bluuebunny/crossref_metadata_2025 huggingface dataset:
Energy-Dependent Accumulation of Norfloxacin and Porin Expression in Clinical Isolates of
Klebsiella pneumoniae
and Relationship to Extended-Spectrum β-Lactamase Production
Authors: Martínez-Martínez Luis , Pascual Alvaro , Conejo María del Carmen , García Isabel , Joyanes Providencia , Doménech-Sánchez Antonio , Benedí Vicente Javier
Published: November 2002

The relationships between porin deficiency, active efflux of fluoroquinolones, and extended-spectrum β-lactamase (ESBL) production were determined for 53 clinical isolates of
Klebsiella pneumoniae
. Thirty-two ESBL-positive strains (including 22 strains expressing porins and 10 strains lacking porins) and 21 ESBL-negative strains were evaluated. Active efflux of norfloxacin was defined as a ≥50% increase in the accumulation of norfloxacin in the presence of carbonyl cyanide
m
-chlorophenylhydrazone (CCCP) in comparison with the corresponding basal value in the absence of CCCP. The quinolone resistance-determining regions of both
gyrA
and
parC
from 13 strains, representing all isolates with different porin profiles and with or without active efflux, were determined. Porin loss was significantly more common among ESBL-positive strains (10 of 32 [31.2%]) than among ESBL-negative strains (0 of 2 [0%]) (
P
< 0.01). Active efflux was observed in 7 of 10 (70%) strains lacking porins and in 4 of 43 (9.3%) strains producing porins (
P
< 0.001). The 11 strains showing active efflux corresponded to 3 of 21 (14.3%) ESBL-negative strains and 8 of 32 (25.5%) ESBL-positive strains (
P
> 0.05). Basal values of norfloxacin accumulation were higher in strains lacking active efflux than in those that had this mechanism (
P
< 0.05). In the absence of topoisomerase changes, the contribution of either porin loss or active efflux to fluoroquinolone resistance in
K. pneumoniae
was negligible. It is concluded that among
K. pneumoniae
strains of clinical origin, porin loss was observed only in those producing ESBL, and that a significant number of porin-deficient strains also expressed active efflux of norfloxacin. In terms of fluoroquinolone resistance, both mechanisms are significant only in the presence of topoisomerase modifications.
Intramuscular Bioavailability and Clinical Efficacy of Artesunate in Gabonese Children with Severe Malaria
Authors: Nealon Claire , Dzeing Arnaud , Müller-Römer Ulrich , Planche Timothy , Sinou Veronique , Kombila Maryvonne , Kremsner Peter G. , Parzy Daniel , Krishna Sanjeev
Published: November 2002

Artesunate (ARS) is a water-soluble artemisinin derivative that is a potential alternative to quinine for the treatment of severe childhood malaria. We studied the pharmacokinetics and bioavailability of ARS given by the intramuscular (i.m.) route in an open crossover study design. Fourteen children were randomized to receive intravenous (i.v.) ARS in a loading dose (2.4 mg/kg of body weight) followed 12 h later by an i.m. dose (1.2 mg/kg) (group I), and 14 children were randomized to receive i.m. ARS (2.4 mg/kg) followed by an i.v. dose of ARS (1.2 mg/kg) (group II). We carried out a two-compartment analysis of ARS and dihydroartemisinin (DHA; the principal antimalarial metabolite) levels in 21 children (groups I and II combined). Absorption of i.m. ARS was rapid, with the maximum concentration of DHA in serum being achieved in less than 1 h in most children (median time to the maximum concentration of drug in serum, 35.1 min; range, 10.8 to 71.9 min). The absolute bioavailability of DHA was a median of 86.4% (range, 11.4 to 462.1%), the median steady-state volume of distribution was 1.3 liters/kg (range, 0.5 to 7.9 liters/kg), and the median clearance was 0.028 liters/kg/min (range, 0.001 to 1.58 liters/kg/min). There were no major adverse events attributable to ARS. Parasite clearance kinetics were comparable between the two treatment groups. These results support the use of i.m. ARS in children with severe malaria.
Emergence of Tetracycline Resistance in
Helicobacter pylori
: Multiple Mutational Changes in 16S Ribosomal DNA and Other Genetic Loci
Authors: Dailidiene Daiva , Bertoli M. Teresita , Miciuleviciene Jolanta , Mukhopadhyay Asish K. , Dailide Giedrius , Pascasio Mario Alberto , Kupcinskas Limas , Berg Douglas E.
Published: November 2002

Tetracycline is useful in combination therapies against the gastric pathogen
Helicobacter pylori
. We found 6 tetracycline-resistant (Tet
r
) strains among 159 clinical isolates (from El Salvador, Lithuania, and India) and obtained the following four results: (i) 5 of 6 Tet
r
isolates contained one or two nucleotide substitutions in one part of the primary tetracycline binding site in 16S rRNA (AGA
965-967
[
Escherichia coli
coordinates] changed to gGA, AGc, guA, or gGc [lowercase letters are used to represent the base changes]), whereas the sixth (isolate Ind75) retained AGA
965-967
; (ii) PCR products containing mutant 16S ribosomal DNA (rDNA) alleles transformed recipient strains to Tet
r
phenotypes, but transformants containing alleles with single substitutions (gGA and AGc) were less resistant than their Tet
r
parents; (iii) each of 10 Tet
r
mutants of reference strain 26695 (in which mutations were induced with metronidazole, a mutagenic anti-
H. pylori
agent) contained the normal AGA
965-967
sequence; and (iv) transformant derivatives of Ind75 and of one of the Tet
r
26695 mutants that had acquired mutant rDNA alleles were resistant to tetracycline at levels higher than those to which either parent strain was resistant. Thus, tetracycline resistance in
H. pylori
results from an accumulation of changes that may affect tetracycline-ribosome affinity and/or other functions (perhaps porins or efflux pumps). We suggest that the rarity of tetracycline resistance among clinical isolates reflects this need for multiple mutations and perhaps also the deleterious effects of such mutations on fitness. Formally equivalent mutations with small but additive effects are postulated to contribute importantly to traits such as host specificity and virulence and to
H. pylori
's great genetic diversity.
Therapeutic Efficacies of Artesunate-Sulfadoxine-Pyrimethamine and Chloroquine-Sulfadoxine-Pyrimethamine in Vivax Malaria Pilot Studies: Relationship to
Plasmodium vivax dhfr
Mutations
Authors: Tjitra Emiliana , Baker Joanne , Suprianto Sri , Cheng Qin , Anstey Nicholas M.
Published: November 2002

