bluuebunny/crossref_metadata_2025 huggingface dataset:
Safety, Pharmacokinetics, Pharmacodynamics, and Plasma Lipoprotein Distribution of Eritoran (E5564) during Continuous Intravenous Infusion into Healthy Volunteers
Authors: Rossignol Daniel P. , Wasan Kishor M. , Choo Eugene , Yau Edwin , Wong Nancy , Rose Jeffrey , Moran Jeffrey , Lynn Melvyn
Published: August 2004

Eritoran, a structural analogue of the lipid A portion of lipopolysaccharide (LPS), is an antagonist of LPS in animal and human endotoxemia models. Previous studies have shown that low doses (350 to 3,500 μg) of eritoran have demonstrated a long pharmacokinetic half-life but a short pharmacodynamic half-life. The present study describes the safety, pharmacokinetics and pharmacodynamics, and lipid distribution profile of eritoran during and after a 72-h intravenous infusion of 500, 2,000, or 3,500 μg/h into healthy volunteers. Except for the occurrence of phlebitis, eritoran administration over 72 h was safe and well tolerated. Eritoran demonstrated a slow plasma clearance (0.679 to 0.930 ml/h/kg of body weight), a small volume of distribution (45.6 to 49.8 ml/kg), and a relatively long half-life (50.4 to 62.7 h). In plasma, the majority (∼55%) of eritoran was bound to high-density lipoproteins. During infusion and for up to 72 h thereafter, ex vivo response of blood to 1- or 10-ng/ml LPS was inhibited by ≥85%, even when the lowest dose of eritoran (500 μg/h) was infused. Inhibition of response was dependent on eritoran dose and the concentration of LPS used as an agonist. Finally, in vitro analysis with purified lipoprotein and protein fractions from plasma obtained from healthy volunteers indicated that eritoran is inactivated by high-density but not low-density lipoproteins, very-low-density lipoproteins, or albumin. From these results, we conclude that up to 252 mg of eritoran can be safely infused into normal volunteers over 72 h and even though it associates extensively with high-density lipoproteins, antagonistic activity is maintained, even after infusion ceases.
Antiplasmodial Chalcones Inhibit Sorbitol-Induced Hemolysis of
Plasmodium falciparum
-Infected Erythrocytes
Authors: Go Mei-Lin , Liu Mei , Wilairat Prapon , Rosenthal Philip J. , Saliba Kevin J. , Kirk Kiaran
Published: August 2004

A series of alkoxylated and hydroxylated chalcones previously reported to have antiplasmodial activities in vitro were investigated for their effects on the new permeation pathways induced by the malaria parasite in the host erythrocyte membrane. Of 21 compounds with good antiplasmodial activities (50% inhibitory concentrations [IC
50
s], ≤20 μM), 8 members were found to inhibit sorbitol-induced lysis of parasitized erythrocytes to a significant extent (≤40% of control values) at a concentration (10 μM) that was close to their antiplasmodial IC
50
s. Qualitative structure-activity analysis suggested that activity was governed to a greater extent by a substitution on ring B than on ring A of the chalcone template. Most of the active compounds had methoxy or dimethoxy groups on ring B. Considerable variety was permitted on ring A in terms of the electron-donating or -withdrawing property. Lipophilicity did not appear to be an important determinant for activity. Although they are not exceptionally potent as inhibitors (lowest IC
50
, 1.9 μM), the chalcones compare favorably with other more potent inhibitors in terms of their selective toxicities against plasmodia and their neutral character.
Treatment of Experimental Visceral Leishmaniasis with Amphotericin B in Stable Albumin Microspheres
Authors: Sánchez-Brunete J. A. , Dea M. A. , Rama S. , Bolás F. , Alunda J. M. , Raposo R. , Méndez M. T. , Torrado-Santiago S. , Torrado J. J.
Published: August 2004

Hydrophilic albumin microspheres are proposed as a new delivery system for amphotericin B (AMB; AMB microspheres). The acute toxicity of AMB microspheres was lower than that of the AMB-deoxycholate (AMB-Doc) reference formulation in hamsters. Lethal doses in healthy and infected animals were improved at least eight times. Intravenous bolus administration of doses of AMB microspheres up to 40 mg/kg of body weight did not produce acute symptoms of toxicity. The efficacy of this new formulation was tested against
Leishmania infantum-
infected hamsters at doses of 2, 10, 20, and 40 mg/kg. With the 2-mg/kg dose, the activity of AMB, as assessed through the parasite load reductions in the liver and spleen and the evolution of antibody levels, was also improved (
P
< 0.05) by use of the AMB microsphere system. At the higher doses of 10, 20, and 40 mg/kg, reductions in parasite levels of more than 99% were achieved in the liver and spleen after the administration of AMB microspheres. A pharmacokinetic study was performed to study the serum, liver, and spleen AMB concentrations after administration of AMB microspheres and the reference formulation. Interestingly, a significant accumulation of AMB in the spleen and liver was observed after AMB microsphere administration. Our results suggest that this new formulation is a promising alternative to the conventional AMB-Doc formulation for the treatment of visceral leishmaniasis.
Genome-Wide Transcriptional Profiling of the
Escherichia coli
Response to a Proline-Rich Antimicrobial Peptide
Authors: Tomasinsig Linda , Scocchi Marco , Mettulio Romina , Zanetti Margherita
Published: August 2004

