bluuebunny/crossref_metadata_2025 huggingface dataset:
New
lnu
(C) Gene Conferring Resistance to Lincomycin by Nucleotidylation in
Streptococcus agalactiae
UCN36
Authors: Achard Adeline , Villers Corinne , Pichereau Vianney , Leclercq Roland
Published: June 2005

Streptococcus agalactiae
UCN36 was resistant to lincomycin (MIC = 16 μg/ml) but susceptible to clindamycin (MIC = 0.12 μg/ml) and erythromycin (MIC = 0.06 μg/ml). A 4-kb HindIII fragment was cloned from
S. agalactiae
UCN36 total DNA on plasmid pUC18 and introduced into
Escherichia coli
AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of
S. agalactiae
UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS
1
. The downstream ORF,
lnu
(C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuA
N2
, LnuA, and LnuA′ lincosamide nucleotidyltransferases reported for
Bacteroides
and
Staphylococcus
, respectively. Crude lysates of
E. coli
AG100A containing the cloned
lnu
(C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl
2
. Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin.
Antiviral Effect of Oral Administration of Tenofovir Disoproxil Fumarate in Woodchucks with Chronic Woodchuck Hepatitis Virus Infection
Authors: Menne Stephan , Cote Paul J. , Korba Brent E. , Butler Scott D. , George Andrea L. , Tochkov Ilia A. , Delaney William E. , Xiong Shelly , Gerin John L. , Tennant Bud C.
Published: June 2005

Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue approved for treatment of human immunodeficiency virus (HIV) infection. TDF also has been shown in vitro to inhibit replication of wild-type hepatitis B virus (HBV) and lamivudine-resistant HBV mutants and to inhibit lamivudine-resistant HBV in patients and HBV in patients coinfected with the HIV. Data on the in vivo efficacy of TDF against wild-type virus in non-HIV-coinfected or lamivudine-naïve chronic HBV-infected patients are lacking in the published literature. The antiviral effect of oral administration of TDF against chronic woodchuck hepatitis virus (WHV) infection, an established and predictive animal model for antiviral therapy, was evaluated in a placebo-controlled, dose-ranging study (doses, 0.5 to 15.0 mg/kg of body weight/day). Four weeks of once-daily treatment with TDF doses of 0.5, 1.5, or 5.0 mg/kg/day reduced serum WHV viremia significantly (0.2 to 1.5 log reduction from pretreatment level). No effects on the levels of anti-WHV core and anti-WHV surface antibodies in serum or on the concentrations of WHV RNA or WHV antigens in the liver of treated woodchucks were observed. Individual TDF-treated woodchucks demonstrated transient declines in WHV surface antigen serum antigenemia and, characteristically, these woodchucks also had transient declines in serum WHV viremia, intrahepatic WHV replication, and hepatic expression of WHV antigens. No evidence of toxicity was observed in any of the TDF-treated woodchucks. Following drug withdrawal there was prompt recrudescence of WHV viremia to pretreatment levels. It was concluded that oral administration of TDF for 4 weeks was safe and effective in the woodchuck model of chronic HBV infection.
Antibacterial Properties of Some Cyclic Organobismuth(III) Compounds
Authors: Kotani Toshiaki , Nagai Daisuke , Asahi Kensuke , Suzuki Hitomi , Yamao Fumiaki , Kataoka Nobumasa , Yagura Tatsuo
Published: June 2005

Bismuth compounds are known for their low levels of toxicity in mammals, and various types of bismuth salts have been used to treat medical disorders. As part of our program to probe this aspect of bismuth chemistry, cyclic organobismuth compounds 1 to 8 bearing a nitrogen or sulfur atom as an additional ring member have been synthesized, and their antimicrobial activities against five standard strains of gram-negative and gram-positive bacteria were assessed. The eight-membered-ring compounds, compounds 1 to 3, exhibited MICs of less than 0.5 μg/ml againstStaphylococcus aureusand were more active than the six-membered ones, compounds 5 to 8 (MICs, 4.0 to 16 μg/ml). The gram-positive bacteria (Staphylococcus aureus,Bacillus subtilis, andEnterococcus faecalis) were more susceptible to both types of ring compounds than the gram-negative ones (Escherichia coliandPseudomonas aeruginosa). Treatment with polymyxin B nonapeptide increased the susceptibility ofE. colito cyclic organobismuth compounds, indicating the low permeability of the outer membrane of gram-negative bacteria to the compounds. Compound 1 also had activity against methicillin-resistantS. aureus, which had an MIC for 90% of the hospital stock strains of 1.25 μg/ml. The killing curves forS. aureustreated with compound 1 or 3 revealed a static effect at a low dose (2× the MIC). However, whenS. aureuswas treated with 10× the MIC of compound 1 or 3, there was an approximately 3-log reduction in the viable cell number after 48 h of treatment. Electron microscopic inspection demonstrated a considerable increase in the size ofS. aureusand the proportion of cells undergoing cell division after treatment with compound 1 at 0.5× the MIC.
Short-Course Gentamicin in Combination with Daptomycin or Vancomycin against
Staphylococcus aureus
in an In Vitro Pharmacodynamic Model with Simulated Endocardial Vegetations
Authors: Tsuji Brian T. , Rybak Michael J.
Published: June 2005

