bluuebunny/crossref_metadata_2025_split huggingface dataset:
Diversity and Evolution of the Class A Chromosomal Beta-Lactamase Gene inKlebsiella pneumoniae
Authors: Hæggman S. , Löfdahl S. , Paauw A. , Verhoef J. , Brisse S. 
Published: June 2004

ABSTRACTWe investigated the diversity of the chromosomal class A beta-lactamase gene inKlebsiella pneumoniaein order to study the evolution of the gene. A 789-bp portion was sequenced in a panel of 28 strains, representative of three phylogenetic groups, KpI, KpII, and KpIII, recently identified inK. pneumoniaeand of different chromosomal beta-lactamase variants previously identified. Three groups of sequences were found, two of them corresponding to the families SHV (pI 7.6) and LEN (pI 7.1), respectively, and one, more heterogeneous, corresponding to a new family that we named OKP (for otherK. pneumoniaebeta-lactamase). Levels of susceptibility to ampicillin, cefuroxime, cefotaxime, ceftazidime, and aztreonam and inhibition by clavulanic acid were similar in the three groups. One new SHV variant, seven new LEN variants, and four OKP variants were identified. The OKP variants formed two subgroups based on nucleotide sequences, one with pIs of 7.8 and 8.1 and the other with pIs of 6.5 and 7.0. The nucleotide sequences of the housekeeping genesgyrA, coding for subunit A of gyrase, andmdh, coding for malate dehydrogenase, were also determined. Phylogenetic analysis of the three genes studied revealed parallel evolution, with the SHV, OKP, and LEN beta-lactamase families corresponding to the phylogenetic groups KpI, KpII, and KpIII, respectively. This correspondence was fully confirmed for 34 additional strains in PCR assays specific for the three beta-lactamase families. We estimated the time since divergence of the phylogenetic groups KpI and KpIII at between 6 and 28 million years, confirming the ancient presence of the beta-lactamase gene in the genome ofK. pneumoniae.
Efflux Pump-Mediated Intrinsic Drug Resistance in
Mycobacterium smegmatis
Authors: Li Xian-Zhi , Zhang Li , Nikaido Hiroshi 
Published: June 2004

ABSTRACT

The
Mycobacterium smegmatis
genome contains many genes encoding putative drug efflux pumps. Yet with the exception of
lfrA
, it is not clear whether these genes contribute to the intrinsic drug resistance of this organism. We showed first by reverse transcription (RT)-PCR that several of these genes, including
lfrA
as well as the homologues of
Mycobacterium tuberculosis
Rv1145, Rv1146, Rv1877, Rv2846c (
efpA
), and Rv3065 (
mmr
and
emrE
), were expressed at detectable levels in the strain mc
2
155. Null mutants each carrying an in-frame deletion of these genes were then constructed in
M. smegmatis
. The deletions of the
lfrA
gene or
mmr
homologue rendered the mutant more susceptible to multiple drugs such as fluoroquinolones, ethidium bromide, and acriflavine (two- to eightfold decrease in MICs). The deletion of the
efpA
homologue also produced increased susceptibility to these agents but unexpectedly also resulted in decreased susceptibility to rifamycins, isoniazid, and chloramphenicol (two- to fourfold increase in MICs). Deletion of the Rv1877 homologue produced some increased susceptibility to ethidium bromide, acriflavine, and erythromycin. The upstream region of
lfrA
contained a gene encoding a putative TetR family transcriptional repressor, dubbed LfrR. The deletion of
lfrR
elevated the expression of
lfrA
and produced higher resistance to multiple drugs. Multidrug-resistant single-step mutants, independent of LfrA and attributed to a yet-unidentified drug efflux pump (here called LfrX), were selected in vitro and showed decreased accumulation of norfloxacin, ethidium bromide, and acriflavine in intact cells. Finally, use of isogenic β-lactamase-deficient strains showed the contribution of LfrA and LfrX to resistance to certain β-lactams in
M. smegmatis
.
Model System for High-Throughput Screening of Novel Human Immunodeficiency Virus Protease Inhibitors in
Escherichia coli
Authors: Cheng Ting-Jen , Brik Ashraf , Wong Chi-Huey , Kan Chen-Chen 
Published: June 2004

