthesis of peptidoglycan precursors ending with
d
-alanyl-
d
-lactate and harbored a chromosomal
vanD
gene cluster which was not transferable by conjugation to other enterococci. VanX
D
activity, which is not required in the absence of
d
-Ala-
d
-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanY
D
activity in the membrane fractions was inhibited by penicillin G. The mutations E
13
G in the region of
d
-alanine:
d
-alanine ligase (which is implicated in
d
-Ala1 binding in A902) and S
319
N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of
d
-Ala-
d
-Ala-containing peptidoglycan precursors. The same 7-bp insertion in

vanS
D

of BM4539 and BM4540 and a 1-bp deletion in

vanS
D

of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance. Strain BM4538, with a functional VanS
D
, had a G
140
E mutation in VanR
D
that could be responsible for constitutive glycopeptide resistance. This would represent the first example of constitutive
van
gene expression due to a mutation in the structural gene for a VanR transcriptional activator. Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the
ddl
housekeeping gene and in the acquired

vanS
D

or

vanR
D

gene that lead to constitutive resistance to vancomycin.
Human Immunodeficiency Virus Type 1 Protease Inhibitors Block Toll-Like Receptor 2 (TLR2)- and TLR4-Induced NF-κB Activation
Authors: Equils Ozlem , Shapiro Alan , Madak Zeynep , Liu Chunren , Lu Daning
Published: September 2004

Coinfections with opportunistic and pathogenic bacteria induce human immunodeficiency virus (HIV) replication through microbial antigen activation of NF-κB. Here, we assessed whether HIV type 1 protease inhibitors (PI) block microbial antigen activation of NF-κB. Human microvessel endothelial cells were transiently transfected with either endothelial cell-leukocyte adhesion molecule NF-κB luciferase or interleukin 6 (IL-6) promoter luciferase constructs by using FuGENE 6, and they were treated with PI (nelfinavir, ritonavir, or saquinavir) prior to stimulation with the Toll-like receptor 4 (TLR4) and TLR2 ligands, with lipopolysaccharide (LPS), solubleMycobacterium tuberculosisfactor, orStaphylococcus epidermidisphenol-soluble modulin, respectively, or with tumor necrosis factor alpha (TNF-α). Luciferase activity was measured by using a Promega luciferase kit. TNF-α release from the supernatant was measured by enzyme-linked immunosorbent assay. Cell death was assessed by lactate dehydrogenase assay. We observed that PI pretreatment blocked the TLR2- and TLR4- as well as the TNF-α-mediated NF-κB activation, in a dose-dependent manner. PI pretreatment also blocked the LPS-induced IL-6 promoter transactivation and TNF-α secretion. These data suggest that PI block HIV replication not only by inhibiting the HIV protease but also by blocking the TLR- and TNF-α-mediated NF-κB activation and proinflammatory cytokine production. These findings may help explain the immunomodulatory effects of PI, and they suggest an advantage for PI-containing drug regimens in the treatment of HIV-infected patients who are coinfected with opportunistic and pathogenic bacteria.
Long Depletion Time of Enrofloxacin in Rainbow Trout (
Oncorhynchus mykiss
)
Authors: Lucchetti Dario , Fabrizi Laura , Guandalini Emilio , Podestà Elisabetta , Marvasi Luigi , Zaghini Anna , Coni Ettore
Published: September 2004

