bluuebunny/crossref_metadata_2025 huggingface dataset:
RWJ-54428 (MC-02,479), a New Cephalosporin with High Affinity for Penicillin-Binding Proteins, Including PBP 2a, and Stability to Staphylococcal Beta-Lactamases
Authors: Malouin Francois , Blais Johanne , Chamberland Suzanne , Hoang Monica , Park Craig , Chan Christin , Mathias Kristina , Hakem Samia , Dupree Kelly , Liu Eric , Nguyen Tien , Dudley Michael N.
Published: January 2003

RWJ-54428 (MC-02,479) is a new cephalosporin active against gram-positive bacteria, including methicillin-resistant
Staphylococcus aureus
(MRSA). The potency of this new cephalosporin against MRSA is related to a high affinity for penicillin-binding protein 2a (PBP 2a), as assessed in a competition assay using biotinylated ampicillin as the reporter molecule. RWJ-54428 had high activity against MRSA strains COL and 67-0 (MIC of 1 μg/ml) and also showed affinity for PBP 2a, with a 50% inhibitory concentration (IC
50
) of 0.7 μg/ml. RWJ-54428 also displayed excellent affinity for PBP 5 from
Enterococcus hirae
R40, with an IC
50
of 0.8 μg/ml and a MIC of 0.5 μg/ml. The affinity of RWJ-54428 for PBPs of β-lactam-susceptible
S. aureus
(MSSA), enterococci (
E. hirae
), and
Streptococcus pneumoniae
showed that the good affinity of RWJ-54428 for MRSA PBP 2a and
E. hirae
PBP 5 does not compromise its binding to susceptible PBPs. RWJ-54428 showed stability to hydrolysis by purified type A β-lactamase isolated from
S. aureus
PC1. In addition, RWJ-54428 displayed low MICs against strains of
S. aureus
bearing the four classes of staphylococcal β-lactamases, including β-lactamase hyperproducers. The frequency of isolation of resistant mutants to RWJ-54428 from MRSA strains was very low. In summary, RWJ-54428 has high affinity to multiple PBPs and is stable to β-lactamase, properties that may explain our inability to find resistance by standard methods. These data are consistent with its excellent activity against β-lactam-resistant gram-positive bacteria.
AcrAB Multidrug Efflux Pump Is Associated with Reduced Levels of Susceptibility to Tigecycline (GAR-936) in
Proteus mirabilis
Authors: Visalli Melissa A. , Murphy Ellen , Projan Steven J. , Bradford Patricia A.
Published: January 2003

Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with the notable exception of the
Proteeae
. A study was performed to identify the mechanism responsible for the reduced susceptibility to tigecycline in
Proteus mirabilis
. Two independent transposon insertion mutants of
P. mirabilis
that had 16-fold-increased susceptibility to tigecycline were mapped to the
acrB
gene homolog of the
Escherichia coli
AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the parental wild-type
acrRAB
efflux gene cluster. The AcrAB transport system appears to be associated with the intrinsic reduced susceptibility to tigecycline in
P. mirabilis
.
Isoniazid Activation Defects in Recombinant
Mycobacterium tuberculosis
Catalase-Peroxidase (KatG) Mutants Evident in InhA Inhibitor Production
Authors: Wei Chih-Jen , Lei Benfang , Musser James M. , Tu Shiao-Chun
Published: January 2003

Mycobacterium tuberculosis
KatG catalyzes the activation of the antitubercular agent isoniazid to yield an inhibitor targeting enoyl reductase (InhA). However, no firm biochemical link between many KatG variants and isoniazid resistance has been established. In the present study, six distinct KatG variants identified in clinical
Mycobacterium tuberculosis
isolates resistant to isoniazid were generated by site-directed mutagenesis, and the recombinant mutant proteins (KatG
A110V
, KatG
A139P
, KatG
S315N
, KatG
L619P
, KatG
L634F
, and KatG
D735A
) were purified and characterized with respect to their catalase-peroxidase activities (in terms of
k
cat
/

K
m

), rates of free-radical formation from isoniazid oxidation, and, moreover, abilities to activate isoniazid. The A110V amino acid replacement did not result in significant alteration of KatG activities except that the peroxidase activity was enhanced. The other mutations, however, resulted in modestly reduced catalase and peroxidase catalytic efficiencies and, for the four mutants tested, significantly lower activities to oxidize isoniazid. Compared to the wild-type enzyme, the ability of the KatG
L634F
, KatG
A139P
, and KatG
D735A
variants to activate isoniazid decreased by 36%, 76%, and 73%, respectively, whereas the KatG
S315N
and KatG
L619P
variants completely lost their abilities to convert isoniazid into the InhA inhibitor. In addition, the inclusion of exogenous Mn
2+
to the isoniazid activation reaction mix significantly improved the ability of wild-type and KatG mutants to produce the InhA inhibitor.
Genome-Wide Screening ofSaccharomyces cerevisiaeTo Identify Genes Required for Antibiotic Insusceptibility of Eukaryotes
Authors: Blackburn Alexandra S. , Avery Simon V.
Published: January 2003