ABSTRACT

Novel human immunodeficiency virus (HIV) protease inhibitors are urgently needed for combating the drug-resistance problem in the fight against AIDS. To facilitate lead discovery of HIV protease inhibitors, we have developed a safe, convenient, and cost-effective
Escherichia coli
-based assay system. This
E. coli
-based system involves coexpression of an engineered β-galactosidase as an HIV protease substrate and the HIV protease precursor comprising the transframe region and the protease domain. Autoprocessing of the HIV protease precursor releases the mature HIV protease. Subsequently, the HIV protease cleaves β-galactosidase, resulting in a loss of the β-galactosidase activity, which can be detected in high-throughput screens. Using Food and Drug Administration-approved HIV protease inhibitors, this
E. coli
-based system is validated as a surrogate screening system for identifying inhibitors that not only possess inhibitory activity against HIV protease but also have solubility and permeability for in vivo activity. The usefulness of the
E. coli
-based system was demonstrated with the identification of a novel HIV protease inhibitor from a library of compounds that were prepared by an amide-forming reaction with transition-state analog cores. A novel inhibitor with a sulfonamide core of amprenavir, E2, has shown good correlation with the in vitro enzymatic assay and in vivo
E. coli
-based system. This system can also be used to generate drug resistance profiles that could be used to suggest therapeutic uses of HIV protease inhibitors to treat the drug-resistant HIV strains. This simple yet efficient
E. coli
system not only represents a screening platform for high-throughput identification of leads targeting the HIV proteases but also can be adapted to all other classes of proteases.
Oral Efficacy of a Respiratory Syncytial Virus Inhibitor in Rodent Models of Infection
Authors: Cianci Christopher , Genovesi Eugene V. , Lamb Lucinda , Medina Ivette , Yang Zheng , Zadjura Lisa , Yang Hyekyung , D'Arienzo Celia , Sin Ny , Yu Kuo-Long , Combrink Keith , Li Zhufang , Colonno Richard , Meanwell Nicholas , Clark Junius , Krystal Mark 
Published: June 2004

ABSTRACTBMS-433771 is a potent inhibitor of respiratory syncytial virus (RSV) replication in vitro. Mechanism of action studies have demonstrated that BMS-433771 halts virus entry through inhibition of F protein-mediated membrane fusion. BMS-433771 also exhibited in vivo efficacy following oral administration in a mouse model of RSV infection (C. Cianci, K. Y. Yu, K. Combrink, N. Sin, B. Pearce, A. Wang, R. Civiello, S. Voss, G. Luo, K. Kadow, E. Genovesi, B. Venables, H. Gulgeze, A. Trehan, J. James, L. Lamb, I. Medina, J. Roach, Z. Yang, L. Zadjura, R. Colonno, J. Clark, N. Meanwell, and M. Krystal, Antimicrob. Agents Chemother.48:413-422, 2004). In this report, the in vivo efficacy of BMS-433771 against RSV was further examined in the BALB/c mouse and cotton rat host models of infection. By using the Long strain of RSV, prophylactic efficacy via oral dosing was observed in both animal models. A single oral dose, administered 1 h prior to intranasal RSV inoculation, was as effective against infection as a 4-day b.i.d. dosing regimen in which the first oral dose was given 1 h prior to virus inoculation. Results of dose titration experiments suggested that RSV infection was more sensitive to inhibition by BMS-433771 treatment in the BALB/c mouse host than in the cotton rat. This was reflected by the pharmacokinetic and pharmacodynamic analysis of the efficacy data, where the area under the concentration-time curve required to achieve 50% of the maximum response was ∼7.5-fold less for mice than for cotton rats. Inhibition of RSV by BMS-433771 in the mouse is the result of F1-mediated inhibition, as shown by the fact that a virus selected for resistance to BMS-433771 in vitro and containing a single amino acid change in the F1 region was also refractory to treatment in the mouse host. BMS-433771 efficacy against RSV infection was also demonstrated for mice that were chemically immunosuppressed by cyclophosphamide treatment, indicating that compound inhibition of the virus did not require an active host immune response.
A Small Peptide (CEL-1000) Derived from the β-Chain of the Human Major Histocompatibility Complex Class II Molecule Induces Complete Protection against Malaria in an Antigen-Independent Manner
Authors: Charoenvit Yupin , Brice Gary T. , Bacon David , Majam Victoria , Williams Jackie , Abot Esteban , Ganeshan Harini , Sedegah Martha , Doolan Denise L. , Carucci Daniel J. , Zimmerman Daniel H. 
Published: June 2004