Most antimicrobial peptides (AMPs) impair the viability of target bacteria by permeabilizing bacterial membranes. However, the proline-rich AMPs have been shown to kill susceptible organisms without causing significant membrane perturbation and may act by inhibiting the activity of bacterial targets. To gain initial insight into the events that follow interaction of a proline-rich peptide with bacterial cells, we used DNA macroarray technology to monitor transcriptional alterations of
Escherichia coli
in response to challenge with a subinhibitory concentration of the proline-rich Bac7(1-35). Substantial changes in the expression levels of 70 bacterial genes from various functional categories were detected. Among these, 26 genes showed decreased expression, while 44 genes, including genes that are potentially involved in bacterial resistance to antimicrobials, showed increased expression. The generation of a transcriptional response under the experimental conditions used is consistent with the ability of Bac7(1-35) to interact with bacterial components and affect biological processes in this organism.
The Novel Azole R126638 Is a Selective Inhibitor of Ergosterol Synthesis in
Candida albicans
,
Trichophyton
spp., and
Microsporum canis
Authors: Vanden Bossche Hugo , Ausma Jannie , Bohets Hilde , Vermuyten Karen , Willemsens Gustaaf , Marichal Patrick , Meerpoel Lieven , Odds Frank , Borgers Marcel
Published: August 2004

R126638 is a novel triazole with in vitro activity similar to that of itraconazole against dermatophytes,
Candida
spp., and
Malassezia
spp. In animal models of dermatophyte infections, R126638 showed superior antifungal activity. R126638 inhibits ergosterol synthesis in
Candida albicans
,
Trichophyton mentagrophytes
,
Trichophyton rubrum
, and
Microsporum canis
at nanomolar concentrations, with 50% inhibitory concentrations (IC
50
s) similar to those of itraconazole. The decreased synthesis of ergosterol and the concomitant accumulation of 14α-methylsterols provide indirect evidence that R126638 inhibits the activity of CYP51 that catalyzes the oxidative removal of the 14α-methyl group of lanosterol or eburicol. The IC
50
s for cholesterol synthesis from acetate in human hepatoma cells were 1.4 μM for itraconazole and 3.1 μM for R126638. Compared to itraconazole (IC
50
= 3.5 μM), R126638 is a poor inhibitor of the 1α-hydroxylation of 25-hydroxyvitamin D
3
(IC
50
> 10 μM). Micromolar concentrations of R126638 and itraconazole inhibited the 24-hydroxylation of 25-hydroxyvitamin D
3
and the conversion of 1,25-dihydroxyvitamin D
3
into polar metabolites. At concentrations up to 10 μM, R126638 had almost no effect on cholesterol side chain cleavage (CYP11A1), 11β-hydroxylase (CYP11B1), 17-hydroxylase and 17,20-lyase (CYP17), aromatase (CYP19), or 4-hydroxylation of all-
trans
retinoic acid (CYP26). At 10 μM, R126638 did not show clear inhibition of CYP1A2, CYP2A6, CYP2D6, CYP2C8, CYP2C9, CYP2C10, CYP2C19, or CYP2E1. Compared to itraconazole, R126638 had a lower interaction potential with testosterone 6β hydroxylation and cyclosporine hydroxylation, both of which are catalyzed by CYP3A4, whereas both antifungals inhibited the CYP3A4-catalyzed hydroxylation of midazolam similarly. The results suggest that R126638 has promising properties and merits further in vivo investigations for the treatment of dermatophyte and yeast infections.
In Vitro Activity of Caspofungin Combined with Sulfamethoxazole against Clinical Isolates of
Aspergillus
spp
Authors: Yekutiel Aya , Shalit Itamar , Shadkchan Yona , Osherov Nir
Published: August 2004

Caspofungin (CAS) inhibits fungal cell wall synthesis. Sulfamethoxazole (SMX) inhibits folate biosynthesis and is active in vitro against
Aspergillus
spp. We studied the activities of the combination of CAS and SMX against 31
Aspergillus
isolates and compared them with that of SMX combined with amphotericin B (AMB) or itraconazole (ITC). MICs and minimal effective concentrations (MECs) were determined by the NCCLS broth microdilution method. With MIC endpoints, the combination of SMX and CAS showed synergy or synergy to additivity against 29 of 31 isolates. With MEC endpoints, synergy to additivity was found against 12 of 31 isolates and indifference was displayed against the rest of them. SMX in combination with AMB or ITC was not truly synergistic, while synergy to additivity was found for SMX-AMB and SMX-ITC against 17 of 31 and 3 of 12 isolates, respectively. No antagonism was found with any of the drug combinations. Further analysis of the synergy of CAS and SMX was performed by detailed measurement of hyphal length by microscopy and time-dependent 2,3-bis(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2
H
-tetrazolium hydroxide (XTT)-based hyphal damage experiments. With MEC endpoints, the combination of CAS and SMX was characterized by a greater than 50% decrease in hyphal length compared to the hyphal lengths achieved with double the concentration of each drug alone. The XTT-based hyphal damage studies showed a statistically significant (
P
< 0.05) reduction in viability with CAS and SMX in combination compared to the viabilities achieved with double the concentration of each drug alone. These findings support the synergy results found by using MIC endpoints and suggest that visual MEC measurements may not be sufficient to identify the synergistic interactions seen by more sensitive, quantitative methods. Animal models are required to validate the significance of the synergy of CAS and SMX against
Aspergillus
spp. observed in vitro.
Functional and Structural Characterization of the Genetic Environment of an Extended-Spectrum β-Lactamase
bla
VEB
Gene from a
Pseudomonas aeruginosa
Isolate Obtained in India
Authors: Aubert Daniel , Girlich Delphine , Naas Thierry , Nagarajan Shanta , Nordmann Patrice
Published: August 2004