The adverse reactions provoked by many antibiotics in humans are well documented but are generally poorly understood at the molecular level. To elucidate potential genetic defects that could give rise to susceptibility to prokaryote-specific antibiotics in eukaryotes, we undertook genome-wide screens using the yeastSaccharomyces cerevisiaeas a model of eukaryotes; our previous work with a small number of yeast mutants revealed some specific gene functions required for oxytetracycline resistance. Here, the complete yeast deletion strain collection was tested for growth in the presence of a range of antibiotics. The sensitivities of mutants revealed by these screens were validated in independent tests. None of the ∼4,800 defined deletion strains tested were found to be sensitive to amoxicillin, penicillin G, rifampin, or vancomycin. However, two of the yeast mutants were tetracycline sensitive and four were oxytetracycline sensitive; encompassed among the latter were mutants carrying deletions in the same genes that we had characterized previously. Seventeen deletion strains were found to exhibit growth defects in the presence of gentamicin, with MICs for the strains being as low as 32 μg ml−1(the wild type exhibited no growth defects at any gentamicin concentration tested up to 512 μg ml−1). Strikingly, 11 of the strains that were most sensitive to gentamicin carried deletions in genes whose products are all involved in various aspects of vacuolar and Golgi complex (or endoplasmic reticulum) function. Therefore, these and analogous organelles, which are also the principal sites of gentamicin localization in human cells, appear to be essential for normal resistance to gentamicin in eukaryotes. The approach and data described here offer a new route to gaining insight into the potential genetic bases of antibiotic insusceptibilities in eukaryotes.
Tandem Amplification of a 28-Kilobase Region from the
Yersinia enterocolitica
Chromosome Containing the
blaA
Gene
Authors: Seoane Asunción , Sánchez Emilia , García-Lobo Juan M.
Published: January 2003

Most
Yersinia enterocolitica
strains are resistant to β-lactam antibiotics due to the production of one or two chromosomally encoded β-lactamases. Strain Y56 is a
Y. enterocolitica
O:3 serotype natural isolate that is resistant to moderate amounts of penicillins and that produces a single class A β-lactamase. To select mutants with increased levels of resistance to β-lactam antibiotics, strain Y56 was grown on plates containing increasing amounts of ampicillin, and variants resistant to up to 500 μg of ampicillin per ml were obtained. Chromosomal DNA from hyperresistant isolates was analyzed by Southern hybridization with a
blaA
-specific probe to detect gene rearrangements. The use of pulsed-field gel electrophoresis revealed that the increase in the resistance level correlated with the amplification in tandem of a DNA fragment of about 28 kb containing the
blaA
gene. The phenotype of these isolates was not stable, and they recovered the basal low resistance level when the ampicillin used for selection was withdrawn from the growth medium. This loss of resistance was followed by the recovery of the original chromosomal structure. To understand this amplification process, the 28-kb amplification unit was cloned, and the ends were sequenced. The analysis of these sequences did not reveal the presence of either repeats or transposable elements to explain this process. However, we found short sequences similar to some DNA gyrase target sequences that have been described. In addition, we observed that the frequency of appearance of ampicillin-hyperresistant isolates by amplification of the
blaA
locus was lowered in the presence of the gyrase inhibitor novobiocin. These findings suggest that the DNA gyrase could be involved in this amplification event.
Effects of Organic Anion, Organic Cation, and Dipeptide Transport Inhibitors on Cefdinir in the Isolated Perfused Rat Kidney
Authors: Lepsy Christopher S. , Guttendorf Robert J. , Kugler Alan R. , Smith David E.
Published: January 2003

Cefdinir (Omnicef; Abbott Laboratories) is a cephalosporin antibiotic primarily eliminated by the kidney. Nonlinear renal elimination of cefdinir has been previously reported. Cefdinir renal transport mechanisms were studied in the erythrocyte-free isolated perfused rat kidney. Studies were performed with drug-free perfusate and perfusate containing cefdinir alone to establish the baseline physiology and investigate cefdinir renal elimination characteristics. To investigate cefdinir renal transport mechanisms, inhibition studies were conducted by coperfusing cefdinir with inhibitors of the renal organic anion (probenecid), organic cation (tetraethylammonium), or dipeptide (glycylsarcosine) transport system. Cefdinir concentrations in biological samples were determined using reversed-phase high-performance liquid chromatography. Differences between treatments and controls were evaluated using analysis of variance and Dunnett's test. The excretion ratio (ER; the renal clearance corrected for the fraction unbound and glomerular filtration rate) for cefdinir was 5.94, a value indicating net renal tubular secretion. Anionic, cationic, and dipeptide transport inhibitors all significantly affected the cefdinir ER. With probenecid, the ER was reduced to 0.59, clearly demonstrating a significant reabsorptive component to cefdinir renal disposition. This finding was confirmed by glycylsarcosine studies, in which the ER was elevated to 7.95, indicating that reabsorption was mediated, at least in part, by the dipeptide transporter system. The effects of the organic cation tetraethylammonium, in which the ER was elevated to 7.53, were likely secondary in nature. The anionic secretory pathway was found to be the predominant mechanism for cefdinir renal excretion.
Salmonella enterica
Serovar Typhimurium
bla
PER-1
-Carrying Plasmid pSTI1 Encodes an Extended-Spectrum Aminoglycoside 6′-
N
-Acetyltransferase of Type Ib
Authors: Casin Isabelle , Hanau-Berçot Beatrice , Podglajen Isabelle , Vahaboglu Haluk , Collatz Ekkehard
Published: January 2003

We have studied the aminoglycoside resistance gene, which confers high levels of resistance to both amikacin and gentamicin, that is carried by plasmid pSTI1 in the PER-1 β-lactamase-producing strain of
Salmonella enterica
serovar Typhimurium previously isolated in Turkey. This gene, called
aac(6
′

)-Ib
11

, was found in a class 1 integron and codes for a protein of 188 amino acids, a fusion product between the N-terminal moiety (8 amino acids) of the signal peptide of the β-lactamase OXA-1 and the acetyltransferase. The gene lacked a plausible Shine-Dalgarno (SD) sequence and was located 45 nucleotides downstream from a small open reading frame, ORF-18, with a coding capacity of 18 amino acids and a properly spaced SD sequence likely to direct the initiation of
aac(6
′

