bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake and Intracellular Activity of Moxifloxacin in Human Neutrophils and Tissue-Cultured Epithelial Cells
Authors: Pascual Alvaro , García Isabel , Ballesta Sofía , Perea Evelio J.
Published: October 2018

The penetration by moxifloxacin of human neutrophils (polymorphonuclear leukocytes [PMN]) and tissue-cultured epithelial cells (McCoy cells) was evaluated by a fluorometric assay. At extracellular concentrations of 5 mg/liter, the cellular-to-extracellular concentration ratios (C/E) of moxifloxacin in PMN and McCoy cells were 10.9 ± 1.0 and 8.7 ± 1.0, respectively (20 min; 37°C). The uptake of moxifloxacin by PMN was rapid, reversible, nonsaturable (at extracellular concentrations ranging from 1 to 50 μg/ml), and not affected by cell viability. The uptake of moxifloxacin was affected by external pH and the environmental temperature. The incubation of PMN in the presence of sodium fluoride, sodium cyanide, and carbonyl cyanide
m
-chlorophenylhydrazone significantly decreased the C/E of this agent. Neither PMN stimulation nor phagocytosis of opsonized
Staphylococcus aureus
significantly affected the uptake of moxifloxacin by human PMN. This agent, at concentrations of 0.5, 1, and 5 mg/liter, induced a significant reduction in the survival of intracellular
S. aureus
in human PMN. In summary, moxifloxacin reaches much higher intracellular concentrations within phagocytic and nonphagocytic cells than extracellular ones, remaining active inside the neutrophils.
Efficacy of New 5-Nitroimidazoles against Metronidazole-Susceptible and -Resistant
Giardia
,
Trichomonas
, and
Entamoeba
spp
Authors: Upcroft Jacqueline A. , Campbell Raymond W. , Benakli Kamel , Upcroft Peter , Vanelle Patrice
Published: October 2018

The efficacies of 12 5-nitroimidazole compounds and 1 previously described lactam-substituted nitroimidazole with antiparasitic activity, synthesized via S
RN
1 and subsequent reactions, were assayed against the protozoan parasites
Giardia duodenalis
,
Trichomonas vaginalis
, and
Entamoeba histolytica
. Two metronidazole-sensitive lines and two metronidazole-resistant lines of
Giardia
and one line each of metronidazole-sensitive and -resistant
Trichomonas
were tested. All except one of the compounds were as effective or more effective than metronidazole against
Giardia
and
Trichomonas
, but none was as effective overall as the previously described 2-lactam-substituted 5-nitroimidazole. None of the compounds was markedly more effective than metronidazole against
Entamoeba
. Significant cross-resistance between most of the drugs tested and metronidazole was evident among metronidazole-resistant lines of
Giardia
and
Trichomonas
. However, some drugs were lethal to metronidazole-resistant
Giardia
and had minimum lethal concentrations similar to that of metronidazole for drug-susceptible parasites. This study emphasizes the potential in developing new nitroimidazole drugs which are more effective than metronidazole and which may prove to be useful clinical alternatives to metronidazole.
Efficacy of Trovafloxacin in Treatment of Experimental Staphylococcal or Streptococcal Endocarditis
Authors: Entenza J. M. , Vouillamoz J. , Glauser M. P. , Moreillon P.
Published: October 2018

The efficacy of trovafloxacin against
Staphylococcus aureus
and viridans group streptococci was investigated in vitro and in an experimental model of endocarditis. The MICs at which trovafloxacin and ciprofloxacin inhibited 90% of clinical isolates of such bacteria (MIC
90
s) were (i) 0.03 and 2 mg/liter, respectively, for 30 ciprofloxacin-susceptible
S. aureus
isolates, (ii) 32 and 128 mg/liter, respectively, for 20 ciprofloxacin-resistant
S. aureus
isolates, and (iii) 0.25 and 8 mg/liter, respectively, for 28 viridans group streptococci. Rats with aortic vegetations were infected with either of two ciprofloxacin-susceptible but methicillin-resistant
S. aureus
strains (strains COL and P8), one penicillin-susceptible
Streptococcus sanguis
strain, or one penicillin-resistant
Streptococcus mitis
strain. Rats were treated for 3 or 5 days with doses that resulted in kinetics that simulated those achieved in humans with trovafloxacin (200 mg orally once a day), ciprofloxacin (750 mg orally twice a day), vancomycin (1 g intravenously twice a day), or ceftriaxone (2 g intravenously once a day). Against the staphylococci, the activities of both trovafloxacin and ciprofloxacin were equivalent to that of vancomycin, and treatment of endocarditis with these drugs was successful (
P
< 0.05). However, ciprofloxacin selected for resistant derivatives in vitro and in vivo, whereas trovafloxacin was 10 to 100 times less prone than ciprofloxacin to select for resistance in vitro and did not select for resistance in vivo. Against the two streptococcal isolates, trovafloxacin significantly (
P
< 0.05) decreased bacterial counts in the vegetations but was less effective than the control drug, ceftriaxone. Thus, a simulated oral dose of trovafloxacin (200 mg per day) was effective against ciprofloxacin-susceptible staphylococci and was less likely than ciprofloxacin to select for resistance. The simulated oral dose of trovafloxacin also had some activity against streptococcal endocarditis, but optimal treatment of infections caused by such organisms might require higher doses of the drug.
In Vitro Activity of the Ketolide HMR 3647 (RU 6647) for
Legionella
spp., Its Pharmacokinetics in Guinea Pigs, and Use of the Drug To Treat Guinea Pigs with
Legionella pneumophila
Pneumonia
Authors: Edelstein Paul H. , Edelstein Martha A. C.
Published: October 2018

