bluuebunny/crossref_metadata_2025 huggingface dataset:
Five-Day Cefdinir Treatment for Streptococcal Pharyngitis
Authors: Tack Kenneth J. , Henry Daniel C. , Gooch W. Manford , Brink Douglas N. , Keyserling Constance H. , Group The Cefdinir Pharyngitis Study
Published: October 2018

A multicenter, randomized, controlled, investigator-blind study was performed to evaluate the safety and efficacy of oral cefdinir versus oral penicillin V for the treatment of pharyngitis due to group A beta-hemolytic streptococci (GABHS). Patients 13 years of age and older were randomized to receive either oral cefdinir (300 mg twice a day) for 5 days followed by placebo for 5 days or oral penicillin V (250 mg four times a day) for 10 days. Throat cultures were obtained, and signs and symptoms of pharyngitis were recorded at study admission and follow-up visits on study days 11 to 15, 16 to 20, and 25 to 31. Patients kept a diary to record medication intake and their assessment of throat pain at admission and at each day of study treatment. Five hundred fifty-eight patients were enrolled, of whom 432 (77.4%) were clinically and microbiologically evaluable. The GABHS eradication rates 5 to 10 days after completion of therapy were 193 of 218 (88.5%) in the cefdinir group and 176 of 214 (82.2%) in the penicillin group (
P
= 0.053). Clinical cure rates were 89.0 and 84.6%, respectively (
P
= 0.80). By the time of the long-term follow-up visit, 2 to 3 weeks after completion of treatment, 156 of 191 (81.7%) of the assessable cefdinir patients and 152 of 195 (77.9%) of the penicillin patients remained free of GABHS. Both treatments were well tolerated, with adverse reaction rates of 18.3% in the cefdinir study arm and 15.0% in the penicillin study arm (
P
= 0.278). Five-day treatment with cefdinir is safe and effective therapy for GABHS pharyngitis. Based on its twice-a-day dosage and shorter course of therapy, leading to potentially greater patient compliance, cefdinir may be considered for use in the treatment of pharyngitis caused by GABHS.
Pharmacodynamics of Fluconazole in a Murine Model of Systemic Candidiasis
Authors: Louie Arnold , Drusano George L. , Banerjee Partha , Liu Qing-Feng , Liu Weiguo , Kaw Pamela , Shayegani Mehdi , Taber Harry , Miller Michael H.
Published: October 2018

In this study we defined the pharmacodynamic parameter that optimizes outcome in deep-seated
Candida albicans
infections treated with fluconazole. Using a murine model of systemic candidiasis, we conducted single-dose dose-ranging studies with fluconazole to determine the dosage of this drug that resulted in a 50% reduction in fungal densities (50% effective dose [ED
50
]) in kidneys versus the fungal densities in the kidneys of untreated controls. We found that the ED
50
of fluconazole given intraperitoneally was 4.56 mg/kg of body weight/day (95% confidence interval, 3.60 to 5.53 mg/kg/day), and the dose-response relationship was best described by an inhibitory sigmoid maximal effect (
E
max
) curve. To define the pharmacodynamics of fluconazole, we gave dosages lower than, approximating, and higher than the ED
50
of fluconazole (range, 3.5 to 5.5 mg/kg/day, equivalent to the ED
16
to the ED
75
) to various groups of infected animals using three dose-fractionation schedules. For each total dose of fluconazole examined, the dose-fractionation schedules optimized the ratio of the area under the concentration-time curve (AUC) to the MIC (the AUC/MIC ratio), the ratio of the maximum concentration of drug in serum (
C
max
) to the MIC, and the time that the drug remained above the MIC for the infecting
C. albicans
isolate. Similar reductions in fungal densities in kidneys were seen between groups that received the same total dose of fluconazole in one, two, or four equally divided doses. Thus, dose-fractionation studies demonstrated that the pharmacodynamic parameter of fluconazole that best predicted outcome was the AUC/MIC ratio.
CNI-H0294, a Nuclear Importation Inhibitor of the Human Immunodeficiency Virus Type 1 Genome, Abrogates Virus Replication in Infected Activated Peripheral Blood Mononuclear Cells
Authors: Haffar Omar K. , Smithgall Molly D. , Popov Serguei , Ulrich Peter , Bruce A. Gregory , Nadler Steven G. , Cerami Anthony , Bukrinsky Michael I.
Published: October 2018

Active nuclear importation of the human immunodeficiency virus (HIV) type 1 (HIV-1) preintegration complex (PIC) is required for the productive infection of nondividing cells, but it is believed to be dispensable for the infection of proliferating cells, such as activated T lymphocytes. To investigate this question, we exploited the properties of the small arylene bis (methyl ketone) compound CNI-H0294. We have previously shown that this compound associated with the HIV-1 matrix protein nuclear localization sequence and blocked binding of the HIV-1 PIC to yeast karyopherin α. CNI-H0294 abrogated nuclear importation of the HIV-1 genome in macrophages and effectively inhibited infection of nondividing cells. In this study we demonstrate that CNI-H0294 inhibits binding of the HIV-1 PIC to human karyopherin α and reduces nuclear importation of the viral genome in primary peripheral blood mononuclear cells (PBMCs). We also demonstrate that CNI-H0294 inhibits acute infection of PBMC cultures in vitro with a primary isolate of HIV-1 and reduces virus replication and virus load in cultures of endogenously infected PBMCs from seropositive individuals. Thus, as for infection of nondividing, terminally differentiated macrophages, HIV-1 uses active nuclear importation of the virus genome to infect activated CD4+T cells. These results support nuclear importation as a novel target and CNI-H0294 and its derivatives as novel compounds for therapeutic intervention in HIV infection and AIDS.
Sequencing, Disruption, and Characterization of the
Candida albicans
Sterol Methyltransferase (
ERG6
) Gene: Drug Susceptibility Studies in
erg6
Mutants
Authors: Jensen-Pergakes K. L. , Kennedy M. A. , Lees N. D. , Barbuch R. , Koegel C. , Bard M.
Published: October 2018

