bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic Study of the Inflammatory Response in
Streptococcus pneumoniae
Experimental Pneumonia Treated with the Ketolide HMR 3004
Authors: Duong Michel , Simard Marie , Bergeron Yves , Bergeron Michel G.
Published: July 2002

Patients still die from
Streptococcus pneumoniae
pneumonia after initiation of antibiotic therapy, when tissues are sterile and the pneumonia is clearing. There is growing evidence that overwhelming inflammation resulting from toxin release contributes to tissue injury, shock, and death. Monitoring host response may help us understand the consequences of antibiotic therapy for the inflammatory processes that occur in bacterial pneumonia. HMR 3004 is a ketolide that displays excellent in vitro activity against
S. pneumoniae
. In the present experiment, we investigated the chronology of inflammatory events that occur during pneumococcal pneumonia in mice treated with HMR 3004. Infection of mice with 10
7
CFU of living
S. pneumoniae
resulted in 100% mortality within 5 days. HMR 3004 given at 12.5 mg/kg of body weight/dose twice daily from 48 h postinfection achieved complete bacterial clearance from lungs and blood within 36 h and ensured survival of mice. Recruitment of neutrophils and monocytes from blood to lungs was significantly reduced, and nitric oxide release was totally prevented. Interleukin-6 secretion in lungs and blood became rapidly undetectable after initiation of therapy. Histological examination of lung tissue showed protection of interstitium against edema. By controlling bacterial invasion, HMR 3004 led to rapid and profound modifications of the host response in lungs, which may protect mice from deleterious inflammatory reactions.
Use of a Recombinant Strain of
Mycobacterium avium
Expressing β-Galactosidase To Evaluate the Activities of Antimycobacterial Agents inside Macrophages
Authors: Maisetta Giuseppantonio , Batoni Giovanna , Pardini Manuela , Boschi Antonella , Bottai Daria , Esin Semih , Campa Mario , Senesi Sonia
Published: July 2002

A reliable and low-cost method that enables rapid screening of the activity exerted by new antimicrobial agents on intracellularly growing
Mycobacterium avium
has been developed. To this aim, a recombinant (
lacZ
) strain of
M. avium
expressing the
Escherichia coli
β-galactosidase gene was used to evaluate, in murine macrophages, the susceptibility of
M. avium
to common antimycobacterial agents. β-Galactosidase levels, measured in the presence of each of the antibiotics tested, were closely correlated with the number of CFU recovered from the
M. avium lacZ
strain-infected macrophages.
Correlation between In Vitro and In Vivo Activities of GM 237354, a New Sordarin Derivative, against
Candida albicans
in an In Vitro Pharmacokinetic-Pharmacodynamic Model and Influence of Protein Binding
Authors: Aviles P. , Falcoz C. , Guillén M. J. , San Roman R. , Gómez De Las Heras F. , Gargallo-Viola D.
Published: July 2002

The antifungal effect of GM 237354, a sordarin derivative, was studied in an in vitro pharmacokinetic (PK)-pharmacodynamic dynamic system (bioreactor) which reproduces PK profiles observed in a previously described model of drug efficacy against murine systemic candidiasis. Immunocompetent mice infected intravenously with 10
5
CFU of
Candida albicans
were treated with GM 237354 at 2.5, 10, and 40 mg/kg of body weight every 8 h subcutaneously for 7 days. Free concentrations in serum were calculated by multiplying total concentrations measured in vivo by 0.05, the free fraction determined in vitro by equilibrium dialysis. In the bioreactor the inoculum was ≈10
6
CFU/ml; and a one-compartment PK model was used to reproduce the PK profiles of free and total GM 237354 in serum obtained in mice, and clearance of
C. albicans
was measured over 48 h. A good correlation was observed when the in vivo fungal kidney burden and the area under the survival time curve were compared with the in vitro broth “burden,” although only when free in vivo levels in serum were reproduced in vitro. GM 237354 displayed a 3-log decrease effect both in vivo and in vitro. The very few reports available on in vitro-in vivo correlations have been obtained with antibiotics. The good in vitro-in vivo correlation obtained with an antifungal agent shows that the in vitro dynamic system could constitute a powerful investigational tool prior to assessment of the efficacy of an anti-infective agent in animals and humans.
Antimicrobial Susceptibilities of Clinical
Desulfovibrio
Isolates
Authors: Lozniewski A. , Labia R. , Haristoy X. , Mory F.
Published: July 2002

The antimicrobial susceptibilities of 16 clinical isolates of
Desulfovibrio
spp. were determined. All or most isolates were susceptible to imipenem (MIC
90
[MIC at which 90% of the isolates tested were inhibited], 0.5 μg/ml), metronidazole (MIC
90
, 0.25 μg/ml), clindamycin (MIC
90
, 4 μg/ml), and chloramphenicol (MIC
90
, 16 μg/ml) but were resistant or intermediate to penicillin G (MIC
90
, 64 μg/ml), piperacillin (MIC
90
, 256 μg/ml), piperacillin-tazobactam (MIC
90
, 256 μg/ml), cefoxitin (MIC
90
, >256 μg/ml), and cefotetan (MIC
90
, 64 μg/ml). Among isolates with decreased susceptibility to β-lactams (
n
= 15), only six were β-lactamase positive and susceptible to amoxicillin-clavulanate and ticarcillin-clavulanate.
Efficacies of Clarithromycin Regimens against
Mycobacterium xenopi
in Mice
Authors: Lounis Nacer , Truffot-Pernot Chantal , Bentoucha Abdelhalim , Robert Jerome , Ji Baohong , Grosset Jacques
Published: July 2002