Artemisinin-derivative combination therapies (ACT) are highly efficacious against multidrug-resistant
Plasmodium falciparum
malaria. Few efficacy data, however, are available for vivax malaria. With high rates of chloroquine (CQ) resistance in both vivax and falciparum malaria in Papua Province, Indonesia, new combination therapies are required for both species. We recently found artesunate plus sulfadoxine-pyrimethamine (ART-SP) to be highly effective (96%) in the treatment of falciparum malaria in Papua Province. Following a preliminary study of CQ plus sulfadoxine-pyrimethamine (CQ-SP) for the treatment of
Plasmodium vivax
infection, we used modified World Health Organization criteria to evaluate the efficacy of ART-SP for the treatment of vivax malaria in Papua. Nineteen of 22 patients treated with ART-SP could be evaluated on day 28, with no early treatment failures. Adequate clinical and parasitological responses were found by day 14 in all 20 (100%) of the patients able to be evaluated and by day 28 in 17 patients (89.5%). Fever and parasite clearance times were short, with hematological improvement observed in 70.6% of the patients. Double (at positions 58 and 117) and quadruple (at positions 57, 58, 61, and 117) mutations in the
P. vivax
dihydrofolate reductase (PvDHFR) were common in Papuan
P. vivax
isolates (46 and 18%, respectively). Treatment failure with SP-containing regimens was significantly higher with isolates with this PvDHFR quadruple mutation, which included a novel T→M mutation at residue 61 linked to an S→T (but not an S→N) mutation at residue 117. ART-SP ACT resulted in a high cure rate for both major
Plasmodium
species in Papua, though progression of DHFR mutations in both species due to the continued use of SP monotherapy for clinically diagnosed malaria threatens the future utility of this combination.
env
Chimeric Virus Technology for Evaluating Human Immunodeficiency Virus Susceptibility to Entry Inhibitors
Authors: Fikkert Valery , Cherepanov Peter , Van Laethem Kristel , Hantson Anke , Van Remoortel Barbara , Pannecouque Christophe , De Clercq Erik , Debyser Zeger , Vandamme Anne-Mieke , Witvrouw Myriam
Published: November 2002

We describe the development of chimeric virus technology (CVT) for human immunodeficiency virus (HIV) type 1 (HIV-1)
env
genes
gp120
,
gp41
, and
gp160
for evaluation of the susceptibilities of HIV to entry inhibitors. This
env
CVT allows the recombination of
env
sequences derived from different strains into a proviral wild-type HIV-1 clone (clone NL4.3) from which the corresponding
env
gene has been deleted. An HIV-1 strain (strain NL4.3) resistant to the fusion inhibitor T20 (strain NL4.3/T20) was selected in vitro in the presence of T20. AMD3100-resistant strain NL3.4 (strain NL4.3/AMD3100) was previously selected by De Vreese et al. (K. De Vreese et al., J. Virol. 70:689-696, 1996). NL4.3/AMD3100 contains several mutations in its
gp120
gene (De Vreese et al., J. Virol. 70:689-696, 1996), whereas NL4.3/T20 has mutations in both
gp120
and
gp41
. Phenotypic analysis revealed that NL4.3/AMD3100 lost its susceptibility to dextran sulfate, AMD3100, AMD2763, T134, and T140 but not its susceptibility to T20, whereas NL4.3/T20 lost its susceptibility only to the inhibitory effect of T20. The recombination of
gp120
of NL4.3/AMD3100 and
gp41
of NL4.3/T20 or recombination of the
gp160
genes of both strains into a wild-type background reproduced the phenotypic (cross-)resistance profiles of the corresponding strains selected in vitro. These data imply that mutations in
gp120
alone are sufficient to reproduce the resistance profile of NL4.3/AMD3100. The same can be said for
gp41
in relation to NL4.3/T20. In conclusion, we demonstrate the use of
env
CVT as a research tool in the delineation of the region important for the phenotypic (cross-)resistance of HIV strains to entry inhibitors. In addition, we obtained a proof of principle that
env
CVT can become a helpful diagnostic tool in assessments of the phenotypic resistance of clinical HIV isolates to HIV entry inhibitors.
Evolution of Multiresistance in Nontyphoid
Salmonella
Serovars from 1984 to 1998 in Argentina
Authors: Orman Betina E. , Piñeiro Silvia A. , Arduino Sonia , Galas Marcelo , Melano Roberto , Caffer Maria I. , Sordelli Daniel O. , Centrón Daniela
Published: November 2002

Molecular evolution of multiresistance in nontyphoid
Salmonella
spp. was investigated with 155 isolates obtained in Argentina from 1984 to 1998. In 74 isolates obtained from 1984 to 1988 resistance was associated with the presence of Tn
3
, Tn
9
, class I (In0) and II (Tn
7
) integrons, and the
aac
(
3
)
-IIa
gene. Extended-spectrum cephalosporin (ESC) resistance in
Salmonella
spp. emerged in 1989, and 81 isolates resistant to at least one ESC and one aminoglycoside were collected thereafter. Among these, two patterns of antimicrobial resistance mechanisms were found: from 1989 to 1992, resistance was related to the spreading of Tn
1331
and
bla
CTX-M-2
, in addition to the persistence of In0 and Tn
7
. From 1993 to 1998, several integrons were added to the first pattern and three integron groups (IG), namely, IG1 (38% of the isolates), IG2 (51%), and IG3 (11%), were identified. At least two β-lactamase genes were detected in 65% of the isolates (after 1989) by PCR analysis. Furthermore, five β-lactamase genes,
bla

