bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Characterization of Increasing Fluoroquinolone Resistance in
Streptococcus pneumoniae
Isolates in Canada, 1997 to 2005
Authors: Adam Heather J. , Schurek Kristen N. , Nichol Kimberly A. , Hoban Chris J. , Baudry Trish J. , Laing Nancy M. , Hoban Daryl J. , Zhanel George G. 
Published: December 2006

ABSTRACT

Molecular characterization of fluoroquinolone-resistant
Streptococcus pneumoniae
in Canada was conducted from 1997 to 2005. Over the course of the study, 205 ciprofloxacin-resistant isolates were evaluated for ParC and GyrA quinolone resistance-determining region (QRDR) substitutions, substitutions in the full genes of ParC, ParE, and GyrA, reserpine sensitivity, and serotype and by pulsed-field gel electrophoresis. Rates of ciprofloxacin resistance of
S. pneumoniae
increased significantly, from less than 1% in 1997 to 4.2% in 2005. Ciprofloxacin resistance was greatest in people >64 years of age and least in those <16 years of age. Significant increases were also noted in rates of resistance to gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin, to the current rates of 1.6%, 1.0%, 1.1%, and 1.0%, respectively. The most common genotype observed consisted of QRDR substitutions in GyrA (Ser81Phe) and ParC (Ser79Phe). Substitutions outside the QRDR of GyrA, ParC, and ParE were not associated with fluoroquinolone resistance in this study. Overall, 21% of isolates were reserpine-sensitive and were thus assumed to be efflux positive. The ciprofloxacin-resistant isolates belonged to 35 different serotypes, but 10 (19F, 11A, 23F, 6B, 22F, 12F, 6A, 14, 9V, and 19A) accounted for 72% of all isolates. The majority of the isolates were found to be genetically unrelated by pulsed-field gel electrophoresis. Within the observed clusters, there was considerable genetic heterogeneity with regard to fluoroquinolone resistance mechanisms and serotypes. Continued surveillance and molecular analysis of fluoroquinolone-resistant
S. pneumoniae
in Canada are essential for appropriate empirical treatment of infections and early detection of novel resistance mechanisms.
Identification of CTX-M-Type Extended-Spectrum-β-Lactamase Genes Using Real-Time PCR and Pyrosequencing
Authors: Naas Thierry , Oxacelay Cynthia , Nordmann Patrice 
Published: December 2006

ABSTRACT

CTX-M extended-spectrum β-lactamases (ESBLs) are increasingly prevalent worldwide among
Escherichia coli
bacteria, mostly in community-acquired urinary tract infections. Finding a fast and reliable technique for identification of CTX-M enzymes is becoming a challenge for the microbiology laboratory. A fast real-time PCR amplification technique, using degenerated primers specific for all the
bla
CTX-M
alleles, coupled to real-time pyrosequencing was developed. The five CTX-M groups were unambiguously identified by pyrosequencing a 13-bp DNA region. Further sequencing of an additional 16-bp region allowed further division into subgroups. Phylogenetic trees constructed with the entire
bla
CTX-M
genes and with both pyrosequenced regions (29 bp) gave similar results, suggesting that this technique, termed the real-time detection and sequencing method, has a powerful discriminatory ability. This high-throughput technique has been evaluated by screening 48 ESBL-producing
E. coli
isolates recovered from the Bicêtre hospital (France) in 2004. Forty-four of these strains were CTX-M positive by real-time PCR detection and direct pyrosequencing of the PCR products, which identified CTX-M-15 as the main CTX-M-type β-lactamase. Pulsed-field gel electrophoresis analysis of these strains revealed that several clones, of which one CTX-M-15-positive clone was predominant (60%), were identified both in nosocomial and in community-acquired isolates. The combination of real-time PCR with pyrosequencing represents a powerful tool for epidemiological studies of CTX-M producers. This assay has the potential to be used in a diagnostic laboratory since up to 96 bacterial isolates may be screened in less than 3 h.
Library Screen for Inhibitors Targeting Norovirus Binding to Histo-Blood Group Antigen Receptors
Authors: Feng Xizhi , Jiang Xi 
Published: December 2006

