bluuebunny/crossref_metadata_2025_split huggingface dataset:
TEM-109 (CMT-5), a Natural Complex Mutant of TEM-1 β-Lactamase Combining the Amino Acid Substitutions of TEM-6 and TEM-33 (IRT-5)
Authors: Robin F. , Delmas J. , Chanal C. , Sirot D. , Sirot J. , Bonnet R. 
Published: October 2005

ABSTRACT

Escherichia coli
CF349 exhibited a complex β-lactam resistance phenotype, including resistance to amoxicillin and ticarcillin alone and in combination with clavulanate and to some extended-spectrum cephalosporins. The double-disk synergy test was positive. CF349 harbored an 85-kb conjugative plasmid which encoded a β-lactamase of pI 5.9. The corresponding
bla
gene was identified by PCR and sequencing as a
bla
TEM
gene. The deduced protein sequence revealed a new complex mutant of TEM-1 β-lactamase designated TEM-109 (CMT-5). TEM-109 contained both the substitutions Glu104Lys and Arg164His of the expanded-spectrum β-lactamase (ESBL) TEM-6 and Met69Leu of the inhibitor-resistant TEM-33 (IRT-5). TEM-109 exhibited hydrolytic activity against ceftazidime similar to that of TEM-6 (
k
cat
, 56 s
−1
and 105 s
−1
, respectively;

K
m

values, 226 and 247 μM, respectively). The 50% inhibitory concentrations of clavulanate and tazobactam (0.13 μM and 0.27 μM, respectively) were 5- to 10-fold higher for TEM-109 than for TEM-6 (0.01 and 0.06 μM, respectively) but were almost 10-fold lower than those for TEM-33. The characterization of this novel CMT, which exhibits a low level of resistance to inhibitors, highlights the emergence of this new ESBL type.
Anti-Human Immunodeficiency Virus Type 1 Activity of the Nonnucleoside Reverse Transcriptase Inhibitor GW678248 in Combination with Other Antiretrovirals against Clinical Isolate Viruses and In Vitro Selection for Resistance
Authors: Hazen Richard J. , Harvey Robert J. , St. Clair Marty H. , Ferris Robert G. , Freeman George A. , Tidwell Jeffrey H. , Schaller Lee T. , Cowan Jill R. , Short Steven A. , Romines Karen R. , Chan Joseph H. , Boone Lawrence R. 
Published: October 2005

ABSTRACT

GW678248, a novel nonnucleoside reverse transcriptase inhibitor, has been evaluated for anti-human immunodeficiency virus activity in a variety of in vitro assays against laboratory strains and clinical isolates. When GW678248 was tested in combination with approved drugs in the nucleoside and nucleotide reverse transcriptase inhibitor classes or the protease inhibitor class, the antiviral activities were either synergistic or additive. When GW678248 was tested in combination with approved drugs in the nonnucleoside reverse transcriptase inhibitor class, the antiviral activities were either additive or slightly antagonistic. Clinical isolates from antiretroviral drug-experienced patients were selected for evaluation of sensitivity to GW678248 in a recombinant virus assay. Efavirenz (EFV) and nevirapine (NVP) had ≥10-fold increases in their 50% inhibitory concentrations (IC
50
s) for 85% and 98% of the 55 selected isolates, respectively, whereas GW678248 had a ≥10-fold increase in the IC
50
for only 17% of these isolates. Thus, 81 to 83% of the EFV- and/or NVP-resistant viruses from this data set were susceptible to GW678248. Virus populations resistant to GW678248 were selected by in vitro dose-escalating serial passage. Resistant progeny viruses recovered after eight passages had amino acid substitutions V106I, E138K, and P236L in the reverse transcriptase-coding region in one passage series and amino acid substitutions K102E, V106A, and P236L in a second passage series.
Photodynamic Sensitization ofLeishmania amazonensisin both Extracellular and Intracellular Stages with Aluminum Phthalocyanine Chloride for Photolysis In Vitro
Authors: Dutta Sujoy , Ray Debalina , Kolli Bala K. , Chang Kwang-Poo 
Published: October 2005