)-Ib
11

translation. AAC(6′)-Ib
11
had Leu118 and Ser119 as opposed to Gln and Leu or Gln and Ser, respectively, which were observed in all previously described enzymes of this type. We have evaluated the effect of Leu or Gln at position 118 by site-directed mutagenesis of
aac(6
′

)-Ib
11

and two other acetyltransferase gene variants,
aac(6
′

)-Ib
7

and

-Ib
8

, which naturally encode Gln118. Our results show that the combination of Leu118 and Ser119 confers an extended-spectrum aminoglycoside resistance, with the MICs of all aminoglycosides in clinical use, including gentamicin, being two to eight times higher for strains with Leu118 and Ser119 than for those with Gln118 and Ser119.
Efflux Pump Lde Is Associated with Fluoroquinolone Resistance in
Listeria monocytogenes
Authors: Godreuil Sylvain , Galimand Marc , Gerbaud Guy , Jacquet Christine , Courvalin Patrice
Published: January 2003

Five
Listeria monocytogenes
isolates (CLIP 21369, CLIP 73298, CLIP 74811, CLIP 75679, and CLIP 79372) were found to be resistant to fluoroquinolones during the screening for antibiotic resistance of 488
L. monocytogenes
isolates from human cases of listeriosis in France. On the basis of a fourfold or greater decrease in the ciprofloxacin MIC in the presence of reserpine, fluoroquinolone resistance was attributed to active efflux of the drugs. The
lde
gene (
Listeria
drug efflux; formerly
lmo2741
) encodes a 12-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of secondary transporters that displayed 44% identity with PmrA from
Streptococcus pneumoniae
. Insertional inactivation of the
lde
gene in CLIP 21369 indicated that the corresponding protein was responsible for fluoroquinolone resistance and was involved in the level of susceptibility to dyes such as ethidium bromide and acridine orange.
Genetics of Mefloquine Resistance in the Rodent Malaria Parasite
Plasmodium chabaudi
Authors: Cravo Pedro V. L. , Carlton Jane M.-R. , Hunt Paul , Bisoni Laura , Padua Rose Ann , Walliker David
Published: January 2003

The genetic determinants of resistance to mefloquine in malaria parasites are unclear. Some studies have implied that amplification of, or mutations in, the multidrug resistance gene
pfmdr1
in
Plasmodium falciparum
may be involved. Using the rodent malaria model
Plasmodium chabaudi
, we investigated the role of the orthologue of this gene,
pcmdr1
, in a stable mefloquine-resistant mutant, AS(15MF/3), selected from a sensitive clone.
pcmdr1
exists as a single copy gene on chromosome 12 of the sensitive clone. In AS(15MF/3), the gene was found to have undergone duplication, with one copy translocating to chromosome 4. mRNA levels of
pcmdr1
were higher in the mutant than in the parent sensitive clone. A partial genetic map of the translocation showed that other genes in addition to
pcmdr1
had been cotranslocated. The sequences of both copies of
pcmdr1
of AS(15MF/3) were identical to that of the parent sensitive clone. A cross was made between AS(15MF/3) and an unrelated mefloquine-sensitive clone, AJ. Phenotypic and molecular analysis of progeny clones showed that duplication and overexpression of the
pcmdr1
gene was an important determinant of resistance. However, not all mefloquine-resistant progeny contained the duplicated gene, showing that at least one other gene was involved in resistance.
Phenothiazines and Thioxanthenes Inhibit Multidrug Efflux Pump Activity in
Staphylococcus aureus
Authors: Kaatz Glenn W. , Moudgal Varsha V. , Seo Susan M. , Kristiansen Jette E.
Published: January 2003

Efflux-related multidrug resistance (MDR) is a significant means by which bacteria can evade the effects of selected antimicrobial agents. Genome sequencing data suggest that
Staphylococcus aureus
may possess numerous chromosomally encoded MDR efflux pumps, most of which have not been characterized. Inhibition of these pumps, which may restore clinically relevant activity of antimicrobial agents that are substrates for them, may be an effective alternative to the search for new antimicrobial agents that are not substrates. The inhibitory effects of selected phenothiazines and two geometric stereoisomers of the thioxanthene flupentixol were studied using strains of
S. aureus
possessing unique efflux-related MDR phenotypes. These compounds had some intrinsic antimicrobial activity and, when combined with common MDR efflux pump substrates, resulted in additive or synergistic interactions. For
S. aureus
SA-1199B, which overexpresses the NorA MDR efflux pump, and for two additional strains of
S. aureus
having non-NorA-mediated MDR phenotypes, the 50% inhibitory concentration (IC
50
) for ethidium efflux for all tested compounds was between 4 and 15% of their respective MICs. Transport of other substrates was less susceptible to inhibition; the prochlorperazine IC
50
for acriflavine and pyronin Y efflux by SA-1199B was more than 60% of its MIC. Prochlorperazine and
trans
(E)-flupentixol were found to reduce the proton motive force (PMF) of
S. aureus
by way of a reduction in the transmembrane potential. We conclude that the mechanism by which phenothiazines and thioxanthenes inhibit efflux by PMF-dependent pumps is multifactorial and, because of the unbalanced effect of these compounds on the MICs and the efflux of different substrates, may involve an interaction with the pump itself and, to a lesser extent, a reduction in the transmembrane potential.
Molecular Epidemiology of the SRL Pathogenicity Island
Authors: Turner Sally A. , Luck Shelley N. , Sakellaris Harry , Rajakumar Kumar , Adler Ben
Published: January 2003

