bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Increase in Resistance to Ciprofloxacin in
Escherichia coli
Associated with Deletion of the C-Terminal Part of MarR
Authors: Linde Hans-Jörg , Notka Frank , Metz Michaela , Kochanowski Bernd , Heisig Peter , Lehn Norbert 
Published: July 2002

ABSTRACT

We recovered two isolates (EP1 and EP2) of
Escherichia coli
from the same patient that had identical pulsed-field gel electrophoresis patterns but required different MICs of ciprofloxacin (CIP): 16 and 256 mg/liter for EP1 and EP2, respectively. Both isolates had mutations in the quinolone resistance-determining regions of GyrA (Ser83Leu and Asp87Tyr) and ParC (Ser80Ile), but not in those regions of GyrB or ParE. Isolate EP2 was also more resistant to chloramphenicol, tetracyclines, cefuroxime, and organic solvents. A deletion of adenine (A) 1821 was found in
marR
of isolate EP2, which resulted in an 18-amino-acid C-terminal deletion in the MarR protein. The causative relationship between ΔA1821 and the Mar phenotype was demonstrated both by the replacement of the wild-type
marR
by
marR
ΔA1821 in isolate EP1 and by complementation with the wild-type
marR
in
trans
in isolate EP2. In isolate EP2 complemented with wild-type
marR
, susceptibility to chloramphenicol was restored completely, whereas susceptibility to CIP was restored only incompletely. Northern blotting demonstrated increased expression of
marA
and
acrAB
but not of
soxS
in isolate EP2 compared to EP1. In conclusion, the deletion of A1821 in
marR
in the clinical isolate EP2 caused an increase in the MICs of CIP and unrelated antibiotics. Presumably, the C-terminal part of MarR is necessary for proper repressor function.
Pharmacokinetic and Pharmacodynamic Study of the Human Immunodeficiency Virus Protease Inhibitor Amprenavir after Multiple Oral Dosing
Authors: Sadler Brian M. , Gillotin Catherine , Lou Yu , Stein Daniel S. 
Published: July 2002

ABSTRACT

In a dose-ranging study of amprenavir (formerly 141W94), an inhibitor of the protease enzyme of human immunodeficiency virus (HIV) type 1, single-dose and steady-state pharmacokinetic parameters were estimated from plasma samples collected on day 1 and during week 3, respectively. Amprenavir was administered on either a twice-daily (b.i.d.) or three-times-daily dosage schedule to 62 HIV-infected adults, 59 of whom had pharmacokinetic data. Log-log regression analysis (the power model) revealed that the steady-state area under the curve (AUC
ss
) and the maximum, minimum, and average concentrations at steady state (
C
max,ss
,
C
min,ss
, and
C
avg,ss
, respectively) increased in a dose-proportional manner over the 300- to 1,200-mg dose range. Steady-state clearance was dose independent. AUC
ss
/AUC
0→∞
decreased linearly with dose and correlated significantly with treatment-associated decreases in α
1
-acid glycoprotein. After 3 weeks, the dose of 1,200 mg b.i.d. provided a median amprenavir C
min,ss
(0.280 μg/ml) that was higher than the median in vitro 50% inhibitory concentration for clinical HIV isolates (0.023 μg/ml), even after adjustment for protein binding. The median amprenavir
C
min,ss
was also greater than the estimated in vivo trough concentration calculated to yield 90% of the maximum antiviral effect (0.228 μg/ml) over 4 weeks. A pharmacodynamic analysis of the relationship between steady-state pharmacokinetic parameters and safety revealed headache and oral numbness to be the only side effects significantly associated with
C
max
. The pharmacodynamic relationship defined in this study supports the use of 1,200 mg b.i.d. as the approved dose of amprenavir.
Prevalence and Mechanisms of Macrolide Resistance in Invasive and Noninvasive Group B Streptococcus Isolates from Ontario, Canada
Authors: de Azavedo Joyce C. S. , McGavin Mary , Duncan Carla , Low Donald E. , McGeer Allison 
Published: July 2002