Mice were infected intravenously with 3.5 × 10
7
CFU of
Mycobacterium xenopi
and treated with various clarithromycin-containing regimens or left untreated for 4 weeks. All nine of the clarithromycin-containing regimens reduced the CFU counts to the levels below the pretreatment values, indicating that these regimens had a bactericidal effect on
M. xenopi
in mice. The rifampin-isoniazid-ethambutol regimen was significantly less bactericidal than clarithromycin alone or clarithromycin-containing combined regimens.
Staphylococcus aureus
Mutants Selected by BMS-284756
Authors: Discotto L. F. , Lawrence L. E. , Denbleyker K. L. , Barrett J. F.
Published: July 2002

BMS-284756, a novel des-fluoro(6)-quinolone, was used to select for in vitro mutants of
Staphylococcus aureus
ISP794. Step mutants were obtained, and the quinolone resistance-determining regions of four target genes,
gyrA, gyrB, grlA
, and
grlB
, were sequenced. The data suggest that DNA gyrase is the primary target for BMS-284756 in
S. aureus
.
Postantibiotic Effects of ABT-773 and Amoxicillin-Clavulanate against
Streptococcus pneumoniae
and
Haemophilus influenzae
Authors: Neuhauser Melinda M. , Prause Jennifer L. , Danziger Larry H. , Pendland Susan L.
Published: July 2002

This study determined the postantibiotic effect (PAE) of ABT-773 versus that of amoxicillin-clavulanate against clinical isolates of
Streptococcus pneumoniae
and
Haemophilus influenzae
. The PAEs of ABT-773 and amoxicillin-clavulanate ranged from 2.3 to 6.0 h and 0 to 2.2 h against
S. pneumoniae
and from 2.7 to 9.1 h and 0 to 0.8 h against
H. influenzae
, respectively.
Pharmacokinetic Study of Human Immunodeficiency Virus Protease Inhibitors Used in Combination with Amprenavir
Authors: Sadler Brian M. , Gillotin Catherine , Lou Yu , Eron Joseph J. , Lang William , Haubrich Richard , Stein Daniel S.
Published: July 2002

In an open-label, randomized, multicenter, multiple-dose pharmacokinetic study, we determined the steady-state pharmacokinetics of amprenavir with and without coadministration of indinavir, nelfinavir, or saquinavir soft gel formulation in 31 human immunodeficiency virus type 1-infected subjects. The results indicated that amprenavir plasma concentrations were decreased by saquinavir soft gel capsule (by 32% for area under the concentration-time curve at steady state [AUC
ss
] and 37% for peak plasma concentration at steady state [
C
max,ss
]) and increased by indinavir (33% for AUC
ss
). Nelfinavir significantly increased amprenavir minimum drug concentration at steady state (by 189%) but did not affect amprenavir AUC
ss
or
C
max,ss
. Nelfinavir and saquinavir steady-state pharmacokinetics were unchanged by coadministration with amprenavir compared with the historical monotherapy data. Concentrations of indinavir, coadministered with amprenavir, in plasma decreased in both single-dose and steady-state evaluations. The changes in amprenavir steady-state pharmacokinetic parameters, relative to those for amprenavir alone, were not consistent among protease inhibitors, nor were the changes consistent with potential interactions in CYP3A4 metabolism or P-glycoprotein transport. No dose adjustment of either protease inhibitor in any of the combinations studied is needed.
Differential Selection of Multidrug Efflux Mutants by Trovafloxacin and Ciprofloxacin in an Experimental Model of
Pseudomonas aeruginosa
Acute Pneumonia in Rats
Authors: Join-Lambert O. F. , Michéa-Hamzehpour M. , Köhler T. , Chau F. , Faurisson F. , Dautrey S. , Vissuzaine C. , Carbon C. , Pechère J.-C.
Published: July 2002

The ability of trovafloxacin and ciprofloxacin to select efflux mutants in vivo was studied in a model of acute
Pseudomonas aeruginosa
pneumonia in rats. Twelve hours after intratracheal inoculation of 10
6
CFU of
P. aeruginosa
strain PAO1 enmeshed in agar beads, two groups of 12 rats were treated by three intraperitoneal injections of each antibiotic given every 5 h. Dosing regimens were chosen to obtain a comparable area under the concentration-time curve from 0 to infinity/MIC ratio of 27.9 min for trovafloxacin (75 mg/kg of body weight) and of 32.6 min for ciprofloxacin (12.5 mg/kg). Twelve rats were left untreated and served as controls. Rats were sacrificed 12 h after the last injection (34 h after infection) for lung bacteriological studies. Selection of resistant bacteria was determined by plating lung homogenates on Trypticase soy agar plates containing antibiotic. In untreated animals, the frequency of resistant colonies was 10-fold higher than in agar beads. Compared to controls, both treatment regimens resulted in a 2-log reduction of lung bacterial load. The frequency of resistant colonies was 10-fold less with trovafloxacin than with ciprofloxacin at twice the MIC (7.4 × 10
−5
versus 8.4 × 10
−4
, respectively) (
P
< 0.05) and at four times the MIC (6.2 × 10
−4
versus 5.0 × 10
−5
, respectively) (
P
< 0.05). A multidrug resistance phenotype typical of efflux mutants was observed in all 41 randomly tested colonies obtained from treated and untreated rats. In agreement with in vitro results, trovafloxacin and ciprofloxacin preferentially selected MexCD-OprJ and MexEF-OprN overproducers, respectively. These results demonstrate the differential ability of trovafloxacin and ciprofloxacin to select efflux mutants in vivo and highlight the rapid emergence of those mutants, even without treatment.
Nucleotide Sequence and Characterization of a Novel Cefotaxime-Hydrolyzing β-Lactamase (CTX-M-10) Isolated in Spain
Authors: Oliver Antonio , Pérez-Dı́az José Claudio , Coque Teresa M. , Baquero Fernando , Cantón Rafael
Published: July 2002