The activities of HMR 3647, HMR 3004, erythromycin, clarithromycin, and levofloxacin for 97
Legionella
spp. isolates were determined by microbroth dilution susceptibility testing. Growth inhibition of two
Legionella pneumophila
strains grown in guinea pig alveolar macrophages was also determined. The concentrations required to inhibit 50% of strains tested were 0.06, 0.02, 0.25, 0.03, and 0.02 μg/ml for HMR 3647, HMR 3004, erythromycin, clarithromycin, and levofloxacin, respectively. BYEα broth did not significantly inhibit the activities of the drugs tested, as judged by the susceptibility of the control
Staphylococcus aureus
strain; however, when
Escherichia coli
was used as the test strain, levofloxacin activity tested in BYEα broth was fourfold lower. HMR 3647, HMR 3004, erythromycin, and clarithromycin (0.25 and 1 μg/ml) reduced bacterial counts of two
L. pneumophila
strains grown in guinea pig alveolar macrophages by 0.5 to 1 log
10
, but regrowth occurred over a 2-day period. HMR 3647, erythromycin, and clarithromycin appeared to have equivalent intracellular activities which were solely static in nature. HMR 3004 was more active than all drugs tested except levofloxacin. In contrast, levofloxacin (1 μg/ml) was bactericidal against intracellular
L. pneumophila
and significantly more active than the other drugs tested. Therapy studies with HMR 3647 and erythromycin were performed in guinea pigs with
L. pneumophila
pneumonia. When HMR 3647 was given (10 mg/kg of body weight) by the intraperitoneal route to infected guinea pigs, mean peak plasma levels were 1.4 μg/ml at 0.5 h and 1.0 μg/ml at 1 h postinjection. The terminal half-life phase of elimination from plasma was 1.4 h. All 16
L. pneumophila
-infected guinea pigs treated with HMR 3647 (10 mg/kg/dose given intraperitoneally once daily) for 5 days survived for 9 days after antimicrobial therapy, as did all 16 guinea pigs treated with the same dose of HMR 3647 given twice daily. Fourteen of 16 erythromycin-treated (30 mg/kg/dose given intraperitoneally twice daily) animals survived, whereas 0 of 12 animals treated with saline survived. HMR 3647 is effective against
L. pneumophila
in vitro, in infected macrophages, and in a guinea pig model of Legionnaires’ disease. HMR 3647 given once daily should be evaluated as a treatment for Legionnaires’ disease in humans.
Killing Activities of Trovafloxacin Alone and in Combination with β-Lactam Agents, Rifampin, or Vancomycin against
Streptococcus pneumoniae
Isolates with Various Susceptibilities to Extended-Spectrum Cephalosporins at Concentrations Clinically Achievable in Cerebrospinal Fluid
Authors: Fitoussi Frederic , Doit Catherine , Geslin Pierre , Bingen Edouard
Published: October 2018

The killing activities of trovafloxacin alone and in combination with β-lactam agents (extended-spectrum cephalosporins, meropenem), rifampin, or vancomycin were evaluated against 20 genotypically characterized
Streptococcus pneumoniae
isolates for which amoxicillin MICs were ≥4 μg/ml (cefotaxime MICs, ≥4 μg/ml for six strains) at concentrations clinically achievable in cerebrospinal fluid. At 6 h the mean killing activity of trovafloxacin alone (range, 2.6 to 2.9 log
10
CFU/ml) did not vary significantly according to the susceptibility of the strains to β-lactam agents. The activities of trovafloxacin or vancomycin added to the β-lactam agents and the combination trovafloxacin-vancomycin were additive or indifferent. Against the ceftriaxone-resistant isolates, the killing activity of the combination of a β-lactam agent and trovafloxacin did not differ significantly from that of a β-lactam agent and vancomycin.
Molecular Cloning of the
gyrA
and
gyrB
Genes of
Bacteroides fragilis
Encoding DNA Gyrase
Authors: Onodera Yoshikuni , Sato Kenichi
Published: October 2018

The genes encoding the DNA gyrase A and B subunits of
Bacteroides fragilis
were cloned and sequenced. The
gyrA
and
gyrB
genes code for proteins of 845 and 653 amino acids, respectively. These proteins were expressed in
Escherichia coli
, and the combination of GyrA and GyrB exhibited ATP-dependent supercoiling activity. To analyze the role of DNA gyrase in quinolone resistance of
B. fragilis
, we isolated mutant strains by stepwise selection for resistance to increasing concentrations of levofloxacin. We analyzed the resistant mutants and showed that Ser-82 of GyrA, equivalent to resistance hot spot Ser-83 of GyrA in
E. coli
, was in each case replaced with Phe. These results suggest that DNA gyrase is an important target for quinolones in
B. fragilis.
Detection of Rifampin Resistance in
Mycobacterium tuberculosis
by Double Gradient-Denaturing Gradient Gel Electrophoresis
Authors: Scarpellini Paolo , Braglia Sergio , Carrera Paola , Cedri Maura , Cichero Paola , Colombo Alessia , Crucianelli Rosella , Gori Andrea , Ferrari Maurizio , Lazzarin Adriano
Published: October 2018

We applied double gradient-denaturing gradient gel electrophoresis (DG-DGGE) for the rapid detection of rifampin (RMP) resistance from
rpoB
PCR products of
Mycobacterium tuberculosis
isolates and clinical samples. The results of this method were fully concordant with those of DNA sequencing and susceptibility testing analyses. DG-DGGE is a valid alternative to the other methods of detecting mutations for predicting RMP resistance.
Lipoprotein Distribution of a Novel Endotoxin Antagonist, E5531, in Plasma from Human Subjects with Various Lipid Levels
Authors: Wasan Kishor M. , Strobel Frank W. , Parrott Steven C. , Lynn Melvyn , Christ William J. , Hawkins Lynn D. , Rossignol Daniel P.
Published: October 2018