ABSTRACT

Macrolide resistance has been demonstrated in group B streptococcus (GBS), but there is limited information regarding mechanisms of resistance and their prevalence. We determined these in GBS obtained from neonatal blood cultures and vaginal swabs from pregnant women. Of 178 isolates from cases of neonatal GBS sepsis collected from 1995 to 1998, 8 and 4.5% were resistant to erythromycin and clindamycin, respectively, and one isolate showed intermediate penicillin resistance (MIC, 0.25 μg/ml). Of 101 consecutive vaginal or rectal/vaginal isolates collected in 1999, 18 and 8% were resistant to erythromycin and clindamycin, respectively. Tetracycline resistance was high (>80%) among both groups of isolates. Of 32 erythromycin-resistant isolates, 28 possessed the
erm
methylase gene (7
ermB
and 21
ermTR/ermA
) and 4 harbored the
mefA
gene; one isolate harbored both genes. One isolate which was susceptible to erythromycin but resistant to clindamycin (MIC, 4 μg/ml) was found to have the
linB
gene, previously identified only in
Enterococcus faecium
. The
mreA
gene was found in all the erythromycin-resistant strains as well as in 10 erythromycin-susceptible strains. The rate of erythromycin resistance increased from 5% in 1995–96 to 13% in 1998–99, which coincided with an increase in macrolide usage during that time.
Expression of Green Fluorescent Protein as a Marker for Effects of Antileishmanial Compounds In Vitro
Authors: Kamau Sarah W. , Grimm Felix , Hehl Adrian B. 
Published: July 2002

ABSTRACTTransgenicLeishmania infantumpromastigotes, which constitutively express green fluorescent protein (GFP) in their cytoplasm, were used to monitor the effects of antileishmanial compounds in real time. The GFP-based assay provided a reliable measure of drug-induced inhibitory effects on protein expression, resulting in a dynamic picture of the responses of leishmanial promastigotes to the compounds tested.
Penicillin-Binding Proteins in
Leptospira interrogans
Authors: Brenot Audrey , Trott Daren , Girons Isabelle Saint , Zuerner Richard 
Published: July 2002

ABSTRACT

The
Leptospira interrogans ponA
and
pbpB
genes were isolated and characterized.
ponA
and
pbpB
encode the penicillin-binding proteins (PBPs) 1 and 3, respectively. There is little sequence variation between the PBP genes from two
L. interrogans
strains (serovar icterohaemorrhagiae strain Verdun and serovar pomona strain RZ11). The deduced
L. interrogans
PBP 1 and PBP 3 protein sequences from the two strains shared over 50% similarity to homologous proteins from
Escherichia coli
. It was demonstrated for strain Verdun that
ponA
and
pbpB
are transcribed individually from their own promoter. The
ponA
and
pbpB
genes from both strains are separated by 8 to 10 kb and oriented such that their transcription is convergent. The
L. interrogans
PBP 1 and PBP 3 proteins were synthesized in
E. coli
and were modified with ampicillin using a digoxigenin-ampicillin conjugate. These data show that both genes encode functional PBPs.
Gentamicin Increases the Efficacy of Vancomycin against Penicillin-Resistant Pneumococci in the Rabbit Meningitis Model
Authors: Cottagnoud Philippe , Gerber Cynthia M. , Cottagnoud Marianne , Täuber Martin G. 
Published: July 2002

ABSTRACT

In experimental meningitis a single dose of gentamicin (10 mg/kg of body weight) led to gentamicin levels in around cerebrospinal fluid (CSF) of 4 mg/liter for 4 h, decreasing slowly to 2 mg/liter 4 h later. The CSF penetration of gentamicin ranged around 27%, calculated by comparison of areas under the curve (AUC in serum/AUC in CSF). Gentamicin monotherapy (−1.24 log
10
CFU/ml) was inferior to vancomycin monotherapy (−2.54 log
10
CFU/ml) over 8 h against penicillin-resistant pneumococci. However, the combination of vancomycin with gentamicin was significantly superior (−4.48 log
10
CFU/ml) compared to either monotherapy alone. The synergistic activity of vancomycin combined with gentamicin was also demonstrated in vitro in time-kill assays.
In Situ Analysis of Sulfate-Reducing Bacteria Related to
Desulfocapsa thiozymogenes
in the Chemocline of Meromictic Lake Cadagno (Switzerland)
Authors: Tonolla Mauro , Demarta Antonella , Peduzzi Sandro , Hahn Dittmar , Peduzzi Raffaele 
Published: July 2002