The Shigella resistance locus (SRL), which is carried on the SRL pathogenicity island (PAI) in
Shigella flexneri
2a YSH6000, mediates resistance to the antibiotics streptomycin, ampicillin, chloramphenicol, and tetracycline. In the present study, we investigated the distribution and structural variation of the SRL and the SRL PAI in 71
Shigella
isolates and 28 other enteric pathogens by PCR and Southern analysis. The SRL and SRL-related loci, although absent from the other enteric pathogens evaluated in this study, were found to be present in a number of
Shigella
isolates. SRL PAI markers were also present in the majority of strains carrying the SRL and SRL-related loci. PCR linkage studies with six of these strains demonstrated that the SRL is carried on elements similar in structure and organization to the YSH6000 SRL PAI, consistent with the hypothesis that the SRL PAI may be involved in the spread of multiple-antibiotic resistance in these strains.
Fosmidomycin, a Novel Chemotherapeutic Agent for Malaria
Authors: Lell Bertrand , Ruangweerayut Ronnatrai , Wiesner Jochen , Missinou Michel Anoumou , Schindler Andreas , Baranek Thomas , Hintz Martin , Hutchinson David , Jomaa Hassan , Kremsner Peter Gottfried
Published: January 2003

In previous studies, fosmidomycin has been shown to possess activity against
Plasmodium falciparum
in vitro and in the mouse model. It has a novel mode of action through inhibition of 1-deoxy-
d
-xylulose 5-phosphate reductoisomerase, an enzyme of the nonmevalonate pathway of isoprenoid biosynthesis, which is absent in humans. In this open-label, uncontrolled trial, the efficacy and safety of fosmidomycin, in an oral dose of 1,200 mg every 8 h for 7 days, were evaluated in the treatment of acute uncomplicated
Plasmodium falciparum
malaria in 20 adult subjects in Gabon and Thailand. Clinical assessments were performed and thick blood smears were evaluated every 8 h until parasite clearance and resolution of symptoms were achieved; assessments continued at weekly intervals thereafter for the duration of the 28-day followup period. All subjects were clinically and parasitologically cured on day 7 (primary end point). Parasite and fever clearance were rapid, with means of 44 and 41 h, respectively. On day 28, seven out of nine subjects (78%) were cured in Gabon and two out of nine subjects (22%) were cured in Thailand. The drug was well tolerated, although mild gastrointestinal side effects were recorded for five subjects. Analysis of hematological and biochemical parameters showed no clinically significant changes throughout the study. Fosmidomycin is an effective and safe antimalarial drug, although its use as a single agent is restricted by the occurrence of recrudescent infections. However, its role in combination therapy should be explored.
Influence of Macrolide Susceptibility on Efficacies of Clarithromycin and Azithromycin against
Streptococcus pneumoniae
in a Murine Lung Infection Model
Authors: Hoffman Holly L. , Klepser Michael E. , Ernst Erika J. , Petzold C. Rosemarie , Sa'adah Loai Mohammed , Doern Gary V.
Published: January 2003

We evaluated the activities of clarithromycin and azithromycin against 19 isolates of
Streptococcus pneumoniae
using a neutropenic lung infection model. The isolates included five susceptible isolates (clarithromycin and azithromycin MICs, ≤0.12 μg/ml), nine isolates exhibiting low-level,
mefA
-mediated resistance (clarithromycin and azithromycin MICs, 0.5 to 32 μg/ml), and five isolates expressing high-level,
ermB
-mediated macrolide resistance (clarithromycin and azithromycin MICs, ≥64 μg/ml). Infected mice were administered either saline (control), clarithromycin (4, 40, or 200 mg/kg of body weight twice daily or 200 mg/kg once daily), or azithromycin (4, 40, or 200 mg/kg once daily or 40 mg/kg twice daily) by oral gavage for 72 h. Mortality was assessed at regular intervals for 10 days, and survival in each group was compared to that of untreated controls. Animals infected with susceptible isolates demonstrated significant improvement in survival compared to the controls following treatment with either agent at doses of ≥40 mg/kg. In contrast, none of the regimens improved the survival of animals infected with isolates exhibiting high-level macrolide resistance. Among mice infected with strains expressing low-level resistance, significant improvement in survival compared to the controls was noted among isolates treated with clarithromycin at 40 (seven of nine isolates) and 200 (nine of nine isolates) mg/kg twice a day and with azithromycin at 40 (one of nine isolates) and 200 (three of nine isolates) mg/kg once a day. Animals infected with isolates of
S. pneumoniae
exhibiting low-level,
mefA
-mediated macrolide resistance responded to treatment with clarithromycin at rates similar to those observed among mice infected with fully susceptible isolates.
Lamivudine Can Exert a Modest Antiviral Effect against Human Immunodeficiency Virus Type 1 Containing the M184V Mutation
Authors: Quan Yudong , Brenner Bluma G. , Oliveira Maureen , Wainberg Mark A.
Published: January 2003

The M184V mutation in human immunodeficiency virus (HIV) reverse transcriptase is associated with high-level resistance to both (−)2′,3′-dideoxy-3′-thiacytidine (3TC) and (−)2′,3′-dideoxy-5-fluoro-3′-thiacytidine as well as low-level resistance to 2′,3′-dideoxyinosine, 2′,3′-dideoxycytidine, and abacavir. This mutation is also associated with diminished HIV replicative fitness as well as several functional changes in enzyme activity, including diminutions in polymerase processivity, pyrophosphorylysis, and nucleotide primer unblocking. Despite the fact that M184V encodes up to 1,000-fold resistance to 3TC, we asked whether this drug might still display some antiviral effect in regard to viruses containing this mutation. Cell-free assays revealed that high concentrations of 3TC triphosphate (i.e., >100 μM) could affect chain termination and/or inhibit purified reverse transcriptase containing the M184V substitution. This effect became more pronounced with elongation of reverse transcriptase products. In newly infected cells (i.e., peripheral blood mononuclear cells), we found that the amount of full-length reverse transcriptase product was diminished in the presence of 2 to 10 μM 3TC, although no decrease in the first product of the reverse transcriptase reaction, i.e., minus strong-stop DNA, was observed. In the presence of two other HIV inhibitors, e.g., nevirapine and indinavir, 3TC exerted additive effects in tissue culture at concentrations only marginally higher than the 50% inhibitory concentration (IC50). Reverse transcriptases cloned from clinical isolates harboring M184V in the context of multidrug resistance had similar IC50values for 3TC triphosphate compared to reverse transcriptase containing only the M184V mutation. These results suggest that viruses containing M184V can retain a higher degree of sensitivity to 3TC than previously assumed.
Emergence inKlebsiella pneumoniaeof a Chromosome-Encoded SHV β-Lactamase That Compromises the Efficacy of Imipenem
Authors: Poirel Laurent , Héritier Claire , Podglajen Isabelle , Sougakoff Wladimir , Gutmann Laurent , Nordmann Patrice
Published: January 2003