The rise in the frequency of fungal infections and the increased resistance noted to the widely employed azole antifungals make the development of new antifungals imperative for human health. The sterol biosynthetic pathway has been exploited for the development of several antifungal agents (allylamines, morpholines, azoles), but additional potential sites for antifungal agent development are yet to be fully investigated. The sterol methyltransferase gene (
ERG6
) catalyzes a biosynthetic step not found in humans and has been shown to result in several compromised phenotypes, most notably markedly increased permeability, when disrupted in
Saccharomyces cerevisiae
. The
Candida albicans ERG6
gene was isolated by complementation of a
S. cerevisiae erg6
mutant by using a
C. albicans
genomic library. Sequencing of the
Candida ERG6
gene revealed high homology with the
Saccharomyces
version of
ERG6
. The first copy of the
Candida ERG6
gene was disrupted by transforming with the
URA3
blaster system, and the second copy was disrupted by both
URA3
blaster transformation and mitotic recombination. The resulting
erg6
strains were shown to be hypersusceptible to a number of sterol synthesis and metabolic inhibitors, including terbinafine, tridemorph, fenpropiomorph, fluphenazine, cycloheximide, cerulenin, and brefeldin A. No increase in susceptibility to azoles was noted. Inhibitors of the
ERG6
gene product would make the cell increasingly susceptible to antifungal agents as well as to new agents which normally would be excluded and would allow for clinical treatment at lower dosages. In addition, the availability of
ERG6
would allow for its use as a screen for new antifungals targeted specifically to the sterol methyltransferase.
Comparative Activities of Clinafloxacin against Gram-Positive and -Negative Bacteria
Authors: Ednie Lois M. , Jacobs Michael R. , Appelbaum Peter C.
Published: October 2018

Activities of clinafloxacin, ciprofloxacin, levofloxacin, sparfloxacin, trovafloxacin, piperacillin, piperacillin-tazobactam, trimethoprim-sulfamethoxazole, ceftazidime, and imipenem against 354 ciprofloxacin-susceptible and -intermediate-resistant organisms were tested by agar dilution. Clinafloxacin yielded the lowest quinolone MICs (≤0.5 μg/ml against ciprofloxacin-susceptible organisms and ≤16.0 μg/ml against ciprofloxacin-intermediate-resistant organisms) compared to those of levofloxacin, trovafloxacin, and sparfloxacin. Ceftazidime, piperacillin alone or combined with tazobactam, trimethoprim-sulfamethoxazole, and imipenem usually yielded higher MICs against ciprofloxacin-resistant strains.
Antibacterial Activities and Inhibitory Effects of Sitafloxacin (DU-6859a) and Its Optical Isomers against Type II Topoisomerases
Authors: Akasaka Takaaki , Kurosaka Seiko , Uchida Yoko , Tanaka Mayumi , Sato Kenichi , Hayakawa Isao
Published: October 2018

The in vitro inhibitory effects of sitafloxacin (DU-6859a) and its three stereoisomers on bacterial DNA gyrase from
Escherichia coli
, topoisomerase IV from
Staphylococcus aureus
, and topoisomerase II from human placenta were compared. No correlation was observed between the inhibitory activities of quinolones against bacterial type II topoisomerases and those against human topoisomerase II. Sitafloxacin showed the most potent inhibitory activities against bacterial type II topoisomerases and the lowest activity against human type II topoisomerase.
Recombinant Expression and Characterization of the Major β-Lactamase of
Mycobacterium tuberculosis
Authors: Voladri Rama Kishan R. , Lakey David L. , Hennigan Steven H. , Menzies Barbara E. , Edwards Kathryn M. , Kernodle Douglas S.
Published: October 2018

New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis.
Mycobacterium tuberculosis
has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of β-lactam antibiotics. β-Lactamase production appears to be the major mechanism by which
M. tuberculosis
expresses β-lactam resistance. β-Lactamases from the broth supernatant of 3- to 4-week-old cultures of
M. tuberculosis
H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of β-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing β-lactamase activity, respectively, were identified. The β-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the β-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library of
M. tuberculosis
H37Rv with homology to known class A β-lactamases was amplified from chromosomal DNA of
M. tuberculosis
H37Ra by PCR and was overexpressed in
Escherichia coli
. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from
M. tuberculosis
. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid and
m
-aminophenylboronic acid but not by EDTA. We conclude that the major β-lactamase of
M. tuberculosis
is a class A β-lactamase with predominant penicillinase activity. A second, minor β-lactamase with relatively greater cephalosporinase activity is also present.
Evaluation of Endpoints for Antifungal Susceptibility Determinations with LY303366
Authors: Klepser Michael E. , Ernst Erika J. , Ernst Michael E. , Messer Shawn A. , Pfaller Michael A.
Published: October 2018