The purpose of this study was to determine the distribution profile of a novel endotoxin antagonist, [14C]E5531, at 1 μg/ml in plasma samples obtained from fasted human subjects with various lipid and protein concentrations. Our findings suggest that the majority of E5531 binds with high-density lipoproteins (HDLs) independently of plasma lipid and protein levels tested. Furthermore, it appears that an increase in triglyceride-rich lipoprotein (TRL) lipid and protein levels and an increase in low-density lipoprotein (LDL) lipid levels significantly increase TRL plus LDL binding of E5531. However, only an increase in HDL protein levels significantly increases HDL binding of E5531.
Insertion of Mini-IS
605
and Deletion of Adjacent Sequences in the Nitroreductase (
rdxA
) Gene Cause Metronidazole Resistance in
Helicobacter pylori
NCTC11637
Authors: Debets-Ossenkopp Yvette J. , Pot Raymond G. J. , van Westerloo David J. , Goodwin Avery , Vandenbroucke-Grauls Christina M. J. E. , Berg Douglas E. , Hoffman Paul S. , Kusters Johannes G.
Published: October 2018

We found that NCTC11637, the type strain of
Helicobacter pylori
, the causative agent of peptic ulcer disease and an early risk factor for gastric cancer, is metronidazole resistant. DNA transformation, PCR-based restriction analysis, and DNA sequencing collectively showed that the metronidazole resistance of this strain was due to mutation in
rdxA
(gene HP0954 in the full genome sequence of
H. pylori
26695) and that resistance did not depend on mutation in any of the other genes that had previously been suggested: catalase (
katA
), ferredoxin (
fdx
), flavodoxin (
fldA
), pyruvate:flavodoxin oxidoreductase (
por
γδαβ), RecA (
recA
), or superoxide dismutase (
sodB
). This is in accord with another recent study that attributed metronidazole resistance to point mutations in
rdxA
. However, the mechanism of
rdxA
inactivation that we found in NCTC11637 is itself also novel: insertion of mini-IS
605
, one of the endogenous transposable elements of
H. pylori
, and deletion of adjacent DNA sequences including 462 bp of the 851-bp-long
rdxA
gene.
High-Performance Liquid Chromatographic Determination of Ribavirin in Whole Blood To Assess Disposition in Erythrocytes
Authors: Homma Masato , Jayewardene Anura L. , Gambertoglio John , Aweeka Francesca
Published: October 2018

Ribavirin is an antiviral agent used in the treatment of chronic hepatitis C virus infection. One of the limitations associated with the use of ribavirin is a reversible anemia caused by its accumulation in erythrocytes. Therefore, it is of interest to determine ribavirin levels in erythrocytes, as well as in plasma, as these measurements may be predictive of hematotoxicity. In the present study, we describe a high-performance liquid chromatographic (HPLC) assay for ribavirin in whole blood to estimate concentrations of free ribavirin and phosphorylated anabolites in erythrocytes. Since ribavirin exists primarily as phosphorylated anabolites (mono-, di-, and triphosphates) in erythrocytes, whole-blood extracts were initially dephosphorylated with acid phosphatase. The enzyme-treated samples were subjected to phenyl boronic acid column extraction for cleanup. The purified fraction was analyzed by reversed-phase HPLC, which was optimized for determination of ribavirin levels in whole blood. The recoveries of ribavirin from whole blood ranged from 63.1 to 90.7% at concentrations ranging from 1.67 to 40.0 μM. Intra- and interassay variations estimated at these concentrations were 3.2 to 10.4 and 4.7 to 11.7%, respectively. This method was used to quantitate ribavirin in samples both treated and untreated with acid phosphatase to estimate the extent of intracellular phosphorylation in erythrocytes. The method was also used to evaluate the effects of dipyridamole, a nucleoside transporter inhibitor, on ribavirin disposition in erythrocytes in in vitro experiments.
Efficacy of Levofloxacin for Experimental Aortic-Valve Endocarditis in Rabbits Infected with Viridans Group Streptococcus orStaphylococcus aureus
Authors: Chambers Henry F. , Xiang Qing , Liu  , Chow Lucian Liuxin , Hackbarth Corinne
Published: October 2018

Levofloxacin is among the more active fluoroquinolones against streptococci and staphylococci. It is effective against moderately severe infections caused by these organisms, but its efficacy in the treatment of bacteremia and serious infections such as endocarditis is not well defined. We compared the efficacy of levofloxacin to those of standard agents in the rabbit model of aortic-valve endocarditis caused by fluoroquinolone-susceptible strains including a penicillin-susceptible strain ofStreptococcus sanguis, a penicillin-resistant strain ofStreptococcus mitis, a methicillin-resistant strain ofStaphylococcus aureus, and a methicillin-susceptible strain ofS. aureus. Levofloxacin administered intramuscularly at dosages of 20 to 40 mg/kg of body weight twice daily (b.i.d.) was completely ineffective against the penicillin-susceptible strain, with mean vegetation titers after 3 days of therapy not statistically significantly different from those for controls. Levofloxacin was no more effective than penicillin against the penicillin-resistant strain. Levofloxacin administered for 4 days at a dosage of 20 mg/kg b.i.d. was at least as effective as vancomycin administered intravenously at a dosage of 25 mg/kg b.i.d. against the methicillin-resistantS. aureusstrain and was as effective as nafcillin administered intramuscularly at 100 mg three times daily against the methicillin-susceptible strain. Emergence of resistance to levofloxacin in vitro was less likely to occur than resistance to ciprofloxacin, and resistance to levofloxacin was not observed in vivo. Levofloxacin-rifampin combinations were antagonistic in vitro and in vivo. Levofloxacin was highly effective as a single agent against experimental staphylococcal endocarditis but was surprisingly ineffective against streptococcal endocarditis, suggesting that it has a potential role as treatment for seriousS. aureusbut not viridans group streptococcal infections in humans.
In Vitro Activities of Gemifloxacin (SB 265805, LB20304) against Recent Clinical Isolates of
Chlamydia pneumoniae
Authors: Roblin Patricia M. , Reznik Tamara , Kutlin Andrei , Hammerschlag Margaret R.
Published: October 2018