A
Pseudomonas aeruginosa
clinical strain isolated from a patient hospitalized in a New Delhi, India, hospital was resistant to expanded-spectrum cephalosporins, imipenem, and aztreonam. A
bla
VEB-1
-like gene named
bla
VEB-1a
, which codes for the extended-spectrum β-lactamase VEB-1a, was identified. The genetic environment of
bla
VEB-1a
was peculiar: (i) no 5′ conserved sequence (5′-CS) region was present upstream of the β-lactamase gene, whereas
bla
VEB-1
-like genes are usually associated with class 1 integrons; (ii)
bla
VEB-1a
was inserted between two truncated 3′-CS regions in a direct repeat; and (iii) four 135-bp repeated DNA sequences (repeated elements) were located on each side of the
bla
VEB-1a
gene. Expression of the
bla
VEB-1a
gene was driven by a strong promoter located in one of these repeated sequences. In addition, cloning of the β-lactamase content of this
P. aeruginosa
isolate followed by expression in
Escherichia coli
identified the naturally occurring AmpC β-lactamase and a gene encoding an OXA-2-like β-lactamase located in a class 1 integron, In78, in which an insertion sequence, IS
pa7
, was inserted within its 5′-CS region.
Penetration of
Candida
Biofilms by Antifungal Agents
Authors: Al-Fattani Mohammed A. , Douglas L. Julia
Published: August 2004

A filter disk assay was used to investigate the penetration of antifungal agents through biofilms containing single and mixed-species biofilms containing
Candida
. Fluconazole permeated all single-species
Candida
biofilms more rapidly than flucytosine. The rates of diffusion of either drug through biofilms of three strains of
Candida albicans
were similar. However, the rates of drug diffusion through biofilms of
C. glabrata
or
C. krusei
were faster than those through biofilms of
C. parapsilosis
or
C. tropicalis
. In all cases, after 3 to 6 h the drug concentration at the distal edge of the biofilm was very high (many times the MIC). Nevertheless, drug penetration failed to produce complete killing of biofilm cells. These results indicate that poor antifungal penetration is not a major drug resistance mechanism for
Candida
biofilms. The abilities of flucytosine, fluconazole, amphotericin B, and voriconazole to penetrate mixed-species biofilms containing
C. albicans
and
Staphylococcus epidermidis
(a slime-producing wild-type strain, RP62A, and a slime-negative mutant, M7) were also investigated. All four antifungal agents diffused very slowly through these mixed-species biofilms. In most cases, diffusion was slower with biofilms containing
S. epidermidis
RP62A, but amphotericin B penetrated biofilms containing the M7 mutant more slowly. However, the drug concentrations reaching the distal edges of the biofilms always substantially exceeded the MIC. Thus, although the presence of bacteria and bacterial matrix material undoubtedly retarded the diffusion of the antifungal agents, poor penetration does not account for the drug resistance of
Candida
biofilm cells, even in these mixed-species biofilms.
Expression of the RND-Type Efflux Pump AdeABC in
Acinetobacter baumannii
Is Regulated by the AdeRS Two-Component System
Authors: Marchand Isabelle , Damier-Piolle Laurence , Courvalin Patrice , Lambert Thierry
Published: August 2004

The AdeABC pump of
Acinetobacter baumannii
BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The
adeA
,
adeB
, and
adeC
genes are contiguous and adjacent to
adeS
and
adeR
, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to
adeAB
and
adeC
were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of
adeS
led to aminoglycoside susceptibility. Transcripts corresponding to
adeAB
were not detected in susceptible
A. baumannii
CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr153→Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro116→Leu in AdeR at the first residue of the α
5
helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild
A. baumannii
due to stringent control by the AdeRS two-component system.
In Vitro Antimicrobial Susceptibilities of
Streptococcus pneumoniae
Clinical Isolates Obtained in Canada in 2002
Authors: Powis Jeff , McGeer Allison , Green Karen , Vanderkooi Otto , Weiss Karl , Zhanel George , Mazzulli Tony , Kuhn Magdalena , Church Deirdre , Davidson Ross , Forward Kevin , Hoban Daryl , Simor Andrew , Low Donald E.
Published: August 2004

Empirical treatment is best guided by current surveillance of local resistance patterns. The goal of this study is to characterize the prevalence of antimicrobial nonsusceptibility within pneumococcal isolates from Canada. The Canadian Bacterial Surveillance Network is comprised of laboratories from across Canada. Laboratories collected a defined number of consecutive clinical and all sterile site isolates of
S. pneumoniae
in 2002. In vitro susceptibility testing was performed by broth microdilution with NCCLS guidelines. Rates of nonsusceptibility were compared to previously published reports from the same network. A total of 2,539 isolates were tested. Penicillin nonsusceptibility increased to 15% (8.5% intermediate, 6.5% resistant) compared to 12.4% in 2000 (
P
≤ 0.025, χ
2
). Only 32 (1.3%) isolates had an amoxicillin MIC of ≥4 μg/ml and only 2 of 32 cerebrospinal fluid isolates had an intermediate susceptibility to ceftriaxone by meningeal interpretive criteria (MIC = 1 μg/ml). A total of 354 (13.9%) isolates were macrolide nonsusceptible (46.3% MLS
B
, 56.7% M phenotype), increasing from 11.4% in 2000 (
P
≤ 0.0075, χ
2
). Only 13 (<1%) isolates had a telithromycin MIC of >1 μg/ml. Ciprofloxacin nonsusceptibility (defined as an MIC of ≥4 μg/ml) increased to 2.7% compared to 1.4% in 2000 (
P
≤ 0.0025, χ
2
) and was primarily found in persons ≥18 years old (98.5%). Nonsusceptibility to penicillin, macrolides, and fluoroquinolones is increasing in Canada. Nonsusceptibility to amoxicillin and ceftriaxone remains uncommon. Newer antimicrobials such as telithromycin and respiratory fluoroquinolones have excellent in vitro activity.
Posaconazole and Amphotericin B Combination Therapy against
Cryptococcus neoformans
Infection
Authors: Barchiesi Francesco , Spreghini Elisabetta , Schimizzi Anna M. , Maracci Monia , Giannini Daniele , Carle Flavia , Scalise Giorgio
Published: August 2004