The international production of farmed fish has been growing continuously over recent years. Until now few veterinary drugs have been approved by the European Union for use in aquaculture, and this has favored the off-label use of products authorized for use in food-producing animal species different from fishes among fish farmers. Adequate field studies are lacking, especially for those species called minor species which are consumed extensively only in some European countries. In the present investigation we studied the depletion of the fluoroquinolone antibacterial enrofloxacin over time in a minor species, the rainbow trout (
Oncorhynchus mykiss
), reared on a real fish farm and treated with medicated feed (10 mg kg of trout body weight
−1
day
−1
). Edible tissue samples (muscle plus skin in natural proportions) and fish bone samples were analyzed for enrofloxacin and for its major metabolite, ciprofloxacin, by high-performance liquid chromatography with fluorescence detection at different times after the end of treatment. Our results show that at 500°C-day (in which degree-days are calculated by multiplying the mean daily water temperature by the total number of days on which the temperature was measured), which is the minimum withdrawal period established by European Economic Commission Directive No. 82/2001 for any type of product administered off-label, edible trout tissues might still contain about 170 μg of enrofloxacin kg
−1
, whereas the maximum residue level for enrofloxacin plus ciprofloxacin is set at 100 μg kg
−1
. To our knowledge, no studies of the depletion of enrofloxacin in rainbow trout have been performed. On the basis of the data obtained in the present study, we suggest a more appropriate withdrawal time of 816°C-day for the sum of enrofloxacin plus ciprofloxacin levels in rainbow trout muscle plus skin tissues.
Daptomycin Is Highly Efficacious against Penicillin-Resistant and Penicillin- and Quinolone-Resistant Pneumococci in Experimental Meningitis
Authors: Cottagnoud Philippe , Pfister Marc , Acosta Fernando , Cottagnoud Marianne , Flatz Lukas , Kühn Felix , Müller Hans-Peter , Stucki Armin
Published: September 2004

The penetration of daptomycin, a new lipopeptide antibiotic, into inflamed meninges ranged between 4.37 and 7.53% (mean, 5.97%). Daptomycin was very efficacious in the treatment of experimental pneumococcal meningitis, producing a decrease of −1.20 ± 0.32 Δlog
10
CFU/ml · h in the bacterial titer of
Streptococcus pneumoniae
against a penicillin-resistant strain and of −0.97 ± 0.32 Δlog
10
CFU/ml · h against a penicillin- and quinolone-resistant strain found in cerebrospinal fluid (CSF). For both strains, daptomycin was significantly superior to the standard regimen of a combination of ceftriaxone with vancomycin, sterilizing 9 of 10 CSF samples after 4 h. In vitro, daptomycin produced highly bactericidal activity in concentrations above the MIC.
Mechanism of Resistance to Several Antimicrobial Agents inSalmonellaClinical Isolates Causing Traveler's Diarrhea
Authors: Cabrera Roberto , Ruiz Joaquím , Marco Francesc , Oliveira Inés , Arroyo Margarita , Aladueña Ana , Usera Miguel A. , Jiménez De Anta M. Teresa , Gascón Joaquím , Vila Jordi
Published: September 2004

The evolution of antimicrobial resistance inSalmonellaisolates causing traveler's diarrhea (TD) and their mechanisms of resistance to several antimicrobial agents were analyzed. From 1995 to 2002, a total of 62Salmonellastrains were isolated from stools of patients with TD. The antimicrobial susceptibility to 12 antibiotics was determined, and the molecular mechanisms of resistance to several of them were detected as well. The highest levels of resistance were found against tetracycline and ampicillin (21 and 19%, respectively), followed by resistance to nalidixic acid (16%), which was mainly detected from 2000 onward. Molecular mechanisms of resistance were analyzed in 16 isolates. In these isolates, which were resistant to ampicillin, two genes encoding β-lactamases were detected:oxa-1(one isolate) andtem-like (seven isolates [in one strain concomitantly with acarb-2]). Resistance to tetracycline was mainly related totetA(five cases) and totetBandtetG(one case each). Resistance to chloramphenicol was related to the presence of thefloRandcmlAgenes and to chloramphenicol acetyltransferase activity in one case each. Different genes encoding dihydrofolate-reductases (dfrA1,dfrA12,dfrA14, anddfrA17) were detected in trimethoprim-resistant isolates. Resistance to nalidixic acid was related to the presence of mutations in the amino acid codons 83 or 87 of thegyrAgene. Further surveillance of theSalmonellaspp. causing TD is needed to detect trends in their resistance to antimicrobial agents, as we have shown in our study with nalidixic acid. Moreover, such studies will lead to better treatment and strategies to prevent and limit their spread.
A 7-Deaza-Adenosine Analog Is a Potent and Selective Inhibitor of Hepatitis C Virus Replication with Excellent Pharmacokinetic Properties
Authors: Olsen David B. , Eldrup Anne B. , Bartholomew Linda , Bhat Balkrishen , Bosserman Michele R. , Ceccacci Alessandra , Colwell Lawrence F. , Fay John F. , Flores Osvaldo A. , Getty Krista L. , Grobler Jay A. , LaFemina Robert L. , Markel Eric J. , Migliaccio Giovanni , Prhavc Marija , Stahlhut Mark W. , Tomassini Joanne E. , MacCoss Malcolm , Hazuda Daria J. , Carroll Steven S.
Published: September 2004

Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2′-
C
-methyl-adenosine and 2′-
C
-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2′-
C
-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5′-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2′-
C
-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2′-
C
-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2′-
C
-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.
Mutant Prevention Concentrations for Single-Step Fluoroquinolone-Resistant Mutants of Wild-Type, Efflux-Positive, or ParC or GyrA Mutation-Containing
Streptococcus pneumoniae
Isolates
Authors: Smith Heather J. , Walters Michael , Hisanaga Tamiko , Zhanel George G. , Hoban Daryl J.
Published: September 2004

Three fluoroquinolone-susceptible and five fluoroquinolone-resistant (two with ParC Ser79Phe mutations, one with a GyrA Ser81Phe mutation, and two that were efflux positive)
Streptococcus pneumoniae
isolates were exposed to one, two, four, eight, and sixteen times the MICs of ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin. Mutational frequencies were calculated at each multiple of the MIC for which growth was observed. Mutant prevention concentrations (MPCs) and the multiple of the MIC at the MPC (MP
MIC
) were evaluated. All resulting mutants were sequenced for quinolone resistance-determining region changes in GyrA and ParC and were evaluated for reserpine-sensitive efflux. The MPC order was generally ciprofloxacin > levofloxacin > gatifloxacin > moxifloxacin > gemifloxacin. The MP
MIC
order varied depending on the genetic constitution of the original isolates from which the mutants were generated. For those mutants created from fluoroquinolone-susceptible isolates (those that had wild-type ParC and GyrA and were efflux negative), the MP
MIC
order was ciprofloxacin = moxifloxacin > gemifloxacin > levofloxacin > gatifloxacin. The MP
MIC
s of each fluoroquinolone for mutants created from isolates with a ParC mutation (with wild-type GyrA and efflux negative) were similar. A similar occurrence was observed with the mutants created from the efflux-positive isolates (with wild-type ParC and GyrA). The MP
MIC
order for the mutants created from the isolate with a GyrA mutation (with wild-type ParC and efflux negative) was ciprofloxacin = gemifloxacin > levofloxacin = moxifloxacin > gatifloxacin. Gatifloxacin, levofloxacin, and moxifloxacin may be intrinsically more able to prevent the development of resistance by fluoroquinolone-susceptible isolates, isolates that are efflux positive, or isolates that carry a GyrA mutation. However, once a ParC mutation is present, the MPC increases dramatically for all fluoroquinolones.
Efficacy of PLD-118, a Novel Inhibitor of
Candida
Isoleucyl-tRNA Synthetase, against Experimental Oropharyngeal and Esophageal Candidiasis Caused by Fluconazole-Resistant
C. albicans
Authors: Petraitis Vidmantas , Petraitiene Ruta , Kelaher Amy M. , Sarafandi Alia A. , Sein Tin , Mickiene Diana , Bacher John , Groll Andreas H. , Walsh Thomas J.
Published: September 2004