A cefotaxime-resistant, ceftazidime-susceptible
Escherichia coli
isolate was obtained from a patient with sepsis in 1997, from which a β-lactamase with a pI of 8.1 was cloned. Cephaloridine and cefotaxime relative hydrolysis rates were 167 and 81, respectively (penicillin G rate = 100), whereas ceftazidime hydrolysis was not detected. The nucleotide sequence revealed a
bla
gene related to that coding for CTX-M-3. Despite 21 nucleotide substitutions, only 2 determined amino acid changes (Ala27Val and Arg38Gln). The amino acid sequence identity between this enzyme, designated CTX-M-10, and the chromosomal β-lactamase of
Kluyvera ascorbata
was 81%.
Macrolide Resistance in
Peptostreptococcus
spp. Mediated by
ermTR
: Possible Source of Macrolide-Lincosamide-Streptogramin B Resistance in
Streptococcus pyogenes
Authors: Reig Milagro , Galan Juan-Carlos , Baquero Fernando , Perez-Diaz Jose Claudio
Published: July 2002

Eighty percent (21 of 26) of macrolide-resistant
Peptostreptococcus
strains studied harbored the
ermTR
gene. This methyltransferase gene is also the most frequently found gene among macrolide-lincosamide-streptogramin B-resistant
Streptococcus pyogenes
strains. Transfer of the
ermTR
gene from
Peptostreptococcus magnus
to macrolide-susceptible
S. pyogenes
strains indicates that this resistance determinant may circulate among gram-positive aerobic and anaerobic species of the oropharyngeal bacterial flora.
Quinolone Resistance in Staphylococci: Activities of New Nonfluorinated Quinolones against Molecular Targets in Whole Cells and Clinical Isolates
Authors: Roychoudhury Siddhartha , Catrenich Carl E. , McIntosh Eric J. , McKeever Helana D. , Makin Kelly M. , Koenigs Paula M. , Ledoussal Benoit
Published: July 2002

The activity of three new, 8-methoxy-nonfluorinated quinolones (NFQs) against multiple-drug-resistant staphylococci was investigated. First, using
Staphylococcus aureus
strains containing point mutations in the serine 84–80 hot spots of the target genes (
gyrA
and
grlA
), cell growth inhibition potencies of the NFQs as a result of DNA gyrase and topoisomerase IV inhibition were estimated and compared with those of known fluoroquinolones. The NFQs and clinafloxacin showed higher affinities toward both the targets than ciprofloxacin, trovafloxacin and gatifloxacin. Furthermore, the ratio of the calculated affinity parameter for DNA gyrase to that for topoisomerase IV was lower in the case of the NFQs, clinafloxacin, and gatifloxacin than in the case of ciprofloxacin and trovafloxacin. These results suggest that the former group of quinolones is better able to exploit both the targets. Next, using clinical isolates of methicillin-resistant
S. aureus
(MRSA;
n
= 34) and coagulase-negative staphylococci (CoNS;
n
= 24), the NFQs and clinafloxacin were shown to be more potent (MIC at which 90% of the isolates are inhibited [MIC
90
] = 2 μg/ml for MRSA and 0.5 μg/ml for CoNS) than ciprofloxacin, trovafloxacin, and gatifloxacin (MIC
90
= 16 to >64 μg/ml for MRSA and 4 to >32 μg/ml for CoNS). Bactericidal kinetics experiments, using two MRSA isolates, showed that exposure to the NFQs at four times the MIC reduced the bacterial counts (measured in CFU per milliliter) by ≥3 log units in 2 to 4 h. Overall, the NFQs and clinafloxacin were less susceptible than the other quinolones to existing mechanisms of quinolone resistance in staphylococci.
In Vitro and in Vivo Combinations of Cefotaxime and Minocycline against
Aeromonas hydrophila
Authors: Ko Wen-Chien , Lee Hsin-Chun , Chuang Yin-Ching , Ten Shih-Hua , Su Chia-Yang , Wu Jiunn-Jong
Published: July 2002

The activities of cefotaxime and minocycline against
Aeromonas hydrophila
were investigated. Cefotaxime (4 times the MIC) plus minocycline (0.75 times the MIC) elicited an inhibitory effect for 48 h in a time-kill study, and more infected mice treated with both drugs survived (91%) than survived after treatment with cefotaxime (9%) or minocycline (44%) alone, suggesting that cefotaxime and minocycline act synergistically against
A. hydrophila
.
Pharmacodynamic Assessment of Gatifloxacin against
Streptococcus pneumoniae
Authors: Mattoes Holly M. , Banevicius Maryanne , Li Dadong , Turley Christina , Xuan Dawei , Nightingale Charles H. , Nicolau David P.
Published: July 2002

The pharmacodynamic parameters of peak serum drug concentration/MIC (peak/MIC) ratio and the area under the curve (AUC)/MIC ratio have been used to characterize in vivo drug exposure and its relationship to bacterial killing for the fluoroquinolones. Our study objectives were to describe the pharmacodynamic relationship between gatifloxacin exposure and outcome as assessed by bacterial density and survival in an immunocompromised murine thigh model of pneumococcal infection and to assess the relationship between drug exposure and these outcomes in an immunocompetent host. ICR mice were rendered neutropenic, and thigh infection was induced by intramuscular administration of 0.1 ml of 10
5
to 10
7
CFU of
Streptococcus pneumoniae
/ml. Mice received 1 to 5 mg of uranyl nitrate/kg of body weight at day −3 and were randomized to receive 10 to 80 mg of gatifloxacin/kg every 6 to 24 h orally, starting at 2 h postinoculation. Bacterial density studies were completed 24 h after initiation of therapy, and survival was assessed after 4 days of treatment. MICs for clinical isolates (
n
= 8) ranged from 0.25 to 1.0 μg/ml. Correlations were assessed between the change in bacterial density, as well as survival, and the AUC/MIC ratio, peak/MIC ratio, and the duration of time that serum drug concentration remained above the MIC. The best predictor of bacterial response was the AUC/MIC ratio for both outcome measures. There was greater efficacy, as measured by a decrease in log change in CFU as well as by survival data, in the immunocompetent mice compared to the immunocompromised mice. These data demonstrate (i) the appropriateness of the AUC/MIC ratio as a dynamic predictor of response to pneumococcal infection for the fluoroquinolones, (ii) that gatifloxacin AUC/MIC ratios of 30 to 40 appear to optimize bactericidal activity and survival in this model, and (iii) that immunocompetency of the host plays a role in efficacy.
Discrimination of SHV β-Lactamase Genes by Restriction Site Insertion-PCR
Authors: Chanawong Aroonwadee , M'Zali Fatima Hannachi , Heritage John , Lulitanond Aroonlug , Hawkey Peter Michael
Published: July 2002