We compared the in vitro activity of gemifloxacin, a new quinolone antibiotic, to the activities of levofloxacin, moxifloxacin, trovafloxacin, erythromycin, and doxycycline against 20 isolates of
Chlamydia pneumoniae
. Gemifloxacin was the most active quinolone tested, with a MIC at which 90% of the isolates are inhibited and a minimal bactericidal concentration at which 90% of strains tested are killed of 0.25 μg/ml, but this activity was less than those of doxycycline and erythromycin.
Interaction between Fluconazole and Amphotericin B in Mice with Systemic Infection Due to Fluconazole-Susceptible or -Resistant Strains of
Candida albicans
Authors: Louie Arnold , Banerjee Partha , Drusano George L. , Shayegani Mehdi , Miller Michael H.
Published: October 2018

The interaction between fluconazole (Flu) and amphotericin B (AmB) was evaluated in a murine model of systemic candidiasis for one Flu-susceptible strain (MIC, 0.5 μg/ml), two strains with intermediate Flu resistance (Flu mid-resistant strains) (MIC, 64 and 128 μg/ml), and one highly Flu-resistant strain (MIC, 512 μg/ml) of
Candida albicans
. Differences in fungal densities in kidneys of infected mice after 24 h of therapy and in survival rates at 62 days of mice treated with an antifungal drug or a combination of antifungal drugs for 4 days were compared. For the Flu-susceptible and Flu mid-resistant strains, the combination of Flu and AmB was antagonistic, as shown by both quantitative culture results and survival. The interaction was additive for the highly Flu-resistant strain. These results suggest that the combination of Flu and AmB should be used with caution in infections due to fungi that are usually susceptible to both antifungal agents and as empirical antifungal drug therapy.
Automated Thermal Cycling Is Superior to Traditional Methods for Nucleotide Sequencing of
bla
SHV
Genes
Authors: Bradford Patricia A.
Published: October 2018

Genes encoding SHV-1 and SHV-2 were sequenced by different methods. Nucleotide sequencing of the coding strand by standard dideoxy-chain termination methods resulted in errors in the interpretation of the nucleotide sequence and the derived amino acid sequence in two main regions which corresponded to nucleotide and amino acid changes that had been reported previously. The automated thermal cycling method was clearly superior and consistently resulted in the correct sequences for these genes.
Gatifloxacin Activity against Quinolone-Resistant Gyrase: Allele-Specific Enhancement of Bacteriostatic and Bactericidal Activities by the C-8-Methoxy Group
Authors: Lu Tao , Zhao Xilin , Drlica Karl
Published: October 2018

Antibacterial activities of gatifloxacin (AM1155), a new C-8-methoxy fluoroquinolone, and two structurally related compounds, AM1121 and ciprofloxacin, were studied with an isogenic set of ten quinolone-resistant,
gyrA
(gyrase) mutants of
Escherichia coli
. To compare the effect of each mutation on resistance, the mutant responses were normalized to those of wild-type cells. Alleles exhibiting the most resistance to growth inhibition mapped in α-helix 4, which is thought to lie on a GyrA dimer surface that interacts with DNA. The C-8-methoxy group lowered the resistance due to these mutations more than it lowered resistance arising from several
gyrA
alleles located outside α-helix 4. These data are consistent with α-helix 4 being a distinct portion of the quinolone-binding site of GyrA. A helix change to proline behaved more like nonhelix alleles, indicating that helix perturbation differs from the other changes at helix residues. Addition of a
parC
(topoisomerase IV) resistance allele revealed that the C-8-methoxy group also facilitated attack of topoisomerase IV. When lethal effects were measured at a constant multiple of the minimum inhibitory concentration for each fluoroquinolone to normalize for differences in bacteriostatic action, gatifloxacin was more potent than the C-8-H compounds, both in the presence and absence of protein synthesis (an exception was observed when alanine was substituted for aspartic acid at position 82). Collectively, these data show that the C-8-methoxy group contributes to the enhanced activity of gatifloxacin against resistant gyrase and wild-type topoisomerase IV.
Characterization of a
Pseudomonas aeruginosa
Efflux Pump Contributing to Aminoglycoside Impermeability
Authors: Westbrock-Wadman Shannon , Sherman David R. , Hickey Mark J. , Coulter Silvija N. , Zhu Ya Qi , Warrener Paul , Nguyen Lisa Y. , Shawar Ribhi M. , Folger Kim R. , Stover C. Kendall
Published: October 2018

Pseudomonas aeruginosa
can employ many distinct mechanisms of resistance to aminoglycoside antibiotics; however, in cystic fibrosis patients, more than 90% of aminoglycoside-resistant
P. aeruginosa
isolates are of the impermeability phenotype. The precise molecular mechanisms that produce aminoglycoside impermeability-type resistance are yet to be elucidated. A subtractive hybridization technique was used to reveal gene expression differences between PAO1 and isogenic, spontaneous aminoglycoside-resistant mutants of the impermeability phenotype. Among the many genes found to be up-regulated in these laboratory mutants were the
amrAB
genes encoding a recently discovered efflux system. The
amrAB
genes appear to be the same as the recently described
mexXY
genes; however, the resistance profile that we see in
P. aeruginosa
is very different from that described for
Escherichia coli
with
mexXY
. Direct evidence for AmrAB involvement in aminoglycoside resistance was provided by the deletion of
amrB
in the PAO1-derived laboratory mutant, which resulted in the restoration of aminoglycoside sensitivity to a level nearly identical to that of the parent strain. Furthermore, transcription of the
amrAB
genes was shown to be up-regulated in
P. aeruginosa
clinical isolates displaying the impermeability phenotype compared to a genotypically matched sensitive clinical isolate from the same patient. This suggests the possibility that AmrAB-mediated efflux is a clinically relevant mechanism of aminoglycoside resistance. Although it is unlikely that hyperexpression of AmrAB is the sole mechanism conferring the impermeability phenotype, we believe that the Amr efflux system can contribute to a complex interaction of molecular events resulting in the aminoglycoside impermeability-type resistance phenotype.
Antibiotic-Induced Cell Wall Fragments ofStaphylococcus aureusIncrease Endothelial Chemokine Secretion and Adhesiveness for Granulocytes
Authors: van Langevelde P. , Ravensbergen E. , Grashoff P. , Beekhuizen H. , Groeneveld P. H. P. , van Dissel J. T.
Published: October 2018