The ability to maximize bactericidal activity while minimizing toxicity is a therapeutic goal in the treatment of infective endocarditis. We evaluated the impact of administering short-course regimens of gentamicin in combination with daptomycin or vancomycin against one methicillin-susceptible (MSSA 1199) and one methicillin-resistant (MRSA 494)
Staphylococcus aureus
isolate using an in vitro pharmacodynamic model with simulated endocardial vegetations over 96 h. Human therapeutic dosing regimens for daptomycin (6 and 8 mg/kg of body weight), vancomycin, and gentamicin were simulated. Short-course combination regimens involving gentamicin were administered either as a single 5-mg/kg dose or three 1-mg/kg doses for only the first 24 h and compared to the regimens administered for the full 96-h duration. For all experiments, physiologic conditions of albumin, calcium, and pH were simulated. Both regimens of daptomycin achieved 99.9% kill by 32 h and maintained bactericidal activity against both isolates, which was significantly different from vancomycin, which displayed bacteriostatic activity (
P
< 0.05). The effects of all short-course regimens of gentamicin were equal to those of the full-duration regimens in combination with daptomycin. Adding three doses of gentamicin (1 mg/kg) to daptomycin resulted in enhancement and bactericidal activity at 24 h against both MRSA and MSSA. The addition of a single dose of gentamicin (5 mg/kg) enhanced or improved the activity of daptomycin and resulted in early bactericidal activity at 4 h against both isolates. The addition of three doses of gentamicin (1 mg/kg) did not improve the activity of vancomycin. However, the addition of a single 5-mg/kg dose of gentamicin to vancomycin resulted in early enhancement at 4 h and 99.9% kill at 32 h for MRSA. These results suggest that a single high dose of gentamicin in combination with daptomycin or vancomycin may be of utility to maximize synergistic and bactericidal activity and minimize toxicity. Further investigation is warranted.
Constitutive
soxR
Mutations Contribute to Multiple-Antibiotic Resistance in Clinical
Escherichia coli
Isolates
Authors: Koutsolioutsou Anastasia , Peña-Llopis Samuel , Demple Bruce
Published: June 2005

The
soxRS
regulon of
Escherichia coli
and
Salmonella enterica
is induced by redox-cycling compounds or nitric oxide and provides resistance to superoxide-generating agents, macrophage-generated nitric oxide, antibiotics, and organic solvents. We have previously shown that constitutive expression of
soxRS
can contribute to quinolone resistance in clinically relevant
S. enterica
. In this work, we have carried out an analysis of the mechanism of constitutive
soxS
expression and its role in antibiotic resistance in
E. coli
clinical isolates. We show that constitutive
soxS
expression in three out of six strains was caused by single point mutations in the
soxR
gene. The mutant SoxR proteins contributed to the multiple-antibiotic resistance phenotypes of the clinical strains and were sufficient to confer multiple-antibiotic resistance in a fresh genetic background. In the other three clinical isolates, we observed, for the first time, that elevated
soxS
expression was not due to mutations in
soxR
. The mechanism of such increased
soxS
expression remains unclear. The same
E. coli
clinical isolates harbored polymorphic
soxR
and
soxS
DNA sequences, also seen for the first time.
Macrolide Resistance in
Campylobacter jejuni
and
Campylobacter coli
: Molecular Mechanism and Stability of the Resistance Phenotype
Authors: Gibreel Amera , Kos Veronica N. , Keelan Monika , Trieber Cathy A. , Levesque Simon , Michaud Sophie , Taylor Diane E.
Published: June 2005

A collection of 23 macrolide-resistant
Campylobacter
isolates from different geographic areas was investigated to determine the mechanism and stability of macrolide resistance. The isolates were identified as
Campylobacter jejuni
or
Campylobacter coli
based on the results of the hippurate biochemical test in addition to five PCR-based genotypic methods. Three point mutations at two positions within the peptidyl transferase region in domain V of the 23S rRNA gene were identified. About 78% of the resistant isolates exhibited an A→G transition at
Escherichia coli
equivalent base 2059 of the 23S rRNA gene. The isolates possessing this mutation showed a wide range of erythromycin and clarithromycin MICs. Thus, this mutation may incur a greater probability of treatment failure in populations infected by resistant
Campylobacter
isolates. Another macrolide-associated mutation (A→C transversion), at
E. coli
equivalent base 2058, was detected in about 13% of the isolates. An A→G transition at a position cognate with
E. coli
23S rRNA base 2058, which is homologous to the A2142G mutation commonly described in
Helicobacter pylori
, was also identified in one of the
C. jejuni
isolates examined. In the majority of
C. jejuni
isolates, the mutations in the 23S rRNA gene were homozygous except in two cases where the mutation was found in two of the three copies of the target gene. Natural transformation demonstrated the transfer of the macrolide resistance phenotype from a resistant
Campylobacter
isolate to a susceptible
Campylobacter
isolate. Growth rates of the resulting transformants containing A-2058→C or A-2059→G mutations were similar to that of the parental isolate. The erythromycin resistance of six of seven representative isolates was found to be stable after successive subculturing in the absence of erythromycin selection pressure regardless of the resistance level, the position of the mutation, or the number of the mutated copies of the target gene. One
C. jejuni
isolate showing an A-2058→G mutation, however, reverted to erythromycin and clarithromycin susceptibility after 55 subcultures on erythromycin-free medium. Investigation of ribosomal proteins L4 and L22 by sequence analysis in five representative isolates of
C. jejuni
and
C. coli
demonstrated no significant macrolide resistance-associated alterations in either the L4 or the L22 protein that might explain either macrolide resistance or enhancement of the resistance level.
Population Pharmacokinetics of Cefepime in the Neonate
Authors: Capparelli Edmund , Hochwald Christine , Rasmussen Maynard , Parham Amy , Bradley John , Moya Fernando
Published: June 2005