We have previously reported poor correlation between the in vitro fungicidal activity of LY303366 and MIC results in RPMI medium based upon the manufacturer’s suggested susceptibility endpoint, lack of visual growth. Additionally, we have noted a significant trailing effect with LY303366 when MICs are determined in RPMI medium. These observations have led us to evaluate an alternative susceptibility endpoint for LY303366, an 80% reduction in growth compared with control (similar to that utilized for azoles). Two isolates each of
Candida albicans
,
Candida glabrata
, and
Candida tropicalis
were selected for testing. MICs were determined for LY303366 in RPMI 1640 medium buffered with morpholinepropanesulfonic acid. MICs were determined with suggested (MIC
100
) and experimental (MIC
80
) endpoints. The minimal fungicidal concentration (MFC) of LY303366 for each isolate was also determined. Time-kill curves were determined in RPMI medium with each isolate at concentrations of LY303366 ranging from 0.125 to 16× MIC
80
to assess the correlation between MIC
80
and fungicidal activity. Lastly, fungi exposed to LY303366 were examined via scanning electron microscope (SEM) for evidence of drug-induced ultrastructure change. MIC
80
s for test isolates ranged from 0.015 to 0.12 μg/ml and were consistently three to five wells less than MIC
100
s. Good correlation was observed between fungicidal activity, as assessed by kill curves, and the MIC
80
. SEM data revealed significant ultrastructure changes induced by LY303366 even at sub-MIC
80
s. Based on our results demonstrating better correlation between MIC
80
and fungicidal activity, i.e., time-kill curves and MFCs, we suggest that 80% reduction in visible growth be utilized as the endpoint for susceptibility determinations with LY303366 in RPMI medium.
Chondrotoxicity and Toxicokinetics of Sparfloxacin in Juvenile Rats
Authors: Stahlmann Ralf , Zippel Uta , Förster Christian , Schwabe Rudolf , Shakibaei Mehdi , Merker Hans-Joachim , Borner Klaus
Published: October 2018

Sparfloxacin is a fluoroquinolone with improved antibacterial activity against gram-positive pathogens. Like other quinolones, use of this drug is contraindicated in children and adolescents because of its potential chondrotoxicity in juveniles. We performed histological and immunohistochemical studies on the knee joint cartilage in 5-week-old rats after treatment with 600 or 1,800 mg of sparfloxacin/kg of body weight. Treatment with single or multiple oral doses of 600 mg of sparfloxacin/kg was not sufficient to induce joint cartilage lesions. However, five of eight rats treated with a single oral dose of 1,800 mg of sparfloxacin/kg of body weight showed typical cartilage lesions in the femoral part of the knee joint. The concentrations of the drug in plasma measured 0.25, 0.75, 1.5, 3, 6, 12, and 24 h after the administration of an oral dose of 600 mg of sparfloxacin/kg were 6.3 ± 1.8, 9.2 ± 1.7, 9.6 ± 2.7, 13.0 ± 1.8, 12.3 ± 1.6, 3.4 ± 0.4, and 0.30 ± 0.20 mg/liter, respectively (mean ± standard deviation [SD];
n
= 5 to 6 per group). The concentrations in plasma measured 0.75, 1.5, 3, 6, 24, and 48 h after the administration of an oral dose of 1,800 mg of sparfloxacin/kg were 10.9 ± 1.5, 15.9 ± 1.6, 19.1 ± 1.7, 14.9 ± 3.1, 4.1 ± 0.6, and 0.46 ± 0.37 mg/liter, respectively (mean ± SD;
n
= 3 to 4 per group). The concentrations of sparfloxacin in joint cartilage were significantly higher at all time points studied (114.8 ± 80, 99.4 ± 31.5, 84.9 ± 16.8, 44.4 ± 13.9, and 14.2 ± 4.8 mg of sparfloxacin/kg at 1.5, 3, 6, 24, and 48 h after the administration of 1,800 mg/kg, respectively). The range of concentrations in bone were similar to the range of concentrations in cartilage (peak, 115 ± 12 mg/kg after 3 h). Our data indicate that chondrotoxic doses of sparfloxacin in juvenile rats are approximately 300 times higher than the doses of sparfloxacin used therapeutically (1,800 versus approximately 6 mg/kg of body weight), but due to species differences in kinetics, concentrations in plasma differ by a factor of only approximately 15. More data on quinolone concentrations in cartilage from animals and humans could provide a better basis for a reasonable risk assessment.
Roxithromycin Inhibits Cytokine Production by and Neutrophil Attachment to Human Bronchial Epithelial Cells In Vitro
Authors: Kawasaki Shin , Takizawa Hajime , Ohtoshi Takayuki , Takeuchi Naonobu , Kohyama Tadashi , Nakamura Hidenori , Kasama Tsuyoshi , Kobayashi Kazuo , Nakahara Kazuhiko , Morita Yutaka , Yamamoto Kazuhiko
Published: October 2018

We evaluated the effect of roxithromycin on cytokine production and neutrophil attachment to human airway epithelial cells. Roxithromycin suppressed production of interleukin 8 (IL-8), IL-6, and granulocyte-macrophage colony-stimulating factor. It inhibited neutrophil adhesion to epithelial cells. Roxithromycin modulates local recruitment and activation of inflammatory cells, which may have relevance to its efficacy in airway diseases.
Glucuronidation of 3′-Azido-3′-Deoxythymidine (Zidovudine) by Human Liver Microsomes: Relevance to Clinical Pharmacokinetic Interactions with Atovaquone, Fluconazole, Methadone, and Valproic Acid
Authors: Trapnell Carol Braun , Klecker Raymond W. , Jamis-Dow Carlos , Collins Jerry M.
Published: October 2018

Zidovudine (3′-azido-3′-deoxythymidine [AZT]), an antiviral nucleoside analog effective in the treatment of human immunodeficiency virus infection, is primarily metabolized to an inactive glucuronide form, GAZT, via uridine-5′-diphospho-glucuronosyltransferase (UGT) enzymes. UGT enzymes exist as different isoforms, each exhibiting substrate specificity. Published clinical studies have shown that atovaquone, fluconazole, methadone, and valproic acid decreased GAZT formation, presumably due to UGT inhibition. The effect of these drugs on AZT glucuronidation was assessed in vitro by using human hepatic microsomes to begin understanding in vitro-in vivo correlations for UGT metabolism. The concentrations of each drug studied were equal to those reported with the usual clinical doses and at concentrations at least 10 times higher than would be expected with these doses. High-performance liquid chromatography was used to assess the respective metabolism and formation of AZT and GAZT. All four drugs exhibited concentration-dependent inhibition of AZT glucuronidation. The respective concentrations of atovaquone and methadone which caused 50% inhibition of GAZT were >100 and 8 μg/ml, well above their usual clinical concentrations. Fluconazole and valproic acid exhibited 50% inhibition of GAZT at 50 and 100 μg/ml, which are within the clinical ranges of 10 to 100 and 50 to 100 μg/ml, respectively. These data suggest that inhibition of AZT glucuronidation may be more clinically significant with concomitant fluconazole and valproic acid. Factors such as inter- and intraindividual pharmacokinetic variability and changes in AZT intracellular concentrations should be considered as other mechanisms responsible for changes in AZT pharmacokinetics with concomitant therapies.
Effects of DU-6859a, a New Quinolone Antimicrobial, on Theophylline Metabolism in In Vitro and In Vivo Studies
Authors: Niki Yoshihito , Itokawa Kenichi , Okazaki Osamu
Published: October 2018