To investigate the effects of posaconazole (POS) and amphotericin B (AMB) combination therapy in cryptococcal infection, we established an experimental model of systemic cryptococcosis in CD1 mice by intravenous injection of three distinct clinical isolates of
Cryptococcus neoformans
. Therapy was started 24 h after the infection and continued for 10 consecutive days. POS was given at 3 and 10 mg/kg of body weight/day, while AMB was given at 0.3 mg/kg/day. Combination therapy consisted of POS given at a low (combo 3) or at a high (combo 10) dose plus AMB. Survival studies showed that combo 3 was significantly more effective than POS at 3 mg/kg for two isolates tested (
P
value, ≤0.001), while combo 10 was significantly more effective than POS at 10 mg/kg for all three isolates (
P
values ranging from <0.001 to 0.005). However, neither combination regimen was more effective than AMB alone. For two isolates, combination therapy was significantly more effective than each single drug at reducing the fungal burden in the brain (
P
values ranging from 0.001 to 0.015) but not in the lungs. This study demonstrates that the major impact of POS and AMB combination therapy is on brain fungal burden rather than on survival.
In Vitro Activities of Voriconazole in Combination with Three Other Antifungal Agents against
Candida glabrata
Authors: Barchiesi Francesco , Spreghini Elisabetta , Maracci Monia , Fothergill Annette W. , Baldassarri Isabella , Rinaldi Michael G. , Scalise Giorgio
Published: August 2004

Candida glabrata
has recently emerged as a significant pathogen involved in both superficial and deep-seated infections. In the present study, a checkerboard broth microdilution method was performed to investigate the in vitro activities of voriconazole (VOR) in combination with terbinafine (TRB), amphotericin B (AMB), and flucytosine (5FC) against 20 clinical isolates of
C. glabrata
. Synergy, defined as a fractional inhibitory concentration (FIC) index of ≤0.50, was observed in 75% of VOR-TRB, 10% of VOR-AMB, and 5% of VOR-5FC interactions. None of these combinations yielded antagonistic interactions (FIC index > 4). When synergy was not achieved, there was still a decrease in the MIC of one or both drugs used in the combination. In particular, the MICs were reduced to ≤1.0 μg/ml as a result of the combination for all isolates for which the AMB MIC at the baseline was ≥2.0 μg/ml. By a disk diffusion assay, the halo diameters produced by antifungal agents in combination were greater that those produced by each drug alone. Finally, killing curves showed that VOR-AMB exhibited synergistic interactions, while VOR-5FC sustained fungicidal activities against
C. glabrata
. These studies demonstrate that the in vitro activity of VOR against this important yeast pathogen can be enhanced upon combination with other drugs that have different modes of action or that target a different step in the ergosterol pathway. Further studies are warranted to elucidate the potential beneficial effects of such combination regimens in vivo.
Double-Blind, Randomized Study of the Efficacy and Safety of Oral Pharmacokinetically Enhanced Amoxicillin-Clavulanate (2,000/125 Milligrams) versus Those of Amoxicillin-Clavulanate (875/125 Milligrams), Both Given Twice Daily for 7 Days, in Treatment of Bacterial Community-Acquired Pneumonia in Adults
Authors: File T. M. , Lode H. , Kurz H. , Kozak R. , Xie H. , Berkowitz E.
Published: August 2004

This randomized, double-blind, noninferiority trial was designed to demonstrate that pharmacokinetically enhanced amoxicillin-clavulanate (2,000/125 mg) was at least as effective clinically as amoxicillin-clavulanate 875/125 mg, both given twice daily for 7 days, in the treatment of community-acquired pneumonia in adults. In total, 633 clinically and radiologically confirmed community-acquired pneumonia patients (intent-to-treat population) were randomized to receive either oral amoxicillin-clavulanate 2,000/125 mg (
n
= 322) or oral amoxicillin-clavulanate 875/125 mg (
n
= 311). At screening, 160 of 633 (25.3%) patients had at least one typical pathogen isolated from expectorated or invasive sputum samples or blood culture (bacteriology intent-to-treat population).
Streptococcus pneumoniae
(58 of 160, 36.3%), methicillin-susceptible
Staphylococcus aureus
(34 of 160, 21.3%), and
Haemophilus influenzae
(33 of 160, 20.6%) were the most common typical causative pathogens isolated in both groups in the bacteriology intent-to-treat population. Clinical success in the clinical per protocol population at test of cure (days 16 to 37), the primary efficacy endpoint, was 90.3% (223 of 247) for amoxicillin-clavulanate 2,000/125 mg and 87.6% (198 of 226) for amoxicillin-clavulanate 875/125 mg (treatment difference, 2.7; 95% confidence interval, −3.0, 8.3). Bacteriological success at test of cure in the bacteriology per protocol population was 86.6% (58 of 67) for amoxicillin-clavulanate 2,000/125 mg and 78.4% (40 of 51) for amoxicillin-clavulanate 875/125 mg (treatment difference, 8.1%; 95% confidence interval, −5.8, 22.1). Both therapies were well tolerated. Amoxicillin-clavulanate 2,000/125 mg twice daily was shown to be as clinically effective as amoxicillin-clavulanate 875/125 mg twice daily for 7 days in the treatment of adult patients with community-acquired pneumonia, without a noted increase in the reported rate of adverse events.
Plasmid-Encoded Multidrug Efflux Pump Conferring Resistance to Olaquindox in
Escherichia coli
Authors: Hansen Lars Hestbjerg , Johannesen Elsebetta , Burmølle Mette , Sørensen Anders Hay , Sørensen Søren J.
Published: August 2004