ABSTRACT
Human noroviruses (NVs) are a common cause of nonbacterial gastroenteritis. The disease is difficult to control due to its widespread nature and the lack of antivirals or vaccines against NVs. The recent identification of human histo-blood group antigens (HBGAs) as NV receptors opens a new way for the discovery and design of antivirals against NVs. A saliva-based enzyme immune assay (EIA) was used to screen a synthetic-compound library for inhibition of the binding of norovirus-like particles to HBGA receptors. Among 5,000 compounds tested in the first round of screening, 153 compounds exhibited >50% inhibition of the binding of VA387 (an NV that binds to A, B, and H epitopes) to the A antigen in saliva at ∼50 μg/ml, and 14 of the 153 compounds revealed strong inhibition, with a 50% effective concentration of <15 μM. Ten and 11 of the 14 compounds also revealed inhibition of the binding of VA387 to the B and H antigens, respectively. Seven and 6 of the 14 compounds also blocked the binding of the prototype Norwalk virus (A and H binder) to the A and H antigens, respectively. One compound significantly inhibited the binding of MOH (A and B binder) to the A and B antigens, but no compound revealed any inhibitory effect on the binding of a Lewis binding strain (VA207) to the Lewis antigens. The EIA is a high-throughput method for large-scale library screening for antivirals against NVs. Studies to further characterize the lead compounds and to screen additional compounds for other NVs are ongoing in our laboratory.
Multidrug Resistance Conferred by Novel DNA Polymerase Mutations in Human Cytomegalovirus Isolates
Authors: Scott Gillian M. , Weinberg Adriana , Rawlinson William D. , Chou Sunwen 
Published: December 2006

ABSTRACT
The emergence of antiviral-resistant cytomegalovirus (CMV) strains is a continuing clinical problem, with increased numbers of immunocompromised patients given longer-duration antiviral prophylaxis. Two previously unrecognized CMV DNA polymerase mutations (N408K and A834P) identified separately and together in at-risk lung and kidney transplant recipients and a third mutation (L737M) identified in a liver transplant recipient were characterized by marker transfer to antiviral-sensitive laboratory strains AD169 and Towne. Subsequent phenotypic analyses of recombinant strains demonstrated the ability of mutation N408K to confer ganciclovir (GCV) and cidofovir (CDV) resistance and of mutation A834P to confer GCV, foscarnet, and CDV resistance. Mutation L737M did not confer resistance to any of the antiviral agents tested. A recombinant strain containing both N408K and A834P demonstrated increased GCV and CDV resistance compared to the levels of resistance of the virus containing only the A834P mutation. The addition of mutation N408K in combination with A834P also partially reconstituted the replication impairment of recombinant virus containing only A834P. This suggests that perturbation of both DNA polymerization (A834P) and exonuclease (N408K) activities contributes to antiviral resistance and altered replication kinetics in these mutant strains. The identification of these multidrug-resistant CMV strains in at-risk seronegative recipients of organs from seropositive donors suggests that improved prophylactic and treatment strategies are required. The additive effect of multiple mutations on antiviral susceptibility suggests that increasing antiviral-resistant phenotypes can result from different virus-antiviral interactions.
Effects of Inoculum Size and Incubation Time on Broth Microdilution Susceptibility Testing of Lactic Acid Bacteria
Authors: Egervärn Maria , Lindmark Hans , Roos Stefan , Huys Geert , Lindgren Sven 
Published: December 2006

ABSTRACT

Inoculum size and incubation time were varied during broth microdilution testing of the susceptibilities of 35 strains of lactic acid bacteria to six antibiotics. An increase in either parameter resulted in elevated MICs for all species. An inoculum of 3 × 10
5
CFU/ml is recommended to assess the antibiotic susceptibilities of these bacteria by using broth microdilution.
Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors
Authors: Sluis-Cremer Nicolas , Sheen Chih-Wei , Zelina Shannon , Torres Pedro S. Argoti , Parikh Urvi M. , Mellors John W. 
Published: December 2006