PLD-118, formerly BAY 10-8888, is a synthetic antifungal derivative of the naturally occurring β-amino acid cispentacin. We studied the activity of PLD-118 in escalating dosages against experimental oropharyngeal and esophageal candidiasis (OPEC) caused by fluconazole (FLC)-resistant
Candida albicans
in immunocompromised rabbits. Infection was established by fluconazole-resistant (MIC > 64 μg/ml) clinical isolates from patients with refractory esophageal candidiasis. Antifungal therapy was administered for 7 days. Study groups consisted of untreated controls; animals receiving PLD-118 at 4, 10, 25, or 50 mg/kg of body weight/day via intravenous (i.v.) twice daily (BID) injections; animals receiving FLC at 2 mg/kg/day via i.v. BID injections; and animals receiving desoxycholate amphotericin B (DAMB) i.v. at 0.5 mg/kg/day. PLD-118- and DAMB-treated animals showed a significant dosage-dependent clearance of
C. albicans
from the tongue, oropharynx, and esophagus in comparison to untreated controls (
P
≤ 0.05,
P
≤ 0.01,
P
≤ 0.001, respectively), while FLC had no significant activity. PLD-118 demonstrated nonlinear plasma pharmacokinetics across the investigated dosage range, as was evident from a dose-dependent increase in plasma clearance and a dose-dependent decrease in the area under the plasma concentration-time curve. The biochemical safety profile was similar to that of FLC. In summary, PLD-118 demonstrated dosage-dependent antifungal activity and nonlinear plasma pharmacokinetics in treatment of experimental FLC-resistant oropharyngeal and esophageal candidiasis.
DB75, a Novel Trypanocidal Agent, Disrupts Mitochondrial Function in
Saccharomyces cerevisiae
Authors: Lanteri Charlotte A. , Trumpower Bernard L. , Tidwell Richard R. , Meshnick Steven R.
Published: September 2004

The aromatic diamidines represent a class of compounds with broad-spectrum antimicrobial activity; however, their development is hindered by a lack of understanding of their mechanism of antimicrobial action. DB75 [2,5-bis(4-amidinophenyl)furan] is a trypanocidal aromatic diamidine that was originally developed as a structural analogue of the antitrypanosomal agent pentamidine. DB289, a novel orally active prodrug of DB75, is undergoing phase IIb clinical trials for early-stage human African trypanosomiasis,
Pneumocystis jiroveci carinii
pneumonia, and malaria. The purpose of this study was to investigate mechanisms of action of DB75 using
Saccharomyces cerevisiae
as a model organism. The results of this investigation suggest that DB75 inhibits mitochondrial function. Yeast cells relying upon mitochondrial metabolism for energy production are especially sensitive to DB75. DB75 localizes (by fluorescence) within the mitochondria of living yeast cells and collapses the mitochondrial membrane potential in isolated yeast mitochondria. Furthermore, addition of DB75 to yeast cells or isolated rat liver mitochondria results in immediate uncoupling of oxidative phosphorylation and subsequent inhibition of respiration. We conclude that the mitochondrion is a cellular target of DB75 in yeast cells and anticipate that the results of this study will aid in the target-based design of new antimicrobial aromatic diamidines.
Rifalazil Treats and Prevents Relapse of
Clostridium difficile
-Associated Diarrhea in Hamsters
Authors: Anton Pauline M. , O'Brien Michael , Kokkotou Efi , Eisenstein Barry , Michaelis Arthur , Rothstein David , Paraschos Sophia , Kelly Ciáran P. , Pothoulakis Charalabos
Published: September 2004