In vitro and in vivo studies were conducted to investigate the drug interaction between a new quinolone antimicrobial, DU-6859a, and theophylline (TP). The effect of DU-6859a on TP metabolism was evaluated in vitro by measuring the rate of TP metabolite formation by using human liver microsomes. DU-6859a inhibited the metabolism of TP, especially the formation of 1-methylxanthine, in vitro, but to a lesser extent than other drugs that are known to interact with TP. TP was administered alone (200 mg twice a day [b.i.d.] for 9 days) or in combination with DU-6859a (50 or 100 mg b.i.d. for 5 days) to six healthy subjects. DU-6859a administered at a dose of 50 mg resulted in no changes in serum TP concentrations, and slight increases in serum TP concentrations were observed at a dose of 100 mg. Moreover, the administration of 100 mg of DU-6859a resulted in decreases in all urinary TP metabolites, with significant differences. It appears that although DU-6859a has a weak inhibitory effect on TP metabolism in vitro, its concomitant use with TP at clinical dosage levels does not cause any adverse effects, showing only a slight increase in blood TP concentrations and a decrease in urinary metabolites.
Characterization of a Staphylococcal Plasmid Related to pUB110 and Carrying Two Novel Genes,
vatC
and
vgbB
, Encoding Resistance to Streptogramins A and B and Similar Antibiotics
Authors: Allignet Jeanine , Liassine Nadia , El Solh Névine
Published: October 2018

We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a
Staphylococcus cohnii
subsp.
cohnii
strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i)
vatC
, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii)
vgbB
, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to
repB
, which encodes a replication protein, and to
pre
(
mob
), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (
dso
,

sso
U

) and the recombination site, RS
A
. The antibiotic resistance genes
repB
and
pre
(
mob
) carried by each of these plasmids were found in the same transcriptional orientation.
In Vivo Antibacterial Activities of Sanfetrinem Cilexetil, a New Oral Tricyclic Antibiotic
Authors: Tamura Shinobu , Miyazaki Shuichi , Tateda Kazuhiro , Ohno Akira , Ishii Yoshikazu , Matsumoto Tetsuya , Furuya Nobuhiko , Yamaguchi Keizo
Published: October 2018

The in vivo antibacterial activities of a new oral trinem, sanfetrinem cilexetil (a prodrug of sanfetrinem), were evaluated in comparison with those of cefdinir and amoxicillin. Sanfetrinem cilexetil showed potent efficacy against experimental murine septicemia caused by
Staphylococcus aureus
,
Streptococcus pyogenes
, and
Escherichia coli
and against murine respiratory infections caused by
Streptococcus pneumoniae
. Likewise, in murine models of respiratory infection by penicillin-susceptible and penicillin-resistant
S. pneumoniae
, sanfetrinem cilexetil was more effective than amoxicillin in reducing the number of bacteria in infected lungs. These results were reflected in its potent in vitro activity and high levels in plasma.
Immunomodulating Properties of the Antibiotic Novobiocin in Human Monocytes
Authors: Lührmann Anja , Thölke Jürgen , Behn Ingrid , Schumann Jens , Tiegs Gisa , Hauschildt Sunna
Published: October 2018

We show that the coumeromycin antibiotic novobiocin, a potent inhibitor of ADP ribosylation, prevents lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, and IL-10 secretion in human peripheral blood mononuclear cells. It shares these cytokine-suppressing properties with other inhibitors of ADP ribosylation. We found that novobiocin prevents TNF-α production by inhibiting translation of the TNF-α mRNA. Elevated TNF-α levels in mice treated withd-galactosamine (GalN)-LPS or GalN-TNF were not reduced by novobiocin; however, the drug exhibited hepatoprotective properties. Novobiocin causes downregulation of the surface molecules on monocytes, among which CD14 was the most affected. The diminished expression of surface molecules was not observed on T and B lymphocytes. Similar to other inhibitors of ADP ribosylation, novobiocin prevents LPS-induced phosphate labelling of γ-actins.
Molecular Cloning and Functional Analysis of a Novel Tetracycline Resistance Determinant,
tet
(V), from
Mycobacterium smegmatis
Authors: De Rossi Edda , Blokpoel Marian C. J. , Cantoni Rita , Branzoni Manuela , Riccardi Giovanna , Young Douglas B. , De Smet Koen A. L. , Ciferri Orio
Published: October 2018

The nucleotide sequence and mechanism of action of a tetracycline resistance gene from
Mycobacterium smegmatis
were determined. Analysis of a 2.2-kb sequence fragment showed the presence of one open reading frame, designated
tet
(V), encoding a 419-amino-acid protein (molecular weight, 44,610) with at least 10 transmembrane domains. A database search showed that the gene is homologous to membrane-associated antibiotic efflux pump proteins but not to any known tetracycline efflux pumps. The steady-state accumulation level of tetracycline by
M. smegmatis
harboring a plasmid carrying the
tet
(V) gene was about fourfold lower than that of the parental strain. Furthermore, the energy uncoupler carbonyl cyanide
m
-chlorophenylhydrazone blocked tetracycline efflux in deenergized cells. These results suggest that the
tet
(V) gene codes for a drug antiporter which uses the proton motive force for the active efflux of tetracycline. By primer-specific amplification the gene appears to be restricted to
M. smegmatis
and
M. fortuitum
.
Paclitaxel Arrests Growth of Intracellular
Toxoplasma gondii
Authors: Estes Randee , Vogel Nicolas , Mack Douglas , McLeod Rima
Published: October 2018