CTX-M-

2

,
bla
OXA-9
,
bla
OXA-2
,
bla
TEM-1
, and
bla
PER-2
, were found in two isolates. The
bla
CTX-M-2
gene was found in several complex
sulI
-type integrons with different rearrays within the variable region of class I integrons, suggesting evolution of these integrons in nontyphoid
Salmonella
. In conclusion, progressive acquisition and accumulation of plasmid-mediated resistance determinants occurred from 1984 to 1998 in nontyphoid
Salmonella
isolates of the most prevalent serovars from Argentina. It is suggested that antimicrobial resistance mechanisms in these bacteria may have been the consequence of plasmid exchange between
Salmonella enterica
serovar Typhimurium and
Escherichia coli
or
Shigella flexneri
and/or spreading of mobile elements from the nosocomial environment.
Amino Acid Substitutions at Ambler Position Gly238 in the SHV-1 β-Lactamase: Exploring Sequence Requirements for Resistance to Penicillins and Cephalosporins
Authors: Hujer Andrea M. , Hujer Kristine M. , Helfand Marion S. , Anderson Vernon E. , Bonomo Robert A.
Published: November 2002

Site saturation mutagenesis of the 238 position in the SHV β-lactamase was performed to identify the complete sequence requirements needed for the extended spectrum β-lactamase (ESBL) phenotype. MICs (in micrograms per milliliter) in an isogenic background,
Escherichia coli
DH10B, demonstrated that the Gly238Ala mutation conferred the most resistance to penicillins and cephalosporins. The absolute increase in resistance was greatest against cefotaxime for the Gly238Ala mutant (0.06 to 8 μg/ml). Except for the strain possessing the Gly238Pro β-lactamase, ceftazidime MICs were also elevated. None of the mutant SHV β-lactamases were expressed in as great an amount as the wild-type β-lactamase. Kinetic analysis of the Gly238Ala mutant revealed that penicillin and cephalosporin substrates have a lower

K
m

for the enzyme because of this mutation. Ampicillin and piperacillin MICs were inversely proportional to the side chain volume of the amino acid in cases larger than Ser, suggesting that steric considerations may be a primary requirement for penicillin resistance. Secondary structural effects explain increased resistance to oxyiminocephalosporins. Based upon this study, we anticipate that additional mutations of Gly238 in the SHV β-lactamase will continue to be discovered with an ESBL (ceftazidime or cefotaxime resistant) phenotype.
Structural Basis for Imipenem Inhibition of Class C β-Lactamases
Authors: Beadle Beth M. , Shoichet Brian K.
Published: November 2002

To determine how imipenem inhibits the class C β-lactamase AmpC, the X-ray crystal structure of the acyl-enzyme complex was determined to a resolution of 1.80 Å. In the complex, the lactam carbonyl oxygen of imipenem has flipped by approximately 180° compared to its expected position; the electrophilic acyl center is thus displaced from the point of hydrolytic attack. This conformation resembles that of imipenem bound to the class A enzyme TEM-1 but is different from that of moxalactam bound to AmpC.
Biochemical Characterization of TEM-92 Extended-Spectrum β-Lactamase, a Protein Differing from TEM-52 in the Signal Peptide
Authors: Perilli Mariagrazia , Segatore Bernardetta , De Massis Maria Rosaria , Pagani Laura , Luzzaro Francesco , Rossolini Gian Maria , Amicosante Gianfranco
Published: November 2002

A
bla
TEM-92
gene was cloned from a
Proteus mirabilis
isolate and expressed in
Escherichia coli
. Production of the enzyme caused reduction of susceptibility to penicillins and narrow- to expanded-spectrum cephalosporins but not to moxalactam and cephamycins. Determination of kinetic parameters with the purified enzyme revealed hydrolysis of expanded-spectrum cephalosporins, while cephamycins, moxalactam, and aztreonam were very poorly or not hydrolyzed. Clavulanate and penicillanic acid sulfones acylated TEM-92 slowly, and deacylation occurred at measurable rates.
AcrAB Efflux System: Expression and Contribution to Fluoroquinolone Resistance in
Klebsiella
spp
Authors: Mazzariol Annarita , Zuliani Jessica , Cornaglia Giuseppe , Rossolini Gian Maria , Fontana Roberta
Published: November 2002

Seven
Klebsiella pneumoniae
and four
Klebsiella oxytoca
clinical isolates with different levels of resistance to ciprofloxacin were studied. Mutations in the topoisomerase genes were found in almost all strains, but the contribution of a multidrug efflux system homologous to AcrAB in
Escherichia coli
was also observed. Overexpression of this efflux system was demonstrated by immunoblotting with antibodies against
E. coli
AcrA.
Erythromycin-Resistant Pharyngeal Isolates of
Streptococcus pyogenes
Recovered in Italy
Authors: Dicuonzo Giordano , Fiscarelli Ersilia , Gherardi Giovanni , Lorino Giulia , Battistoni Fabrizio , Landi Simona , De Cesaris Marina , Petitti Tommasangelo , Beall Bernard
Published: November 2002

Three classes of macrolide resistance phenotypes and three different erythromycin resistance determinants were found among 127 erythromycin-resistant group A streptococcal (GAS) isolates recovered from 355 (35.8%) pediatric pharyngitis patients in Rome, Italy. According to
emm
and
sof
sequence typing results, erythromycin-resistant isolates comprised 11 different clonal types. Remarkably, 126 of the 127 macrolide-resistant isolates were serum opacity factor (
sof
) gene positive. These data suggest a strong association between macrolide resistance and the presence of
sof
among GAS isolates recovered from Italian pediatric pharyngitis patients.
Prevalence of Clinical Isolates of
Escherichia coli
Producing Inhibitor-Resistant β-Lactamases at a University Hospital in Barcelona, Spain, over a 3-Year Period
Authors: Miró Elisenda , Navarro Ferran , Mirelis Beatriz , Sabaté Montserrat , Rivera Alba , Coll Pere , Prats Guillem
Published: November 2002