Newborn infants cared for in neonatal intensive care units may develop nosocomial infections. Cefepime, a “fourth-generation” cephalosporin (i.e., with activity against virtually all of the chromosomal-beta-lactamase-producing and many extended-spectrum-beta-lactamase-producing organisms), provides excellent activity against many gram-negative pathogens resistant to expanded-spectrum cephalosporins currently used to treat neonatal infections. The purpose of this study was to determine the pharmacokinetics of cefepime in this population to optimize dosing and minimize potential adverse events. Premature and term infants <4 months of age hospitalized in two neonatal intensive care units were studied. Limited pharmacokinetic (PK) sampling occurred following a dose of cefepime at 50 mg/kg of body weight infused over 30 min. Population pharmacokinetic parameters were determined using the program NONMEM. Fifty-five infants were enrolled. Their average (± standard deviation) gestational age at birth was 30.5 ± 5.3 weeks, and their average postnatal age at PK evaluation was 14.5 ± 14.7 days. In the final PK model, cefepime clearance (CL) was strongly associated with serum creatinine (SCr) (CL [ml/min/kg] = 0.26 + 0.59/SCr). The volume of distribution for infants with a postconceptional age of <30 weeks was larger than that for infants with a postconceptional age of >30 weeks (0.51 versus 0.39 liter/kg, respectively). The Bayesian analysis-predicted cefepime trough concentration at a dose of 50 mg/kg every 12 h for infants ≤14 days of age was 29.9 ± 16.6 μg/ml. Cefepime, dosed at 30 mg/kg/dose every 12 h for infants less than 14 days of age, regardless of gestational age, should provide antibiotic exposure equivalent to or greater than 50 mg/kg every 8 h in older infants and children.
Novel Approach to Mapping of Resistance Mutations in Whole Genomes by Using Restriction Enzyme Modulation of Transformation Efficiency
Authors: Lerner Claude G. , Kakavas Stephan J. , Wagner Christian , Chang Richard T. , Merta Philip J. , Ruan Xiaoan , Metzger Randy E. , Beutel Bruce A.
Published: June 2005

Restriction enzyme modulation of transformation efficiencies (REMOTE) is a method that makes use of genome restriction maps and experimentally observed differences in transformation efficiencies of genomic DNA restriction digests to discover the location of mutations in genomes. The frequency with which digested genomic DNA from a resistant strain transforms a susceptible strain to resistance is primarily determined by the size of the fragment containing the resistance mutation and the distance of the mutation to the end of the fragment. The positions of restriction enzyme cleavage sites immediately flanking the resistance mutation define these parameters. The mapping procedure involves a process of elimination in which digests that transform with high frequency indicate that the restriction enzyme cleavage sites are relatively far away from the mutation, while digests that transform with low frequency indicate that the sites are close to the mutation. The transformation data are compared computationally to the genome restriction map to identify the regions that best fit the data. Transformations with PCR amplicons encompassing candidate regions identify the resistance locus and enable identification of the mutation. REMOTE was developed using
Haemophilus influenzae
strains with mutations in
gyrA
,
gyrB
, and
rpsE
that confer resistance to ciprofloxacin, novobiocin, and spectinomycin, respectively. We applied REMOTE to identify mutations that confer resistance to two novel antibacterial compounds. The resistance mutations were found in genes that can decrease the intracellular concentration of compounds:
acrB
, which encodes a subunit of the AcrAB-TolC efflux pump; and
fadL
, which encodes a long-chain fatty acid transporter.
Structure-Based Phylogeny of the Metallo-β-Lactamases
Authors: Garau Gianpiero , Di Guilmi Anne Marie , Hall Barry G.
Published: June 2005

The metallo-β-lactamases fall into two groups: Ambler class B subgroups B1 and B2 and Ambler class B subgroup B3. The two groups are so distantly related that there is no detectable sequence homology between members of the two different groups, but homology is clearly detectable at the protein structure level. The multiple structure alignment program MAPS has been used to align the structures of eight metallo-β-lactamases and five structurally homologous proteins from the metallo-β-lactamase superfamily, and that alignment has been used to construct a phylogenetic tree of the metallo-β-lactamases. The presence of genes from
Eubacteria
,
Archaebacteria
, and
Eukaryota
on that tree is consistent with a very ancient origin of the metallo-β-lactamase family.
Drug-Induced Regulation of the
MDR1
Promoter in
Candida albicans
Authors: Harry Jo Beth , Oliver Brian G. , Song Jia L. , Silver Peter M. , Little John T. , Choiniere Jake , White Theodore C.
Published: June 2005

Resistance of
Candida albicans
to azole antifungal drugs is mediated by two types of efflux pumps, encoded by the
MDR1
gene and the
CDR
gene family.
MDR1
mRNA levels in a susceptible clinical isolate are induced by benomyl (BEN) but not by other drugs previously shown to induce
MDR1
. To monitor
MDR1
expression under several conditions, the
MDR1
promoter was fused to the
Renilla reniformis
luciferase reporter gene (
RLUC
). The promoter was monitored for its responses to four oxidizing agents, five toxic hydrophobic compounds, and an alkylating agent, all shown to induce major facilitator pumps in other organisms. Deletion constructs of the
MDR1
promoter were used to analyze the basal transcription of the promoter and its responses to the toxic compound BEN and the oxidizing agent
tert
-butyl hydrogen peroxide (T-BHP). The
cis
-acting elements in the
MDR1
promoter responsible for induction by BEN were localized between −399 and −299 upstream of the start codon. The
cis
-acting elements responsible for
MDR1
induction by T-BHP were localized between −601 and −500 upstream of the start codon. The T-BHP induction region contains a sequence that resembles the
YAP1
-responsive element (YRE) in
Saccharomyces cerevisiae
. This
Candida
YRE was placed upstream of a noninducible promoter in the luciferase construct, resulting in an inducible promoter. Inversion or mutation of the 7-bp YRE eliminated induction. Many of the drugs used in this analysis induce the
MDR1
promoter at concentrations that inhibit cell growth. These analyses define
cis
-acting elements responsible for drug induction of the
MDR1
promoter.
Multiple-Antibiotic Resistance in
Salmonella enterica
Serotype Paratyphi B Isolates Collected in France between 2000 and 2003 Is Due Mainly to Strains Harboring
Salmonella
Genomic Islands 1, 1-B, and 1-C
Authors: Weill François-Xavier , Fabre Laëtitia , Grandry Bernadette , Grimont Patrick A. D. , Casin Isabelle
Published: June 2005