Restriction site insertion-PCR (RSI-PCR) is a simple, rapid technique for detection of point mutations. This technique exploits primers with one to three base mismatches near the 3′ end to modulate a restriction site. We have developed this technique to identify described mutations of the
bla
SHV
genes for differentiation of SHV variants that cannot be distinguished easily by other techniques. To validate this method, eight standard strains were used, each producing a different SHV β-lactamase: SHV-1, SHV-2, SHV-3, SHV-4, SHV-5, SHV-6, SHV-8, and SHV-18. Mismatch primers were designed to detect mutations affecting amino acids at positions 8 (
Ssp
I), 179 (
Hin
fI), 205 (
Pst
I), 238 (Gly→Ala) (
Bsr
I), and 240 (
Nru
I) of
bla
SHV
genes. All amplimers of the
bla
SHV
genes used in this study yielded the predicted restriction endonuclease digestion products. In addition, this study also makes theoretical identification of
bla
SHV-6
,
bla
SHV-8
, and 12 novel
bla
SHV
variants using the PCR-restriction fragment length polymorphism (RFLP) technique possible. By using a combination of PCR-RFLP and RSI-PCR techniques, up to 27 SHV variants can now be distinguished rapidly and reliably. These simple techniques are readily applied to epidemiological studies of the SHV β-lactamases and may be extended to the characterisation of other resistance determinants.
Isolation of Rat Dihydrofolate Reductase Gene and Characterization of Recombinant Enzyme
Authors: Wang Yangzhou , Bruenn Jeremy A. , Queener Sherry F. , Cody Vivian
Published: July 2002

While assays of many antifolate inhibitors for dihydrofolate reductase (DHFR) have been performed using rat DHFR as a target, neither the sequence nor the structure of rat DHFR is known. Here, we report the isolation of the rat DHFR gene through screening of a rat liver cDNA library. The rat liver DHFR gene has an open reading frame of 561 bp encoding a protein of 187 amino acids. Comparisons of the rat enzyme with those from other species indicate a high level of conservation at the primary sequence level and more so for the amino acid residues comprising the active site of the enzyme. Expression of the rat DHFR gene in bacteria produced a recombinant protein with high enzymatic activity. The recombinant protein also paralleled the human enzyme with respect to the inhibition by most of the antifolates tested with PT652 and PT653 showing a reversal in their patterns. Our results indicated that rat DHFR can be used as a model to study antifolate compounds as potential drug candidates. However, variations between rat and human DHFR enzymes, coupled with unique features in the inhibitors, could lead to the observed differences in enzyme sensitivity and selectivity.
Efficacies of ABT-773, a New Ketolide, against Experimental Bacterial Infections
Authors: Mitten M. J. , Meulbroek J. , Nukkala M. , Paige L. , Jarvis K. , Oleksijew A. , Tovcimak A. , Hernandez L. , Alder J. D. , Ewing P. , Or Y. S. , Ma Z. , Nilius A. M. , Mollison K. , Flamm R. K.
Published: July 2002

ABT-773 is a novel ketolide effective against antibacterial-resistant respiratory tract pathogens. The pharmacokinetic profile of ABT-773 was studied in rats and consisted of a mean peak concentration in plasma of 1.07 μg/ml and an area under the concentration-time curve (AUC) of 12.03 μg · h/ml when the compound was delivered at a dose of 25 mg/kg of body weight. It concentrated in rat lung tissue, with a lung tissue-to-plasma ratio of 29 based on the AUC. In acute systemic infections in mice, ABT-773 showed efficacy against macrolide-susceptible strains of
Staphylococcus aureus
,
Streptococcus pneumoniae
,
S. pyogenes
, and
Listeria monocytogenes
. Additionally, ABT-773 improved the survival of mice infected with resistant
S. pneumoniae
containing either the
ermB
gene, the
mefE
gene, or altered penicillin binding protein genes. In a rat lung model of infection, ABT-773 demonstrated 50% effective doses lower than those of comparator macrolides when evaluated against the following strains of
S. pneumoniae
: a macrolide-lincosamide-streptogramin B-susceptible strain, an
ermB
strain, and an
mefE
strain
.
ABT-773 was also effective against
Haemophilus influenzae
lung infections in rats. Thus, ABT-773 may prove to be a useful new antibacterial agent for the treatment of respiratory tract infections.
Increased Glycan Chain Length Distribution and Decreased Susceptibility to Moenomycin in a Vancomycin-Resistant
Staphylococcus aureus
Mutant
Authors: Komatsuzawa Hitoshi , Ohta Kouji , Yamada Sakuo , Ehlert Kerstin , Labischinski Harald , Kajimura Junko , Fujiwara Tamaki , Sugai Motoyuki
Published: July 2002