Antibiotics release inflammatory fragments, such as lipoteichoic acid (LTA) and peptidoglycan (PG), from the cell wall ofStaphylococcus aureus. In this study, we exposedS. aureuscultures to a number of β-lactam antibiotics (imipenem, flucloxacillin, and cefamandole) and protein synthesis-inhibiting antibiotics (erythromycin, clindamycin, and gentamicin) and investigated whether supernatants of these cultures differ in their capacity to stimulate endothelial cells (EC). After 24 h of incubation, endothelial adhesiveness for leukocytes, surface expression of various adhesion molecules, and secretion of the chemokines interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) were measured. Supernatants of β-lactam-exposed cultures (designated β-lactam supernatants) enhanced the adhesiveness of EC for granulocytes, whereas those of protein synthesis-inhibiting antibiotic-exposed cultures (designated protein synthesis-inhibitor supernatants) did not. This hyperadhesiveness coincided with a higher intercellular adhesion molecule-1 expression on the surface of the stimulated EC. In addition, EC stimulated with β-lactam supernatants secreted significantly higher concentrations of the chemokines IL-8 and MCP-1 than those stimulated with protein synthesis-inhibitor supernatants. The finding that the concentrations of LTA and PG in β-lactam supernatants were much higher than those in protein synthesis-inhibitor supernatants suggests that the observed differences in stimulatory effect between these supernatants are a result of differences in the release of cell wall fragments, although the presence of other stimulatory factors in the supernatants cannot be excluded. In conclusion, our results argue for a release of LTA and PG fromS. aureusafter exposure to β-lactam antibiotics that enhances the development of a systemic inflammatory response by stimulating EC such that adhesiveness for granulocytes is increased and large amounts of IL-8 and MCP-1 are secreted.
Antimicrobial Resistance of Diarrheagenic
Escherichia coli
Isolated from Children under the Age of 5 Years from Ifakara, Tanzania
Authors: Vila Jordi , Vargas Martha , Casals Climent , Urassa Honorato , Mshinda Hassan , Schellemberg David , Gascon Joaquim
Published: October 2018

Diarrhea caused by multidrug-resistant bacteria is an important public health problem among children in developing countries. The prevalence and antimicrobial susceptibility of diarrheagenic
Escherichia coli
in 346 children under 5 years of age in Ifakara, Tanzania, were studied. Thirty-eight percent of the cases of diarrhea were due to multiresistant enterotoxigenic
E. coli
, enteroaggregative
E. coli
, or enteropathogenic
E. coli
. Strains of all three
E. coli
categories showed high-level resistance to ampicillin, tetracycline, co-trimoxazole, and chloramphenicol but were highly susceptible to quinolones. Guidelines for appropriate use of antibiotics in developing countries need updating.
A Broad-Spectrum Microbicide with Virucidal Activity against Sexually Transmitted Viruses
Authors: Howett M. K. , Neely E. B. , Christensen N. D. , Wigdahl B. , Krebs F. C. , Malamud D. , Patrick S. D. , Pickel M. D. , Welsh P. A. , Reed C. A. , Ward M. G. , Budgeon L. R. , Kreider J. W.
Published: October 2018

Sodium dodecyl sulfate (SDS), an alkyl sulfate surfactant derived from an organic alcohol, possesses surfactant properties but also denatures and unfolds both monomeric and subunit proteins. In preliminary experiments, we demonstrated that SDS is a potent inactivator of herpes simplex virus type 2 and human immunodeficiency virus type 1 at concentrations comparable to those used for the surfactant nonoxynol-9. We hypothesized that SDS might be capable of denaturing the capsid proteins of nonenveloped viruses. In this report, we demonstrate inactivation of rabbit, bovine, and human papillomaviruses after brief treatment with dilute solutions of SDS. Effective concentrations were nontoxic to rabbit skin and to split-thickness grafts of human foreskin epithelium. This is the first report of a microbicidal surfactant that will inactivate papillomaviruses. We propose that SDS is now a candidate microbicide for formulation and testing with humans.
Pharmacokinetics of Mefloquine Combined with Artesunate in Children with Acute Falciparum Malaria
Authors: Price Ric , Simpson Julie A. , Teja-Isavatharm Paktiya , Than Myint Myint , Luxemburger Christine , Gray Heppner D. , Chongsuphajaisiddhi Tan , Nosten François , White Nicholas J.
Published: October 2018

Combining artemisinin or a derivative with mefloquine increases cure rates in falciparum malaria patients, reduces transmission, and may slow the development of resistance. The combination of artesunate, given for 3 days, and mefloquine is now the treatment of choice for uncomplicated multidrug-resistant falciparum malaria acquired on the western or eastern borders of Thailand. To optimize mefloquine administration in this combination, a prospective study of mefloquine pharmacokinetics was conducted with 120 children (4 to 15 years old) with acute uncomplicated falciparum malaria, who were divided into four age- and sex-matched groups. The patients all received artesunate (4 mg/kg of body weight/day orally for 3 days and mefloquine as either (i) a single dose (25 mg/kg) on day 2 with food, (ii) a split dose (15 mg/kg on day 2 and 10 mg/kg on day 3) with food, (iii) a single dose (25 mg/kg) on day 0 without food, or (iv) a single dose (25 mg/kg) on day 2 without food. Delaying administration of mefloquine until day 2 was associated with a mean (95% confidence interval) increase in estimated oral bioavailability of 72% (36 to 109%). On day 2 coadministration with food did not increase mefloquine absorption significantly, and there were no significant differences between patients receiving split- and single-dose administration. In combination with artesunate, mefloquine administration should be delayed until the second or third day after presentation.
Antisense Oligonucleotide Inhibition of Hepatitis C Virus (HCV) Gene Expression in Livers of Mice Infected with an HCV-Vaccinia Virus Recombinant
Authors: Zhang Hong , Hanecak Ronnie , Brown-Driver Vickie , Azad Raana , Conklin Boyd , Fox Maureen C. , Anderson Kevin P.
Published: October 2018

Hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis worldwide. Current treatments are not curative for most infected individuals, and there is an urgent need for both novel therapeutic agents and small-animal models which can be used to evaluate candidate drugs. A small-animal model of HCV gene expression was developed with recombinant vaccinia virus vectors. VHCV-IRES (internal ribosome entry site) is a recombinant vaccinia viral vector containing the HCV 5′ nontranslated region (5′-NTR) and a portion of the HCV core coding region fused to the firefly luciferase gene. Intraperitoneal injection of VHCV-IRES produced high levels of luciferase activity in the livers of BALB/c mice. Antisense oligonucleotides complementary to the HCV 5′-NTR and translation initiation codon regions were then evaluated for their effects on the expression of these target HCV sequences in BALB/c mice infected with the vaccinia virus vector. Treatment of VHCV-IRES-infected mice with 20-base phosphorothioate oligonucleotides complementary to the sequence surrounding the HCV initiation codon (nucleotides 330 to 349) specifically reduced luciferase expression in the livers in a dose-dependent manner. Inhibition of HCV reporter gene expression in this small-animal model suggests that antisense oligonucleotides may provide a novel therapy for treatment of chronic HCV infection.
Primary Targets of Fluoroquinolones in
Streptococcus pneumoniae
Authors: Fukuda Hideyuki , Hiramatsu Keiichi
Published: October 2018

Mutants of wild-type
Streptococcus pneumoniae
IID553 with mutations in
parC
were obtained by selection with trovafloxacin, levofloxacin, norfloxacin, and ciprofloxacin. All of the
parC
mutants were cross-resistant to the selecting agents but were not resistant to gatifloxacin and sparfloxacin. On the other hand,
gyrA
mutants were isolated by selection with gatifloxacin and sparfloxacin. The
gyrA
mutants were cross-resistant to gatifloxacin and sparfloxacin but were not resistant to the other fluoroquinolones tested. These results suggest that in wild-type
S. pneumoniae
the primary target of trovafloxacin, levofloxacin, norfloxacin, and ciprofloxacin is topoisomerase IV, whereas the primary target of gatifloxacin and sparfloxacin is DNA gyrase.
Italian Survey on Comparative Levofloxacin Susceptibility in 334 Clinical Isolates of
Pseudomonas aeruginosa
Authors: Segatore Bernardetta , Setacci Domenico , Perilli Mariagrazia , Franceschini Nicola , De Santis Annalisa , Marchetti Federico , Amicosante Gianfranco
Published: October 2018

A national survey on susceptibility patterns of 334
Pseudomonas aeruginosa
isolates from intensive care units and hematology and oncology wards from 13 Italian hospitals compared the in vitro activity of levofloxacin, an injectable oral fluoroquinolone, to those of ciprofloxacin, ofloxacin, ceftazidime, imipenem, amikacin, and gentamicin. Amikacin and imipenem had the best susceptibility profiles. The activity of levofloxacin was superior to those of the other quinolones and was comparable to that of ceftazidime. The effect of levofloxacin in vitro on
P. aeruginosa
clinical isolates suggests that further clinical investigations are warranted.
Molecular Diversity and Evolutionary Relationships of Tn
1546
-Like Elements in Enterococci from Humans and Animals
Authors: Willems Rob J. L. , Top Janetta , van den Braak Nicole , van Belkum Alex , Mevius Dik J. , Hendriks Giel , van Santen-Verheuvel Marga , van Embden Jan D. A.
Published: October 2018

We report on a detailed study on the molecular diversity and evolutionary relationships of Tn
1546
-like elements in vancomycin-resistant enterococci (VRE) from humans and animals. Restriction fragment length polymorphism (RFLP) analysis of the VanA transposon of 97 VRE revealed seven different Tn
1546
types. Subsequent sequencing of the complete VanA transposons of 13 VRE isolates representing the seven RFLP types followed by sequencing of the identified polymorphic regions in 84 other VanA transposons resulted in the identification of 22 different Tn
1546
derivatives. Differences between the Tn
1546
types included point mutations in
orf1
,
vanS
,
vanA
,
vanX
, and
vanY
. Moreover, insertions of an IS
1216V
-IS
3
-like element in
orf1
, of IS
1251
in the
vanS-vanH
intergenic region, and of IS
1216V
in the
vanX-vanY
intergenic region were found. The presence of insertion sequence elements was often associated with deletions in Tn
1546
. Identical Tn
1546
types were found among isolates from humans and farm animals in The Netherlands, suggesting the sharing of a common vancomycin resistance gene pool. Application of the genetic analysis of Tn
1546
to VRE isolates causing infections in hospitals in Oxford, United Kingdom, and Chicago, Ill., suggested the possibility of the horizontal transmission of the vancomycin resistance transposon. The genetic diversity in Tn
1546
combined with epidemiological data suggest that the DNA polymorphism among Tn
1546
variants can successfully be exploited for the tracing of the routes of transmission of vancomycin resistance genes.
Pharmacokinetics and Urinary Excretion of Amikacin in Low-Clearance Unilamellar Liposomes after a Single or Repeated Intravenous Administration in the Rhesus Monkey
Authors: Fielding Robert M. , Moon-Mcdermott Lotus , Lewis Ramilla O. , Horner Michelle J.
Published: October 2018