AKlebsiella pneumoniaeisolate was identified that had reduced susceptibility to several expanded-spectrum cephalosporins and imipenem. That isolate produced a chromosome-encoded SHV-type β-lactamase, SHV-38, that had an alanine to valine substitution in position Ambler 146 compared to β-lactamase SHV-1. The kinetic parameters for purified β-lactamases SHV-38 and SHV-1 showed that the hydrolytic spectrum of SHV-38 included only ceftazidime and imipenem. This report is the first example of an SHV-type β-lactamase capable of hydrolyzing imipenem.
Selection of High-Level Resistance to Human Immunodeficiency Virus Type 1 Protease Inhibitors
Authors: Watkins Terri , Resch Wolfgang , Irlbeck David , Swanstrom Ronald
Published: January 2003

Protease inhibitors represent some of the most potent agents available for therapeutic strategies designed to inhibit human immunodeficiency virus type 1 (HIV-1) replication. Under certain circumstances the virus develops resistance to the inhibitor, thereby negating the benefits of this therapy. We have carried out selections for high-level resistance to each of three protease inhibitors (indinavir, ritonavir, and saquinavir) in cell culture. Mutations accumulated over most of the course of the increasing selective pressure. There was significant overlap in the identity of the mutations selected with the different inhibitors, and this gave rise to high levels of cross-resistance. Virus particles from the resistant variants all showed defects in processing at the NC/p1 protease cleavage site in Gag. Selections with pairs of inhibitors yielded similar patterns of resistance mutations. A virus that could replicate at near-toxic levels of the three protease inhibitors combined was selected. Theprosequence of this virus was similar to that of the viruses that had been selected for high-level resistance to each of the drugs singly. Finally, a molecular clone carrying the eight most common resistance mutations seen in these selections was characterized. The sequence of this virus was relatively stable during selection for revertants in spite of displaying poor processing at the NC/p1 site and having significantly reduced fitness. These results reveal patterns of drug resistance that extend to near the limits of attainable selective pressure with these inhibitors and confirm the patterns of cross-resistance for these three inhibitors and the attenuation of virion protein processing and fitness that accompanies high-level resistance.
In Vitro and In Vivo Pharmacokinetics-Pharmacodynamics of GV143253A, a Novel Trinem
Authors: Ferrari Livia , Iavarone Laura , Braggio Simone , Di Modugno Enza
Published: January 2003

The aim of the present study was to characterize the pharmacokinetic-pharmacodynamic relationship of GV143253A, a novel trinem anti-methicillin-resistantStaphylococcus aureus(MRSA) agent active against gram-positive cocci, including multidrug-resistant clinical isolates. An in vitro pharmacodynamic study with methicillin-susceptibleS. aureus(MSSA) and MRSA has shown that the duration of exposure to GV143253A rather than its concentration is the major determinant of the extent of bacterial killing. The in vitro findings were confirmed by use of a neutropenic murine model of thigh infection caused by MSSA ATCC 25923. From the dose-response curves, the static doses extrapolated for three different dosing intervals showed that more frequent dosing of GV143253A was more effective than less frequent dosing. A pharmacokinetic-pharmacodynamic analysis demonstrated that only the time during which the drug concentration exceeded the MIC (t>MIC) correlated with in vivo GV143253A activity. The value oft>MIC required to achieve a bacteriostatic effect in a thigh infection of neutropenic animals was 20% (95% confidence interval [CI], 18 to 22%) of the dosing interval. This result is similar to those reported in the literature for carbapenems and for GV104326A, another novel trinem compound. In addition, in order to compare the therapeutic efficacy of GV143253A to that of vancomycin in a thigh infection caused by MRSA in immunocompetent mice, suitable dosing regimens were designed on the basis of previous pharmacokinetic-pharmacodynamic findings for GV143253A and on the human pharmacokinetic profile of the glycopeptide. Although the pharmacokinetic profiles of the two agents were completely different, GV143253A showed good efficacy comparable to that of vancomycin, reducing by 4 log units the bacterial counts in the thighs of treated mice relative to untreated infected animals after 48 h of therapy. The results suggest that if the time of exposure to the pathogen above the MIC is at least 30% of the dosing interval, GV143253A could have a role in the clinical treatment of infections caused by MRSA, which is difficult to eradicate with current antibiotics.
Interferons, Interferon Inducers, and Interferon-Ribavirin in Treatment of Flavivirus-Induced Encephalitis in Mice
Authors: Leyssen Pieter , Drosten Christian , Paning Marcus , Charlier Nathalie , Paeshuyse Jan , De Clercq Erik , Neyts Johan
Published: January 2003