About 7% of 7,252 nonduplicated clinical
Escherichia coli
strains from a Spanish hospital showed reduced susceptibility to amoxicillin-clavulanate. Of these, 0.37% produced the IRTs TEM-30, TEM-31, TEM-33, TEM-34, TEM-37, TEM-40, TEM-51, and TEM-54; 5.3% were probable class C β-lactamase overproducers; 0.8% were probable TEM-1 hyperproducers; 0.18% produced OXA-30; 0.15% overexpressed SHV-1; and 0.03% produced a PSE-1 enzyme.
In Vitro Activities of Garenoxacin (BMS 284756) against 108 Clinical Isolates of
Gardnerella vaginalis
Authors: Goldstein Ellie J. C. , Citron Diane M. , Merriam C. Vreni , Warren Yumi A. , Tyrrell Kerin L. , Fernandez Helen T.
Published: November 2002

Garenoxacin (BMS 284756) was active against 105 of 108 (97%) recent clinical
Gardnerella vaginalis
isolates at ≤2 μg/ml by using the reference agar dilution method for anaerobes. Twenty-eight percent of isolates (31 of 108) were resistant to metronidazole, and 44% were resistant to doxycycline. All were susceptible to clindamycin and ampicillin-sulbactam.
In Vitro Killing Effect of Moxifloxacin on Clinical Isolates of
Stenotrophomonas maltophilia
Resistant to Trimethoprim-Sulfamethoxazole
Authors: Giamarellos-Bourboulis Evangelos J. , Karnesis Lazaros , Galani Irene , Giamarellou Helen
Published: November 2002

The time-kill effect of moxifloxacin on 20 genetically distinct isolates of
Stenotrophomonas maltophilia
resistant to trimethoprim-sulfamethoxazole was studied. The majority (80%) were killed by a concentration equivalent to four times the MIC; the MIC induced a transient decrease in bacterial counts at 4 h, followed by regrowth. No effect was detected in four isolates. These results merit further clinical consideration.
Phenotypic and Genotypic Analysis of Biologically Cloned Human Immunodeficiency Virus Type 1 Isolates from Patients Treated with Zidovudine and Lamivudine
Authors: Stoeckli Thomas C. , MaWhinney Samantha , Uy Jonathan , Duan Chengying , Lu Jing , Shugarts David , Kuritzkes Daniel R.
Published: November 2002

Mutations at reverse transcriptase codons 44, 118, 207, and 208 were significantly correlated with reduced zidovudine susceptibility in biologically cloned human immunodeficiency virus type 1 (HIV-1) isolates. Sequences from the Stanford HIV RT and Protease Sequence Database showed that these mutations were more common in HIV-1 isolates from patients treated with zidovudine and lamivudine than in patients not treated with these drugs.
Structure-Activity Relationship of a New Class of Anti-Hepatitis B Virus Agents
Authors: Mehta Anand , Conyers Bertha , Tyrrell D. L. J. , Walters Kathie-Anne , Tipples Graham A. , Dwek Raymond A. , Block Timothy M.
Published: November 2002

N
-Nonyl-deoxy-galactonojirimycin (
N
-nonyl-DGJ) has been shown to reduce the amount of hepatitis B virus (HBV) produced by tissue cultures under conditions where cell viability is not affected. We show here that the compound
N
-nonyl-DGJ was effective against lamivudine-resistant HBV mutants bearing the YMDD motif in the polymerase gene, consistent with the compound's activity being distinct from those of nucleoside inhibitors. To better understand the chemical structures that influence its antiviral activity, a series of imino sugar derivatives were made and tested for their antiviral activity against HBV. This work suggests that the antiviral activity of the alkovirs requires an alkyl chain length of at least eight carbons but that the galactose-based head group can be modified with little or no loss in activity.
High Concentrations of Nelfinavir as an Independent Risk Factor for Lipodystrophy in Human Immunodeficiency Virus-Infected Patients
Authors: Tréluyer Jean-Marc , Morini Jean-Pierre , Dimet Jérome , Gorin Isabelle , Rey Elisabeth , Deleuze Jean , Ceccaldi Pierre-François , Escande Jean-Paul , Pons Gérard , Dupin Nicolas
Published: November 2002

To assess the relationship between antiretroviral drug exposure and lipodystrophy, 69 human immunodeficiency virus type 1-infected patients receiving nelfinavir were investigated cross-sectionally. Lipodystrophy was defined by patients' self-report. Nelfinavir trough concentrations in plasma were significantly related to overall lipodystrophy and peripheral fat wasting scores and appeared to be an independent risk factor for lipodystrophy
Uptake and Intracellular Activity of Linezolid in Human Phagocytes and Nonphagocytic Cells
Authors: Pascual Álvaro , Ballesta Sofía , García Isabel , Perea Evelio J.
Published: November 2002

The intracellular penetration and activity of linezolid in human polymorphonuclear leukocytes and tissue-cultured cells (McCoy) were evaluated. Linezolid reached intracellular concentrations slightly greater than extracellular ones in both types of cell. The uptake was rapid and not saturable and was affected by environmental temperature and cell viability. Linezolid showed slight intracellular activity againstStaphylococcus epidermidisat high extracellular concentrations.
New Automated Method for Determining Postantifungal Effect of Amphotericin B againstCandidaSpecies: Effects of Concentration, Exposure Time, and Area under the Curve
Authors: Chryssanthou Erja , Cars Otto , Sjölin Jan
Published: November 2002