Although vancomycin and metronidazole effectively treat
Clostridium difficile
-associated diarrhea and colitis (CDAD), their use is associated with a high incidence of relapsing
C. difficile
infection. Rifalazil is a new benzoxazinorifamycin that possesses activity against
Mycobacterium tuberculosis
and gram-positive bacteria. Here we compared rifalazil and vancomycin for effectiveness in preventing or treating clindamycin-induced cecitis in a hamster model of CDAD. Golden Syrian hamsters were injected subcutaneously with clindamycin phosphate (10 mg/kg), followed 24 h later by
C. difficile
gavage. Hamsters received by gavage for 5 days vehicle, vancomycin (50 mg/kg), or rifalazil (20 mg/kg) either simultaneously with (prophylactic protocol) or 24 h after
C. difficile
administration (treatment protocol). While all vehicle-administered animals became moribund within 48 h of
C. difficile
administration, no rifalazil- or vancomycin-treated animals in either protocol showed signs of morbidity after 7 days. Ceca of rifalazil-treated animals showed absence of epithelial cell damage, significantly reduced congestion and edema, and less, but not statistically significantly less, neutrophil infiltration compared to those of vehicle-treated animals. In contrast, vancomycin-treated animals demonstrated severe epithelial cell damage and mildly reduced congestion and edema. Moreover, hamsters relapsed and tested
C. difficile
toxin positive (by enzyme-linked immunosorbent assay) 10 to 15 days after discontinuation of vancomycin treatment. None of the rifalazil-treated hamsters showed signs of disease or presence of toxins in their feces 30 days after discontinuation of treatment. Our results indicate that once daily rifalazil may be superior to vancomycin for curative treatment of CDAD.
Tetracycline Resistance inChlamydia suisMediated by Genomic Islands Inserted into the Chlamydialinv-Like Gene
Authors: Dugan Jae , Rockey Daniel D. , Jones Loren , Andersen Arthur A.
Published: September 2004

Many strains ofChlamydia suis, a pathogen of pigs, express a stable tetracycline resistance phenotype. We demonstrate that this resistance pattern is associated with a resistance gene,tet(C), in the chlamydial chromosome. Four related genomic islands were identified in seven tetracycline-resistantC. suisstrains. All resistant isolates carry the structural genetet(C) and the tetracycline repressor genetetR(C). The islands share significant nucleotide sequence identity with resistance plasmids carried by a variety of different bacterial species. Three of the fourtet(C) islands also carry a novel insertion sequence that is homologous to the IS605family of insertion sequences. In each strain, the resistance gene and associated sequences are recombined into an identical position in a gene homologous to theinvgene of the yersiniae. These genomic islands represent the first examples of horizontally acquired DNA integrated into a natural isolate of chlamydiae or within any other obligate intracellular bacterium.
Mechanisms of Resistance in Multiple-Antibiotic-Resistant
Escherichia coli
Strains of Human, Animal, and Food Origins
Authors: Sáenz Yolanda , Briñas Laura , Domínguez Elena , Ruiz Joaquim , Zarazaga Myriam , Vila Jordi , Torres Carmen
Published: September 2004

Seventeen multiple-antibiotic-resistant nonpathogenic
Escherichia coli
strains of human, animal, and food origins showed a wide variety of antibiotic resistance genes, many of them carried by class 1 and class 2 integrons. Amino acid changes in MarR and mutations in
marO
were identified for 15 and 14
E. coli
strains, respectively.
Determination of Susceptibilities of 26Leptospirasp. Serovars to 24 Antimicrobial Agents by a Broth Microdilution Technique
Authors: Murray Clinton K. , Hospenthal Duane R.
Published: September 2004

The MICs of 24 antimicrobials for 26Leptospiraspp. serovars were determined using a broth microdilution technique. The MICs at which 90% of isolates tested were inhibited (MIC90s) of cefepime, imipenem-cilastatin, erythromycin, clarithromycin, and telithromycin were all ≤0.01 μg/ml. The MIC90s of amoxicillin, aztreonam, cefdinir, chloramphenicol, and penicillin G were ≥3.13 μg/ml. Many antimicrobials have excellent in vitro activity againstLeptospira.
Correlation between In Vitro Susceptibility of
Scedosporium apiospermum
to Voriconazole and In Vivo Outcome of Scedosporiosis in Guinea Pigs
Authors: Capilla Javier , Guarro Josep
Published: September 2004

