bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Hypersusceptibility of Human Immunodeficiency Virus Type 1 Subtype C Protease to Lopinavir
Authors: Gonzalez Luis M. F. , Brindeiro Rodrigo M. , Tarin Michelle , Calazans Alexandre , Soares Marcelo A. , Cassol Sharon , Tanuri Amilcar
Published: August 2003

In order to characterize the impact of genetic polymorphisms on the susceptibility of subtype C strains of human immunodeficiency virus type 1 to protease inhibitors (PIs), a subtype B protease that originated from an infectious clone was modified through site-directed mutagenesis to include the amino acid residue signatures of subtype C viruses (I15V, M36I, R41K, H69K, L89 M) with (clone C6) or without (clone C5) an I93L polymorphism present as a molecular signature of the worldwide subtype C protease. Their susceptibilities to commercially available PIs were measured by a recombinant virus phenotyping assay. We could not detect any differences in the 50% inhibitory concentration (IC
50
s) of amprenavir, indinavir, ritonavir, saquinavir, and nelfinavir for the clones analyzed. However, we did observe hypersusceptibility to lopinavir solely in clone C6, which includes the I93L substitution (a 2.6-fold decrease in the IC
50
compared to that for the subtype B reference strain). The same phenotypic behavior was observed for 11 Brazilian and South African clinical isolates tested, in which only subtype C isolates carrying the I93L mutation presented significant hypersusceptibility to lopinavir.
Deciphering Tuberactinomycin Biosynthesis: Isolation, Sequencing, and Annotation of the Viomycin Biosynthetic Gene Cluster
Authors: Thomas Michael G. , Chan Yolande A. , Ozanick Sarah G.
Published: August 2003

The tuberactinomycin antibiotics are essential components in the drug arsenal against
Mycobacterium tuberculosis
infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant
Staphylococcus aureus
strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, viomycin, from
Streptomyces
sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives.
Role of AcrR and RamA in Fluoroquinolone Resistance in Clinical
Klebsiella pneumoniae
Isolates from Singapore
Authors: Schneiders T. , Amyes S. G. B. , Levy S. B.
Published: August 2003

The MICs of ciprofloxacin for 33 clinical isolates of
K. pneumoniae
resistant to extended-spectrum cephalosporins from three hospitals in Singapore ranged from 0.25 to >128 μg/ml. Nineteen of the isolates were fluoroquinolone resistant according to the NCCLS guidelines. Strains for which the ciprofloxacin MIC was ≥0.5 μg/ml harbored a mutation in DNA gyrase A (Ser83→Tyr, Leu, or IIe), and some had a secondary Asp87→Asn mutation. Isolates for which the MIC was 16 μg/ml possessed an additional alteration in ParC (Ser80→IIe, Trp, or Arg). Tolerance of the organic solvent cyclohexane was observed in 10 of the 19 fluoroquinolone-resistant strains; 3 of these were also pentane tolerant. Five of the 10 organic solvent-tolerant isolates overexpressed AcrA and also showed deletions within the
acrR
gene. Complementation of the mutated
acrR
gene with the wild-type gene decreased AcrA levels and produced a two- to fourfold reduction in the fluoroquinolone MICs. None of the organic solvent-tolerant clinical isolates overexpressed another efflux-related gene,
acrE.
While
marA
and
soxS
were not overexpressed, another
marA
homologue,
ramA
, was overexpressed in 3 of 10 organic solvent-tolerant isolates. These findings indicate that multiple target and nontarget gene changes contribute to fluoroquinolone resistance in
K. pneumoniae.
Besides AcrR mutations,
ramA
overexpression (but not
marA
or
soxS
overexpression) was related to increased AcrAB efflux pump expression in this collection of isolates.
Molecular Characterization of a New Class 3 Integron in
Klebsiella pneumoniae
Authors: Correia Mário , Boavida Filipa , Grosso Filipa , Salgado M. J. , Lito L. M. , Cristino J. Melo , Mendo Sónia , Duarte Aida
Published: August 2003

Klebsiella pneumoniae
FFUL 22K was isolated in April 1999 from the urine of an intensive care unit patient in Portugal. The strain showed an extended-spectrum cephalosporin resistance profile. A typical synergistic effect between cefotaxime or cefepime and clavulanic acid was observed. An
Escherichia coli
transformant displayed a similar resistance phenotype and harbored a ca. 9.4-kb plasmid (p22K9). Cloning experiments revealed that the extended-spectrum β-lactamase was encoded by
bla
GES-1
, previously described in class 1 integrons from
K. pneumoniae
ORI-1 and
Pseudomonas aeruginosa
Pa695. Further sequence analysis demonstrated that the
bla
GES-1
gene cassette was located on a new class 3 integron. The integron was 2,863 bp long and consisted of an
intI3
integrase gene, an
attI3
recombination site, two promoter regions, and two gene cassettes. The IntI3 integrase was 98.8% identical to that of
Serratia marcescens
AK9373. The
bla
GES-1
gene cassette was inserted at the
attI3
site. The second gene cassette was the result of a fusion event between
bla
OXA-10
-type and
aac(6′)-Ib
gene cassettes and conferred resistance to kanamycin. This is the second class 3 integron reported and the first time that the
bla
GES-1
gene cassette has been found on an integron belonging to this class, highlighting the considerable heterogeneity of their genetic environment and the spread of gene cassettes among different classes of integrons.
Presence of the
tet
(O) Gene in Erythromycin- and Tetracycline-Resistant Strains of
Streptococcus pyogenes
and Linkage with either the
mef
(A) or the
erm
(A) Gene
Authors: Giovanetti Eleonora , Brenciani Andrea , Lupidi Remo , Roberts Marilyn C. , Varaldo Pietro E.
Published: August 2003