We report here the first gene-encoded resistance mechanism to the swine growth enhancer olaquindox. The genetic elements involved in resistance to olaquindox were subcloned and sequenced from a conjugative plasmid isolated from
Escherichia coli
. The subcloned fragment contained two open reading frames,
oqxA
and
oqxB
, that are homologous to several resistance-nodulation-cell-division family efflux systems from different species. The putative protein sequences were aligned to both experimentally verified and putative efflux pumps. We show that
oqxA
and
oqxB
are expressed in
E. coli
. Plasmids containing the
oqxAB
genes yielded high (>128 μg/ml) resistance to olaquindox in
E. coli
, whereas strains containing the control plasmid showed low resistance to the drug (8 μg/ml). The
oqxAB
-encoded pump also conferred high (>64 μg/ml) resistance to chloramphenicol. We demonstrate that the subcloned fragment conferred H
+
-dependent ethidium efflux abilities to
E. coli
strain N43. In addition, we show that the efflux system is dependent on the host TolC outer membrane protein when expressed in
E. coli
.
In Vitro Activity of the New Quinolone WCK 771 against Staphylococci
Authors: Jacobs Michael R. , Bajaksouzian Saralee , Windau Anne , Appelbaum Peter C. , Patel Mahesh V. , Gupte Shrikant V. , Bhagwat Sachin S. , De Souza Noel J. , Khorakiwala Habil F.
Published: August 2004

The activity of WCK 771, an experimental quinolone developed to overcome quinolone resistance in staphylococci and other bacteria, was determined against quinolone-susceptible and -resistant
Staphylococcus aureus
and
S. epidermidis
. WCK 771 MICs for 50 and 90% of the strains tested (MIC
50
and MIC
90
, respectively) were 0.008 and 0.015 μg/ml for
S. aureus
(
n
= 43) and 0.015 and 0.03 μg/ml for
S. epidermidis
(
n
= 44) for quinolone-susceptible isolates, compared to ciprofloxacin values of 0.12 and 0.25 μg/ml and 0.25 and 0.5 μg/ml, respectively. Values for levofloxacin were 0.12 and 0.25 μg/ml and 0.12 and 0.25 μg/ml, those for clinafloxacin were 0.015 and 0.03 μg/ml and 0.015 and 0.03 μg/ml, those for moxifloxacin were 0.03 and 0.06 μg/ml and 0.06 and 0.12 μg/ml, and those for gatifloxacin were 0.06 and 0.12 μg/ml and 0.12 and 0.25 μg/ml, respectively. The WCK 771 MIC
50
and MIC
90
, respectively, were 0.5 and 1 μg/ml for both species of staphylococci (
n
= 73 for
S. aureus
,
n
= 70 for
S. epidermidis
) for isolates highly resistant to ciprofloxacin (MIC
50
and MIC
90
, >32 and >32 μg/ml, respectively). Values for levofloxacin were 8 and 32 μg/ml and 8 and 32 μg/ml, those for clinafloxacin were 1 and 2 μg/ml and 0.5 and 2 μg/ml, those for moxifloxacin 4 and >4 μg/ml and 4 and >4 μg/ml, and those for gatifloxacin were 4 and >4 μg/ml and 2 and >4 μg/ml, respectively. WCK 771 and clinafloxacin demonstrated MICs of 1 μg/ml against three vancomycin-intermediate strains. WCK 771 showed concentration-independent killing for up to 24 h at 2, 4, and 8 times the MICs against quinolone-resistant staphylococci and was also bactericidal after 8 h for high-density inocula (10
8
CFU/ml) of quinolone-resistant strains at 5 μg/ml, whereas moxifloxacin at 7.5 μg/ml was bacteriostatic. WCK 771 was not a substrate of the NorA efflux pump as evident from the similar MICs against both an efflux-positive and an efflux-negative strain. Overall, WCK 771 was the most potent quinolone tested against the staphylococci tested, regardless of quinolone susceptibility.
Novel Murine Model of Pneumococcal Pneumonia: Use of Temperature as a Measure of Disease Severity To Compare the Efficacies of Moxifloxacin and Levofloxacin
Authors: Bast Darrin J. , Yue M. , Chen X. , Bell D. , Dresser L. , Saskin R. , Mandell L. A. , Low D. E. , de Azavedo Joyce C. S.
Published: August 2004

Surface temperature measured by an infrared temperature-scanning thermometer was used to evaluate disease severity and predict imminent death in a murine model of pneumococcal pneumonia. We showed that a decrease in temperature was associated with increasing severity of disease and concomitant histological changes and also that a temperature of 30°C or less was a predictor of death. Furthermore, viable bacterial counts in the lungs of mice euthanized at a temperature of ≤ 30°C were not significantly different from those seen in the lungs of mice allowed to die without intervention. These data support temperature change as a more subtle indicator of outcome than death and demonstrate that this could be used as a reliable end point for euthanasia. To test the utility of our model in a drug trial, we examined the efficacies of moxifloxacin and levofloxacin by using temperature as a measure of disease severity prior to and during treatment. Regardless of the antibiotic used, mice assessed as moderately ill (temperature ≥ 32°C) at the start of treatment had better clinical and bacteriological outcomes than mice assessed as severely ill (temperature < 32°C). However, moxifloxacin offered better protection and greater bacterial clearance than did levofloxacin in all infected mice independent of disease severity. This model not only allows a more subtle evaluation of drug efficacy but also ensures a better degree of standardization and a more humane approach to drug efficacy studies involving animals.
Adherence of
Candida albicans
to Silicone Induces Immediate Enhanced Tolerance to Fluconazole
Authors: Mateus Carolina , Crow Sidney A. , Ahearn Donald G.
Published: August 2004