Addition of paclitaxel (Taxol) at a concentration of 1 μM to
Toxoplasma gondii
-infected human foreskin fibroblasts arrested parasite multiplication. Division of the
T. gondii
tachyzoite nucleus was inhibited, leading to syncytium-like parasite structures within the fibroblasts by 24 h after infection and treatment of the cultures. By 4 days after infection and treatment of the cultures with paclitaxel, this inhibition was irreversible, since the arrested intracellular form was incapable of leaving the host cell, infecting new cells, and initiating the growth of tachyzoites with normal morphology. Specifically, when paclitaxel was added to infected cells for 4 days and then removed by washing and the infected, paclitaxel-treated cells were cultured for 4 more days, there were no remaining
T. gondii
organisms with normal morphology. Syncytium-like structures in the cultures that were infected and treated with paclitaxel for 8 days were similar in appearance to those in preparations of infected paclitaxel-treated fibroblasts that had been cultured for 24 to 48 h. Pretreatment of the tachyzoites for 1 h with paclitaxel followed by the removal of the paclitaxel by repeatedly centrifuging and resuspending the parasites in fresh medium without paclitaxel and then adding fresh medium prior to culture of the parasites with fibroblasts did not prevent their invasion of fibroblasts but did affect their subsequent ability to replicate within fibroblasts. Pretreatment of the fibroblasts with paclitaxel also diminished subsequent replication of
T. gondii
in such host cells after 8 days. Thus, paclitaxel alters the ability of
T. gondii
to replicate in host cells. Inhibition of parasite microtubules by such compounds at concentrations which do not interfere with the function of host cell microtubules may be useful for development of novel medicines to treat
T. gondii
infections in the future.
Mode of Action of (1′
S
,2′
R
)-9-{[1′,2′-Bis(hydroxymethyl) cycloprop-1′-yl]methyl}guanine (A-5021) against Herpes Simplex Virus Type 1 and Type 2 and Varicella-Zoster Virus
Authors: Ono Nobukazu , Iwayama Satoshi , Suzuki Katsuya , Sekiyama Takaaki , Nakazawa Harumi , Tsuji Takashi , Okunishi Masahiko , Daikoku Tohru , Nishiyama Yukihiro
Published: October 2018

The mode of action of (1′
S
,2′
R
)-9-{[1′,2′-bis(hydroxymethyl)cycloprop-1′-yl]methyl}guanine (A-5021) against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) was studied. A-5021 was monophosphorylated at the 2′ site by viral thymidine kinases (TKs). The 50% inhibitory values for thymidine phosphorylation of A-5021 by HSV-1 TK and HSV-2 TK were comparable to those for penciclovir (PCV) and lower than those for acyclovir (ACV). Of these three agents, A-5021 inhibited VZV TK most efficiently. A-5021 was phosphorylated to a mono-, di-, and triphosphate in MRC-5 cells infected with HSV-1, HSV-2, and VZV. A-5021 triphosphate accumulated more than ACV triphosphate but less than PCV triphosphate in MRC-5 cells infected with HSV-1 or VZV, whereas HSV-2-infected MRC-5 cells had comparable levels of A-5021 and ACV triphosphates. The intracellular half-life of A-5021 triphosphate was considerably longer than that of ACV triphosphate and shorter than that of PCV triphosphate. A-5021 triphosphate competitively inhibited HSV DNA polymerases with respect to dGTP. Inhibition was strongest with ACV triphosphate, followed by A-5021 triphosphate and then (
R
,
S
)-PCV triphosphate. A DNA chain elongation experiment revealed that A-5021 triphosphate was incorporated into DNA instead of dGTP and terminated elongation, although limited chain extension was observed. Thus, the strong antiviral activity of A-5021 appears to depend on a more rapid and stable accumulation of its triphosphate in infected cells than that of ACV and on stronger inhibition of viral DNA polymerase by its triphosphate than that of PCV.
Antimicrobial Resistance of Clinical Strains of
Campylobacter jejuni
subsp.
jejuni
Isolated from 1985 to 1997 in Quebec, Canada
Authors: Gaudreau Christiane , Gilbert Huguette
Published: October 2018

The antimicrobial resistance of 158
Campylobacter jejuni
strains isolated from humans in Quebec, Canada, from 1995 to 1997 was compared to the resistance of 47 and 86 strains of
C. jejuni
isolated in 1985 and 1986 and in 1992 and 1993, respectively. Of the 291
C. jejuni
strains tested, no strain was resistant to erythromycin. Compared to the
C. jejuni
strains isolated in 1985 and 1986, the
C. jejuni
strains isolated in 1992 and 1993 were more resistant to tetracycline (40.7 versus 19.1%, respectively;
P
= 0.01) but not to nalidixic acid or ciprofloxacin (
P
> 0.05). Compared to the
C. jejuni
strains isolated in 1992 and 1993 and in 1985 and 1986, the
C. jejuni
strains isolated from 1995 to 1997 were more resistant to tetracycline (55.7% versus 40.7 and 19.1%, respectively;
P
= 0.03 and
P
< 0.001, respectively) to nalidixic acid (13.9% versus 4.7 and 0%, respectively;
P
= 0.02 and
P
= 0.007, respectively), and to ciprofloxacin (12.7% versus 3.5 and 0%, respectively;
P
= 0.02 and
P
= 0.009, respectively).
Long-Term Therapy with the Guanine Nucleoside Analog Penciclovir Controls Chronic Duck Hepatitis B Virus Infection In Vivo
Authors: Lin Enjarn , Luscombe Carolyn , Colledge Danni , Wang Yan Yan , Locarnini Stephen
Published: October 2018