A vancomycin-resistant
Staphylococcus aureus
mutant, COL-VR1 (MIC, 16 μg/ml), was isolated from methicillin-resistant
S. aureus
COL by exposure to vancomycin. COL-VR1 also showed decreased susceptibility to teicoplanin (8-fold), methicillin (2-fold), macarbomycin (8-fold), and moenomycin (16-fold). Macarbomycin and moenomycin are thought to directly inhibit transglycosylase activity. Characterization of the mutant revealed a thickened cell wall and suppression of penicillin-induced lysis, although the amounts of the five penicillin-binding proteins (PBPs 1, 2, 3, 4, and 2′) and the profiles of peptidoglycan hydrolases were not altered. Analysis of muropeptide profile and glycan chain length distribution by reversed-phase high-pressure liquid chromatography revealed slightly decreased peptide cross-linking and an increased average glycan chain length compared to those of the parent. These results together suggest that a transglycosylase activity was enhanced in the mutant. This may represent a novel mechanism of glycopeptide resistance in
S. aureus
.
In Vitro Activity of Cephalosporin RWJ-54428 (MC-02479) against Multidrug-Resistant Gram-Positive Cocci
Authors: Johnson Alan P. , Warner Marina , Carter Michael , Livermore David M.
Published: July 2002

RWJ-54428 (MC-02479) is a novel cephalosporin that binds to penicillin-binding protein (PBP) PBP 2′ (PBP 2a) of methicillin-resistant staphylococci. Its in vitro activity was assessed against 472 gram-positive cocci, largely selected as epidemiologically unrelated isolates with multidrug resistance. The MIC at which 50% of isolates are inhibited (MIC
50
) and MIC
90
of RWJ-54428 for methicillin-resistant
Staphylococcus aureus
(MRSA) were 1 and 2 μg/ml, respectively, whereas they were 0.5 and 0.5 μg/ml, respectively, for methicillin-susceptible
S. aureus
. The MIC
50
and MIC
90
were 1 and 4 μg/ml, respectively, for methicillin-resistant coagulase-negative staphylococci (MRCoNS), whereas they were 0.25 and 1 μg/ml, respectively, for methicillin-susceptible isolates. The highest MICs for MRSA and MRCoNS isolates were 2 and 4 μg/ml, respectively. The MIC
50
and MIC
90
of RWJ-54428 for
Enterococcus faecalis
were 0.5 and 1 μg/ml, respectively, but they were 4 and 8 μg/ml, respectively, for
Enterococcus faecium
. For penicillin-susceptible, -intermediate, and -resistant pneumococci, the MIC
90
s of RWJ-54428 were 0.03, 0.25, and 0.5 μg/ml, respectively, with the highest MIC for a pneumococcus being 1 μg/ml, recorded for a strain for which penicillin and cefotaxime MICs were 8 and 4 μg/ml. MICs for Lancefield group A, B, C, and G streptococci were ≤0.008 μg/ml; those for viridans group streptococci, including isolates not susceptible to penicillin, were from 0.015 to 0.5 μg/ml. RWJ-54428 did not select resistant mutants of MRSA or enterococci in challenge experiments and has the potential to be useful for the treatment of infections caused by gram-positive cocci.
Selection of Zone Size Interpretive Criteria for Disk Diffusion Susceptibility Tests of Three Antibiotics against
Streptococcus pneumoniae,
Using the New Guidelines of the National Committee for Clinical Laboratory Standards
Authors: Fuchs Peter C. , Barry Arthur L. , Brown Steven D.
Published: July 2002

Disk diffusion and broth microdilution susceptibility tests were performed with cefotaxime, ceftriaxone, telithromycin, and erythromycin (control) against 407 selected isolates of
Streptococcus pneumoniae
. Scattergrams were prepared from the results of these tests, and the current NCCLS guidelines for setting disk diffusion test interpretive criteria were applied. Erythromycin zone diameter breakpoints were confirmed. Telithromycin interpretive criteria for the disk test could be easily set with acceptable discrepancy rates. For cefotaxime and ceftriaxone, the minor discrepancy rates for MICs in the intermediate category ± 1 dilution were far in excess of the acceptable 40% limit, i.e., 52 and 71%, respectively. We conclude that the 30-μg disk of these two drugs cannot be reliably used to test pneumococci.
Cleavable-Complex Formation by Wild-Type and Quinolone-Resistant
Streptococcus pneumoniae
Type II Topoisomerases Mediated by Gemifloxacin and Other Fluoroquinolones
Authors: Yague Genoveva , Morris Julia E. , Pan Xiao-Su , Gould Katherine A. , Fisher L. Mark
Published: July 2002

Gemifloxacin is a recently developed fluoroquinolone with potent activity against
Streptococcus pneumoniae
. We show that the drug is more active than moxifloxacin, gatifloxacin, levofloxacin, and ciprofloxacin against
S. pneumoniae
strain 7785 (MICs, 0.03 to 0.06 μg/ml versus 0.25, 0.25, 1, and 1 to 2 μg/ml, respectively) and against isogenic quinolone-resistant
gyrA
-
parC
mutants (MICs, 0.5 to 1 μg/ml versus 2 to 4, 2 to 4, 16 to 32, and 64 μg/ml, respectively). Gemifloxacin was also the most potent agent against purified
S. pneumoniae
DNA gyrase and topoisomerase IV in both catalytic inhibition and DNA cleavage assays. The drug concentrations that inhibited DNA supercoiling or DNA decatenation by 50% (IC
50
s) were 5 to 10 and 2.5 to 5.0 μM, respectively. Ciprofloxacin and levofloxacin were some four- to eightfold less active against either enzyme; moxifloxacin and gatifloxacin showed intermediate activities. In assays of drug-mediated DNA cleavage by gyrase and topoisomerase IV, the same order of potency was seen: gemifloxacin > moxifloxacin > gatifloxacin > levofloxacin ≈ ciprofloxacin. For gemifloxacin, the drug concentrations that caused 25% linearization of the input DNA by gyrase and topoisomerase IV were 2.5 and 0.1 to 0.3 μM, respectively; these values were 4-fold and 8- to 25-fold lower than those for moxifloxacin, respectively. Each drug induced DNA cleavage by gyrase at the same spectrum of sites but with different patterns of intensity. Finally, for enzymes reconstituted with quinolone-resistant GyrA S81F or ParC S79F subunits, although cleavable-complex formation was reduced by at least 8- to 16-fold for all the quinolones tested, gemifloxacin was the most effective; e.g., it was 4- to 16-fold more active than the other drugs against toposiomerase IV with the ParC S79F mutation. It appears that the greater potency of gemifloxacin against both wild-type and quinolone-resistant
S. pneumoniae
strains arises from enhanced stabilization of gyrase and topoisomerase IV complexes on DNA.
Comparison of In Vitro Activities of ABT-773 and Telithromycin against Macrolide-Susceptible and -Resistant Streptococci and Staphylococci
Authors: Shortridge Virginia D. , Zhong Ping , Cao Zhensheng , Beyer Jill M. , Almer Laurel S. , Ramer Nancy C. , Doktor Stella Z. , Flamm Robert K.
Published: July 2002