Liposomal aminoglycosides have been shown to have activity against intracellular infections, such as those caused by
Mycobacterium avium
. Amikacin in small, low-clearance liposomes (MiKasome) also has curative and prophylactic efficacies against
Pseudomonas aeruginosa
and
Klebsiella pneumoniae
. To develop appropriate dosing regimens for low-clearance liposomal amikacin, we studied the pharmacokinetics of liposomal amikacin in plasma, the level of exposure of plasma to free amikacin, and urinary excretion of amikacin after the administration of single-dose (20 mg/kg of body weight) and repeated-dose (20 mg/kg eight times at 48-h intervals) regimens in rhesus monkeys. The clearance of liposomal amikacin (single-dose regimen, 0.023 ± 0.003 ml min
−1
kg
−1
; repeated-dose regimen, 0.014 ± 0.001 ml min
−1
kg
−1
) was over 100-fold lower than the creatinine clearance (an estimate of conventional amikacin clearance). Half-lives in plasma were longer than those reported for other amikacin formulations and declined during the elimination phase following administration of the last dose (from 81.7 ± 27 to 30.5 ± 5 h). Peak and trough (48 h) levels after repeated dosing reached 728 ± 72 and 418 ± 60 μg/ml, respectively. The levels in plasma remained >180 μg/ml for 6 days after the administration of the last dose. The free amikacin concentration in plasma never exceeded 17.4 ± 1 μg/ml and fell rapidly (half-life, 1.47 to 1.85 h) after the administration of each dose of liposomal amikacin. This and the low volume of distribution (45 ml/kg) indicate that the amikacin in plasma largely remained sequestered in long-circulating liposomes. Less than half the amikacin was recovered in the urine, suggesting that the level of renal exposure to filtered free amikacin was reduced, possibly as a result of intracellular uptake or the metabolism of liposomal amikacin. Thus, low-clearance liposomal amikacin could be administered at prolonged (2- to 7-day) intervals to achieve high levels of exposure to liposomal amikacin with minimal exposure to free amikacin.
Treatment of Murine Fusariosis with SCH 56592
Authors: Lozano-Chiu M. , Arikan S. , Paetznick V. L. , Anaissie E. J. , Loebenberg D. , Rex J. H.
Published: October 2018

Doses of 10 to 100 mg of the azole antifungal agent SCH 56592/kg of body weight/day were studied in immunocompetent mice as therapy for systemic infection by
Fusarium solani
. Treatment was begun 1 h after intravenous infection and continued daily for 4 or 13 doses. Prolongation of survival and organ clearance were dependent on both the dose and the duration of SCH 56592 therapy, with the best results seen at 50 and 100 mg/kg/day. The results at the highest doses of SCH 56592 used (50 or 100 mg/kg/day) were comparable to those obtained with amphotericin B at 1 mg/kg/day. SCH 56592 has potential for therapy of systemic infections caused by
F. solani
.
Penetration of Meropenem in Lung, Bronchial Mucosa, and Pleural Tissues
Authors: Byl Baudouin , Jacobs Frédérique , Roucloux Ingrid , de Franquen Philippe , Cappello Matteo , Thys Jean-Pierre
Published: October 2018

Lung, bronchial mucosa, and pleural tissue samples were obtained from 14 patients undergoing lung surgery 1 to 5 h after administration of 1 g of meropenem. The mean (range) peak concentrations of meropenem were 3.9 (0.2 to 8.2), 6.6 (3.0 to 13.3), and 2.8 (0.6 to 7.8) mg/kg of tissue, respectively, exceeding the MICs at which 90% of isolates are inhibited for most respiratory pathogens.
Antibiotic Resistance Conferred by a Conjugative Plasmid and a Class I Integron in
Vibrio cholerae
O1 El Tor Strains Isolated in Albania and Italy
Authors: Falbo Vincenzo , Carattoli Alessandra , Tosini Fabio , Pezzella Cristina , Dionisi Anna Maria , Luzzi Ida
Published: October 2018

Multidrug-resistant
Vibrio cholerae
O1 El Tor strains isolated during the 1994 outbreak of cholera in Albania and Italy were characterized for the molecular basis of antibiotic resistance. All strains were found to be resistant to tetracycline, streptomycin, spectinomycin, trimethoprim, sulfathiazole, and the vibriostatic compound O/129 (2,4-diamino-6,7-diisopropylteridine). Resistance genes were self-transferable by a conjugative plasmid of about 60 MDa, with the exception of spectinomycin resistance, which was conferred by the
aadA1
gene cassette located in the bacterial chromosome within a class 1 integron. The resistance to trimethoprim and O/129 was conferred by the
dfrA1
gene, which was present on the plasmid. Although the
dfrA1
gene is known to be borne on an integron cassette, class 1, 2, or 3
intI
genes were not detected as part of the plasmid DNA from the strains studied.
Emergence of Fosfomycin-Resistant Isolates of Shiga-Like Toxin-Producing
Escherichia coli
O26
Authors: Horii Toshinobu , Kimura Taku , Sato Kumiko , Shibayama Keigo , Ohta Michio
Published: October 2018

We evaluated the susceptibilities of 129 Shiga-like toxin-producing
Escherichia coli
(STEC) isolates to various antibiotics. The numbers of isolates for which MICs were high (≧128 μg/ml) were as follows: 5 for fosfomycin, 14 for ampicillin, 1 for cefaclor, 6 for kanamycin, 22 for tetracycline, and 2 for doxycycline. For two isolates of STEC O26 MICs of fosfomycin were high (1,024 and 512 μg/ml, respectively). Conjugation experiments and glutathione
S
-transferase assays suggested that the fosfomycin resistance in these isolates was determined not by a plasmid but chromosomally. The amount of active intracellular fosfomycin in these STEC isolates was 100- to 200-fold less than that in
E. coli
C600 harboring pREFTT47B408 in the presence of either
l
-α-glycerophosphate or glucose-6-phosphate. Cloning, sequencing, and Northern blot analysis demonstrated that the transcriptional level of the
murA
gene encoding UDP-
N
-acetylglucosamine enolpyruvoyl transferase in these isolates was greater than that in
E. coli
C600. Our results suggest that the fosfomycin resistance in these STEC isolates is due to concurrent effects of alteration of the
glpT
and/or
uhp
transport systems and of the enhanced transcription of the
murA
gene.
High-Frequency, In Vitro Reversible Switching of
Candida lusitaniae
Clinical Isolates from Amphotericin B Susceptibility to Resistance
Authors: Yoon Stephanie A. , Vazquez Jose A. , Steffan Paul E. , Sobel Jack D. , Akins Robert A.
Published: October 2018