Wild-type and efflux pump-deficient cells of
Candida albicans
adhering to silicone were compared with planktonic cells by flow cytometry for their relative resistance to fluconazole (FCZ). Flow cytometry data on cells carrying a fusion of green fluorescent protein to efflux pump promoters confirmed that enhanced tolerance of attached cells to FCZ was due in part to increased expression of
CaMDR1
and
CDR1
promoters. Within 2 h of their attachment to silicone, the adherent cells demonstrated levels of FCZ tolerance shown by cells from 24-h biofilms. Following their mechanical detachment, this subset of cells retained a four- to eightfold increase in tolerance compared with the tolerance of planktonic cells for at least two generations. Enhanced efflux pump tolerance to FCZ appeared to be induced within the initial 15 min of attachment in a subset of cells that were firmly attached to the substrata.
Human Tear Lipocalin Exhibits Antimicrobial Activity by Scavenging Microbial Siderophores
Authors: Fluckinger Maria , Haas Hubertus , Merschak Petra , Glasgow Ben J. , Redl Bernhard
Published: August 2004

Human tear lipocalin (TL; also known as Lcn1) is a secretory protein present in large amounts in fluids that cover epithelial surfaces such as tears and respiratory secretions. It is supposed to act as a physiological scavenger of hydrophobic, potentially harmful molecules, but there is evidence that it also inhibits bacterial growth. In the present study, we reconsidered the possibility that TL might interfere with microbial growth by scavenging of siderophores, as described for human neutrophil gelatinase-associated lipocalin (NGAL). Indeed, our experiments revealed that TL binds to microbial siderophores with high affinities. In contrast to NGAL, which was shown to have some specificity for bacterial catecholate-type siderophores, TL binds to a broad array of siderophores, including bacterial catecholate-type enterobactin and hydroxamate-type desferrioxamine B, and all major classes of fungal siderophores. By adding exogenous TL, bacterial and fungal growth could be inhibited under iron-limiting conditions. Thus, TL might be a novel member of the innate immune system especially involved in mucosal defense against fungal infections.
Screening and Characterization of Mutations in Isoniazid-Resistant
Mycobacterium tuberculosis
Isolates Obtained in Brazil
Authors: Cardoso Rosilene Fressatti , Cooksey Robert C. , Morlock Glenn P. , Barco Patricia , Cecon Leticia , Forestiero Francisco , Leite Clarice Q. F. , Sato Daisy N. , Shikama Maria de Lourdes , Mamizuka Elsa M. , Hirata Rosario D. C. , Hirata Mario H.
Published: August 2004

We investigated mutations in the genes
katG
,
inhA
(regulatory and structural regions), and
kasA
and the
oxyR-ahpC
intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible
Mycobacterium tuberculosis
isolates obtained in two states in Brazil: São Paulo and Paraná. PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of
katG
, the regulatory region and codons 16 and 94 of
inhA
,
kasA
, and the
oxyR-ahpC
intergenic region. DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles. Mutations in
katG
were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported
katG
mutations. Mutations in the
inhA
promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had
inhA
structural gene mutations, and 10.3% had mutations in the
oxyR-ahpC
intergenic region (one, nucleotide −48, previously unreported). Polymorphisms in the
kasA
gene occurred in both INH-resistant and INH-susceptible isolates. The most frequent polymorphism encoded a G
269
A substitution. Although KatG

315

substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil. Since ca. 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance.
Bactericidal Effects of a Fusion Protein of Llama Heavy-Chain Antibodies Coupled to Glucose Oxidase on Oral Bacteria
Authors: Szynol A. , de Soet J. J. , Sieben-van Tuyl E. , Bos J. W. , Frenken L. G.
Published: August 2004

Enzymes such as lactoperoxidase and glucose oxidase (GOx) are used as antimicrobial agents in oral care products. Their low specificities and substantiveness can be reduced by covalent coupling of antimicrobial molecules to antibodies. Variable domains (VHH) derived from llama heavy-chain antibodies are particularly suited for such an approach. The antibodies are composed solely of heavy-chain dimers; therefore, production of active fusion proteins by using molecular biology-based techniques is less complicated than production by use of conventional antibodies. In this study, a fusion protein consisting of VHHand GOx was constructed and expressed bySaccharomyces cerevisiae. A llama was immunized withStreptococcus mutansstrain HG982. Subsequently, B lymphocytes were isolated and cDNA fragments encoding the VHHfragments were obtained by reverse transcription-PCR. After construction of a VHHlibrary inEscherichia coliand screening of the library against mutans group streptococci andStreptococcus sanguinisstrains, we found two VHHfragments with high specificities forS. mutansstrains. A GOx gene was linked to the two VHHgenes and cloned intoS. cerevisiaeyeasts. The yeasts expressed and secreted the recombinant proteins into the growth medium. The test of binding of fusion proteins to oral bacteria through their VHHfragments showed thatS. mutanshad been specifically targeted by GOx-S120, one of the fusion protein constructs. A low concentration of the fusion protein was also able to selectively killS. mutanswithin 20 min in the presence of lactoperoxidase and potassium iodide. These findings demonstrate that the fusion protein GOx-VHHis potentially valuable in the selective killing of target bacteria such asS. mutans.
A Mucoadhesive Polymer Extracted from Tamarind Seed Improves the Intraocular Penetration and Efficacy of Rufloxacin in Topical Treatment of Experimental Bacterial Keratitis
Authors: Ghelardi Emilia , Tavanti Arianna , Davini Paola , Celandroni Francesco , Salvetti Sara , Parisio Eva , Boldrini Enrico , Senesi Sonia , Campa Mario
Published: August 2004