The activity of a new ketolide, ABT-773, was compared to the activity of the ketolide telithromycin (HMR-3647) against over 600 gram-positive clinical isolates, including 356
Streptococcus pneumoniae
, 167
Staphylococcus aureus
, and 136
Streptococcus pyogenes
isolates. Macrolide-susceptible isolates as well as macrolide-resistant isolates with ribosomal methylase (Erm), macrolide efflux (Mef), and ribosomal mutations were tested using the NCCLS reference broth microdilution method. Both compounds were extremely active against macrolide-susceptible isolates, with the minimum inhibitory concentrations at which 90% of the isolates tested were inhibited (MIC
90
s) for susceptible streptococci and staphylococci ranging from 0.002 to 0.03 μg/ml for ABT-773 and 0.008 to 0.06 μg/ml for telithromycin. ABT-773 had increased activities against macrolide-resistant
S. pneumoniae
(Erm MIC
90
, 0.015 μg/ml; Mef MIC
90
, 0.12 μg/ml) compared to those of telithromycin (Erm MIC
90
, 0.12 μg/ml; Mef MIC
90
, 1 μg/ml). Both compounds were active against strains with rRNA or ribosomal protein mutations (MIC
90
, 0.12 μg/ml). ABT-773 was also more active against macrolide-resistant
S. pyogenes
(ABT-773 Erm MIC
90
, 0.5 μg/ml; ABT-773 Mef MIC
90
, 0.12 μg/ml; telithromycin Erm MIC
90
, >8 μg/ml; telithromycin Mef MIC
90
, 1.0 μg/ml). Both compounds lacked activity against constitutive macrolide-resistant
Staphylococcus aureus
but had good activities against inducibly resistant
Staphylococcus aureus
(ABT-773 MIC
90
, 0.06 μg/ml; telithromycin MIC
90
, 0.5 μg/ml). ABT-773 has superior activity against macrolide-resistant streptococci compared to that of telithromycin.
Risk Factors for Piperacillin-Tazobactam-Resistant
Pseudomonas aeruginosa
among Hospitalized Patients
Authors: Harris Anthony D. , Perencevich Eli , Roghmann Mary-Claire , Morris Glenn , Kaye Keith S. , Johnson Judith A.
Published: July 2002

Antimicrobial resistance is an emerging problem with
Pseudomonas aeruginosa
. This study determined risk factors for the recovery of piperacillin-tazobactam-resistant
P. aeruginosa
from clinical cultures from hospitalized patients. A case-control study design was used to compare two groups of case patients with control patients. The first group of case patients was defined by nosocomial isolation of piperacillin-tazobactam-resistant
P. aeruginosa
, and the second group of cases yielded piperacillin-tazobactam-susceptible
P. aeruginosa
. Controls were selected in a 6:1 ratio from the same medical or surgical services among which piperacillin-tazobactam-resistant
P. aeruginosa
arose in patients. Risk factors analyzed included antimicrobial drug exposure, comorbid conditions, and demographics. Bivariate and multivariable analyses were performed. Piperacillin-tazobactam-resistant
P. aeruginosa
was isolated from 179 patients, and piperacillin-tazobactam-susceptible
P. aeruginosa
was isolated from 624 patients over a 2.5-year period. Piperacillin-tazobactam (odds ratio [OR] = 6.82; 95% confidence interval [CI], 4.56 to 10.21), imipenem (OR = 2.42; 95% CI, 1.19 to 4.94), aminoglycosides (OR = 2.18; 95% CI, 1.44 to 3.28), vancomycin (OR = 1.87; 95% CI, 1.21 to 2.89), and broad-spectrum cephalosporins (OR = 2.38; 95% CI, 1.45 to 3.88) were the antibiotics associated with the isolation of piperacillin-tazobactam-resistant
P. aeruginosa
. Exposure to vancomycin (OR = 1.53; 95% CI, 1.13 to 2.06) or ampicillin-sulbactam (OR = 2.28; 95% CI, 1.62 to 3.21) was associated with recovery of piperacillin-tazobactam-susceptible
P. aeruginosa
. In this study, antibiotics associated with piperacillin-tazobactam-susceptible
P. aeruginosa
were different from antibiotics associated with piperacillin-tazobactam-resistant
P. aeruginosa
. Piperacillin-tazobactam was a strong risk factor for piperacillin-tazobactam-resistant
P. aeruginosa
. Our results suggest that the nosocomial isolation of piperacillin-tazobactam-resistant
P. aeruginosa
may be affected by multiple antibiotics.
Novel Use of a Guanosine Prodrug Approach To Convert 2',3'-Didehydro-2',3'-Dideoxyguanosine into a Viable Antiviral Agent
Authors: Ray Adrian S. , Yang Zhenjun , Chu Chung K. , Anderson Karen S.
Published: July 2002