ABSTRACT

Comparative sequence analysis of a 16S rRNA gene clone library from the chemocline of the meromictic Lake Cadagno (Switzerland) retrieved two clusters of sequences resembling sulfate-reducing bacteria within the family
Desulfovibrionaceae
. In situ hybridization showed that, similar to sulfate-reducing bacteria of the family
Desulfobacteriaceae
, bacteria of one cluster with similarity values to the closest cultured relatives of between 92.6 and 93.1% resembled free cells or cells loosely attached to other cells or debris. Bacteria of the second cluster closely related to
Desulfocapsa thiozymogenes
DSM7269 with similarity values between 97.9 and 98.4% were generally associated with aggregates of different small-celled phototrophic sulfur bacteria, suggesting a potential interaction between the two groups of bacteria.
Optimization of Annealing Temperature To Reduce Bias Caused by a Primer Mismatch in Multitemplate PCR
Authors: Ishii Kousuke , Fukui Manabu 
Published: July 2002

ABSTRACT

To reduce PCR bias derived from a primer mismatch, the effect of the annealing temperature on the product ratio was investigated by denaturing gradient gel electrophoresis analysis of PCR products from a mixture of perfect-match and one-mismatch templates. These templates were generated by PCR from
Pediococcus acidilactici
for one mismatch and
Micrococcus luteus
for the perfect match. PCRs showed that the bias was reduced at lower temperatures. An environmental sample was also examined.
New Spatially Explicit Method for Detecting Extracellular Protease Activity in Biofilms
Authors: Francoeur Steven N. , Wetzel Robert G. , Neely Robert K. 
Published: July 2002

ABSTRACTA novel method of detecting extracellular protease activity at biofilm-substratum interfaces was developed. This method utilizes fluorescent molecules bound to cellulose substrata with a lectin. Extracellular proteases degrade the lectin and release the fluorochrome into solution. This new technique and a standard dissolved-substrate assay detected similar responses of biofilm extracellular protease activity to experimental manipulation of N supply. Combination of this technique with confocal scanning laser microscopy allowed direct visualization of microspatial patterns of bacterial distribution and extracellular protease activity at the biofilm-substratum interface.
Essential Role for the
Legionella pneumophila
Rep Helicase Homologue in Intracellular Infection of Mammalian Cells
Authors: Harb Omar S. , Abu Kwaik Yousef 
Published: July 2002

ABSTRACT

We have previously isolated 32 mutants of
Legionella pneumophila
that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (
mil
) loci. In this study we characterized the
mil
mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar to
rep
helicase genes of other bacteria. Since helicase mutants of
Escherichia coli
are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the
L. pneumophila
phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the
L. pneumophila
phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in the
rep
helicase homologue mutant, rendering it defective in intracellular replication.
Molecular Characterization of a New Variant of Toxin-Coregulated Pilus Protein (TcpA) in a Toxigenic Non-O1/Non-O139 Strain of
Vibrio cholerae
Authors: Nandi Bisweswar , Nandy Ranjan K. , Vicente Ana C. P. , Ghose Asoke C. 
Published: July 2002

ABSTRACT

A toxigenic non-O1/non-O139 strain of
Vibrio cholerae
(10259) was found to contain a new variant of the toxin-coregulated pilus (TCP) protein gene (
tcpA
) as determined by PCR and Southern hybridization experiments. Nucleotide sequence analysis data of the new
tcpA
gene in strain 10259 (O53) showed it to be about 74 and 72% identical to those of O1 classical and El Tor biotype strains, respectively. The predicted amino acid sequence of the 10259 TcpA protein shared about 81 and 78% identity with the corresponding sequences of classical and El Tor TcpA strains, respectively. An antiserum raised against the TCP of a classical strain, O395, although it recognized the TcpA protein of strain 10259 in an immunoblotting experiment, exhibited considerably less protection against 10259 challenge compared to that observed against the parent strain. Incidentally, the
tcpA
sequences of two other toxigenic non-O1/non-O139 strains (V2 and S7, both belonging to the serogroup O37) were determined to be almost identical to that of classical
tcpA
. Further,
tcpA
of another toxigenic non-O1/non-O139 strain V315-1 (O nontypeable) was closely related to that of El Tor
tcpA
. Analysis of these results with those already available in the literature suggests that there are at least four major variants of the
tcpA
gene in
V. cholerae
which probably evolved in parallel from a common ancestral gene. Existence of highly conserved as well as hypervariable regions within the sequence of the TcpA protein would also predict that such evolution is under the control of considerable selection pressure.
Evidence of Involvement of the Mannose Receptor in Adhesion ofBorrelia burgdorferito Monocyte/Macrophages
Authors: Cinco Marina , Cini Barbara , Murgia Rossella , Presani Gianni , Prodan Mario , Perticarari Sandra 
Published: July 2002