This study was conducted to investigate the occurrence of multiple-antibiotic resistance among 261 clinical isolates of
Salmonella enterica
serotype Paratyphi B strains collected between 2000 and 2003 through the network of the French National Reference Center for
Salmonella
. The 47 multidrug-resistant (MDR) isolates identified (18%), were characterized on the basis of the presence of several resistance genes (
bla
TEM
,
bla
PSE-1
,
bla
CTX-M
,
floR
,
aadA2
,
qacEΔ1
, and
sul1
), the presence of
Salmonella
genomic island 1 (SGI1) by PCR mapping and hybridization, and the clonality of these isolates by several molecular (ribotyping, IS
200
profiling, and pulsed-field gel electrophoresis [PFGE]) and phage typing methods. The results of PCR and Southern blot experiments indicated that 39 (83%) of the 47
S. enterica
serotype Paratyphi B biotype Java MDR isolates possessed the SGI1 cluster (MDR/SGI1). Among these 39 MDR/SGI1 isolates, only 3 contained variations in SGI1, SGI1-B (
n
= 1) and SGI1-C (
n
= 2). The 39 MDR/SGI1 isolates showed the same specific PstI-IS
200
profile 1, which contained seven copies from 2.6 to 18 kb. Two PstI ribotypes were found in MDR/SGI1 isolates, RP1 (
n
= 38) and RP6 (
n
= 1). Ribotype RP1 was also found in two susceptible strains. Analysis by PFGE using XbaI revealed that all the MDR/SGI1 isolates were grouped in two related clusters, with a similarity percentage of 82%. Isolation of MDR/SGI1 isolates in France was observed mainly between the second quarter of 2001 and the end of 2002. The source of the contamination has not been identified to date. A single isolate possessing the extended-spectrum β-lactamase
bla
CTX-M-15
gene was also identified during the study.
Pharmacokinetics and Short-Term Safety of 873140, a Novel CCR5 Antagonist, in Healthy Adult Subjects
Authors: Adkison Kimberly K. , Shachoy-Clark Anne , Fang Lei , Lou Yu , O'Mara Kathy , Berrey M. Michelle , Piscitelli Stephen C.
Published: June 2005

873140 is a novel CCR5 antagonist with potent in vitro anti-human immunodeficiency virus (HIV) activity. This study was a double-blind, randomized, placebo-controlled, single- and repeat-dose escalation investigation of the safety, pharmacokinetics, and food effect of 873140 in 70 adult subjects. During single-dose escalation, three cohorts (each composed of 10 subjects, with 8 subjects receiving the active drug and 2 subjects receiving the placebo [8 active and 2 placebo]) received doses of 50, 200, 400, 800, and 1,200 mg after an overnight fast, or 400 mg plus a standard high-fat breakfast in an alternating panel design. During repeat-dose escalation, four cohorts (each with 8 active and 2 placebo) received doses of 200, 400, 600, or 800 mg every 12 h (BID) for 8 days. Laboratory safety tests, vital signs, and electrocardiograms (ECGs) were performed at regular intervals, and blood samples were obtained for pharmacokinetics. Single and repeat doses of 50 mg to 800 mg were well tolerated, with no serious adverse events and no grade 3 or 4 adverse events. The mild-to-moderate side effects were primarily gastrointestinal and included abdominal cramping, nausea, and diarrhea. No specific trends in laboratory parameters or clinically significant ECG changes were noted. Plasma 873140 concentrations increased rapidly; the median time to maximum concentration of drug in serum was 1.75 to 5 h. The median area under the plasma concentration-time profile (AUC) and the maximum concentration of drug in serum (
C
max
) ranged from 127 ng · h/ml and 24 ng/ml at 200 mg BID to 329 ng · h/ml and 100 ng/ml at 800 mg BID, respectively. Food consumption increased the AUC and
C
max
by a mean of 1.7- and 2.2-fold, respectively. The pharmacokinetic and safety profile supports the continued investigation of 873140 with HIV-infected subjects.
Sixteen Homologs of the Mex-Type Multidrug Resistance Efflux Pump in
Bacteroides fragilis
Authors: Ueda Ohmi , Wexler Hannah M. , Hirai Kaname , Shibata Yukinaga , Yoshimura Fuminobu , Fujimura Setsuo
Published: June 2005

Sixteen homologs of multidrug resistance efflux pump operons of the resistance-nodulation-cell division (RND) family were found in the
Bacteroides fragilis
genome sequence by homology searches. Disruption mutants were made to the
mexB
homologs of the four genes most similar to
Pseudomonas aeruginosa mexB
. Reverse transcription-PCR was conducted and indicated that the genes were transcribed in a polycistronic fashion and that the promoter was upstream of
bmeA
(the
mexA
homolog). One of these disruption mutants (in
bmeB
, the
mexB
homolog) was more susceptible than the parental strain to certain cephems, polypeptide antibiotics, fusidic acid, novobiocin, and puromycin. The gene for this homolog and the adjacent upstream gene,
bmeA
, were cloned in a hypersensitive
Escherichia coli
host. The resultant transformants carrying
B. fragilis bmeAB
were more resistant to certain agents; these agents also had lower MICs for the
B. fragilis bmeB
disruption mutants than for the parental strain. The putative efflux pump operon is composed of
bmeA
,
bmeB
, and
bmeC
(a putative outer membrane channel protein homologous with OprM). Addition of the efflux pump inhibitors, carbonyl cyanide
m
-chlorophenylhydrazone (a proton conductor that eliminates the energy source) and Phe-Arg β-naphthylamide (MC-207,110) (the first specific inhibitor described for RND pumps in
P. aeruginosa
), resulted in lowered MICs in the parental strain but not in the
bmeB
disruption mutant, indicating that the
bmeB
pump is affected by these inhibitors. This is the first description of RND type pumps in the genus
Bacteroides
.
Imipenem for Treatment of Tuberculosis in Mice and Humans
Authors: Chambers Henry F. , Turner Joan , Schecter Gisela F. , Kawamura Masae , Hopewell Philip C.
Published: June 2005