ABSTRACTLeishmania amazonensis, a causative agent of cutaneous leishmaniasis, is susceptible in vitro to light-mediated cytolysis in the presence of or after pretreatment with the photosensitizer aluminum phthalocyanine chloride. Cytolysis of both promastigotes and axenic amastigotes required less photosensitizer (e.g., one μg · ml−1) and a lower light dose (e.g., 1.5 J · cm−2) than did the mammalian cells examined for comparison. Exposure ofLeishmaniacells to the photosensitizer alone had little effect on their viability, as judged from their motility, growth, and/or retention of green fluorescent proteins genetically engineered for episomal expression. Fluorimetric assays for cell-associated and released green fluorescence proteins proved to be even more sensitive for the evaluation of cell viability than microscopy for the evaluation of motility and/or integrity. Axenic amastigotes pretreated with the photosensitizer infected macrophages of the J774 line but were lysed intracellularly when the infected cells were exposed to light. Addition of the photosensitizer to the already infected cells produced no effect on their intracellular parasites. However, light irradiation lysed these macrophages and also those infected with parasites preincubated with the photosensitizer at a concentration of 5 μg · ml−1or higher. PhotosensitizedLeishmaniacells are highly susceptible to cytolysis, apparently due to the generation of reactive oxidative species on light illumination, suggestive of inefficiency of their antioxidant mechanisms. Efficient delivery of photosensitizers to intracellularLeishmaniais expected to increase their therapeutic potentials against leishmaniasis.
Identification of a Dithiazoline Inhibitor of
Escherichia coli
l
,
d
-Carboxypeptidase A
Authors: Baum Ellen Z. , Crespo-Carbone Steven M. , Foleno Barbara , Peng Sean , Hilliard Jamese J. , Abbanat Darren , Goldschmidt Raul , Bush Karen 
Published: October 2005

ABSTRACT

The enzyme
l
,
d
-carboxypeptidase A is involved in the recycling of bacterial peptidoglycan and is essential in
Escherichia coli
during stationary phase. By high-throughput screening, we have identified a dithiazoline inhibitor of the enzyme with a 50% inhibitory concentration of 3 μM. The inhibitor appeared to cause lysis of
E. coli
during stationary phase, behavior that is similar to a previously described deletion mutant of
l
,
d
-carboxypeptidase A (M. F. Templin, A. Ursinus, and J.-V. Holtje, EMBO J.
18:
4108-4117, 1999). As much as a one-log drop in CFU in stationary phase was observed upon treatment of
E. coli
with the inhibitor, and the amount of intracellular tetrapeptide substrate increased by approximately 33%, consistent with inhibition of the enzyme within bacterial cells. Stationary-phase targets such as
l
,
d
-carboxypeptidase A are largely underrepresented as targets of the antibiotic armamentarium but provide potential opportunities to interfere with bacterial growth and persistence.
Susceptibilities of Antiviral-Resistant Influenza Viruses to Novel Neuraminidase Inhibitors
Authors: Mishin Vasiliy P. , Hayden Frederick G. , Gubareva Larisa V. 
Published: October 2005

ABSTRACTThe susceptibilities of five zanamivir-resistant and six oseltamivir-resistant influenza viruses were assessed against four neuraminidase (NA) inhibitors, including peramivir and A-315675, by a fluorometric NA activity inhibition assay. The enzyme activity of a majority of the variants was effectively inhibited by either A-315675 or both peramivir and A-315675 (50% inhibitory concentration, <10 nM). A novel oseltamivir-resistant influenza virus B variant carrying substitution at residue 198 (Asp→Asn) (N2 numbering) retained susceptibility to peramivir and A-315675. In vivo, the Asn198 variant showed no apparent fitness impairment as judged by its recovery on day 5 from the nasal washes of ferrets coinfected with equal doses of the wild-type virus and the Asn198 variant. Based on the sequence analysis of the virus in the nasal washes, oseltamivir treatment (5 mg/kg twice daily for 5 days) did not provide growth advantage to the Asn198 variant. Nevertheless, treatment with A-315675 (prodrug A-322278) reduced the number of the animals (two of seven) shedding the Asn198 variant. These studies indicate that different patterns of susceptibility and cross-resistance between NA inhibitors may prove important if antiviral resistance to zanamivir and oseltamivir were to emerge.
Evaluation of Antibiotic Treatment against “
Candidatus
Helicobacter suis” in a Mouse Model
Authors: Hellemans A. , Decostere A. , Haesebrouck F. , Ducatelle R. 
Published: October 2005