ABSTRACTThe K70E mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has become more prevalent in clinical samples, particularly in isolates derived from patients for whom triple-nucleoside regimens that include tenofovir (TNV), abacavir, and lamivudine (3TC) failed. To elucidate the molecular mechanism by which this mutation confers resistance to these nucleoside RT inhibitors (NRTI), we conducted detailed biochemical analyses comparing wild-type (WT), K70E, and K65R HIV-1 RT. Pre-steady-state kinetic experiments demonstrate that the K70E mutation in HIV-1 RT allows the enzyme to discriminate between the natural deoxynucleoside triphosphate substrate and the NRTI triphosphate (NRTI-TP). Compared to the WT enzyme, K70E RT showed 2.1-, 2.3-, and 3.5-fold-higher levels of resistance toward TNV-diphosphate, carbovir-TP, and 3TC-TP, respectively. By comparison, K65R RT demonstrated 12.4-, 12.0-, and 13.1-fold-higher levels of resistance, respectively, toward the same analogs. NRTI-TP discrimination by the K70E (and K65R) mutation was primarily due to decreased rates of NRTI-TP incorporation and not to changes in analog binding affinity. The K65R and K70E mutations also profoundly impaired the ability of RT to excise 3′-azido-2′,3′-dideoxythymidine monophosphate (AZT-MP) and other NRTI-MP from the 3′ end of a chain-terminated primer. When introduced into an enzyme with the thymidine analog mutations (TAMs) M41L, L210W, and T215Y, the K70E mutation inhibited ATP-mediated excision of AZT-MP. Taken together, these findings indicate that the K70E mutation, like the K65R mutation, reduces susceptibility to NRTI by selectively decreasing NRTI-TP incorporation and is antagonistic to TAM-mediated nucleotide excision.
Effect of Anaerobic Growth on Quinolone Lethality with
Escherichia coli
Authors: Malik Muhammad , Hussain Syed , Drlica Karl 
Published: December 2006

ABSTRACT

Quinolone activity against
Escherichia coli
was examined during aerobic growth, aerobic treatment with chloramphenicol, and anaerobic growth. Nalidixic acid, norfloxacin, ciprofloxacin, and PD161144 were lethal for cultures growing aerobically, and the bacteriostatic activity of each quinolone was unaffected by anaerobic growth. However, lethal activity was distinct for each quinolone with cells treated aerobically with chloramphenicol or grown anaerobically. Nalidixic acid failed to kill cells under both conditions; norfloxacin killed cells when they were grown anaerobically but not when they were treated with chloramphenicol; ciprofloxacin killed cells under both conditions but required higher concentrations than those required with cells grown aerobically; and PD161144, a C-8-methoxy fluoroquinolone, was equally lethal under all conditions. Following pretreatment with nalidixic acid, a shift to anaerobic conditions or the addition of chloramphenicol rapidly blocked further cell death. Formation of quinolone-gyrase-DNA complexes, observed as a sodium dodecyl sulfate (SDS)-dependent drop in cell lysate viscosity, occurred during aerobic and anaerobic growth and in the presence and in the absence of chloramphenicol. However, lethal chromosome fragmentation, detected as a drop in viscosity in the absence of SDS, occurred with nalidixic acid treatment only under aerobic conditions in the absence of chloramphenicol. With PD161144, chromosome fragmentation was detected when the cells were grown aerobically and anaerobically and in the presence and in the absence of chloramphenicol. Thus, all quinolones tested appear to form reversible bacteriostatic complexes containing broken DNA during aerobic growth, during anaerobic growth, and when protein synthesis is blocked; however, the ability to fragment chromosomes and to rapidly kill cells under these conditions depends on quinolone structure.
In Vitro-Clinical Correlations for Amphotericin B Susceptibility in AIDS-Associated Cryptococcal Meningitis
Authors: Larsen R. A. , Bauer M. , Brouwer A. E. , Sanchez A. , Thomas A. M. , Rajanuwong A. , Chierakul W. , Peacock S. J. , Day N. , White N. J. , Rinaldi M. G. , Harrison T. S. 
Published: December 2006

ABSTRACT

Reliable measures of antifungal drug susceptibility are needed. We tested the susceptibility of
Cryptococcus neoformans
from patients treated with amphotericin B. In vitro susceptibility employed a modified broth macrodilution method. We demonstrate a strong correlation between the quantitative measures of in vitro amphotericin B susceptibility and the quantitative response observed in patients.
Voriconazole Concentration in Human Aqueous Humor and Plasma during Topical or Combined Topical and Systemic Administration for Fungal Keratitis
Authors: Thiel Michael A. , Zinkernagel Annelies S. , Burhenne Jürgen , Kaufmann Claude , Haefeli Walter E. 
Published: December 2006