Transient kinetic studies with human immunodeficiency virus (HIV) type 1 reverse transcriptase suggest that nucleotide analogs containing the 2',3'-didehydro-2',3'-dideoxy ribose ring structure present in D4T (stavudine) triphosphate are among the most effective alternative substrates. For unclear reasons, however, the corresponding purine nucleoside, 2',3'-didehydro-2',3'-dideoxyguanosine (D4G), was found to be inactive in cell culture. We have found that the previously reported lack of activity of D4G is primarily due to solution instability, and in this report we describe a novel use of a guanosine prodrug approach to stabilize the nucleoside. D4G was modified at the 6 position of the purine ring to contain a cyclopropylamino group yielding the prodrug, cyclo-D4G. An evaluation of cyclo-D4G revealed that the prodrug possessed anti-HIV activity. In addition, cyclo-D4G had increased stability, lipophilicity, and solubility, as well as decreased toxicity relative to D4G, suggesting that further study is warranted.
In Vitro and In Vivo Activities of a Novel Cephalosporin, BMS-247243, against Organisms other than Staphylococci
Authors: Clark Junius , Fung-Tomc Joan C. , Minassian Beatrice , Tsai Yuan-Hwang , Yang Hyekyung , Huczko Elizabeth , Kolek Benjamin , Chaniewski Susan , Ferraro Cheryl , Drain Rebecca , Gradelski Elizabeth , Bonner Daniel P.
Published: July 2002

BMS-247243, a novel cephalosporin inhibitory for methicillin-resistant staphylococci, primarily has activity against gram-positive bacteria. The activities of BMS-247243, cefotaxime, and ceftriaxone against streptococci and
Streptococcus pneumoniae
were similar. BMS-247243 inhibits
Enterococcus faecalis
but not
Enterococcus faecium
. BMS-247243 also inhibits many inherently vancomycin-resistant species (
Leuconstoc
,
Lactobacillus
,
Pediococcus
) and anaerobic gram-positive bacteria.
In Vitro Activities of a New Des-Fluoroquinolone, BMS 284756, and Seven Other Antimicrobial Agents against 151 Isolates of
Eikenella corrodens
Authors: Goldstein Ellie J. C. , Citron Diane M. , Merriam C. Vreni , Warren Yumi A. , Tyrrell Kerin L. , Fernandez Helen
Published: July 2002

The des-fluoroquinolone BMS 284756 was active in vitro against all 151 clinical strains of
Eikenella corrodens
at a MIC of ≤0.25 μg/ml and was comparable in activity to moxifloxacin and levofloxacin. The MIC at which 90% of the isolates were inhibited by penicillin G was 2 μg/ml; MICs for 8.6% of the strains (13 of 151) were ≥4 μg/ml, including for two beta-lactamase-producing isolates. Amoxicillin-clavulanate and ampicillin-sulbactam inhibited all strains at a MIC of ≤1 μg/ml.
Candida albicans
Sterol C-14 Reductase, Encoded by the
ERG24
Gene, as a Potential Antifungal Target Site
Authors: Jia N. , Arthington-Skaggs B. , Lee W. , Pierson C. A. , Lees N. D. , Eckstein J. , Barbuch R. , Bard M.
Published: July 2002

The incidence of fungal infections has increased dramatically, which has necessitated additional and prolonged use of the available antifungal agents. Increased resistance to the commonly used antifungal agents, primarily the azoles, has been reported, thus necessitating the discovery and development of compounds that would be effective against the major human fungal pathogens. The sterol biosynthetic pathway has proved to be a fertile area for antifungal development, and steps which might provide good targets for novel antifungal development remain. The sterol C-14 reductase, encoded by the
ERG24
gene, could be an effective target for drug development since the morpholine antifungals, inhibitors of Erg24p, have been successful in agricultural applications. The
ERG24
gene of
Candida albicans
has been isolated by complementation of a
Saccharomyces cerevisiae erg24
mutant. Both copies of the
C. albicans ERG24
gene have been disrupted by using short homologous regions of the
ERG24
gene flanking a selectable marker. Unlike
S. cerevisiae
, the
C. albicans ERG24
gene was not required for growth, but
erg24
mutants showed several altered phenotypes. They were demonstrated to be slowly growing, with doubling times at least twice that of the wild type. They were also shown to be significantly more sensitive to an allylamine antifungal and to selected cellular inhibitors including cycloheximide, cerulenin, fluphenazine, and brefeldin A. The
erg24
mutants were also slightly resistant to the azoles. Most importantly,
erg24
mutants were shown to be significantly less pathogenic in a mouse model system and failed to produce germ tubes upon incubation in human serum. On the basis of these characteristics, inhibitors of Erg24p would be effective against
C. albicans
.
Pharmacokinetics of Amino Acid Phosphoramidate Monoesters of Zidovudine in Rats
Authors: Song Heng , Griesgraber George W. , Wagner Carston R. , Zimmerman Cheryl L.
Published: July 2002