The postantifungal effect (PAFE) of amphotericin B was determined with a BacT/Alert automated system, which is based on the colorimetric detection of CO2. The levels of accuracy and precision of the automated method were high. Longer PAFEs were obtained forCandidaalbicans(P< 0.001),C. glabrata(P< 0.01), andC. krusei(P< 0.01) at increasing amphotericin B concentrations and exposure times.
In Vitro Activity of Moxifloxacin against Bacteria Isolated from Odontogenic Abscesses
Authors: Sobottka Ingo , Cachovan Georg , Stürenburg Enno , Ahlers M. Oliver , Laufs Rainer , Platzer Ursula , Mack Dietrich
Published: November 2002

We evaluated the antimicrobial susceptibility of 87 pathogens isolated from 37 patients with odontogenic abscesses. The most prevalent bacteria were viridans group streptococci and
Prevotella
species. Considering all bacterial isolates, 100% were susceptible to amoxicillin-clavulanic acid, 98% were susceptible to moxifloxacin and to levofloxacin, 76% were susceptible to doxycycline, 75% were susceptible to clindamycin, and 69% were susceptible to penicillin.
Postantibiotic Effect and Delay of Regrowth in Strains Carrying Mutations That Save Proteins or RNA
Authors: Dolcino Marzia , Zoratti Alberto , Debbia Eugenio A. , Schito Gian Carlo , Marchese Anna
Published: November 2002

The postantibiotic effect (PAE) values found for proteinase-defective (Lon
−
)
Escherichia coli
and RNase-defective
E. coli
exposed to antibiotics were reduced (31 to 60% and 35 to 50%, respectively) in comparison with the control (AB1157), and in the
recA13
mutant these values were about 0.4 h with all drugs. Nalidixic acid, under anaerobic conditions, induced no PAE (0 to 0.1 h) in AB1157. A delay in regrowth (0.2 to 0.26 h) was noted with
dnaA46
(Ts),
gyrA43
(Ts), and
gyrB41
(Ts) mutants cultured for 2 h at 43°C. These findings suggest that when proteins and RNA are saved, the cell rapidly resumes the original growth rate.
Novel Variant (
bla
VIM-4
) of the Metallo-β-Lactamase Gene
bla
VIM-1
in a Clinical Strain of
Pseudomonas aeruginosa
Authors: Pournaras Spyros , Tsakris Athanassios , Maniati Maria , Tzouvelekis Leonidas S. , Maniatis Antonios N.
Published: November 2002

A
Pseudomonas aeruginosa
isolate highly resistant to carbapenems was collected from a patient with postsurgical cerebrospinal infection in Greece. The isolate carried a class 1 integron that contained as a sole cassette the gene
bla
VIM-4
, a novel variant of
bla
VIM-1
, with one nucleotide difference resulting in a Ser-to-Arg change at amino acid position 175 of the VIM-1 enzyme. This is the first detection of a VIM-1 variant after its appearance in Italy.
Pharmacodynamic Modeling of Clarithromycin against Macrolide-Resistant [PCR-Positive
mef
(A) or
erm
(B)]
Streptococcus pneumoniae
Simulating Clinically Achievable Serum and Epithelial Lining Fluid Free-Drug Concentrations
Authors: Noreddin Ayman M. , Roberts Danielle , Nichol Kim , Wierzbowski Aleksandra , Hoban Daryl J. , Zhanel George G.
Published: November 2002

The association between macrolide resistance mechanisms and clinical outcomes remains understudied. The present study, using an in vitro pharmacodynamic model, assessed clarithromycin (CLR) activity against
mef
(A)-positive and
erm
(B)-negative
Streptococcus pneumoniae
isolates by simulating free-drug concentrations in serum and both total (protein-bound and free) and free drug in epithelial lining fluid (ELF). Five
mef
(A)-positive and
erm
(B)-negative strains, one
mef
(A)-negative and
erm
(B)-positive strain, and a control [
mef
(A)-negative and
erm
(B)-negative] strain of
S. pneumoniae
were tested. CLR was modeled using a one-compartment model, simulating a dosage of 500 mg, per os, twice a day (in serum, free-drug

C
p

maximum of 2 μg/ml,
t
1/2
of 6 h; in ELF,
C
ELF(total)
maximum of 35μg/ml,
t
1/2
of 6 h; C
ELF(free)
maximum of 14 μg/ml,
t
1/2
of 6 h). Starting inocula were 10
6
CFU/ml in Mueller-Hinton broth with 2% lysed horse blood. With sampling at 0, 4, 8, 12, 20, and 24 h, the extent of bacterial killing was assessed. Achieving CLR T/MIC values of ≥90% (AUC
0-24
/MIC ratio, ≥61) resulted in bacterial eradication, while T>MIC values of 40 to 56% (AUC
0-24
/MIC ratios of ≥30.5 to 38) resulted in a 1.2 to 2.0 log
10
CFU/ml decrease at 24 h compared to that for the initial inoculum. CLR T/MIC values of ≤8% (AUC
0-24
/MIC ratio, ≤17.3) resulted in a static effect or bacterial regrowth. The high drug concentrations in ELF that were obtained clinically with CLR may explain the lack of clinical failures with
mef
(A)-producing
S. pneumoniae
strains, with MICs up to 8 μg/ml. However,
mef
(A) isolates for which MICs are ≥16 μg/ml along with
erm
(B) may result in bacteriological failures.
Promoters
P3
,
Pa
/
Pb
,
P4
, and
P5
Upstream from
bla
TEM
Genes and Their Relationship to β-Lactam Resistance
Authors: Lartigue Marie Frédérique , Leflon-Guibout Véronique , Poirel Laurent , Nordmann Patrice , Nicolas-Chanoine Marie-Hélène
Published: November 2002

Using an isogenic system, we have determined the impact that the four promoters known to control
bla
TEM
gene expression have on β-lactamase activity. For both TEM-1 and TEM-30, this activity gradually increased in relation to the presence of promoters
P3
,
Pa/Pb
, and
P4
upstream of the corresponding gene. Promoter
P5
, only found upstream of the
bla
TEM-1B
gene, was related to the highest expression of this gene.
Chromosome-Encoded Ambler Class A β-Lactamase of
Kluyvera georgiana
, a Probable Progenitor of a Subgroup of CTX-M Extended-Spectrum β-Lactamases
Authors: Poirel Laurent , Kämpfer Peter , Nordmann Patrice
Published: November 2002