We have evaluated the efficacy of voriconazole (VRC) in a systemic infection by
Scedosporium apiospermum
in immunodepressed guinea pigs. Animals were infected with two strains; one required a VRC MIC of 0.5 to 1 μg/ml, common for this fungus, and the other required a high MIC (8 μg/ml), unusual in this species. VRC prolonged survival and reduced fungal load in kidney and brain tissues of the animals infected with the first strain but was unable to prolong survival or to reduce fungal load in brain tissue for the latter strain.
Prevalence of Mutations within the Quinolone Resistance-Determining Region of
gyrA
,
gyrB
,
parC
, and
parE
and Association with Antibiotic Resistance in Quinolone-Resistant
Salmonella enterica
Authors: Eaves Deborah J. , Randall Luke , Gray Douglas T. , Buckley Antony , Woodward Martin J. , White Allan P. , Piddock Laura J. V.
Published: September 2004

Salmonella enterica
isolates (
n
= 182) were examined for mutations in the quinolone resistance-determining region of
gyrA
,
gyrB
,
parC
, and
parE
. The frequency, location, and type of GyrA substitution varied with the serovar. Mutations were found in
parC
that encoded Thr57-Ser, Thr66-Ile, and Ser80-Arg substitutions. Mutations in the
gyrB
quinolone resistance-determining region were located at codon Tyr420-Cys or Arg437-Leu. Novel mutations were also found in
parE
encoding Glu453-Gly, His461-Tyr, Ala498-Thr, Val512-Gly, and Ser518-Cys. Although it is counterintuitive, isolates with a mutation in both
gyrA
and
parC
were more susceptible to ciprofloxacin than were isolates with a mutation in
gyrA
alone.
In Vitro Killing of Community-Associated Methicillin-Resistant
Staphylococcus aureus
with Drug Combinations
Authors: Shelburne Samuel A. , Musher Daniel M. , Hulten Kristina , Ceasar Heather , Lu Michael Y. , Bhaila Imran , Hamill Richard J.
Published: September 2004

This study employs time-kill techniques to examine the most common drug combinations used in the therapy of methicillin-resistant
Staphylococcus aureus
(MRSA) infections, vancomycin plus either gentamicin or rifampin. Community-associated MRSA were more likely to be synergistically inhibited by combinations of vancomycin and gentamicin versus vancomycin alone compared to inhibition associated with hospital-acquired strains.
Alterations of Penicillin-Binding Proteins 1A, 2X, and 2B in
Streptococcus pneumoniae
Isolates for Which Amoxicillin MICs Are Higher than Penicillin MICs
Authors: Kosowska K. , Jacobs M. R. , Bajaksouzian S. , Koeth L. , Appelbaum P. C.
Published: September 2004

Penicillin-binding proteins (PBPs) of 15 selected penicillin- and amoxicillin-resistant
Streptococcus pneumoniae
isolates (MICs of 2 to 8 and 8 to 16 μg/ml, respectively) were studied. In addition to typical changes in PBPs 1A and 2X, these strains had 10 unique changes in PBP 2B, including a
618
A-G substitution, which may be the key alteration associated with amoxicillin resistance.
Peptide Nucleic Acid-Mediated Competitive PCR Clamping for Detection of Rifampin-Resistant
Mycobacterium tuberculosis
Authors: Iwamoto Tomotada , Sonobe Toshiaki
Published: September 2004

Peptide nucleic acid-mediated competitive PCR clamping, which can selectively amplify mutant alleles, was developed to detect mutations in four codons (513, 516, 526, and 531) of the
rpoB
gene in
Mycobacterium tuberculosis
strains. This simple method successfully identified the mutations in all 40 of the
M. tuberculosis
strains tested.
Antipneumococcal Activity of LBM415, a New Peptide Diformylase Inhibitor, Compared with Those of Other Agents
Authors: Ednie Lois M. , Pankuch Glenn , Appelbaum Peter C.
Published: September 2004