Chemotherapy of tuberculosis caused by multiple-drug-resistant (MDR) strains is problematic because of choices limited to relatively inefficacious and toxic drugs. Some beta-lactam antibiotics are active against
Mycobacterium tuberculosis
in vitro. We investigated the efficacy of imipenem in a mouse model of tuberculosis and in humans with MDR tuberculosis. Mice infected with
M. tuberculosis
strain H37Rv were treated with isoniazid or imipenem. Residual organisms in lung and spleen and survival of imipenem-treated mice were compared to those of untreated or isoniazid-treated mice. Ten patients with MDR tuberculosis also were treated with imipenem in combination with other first- or second-line agents; elimination of
M. tuberculosis
from sputum samples was measured by quantitative culture. Although it was less effective than isoniazid, imipenem significantly reduced the numbers of
M. tuberculosis
organisms in lungs and spleens and improved survival of mice. Eight of 10 patients with numerous risk factors for poor outcomes responded to imipenem combination therapy with conversion of cultures to negative. Seven remained culture-negative off of therapy. There were two deaths, one of which was due to active tuberculosis. Organisms were eliminated from the sputa of responders at a rate of 0.35 log
10
CFU/ml/week. Relapse upon withdrawal of imipenem and development of resistance to imipenem in a nonresponder suggest that imipenem exerts antimycobacterial activity in humans infected with
M. tuberculosis
. Imipenem had antimycobacterial activity both in a mouse model and in humans at high risk for failure of treatment for MDR tuberculosis.
Helicobacter pylori
Accumulates Photoactive Porphyrins and Is Killed by Visible Light
Authors: Hamblin Michael R. , Viveiros Jennifer , Yang Changming , Ahmadi Atosa , Ganz Robert A. , Tolkoff M. Joshua
Published: June 2005

Helicobacter pylori
colonizes the mucus layer of the human stomach and duodenum, causes chronic gastritis, gastric ulcer, and is a risk factor for gastric adenocarcinoma. There is a 20% failure rate in antibiotic therapy, which is increasingly due to antibiotic resistance and necessitates the search for alternative antimicrobial methods. We have discovered that
H. pylori
when cultured in liquid medium, accumulates significant quantities of coproporphyrin and protoporphyrin IX, both in the cells and secreted into the medium. These photoactive porphyrins lead to cell death (up to 5 logs) by photodynamic action upon illumination with low doses of visible light, with blue/violet light being most efficient. The degree of killing increases with the age of the culture and is greater than that found with
Propionibacterium acnes
(another bacterium known to be photosensitive due to porphyrin accumulation). Both virulent and drug-resistant strains are killed. The data suggest that phototherapy might be used to treat
H. pylori
infection in the human stomach.
Safety, Pharmacokinetics, and Efficacy of (+/−)-β-2′,3′-Dideoxy-5-Fluoro-3′-Thiacytidine with Efavirenz and Stavudine in Antiretroviral-Naïve Human Immunodeficiency Virus-Infected Patients
Authors: Herzmann C. , Arastèh K. , Murphy R. L. , Schulbin H. , Kreckel P. , Drauz D. , Schinazi R. F. , Beard A. , Cartee L. , Otto M. J.
Published: June 2005

Racivir [RCV; (+/−)-β-2′,3′-dideoxy-5-fluoro-3′-thiacytidine], a 50:50 racemic mixture of the two β nucleoside enantiomers, is currently in development for the treatment of human immunodeficiency virus type 1 (HIV-1) infections. RCV was administered once a day orally for 14 days at doses of 200, 400, or 600 mg in combination with stavudine and efavirenz to HIV-1-infected treatment-naïve male volunteers in a phase Ib/IIa study. Six volunteers at each dose were monitored for a total of 35 days for tolerance, pharmacokinetics, and plasma HIV RNA levels. RCV in combination with stavudine and efavirenz was well tolerated at all doses tested. Pharmacokinetic parameters were dose proportional, and the maximum concentration of drug in serum at all doses exceeded the 90% effective concentration for wild-type HIV-1. Viral loads dropped as expected in all dosage groups, with mean reductions from 1.13 to 1.42 log
10
by day 4 and 2.02 to 2.43 log
10
by day 14. HIV RNA levels remained suppressed for more than 2 weeks in the absence of any additional therapy, with mean viral loads ranging from 2.1 to 2.6 log
10
below baseline through day 28. By day 35, HIV RNA levels began to increase but still remained >1 log
10
below baseline levels.
Genetic Approach To Study the Relationship between Penicillin-Binding Protein 3 Mutations and
Haemophilus influenzae
β-Lactam Resistance by Using Site-Directed Mutagenesis and Gene Recombinants
Authors: Osaki Yumi , Sanbongi Yumiko , Ishikawa Midori , Kataoka Hiroshi , Suzuki Takahisa , Maeda Kumiko , Ida Takashi
Published: June 2005