Sixty-three recent Italian clinical isolates of
Streptococcus pyogenes
resistant to both erythromycin (MICs ≥ 1 μg/ml) and tetracycline (MICs ≥ 8 μg/ml) were genotyped for macrolide and tetracycline resistance genes. We found 19 isolates carrying the
mef
(A) and the
tet
(O) genes; 25 isolates carrying the
erm
(A) and
tet
(O) genes; and 2 isolates carrying the
erm
(A),
tet
(M), and
tet
(O) genes. The resistance of all
erm
(A)-containing isolates was inducible, but the isolates could be divided into two groups on the basis of erythromycin MICs of either >128 or 1 to 4 μg/ml. The remaining 17 isolates included 15 isolates carrying the
erm
(B) gene and 2 isolates carrying both the
erm
(B) and the
mef
(A) genes, with all 17 carrying the
tet
(M) gene. Of these, 12 carried Tn
916
-Tn
1545
-like conjugative transposons. Conjugal transfer experiments demonstrated that the
tet
(O) gene moved with and without the
erm
(A) gene and with the
mef
(A) gene. These studies, together with the results of pulsed-field gel electrophoresis experiments and hybridization assays with DNA probes specific for the
tet
(O),
erm
(A), and
mef
(A) genes, suggested a linkage of
tet
(O) with either
erm
(A) or
mef
(A) in erythromycin- and tetracycline-resistant
S. pyogenes
isolates. By amplification and sequencing experiments, we detected the
tet
(O) gene ca. 5.5 kb upstream from the
mef
(A) gene. This is the first report demonstrating the presence of the
tet
(O) gene in
S. pyogenes
and showing that it may be linked with another gene and can be moved by conjugation from one chromosome to another.
Biological Costs and Mechanisms of Fosfomycin Resistance in
Escherichia coli
Authors: Nilsson Annika I. , Berg Otto G. , Aspevall Olle , Kahlmeter Gunnar , Andersson Dan I.
Published: August 2003

Fosfomycin is a cell wall inhibitor used mainly for the treatment of uncomplicated lower urinary tract infections. As shown here, resistance to fosfomycin develops rapidly in
Escherichia coli
under experimental conditions, but in spite of the relatively high mutation rate in vitro, resistance in clinical isolates is rare. To examine this apparent contradiction, we mathematically modeled the probability of resistance development in the bladder during treatment. The modeling showed that during a typical episode of urinary tract infection, the probability of resistance development was high (>10
−2
). However, if resistance was associated with a reduction in growth rate, the probability of resistance development rapidly decreased. To examine if fosfomycin resistance causes a reduced growth rate, we isolated in vitro and in vivo a set of resistant strains. We determined their resistance mechanisms and examined the effect of the different resistance mutations on bacterial growth in the absence and presence of fosfomycin. The types of mutations found in vitro and in vivo were partly different. Resistance in the mutants isolated in vitro was caused by
ptsI
,
cyaA
,
glpT
,
uhpA/T
, and unknown mutations, whereas no
cyaA
or
ptsI
mutants could be found in vivo. All mutations caused a decreased growth rate both in laboratory medium and in urine, irrespective of the absence or presence of fosfomycin. According to the mathematical model, the reduced growth rate of the resistant strains will prevent them from establishing in the bladder, which could explain why fosfomycin resistance remains rare in clinical isolates.
Molecular and Biochemical Characterization of OXA-45, an Extended-Spectrum Class 2d′ β-Lactamase inPseudomonas aeruginosa
Authors: Toleman Mark A. , Rolston Kenneth , Jones Ronald N. , Walsh Timothy R.
Published: August 2003

As part of the CANCER Antimicrobial Surveillance Program in North America, a clinical strain ofPseudomonas aeruginosa, strain 07-406, isolated in Texas was found to be resistant to all antimicrobials except polymyxin B. Genetic analysis of this isolate identified two unique extended-spectrum β-lactamase genes. One,blaVIM-7, encoded a metallo-β-lactamase (unpublished data), and the other,blaOXA-45, described here, encoded a class D extended-spectrum β-lactamase.blaOXA-45was isolated on aSau3A1 genomic fragment of 1.8 kb and encodes a protein of 264 amino acids with the highest identities to OXA-18 (65.9%), OXA-9 (42.8%), OXA-22 (40.2%), OXA-12 (38.6%), and OXA-29 (35.2%) but weak identities with other class D β-lactamases.blaOXA-45was found to be harbored on a 24-kb plasmid in a region that displays high identities with a section of the 43-kb genomic island ofSalmonella entericaserovar Typhimurium DT104. Biochemically OXA-45 is most similar to OXA-18 in its substrate profile and inhibition by clavulanic acid and is a member of the 2d′ class of β-lactamases.
Extended-Spectrum β-Lactamases in
Enterobacteriaceae
in Buenos Aires, Argentina, Public Hospitals
Authors: Quinteros M. , Radice M. , Gardella N. , Rodriguez M. M. , Costa N. , Korbenfeld D. , Couto E. , Gutkind G.
Published: August 2003

Resistance to extended-spectrum cephalosporins is often associated with plasmid encoded extended spectrum β-lactamases (ESBL). In order to evaluate the prevalence and diversity of ESBLs in enterobacteria in our city, a 1-month-period survey was carried out from April to May 2000. Extended-spectrum-cephalosporin-resistant strains, isolated from inpatient clinical specimens other than stools, were collected among 17 participating hospitals. From a total of 427 enterobacterial strains that were collected during this period, 39 were extended-spectrum cephalosporin resistant. The National Committee for Clinical Laboratory Standards' Screening and Confirmatory Tests for ESBL production were performed using cefotaxime and ceftazidime; cefepime and cefepime-clavulanic acid-containing disks were included. β-Lactamases were characterized by isoelectric focusing and PCR amplification using specific primers. Three different ESBLs were detected: SHV-related (4 isolates), PER-2-type (9 isolates), and CTX-M-2-related (26 isolates). Sequencing of the corresponding genes confirmed CTX-M-2 in 19 of 21 and CTX-M-31 (an allelic variant) in the remaining 2 of 21. CTX-M-2 (or its variant) was detected in all
Escherichia coli
,
Enterobacter aerogenes
,
Serratia marcescens
,
Proteus mirabilis
, and
Providencia stuartii
strains, while PER-2 was detected in
Enterobacter cloacae
,
E. aerogenes
, and
Klebsiella pneumoniae
; SHV-related ESBL were found only in
K. pneumoniae
. These results clearly show that CTX-M-2 is the most prevalent ESBL produced by enterobacterial species isolated from public hospitals in Buenos Aires.
Microcin C51 Plasmid Genes: Possible Source of Horizontal Gene Transfer
Authors: Fomenko Dmitri E. , Metlitskaya Anastazia Z. , Péduzzi Jean , Goulard Christophe , Katrukha Genrikh S. , Gening Leonid V. , Rebuffat Sylvie , Khmel Inessa A.
Published: August 2003