Ducks congenitally infected with duck hepatitis B virus (DHBV) were treated with the antiviral guanine nucleoside analog penciclovir for 12 or 24 weeks at a dosage of 10 mg/kg of body weight per day. By the completion of both 12 and 24 weeks of therapy, molecular hybridization studies of the liver tissue revealed that the viral DNA, RNA, and protein levels were significantly reduced compared to those in the placebo-treated controls. Penciclovir treatment for 12 or 24 weeks was not associated with any toxicity, establishing the efficacy and safety of long-term penciclovir therapy in chronic DHBV infection.
Association of a Thr-371 Substitution in a Conserved Amino Acid Motif of Penicillin-Binding Protein 1A with Penicillin Resistance of
Streptococcus pneumoniae
Authors: Asahi Yasuko , Ubukata Kimiko
Published: October 2018

We determined the nucleotide sequence between 1,903 and 3,097 bp of
pbp1a
, which encodes the transpeptidase domain of PBP 1A, from clinical isolates of penicillin-resistant
Streptococcus pneumoniae
(PRSP) serotypes 19 (
n
= 8), 6 (
n
= 9), 23 (
n
= 6), and 14 (
n
= 2) and two penicillin-susceptible
S. pneumoniae
(PSSP) isolates. These serotyped PRSP strains were isolated predominantly in Japan from 1993 through 1997. The 25 resistant strains were classified into five groups on the basis of the extent of sequence differences. Strains in groups I (
n
= 5; serotype 6), II (
n
= 3; serotype 19), and III (
n
= 12; different serotypes) had sequences highly homologous to the sequence of
pbp1a
of PRSP strains from South Africa, Spain, and the United States. The group IV strain (
n
= 1; serotype 14) had unique deletions from or insertions in the sequences. The sequences of group V strains (
n
= 4; serotypes 6 and 23) had relatively few differences from the sequences of the PSSP strains. For strains (
n
= 18) for which the threonine at codon 371 (Thr-371) in a conserved STMK motif of PBP 1A was substituted with an alanine or a serine (in addition to having altered
pbp2x
and
pbp2b
genes), penicillin MICs were ≧1.0 μg/ml. The PBPs 1A of these strains showed decreased affinities for [
3
H]benzylpenicillin and slightly faster mobilities on sodium dodecyl sulfate-polyacrylamide gels. In contrast, for strains (
n
= 4) without a substitution at Thr-371 in PBP 1A but with mutations in both
pbp2x
and
pbp2b
, penicillin MICs were 0.125 to 0.25 μg/ml, and the affinities of their PBPs 1A were similar to that of PSSP PBPs 1A. Furthermore, for the Thr-371-substituted strains (
n
= 3) with altered
pbp2x
genes but native
pbp2b
genes, penicillin MICs were 0.125 to 0.25 μg/ml. These results suggest that amino acid substitution of Thr-371 contributes to the development of penicillin resistance in PRSP strains with altered
pbp2x
and
pbp2b
genes.
Sordarins: A New Class of Antifungals with Selective Inhibition of the Protein Synthesis Elongation Cycle in Yeasts
Authors: Domínguez Juan Manuel , Kelly Valerie A. , Kinsman Oonagh S. , Marriott Michael S. , Gómez de las Heras Federico , Martín J. Julio
Published: October 2018

GR135402, a sordarin derivative, was isolated in an antifungal screening program. GR135402, sordarin, and derivatives of both compounds were evaluated for their ability to inhibit cell-free translational systems from five different pathogenic fungi (
Candida albicans
,
Candida glabrata
,
Candida krusei
,
Candida parapsilosis
, and
Cryptococcus neoformans
). The activity profile of GR135402 is extended to other chemical compounds derived from sordarin. Experimental results indicate that sordarin analogs exert their antifungal effects by specifically inhibiting the protein synthesis elongation cycle in yeasts but do not affect protein synthesis machinery in mammalian systems. Intrinsically resistant strains owe their resistance to differences in the molecular target of sordarins in these strains. Preliminary studies performed to elucidate the mode of action of this new class of antifungal agents have shown that the putative target of sordarins is one of the protein synthesis elongation factors.
Rapid Ganciclovir Susceptibility Assay Using Flow Cytometry for Human Cytomegalovirus Clinical Isolates
Authors: McSharry James J. , Lurain Nell S. , Drusano George L. , Landay Alan L. , Notka Mostafa , O’Gorman Maurice R. G. , Weinberg Adriana , Shapiro Howard M. , Reichelderfer Patricia S. , Crumpacker Clyde S.
Published: October 2018

Rapid, quantitative, and objective determination of the susceptibilities of human cytomegalovirus (HCMV) clinical isolates to ganciclovir has been assessed by an assay that uses a fluorochrome-labeled monoclonal antibody to an HCMV immediate-early antigen and flow cytometry. Analysis of the ganciclovir susceptibilities of 25 phenotypically characterized clinical isolates by flow cytometry demonstrated that the 50% inhibitory concentrations (IC
50
s) of ganciclovir for 19 of the isolates were between 1.14 and 6.66 μM, with a mean of 4.32 μM (±1.93) (sensitive; IC
50
less than 7 μM), the IC
50
s for 2 isolates were 8.48 and 9.79 μM (partially resistant), and the IC
50
s for 4 isolates were greater than 96 μM (resistant). Comparative analysis of the drug susceptibilities of these clinical isolates by the plaque reduction assay gave IC
50
s of less than 6 μM, with a mean of 2.88 μM (±1.40) for the 19 drug-sensitive isolates, IC
50
s of 6 to 8 μM for the partially resistant isolates, and IC
50
s of greater than 12 μM for the four resistant clinical isolates. Comparison of the IC
50
s for the drug-susceptible and partially resistant clinical isolates obtained by the flow cytometry assay with the IC
50
s obtained by the plaque reduction assay showed an acceptable correlation (
r
2
= 0.473;
P
= 0.001), suggesting that the flow cytometry assay could substitute for the more labor-intensive, subjective, and time-consuming plaque reduction assay.
Effect of Disposition of Mannich Antimalarial Agents on Their Pharmacology and Toxicology
Authors: Ruscoe J. E. , Tingle M. D. , O’Neill P. M. , Ward S. A. , Park B. K.
Published: October 2018