The MICs of LBM415, a new peptide diformylase inhibitor, were evaluated and ranged from 0.03 to 4.0 μg/ml for 300 pneumococci, irrespective of their β-lactam, macrolide, and quinolone susceptibilities. By comparison, vancomycin, teicoplanin, linezolid, and quinupristin-dalfopristin were also active, with MICs ≤2.0 μg/ml. Gatifloxacin and moxifloxacin were the most active quinolones tested, while the MICs of the β-lactams rose with those of penicillin G. LBM415 at two times the MIC was bactericidal (99.9% killing) against six strains after 24 h.
Antistaphylococcal Activity of LBM415, a New Peptide Deformylase Inhibitor, Compared with Those of Other Agents
Authors: Credito Kim , Lin Gengrong , Ednie Lois M. , Appelbaum Peter C.
Published: September 2004

The MICs of LBM415, a new peptide diformylase inhibitor, were ≤0.06 to 4.0 μg/ml for 258 isolates of
Staphylococcus aureus
and coagulase-negative staphylococci. LBM415 MICs were similar irrespective of whether the strains were methicillin susceptible or resistant. All strains were also susceptible to vancomycin, linezolid, ranbezolid, daptomycin, oritavancin, and quinupristin-dalfopristin. LBM415 at the MIC was bacteriostatic after 24 h.
Antistaphylococcal Activity of CB-181963 (CAB-175), an Experimental Parenteral Cephalosporin
Authors: Hoellman Dianne B. , Pankuch Glenn A. , Appelbaum Peter C.
Published: September 2004

Among 265 methicillin-susceptible and -resistant staphylococci, CB-181963 (CAB-175) had a 50% minimum inhibitory concentration of 2 μg/ml and a 90% minimum inhibitory concentration of 4 μg/ml. All strains except two vancomycin-resistant
S. aureus
and 5 vancomycin-intermediate
S. aureus
strains were also susceptible to vancomycin and teicoplanin, and all were susceptible to linezolid, ranbezolid, tigecycline, and quinupristin-dalfopristin. Most methicillin-resistant strains were levofloxacin resistant. CB-181963 was bactericidal against all six methicillin-resistant strains at four times the MIC after 24 h.
Resistance Phenotypes and Genotypes of Erythromycin-Resistant
Streptococcus pneumoniae
Isolates in Beijing and Shenyang, China
Authors: Tiemei Zhao , Xiangqun Fang , Youning Liu
Published: September 2004

Of a total of 192
Streptococcus pneumoniae
isolates, 149 (77.6%) were not susceptible to erythromycin. Of these 149 isolates, 117 (79.1%) contained the
erm
(B) gene, 16 (10.8%) contained the
mef
(A) gene, and 15 (10.1%) harbored both the
erm
(B) and
mef
(A) genes.
CARB-9, a Carbenicillinase Encoded in the VCR Region of
Vibrio cholerae
Non-O1, Non-O139 Belongs to a Family of Cassette-Encoded β-Lactamases
Authors: Petroni Alejandro , Melano Roberto G. , Saka Héctor A. , Garutti Alicia , Mange Laura , Pasterán Fernando , Rapoport Melina , Miranda Mariana , Faccone Diego , Rossi Alicia , Hoffman Paul S. , Galas Marcelo F.
Published: September 2004

The gene
bla
CARB-9
was located in the
Vibrio cholerae
super-integron, but in a different location relative to
bla
CARB-7
. CARB-9 (pI 5.2) conferred β-lactam MICs four to eight times lower than those conferred by CARB-7, differing at Ambler's positions V97I, L124F, and T228K. Comparison of the genetic environments of all reported
bla
CARB
genes indicated that the CARB enzymes constitute a family of cassette-encoded β-lactamases.