Microcin C51 (MccC51) is an antimicrobial nucleotide-heptapeptide produced by a natural
Escherichia coli
strain. A 5.7-kb fragment of the pC51 plasmid carrying the genes involved in MccC51 production, secretion, and self-immunity was sequenced, and the genes were characterized. The sequence of the MccC51 gene cluster is highly similar to that of the MccC7 gene. Recombinant plasmids carrying different combinations of the
mcc
genes involved in the MccC51 production or immunity were constructed to characterize their functional roles. The
mccA
,
mccB
,
mccD
, and
mccE
genes are involved in MccC51 production, while the
mccC
and
mccE
genes are responsible for immunity to MccC51. The
mcc
gene cluster is flanked by 44-bp direct repeats. Amino acid sequence comparisons allowed us to propose functions for each Mcc polypeptide in MccC51 biosynthesis. Plasmid pUHN containing the cloned
mccA
,
mccB
,
mccC
, and
mccE
genes, but lacking
mccD
, directed the synthesis of MccC51p, a substance chemically related to MccC51. MccC51p exhibited weak antibiotic activity against
E. coli
and was toxic to the producing cells. The immunity to exogenous MccC51 determined by the
mccC
and
mccE
genes did not overcome the toxic action of MccC51p on the producing cells. The G+C content of the MccC51 operon, markedly lower than that of the
E. coli
genome, and the presence of direct repeats suggest the possibility of horizontal transfer of this gene cluster.
Intracellular Expression of Peptide Fusions for Demonstration of Protein Essentiality in Bacteria
Authors: Benson R. Edward , Gottlin Elizabeth B. , Christensen Dale J. , Hamilton Paul T.
Published: August 2003

We describe a “protein knockout” technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione
S
-transferase in
Escherichia coli.
Expression of peptide fusions directed against
E. coli
DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growth-inhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.
Epidemiological Risk Factors for Isolation of Ceftriaxone-Resistant versus -Susceptible
Citrobacter freundii
in Hospitalized Patients
Authors: Kim Peter W. , Harris Anthony D. , Roghmann Mary-Claire , Morris J. Glenn , Strinivasan Arjun , Perencevich Eli N.
Published: August 2003

Antimicrobial resistance is an emerging problem among nosocomial bacteria. Risk factors for the recovery of ceftriaxone-resistant (CRCF) or -susceptible (CSCF)
Citrobacter freundii
in clinical cultures from hospitalized patients were determined by using a case-case-control study design. CRCF was isolated from 43 patients (case group 1) and CSCF was isolated from 87 patients (case group 2) over a 3-year period. Risk factors for CRCF were exposure to imipenem (odds ratio [OR], 7.5; 95% confidence interval [CI], 1.2 to 45.4), broad-spectrum cephalosporins (OR, 6.9; 95% CI, 1.8 to 26.7), vancomycin (OR, 3.0; 95% CI, 1.2 to 7.4), or piperacillin-tazobactam (OR, 2.6; 95% CI, 1.1 to 6.2), as well as hospital length of stay ≥1 week (OR, 3.6; 95% CI, 1.3 to 10.2) and intensive care unit (ICU) stay (OR, 2.6; 95% CI, 1.1 to 6.2). Risk factors for CSCF were peripheral vascular disease (OR, 23.2; 95% CI, 4.3 to 124.6), AIDS (OR, 9.5; 95% CI, 1.6 to 55.5), cerebrovascular disease (OR, 4.2; 95% CI, 1.6 to 10.8), and ICU stay (OR, 3.1; 95% CI, 1.8 to 5.4).
Optimal Sampling Schedule Design for Populations of Patients
Authors: Tam Vincent H. , Preston Sandra L. , Drusano G. L.
Published: August 2003

Generation of pharmacodynamic relationships in the clinical arena requires estimation of pharmacokinetic parameter values for individual patients. When the target population is severely ill, the ability to obtain traditional intensive blood sampling schedules is curtailed. Population modeling guided by optimal sampling theory has provided robust estimates of individual patient pharmacokinetic parameter values. Because of the wide range of parameter values seen in this circumstance, it is important to know how the range of parameter values in the population affects the timing of the optimal samples. We describe a new, simple technique to obtain optimal samples for a population of patients. This technique uses the nonparametric distribution associated with a nonparametric adaptive grid population pharmacokinetic analysis. We used the distribution from an analysis of 58 patients receiving levofloxacin for nosocomial pneumonia at a dose of 750 mg. The collection of parameter vectors and their associated probabilities were entered into a D-optimal design evaluation by using ADAPT II. The sampling times, weighted for their probabilities, were displayed in a frequency histogram (an expression of how system information varies with time for the population). Such an explicit expression of the time distribution of information allows rational sampling design that is robust not only for the population mean vector, as in traditional D-optimal design theory, but also for large portions of the total population. For levofloxacin, one reasonable six-sample design would be 1.5, 2, 2.25, 4, 4.75, and 24 h after starting a 90-min infusion. Such sampling designs allow informative population pharmacokinetic analysis with precise and unbiased estimates after the maximal a posteriori probability Bayesian step. This allows the highest probability of delineating a pharmacodynamic relationship.
Resistance to the Peptidyl Transferase Inhibitor Tiamulin Caused by Mutation of Ribosomal Protein L3
Authors: Bøsling Jacob , Poulsen Susan M. , Vester Birte , Long Katherine S.
Published: August 2003