ABSTRACTCEL-1000 (DGQEEKAGVVSTGLIGGG) is a novel potential preventative and therapeutic agent. We report that CEL-1000 confers a high degree of protection againstPlasmodiumsporozoite challenge in a murine model of malaria, as shown by the total absence of blood stage infection following challenge with 100 sporozoites (100% protection) and by a substantial reduction (400-fold) of liver stage parasite RNA following challenge with 50,000 sporozoites. CEL-1000 protection was demonstrated in A/J (H-2a) and C3H/HeJ (H-2k) mice but not in BALB/c (H-2d) or CAF1 (A/J × BALB/c F1hybrid) mice. In CEL-1000-treated and protected mice, high levels of gamma interferon (IFN-γ) in serum and elevated frequencies of hepatic and splenic CD4+IFN-γ-positive T cells were detected 24 h after administration of an additional dose of CEL-1000. Treatment of A/J mice that received CEL-1000 with antibodies against IFN-γ just prior to challenge abolished the protection, and a similar treatment with antibodies against CD4+T cells partially reduced the level of protection, while treatment with control antibodies or antibodies specific for interleukin-12 (IL-12), CD8+T cells, or NK cells had no effect. Our data establish that the protection induced by CEL-1000 is dependent on IFN-γ and is partially dependent on CD4+T cells but is independent of CD8+T cells, NK cells, and IL-12 at the effector phase and does not induce a detectable antibody response.
Extra Copies of the
Aspergillus fumigatus
Squalene Epoxidase Gene Confer Resistance to Terbinafine: Genetic Approach to Studying Gene Dose-Dependent Resistance to Antifungals in
A. fumigatus
Authors: Liu Wei , May Gregory S. , Lionakis Michail S. , Lewis Russell E. , Kontoyiannis Dimitrios P. 
Published: June 2004

ABSTRACT

With the increasing use of antifungals such as amphotericin B, itraconazole, voriconazole, caspofungin, and terbinafine (TRB) in patients at high risk for invasive aspergillosis, resistance of
Aspergillus fumigatus
to these agents will ultimately emerge. Due to the limited availability of molecular genetics for
A. fumigatus
, few studies have addressed its mechanisms of resistance to antifungals. We transformed
A. fumigatus
protoplasts with a
pyrG
-based
A. fumigatus
genomic DNA library (constructed in the multicopy nonintegrating vector pRG3-AMA1-NotI, which also has the
pyr-4
gene for selection). We obtained one

pyrG
+

transformant that grew in medium containing a fungicidal concentration (0.625 μg/ml) of TRB. To determine whether TRB resistance in that transformant was plasmid dependent, we evicted the plasmid and found concomitant loss of uracil prototrophy and TRB resistance. DNA sequence analysis identified the gene responsible for TRB resistance as the
A. fumigatus
squalene epoxidase gene (
ERG1
), which encodes the target enzyme of TRB. Authentic
A. fumigatus ERG1
, amplified from the genome and cloned into pRG3-AMA1-NotI, also conferred TRB-specific resistance. This molecular approach has the potential to enhance our knowledge of the mechanisms of
A. fumigatus
resistance to modern antifungals.
In Vitro Effects of Resveratrol on the Viability and Infectivity of the Microsporidian
Encephalitozoon cuniculi
Authors: Leiro José , Cano Ernesto , Ubeira Florencio M. , Orallo Francisco , Sanmartín Manuel L. 
Published: June 2004