To clarify the relationship between mutations commonly found for penicillin-binding protein 3 (PBP 3) of β-lactamase-nonproducing ampicillin-resistant (BLNAR)
Haemophilus influenzae
isolates and β-lactam resistance, single and multiple amino acid mutations at positions 377, 385, 389, 517, and 526 were introduced into PBP 3 of a β-lactam-susceptible Rd strain by site-directed mutagenesis. Twelve isogenic recombinant strains were challenged with nine β-lactam antibiotics. Replacement of the asparagine at position 526 with lysine (N526K) increased the resistance to imipenem eightfold and increased the resistance to various cephalosporins two- to eightfold. Substitution of threonine for serine at position 385 (S385T) and/or substitution of phenylalanine for leucine at position 389 (L389F), in addition to the N526K mutation, led to two- to fourfold additional increases in cephalosporin resistance. An isoleucine-to-methionine substitution at position 377 did not change the antibiotic sensitivity of any of the recombinant strains also carrying other PBP 3 mutations tested. Thirty-six clinical isolates carrying a PBP 3 gene (
ftsI
) with the S385T, L389F, R517H, and/or N526K mutation were chosen from among 279 clinical isolates collected in Japan, and the isolates were grouped into six classes on the basis of the patterns of the four mutations in PBP 3. Rd recombinants were made with each of the
ftsI
genes. The levels of resistance to β-lactams varied between recombinants of different classes but were comparable for those of the same class. The levels of resistance to cephalosporins of these recombinants were similar to those of the parent clinical isolates, while those to ampicillin and carbapenems were lower. These results indicate that resistance to β-lactams, at least to cephalosporins, depends in large part on the PBP 3 mutations R517H, N526K, S385T, and L389F.
Amino Acid Substitution inTrichophyton rubrumSqualene Epoxidase Associated with Resistance to Terbinafine
Authors: Osborne Colin S. , Leitner Ingrid , Favre Bertrand , Ryder Neil S.
Published: June 2005

There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequentialTrichophyton rubrumisolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA fromT. rubrumrevealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in theCandida albicansSE gene (L398F) and expression of this gene inSaccharomyces cerevisiaeconferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in theseT. rubrumclinical isolates appears to be due to a single amino acid substitution in SE.
Antimicrobial and Chemoattractant Activity, Lipopolysaccharide Neutralization, Cytotoxicity, and Inhibition by Serum of Analogs of Human Cathelicidin LL-37
Authors: Ciornei Cristina D. , Sigurdardóttir Thorgerdur , Schmidtchen Artur , Bodelsson Mikael
Published: June 2005

Antimicrobial peptides have been evaluated in vitro and in vivo as alternatives to conventional antibiotics. Apart from being antimicrobial, the native human cathelicidin-derived peptide LL-37 (amino acids [aa] 104 to 140 of the human cathelicidin antimicrobial peptide) also binds and neutralizes bacterial lipopolysaccharide (LPS) and might therefore have beneficial effects in the treatment of septic shock. However, clinical trials have been hampered by indications of toxic effects of LL-37 on mammalian cells and evidence that its antimicrobial effects are inhibited by serum. For the present study, LL-37 was compared to two less hydrophobic fragments obtained by N-terminal truncation, named 106 (aa 106 to 140) and 110 (aa 110 to 140), and to a previously described more hydrophobic variant, the 18-mer LLKKK, concerning antimicrobial properties, lipopolysaccharide neutralization, toxicity against human erythrocytes and cultured vascular smooth muscle cells, chemotactic activity, and inhibition by serum. LL-37, fragments 106 and 110, and the 18-mer LLKKK inhibited the growth ofEscherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus, andCandida albicansin a radial diffusion assay, inhibited lipopolysaccharide-induced vascular nitric oxide production, and attracted neutrophil granulocytes similarly. While fragments 106 and 110 caused less hemolysis and DNA fragmentation in cultured cells than did LL-37, the 18-mer LLKKK induced severe hemolysis. The antibacterial effect of fragments 106 and 110 was not affected by serum, while the effect of LL-37 was reduced. We concluded that the removal of N-terminal hydrophobic amino acids from LL-37 decreases its cytotoxicity as well as its inhibition by serum without negatively affecting its antimicrobial or LPS-neutralizing action. Such LL-37-derived peptides may thus be beneficial for the treatment of patients with sepsis.
Cryptococcus neoformans
Resistance to Echinocandins: (1,3)β-Glucan Synthase Activity Is Sensitive to Echinocandins
Authors: Maligie Marybeth A. , Selitrennikoff Claude P.
Published: June 2005

(1,3)β-
d
-Glucan synthase (EC 2.4.1.34. UDP-glucose: 1,3-β-
d
-glucan 3-β-glucosyltransferase) uses UDP-glucose as substrate and catalyzes the polymerization of glucose ([1,3]-β-linkages) to form the major carbohydrate component of the fungal cell wall. We have optimized in vitro assay conditions for (1,3)β-glucan synthase activity from
Cryptococcus neoformans
. Cells lysed in 50 mM Tris, pH 7.75, containing 20% glycerol, 2 mM NaF, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl
2
, 0.1% protease and phosphatase inhibitor cocktails, and 60 μM GTPγS produced maximum specific activity in vitro. We tested in vitro
C. neoformans
(1,3)β-glucan synthase activity against the (1,3)β-glucan synthase inhibitors, caspofungin and cilofungin, and have determined that (1,3)β-glucan synthase activity is very sensitive (apparent

K
i

of 0.17 ± 0.02 μM and 22 ± 5.7 μM, respectively) to these echinocandins. Taken together with high MICs for
C. neoformans
(caspofungin MIC, 16 μg/ml; cilofungin MIC, 64 μg/ml), our results indicate that
C. neoformans
is resistant to caspofungin and cilofungin by a mechanism(s) unrelated to (1,3)β-glucan synthase resistance.
3-Aminooxy-1-Aminopropane and Derivatives Have an Antiproliferative Effect on Cultured
Plasmodium falciparum
by Decreasing Intracellular Polyamine Concentrations
Authors: Das Gupta Robin , Krause-Ihle Tanja , Bergmann Bärbel , Müller Ingrid B. , Khomutov Alex R. , Müller Sylke , Walter Rolf D. , Lüersen Kai
Published: June 2005