Bacterial keratitis is a serious infectious ocular disease requiring prompt treatment to prevent frequent and severe visual disabilities. Standard treatment of bacterial keratitis includes topical administration of concentrated antibiotic solutions repeated at frequent intervals in order to reach sufficiently high drug levels in the corneal tissue to inhibit bacterial growth. However, this regimen has been associated with toxicity to the corneal epithelium and requires patient hospitalization. In the present study, a mucoadhesive polymer extracted from tamarind seeds was used for ocular delivery of 0.3% rufloxacin in the treatment of experimental
Pseudomonas aeruginosa
and
Staphylococcus aureus
keratitis in rabbits. The polysaccharide significantly increased the intra-aqueous penetration of rufloxacin in both infected and uninfected eyes. Rufloxacin delivered by the polysaccharide reduced
P. aeruginosa
and
S. aureus
in the cornea at a higher rate than that obtained by rufloxacin alone. In particular, use of the polysaccharide allowed a substantial reduction of
S. aureus
in the cornea to be achieved even when the time interval between drug administrations was extended. These results suggest that the tamarind seed polysaccharide prolongs the precorneal residence times of antibiotics and enhances drug accumulation in the cornea, probably by reducing the washout of topically administered drugs. The tamarind seed polysaccharide appears to be a promising candidate as a vehicle for the topical treatment of bacterial keratitis.
Sequence-Selective Recognition of Extended-Spectrum β-Lactamase GES-2 by a Competitive, Peptide Nucleic Acid-Based Multiplex PCR Assay
Authors: Weldhagen Gerhard F.
Published: August 2004

Extended-spectrum β-lactamases (ESBLs) in
Pseudomonas aeruginosa
, such as GES-2, which compromises the efficacy of imipenem, tend to be geographically restricted. The CC-to-AA base pair substitution at positions 493 and 494 of the
bla
GES-2
-coding region distinguishes this ESBL from
bla
GES-1
and the
bla
IBC
-type genes, making it an ideal target for the development of a novel sequence-specific, peptide nucleic acid (PNA)-based multiplex PCR detection method. By using two primer pairs in conjunction with a PNA probe, this method provided an accurate means of identification of
bla
GES-2
compared to standard PCR and gene sequencing techniques when it was used to test 100
P. aeruginosa
clinical isolates as well as previously published, well-described control strains encompassing all presently known genes in the
bla
GES-IBC
ESBL family. This novel method has the potential to be used in large-scale, cost-effective screening programs for specific or geographically restricted ESBLs.
Genes Required for Intrinsic Multidrug Resistance in
Mycobacterium avium
Authors: Philalay Julie S. , Palermo Christine O. , Hauge Kirsten A. , Rustad Tige R. , Cangelosi Gerard A.
Published: August 2004

Genes required for intrinsic multidrug resistance by
Mycobacterium avium
were identified by screening a library of transposon insertion mutants for the inability to grow in the presence of ciprofloxacin, clarithromycin, and penicillin at subinhibitory concentrations. Two genes,
pks12
and Maa2520, were disrupted in multiple drug-susceptible mutants. The
pks12
gene (Maa1979), which may be cotranscribed with a downstream gene (Maa1980), is widely conserved in the actinomycetes. Its ortholog in
Mycobacterium tuberculosis
is a polyketide synthase required for the synthesis of dimycocerosyl phthiocerol, a major cell wall lipid. Mutants of
M. avium
with insertions into
pks12
exhibited altered colony morphology and were drug susceptible, but they grew as well as the wild type did in vitro and intracellularly within THP-1 cells. A
pks12
mutant of
M. tuberculosis
was moderately more susceptible to clarithromycin than was its parent strain; however, susceptibility to ciprofloxacin and penicillin was not altered.
M. avium
complex (MAC) and
M. tuberculosis
appear to have different genetic mechanisms for resisting the effects of these antibiotics, with
pks12
playing a relatively more significant role in MAC. The second genetic locus identified in this study, Maa2520, is a conserved hypothetical gene with orthologs in
M. tuberculosis
and
Mycobacterium leprae
. It is immediately upstream of Maa2521, which may code for an exported protein. Mutants with insertions at this locus were susceptible to multiple antibiotics and slow growing in vitro and were unable to survive intracellularly within THP-1 cells. Like
pks12
mutants, they exhibited increased Congo red binding, an indirect indication of cell wall modifications. Maa2520 and
pks12
are the first genes to be linked by mutation to intrinsic drug resistance in MAC.
Effect of Ertapenem Protein Binding on Killing of Bacteria
Authors: Nix David E. , Matthias Kathryn R. , Ferguson Emily C.
Published: August 2004

The effect of protein binding on the antimicrobial activity of ertapenem was evaluated using the bacterial kill rate and concentration-response studies. Various proportions of human serum were utilized to determine the total and free-drug concentrations using a validated high-performance liquid chromatography assay. The MICs and kill curves were determined for test isolates of
Enterobacter cloacae
and
Staphylococcus aureus
at various percentages of human serum. The killing of bacteria was analyzed in relation to the free and total concentrations of ertapenem at various proportions of human serum. It was determined that unbound ertapenem was responsible for the antimicrobial activity against the test isolates.
Disruption of the
Candida albicans CYB5
Gene Results in Increased Azole Sensitivity
Authors: Rogers K. M. , Pierson C. A. , Culbertson N. T. , Mo C. , Sturm A. M. , Eckstein J. , Barbuch R. , Lees N. D. , Bard M.
Published: August 2004