The use of the antimalarial agent amodiaquine has been curtailed due to drug-induced idiosyncratic reactions. These have been attributed to the formation of a protein-reactive quinoneimine species via oxidation of the 4-aminophenol group. Therefore, the effects of chemical modifications on the disposition of amodiaquine in relation to its metabolism, distribution, and pharmacological activity have been investigated. The inclusion of a group at the C-5′ position of amodiaquine reduced or eliminated bioactivation, as determined by glutathione conjugate formation in vivo. This can be seen in two series of C-5′-substituted compounds: the bis-Mannich antimalarial agents, including cycloquine and pyronaridine, and mono-Mannich antimalarial agents containing a 5′-chlorophenyl group (tebuquine and 5′-ClPAQ). Chemical substitution at the C-5′ position also resulted in compounds which underwent slower elimination (<5% of the dose excreted into bile and urine, compared with 50% for amodiaquine) and increased levels of accumulation in tissue (10% of the dose in the liver at 48 h compared with 1% with amodiaquine). This may be due to an increase in either the lipophilicity or the basicity of the analogs and may reflect the lack of metabolic clearance for these compounds. The alteration in the disposition following the introduction of the C-5′ substituent resulted in an increased duration of antimalarial activity in the mouse compared with that for amodiaquine. While this is desirable in the treatment of malaria, repeated administration for prophylaxis may induce toxicity through accumulation. Therefore, by simple chemical modification it is possible to block the bioactivation of amodiaquine while maintaining and in some cases extending the duration of antimalarial activity.
Activity of Liposomal Amphotericin B with Prolonged Circulation in Blood versus Those of AmBisome and Fungizone against Intracellular
Candida albicans
in Murine Peritoneal Macrophages
Authors: van Etten Els W. M. , van Vianen Wim , Hak Janneke , Bakker-Woudenberg Irma A. J. M.
Published: October 2018

Activity against intracellular
Candida albicans
was assessed in
C. albicans
-infected murine peritoneal macrophages exposed to long-circulating pegylated amphotericin B liposomes (PEG-AMB-LIP), AmBisome, or Fungizone. The level of antifungal activity of Fungizone is much higher than that of AmBisome or PEG-AMB-LIP, while PEG-AMB-LIP and AmBisome show equivalent activity levels. Previous exposure of uninfected macrophages to PEG-AMB-LIP or AmBisome is advantageous for intracellular antifungal activity.
Mixture-Based Heterocyclic Combinatorial Positional Scanning Libraries: Discovery of Bicyclic Guanidines Having Potent Antifungal Activities against
Candida albicans
and
Cryptococcus neoformans
Authors: Blondelle Sylvie E. , Crooks Ema , Ostresh John M. , Houghten Richard A.
Published: October 2018

A mixture-based synthetic combinatorial library of more than 100,000 bicyclic guanidines was generated in a positional scanning format and assayed for activity against
Candida albicans
. Potent individual bicyclic guanidines were directly identified following the screening of the library. Time-kill curve studies indicated bactericidal activities for the individual bicyclic guanidines. These compounds also showed potent activity against
Cryptococcus neoformans
. These studies demonstrate the value of using mixture-based combinatorial positional scanning libraries made up of heterocyclic compounds for the rapid identification of novel classes of antifungal compounds.
Detection of a Streptomycin/Spectinomycin Adenylyltransferase Gene (
aadA
) in
Enterococcus faecalis
Authors: Clark Nancye C. , Olsvik Ørjan , Swenson Jana M. , Spiegel Carol A. , Tenover Fred C.
Published: October 2018

Genes encoding streptomycin/spectinomycin adenylyltransferases [ANT(3")(9)] have been reported to exist in gram-negative organisms and
Staphylococcus aureus
. During a study of high-level aminoglycoside resistance in enterococci, we encountered an isolate of
Enterococcus faecalis
that was streptomycin resistant but did not appear to contain the 6′-adenylyltransferase gene (
aadE
) when examined by PCR with specific primers. Phosphocellulose paper binding assays indicated the presence of an ANT(3")(9) enzyme. Streptomycin and spectinomycin MICs of 4,000 and 8,000 μg/ml, respectively, were observed for the isolate. PCR primers corresponding to a highly conserved region of the
aadA
gene were used to amplify a specific 284-bp product. The product hybridized with a digoxigenin-labeled PCR product from
E. coli
C600(pHP45Ω) known to contain the
aadA
gene. The
aadA
gene was transferred via filter matings from the
E. faecalis
donor to
E. faecalis
JH2-2. PCR primers designed for analysis of integrons were used to amplify a 1-kb product containing the
aadA
gene, which was cloned into the vector pCRII and transformed into
Escherichia coli
DH5-α competent cells.
d
-Rhodamine dye terminator cycle sequencing was used to determine the gene sequence, which was compared to previously reported sequences of
aadA
genes. We found the
aadA
gene in
E. faecalis
to be identical to the
aadA
genes reported by Sundström et al. for
E. coli
plasmid R6-5 (L. Sundström, P. Rådström, G. Swedberg, and O. Sköld, Mol. Gen. Genet. 213:191–201, 1988), by Fling et al. for the
aadA
within transposon Tn
7
(M. E. Fling, J. Kopf, and C. Richards, Nucleic Acids Res. 13:7095–7106, 1985), and by Hollingshead and Vapnek for
E. coli
R538-1 (S. Hollingshead and D. Vapnek, Plasmid 13:17–30, 1985). Previous reports of the presence of the
aadA
gene in enterococci appear to be erroneous and probably describe an
aadE
gene, since the isolates were reported to be susceptible to spectinomycin.
ADP-Ribosylation as an Intermediate Step in Inactivation of Rifampin by a Mycobacterial Gene
Authors: Quan Selwyn , Imai Tamae , Mikami Yuzuru , Yazawa Katsukiyo , Dabbs Eric R. , Morisaki Naoko , Iwasaki Shigeo , Hashimoto Yuichi , Furihata Kazuo
Published: October 2018