We evaluated the prophylactic and therapeutic efficacy of interferon α-2b, pegylated interferon α-2b, poly(I · C), and Ampligen against Modoc virus encephalitis in an animal model for flavivirus infections. All compounds significantly delayed virus-induced morbidity (paralysis) and mortality (due to progressive encephalitis). Viral load (as measured on day 7 postinfection) was significantly reduced by 80 to 100% in the serum, brain, and spleen in mice that had been treated with either interferon α-2b, pegylated interferon α-2b, poly(I · C), or Ampligen. We also studied whether a combination of interferon α-2b and ribavirin (presently the standard therapy for the treatment of infections with hepatitis C virus) would be more effective than treatment with interferon alone. However, ribavirin did not enhance the inhibitory effect of interferon therapy in this animal model for flavivirus infections.
Rapid In Vivo Screening of Experimental Drugs for Tuberculosis Using Gamma Interferon Gene-Disrupted Mice
Authors: Lenaerts Anne J. M. , Gruppo Veronica , Brooks Jason V. , Orme Ian M.
Published: January 2003

We have developed a rapid new in vivo method for screening experimental drugs for their activity against
Mycobacterium tuberculosis
by using the gamma interferon gene-disrupted (GKO) C57BL/6 mouse. Due to the rapid growth of the infection, statistical differences indicating positive efficacy of active compounds can be seen after only 8 days of treatment. To validate this model, several fluoroquinolones, including ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin, were tested in parallel.
Nonconjugative Transposition of the
vanB
-Containing Tn
5382
-Like Element in
Enterococcus faecium
Authors: Dahl Kristin H. , Røkenes Torunn P. , Lundblad Eirik W. , Sundsfjord Arnfinn
Published: January 2003

The
vanB
2 operon encoding glycopeptide resistance is an integral part of the putative conjugative transposon Tn
5382
. Characterization of clinical glycopeptide resistant derivatives from an epidemic ampicillin-resistant
Enterococcus faecium
strain showed precise chromosomal or plasmid insertions of a
vanB2
-containing Tn
5382
-like element. Conjugative transposition of the Tn
5382
-like element was not demonstrated in retransfer studies.
Clinical Isolates of
Enterobacteriaceae
Producing Extended-Spectrum β-Lactamases: Prevalence of CTX-M-3 at a Hospital in China
Authors: Wang Hui , Kelkar Swathi , Wu Weiyuan , Chen Minjun , Quinn John P.
Published: January 2003

The prevalence of extended-spectrum β-lactamase-producing strains was demonstrated in 5 of 44 (11.4%)
Escherichia coli
, 17 of 43 (39.5%)
Klebsiella pneumoniae
, 3 of 50 (6.0%)
Enterobacter cloacae
, and 2 of 25 (8.0%)
Citrobacter freundii
strains at a teaching hospital in China. Nineteen of these 27 strains expressed CTX-M-3 β-lactamase (pI 8.6). A subset of the clinical isolates expressing the CTX-M-3 enzyme, tested by pulsed-field gel electrophoresis, revealed multiple clones. Five isolates expressed a novel enzyme, SHV-43 (pI 8.0), which had two substitutions (Leu113Phe and Thr149Ser) compared with SHV-1.
Genetic Basis of Antibiotic Resistance in
Streptococcus agalactiae
Strains Isolated in a French Hospital
Authors: Poyart Claire , Jardy Laurence , Quesne Gilles , Berche Patrick , Trieu-Cuot Patrick
Published: January 2003

The genetic basis of antibiotic resistance in 113 unrelated group B streptococci was studied by PCR. Ninety-four strains were resistant to tetracycline-minocycline, and
tet
(M) was detected in 85% of these isolates. Seventeen erythromycin-resistant strains contained the
erm
(B),
erm
(TR), or
mef
(A) gene. Eleven strains exhibited high-level resistance to kanamycin due to the presence of the
aphA3
gene; eight of these strains were also highly resistant to streptomycin;
aad
-
6-
related sequences were detected in seven strains.
Conjugative Plasmid Conferring Resistance to Olaquindox
Authors: Sørensen Anders Hay , Hansen Lars Hestbjerg , Johannesen Elsebetta , Sørensen Søren J.
Published: January 2003

A conjugative plasmid, pOLA52, conferring resistance to the antibiotic growth promoter olaquindox has been isolated from
Escherichia coli
from swine manure. It also confers resistance to ampicillin and chloramphenicol and has a high frequency of transfer between strains of
E. coli
. Plasmid-borne olaquindox resistance has not been demonstrated before.
Growth-Inhibitory Effects of Artesunate, Pyrimethamine, and Pamaquine againstBabesia equiandBabesia caballiin In Vitro Cultures
Authors: Nagai Akiko , Yokoyama Naoaki , Matsuo Tomohide , Bork Sabine , Hirata Haruyuki , Xuan Xuenan , Zhu Yinchang , Claveria Florencia G. , Fujisaki Kozo , Igarashi Ikuo
Published: January 2003

Three antimalarial drugs, artesunate, pyrimethamine, and pamaquine, were evaluated for their growth-inhibitory effects againstBabesia equiandBabesia caballiin in vitro culture.B. equiwas more resistant to pyrimethamine thanB. caballi. B. equiwas also found to be more sensitive to artesunate and pamaquine thanB. caballi. Of the three compounds, pyrimethamine gave the most promise for in vivo effectiveness.
Molecular Epidemiology of Penicillin-Resistant and Ciprofloxacin-Resistant
Streptococcus pneumoniae
in Canada
Authors: Nichol Kimberly A. , Zhanel George G. , Hoban Daryl J.
Published: January 2003

Eighty-nine penicillin- and ciprofloxacin-resistant
Streptococcus pneumoniae
isolates were evaluated by serotyping and pulsed-field gel electrophoresis. Although penicillin-resistant isolates demonstrated considerable homogeneity, resistance to ciprofloxacin did not correlate with a reduction in genotypic variability. These results suggest that, unlike that of penicillin resistance, the spread of
S. pneumoniae
ciprofloxacin resistance in Canada is currently not attributable to clonal dissemination.
Daily Variations in Ceftriaxone Pharmacokinetics in Rats
Authors: Rebuelto M. , Ambros L. , Rubio M.
Published: January 2003