The intraerythrocytic development of
Plasmodium falciparum
correlates with increasing levels of the polyamines putrescine, spermidine, and spermine in the infected red blood cells; and compartmental analyses revealed that the majority is associated with the parasite. Since depletion of cellular polyamines is a promising strategy for inhibition of parasite proliferation, new inhibitors of polyamine biosynthesis were tested for their antimalarial activities. The ornithine decarboxylase (ODC) inhibitor 3-aminooxy-1-aminopropane (APA) and its derivatives CGP 52622A and CGP 54169A as well as the
S
-adenosylmethionine decarboxlyase (AdoMetDC) inhibitors CGP 40215A and CGP 48664A potently affected the bifunctional
P. falciparum
ODC-AdoMetDC, with

K
i

values in the low nanomolar and low micromolar ranges, respectively. Furthermore, the agents were examined for their in vitro plasmodicidal activities in 48-h incubation assays. APA, CGP 52622A, CGP 54169A, and CGP 40215A were the most effective, with 50% inhibitory concentrations below 3 μM. While the effects of the ODC inhibitors were completely abolished by the addition of putrescine, growth inhibition by the AdoMetDC inhibitor CGP 40215A could not be antagonized by putrescine or spermidine. Moreover, CGP 40215A did not affect the cellular polyamine levels, indicating a mechanism of action against
P. falciparum
independent of polyamine synthesis. In contrast, the ODC inhibitors led to decreased cellular putrescine and spermidine levels in
P. falciparum
, supporting the fact that they exert their antimalarial activities by inhibition of the bifunctional ODC-AdoMetDC.
Frequency of Spontaneous Mutations That Confer Antibiotic Resistance in
Chlamydia
spp
Authors: Binet Rachel , Maurelli Anthony T.
Published: June 2005

Mutations in rRNA genes (
rrn
) that confer resistance to ribosomal inhibitors are typically recessive or weakly codominant and have been mostly reported for clinical strains of pathogens possessing only one or two
rrn
operons, such as
Helicobacter pylori
and
Mycobacterium
spp. An analysis of the genome sequences of several members of the
Chlamydiaceae
revealed that these obligate intracellular bacteria harbor only one or two sets of rRNA genes. To study the contribution of rRNA mutations to the emergence of drug resistance in the
Chlamydiaceae
, we used the sensitivities of
Chlamydia trachomatis
L2 (two
rrn
operons) and
Chlamydophila psittaci
6BC (one
rrn
operon) to the aminoglycoside spectinomycin as a model. Confluent cell monolayers were infected in a plaque assay with about 10
8
wild-type infectious particles and then treated with the antibiotic. After a 2-week incubation time, plaques formed by spontaneous spectinomycin-resistant (Spc
r
) mutants appeared with a frequency of 5 × 10
−5
for
C. psittaci
6BC. No Spc
r
mutants were isolated for
C. trachomatis
L2, although the frequencies of rifampin resistance were in the same range for both strains (i.e., 10
−7
). The risk of emergence of
Chlamydia
strains resistant to tetracyclines and macrolides, the ribosomal drugs currently used to treat chlamydial infections, is discussed.
Human Volunteers Receiving
Escherichia coli
Phage T4 Orally: a Safety Test of Phage Therapy
Authors: Bruttin Anne , Brüssow Harald
Published: June 2005

Fifteen healthy adult volunteers received in their drinking water a lower
Escherichia coli
phage T4 dose (10
3
PFU/ml), a higher phage dose (10
5
PFU/ml), and placebo. Fecal coliphage was detected in a dose-dependent way in volunteers orally exposed to phage. All volunteers receiving the higher phage dose showed fecal phage 1 day after exposure; this prevalence was only 50% in subjects receiving the lower phage dose. No fecal phage was detectable a week after a 2-day course of oral phage application. Oral phage application did not cause a decrease in total fecal
E. coli
counts. In addition, no substantial phage T4 replication on the commensal
E. coli
population was observed. No adverse events related to phage application were reported. Serum transaminase levels remained in the normal range, and neither T4 phage nor T4-specific antibodies were observed in the serum of the subjects at the end of the study. This is, to our knowledge, the first safety test in the recent English literature which has measured the bioavailability of oral phage in humans and is thus a first step to the rational evaluation of phage therapy for diarrheal diseases.
Colonization and Resistance Dynamics of Gram-Negative Bacteria in Patients during and after Hospitalization
Authors: Filius P. Margreet G. , Gyssens Inge C. , Kershof Irma M. , Roovers Patty J. E. , Ott Alewijn , Vulto Arnold G. , Verbrugh Henri A. , Endtz Hubert P.
Published: June 2005