The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistantEscherichia colimutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.
The Aminoglycoside 6′-
N
-Acetyltransferase Type Ib Encoded by Tn
1331
Is Evenly Distributed within the Cell's Cytoplasm
Authors: Dery Ken J. , Søballe Britta , Witherspoon Mavee S. L. , Bui Duyen , Koch Robert , Sherratt David J. , Tolmasky Marcelo E.
Published: August 2003

The multiresistance transposon Tn
1331
, which mediates resistance to several aminoglycosides and β-lactams, includes the
aac(6′)-Ib
,
aadA1
,
bla
OXA-9
, and
bla
TEM-1
genes. The nucleotide sequence of
aac(6′)-Ib
includes a region identical to that of the
bla
TEM-1
gene. This region encompasses the promoter and the initiation codon followed by 15 nucleotides. Since there were three possible translation initiation sites, the amino acid sequence at the N terminus of the aminoglycoside 6′-
N
-acetyltransferase type Ib [AAC(6′)-Ib] was determined and was found to be SIQHF. This result indicated that
aac(6′)-Ib
includes a translational fusion: the first five amino acids of the leader peptide of the TEM β-lactamase are fused to the rest of the AAC(6′)-Ib protein. This gene fusion could have formed during the genesis of Tn
1331
as a consequence of the generation of a 520-nucleotide duplication (M. E. Tolmasky, Plasmid
24:
218-226, 1990). An identical gene isolated from a
Serratia marcescens
strain has been previously described (G. Tran van Nhieu and E. Collatz, J. Bacteriol.
169:
5708-5714, 1987). Extraction of the periplasmic proteins of
E. coli
harboring
aac(6′)-Ib
by spheroplast formation showed that most of the AAC(6′)-Ib protein is present in the cytoplasm. A genetic fusion to
phoA
confirmed these results. AAC(6′)-Ib was shown to be evenly distributed inside the cell's cytoplasm by fluorescent microscopy with an AAC(6′)-Ib-cyan fluorescent protein fusion.
Signature Gene Expression Profiles Discriminate between Isoniazid-, Thiolactomycin-, and Triclosan-Treated
Mycobacterium tuberculosis
Authors: Betts Joanna C. , McLaren Alistair , Lennon Mark G. , Kelly Fiona M. , Lukey Pauline T. , Blakemore Steve J. , Duncan Ken
Published: August 2003

Genomic technologies have the potential to greatly increase the efficiency of the drug development process. As part of our tuberculosis drug discovery program, we used DNA microarray technology to profile drug-induced effects in
Mycobacterium tuberculosis
. Expression profiles of
M. tuberculosis
treated with compounds that inhibit key metabolic pathways are required as references for the assessment of novel antimycobacterial agents. We have studied the response of
M. tuberculosis
to treatment with the mycolic acid biosynthesis inhibitors isoniazid, thiolactomycin, and triclosan. Thiolactomycin targets the β-ketoacyl-acyl carrier protein (ACP) synthases KasA and KasB, while triclosan inhibits the enoyl-ACP reductase InhA. However, controversy surrounds the precise mode of action of isoniazid, with both InhA and KasA having been proposed as the primary target. We have shown that although the global response profiles of isoniazid and thiolactomycin are more closely related to each other than to that of triclosan, there are differences that distinguish the mode of action of these two drugs. In addition, we have identified two groups of genes, possibly forming efflux and detoxification systems, through which
M. tuberculosis
may limit the effects of triclosan. We have developed a mathematical model, based on the expression of 21 genes, which is able to perfectly discriminate between isoniazid-, thiolactomycin-, or triclosan-treated
M. tuberculosis
. This model is likely to prove invaluable as a tool to improve the efficiency of our drug development programs by providing a means to rapidly confirm the mode of action of thiolactomycin analogues or novel InhA inhibitors as well as helping to translate enzyme activity into whole-cell activity.
Antiviral Effects of Geranylgeranylacetone: Enhancement of MxA Expression and Phosphorylation of PKR during Influenza Virus Infection
Authors: Unoshima Masako , Iwasaka Hideo , Eto Junko , Takita-Sonoda Yoshiko , Noguchi Takayuki , Nishizono Akira
Published: August 2003

A cyclic polyisoprenoid compound, geranylgeranylacetone (GGA), has been used as antiulcer drug. GGA is also a potent inducer of heat shock proteins (HSPs). HSPs are considered to induce an antiviral effect; however, the detailed mechanism is unknown. To determine whether GGA might show antiviral activity and what the mechanism is, the effect of GGA against influenza virus (strain PR8) infection in vivo and in vitro was investigated. The results demonstrated that GGA treatment strongly suppressed the deleterious consequences of PR8 replication and was accompanied by an increase in HSP70 gene expression in mice. Results from in vitro analyses demonstrated that GGA significantly inhibited the synthesis of PR8-associated proteins and prominently enhanced expression of human myxovirus resistance 1 (MxA) followed by increased HSP70 transcription. Moreover, GGA augmented the expression of an interferon-inducible double-strand RNA-activated protein kinase (PKR) gene and promoted PKR autophosphorylation and concomitantly α subunit of eukaryotic initiation factor 2 phosphorylation during PR8 infection. It is proposed that GGA-induced HSP70 has potent antiviral activity by enhancement of antiviral factors and can clinically achieve protection from influenza virus infection.
New
Klebsiella oxytoca
β-Lactamase Genes
bla
OXY-3
and
bla
OXY-4
and a Third Genetic Group of
K. oxytoca
Based on
bla
OXY-3
Authors: Granier Sophie A. , Leflon-Guibout Véronique , Goldstein Fred W. , Nicolas-Chanoine Marie-Hélène
Published: August 2003