ABSTRACT

“
Helicobacter
heilmannii
” (proposed name) type 1 colonizes
the human stomach. It has been shown to be identical to“
Candidatus
Helicobacter suis,” a
Helicobacter
species colonizing the stomachs of >60%
of slaughter pigs. This bacterium has not been isolated in vitro until
now. Antibiotic susceptibility testing of “
Candidatus
Helicobacter suis” has not been carried out so far. For the
present study, a mouse model was adopted to evaluate the antibiotic
susceptibility of this organism. Mice infected with“
Candidatus
Helicobacter suis” were treated
with amoxicillin and omeprazole, a therapy which is used to treat
H. heilmannii
infections in humans. Two different isolates of“
Candidatus
Helicobacter suis” were tested.
The excretion of bacterial DNA was assessed during treatment, using PCR
on fecal samples. At the end of the experiment, 8 days after the
cessation of treatment, the presence of infection was evaluated using a
urease test and a PCR test on stomach samples. A marked decrease in the
excretion of bacterial DNA was observed a few days after the onset of
treatment, and the level remained low until the end of the experiment.
A difference in susceptibility between the two“
Candidatus
Helicobacter suis” isolates was
pointed out. The in vivo mouse model infected with“
Candidatus
Helicobacter suis” will be useful
for further screening of potential therapeutic
regimens.
Pharmacokinetics, Safety, and Tolerability of Caspofungin in Children and Adolescents
Authors: Walsh Thomas J. , Adamson Peter C. , Seibel Nita L. , Flynn Patricia M. , Neely Michael N. , Schwartz Cindy , Shad Aziza , Kaplan Sheldon L. , Roden Maureen M. , Stone Julie A. , Miller Alisha , Bradshaw Susan K. , Li Susan X. , Sable Carole A. , Kartsonis Nicholas A. 
Published: October 2005

ABSTRACT

Caspofungin is a parenteral antifungal that inhibits beta-1,3-
d
-glucan synthesis. Although licensed for adult use, the appropriate caspofungin dosing regimen in pediatric patients is not yet known. We therefore investigated the pharmacokinetics and safety of caspofungin in pediatric patients. Thirty-nine children (ages 2 to 11 years) and adolescents (ages 12 to 17 years) with neutropenia were administered caspofungin using either a weight-based regimen (1 mg/kg of body weight/day) or a body surface area regimen (50 mg/m
2
/day or 70 mg/m
2
/day). Plasma samples for caspofungin profiles were collected on days 1 and 4. These results were compared to those from adults treated with either 50 or 70 mg/day for mucosal candidiasis. In children receiving 1 mg/kg/day (maximum, 50 mg/day), the area under the concentration-time curve over 24 h (AUC
0-24
) was significantly smaller (46% after multiple doses) than that observed in adults receiving 50 mg/day (
P
< 0.001). In children and adolescents receiving 50 mg/m
2
/day (maximum, 70 mg/day), the AUC
0-24
following multiple doses was similar to that for the exposure in adults receiving 50 mg/day. The AUC
0-24
and concentration trough (at 24 h) in pediatric patients receiving the 50-mg/m
2
daily regimen were consistent across the range of ages. Caspofungin was generally well tolerated in this study. None of the patients developed a serious drug-related adverse event or were discontinued for toxicity. These results demonstrate that caspofungin at 1 mg/kg/day in pediatric patients is suboptimal. Caspofungin administration at 50 mg/m
2
/day provides a comparable exposure to that of adult patients treated with 50 mg/day.
High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme
Authors: Cancio Reynel , Silvestri Romano , Ragno Rino , Artico Marino , De Martino Gabriella , La Regina Giuseppe , Crespan Emmanuele , Zanoli Samantha , Hübscher Ulrich , Spadari Silvio , Maga Giovanni 
Published: October 2005

ABSTRACT
Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation.
Prior Antimicrobial Therapy and Risk for Hospital-Acquired
Candida glabrata
and
Candida krusei
Fungemia: a Case-Case-Control Study
Authors: Lin Michael Y. , Carmeli Yehuda , Zumsteg Jennifer , Flores Ernesto L. , Tolentino Jocelyn , Sreeramoju Pranavi , Weber Stephen G. 
Published: October 2005

ABSTRACT

The incidence of infections caused by
Candida glabrata
and
Candida krusei
, which are generally more resistant to fluconazole than
Candida albicans
, is increasing in hospitalized patients. However, the extent to which prior exposure to specific antimicrobial agents increases the risk of subsequent
C. glabrata
or
C. krusei
candidemia has not been closely studied. A retrospective case-case-control study was performed at a university hospital. From 1998 to 2003, 60 patients were identified with hospital-acquired non-
C. albicans
candidemia (
C. glabrata
or
C. krusei
; case group 1). For comparison, 68 patients with
C. albicans
candidemia (case group 2) and a common control group of 121 patients without candidemia were studied. Models were adjusted for demographic and clinical risk factors, and the risk for candidemia associated with exposure to specific antimicrobial agents was assessed. After adjusting for both nonantimicrobial risk factors and receipt of other antimicrobial agents, piperacillin-tazobactam (odds ratio [OR], 4.15; 95% confidence interval [CI], 1.04 to 16.50) and vancomycin (OR, 6.48; CI, 2.20 to 19.13) were significant risk factors for
C. glabrata
or
C. krusei
candidemia. For
C. albicans
candidemia, no specific antibiotics remained a significant risk after adjusted analysis. Prior fluconazole use was not significantly associated with either
C. albicans
or non-
C. albicans
(
C. glabrata
or
C. krusei
) candidemia. In this single-center study, exposure to antibacterial agents, specifically vancomycin or piperacillin-tazobactam, but not fluconazole, was associated with subsequent hospital-acquired
C. glabrata
or
C. krusei
candidemia. Further studies are needed to prospectively analyze specific antimicrobial risks for nosocomial candidemia across multiple hospital centers.
Antibacterial Activity of Human Neutrophil Defensin HNP-1 Analogs without Cysteines
Authors: Varkey Jobin , Nagaraj Ramakrishnan 
Published: October 2005