The aim of this study was to determine whether the time of day ceftriaxone was administered modified its pharmacokinetics. Ceftriaxone was given intraperitoneally at either 0400, 1000, 1600, and 2200 h to Sprague-Dawley rats synchronized under a light-dark cycle of 12 h of light and 12 h of dark. Pharmacokinetic parameters were analyzed for the presence of a 24-h rhythm. Results showed significant daily variations (
P
< 0.05) in ceftriaxone clearance, with the highest values during the dark phase. It is concluded that time-dependent variations in ceftriaxone pharmacokinetics may affect the therapeutic efficacy of current once-daily dosing schedules.
Efficacy of Amphotericin B or Itraconazole in a Murine Model of Central Nervous System
Aspergillus
Infection
Authors: Chiller Tom M. , Sobel Raymond A. , Luque Javier Capilla , Clemons Karl V. , Stevens David A.
Published: January 2003

Given the greater than 90% lethality of clinical central nervous system (CNS) aspergillosis despite current therapies, there is a need for an animal model to study therapeutic strategies. We previously established a model of CNS aspergillosis by intracerebral infection and report here the results of treatment with the two therapies with the greatest clinical experience, i.e., treatments with amphotericin B (AMB) and itraconazole (ITZ). Mice were given cyclophosphamide to produce pancytopenia. AMB was given intraperitoneally (i.p.; 3 mg/kg of body weight) or intravenously (i.v.; 0.8 mg/kg) once daily. ITZ in cyclodextrin was given by gavage once daily at a dose of 100 mg/kg or twice daily at 50 mg/kg. Treatments were started at day 1 postinfection and given for 10 days. At day 15, survivors were euthanatized. Ninety percent of the mice given no treatment died by day 6, and 100% died by day 10. Mice treated with AMB either i.p. or i.v. had 40% survival. Mice treated with ITZ either once or twice per day had a median survival time of 10 days, compared with 4 days for control animals, but a survival rate of only 10%. AMB and ITZ prolonged survival (
P
, <0.0001 to <0.05) compared with controls. Brains from surviving mice had CFU of
Aspergillus fumigatus
. This model can be used to compare newer antifungals and to study combination therapy or immunotherapy to find better therapeutic alternatives.
Levofloxacin and Ciprofloxacin In Vitro Activities against 4,003 Clinical Bacterial Isolates Collected in 24 Italian Laboratories
Authors: Gesu Giovanni Pietro , Marchetti Federico , Piccoli Laura , Cavallero Annalisa
Published: January 2003

Levofloxacin showed comparable in vitro susceptibility to ciprofloxacin among
Enterobacteriaceae
,
Pseudomonas aeruginosa
, enterococci, and
Staphylococcus aureus
, while greater susceptibility was observed in
Stenotrophomonas maltophilia
and
Staphylococcus epidermidis
, mainly when oxacillin resistant. The susceptibility of
Streptococcus pneumoniae
to levofloxacin reached 99%.
Pharmacokinetics of Florfenicol in Healthy Pigs and in Pigs Experimentally Infected with
Actinobacillus pleuropneumoniae
Authors: Liu Jianzhong , Fung Ki-Fai , Chen Zhangliu , Zeng Zhenling , Zhang Jie
Published: January 2003

A comparative in vivo pharmacokinetic study of florfenicol was conducted in 18 crossbred pigs infected with
Actinobacillus pleuropneumoniae
following intravenous (i.v.), intramuscular (i.m.), or oral (p.o.) administration of a single dose of 20 mg/kg. The disease model was confirmed by clinical signs, X rays, pathohistologic examinations, and organism isolation. Florfenicol concentrations in plasma were determined by a validated high-performance liquid chromatography method with UV detection at a wavelength of 223 nm. Pharmacokinetic parameters were calculated by using the MCPKP software (Research Institute of Traditional Chinese Veterinary Medicine, Lanzhou, China). The disposition of florfenicol after a single i.v. bolus was described by a two-compartment model with values for the half-life at α phase (
t
1/2α
), the half-life at β phase (
t
1/2β
), the area under the concentration-time curve (AUC
0-∞
), and the volume of distribution at steady state (
V
ss
) of 0.37 h, 2.91 h, 64.86 μg · h/ml, and 1.2 liter/kg, respectively. The concentration-time data fitted the one-compartment (after i.m.) and two-compartment (after p.o.) models with first-order absorption. The values for the maximum concentration of drug in serum (
C
max
),
t
1/2α
,
t
1/2β
, and bioavailability after i.m. and p.o. dosing were 4.00 and 8.11 μg/ml, 0.12 and 3.91 h, 13.88 and 16.53 h, and 122.7 and 112.9%, respectively, for the two models. The study showed that florfenicol was absorbed quickly and completely, distributed widely, and eliminated slowly in the infected pigs, and there was no statistically significant difference between the pharmacokinetic profiles for the infected and healthy pigs.
Serotype Distribution and Antimicrobial Resistance of
Streptococcus pneumoniae
Isolated in Algiers, Algeria
Authors: Ramdani-Bouguessa Nadjia , Rahal Kheira
Published: January 2003

There are few data on antibiotic resistance of
Streptococcus pneumoniae
in Algeria. Among 309 strains, 34.6% were penicillin G-nonsusceptible
S. pneumoniae
strains (25.2% were intermediate and 9.4% were resistant). Serotypes 1, 5, 14, and 6 were the most frequent in invasive child infections. A multicenter study to standardize the national guidelines is needed.
Risk factors for lower extremity injury: a review of the literature
Authors: Murphy D F , Connolly D A J , Beynnon B D
Published: January 2003