The two genetic groups (oxy-1 and oxy-2) previously identified in the
Klebsiella oxytoca
taxon are recognizable by four independent molecular markers: (i) ERIC-1R profiles, (ii) 16S ribosomal DNA (rDNA) signature sequences, (iii) singular nucleotides in a defined fragment of the
rpoB
gene, and (iv) the type of the strain's
bla
OXY
gene (i.e.,
bla
OXY-1
or
bla
OXY-2
).
K. oxytoca
strains SG266 and SG271 could not be classified into these genetic groups based on their ERIC-1R profile and
bla
OXY
gene sequence. With regard to the gene identity percentages between the
bla
OXY-1
and
bla
OXY-2
gene groups (86.8% ± 0.4%) and within a
bla
OXY
gene group (>99%), it was concluded that the
bla
OXY
gene of strain SG271 was representative of a new
bla
OXY
gene group (
bla
OXY-3
), since the mean identity percentages between it and the two
bla
OXY
gene groups were 85.5% ± 0.2% and 84.4% ± 0.4%, respectively. Since the corresponding percentages were 95.0% ± 0.4% and 86.2% ± 0.3% for strain SG266, it was impossible to classify its
bla
OXY
gene, which was therefore named
bla
OXY-4
. The 16S rDNA signature sequences of the two strains could be determined only after cloning experiments. The SG266 clones displayed the same signature sequence as that of the genetic group oxy-1, whereas the SG271 clones displayed three different 16S rDNA signature sequences that also differed from those of the two genetic groups. Singular nucleotides were found within the
rpoB
sequence of the two strains, allowing for their distinction from the two genetic groups. All of these results, combined with those previously obtained by the ERIC-1R PCR method, indicate that strain SG271 is representative of a new
K. oxytoca
genetic group (oxy-3), whereas strain SG266 could not be classified.
Sulfadoxine-Pyrimethamine in Treatment of Malaria in Western Kenya: Increasing Resistance and Underdosing
Authors: Terlouw Dianne J. , Nahlen Bernard L. , Courval Jeanne M. , Kariuki Simon K. , Rosenberg Oren S. , Oloo Aggrey J. , Kolczak Margarette S. , Hawley William A. , Lal Altaf A. , Kuile Feiko O. ter
Published: August 2003

Between 1993 and 1999, we monitored the efficacy of sulfadoxine-pyrimethamine in 1,175 children aged <24 months receiving 2,789 treatments for falciparum malaria in western Kenya using a widely deployed age-based dose regimen: infants, 125 plus 6.25 mg (sulfadoxine plus pyrimethamine); children aged 12 to 23 months; 250 plus 12.5 mg. Cumulative treatment failure by day 7, defined as early clinical failure by day 3 or presence of parasitemia on day 7, increased from 18% in 1993 to 1994 to 22% in 1997 to 1998 (
P
-trend test = 0.20). Based on body weight, the median dose received was 20 plus 1.00 mg/kg, and 73% of the treatments were given at lower than the recommended target dose of 25 plus 1.25 mg/kg. Underdosing accounted for 26% of cumulative treatment failures. After the dose was increased in 1998 (median, 36 plus 1.8 mg/kg), only 4.2% of patients received less than 25 plus 1.25 mg/kg and there was no association with treatment failure. However, the proportion of cumulative treatment failure continued to increase to 27% by 1999 (
P
-trend test = 0.03). These results raise concern about the longevity of sulfadoxine-pyrimethamine in these settings. Underdosing may have contributed to the rate at which sulfadoxine-pyrimethamine resistance developed in this area. Treatment guidelines should ensure that adequate doses are given from the initial deployment of antimalarials onward.
Aerosolized Cidofovir Is Retained in the Respiratory Tract and Protects Mice against Intranasal Cowpox Virus Challenge
Authors: Roy Chad J. , Baker Robert , Washburn Kenneth , Bray Mike
Published: August 2003

We employed a murine model to test the concept of using an aerosolized, long-acting antiviral drug to protect humans against smallpox. We previously showed that a low dose of aerosolized cidofovir (HPMPC [Vistide]) was highly protective against subsequent aerosolized cowpox virus challenge and was more effective than a much larger dose of drug given by injection, suggesting that aerosolized cidofovir is retained in the lung. Because the nephrotoxicity of cidofovir is a major concern in therapy, delivering the drug directly to the respiratory tract might be an effective prophylactic strategy that maximizes the tissue concentration at the site of initial viral replication, while minimizing its accumulation in the kidneys. In the present study, we found that treating mice with aerosolized
14
C-labeled cidofovir (
14
C-cidofovir) resulted in the prolonged retention of radiolabeled drug in the lungs at levels greatly exceeding those in the kidneys. In contrast, subcutaneous injection produced much higher concentrations of
14
C-cidofovir in the kidneys than in the lungs over the 96-h time course of the study. As further evidence of the protective efficacy of aerosolized cidofovir, we found that aerosol treatment before or after infection was highly protective in mice challenged intranasally with cowpox virus. All or nearly all mice that were treated once by aerosol, from 2 days before to 2 days after challenge, survived intranasal infection, whereas all placebo-treated animals died.
Insertion Sequence IS
Ecp1B
Is Involved in Expression and Mobilization of a
bla
CTX-M
β-Lactamase Gene
Authors: Poirel Laurent , Decousser Jean-Winoc , Nordmann Patrice
Published: August 2003