ABSTRACT

Voriconazole (VRC) is an antifungal drug that effectively treats keratitis caused by yeasts and molds when administered orally. We retrospectively evaluated clinical outcomes and plasma and aqueous humor drug concentrations in five patients with fungal keratitis and one patient with posttraumatic endophthalmitis who were treated with VRC. VRC was administered either topically (1% eye drops every hour) or orally (400 mg twice a day). Plasma and aqueous humor samples from affected eyes were taken 12 h after oral administration or 1 h after eye drop application. The drug concentration was measured by liquid chromatography with UV or mass spectrometric detection. All six patients responded well to VRC treatment. The VRC concentration ranged from 2.93 to 3.40 mg/liter in the aqueous humor and from 3.20 to 4.20 mg/liter in the plasma after combined oral and topical treatment. Topical administration alone resulted in highly variable trough VRC concentrations of 0.61 to 3.30 mg/liter in the aqueous humor. VRC concentrations were above the MIC for
Candida albicans Aspergillus fumigatus
and clinical improvement was seen in all four patients with
C. albicans
and
A. fumigatus
keratitis. Combined orally and topically administered VRC resulted in aqueous humor drug concentrations of ≥2.93 mg/liter, which is above the VRC MIC for most fungi. Topical VRC treatment resulted in an aqueous humor drug concentration >0.61 mg/liter, which is above the MIC for most
Candida
species. The results from this small series of patients suggest that both topical and combined systemic and topical VRC therapy can be effective in treating fungal keratitis. Furthermore, the data provide preliminary support for initiation of VRC treatment with a combined topical and systemic administration until the causative fungus and its MIC are identified.
Penetration of Atazanavir in Seminal Plasma of Men Infected with Human Immunodeficiency Virus Type 1
Authors: van Leeuwen E. , ter Heine R. , van der Veen F. , Repping S. , Beijnen J. H. , Prins J. M. 
Published: December 2006

ABSTRACT
In this exploratory study, the human immunodeficiency virus (HIV) protease inhibitor atazanavir was detected in seminal plasma in 15 out of 15 HIV-infected men taking an atazanavir-containing regimen. However, this penetration was limited and variable, and the median seminal/blood plasma ratio was only 0.1.
Activity of Ribavirin against Hantaan Virus Correlates with Production of Ribavirin-5′-Triphosphate, Not with Inhibition of IMP Dehydrogenase
Authors: Sun Yanjie , Chung Dong-Hoon , Chu Yong-Kyu , Jonsson Colleen B. , Parker William B. 
Published: December 2006

ABSTRACT
Ribavirin (RBV) is a broad-spectrum antiviral agent that inhibits the production of infectious Hantaan virus (HTNV). Although the mechanism of action of RBV against HTNV is not understood, RBV is metabolized in human cells to both RBV-5′-monophosphate, which inhibits IMP dehydrogenase, resulting in a decrease in intracellular GTP levels, and RBV-5′-triphosphate (RBV-TP), which could selectively interact with the viral RNA polymerase. To elucidate which activity of RBV was most important to its anti-HTNV activity, the mechanism of action of RBV was studied in Vero E6 cells. Incubation with 10 to 40 μg/ml RBV resulted in a small decrease in GTP levels that was not dose dependent. Increasing the RBV concentration from 10 to 40 μg/ml resulted in a decrease in viral RNA (vRNA) levels and an increase in RBV-TP formation. Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, also resulted in a decrease in vRNA levels; however, treatment with MPA resulted in a much greater decrease in GTP levels than that seen with RBV. Treatment with both MPA and RBV resulted in increased reduction of vRNA levels but did not result in enhanced depression of GTP levels. Although guanosine prevented the depression in GTP levels caused by RBV, guanosine only partially prevented the effect of RBV on vRNA levels. These results suggest that the inhibition of IMP dehydrogenase by RBV is of secondary importance to the inhibition of vRNA replication by RBV and that the interaction of RBV-TP with the viral polymerase is the primary action of RBV.
Inhibitors of Nonhousekeeping Functions of the Apicoplast Defy Delayed Death in
Plasmodium falciparum
Authors: Ramya T. N. C. , Mishra Satyendra , Karmodiya Krishanpal , Surolia Namita , Surolia Avadhesha 
Published: December 2006

ABSTRACT

Targeting of apicoplast replication and protein synthesis in the apicomplexan
Toxoplasma gondii
has conventionally been associated with the typical “delayed death” phenotype, characterized by the death of parasites only in the generation following drug intervention. We demonstrate that antibiotics like clindamycin, chloramphenicol, and tetracycline, inhibitors of prokaryotic protein synthesis, invoke the delayed death phenotype in
Plasmodium falciparum
, too, as evident from a specific reduction of apicoplast genome copy number. Interestingly, however, molecules like triclosan, cerulenin, fops, and NAS-91, inhibitors of the recently discovered fatty acid synthesis pathway, and succinyl acetone, an inhibitor of heme biosynthesis that operates in the apicoplast of the parasite, display rapid and striking parasiticidal effects. Our results draw a clear distinction between apicoplast functions per se and the apicoplast as the site of metabolic pathways, which are required for parasite survival, and thus subserve the development of novel antimalarial therapy.
Molecular Mechanism of Flucytosine Resistance in
Candida lusitaniae
: Contribution of the
FCY2
,
FCY1
, and
FUR1
Genes to 5-Fluorouracil and Fluconazole Cross-Resistance
Authors: Papon Nicolas , Noël Thierry , Florent Martine , Gibot-Leclerc Stéphanie , Jean Dorothée , Chastin Christiane , Villard Jean , Chapeland-Leclerc Florence 
Published: December 2006