ABSTRACTThe mannose receptor (MR) plays an important role in the recognition of some pathogens in nonopsonic phagocytosis and in antigen presentation to T cells. We found thatBorrelia burgdorferi, the agent of Lyme borreliosis, adheres to monocyte-derived macrophages and to rat MR-transfected cells but not to untransfected cells. Antibodies to MR and sugars such as mannose, mannan, fucose, and some lectins significantly lowered the adhesion, confirming participation of the MR in the binding.
Role of Lung Epithelial Cells in Defense against
Klebsiella pneumoniae
Pneumonia
Authors: Cortés Guadalupe , Álvarez Dolores , Saus Carles , Albertí Sebastián 
Published: July 2002

ABSTRACT

The airway epithelium represents a primary site for the entry of pathogenic bacteria into the lungs. It has been suggested for many respiratory pathogens, including
Klebsiella pneumoniae
, that adhesion and invasion of the lung epithelial cells is an early stage of the pneumonia process. We observed that poorly encapsulated
K. pneumoniae
clinical isolates and an isogenic unencapsulated mutant invaded lung epithelial cells more efficiently than highly encapsulated strains independent of the K type. By contrast, the unencapsulated mutant was completely avirulent in a mouse model of pneumonia, unlike the wild-type strain, which produced pneumonia and systemic infection. Furthermore, the unencapsulated mutant bound more epithelially produced complement component C3 than the wild-type strain. Our results show that lung epithelial cells play a key role as a host defense mechanism against
K. pneumoniae
pneumonia, using two different strategies: (i) ingestion and control of the microorganisms and (ii) opsonization of the microorganisms. Capsular polysaccharide avoids both mechanisms and enhances the virulence of
K. pneumoniae
.
Differences in Genotypes ofHelicobacter pylorifrom Different Human Populations
Authors: Kersulyte Dangeruta , Mukhopadhyay Asish K. , Velapatiño Billie , Su WanWen , Pan ZhiJun , Garcia Claudia , Hernandez Virginia , Valdez Yanet , Mistry Rajesh S. , Gilman Robert H. , Yuan Yuan , Gao Hua , Alarcón Teresa , López-Brea Manuel , Balakrish Nair G. , Chowdhury Abhijit , Datta Simanti , Shirai Mutsunori , Nakazawa Teruko , Ally Reidwaan , Segal Isidore , Wong Benjamin C. Y. , Lam S. K. , Olfat Farzad O. , Borén Thomas , Engstrand Lars , Torres Olga , Schneider Roberto , Thomas Julian E. , Czinn Steven , Berg Douglas E. 
Published: July 2002

ABSTRACTDNA motifs at several informative loci in more than 500 strains ofHelicobacter pylorifrom five continents were studied by PCR and sequencing to gain insights into the evolution of this gastric pathogen. Five types of deletion, insertion, and substitution motifs were found at the right end of theH. pylori cagpathogenicity island. Of the three most common motifs, type I predominated in Spaniards, native Peruvians, and Guatemalan Ladinos (mixed Amerindian-European ancestry) and also in native Africans and U.S. residents; type II predominated among Japanese and Chinese; and type III predominated in Indians from Calcutta. Sequences in thecagAgene and invacAm1type alleles of the vacuolating cytotoxin gene (vacA) of strains from native Peruvians were also more like those from Spaniards than those from Asians. These indications of relatedness of Latin American and Spanish strains, despite the closer genetic relatedness of Amerindian and Asian people themselves, lead us to suggest thatH. pylorimay have been brought to the New World by European conquerors and colonists about 500 years ago. This thinking, in turn, suggests thatH. pyloriinfection might have become widespread in people quite recently in human evolution.
In
Lactobacillus plantarum
, Carbamoyl Phosphate Is Synthesized by Two Carbamoyl-Phosphate Synthetases (CPS): Carbon Dioxide Differentiates the Arginine-Repressed from the Pyrimidine-Regulated CPS
Authors: Nicoloff Hervé , Hubert Jean-Claude , Bringel Françoise 
Published: July 2002