The genetic structures (ca. 10-kb DNA fragment) surrounding the plasmid-borne extended-spectrum β-lactamase
bla
CTX-M-19
gene in a
Klebsiella pneumoniae
clinical isolate were determined. This β-lactamase gene was part of a 4,797-bp transposon inserted inside
orf1
of Tn
1721.
Inside this transposon,
bla
CTX-M-19
was bracketed upstream and downstream by insertion sequences IS
E cp1B
and IS
903D,
respectively, and further downstream by a truncated gene encoding an outer membrane protein for iron transport. The single-copy IS
Ecp1B
element was probably involved alone in the mobilization process that led to a 5-bp duplication at the target site of the transposed fragment. This mobilization event probably involved one inverted repeat of IS
E cp1B
and a second sequence farther away, resembling its second inverted repeat. Additionally, IS
Ecp1B
provided −35 and −10 promoter sequences, contributing to the high-level expression of the
bla
CTX-M-19
gene. Southern blot analysis failed to identify a reservoir of IS
Ecp1
-like sequences among a series of gram-negative and gram-positive bacterial species usually found in the skin and intestinal human floras. The ability of IS
Ecp1
-like elements to mobilize and to promote the expression of β-lactamase genes may explain, in part, the current spread of CTX-M-type enzymes worldwide.
In Vitro Activities of New Fluoroquinolones against
Campylobacter jejuni
and
Campylobacter coli
Isolates Obtained from Humans in 1980 to 1982 and 1997 to 2001
Authors: Krausse Rea , Ullmann Uwe
Published: August 2003

The antibacterial activities of three newly developed fluoroquinolones (gatifloxacin, levofloxacin, and moxifloxacin) against a total of 307 gastrointestinal human isolates of
Campylobacter jejuni
and
Campylobacter coli
collected during 1980 to 1982 and 1997 to 2001 were examined and compared to those of ciprofloxacin and the unrelated antibacterial agents, clarithromycin, erythromycin, and tetracycline by using the agar plate dilution method. All of the fluoroquinolones exhibited a good activity against
Campylobacter
, and some of them were more active than ciprofloxacin, the macrolides, and tetracycline. Among the fluoroquinolones, gatifloxacin and moxifloxacin showed the highest anticampylobacter activity, with MICs at which 50% of the isolates tested are inhibited (MIC
50
s) and MIC
90
s of 0.125 and 4 μg/ml, respectively; the MIC
50
for both levofloxacin and ciprofloxacin was 0.25, and the MIC
90
s were 16 and 32 μg/ml, respectively. About 30% of the strains were found to be resistant to at least one fluoroquinolone. Resistance to gatifloxacin occurred in 9.8% of the isolates tested, and resistance to the other fluoroquinolones occurred in 19.9 to 27.4% of the isolates tested; the frequency of cross-resistance was 35.7 to 100%. An increase in fluoroquinolone resistance from 0% in 1980 to 1982 to 11.8 to 29% in 1997 and 1998, 8.2 to 31.8% in 1999 and 2000, and 12.1 to 30.3% in 2001 was found. A total of 61.4 to 73.2% of the
C. jenuni
strains resistant to erythromycin, clarithromycin, and/or tetracycline were susceptible to fluoroquinolones; gatifloxacin showed the highest percentage of inhibition. These results show that the newer fluoroquinolones with their potent activity could be used to treat infections with
C. jejuni
and
C. coli.
However, when these drugs are used, one must consider the increase in resistance and the high cross-resistance to these antimicrobial agents.
Inhibition of Human Immunodeficiency Virus by a New Class of Pyridine Oxide Derivatives
Authors: Stevens Miguel , Pannecouque Christophe , De Clercq Erik , Balzarini Jan
Published: August 2003

A new class of pyridine oxide derivatives as inhibitors of human immunodeficiency virus type 1 (HIV-1) and/or HIV-2 replication in cell culture has been identified. The compounds, which specifically inhibit HIV-1, behave as typical nonnucleoside reverse transcriptase inhibitors (NNRTIs). The most active congener of this group, JPL-133 (UC-B3096), has a 50% effective concentration of 0.05 μg/ml for HIV-1(III
B
) with a selectivity index of approximately 760 in CEM cell cultures. However, the cytostatic activity of most pyridine oxide derivatives highly depended on the nature of the cell line. All compounds, including those pyridine oxide derivatives that inhibit both HIV-1 and HIV-2 replication, select for NNRTI-characteristic mutations in the HIV-1 reverse transcriptase of HIV-infected cell cultures (i.e., Lys103Asn, Val108Ile, Glu138Lys, Tyr181Cys and Tyr188His). These amino acid mutations emerged mostly through transition of guanine to adenine or adenine to guanine in the corresponding codons of the reverse transcriptase (RT) gene. The HIV-1-specific pyridine oxide derivatives lost their antiviral activity against HIV-1 strains containing these mutations in the RT. However, most compounds retained pronounced antiviral potency against virus strains that contained other NNRTI-characteristic RT mutations, such as Leu100Ile and Val179Asp. Furthermore, the complete lack of inhibitory activity of the pyridine oxide derivatives against recombinant HIV-2 RT and partial retention of anti-HIV-1 activity against HIV-1 strains that contain a variety of HIV-1-characteristic mutations suggest that the pyridine oxide derivatives must have a second target of antiviral action independent from HIV-1 RT.
Effects of Ser130Gly and Asp240Lys Substitutions in Extended-Spectrum β-Lactamase CTX-M-9
Authors: Aumeran C. , Chanal C. , Labia R. , Sirot D. , Sirot J. , Bonnet R.
Published: August 2003

In CTX-M-9 extended-spectrum β-lactamases (ESBLs), an S130G mutation induced a 40- to 650-fold increase in 50% inhibitory concentrations but decreased hydrolytic activity against cefotaxime. A D240K mutation did not modify enzymatic efficiency against ceftazidime. Residue K240 could interact with Q270 and therefore not with ceftazidime, in contrast with what was observed with certain TEM/SHV-type ESBLs.
Gentamicin Delivery to
Burkholderia cepacia
Group IIIa Strains via Membrane Vesicles from
Pseudomonas aeruginosa
PAO1
Authors: Allan Nick D. , Beveridge Terry J.
Published: August 2003