The colonization and resistance dynamics of aerobic gram-negative bacteria in the intestinal and oropharyngeal microfloras of patients admitted to intensive care units (ICU) and general wards were investigated during and after hospitalization. A total of 3,316 specimens were obtained from patients upon admission, once weekly during hospitalization, at discharge from the ICU, at discharge from the hospital, and 1 and 3 months after discharge from the hospital. Five colonies per specimen were selected for identification and susceptibility testing. In both patient populations, the gram-negative colonization rates in oropharyngeal specimens increased during hospitalization and did not decrease in the 3 months after discharge. In rectal specimens, colonization rates decreased during hospitalization and increased after discharge. There was a change in species distribution among the dominant microfloras during hospitalization.
Klebsiella
spp.,
Enterobacter
spp.,
Serratia marcescens
, and
Pseudomonas aeruginosa
were isolated more often, whereas the frequency of
Escherichia coli
declined. The percentage of ICU patients colonized with ampicillin- and/or cephalothin-resistant fecal
E. coli
was significantly increased at discharge from the hospital and did not change in the 3 months after discharge. The emergence of multidrug resistance was observed for
E. coli
during patient stays in the ICU. Resistance frequencies in
E. coli
significantly increased with the length of stay in the ICU. For the general ward population, no significant changes in resistance frequencies were found during hospitalization. From a population perspective, the risk of dissemination of resistant gram-negative bacteria into the community through hospitalized patients appears to be low for general ward patients but is noticeably higher among ICU patients.
Efficacy of the Antiadhesin Octyl
O
-(2-Acetamido-2-Deoxy-β-
d
- Galactopyranosyl)-(1-4)-2-
O
-Propyl-β-
d
-Galactopyranoside (Fimbrigal-P) in a Rat Oral Candidiasis Model
Authors: Foldvari M. , Jaafari M. R. , Radhi J. , Segal D.
Published: June 2005

Adherence of
Candida albicans
to buccal epithelial cells via its fimbrial subunit requires the minimal disaccharide sequence β-GalNAc(1-4)-β-galactosidase in host cell receptors asialo-GM1 or asialo-GM2. This and other disaccharides and some of its synthetic derivatives have been shown to inhibit purified fimbrial or pathogen binding in vitro. This study evaluates the in vivo efficacy of the propyl derivative of this disaccharide, octyl
O
-(2-acetamido-2-deoxy-β-
d
-galactopyranosyl)-(1-4)-2-
O
-propyl-β-
d
-galactopyranoside, or Fimbrigal-P, incorporated into a mucoadhesive polymer formulation in a rat oral candidiasis model. Colony counts of microcurette samples from the oral cavity and tongue homogenates were used to estimate the effectiveness of four treatment modalities to reduce oral fungal burden. All treatment modalities (preventative, premixing with the
Candida
inoculant, drinking water, and treatment) significantly reduced fungal burden compared to untreated control animals by day 9; however, the preventative and premixing approaches provided a faster rate of fungal clearance. The low toxicity and immunogenicity of this synthetic carbohydrate and its stability in saliva, as demonstrated by high-performance liquid chromatography, make it a promising candidate for the prevention and treatment of microbial infections in which the pathogen relies on the β-GalNAc(1-4)-β-galactosidase disaccharide to establish adherence.
Identical Penicillin-Binding Domains in Penicillin-Binding Proteins of
Streptococcus pneumoniae
Clinical Isolates with Different Levels of β-Lactam Resistance
Authors: Chesnel Laurent , Carapito Raphaël , Croizé Jacques , Dideberg Otto , Vernet Thierry , Zapun André
Published: June 2005

We have sequenced the penicillin-binding domains of the complete repertoire of penicillin-binding proteins and MurM from 22 clinical isolates of
Streptococcus pneumoniae
that span a wide range of β-lactam resistance levels. Evidence of mosaicism was found in the genes encoding PBP 1a, PBP 2b, PBP 2x, MurM, and, possibly, PBP 2a. Five isolates were found to have identical PBP and MurM sequences, even though the MICs for penicillin G ranged from 0.25 to 2.0 mg/liter. When the sequences encoding PBP 1a, PBP 2b, and PBP 2x from one of these isolates were used to transform laboratory strain R6, the resulting strain had a resistance level higher than that of the less resistant isolates carrying that PBP set but lower than that of the most resistant isolates carrying that PBP set. This result demonstrates that if the R6 strain is arbitrarily defined as the standard genotype, some wild genetic backgrounds can either increase or decrease the PBP-based resistance phenotype.
Antimicrobial Susceptibility of
Streptococcus pneumoniae
in Eight European Countries from 2001 to 2003
Authors: Reinert Ralf René , Reinert Susanne , van der Linden Mark , Cil Murat Y. , Al-Lahham Adnan , Appelbaum Peter
Published: June 2005

Susceptibility testing results for
Streptococcus pneumoniae
isolates (
n
= 2,279) from eight European countries, examined in the PneumoWorld Study from 2001 to 2003, are presented. Overall, 24.6% of
S. pneumoniae
isolates were nonsusceptible to penicillin G and 28.0% were resistant to macrolides. The prevalence of resistance varied widely between European countries, with the highest rates of penicillin G and macrolide resistance reported from Spain and France. Serotype 14 was the leading serotype among penicillin G- and macrolide-resistant
S. pneumoniae
isolates. One strain (PW 158) showed a combination of an efflux type of resistance with a 23S rRNA mutation (A2061G, pneumococcal numbering; A2059G,
Escherichia coli
numbering). Six strains which showed negative results for
mef
(A) and
erm
(B) in repeated PCR assays had mutations in 23S rRNA or alterations in the L4 ribosomal protein (two strains). Fluoroquinolone resistance rates (levofloxacin MIC ≥ 4 μg/ml) were low (Austria, 0%; Belgium, 0.7%; France, 0.9%; Germany, 0.4%; Italy, 1.3%; Portugal, 1.2%; Spain, 1.0%; and Switzerland, 0%). Analysis of quinolone resistance-determining regions showed eight strains with a Ser81 alteration in
gyrA
; 13 of 18 strains showed a Ser79 alteration in
parC
. The clonal profile, as analyzed by multilocus sequence typing (MLST), showed that the 18 fluoroquinolone-resistant strains were genetically heterogeneous. Seven of the 18 strains belonged to new sequence types not hitherto described in the MLST database. Europe-wide surveillance for monitoring of the further spread of these antibiotic-resistant
S. pneumoniae
clones is warranted.