ABSTRACT

Inactivation of the
FCY2
(cytosine permease),
FCY1
(cytosine deaminase), and
FUR1
(uracil phosphoribosyltransferase) genes in
Candida lusitaniae
produced two patterns of resistance to flucytosine. Mutant
fur1
demonstrated resistance to 5-fluorouracil, whereas mutants
fcy1
and
fcy2
demonstrated fluconazole resistance in the presence of subinhibitory flucytosine concentrations.
Markers of Sulfadoxine-Pyrimethamine-Resistant
Plasmodium falciparum
in Placenta and Circulation of Pregnant Women
Authors: Mockenhaupt Frank P. , Bedu-Addo George , Junge Claudia , Hommerich Lena , Eggelte Teunis A. , Bienzle Ulrich 
Published: December 2006

ABSTRACT

Placental sequestration of
Plasmodium falciparum
in pregnancy may impair the usefulness of molecular markers of sulfadoxine-pyrimethamine resistance. In 300 infected, delivering women, the concordance of PCR-restriction fragment length polymorphism-derived parasite resistance alleles in matched samples from placenta and circulation was 83 to 98%. Sulfadoxine-pyrimethamine resistance typing in peripheral blood is reasonably representative of
P. falciparum
infecting pregnant women.
Susceptibility of
Acinetobacter
Strains Isolated from Deployed U.S. Military Personnel
Authors: Hawley Joshua S. , Murray Clinton K. , Griffith Matthew E. , McElmeel M. Leticia , Fulcher Letitia C. , Hospenthal Duane R. , Jorgensen James H. 
Published: December 2006

ABSTRACT

The susceptibilities of 142
Acinetobacter baumannii-calcoaceticus
complex isolates (95 from wounded U.S. soldiers deployed overseas) to 13 antimicrobial agents were determined by broth microdilution. The most active antimicrobial agents (≥95% of isolates susceptible) were colistin, polymyxin B, and minocycline.
Structure and Biosynthesis of Heat-Stable Antifungal Factor (HSAF), a Broad-Spectrum Antimycotic with a Novel Mode of Action
Authors: Yu Fengan , Zaleta-Rivera Kathia , Zhu Xiangcheng , Huffman Justin , Millet Jeffrey C. , Harris Steven D. , Yuen Gary , Li Xing-Cong , Du Liangcheng 
Published: December 2006

ABSTRACT

A screen for antifungal compounds from
Lysobacter enzymogenes
strain C3, a bacterial biological control agent of fungal diseases, has previously led to the isolation of heat-stable antifungal factor (HSAF). HSAF exhibits inhibitory activities against a wide range of fungal species and shows a novel mode of antifungal action by disrupting the biosynthesis of a distinct group of sphingolipids. We have now determined the chemical structure of HSAF, which is identical to that of dihydromaltophilin, an antifungal metabolite with a unique macrocyclic lactam system containing a tetramic acid moiety and a 5,5,6-tricyclic skeleton. We have also identified the genetic locus responsible for the biosynthesis of HSAF in strain C3. DNA sequencing of this locus revealed genes for a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), a sterol desaturase, a ferredoxin reductase, and an arginase. The disruption of the PKS-NRPS gene generated C3 mutants that lost the ability to produce HSAF and to inhibit fungal growth, demonstrating a hybrid PKS-NRPS that catalyzed the biosynthesis of the unique macrolactam system that is found in many biologically active natural products isolated from marine organisms. In addition, we have generated mutants with disrupted sterol desaturase, ferredoxin reductase, and arginase and examined the metabolites produced in these mutants. The work represents the first study of the genetic basis for the biosynthesis of the tetramic acid-containing macrolactams. The elucidation of the chemical structure of HSAF and the identification of the genetic locus for its biosynthesis establish the foundation for future exploitation of this group of compounds as new fungicides or antifungal drugs.