When
Pseudomonas aeruginosa
PAO1 is treated with gentamicin, it releases membrane vesicles containing gentamicin (g-MVs) and peptidoglycan hydrolase, which makes the MVs bactericidal. We evaluate the ability of g-MVs to deliver gentamicin past the intrinsic permeability barrier of group IIIa
Burkholderia cepacia
and show that strain CEP0248 with low resistance to gentamicin is killed but the highly resistant strain C5424 is not. Immunoelectron microscopy revealed that gentamicin was delivered into both strains, suggesting that there might be another mechanism of resistance in C5424.
S
-Adenosyl-
l
-Methionine Inhibitors Δ
24
-Sterol Methyltransferase and Δ
24(28)
-Sterol Methylreductase as Possible Agents against
Paracoccidioides brasiliensis
Authors: Visbal Gonzalo , Alvarez Alvaro , Moreno Belisario , San-Blas Gioconda
Published: August 2003

We studied the antiproliferative effects of three azasterol analogs [piperidyl-2-yl-5α-pregnan-3β,20(
R
)-diol (AZA-1), 22-piperidin-2-yl-pregnan-22(
S
),3β-diol (AZA-2), and 22-piperidin-3-yl-pregnan-22(
S
),3β-diol (AZA-3)] and their effects on the lipid composition of the pathogenic yeastlike phase of the dimorphic fungus
Paracoccidioides brasiliensis
. Inhibition was 100% for AZA-1 at 5 μM, 62% for AZA-2 at 10 μM, and 100% for AZA-3 at 0.5 μM. The analogs inhibited different stages of the sterol biosynthesis pathway.
Discordant Resistance to Kanamycin and Amikacin in Drug-Resistant
Mycobacterium tuberculosis
Authors: Krüüner Annika , Jureen Pontus , Levina Klavdia , Ghebremichael Solomon , Hoffner Sven
Published: August 2003

It is generally thought that there is full cross-resistance in
Mycobacterium tuberculosis
between the aminoglycoside drugs kanamycin and amikacin. However, kanamycin resistance and amikacin susceptibility were seen in 43 of 79 (54%) multidrug-resistant Estonian isolates, indicating that there might be a need to test the resistance of
M. tuberculosis
isolates to both drugs.
Activity of Daptomycin against Recent North American Isolates of
Streptococcus pneumoniae
Authors: Restrepo M. I. , Velez J. A. , McElmeel M. L. , Whitney C. G. , Jorgensen J. H.
Published: August 2003

Daptomycin MICs at which 50% of isolates were inhibited (MIC
50
s) and MIC
90
s determined by the NCCLS broth microdilution method were both 0.25 μg/ml (range, 0.06 to 2 μg/ml) for 350 pneumococcal isolates. MICs determined by E test strips on commercially prepared Mueller-Hinton sheep blood agars with different calcium contents were 2 to 3 dilutions higher than those determined by strips that contained daptomycin plus calcium. Daptomycin zone diameters varied little on the same media.
New Plasmid-Borne Antibiotic Resistance Gene Cluster in
Pasteurella multocida
Authors: Kehrenberg Corinna , Tham Nga Thi Thu , Schwarz Stefan
Published: August 2003

A new antibiotic resistance gene cluster comprising genes for sulfonamide (
sul2
), streptomycin (
strA
-
strB
), and tetracycline [
tetR
-
tet
(H)] resistance was detected on plasmid pVM111 from
Pasteurella multocida
. The
tetR
-
tet
(H) gene region was inserted between
sul2
and
strA
, possibly by illegitimate recombination. Two potential recombination sites of 18 and 25 bp were identified.
A New TEM-Derived Extended-Spectrum β-Lactamase (TEM-91) with an R164C Substitution at the Ω-Loop Confers Ceftazidime Resistance
Authors: Kurokawa Hiroshi , Shibata Naohiro , Doi Yohei , Shibayama Keigo , Kamachi Kazunari , Yagi Tetsuya , Arakawa Yoshichika
Published: August 2003

A new plasmid-mediated TEM-derived extended-spectrum β-lactamase, TEM-91, was identified in a ceftazidime-resistant (MIC, >128 μg per ml)Escherichia colistrain isolated in 1996 in Japan. TEM-91 has three amino acid substitutions, R164C, M184T, and E240K, compared with TEM-1 penicillinase. The isoelectric point (pI),Km, andkcatof TEM-91 for ceftazidime were 5.7, 179 μM, and 29.0 s−1, respectively. TheKiof clavulanic acid for ceftazidime hydrolysis was 30.3 nM.
Effects of 16S rRNA Gene Mutations on Tetracycline Resistance in
Helicobacter pylori
Authors: Gerrits Monique M. , Berning Marco , Van Vliet Arnoud H. M. , Kuipers Ernst J. , Kusters Johannes G.
Published: August 2003

The triple-base-pair 16S rDNA mutation AGA
926-928
→TTC mediates high-level tetracycline resistance in
Helicobacter pylori
. In contrast, single- and double-base-pair mutations mediated only low-level tetracycline resistance and decreased growth rates in the presence of tetracycline, explaining the preference for the TTC mutation in tetracycline-resistant
H. pylori
isolates.
Measurement of Cefaclor and Amoxicillin-Clavulanic Acid Levels in Middle-Ear Fluid in Patients with Acute Otitis Media
Authors: Scaglione F. , Caronzolo D. , Pintucci J. P. , Fraschini F.
Published: August 2003