In vitro studies have demonstrated that water-soluble, nontoxic phosphoramidates of azidothymidine (zidovudine [AZT]) have significant and specific anti-human immunodeficiency virus and anticancer activity. Although polar, these compounds are internalized and processed to the corresponding nucleoside monophosphates. Eight methyl amide and methyl ester phosphoramidate monoesters composed of
d
- or
l
-phenylalanine or tryptophan and AZT were synthesized. The plasma stability and protein binding studies were carried out in vitro. Then in vivo pharmacokinetic evaluations of six of the compounds were conducted. Sprague-Dawley rats received each compound by intravenous bolus dose, and serial blood and urine samples were collected. AZT and phosphoramidate concentrations in plasma and urine were quantitated by high-performance liquid chromatography with UV or fluorescence detection. Pharmacokinetic parameters were calculated by standard noncompartmental means. The plasma half-lives of the phosphoramidates were 10- to 20-fold longer than the half-life of AZT. Although the renal clearances of the phosphoramidates were similar to AZT, their total body clearances were significantly greater than that of AZT. The 3- to 15-fold-larger volume of distribution (
V
ss
) for the phosphoramidates relative to AZT appeared to be dependent on the stereochemistry of the amino acid, with the largest values being associated with the
l
-amino acids. The increased
V
ss
indicates a much greater tissue distribution of the phosphoramidate prodrugs than of AZT. Amino acid phosphoramidate monoesters of AZT have improved pharmacokinetic properties over AZT and significant potential as in vivo pronucleotides.
Comparative Pharmacodynamics of Three Newer Fluoroquinolones versus Six Strains of Staphylococci in an In Vitro Model under Aerobic and Anaerobic Conditions
Authors: Wright David H. , Gunderson Brent W. , Hovde Laurie B. , Ross Gigi H. , Ibrahim Khalid H. , Rotschafer John C.
Published: July 2002

Six strains of staphylococci were exposed to levofloxacin, moxifloxacin, or trovafloxacin in an in vitro pharmacodynamic model under both aerobic and anaerobic conditions. Each agent demonstrated a rapid 3-log
10
kill versus susceptible isolates regardless of condition. Against clinical isolates with reduced susceptibility, regrowth occurred by 24 h and was frequently associated with further increases in MICs.
Quantitative Flow Cytometric Evaluation of Maximal
Cryptosporidium parvum
Oocyst Infectivity in a Neonate Mouse Model
Authors: Delaunay Agnès , Gargala Gilles , Li Xunde , Favennec Loic , Ballet Jean Jacques
Published: July 2002

The importance of waterborne transmission of
Cryptosporidium parvum
to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting
C. parvum
oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.
Field Evaluation of a Semiautomated Method for Rapid and Simple Analysis of Recreational Water Microbiological Quality
Authors: Anglès d'Auriac Marc B. , Roberts Hildegarde , Shaw Terri , Sirevåg Reidun , Hermansen Leonila Fajardo , Berg James D.
Published: July 2002

An early warning system using a rapid enzymatic semiautomated method suitable for fecal coliform detection in recreational waters within 8 h was developed further and evaluated in this study. This rapid method was compared to the standard method followed in the United Kingdom. We used 1,011 samples originating from 206 different locations in Wales. When we assessed the presence or absence of fecal coliforms, targeting very low levels of contamination, we obtained 83.9% agreement between the rapid method and the lauryl sulfate broth-membrane filtration technique, whereas direct confirmation of the samples processed by the rapid method showed 89.3% agreement. Environmental enzymatic background activity was found to be the main limiting factor for this method. Owing to a specific and integrated handling of the results by the software of the instrument, the percentage of false-positive results (a consequence of enzymatic background) was successfully limited to 2.9% by the direct confirmation evaluation. However, 7.8% false-negative results due to “late-growers” had to be accepted in order to produce results within a working day. At present, the method can be used in a more conservative way to assess the environmental threshold of 100 CFU of fecal coliforms per 100 ml in recreational waters. The implications of our findings with regard to the applicability of rapid enzymatic methods are discussed.
Genetic Diversity and Dynamics of
Sinorhizobium meliloti
Populations Nodulating Different Alfalfa Cultivars in Italian Soils
Authors: Carelli Maria , Gnocchi Stefano , Fancelli Silvia , Mengoni Alessio , Paffetti Donatella , Scotti Carla , Bazzicalupo Marco
Published: July 2002

We analyzed the genetic diversity of 531
Sinorhizobium meliloti
strains isolated from nodules of
Medicago sativa
cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of
S. meliloti
. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant.
Population Structure of Rat-Derived
Pneumocystis carinii
in Danish Wild Rats
Authors: Palmer Robert J. , Settnes Osvald P. , Lodal Jens , Wakefield Ann E.
Published: July 2002

The rat model of
Pneumocystis carinii
pneumonia is frequently used to study human
P. carinii
infection, but there are many differences between the rat and human infections. We studied naturally acquired
P. carinii
in wild rats to examine the relevance of the rat model for human infection.
P. carinii
DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived
P. carinii
was found, and these were provisionally named
Pneumocystis carinii
f. sp.
rattus-secundi
,
Pneumocystis carinii
f. sp.
rattus-tertii
, and
Pneumocystis carinii
f. sp.
rattus-quarti
. Our data suggest that low-level carriage of
P. carinii
in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of
P. carinii
. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of
Pneumocystis carinii
f. sp.
carinii
by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the
P. carinii
f. sp.
carinii
population. This study shows that human- and rat-derived
P. carinii
organisms are very different, not only in genetic composition but also in population structure and natural history.
Role of Proteolysis in Determining Potency of
Bacillus thuringiensis
Cry1Ac δ-Endotoxin
Authors: Lightwood Daniel J. , Ellar David J. , Jarrett Paul
Published: July 2002

Bacillus thuringiensis
protein δ-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the
B. thuringiensis
Cry1Ac δ-endotoxin towards
Pieris brassicae
(family: Pieridae) and
Mamestra brassicae
(family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against
P. brassicae
than against
M. brassicae
larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from
P. brassicae
, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of ∼56 kDa, whereas proteases from
M. brassicae
, the non-susceptible insect, generated products with molecular masses of ∼58, ∼40, and ∼20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but
M. brassicae
proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by
M. brassicae
proteases. However, this mutation had little effect on toxicity to
M. brassicae
. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.