Prospective studies on risk factors for lower extremity injury are reviewed. Many intrinsic and extrinsic risk factors have been implicated; however, there is little agreement with respect to the findings. Future prospective studies are needed using sufficient sample sizes of males and females, including collection of exposure data, and using established methods for identifying and classifying injury severity to conclusively determine addtional risk factors for lower extremity injury.
Musculoskeletal manifestations of diabetes mellitus
Authors: Smith L L , Burnet S P , McNeil J D
Published: January 2003

Rheumatic complaints are common in patients with diabetes. Maintaining good glycaemic control by exercise, diet, and medication improves or prevents the development of rheumatic conditions.
Incidence of injury in semi-professional rugby league players
Authors: Gabbett T J
Published: January 2003

Objectives:To investigate the site, nature, cause, and severity of injuries in semi-professional rugby league players.

Methods:The incidence of injury was prospectively studied in one hundred and fifty six semi-professional rugby league players over two competitive seasons. All injuries sustained during matches and training sessions were recorded. Injury data were collected from a total of 137 matches and 148 training sessions. Information recorded included the date and time of injury, site, nature, cause, and severity of injury.

Results:During the two seasons, 1694 playing injuries and 559 training injuries were sustained. The match injury incidence was 824.7 per 1000 player-position game hours and training injury incidence was 45.3 per 1000 training hours. Over 20% of the total training (17.4 per 1000) and playing (168.0 per 1000) injuries sustained were to the thigh and calf. Muscular injuries (haematomas and strains) were the most common type of injury sustained during training (22.0 per 1000, 48.7%) and matches (271.7 per 1000, 32.9%). Playing injuries were most commonly sustained in tackles (382.2 per 1000, 46.3%), while overexertion was the most common cause of training injuries (15.5 per 1000, 34.4%). The majority of playing injuries were sustained in the first half of matches (1013.6 per 1000, 61.5%v635.8 per 1000, 38.5%), whereas training injuries occurred more frequently in the latter stages of the training session (50.0 per 1000, 55.3%v40.5 per 1000, 44.7%). Significantly more training injuries were sustained in the early half of the season, however, playing injuries occurred more frequently in the latter stages of the season.

Conclusions:These results suggest that changes in training and playing intensity impact significantly upon injury rates in semi-professional rugby league players. Further studies investigating the influence of training and playing intensity on injuries in rugby league are warranted.
The effect of glucosamine supplementation on people experiencing regular knee pain
Authors: Braham R , Dawson B , Goodman C
Published: January 2003

Objective:The purpose of this study was to examine the effects of oral glucosamine supplementation on the functional ability and degree of pain felt by individuals who had regular knee pain, most likely due to previous articular cartilage damage, and possibly osteoarthritis.

Methods:Subjects were randomly supplemented with either glucosamine (G) (n=24) or placebo (P) (lactose) (n=22) for 12 weeks at a dose of 2000 mg per day. Over this period, four testing sessions were conducted, with changes in knee pain and function assessed by clinical and functional tests, (joint line palpation, a 3 metre “duck walk” and a repeated, walking stair climb), two questionnaires (the Knee Injury and Osteoarthritis Outcome Score (KOOS) and the Knee Pain Scale (KPS)) and participant subjective evaluations.

Results:The clinical and functional test scores improved with time (main effects: p<0.05, p<0.01) but there were no significant differences between the two groups. The questionnaire results also recorded a significant main effect for time (p<0.05), but the glucosamine group was found to have significantly better KOOS quality of life scores at week eight and 12 (p<0.05), and lower KPS scores (p<0.05) at week eight than the placebo group. On self report evaluations of changes across the 12 week supplementation period, 88% (n=21) of the glucosamine group reported some degree of improvement in their knee pain versus only 17% (n=3) in the placebo group.

Conclusions:These results suggest that glucosamine supplementation can provide some degree of pain relief and improved function in persons who experience regular knee pain, which may be caused by prior cartilage injury and/or osteoarthritis. The trends in the results also suggest that, at a dosage of 2000 mg per day, the majority of improvements are present after eight weeks.
The physiological effects of cycling on tandem and single bicycles
Authors: Seifert J G , Bacharach D W , Burke E R
Published: January 2003

Objective:The purpose of this field study was to compare the physiological responses from cycling on a tandem road bicycle to those from cycling on a single road bicycle.

Methods:Nine pairs of experienced, recreational tandem cyclists rode a tandem or their single bicycle for 5 min at each velocity of 19.3, 22.5, 25.8, and 29.0 kph on a flat, paved surface. Heart rate (HR), rating of perceived exertion (RPE), and lactic acid (LA) data were collected after each interval.

Results:Riding a tandem resulted in lower HR, RPE, and LA mean values across the four velocities compared to the single bicycle. Mean (SD) HR, RPE, and LA for tandem and single bicycles were 126 (20.7)v142 (20.1) bpm, 10.1 (1.7)v11.3 (2.6), and 1.46 (1.0) mM/Lv2.36 (1.7) mM/L, respectively. No position differences were observed between the captain and stoker (front and rear positions) when both were on the tandem. Stokers had significantly lower HR, LA, and RPE values when they rode a tandem compared to a single bicycle. No statistical differences were observed between bicycles for the captains. When on the single bicycle, captains exhibited significantly lower HR, RPE, and LA values than stokers.

Conclusion:Cycling on a tandem resulted in lower physiological stress than when cycling at the same velocity on a single bicycle. Cyclists were able to ride from 4.8–8.0 kph faster on a tandem than on a single bicycle at similar physiological stress. Apparently, stokers can add to power output on a tandem without adding significantly to wind resistance.