Concentrations of cefaclor (CFC) or amoxicillin-clavulanic acid (AMX/CA) in middle-ear fluid collected preserving the stability and clearing the cell contents has been compared to those obtained using the traditional method. Sixty-seven children with effusive otitis media were treated orally with CFC (20 mg/kg of body weight) or AMX/CA (20 mg/kg) (4:1 ratio). The concentrations in cell-free fluid (C−) appear higher than those in the total fluid (C+) (as assayed traditionally).
Functional Cloning and Characterization of a Multidrug Efflux Pump, MexHI-OpmD, from a
Pseudomonas aeruginosa
Mutant
Authors: Sekiya Hiroshi , Mima Takehiko , Morita Yuji , Kuroda Teruo , Mizushima Tohru , Tsuchiya Tomofusa
Published: August 2003

We isolated mutant YM644, which showed elevated resistance to norfloxacin, ethidium bromide, acriflavine, and rhodamine 6G, from
Pseudomonas aeruginosa
YM64, a strain that lacks four major multidrug efflux pumps. The genes responsible for the resistance were
mexHI-opmD.
Elevated ethidium extrusion was observed with cells of YM644 and YM64 harboring a plasmid carrying the genes. Disruption of the genes in the chromosomal DNA of YM644 made the cells sensitive to the drugs.
Multiple Antibiotic Resistance Gene Transfer from Animal to Human Enterococci in the Digestive Tract of Gnotobiotic Mice
Authors: Moubareck C. , Bourgeois N. , Courvalin P. , Doucet-Populaire F.
Published: August 2003

It has been proposed that food animals represent the source of glycopeptide resistance genes present in enterococci from humans. We demonstrated the transfer of
vanA
and of other resistance genes from porcine to human
Enterococcus faecium
at high frequency in the digestive tract of gnotobiotic mice. Tylosin in the drinking water favored colonization by transconjugants.
Novel
Pseudomonas aeruginosa
Gene That Suppresses Tolerance to Carbapenems
Authors: Taniguchi Kazuko , Ono Tsuneko , Murakami Keiji , Viducic Darija , Kayama Sizuo , Hirota Katsuhiko , Nemoto Ken , Miyake Yoichiro
Published: August 2003

A biapenem-tolerant mutant of
Pseudomonas aeruginosa
was isolated by Tn
1737
KH insertion. The survival of the mutant 3 h after the addition of biapenem was about 1,000 times greater than that of the wild type. The mutant was also tolerant to other biapenems, such as imipenem, panipenem, and meropenem.
Efficacy of Linezolid Alone or in Combination with Vancomycin for Treatment of Experimental Endocarditis Due to Methicillin-Resistant
Staphylococcus aureus
Authors: Chiang Fu-Yu , Climo Michael
Published: August 2003

The levels of effectiveness of linezolid, vancomycin, and the combination of linezolid and vancomycin were compared in the rabbit model of endocarditis caused by a clinical methicillin-resistant
Staphylococcus aureus
(MRSA) isolate. Vancomycin alone was more effective than either linezolid alone or the combination of linezolid and vancomycin for the treatment of endocarditis due to MRSA.
Chemotherapeutic Potential of Orally Administered Poly(Lactide-Co-Glycolide) Microparticles Containing Isoniazid, Rifampin, and Pyrazinamide against Experimental Tuberculosis
Authors: ul-Ain Qurrat , Sharma Sadhna , Khuller G. K.
Published: August 2003

Poly(lactide-co-glycolide) (PLG) microspheres (diameter, less than 3 μm) containing isoniazid, rifampin, and pyrazinamide were used in a sustained oral drug delivery system to treat murine tuberculosis. Drug levels above the MIC were observed up to 72 h in plasma and for 9 days in various organs. Relative bioavailability of encapsulated drugs was greater than that of free drugs. Chemotherapy results showed better or equivalent clearance of bacilli in the PLG-drug-administered group (weekly) than with free drugs (daily).
In Vitro Activities of ABT-492, a New Fluoroquinolone, against 155 Aerobic and 171 Anaerobic Pathogens Isolated from Antral Sinus Puncture Specimens from Patients with Sinusitis
Authors: Goldstein Ellie J. C. , Citron Diane M. , Merriam C. Vreni , Warren Yumi A. , Tyrrell Kerin L. , Fernandez Helen T.
Published: August 2003

ABT-492 exhibited excellent in vitro activities against all 326 aerobic and anaerobic antral puncture sinus isolates tested with MICs (in micrograms per milliliter) at which 90% of the isolates tested were inhibited as follows:
Haemophilus influenzae
, 0.001;
Moraxella catarrhalis
, 0.008; and
Streptococcus pneumoniae
, 0.015. It was four- to sixfold more active than other fluoroquinolones, including against levofloxacin-resistant strains of
S. pneumoniae
, methicillin-resistant
Staphylococcus aureus
, and
Prevotella
species.
Postantibiotic Effects of Daptomycin against 14 Staphylococcal and Pneumococcal Clinical Isolates
Authors: Pankuch G. A. , Jacobs M. R. , Appelbaum P. C.
Published: August 2003

Daptomycin mean staphylococcal postantibiotic effects (PAEs) were 1.1 to 6.2 h, with a mean of 2.5 h. The mean pneumococcal PAEs were 1.7 h, ranging between 1.0 and 2.5 h. The staphylococcal and pneumococcal postantibiotic sub-MIC effects at 0.4 times the MIC ranged from 3.0 to >12.0 h and 1.9 to >12.0 h, respectively.
Antitrypanosomal Activities of Fluoroquinolones with Pyrrolidinyl Substitutions
Authors: Nenortas Elizabeth , Kulikowicz Tomasz , Burri Christian , Shapiro Theresa A.
Published: August 2003

Fluoroquinolones with pyrrolidinyl substitutions were tested against
Trypanosoma brucei
and mammalian cells. Bulky substituents at C-7 or a 1-2-bridging thiazolidine ring increased antitrypanosomal activity and selective toxicity. These compounds trap protein-DNA complexes and inhibit nucleic acid biosynthesis in trypanosomes, characteristics of topoisomerase II inhibition.