Sterol synthesis in fungi is an aerobic process requiring molecular oxygen and, for several cytochrome-mediated reactions, aerobically synthesized heme. Cytochrome
b
5
is required for sterol C5-6 desaturation and the encoding gene,
CYB5
, is nonessential in
Saccharomyces cerevisiae
. Cyb5p and Ncp1p (cytochrome P-450 reductase) appear to have overlapping functions in this organism, with disruptions of each alone being viable. The cytochrome P-450 reductase phenotype has also been shown to demonstrate increased sensitivity to azole antifungals. Based on this phenotype, the
CYB5
gene in the human pathogen
Candida albicans
was investigated to determine whether the
cyb5
genotype was viable and would also demonstrate azole sensitivity. Sequential disruption of the
CYB5
alleles by direct transformation resulted in viability, presumably conferred by the presence of a third copy of the
CYB5
gene. Subsequent disruption procedures with a
pMAL2-CYB5
rescue cassette and a
CYB5-URA3
blaster cassette resulted in viable
cyb5
strains with no third copy. The
C. albicans CYB5
gene is concluded to be nonessential. Thus, the essentiality of this gene and whether we observed two or three alleles was dependent upon the gene disruption protocol. The
C. albicans cyb5
strains produced a sterol profile containing low ergosterol levels and sterol intermediates similar to that reported for the
S. cerevisiae cyb5
. The
C. albicans cyb5
shows increased sensitivity to azoles and terbinafine, an inhibitor of squalene epoxidase, and, unexpectedly, increased resistance to morpholines, which inhibit the
ERG2
and
ERG24
gene products. These results indicate that an inhibitor of Cyb5p would not be lethal but would make the cell significantly more sensitive to azole treatment.
In Vivo Imaging of Bioluminescent
Escherichia coli
in a Cutaneous Wound Infection Model for Evaluation of an Antibiotic Therapy
Authors: Jawhara Samir , Mordon Serge
Published: August 2004

A rapid, continuous method for noninvasively monitoring the effectiveness of several antibacterial agents in real time by using a model of wound infection was developed. This study was divided into three steps: (i) construction of a plasmid to transform
Escherichia coli
into a bioluminescent variant, (ii) study of the bioluminescent
E. coli
in vitro as a function of temperature and pH, and (iii) determination of the MIC and the minimal bactericidal concentration of sulfamethoxazole-trimethoprim (SMX-TMP). Finally, the efficacy of SMX-TMP was monitored in vivo in a cutaneous wound model (hairless rat) infected with this bioluminescent bacterium by using a bioluminescence imaging system.
E. coli
was transformed by electroporation with a shuttle vector (pRB474) containing the firefly (
Photinus pyralis
) luciferase gene, resulting in a bioluminescent phenotype. It was found that pH 5.0 was optimal for incorporation of the susbstrate
d
-luciferin for the luciferase reaction. In vitro, when the agar dilution method, standard turbidity assays, and the bioluminescence imaging system were used,
E. coli
(pRB474) proved to be susceptible to SMX-TMP. In vivo, at 4 h, SMX-TMP treatment was already efficient compared to no treatment (
P
= 0.034). At 48 h, no bioluminescence was detected in the wound, demonstrating the susceptibility of
E. coli
to SMX-TMP. In conclusion, this study points out the advantage of using bioluminescence imaging to evaluate the effects of antibiotics for the treatment of acute infections in vivo in a nondestructive and noninvasive manner.
The
mef
(A) Gene Predominates among Seven Macrolide Resistance Genes Identified in Gram-Negative Strains Representing 13 Genera, Isolated from Healthy Portuguese Children
Authors: Ojo K. K. , Ulep C. , Van Kirk N. , Luis H. , Bernardo M. , Leitao J. , Roberts M. C.
Published: August 2004

Of the 176 randomly selected, commensal, gram-negative bacteria isolated from healthy children with low exposure to antibiotics, 138 (78%) carried one or more of the seven macrolide resistance genes tested in this study. These isolates included 79 (91%) isolates from the oral cavity and 59 (66%) isolates from urine samples. The
mef
(A) gene, coding for an efflux protein, was found in 73 isolates (41%) and was the most frequently carried gene. The
mef
(A) gene could be transferred from the donors into a gram-positive
E. faecalis
recipient and a gram-negative
Escherichia coli
recipient. The
erm
(B) gene transferred and was maintained in the
E. coli
transconjugants but was found in 0 to 100% of the
E. faecalis
transconjugants tested, while the other five genes could be transferred only into the
E. coli
recipient. The individual macrolide resistance genes were identified in 3 to 12 new genera. Eight (10%) of the oral isolates and 30 (34%) of the urine isolates for which the MICs were 2 to >500 μg of erythromycin per ml did not hybridize with any of the seven genes and may carry novel macrolide resistance genes.
Azithromycin Inhibits MUC5AC Production Induced by thePseudomonas aeruginosaAutoinducerN-(3-Oxododecanoyl) Homoserine Lactone in NCI-H292 Cells
Authors: Imamura Yoshifumi , Yanagihara Katsunori , Mizuta Yohei , Seki Masafumi , Ohno Hideaki , Higashiyama Yasuhito , Miyazaki Yoshitsugu , Tsukamoto Kazuhiro , Hirakata Yoichi , Tomono Kazunori , Kadota Jun-ichi , Kohno Shigeru
Published: August 2004

The features of chronic airway diseases, including chronic bronchitis, cystic fibrosis, bronchiectasis, and diffuse panbronchiolitis, include chronic bacterial infection and airway obstruction by mucus.Pseudomonas aeruginosais one of the most common pathogens in chronic lung infection, and quorum-sensing systems contribute to the pathogenesis of this disease. The quorum-sensing signal molecule [N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL)] not only regulates bacterial virulence but also is associated with the immune response. In this study, we investigated whether 3O-C12-HSL could stimulate the production of a major mucin core protein, MUC5AC. The effect of a macrolide on MUC5AC production was also studied. 3O-C12-HSL induced NCI-H292 cells to express MUC5AC at both the mRNA and the protein levels in time- and dose-dependent manners. A 15-membered macrolide, azithromycin, inhibited MUC5AC production that was activated by 3O-C12-HSL. 3O-C12-HSL induced extracellular signal-regulated kinase (ERK) 1/2 and I-κB phosphorylation in cells, and this induction was suppressed by azithromycin. 3O-C12-HSL-induced MUC5AC production was blocked by the ERK pathway inhibitor PD98059. Our findings suggest that theP. aeruginosaautoinducer 3O-C12-HSL contributes to excessive mucin production in chronic bacterial infection. Azithromycin seems to reduce this mucin production by interfering with intracellular signal transduction.