A chromosome-encoded β-lactamase gene, cloned and expressed in
Escherichia coli
from
Kluyvera georgiana
reference strain CUETM 4246-74 (DSM 9408), encoded the extended-spectrum β-lactamase KLUG-1, which shared 99% amino acid identity with the plasmid-mediated β-lactamase CTX-M-8. This work provides further evidence that
Kluyvera
spp. may be the progenitor(s) of CTX-M-type β-lactamases.
Antimicrobial Peptides from Human Platelets
Authors: Tang Yi-Quan , Yeaman Michael R. , Selsted Michael E.
Published: November 2002

Platelets share structural and functional similarities with granulocytes known to participate in antimicrobial host defense. To evaluate the potential antimicrobial activities of platelet proteins, normal human platelets were stimulated with human thrombin in vitro. Components of the stimulated-platelet supernatants were purified to homogeneity by reversed-phase high-performance liquid chromatography. Purified peptides with inhibitory activity againstEscherichia coliML35 in an agar diffusion antimicrobial assay were characterized by mass spectrometry, amino acid analysis, and sequence determination. These analyses enabled the identification of seven thrombin-releasable antimicrobial peptides from human platelets: platelet factor 4 (PF-4), RANTES, connective tissue activating peptide 3 (CTAP-3), platelet basic protein, thymosin β-4 (Tβ-4), fibrinopeptide B (FP-B), and fibrinopeptide A (FP-A). With the exception of FP-A and FP-B, all peptides were also purified from acid extracts of nonstimulated platelets. The in vitro antimicrobial activities of the seven released peptides were further tested against bacteria (E. coliandStaphylococcus aureus) and fungi (Candida albicansandCryptococcus neoformans). Each peptide exerted activity against at least two organisms. Generally, the peptides were more potent against bacteria than fungi, activity was greater at acidic pHs, and antimicrobial activities were dose dependent. Exceptions to these observations were observed with PF-4, which displayed a bimodal dose-response relationship in microbicidal assays, and Tβ-4, which had greater activity at alkaline pHs. At concentrations at which they were individually sublethal, PF-4 and CTAP-3 exerted synergistic microbicidal activity againstE. coli. Collectively, these findings suggest a direct antimicrobial role for platelets as they are activated to release peptides in response to trauma or mediators of inflammation.
Construction of a Novel Transposon Mutagenesis System Useful in the Isolation of
Streptococcus parasanguis
Mutants Defective in Fap1 Glycosylation
Authors: Chen Qiang , Wu Hui , Fives-Taylor Paula M.
Published: November 2002

Streptococcus parasanguis
, a primary colonizer of the tooth surface, has long, peritrichous fimbriae. A fimbria-associated protein, Fap1, is identified as an adhesin of
S. parasanguis
FW213. The mature Fap1 protein is glycosylated, and the glycosylation is required for fimbria biogenesis and bacterial adhesion. Little is known about the mechanism of Fap1 glycosylation due to the lack of identifiable mutants. A novel transposon mutagenesis system was established and used to generate a mutant library. Screening of the library with a monoclonal antibody specific for a glycan epitope of Fap1 yielded six mutants with decreased expression levels of surface-associated glycosylated Fap1 protein. Southern blot analyses revealed that three of the mutants had the transposon inserted in the
fap1
locus, whereas the other three mutants had insertions in other genes. Among the latter three mutants, two expressed Fap1 polypeptides on which no glycosylation was detected by glycan-specific antibodies; the other mutant expressed a partially glycosylated Fap1 polypeptide. These data suggest that three mutants were isolated with defects in genes implicated in Fap1 glycosylation.
Induction of Keratinocyte Growth Factor 1 Expression by Lipopolysaccharide Is Regulated by CD-14 and Toll-Like Receptors 2 and 4
Authors: Putnins Edward E. , Sanaie Ali-Reza , Wu Qiang , Firth James D.
Published: November 2002

Periodontal disease is a chronic inflammatory condition that is associated with increased concentrations of gram-negative pathogenic bacteria and epithelial cell proliferation. Regulation of this proliferation is poorly understood but is most likely controlled by locally expressed growth factors. Keratinocyte growth factor 1, an epithelium-specific growth factor, is expressed by gingival fibroblasts, and its expression is regulated in a concentration-dependent manner by lipopolysaccharide. In this study, induction of keratinocyte growth factor 1 protein expression was dependent on gingival fibroblast expression of membrane CD14 (mCD14) and Toll-like receptors 2 and 4. Lipopolysaccharides fromEscherichia coliandPorphyromonas gingivalisinduced membrane expression of CD14 at 1, 3, and 24 h. Specifically, lipopolysaccharide induced low mCD14 expression gingival fibroblasts to express mCD14 at a level consistent with that of high mCD14 expression cells. Functional studies with specific blocking antibodies for CD14 and Toll-like receptors 2 and 4 implicated all of these molecules in signal transduction. The rapid decrease in cell membrane expression of Toll-like receptors 2 and 4 after treatment with lipopolysaccharide was consistent with receptor internalization, and blocking of either of these receptors completely inhibited keratinocyte growth factor 1 protein expression. The transcription factors AP-1 and NF-κB were involved in lipopolysaccharide induction of keratinocyte growth factor 1 mRNA and protein expression. These results suggest that lipopolysaccharide may induce proliferation of periodontal epithelial cells by upregulating keratinocyte growth factor 1 expression via the CD14 and Toll-like receptor signaling pathway.
Mycobacterium bovisBCG Producing Interleukin-18 Increases Antigen-Specific Gamma Interferon Production in Mice
Authors: Biet Franck , Kremer Laurent , Wolowczuk Isabelle , Delacre Myriam , Locht Camille
Published: November 2002