Recent studies have revealed an increase in the incidence of serious infections caused by non-
albicans Candida
species.
Candida lusitaniae
is of special interest because of its sporadic resistance to amphotericin B (AmB). The present in vitro study demonstrated that, unlike other
Candida
species,
C. lusitaniae
isolates frequently generated AmB-resistant lineages form previously susceptible colonies. Cells switching from a resistant colony to a susceptible phenotype were also detected after treatment with either UV light, heat shock, or exposure to whole blood, all of which increased the frequency of switching. In some
C. lusitaniae
lineages, after a cell switched to a resistant phenotype, the resistant phenotype was stable; in other lineages, colonies were composed primarily of AmB-susceptible cells. Although resistant and susceptible lineages were identical in many aspects, their cellular morphologies were dramatically different. Switching mechanisms that involve exposure to antifungals may have an impact on antifungal therapeutic strategies as well as on standardized susceptibility testing of clinical yeast specimens.
Strength and Regulation of the Different Promoters for Chromosomal β-Lactamases of
Klebsiella oxytoca
Authors: Fournier Bénédicte , Gravel Annie , Hooper David C. , Roy Paul H.
Published: October 2018

The two groups of chromosomal β-lactamases from
Klebsiella oxytoca
(OXY-1 and OXY-2) can be overproduced 73- to 223-fold, due to point mutations in the consensus sequences of their promoters. The different versions of promoters from
bla
OXY-1
and
bla
OXY-2
were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene of pKK232-8, and their relative strengths were determined in
Escherichia coli
and in
K. oxytoca
. The three different mutations in the OXY β-lactamase promoters resulted in a 4- to 31-fold increase in CAT activity compared to that of the wild-type promoter. The G→T transversion in the first base of the −10 consensus sequence caused a greater increase in the promoter strength of the wild-type promoter than the two other principal mutations (a G-to-A transition of the fifth base of the −10 consensus sequence and a T-to-A transversion of the fourth base of the −35 sequence). The strength of the promoter carrying a double mutation (transition in the Pribnow box and the transversion in the −35 hexamer) was increased 15- to 61-fold in comparison to that of the wild-type promoter. A change from 17 to 16 bp between the −35 and −10 consensus sequences resulted in a ninefold decrease of the promoter strength. The expression of the
bla
OXY
promoter in
E. coli
differs from that in
K. oxytoca
, particularly for promoters carrying strong mutations. Furthermore, the
bla
OXY
promoter appears not to be controlled by DNA supercoiling or an upstream curved DNA, but it is dependent on the gene copy number.
Ampicillin-Sulbactam and Amoxicillin-Clavulanate Susceptibility Testing of
Escherichia coli
Isolates with Different β-Lactam Resistance Phenotypes
Authors: Oliver Antonio , Pérez-Vázquez María , Martínez-Ferrer Manuel , Baquero Fernando , de Rafael Luis , Cantón Rafael
Published: October 2018

The activities of ampicillin-sulbactam and amoxicillin-clavulanate were studied with 100 selected clinical
Escherichia coli
isolates with different β-lactam susceptibility phenotypes by standard agar dilution and disk diffusion techniques and with a commercial microdilution system (PASCO). A fixed ratio (2:1) and a fixed concentration (clavulanate, 2 and 4 μg/ml; sulbactam, 8 μg/ml) were used in the agar dilution technique. The resistance frequencies for amoxicillin-clavulanate with different techniques were as follows: fixed ratio agar dilution, 12%; fixed concentration 4-μg/ml agar dilution, 17%; fixed ratio microdilution, 9%; and disk diffusion, 9%. Marked discrepancies were found when these results were compared with those obtained with ampicillin-sulbactam (26 to 52% resistance), showing that susceptibility to amoxicillin-clavulanic acid cannot be predicted by testing the isolate against ampicillin-sulbactam. Interestingly, the discrimination between susceptible and intermediate isolates was better achieved with 4 μg of clavulanate per ml than with the fixed ratio. In contrast, amoxicillin susceptibility was not sufficiently restored when 2 μg of clavulanate per ml was used, particularly in moderate (mean β-lactamase activity, 50.8 mU/mg of protein) and high-level (215 mU/mg) TEM-1 β-lactamase producer isolates. Four micrograms of clavulanate per milliliter could be a reasonable alternative to the 2:1 fixed ratio, because most high-level β-lactamase-hyperproducing isolates would be categorized as nonsusceptible, and low- and moderate-level β-lactamase-producing isolates would be categorized as nonresistant. This approach cannot be applied to sulbactam, either with the fixed 2:1 ratio or with the 8-μg/ml fixed concentration, because many low-level β-lactamase-producing isolates would be classified in the resistant category. These findings call for a review of breakpoints for β-lactam–β-lactamase inhibitor combinations.
Molecular Basis of AmpC Hyperproduction in Clinical Isolates of
Escherichia coli
Authors: Nelson E. C. , Elisha B. Gay
Published: October 2018

DNA sequencing data showed that five clinical isolates of
Escherichia coli
with reduced susceptibility to ceftazidime, ceftriaxone, and cefotaxime contain an
ampC
gene that is preceded by a strong promoter. Transcription from the strong promoter was 8- to 18-fold higher than that from the promoter from a susceptible isolate. RNA studies showed that mRNA stability does not play a role in the control of AmpC synthesis.