ABSTRACTThe antibacterial activity of human neutrophil defensin HNP-1 analogs without cysteines has been investigated. A peptide corresponding to the HNP-1 sequence without the six cysteines (HNP-1ΔC) exhibited antibacterial activity toward gram-negative and gram-positive bacteria. Truncated analogs wherein the nine N-terminal residues of HNP-1 and the remaining three cysteines were deleted (HNP-1ΔC18) or the G was replaced with A (HNP-1ΔC18A) also exhibited antibacterial activity. Substantial activity was observed for HNP-1ΔC and HNP-1ΔC18 in the presence of 100 mM NaCl, except in the case ofPseudomonas aeruginosa. The linear peptides were active in the presence of carbonyl cyanidem-chlorophenylhydrazone (CCCP), indicating that proton motive force was not essential for killing of bacteria by the peptides. In fact, in the presence of CCCP, the peptides were active againstP. aeruginosaeven in the presence of 100 mM NaCl. The antibacterial activity of HNP-1ΔC, but not that of the shorter, 18-residue peptides, was attenuated in the presence of serum. The generation of defensins without cysteines would be easier than that of disulfide-linked defensins. Hence, linear defensins could have potential as therapeutic agents.
Pseudomonas aeruginosaAmpR Is a Global Transcriptional Factor That Regulates Expression of AmpC and PoxB β-Lactamases, Proteases, Quorum Sensing, and Other Virulence Factors
Authors: Kong Kok-Fai , Jayawardena Suriya Ravi , Indulkar Shalaka Dayaram , del Puerto Aimee , Koh Chong-Lek , Høiby Niels , Mathee Kalai 
Published: October 2005

ABSTRACTIn members of the familyEnterobacteriaceae,ampC, which encodes a β-lactamase, is regulated by an upstream, divergently transcribed gene,ampR. However, inPseudomonas aeruginosa, the regulation ofampCis not understood. In this study, we compared the characteristics of aP. aeruginosa ampRmutant, PAOampR, with that of an isogenicampR+parent. TheampRmutation greatly altered AmpC production. In the absence of antibiotic, PAOampRexpressed increased basal β-lactamase levels. However, this increase was not followed by a concomitant increase in the PampCpromoter activity. The discrepancy in protein and transcription analyses led us to discover the presence of another chromosomal AmpR-regulated β-lactamase, PoxB. We found that the expression ofP. aeruginosa ampRgreatly altered the β-lactamase production fromampCandpoxBinEscherichia coli: it up-regulated AmpC but down-regulated PoxB activities. In addition, the constitutive PampRpromoter activity in PAOampRindicated that AmpR did not autoregulate in the absence or presence of inducers. We further demonstrated that AmpR is a global regulator because the strain carrying theampRmutation produced higher levels of pyocyanin and LasA protease and lower levels of LasB elastase than the wild-type strain. The increase in LasA levels was positively correlated with the PlasA, PlasI, and PlasRexpression. The reduction in the LasB activity was positively correlated with the PrhlRexpression. Thus, AmpR plays a dual role, positively regulating theampC,lasB, andrhlRexpression levels and negatively regulating thepoxB,lasA,lasI, andlasRexpression levels.
Nystatin Biosynthesis and Transport:
nysH
and
nysG
Genes Encoding a Putative ABC Transporter System in
Streptomyces noursei
ATCC 11455 Are Required for Efficient Conversion of 10-Deoxynystatin to Nystatin
Authors: Sletta Håvard , Borgos Sven E. F. , Bruheim Per , Sekurova Olga N. , Grasdalen Hans , Aune Randi , Ellingsen Trond E. , Zotchev Sergey B. 
Published: October 2005