Mycobacterium smegmatis
DSM43756 inactivates rifampin, and the inactivated antibiotic product recovered from culture medium was ribosylated on the 23-OH group. To study this process, the gene responsible for the inactivation was expressed at high levels by the
lac
promoter in
Escherichia coli
conferring resistance to >500 μg of antibiotic per ml. Cell homogenates generated a novel derivative designated RIP-TAs; in this study, we determined that RIP-TAs is 23-(
O
-ADP-ribosyl)rifampin. Our results indicated that RIP-TAs is an intermediate in the pathway leading to ribosylated rifampin and that the previously characterized gene encodes a mono(ADP-ribosyl)transferase which, however, shows no sequence similarity to other enzymes of this class.
Mechanism of Action and In Vitro Activity of 1′,3′-Dioxolanylpurine Nucleoside Analogues against Sensitive and Drug-Resistant Human Immunodeficiency Virus Type 1 Variants
Authors: Gu Zhengxian , Wainberg Mark A. , Nguyen-Ba Nghe , L’Heureux Lucille , de Muys Jean-Marc , Bowlin Terry L. , Rando Robert F.
Published: October 2018

(−)-β-
d
-1′,3′-Dioxolane guanosine (DXG) and 2,6-diaminopurine (DAPD) dioxolanyl nucleoside analogues have been reported to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1). We have recently conducted experiments to more fully characterize their in vitro anti-HIV-1 profiles. Antiviral assays performed in cell culture systems determined that DXG had 50% effective concentrations of 0.046 and 0.085 μM when evaluated against HIV-1
IIIB
in cord blood mononuclear cells and MT-2 cells, respectively. These values indicate that DXG is approximately equipotent to 2′,3′-dideoxy-3′-thiacytidine (3TC) but 5- to 10-fold less potent than 3′-azido-2′,3′-dideoxythymidine (AZT) in the two cell systems tested. At the same time, DAPD was approximately 5- to 20-fold less active than DXG in the anti-HIV-1 assays. When recombinant or clinical variants of HIV-1 were used to assess the efficacy of the purine nucleoside analogues against drug-resistant HIV-1, it was observed that AZT-resistant virus remained sensitive to DXG and DAPD. Virus harboring a mutation(s) which conferred decreased sensitivity to 3TC, 2′,3′-dideoxyinosine, and 2′,3′-dideoxycytidine, such as a 65R, 74V, or 184V mutation in the viral reverse transcriptase (RT), exhibited a two- to fivefold-decreased susceptibility to DXG or DAPD. When nonnucleoside RT inhibitor-resistant and protease inhibitor-resistant viruses were tested, no change in virus sensitivity to DXG or DAPD was observed. In vitro drug combination assays indicated that DXG had synergistic antiviral effects when used in combination with AZT, 3TC, or nevirapine. In cellular toxicity analyses, DXG and DAPD had 50% cytotoxic concentrations of greater than 500 μM when tested in peripheral blood mononuclear cells and a variety of human tumor and normal cell lines. The triphosphate form of DXG competed with the natural nucleotide substrates and acted as a chain terminator of the nascent DNA. These data suggest that DXG triphosphate may be the active intracellular metabolite, consistent with the mechanism by which other nucleoside analogues inhibit HIV-1 replication. Our results suggest that the use of DXG and DAPD as therapeutic agents for HIV-1 infection should be explored.
Plasmid-Mediated Resistance to Thrombin-Induced Platelet Microbicidal Protein in Staphylococci: Role of the
qacA
Locus
Authors: Kupferwasser Leon Iri , Skurray Ronald A. , Brown Melissa H. , Firth Neville , Yeaman Michael R. , Bayer Arnold S.
Published: October 2018

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of
Staphylococcus aureus
correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of
S. aureus
to tPMP-1 are not fully delineated. The plasmid-encoded staphylococcal gene
qacA
mediates multidrug resistance to multiple organic cations via a proton motive force-dependent efflux pump. We studied whether the
qacA
gene might also confer resistance to cationic tPMP-1. Staphylococcal plasmids encoding
qacA
were found to confer resistance to tPMP-1 in an otherwise susceptible parental strain. Deletions which removed the region containing the
qacA
gene in the
S. aureus
multiresistance plasmid pSK1 abolished tPMP-1 resistance. Resistance to tPMP-1 in the
qacA
-bearing strains was inoculum independent but peptide concentration dependent, with the level of resistance decreasing at higher peptide concentrations for a given inoculum. There was no apparent cross-resistance in
qacA
-bearing strains to other endogenous cationic antimicrobial peptides which are structurally distinct from tPMP-1, including human neutrophil defensin 1, protamine, or the staphylococcal lantibiotics pep5 and nisin. These data demonstrate that the staphylococcal multidrug resistance gene
qacA
also mediates in vitro resistance to cationic tPMP-1.