ABSTRACT

Carbamoyl phosphate (CP) is an intermediate in pyrimidine and arginine biosynthesis. Carbamoyl-phosphate synthetase (CPS) contains a small amidotransferase subunit (GLN) that hydrolyzes glutamine and transfers ammonia to the large synthetase subunit (SYN), where CP biosynthesis occurs in the presence of ATP and CO
2
.
Lactobacillus plantarum
, a lactic acid bacterium, harbors a pyrimidine-inhibited CPS (CPS-P; Elagöz et al., Gene 182:37–43, 1996) and an arginine-repressed CPS (CPS-A). Sequencing has shown that CPS-A is encoded by
carA
(GLN) and
carB
(SYN). Transcriptional studies have demonstrated that
carB
is transcribed both monocistronically and in the
carAB
arginine-repressed operon. CP biosynthesis in
L. plantarum
was studied with three mutants (ΔCPS-P, ΔCPS-A, and double deletion). In the absence of both CPSs, auxotrophy for pyrimidines and arginine was observed. CPS-P produced enough CP for both pathways. In CO
2
-enriched air but not in ordinary air, CPS-A provided CP only for arginine biosynthesis. Therefore, the uracil sensitivity observed in prototrophic wild-type
L. plantarum
without CO
2
enrichment may be due to the low affinity of CPS-A for its substrate CO
2
or to regulation of the CP pool by the cellular CO
2
/bicarbonate level.
Detection of mRNA Transcripts and Active Transcription in Persistent
Mycobacterium tuberculosis
Induced by Exposure to Rifampin or Pyrazinamide
Authors: Hu Yanmin , Mangan Joseph A. , Dhillon Jasvir , Sole Kath M. , Mitchison Denis A. , Butcher Philip D. , Coates Anthony R. M. 
Published: July 2002

ABSTRACT

Mycobacterium tuberculosis
can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase
M. tuberculosis
treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent
M. tuberculosis
has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.
Maturation of IncP Pilin Precursors Resembles the Catalytic Dyad-Like Mechanism of Leader Peptidases
Authors: Eisenbrandt Ralf , Kalkum Markus , Lurz Rudi , Lanka Erich 
Published: July 2002

ABSTRACT

The pilus subunit, the pilin, of conjugative IncP pili is encoded by the
trbC
gene. IncP pilin is composed of 78 amino acids forming a ring structure (R. Eisenbrandt, M. Kalkum, E.-M. Lai, C. I. Kado, and E. Lanka, J. Biol. Chem. 274:22548–22555, 1999). Three enzymes are involved in maturation of the pilin: LepB of
Escherichia coli
for signal peptide removal and a yet-unidentified protease for removal of 27 C-terminal residues. Both enzymes are chromosome encoded. Finally, the inner membrane-associated IncP TraF replaces a four-amino-acid C-terminal peptide with the truncated N terminus, yielding the cyclic polypeptide. We refer to the latter process as “prepilin cyclization.” We have used site-directed mutagenesis of
trbC
and
traF
to unravel the pilin maturation process. Each of the mutants was analyzed for its phenotypes of prepilin cyclization, pilus formation, donor-specific phage adsorption, and conjugative DNA transfer abilities. Effective prepilin cyclization was determined by matrix-assisted laser desorption-ionization–mass spectrometry using an optimized sample preparation technique of whole cells and trans-3-indolyl acrylic acid as a matrix. We found that several amino acid exchanges in the TrbC core sequence allow prepilin cyclization but disable the succeeding pilus assembly. We propose a mechanism explaining how the signal peptidase homologue TraF attacks a C-terminal section of the TrbC core sequence via an activated serine residue. Rather than cleaving and releasing hydrolyzed peptides, TraF presumably reacts as a peptidyl transferase, involving the N terminus of TrbC in the aminolysis of a postulated TraF-acetyl-TrbC intermediate. Under formal loss of a C-terminal tetrapeptide, a new peptide bond is formed in a concerted action, connecting serine 37 with glycine 114 of TrbC.