ABSTRACT

The genes
nysH
and
nysG
, encoding putative ABC-type transporter proteins, are located at the flank of the nystatin biosynthetic gene cluster in
Streptomyces noursei
ATCC 11455. To assess the possible roles of these genes in nystatin biosynthesis, they were inactivated by gene replacements leading to in-frame deletions. Metabolite profile analysis of the
nysH
and
nysG
deletion mutants revealed that both of them synthesized nystatin at a reduced level and produced considerable amounts of a putative nystatin analogue. Liquid chromatography-mass spectrometry and nuclear magnetic resonance structural analyses of the latter metabolite confirmed its identity as 10-deoxynystatin, a nystatin precursor lacking a hydroxyl group at C-10. Washing experiments demonstrated that both nystatin and 10-deoxynystatin are transported out of cells, suggesting the existence of an alternative efflux system(s) for the transport of nystatin-related metabolites. This notion was further corroborated in experiments with the ATPase inhibitor sodium
o
-vanadate, which affected the production of nystatin and 10-deoxynystatin in the wild-type strain and transporter mutants in a different manner. The data obtained in this study suggest that the efflux of nystatin-related polyene macrolides occurs through several transporters and that the NysH-NysG efflux system provides conditions favorable for C-10 hydroxylation.
In Vitro Assessment of Methylene Blue on Chloroquine-Sensitive and -Resistant
Plasmodium falciparum
Strains Reveals Synergistic Action with Artemisinins
Authors: Akoachere Monique , Buchholz Kathrin , Fischer Elisabeth , Burhenne Jürgen , Haefeli Walter E. , Schirmer R. Heiner , Becker Katja 
Published: October 2005

ABSTRACT

Methylene blue (MB) represents a promising antimalarial drug candidate for combination therapies against drug-resistant parasite strains. To support and facilitate the application of MB in future field trials, we studied its antiparasitic effects in vitro. MB is active against all blood stages of both chloroquine (CQ)-sensitive and CQ-resistant
P. falciparum
strains with 50% inhibitory concentration (IC
50
) values in the lower nanomolar range. Ring stages showed the highest susceptibility. As demonstrated by high-performance liquid chromatography-tandem mass spectrometry on different cell culture compartments, MB is accumulated in malarial parasites. In drug combination assays, MB was found to be antagonistic with CQ and other quinoline antimalarials like piperaquine and amodiaquine; with mefloquine and quinine, MB showed additive effects. In contrast, we observed synergistic effects of MB with artemisinin, artesunate, and artemether for all tested parasite strains. Artemisinin/MB combination concentration ratios of 3:1 were found to be advantageous, demonstrating that the combination of artemisinin with a smaller amount of MB can be recommended for reaching maximal therapeutic effects. Our in vitro data indicate that combinations of MB with artemisinin and related endoperoxides might be a promising option for treating drug-resistant malaria and should be studied in future field trials. Resistance development under this drug combination is unlikely to occur.
Phosphinothricin Tripeptide Synthetases in
Streptomyces viridochromogenes
Tü494
Authors: Schwartz Dirk , Grammel Nicolas , Heinzelmann Eva , Keller Ullrich , Wohlleben Wolfgang 
Published: October 2005

ABSTRACT

The tripeptide backbone of phosphinothricin (PT) tripeptide (PTT), a compound with herbicidal activity from
Streptomyces viridochromogenes
, is assembled by three stand-alone peptide synthetase modules. The enzyme PhsA (66 kDa) recruits the PT-precursor
N
-acetyl-demethylphosphinothricin (
N
-Ac-DMPT), whereas the two alanine residues of PTT are assembled by the enzymes PhsB and PhsC (129 and 119 kDa, respectively). During or after assembly, the N-Ac-DMPT residue in the peptide is converted to PT by methylation and deacetylation. Both
phsB
and
phsC
appear to be cotranscribed together with two other genes from a single promoter and they are located at a distance of 20 kb from the gene
phsA
, encoding PhsA, in the PTT biosynthesis gene cluster of
S. viridochromogenes
. PhsB and PhsC represent single nonribosomal peptide synthetase elongation modules lacking a thioesterase domain. Gene inactivations, genetic complementations, determinations of substrate specificity of the heterologously produced proteins, and comparison of PhsC sequence with the amino terminus of the alanine-activating nonribosomal peptide synthetase PTTSII from
S. viridochromogenes
confirmed the role of the two genes in the bialanylation of Ac-DMPT. The lack of an integral thioesterase domain in the PTT assembly system points to product release possibly involving two type II thioesterase genes (
the1
and
the2
) located in the PTT gene cluster alone or in conjunction with an as yet unknown mechanism of product release.