Interleukin-18 (IL-18) and IL-12 play a critical role in the expression of cell-mediated immunity involved in host defense against intracellular pathogens. Both cytokines are produced by macrophages and act in synergy to induce gamma interferon (IFN-γ) production by T, B, and natural killer cells. In the present study, we analyzed both cellular and humoral responses upon infection with IL-18-secreting BCG of BALB/c and C3H/HeJ mice, two strains known to differ in their ability to support the growth of BCG. The cDNA encoding mature IL-18 was fused in frame with the alpha-antigen signal peptide-coding sequence, cloned downstream of the mycobacterialhsp60promoter and expressed in BCG. IL-18 produced by the recombinant BCG strain was functional, as judged by NF-κB-mediated luciferase induction in a tissue culture assay. When susceptible mice were infected with IL-18-producing BCG, their splenocytes were found to produce higher amounts of Th1 cytokines after stimulation with mycobacterial antigens than the splenocytes of mice infected with the nonrecombinant BCG. This was most prominent for IFN-γ, although the mycobacterial antigen-specific secretion of granulocyte-macrophage colony-stimulating factor and IL-10 was also augmented after infection with the recombinant BCG compared to infection with nonrecombinant BCG. In contrast, the immunoglobulin G levels in serum against mycobacterial antigens were lower when the mice were infected with IL-18-producing BCG compared to infection with nonrecombinant BCG. The IL-18 effect was delayed in BALB/c compared to C3H/HeJ mice. These results indicate that the production of IL-18 by recombinant BCG may enhance the immunomodulatory properties of BCG further toward a Th1 profile. This may be particularly useful for immunotherapeutic or prophylactic interventions in which a Th1 response is most desirable.
Effect ofMycobacterium tuberculosis-Specific 10-Kilodalton Antigen on Macrophage Release of Tumor Necrosis Factor Alpha and Nitric Oxide
Authors: Trajkovic Vladimir , Singh Gyanesh , Singh Balwan , Singh Sarman , Sharma Pawan
Published: November 2002

Secreted proteins ofMycobacterium tuberculosisare major targets of the specific immunity in tuberculosis and constitute promising candidates for the development of more efficient vaccines and diagnostic tests. We show here thatM. tuberculosis-specific antigen 10 (MTSA-10, originally designated CFP-10) can bind to the surface of mouse J774 macrophage-like cells and stimulate the secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). MTSA-10 also synergized with gamma interferon (IFN-γ) for the induction of the microbicidal free radical nitric oxide (NO) in J774 cells, as well as in bone marrow-derived and peritoneal macrophages. On the other hand, pretreatment of J774 cells with MTSA-10 markedly reduced NO but not TNF-α or interleukin 10 (IL-10) release upon subsequent stimulation with lipopolysaccharide or the cell lysate ofM. tuberculosis. The presence of IFN-γ during stimulation withM. tuberculosislysate antagonized the desensitizing effect of MTSA-10 pretreatment on macrophage NO production. The activation of protein tyrosine kinases (PTK) and the serine/threonine kinases p38 MAPK and ERK was apparently required for MTSA-10 induction of TNF-α and NO release, as revealed by specific kinase inhibitors. However, only p38 MAPK activity, not PTK or ERK activity, was partly responsible for MTSA-10-mediated macrophage desensitization. The modulation of macrophage function by MTSA-10 suggests a novel mechanism for its involvement in immunopathogenesis of tuberculosis and might have implications for the prevention, diagnosis, and therapy of this disease.
Pseudomonas fluorescens
Encodes the Crohn's Disease-Associated I2 Sequence and T-Cell Superantigen
Authors: Wei Bo , Huang Tiffany , Dalwadi Harnisha , Sutton Christopher L. , Bruckner David , Braun Jonathan
Published: November 2002

Commensal bacteria have emerged as an important disease factor in human Crohn's disease (CD) and murine inflammatory bowel disease (IBD) models. We recently isolated I2, a novel gene segment of microbial origin that is associated with human CD and that encodes a T-cell superantigen. To identify the I2 microorganism, BLAST analysis was used to identify a microbial homologue, PA2885, a novel open reading frame (ORF) in the
Pseudomonas aeruginosa
genome. PCR and Southern analysis identified
Pseudomonas fluorescens
as the originating species of I2, with homologues detectable in 3 of 13 other
Pseudomonas
species. Genomic cloning disclosed a locus containing the full-length I2 gene (
pfiT
) and three other orthologous genes, including a homologue of the
pbrA/pvdS
iron response gene. CD4
+
T-cell responses to recombinant proteins were potent for I2 and
pfiT
, but modest for PA2885.
pfiT
has several features of a virulence factor: association with an iron-response locus, restricted species distribution, and T-cell superantigen bioactivity. These findings suggest roles for
pfiT
and
P. fluorescens
in the pathogenesis of Crohn's disease.
A Glycoconjugate Vaccine forNeisseria meningitidisInduces Antibodies in Human Infants That Afford Protection against Meningococcal Bacteremia in a Neonate Rat Challenge Model
Authors: Mountzouros Kenneth T. , Belanger Kelly A. , Howell Alan P. , Bixler, Garvin S. , Madore Dace V.
Published: November 2002

The functional activities of serum samples from human infants immunized with a glycoconjugate vaccine forNeisseria meningitidisserogroup C were assessed in a complement-mediated antibody-dependent serum bactericidal assay (SBA) and in a neonate rat model of protection from bacteremia. Selective serum samples from individual human infants were combined to make a panel of 11 serum pools to obtain a sufficient volume for testing. Each pool was assayed (i) for the anti-N. meningitidisserogroup C capsular polysaccharide (PS) immunoglobulin G (IgG) concentration as determined by reactivity in a direct-binding enzyme-linked immunosorbent assay, (ii) for bactericidal activity againstN. meningitidisserogroup C strain C11, and (iii) for the ability to reduce bacteremia after passive transfer into a neonate rat model. Representative serum samples from infants who were not previously immunized with anyN. meningitidisserogroup C vaccine served as a negative control. The prepared serum pools ranged in antibody concentration from 0.18 to 17.31 μg of IgG specific forN. meningitidisserogroup C PS per ml. For this serum panel, a direct relationship between concentrations of anti-N. meningitidisserogroup C PS-specific IgG and serum SBA titers (r= 0.9960) was observed. Passive transfer to neonate rats demonstrated the ability of postimmunization serum samples to significantly reduce (≥2-log10reduction compared to control animals) the level of bacteremia following a challenge. Of 79 neonate rats that received ≥0.031 μg of human infant anti-N. meningitidisserogroup C PS IgG, 75 (94.9%) had a ≥2-log10reduction in bacteremia, whereas of the animals that received <0.031 μg of antigen-specific IgG, 10.3% (4 of 39 rats) showed a ≥2-log10reduction in bacteremia. It was concluded that the anti-N. meningitidisserogroup C PS IgG antibody induced by this glycoconjugate vaccine had in vitro functional activity (as determined by a SBA) and also afforded protection against meningococcal bacteremia in an animal model.
Development of Interleukin-12-Producing Capacity throughout Childhood
Authors: Upham John W. , Lee Peter T. , Holt Barbara J. , Heaton Tricia , Prescott Susan L. , Sharp Mary J. , Sly Peter D. , Holt Patrick G.
Published: November 2002

Increasing evidence indicates that the capacity to induce protective Th1 immune responses is impaired in early childhood, an observation that can be partially attributed to deficiencies in antigen-presenting-cell function. Synthesis of interleukin 12 (IL-12), a key Th1-trophic cytokine, is markedly reduced in the neonatal period, though there is a paucity of knowledge concerning the ontogeny of IL-12-synthetic capacity throughout the childhood years. Hence, we examined the production of bioactive IL-12 p70 by circulating mononuclear cells in a population of healthy individuals. As expected, the capacity to synthesize IL-12 p70 in response to either lipopolysaccharide or heat-killedStaphylococcus aureuswas markedly impaired at birth, even after priming of cells with gamma interferon. Surprisingly however, IL-12 p70 synthesis by peripheral blood mononuclear cells from both 5- and 12-year-old children was still substantially below that seen in adults, and this did not appear to be related to excessive production of IL-10. In contrast, dendritic cells from adults and neonates, derived from monocytes with granulocyte-macrophage colony-stimulating factor and IL-4, synthesized equivalent amounts of IL-12 p70 in response to microbial stimulation. This indicates that the impaired capacity for IL-12 synthesis in childhood is not an intrinsic property of circulating mononuclear cells but rather can be readily overcome in response to appropriate maturational stimuli. Because IL-12 arose predominantly from circulating HLA-DR+cells that lacked B-cell- and monocyte-specific markers, we propose that the slow maturation of IL-12-synthetic capacity in the childhood years can be attributed to deficiencies in the number and/or function of dendritic cells.
Antimonial Therapy Induces Circulating Proinflammatory Cytokines in Patients with Cutaneous Leishmaniasis
Authors: Kocyigit Abdurrahim , Gur Selahaddin , Gurel Mehmet S. , Bulut Vedat , Ulukanligil Mustafa
Published: November 2002

The objective of this study was to evaluate the association between antimonial therapy and circulating levels of proinflammatory cytokines in patients with cutaneous leishmaniasis (CL). Patients were treated with conventional chemotherapy by using pentavalent antimonium salts (Glucantime) for 3 weeks. Circulating plasma levels of the proinflammatory cytokines interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) were determined for CL patients and healthy subjects before and 3 weeks after the treatment was started. Plasma IL-1β, IL-6, IL-8, and TNF-α levels were significantly higher for pretreatment CL patients than for healthy subjects. Proinflammatory cytokines significantly increased after 21 days postinfection compared to levels for the pretreatment patients. These increments were approximately 3-fold for IL-1β and TNF-α levels, 10-fold for IL-6 levels, and 20-fold for IL-8 levels in patients with CL. Taken together these results indicate that circulating proinflammatory cytokine levels were increased in patients with CL as a consequence of host defense strategies, and antimonial therapy may induce these cytokines by affecting the macrophage or other components of the host defense system.
Role of β-d-Galactofuranose inLeishmania majorMacrophage Invasion
Authors: Suzuki Erika , Tanaka Ameria K. , Toledo Marcos S. , Takahashi Helio K. , Straus Anita H.
Published: November 2002

The role of glycosylinositol phospholipid 1 (GIPL-1) ofLeishmania(Leishmania)majorin the interaction of promastigotes and amastigotes with macrophages was analyzed. Monoclonal antibody MEST-1, which recognizes glycolipids containing terminal galactofuranose (Galf) residues (E. Suzuki, M. S. Toledo, H. K. Takahashi, and A. H. Straus, Glycobiology7:463-468, 1997), was used to detect GIPL-1 inLeishmaniaby indirect immunofluorescence and to analyze its role in macrophage infectivity.L. majorpromastigotes showed intense fluorescence with MEST-1, and GIPL-1 was detected in both amastigote and promastigote forms by high-performance thin-layer chromatography immunostaining by using MEST-1. Delipidation ofL. majorpromastigotes with isopropanol-hexane-water eliminated the MEST-1 reactivity, confirming that only GIPL-1 is recognized in either amastigotes or promastigotes of this species. The biological role of GIPL-1 in the ability ofL. majorto invade macrophages was studied by using either Fab fragments of MEST-1 or methylglycosides. Preincubation of parasites with Fab fragments reduced macrophage infectivity in about 80% of the promastigotes and 30% of the amastigotes. Preincubation of peritoneal macrophages withp-nitrophenyl-β-galactofuranoside (10 mM) led to significant (∼80%) inhibition of promastigote infectivity. These data suggest that a putative new receptor recognizing β-d-Galf is associated withL. majormacrophage infectivity and that GIPL-1 containing a terminal Galf residue is involved in theL. major-macrophage interaction.