ABSTRACT

Microsporidians of the genus
Encephalitozoon
are an important cause of disease in immunocompromised patients, and there are currently no completely effective treatments. The present study investigated the viability and infectivity of spores of
Encephalitozoon cuniculi
that had been exposed to resveratrol (RESV), a natural phytoalexin found in grapes and red wine. RESV at 50 μM showed significant sporicidal activity, and at 10 to 50 μM it reduced the capacity of the spores to infect dog kidney epithelial cells of the MDCK line. At 10 μM RESV also significantly inhibited intracellular development of the parasite, without affecting host cell viability. These results suggest that RESV may be useful in the treatment of
Encephalitozoon
infections.
Salmonella
Genomic Island 1 Multidrug Resistance Gene Clusters in
Salmonella enterica
Serovar Agona Isolated in Belgium in 1992 to 2002
Authors: Doublet Benoît , Butaye Patrick , Imberechts Hein , Boyd David , Mulvey Michael R. , Chaslus-Dancla Elisabeth , Cloeckaert Axel 
Published: June 2004

ABSTRACT

Salmonella
genomic island 1 (SGI1) harbors a multidrug resistance (MDR) gene cluster which is a complex class 1 integron. Variant SGI1 MDR gene clusters conferring different MDR profiles have also been identified in several
Salmonella enterica
serovars and classified as SGI1-A to -F. A retrospective study was undertaken to characterize MDR regions from serovar Agona strains harboring SGI1 isolated from poultry in Belgium between 1992 and 2002. A total of 171 serovar Agona strains, displaying resistance to at least one antibiotic, were studied for the presence of SGI1. SGI1 was detected in 94 serovar Agona strains. The most prevalent variant was SGI1-A (85%), which harbors within the SGI1 complex class 1 integron a common region (CR1) containing orf513, a putative transposase gene, adjacent to the
dfrA10
trimethoprim resistance gene. A new variant SGI1 named SGI1-G was identified in two strains. It consisted of the
pse-1
gene cassette, as in SGI1-B, but with additional insertion of the orf513/
dfrA10
region structure. Seven strains displaying the typical SGI1 MDR profile (Ap Cm Ff Sm Sp Su Tc) showed genetic variation at the 3′ end of SGI1. These strains harbored the insertion of the CR1 containing orf513 as in SGI1-A, -D, and -G. However, downstream the right end of CR1, they presented different 7.4- to 8.5-kb deletions of the SGI1 3′ end that extended to the chromosomal genes
yieE
and
yieF
. These results suggest a possible role of CR1 in deletion formation, as has been reported for some insertion sequences. Pulsed-field gel electrophoresis analysis showed that all the serovar Agona SGI1-carrying strains belonged to a single clone. Thus, SGI1 is largely encountered in serovar Agona strains isolated from poultry in Belgium, the most prevalent variant being SGI1-A. SGI1 MDR region undergoes recombinational events resulting in a diversity of MDR gene clusters.
A Chimeric Peptide Composed of a Dermaseptin Derivative and an RNA III-Inhibiting Peptide Prevents Graft-Associated Infections by Antibiotic-Resistant Staphylococci
Authors: Balaban Naomi , Gov Yael , Giacometti Andrea , Cirioni Oscar , Ghiselli Roberto , Mocchegiani Federico , Orlando Fiorenza , D'Amato Giuseppina , Saba Vittorio , Scalise Giorgio , Bernes Sabina , Mor Amram 
Published: June 2004

ABSTRACT

Staphylococcal bacteria are a prevalent cause of infections associated with foreign bodies and indwelling medical devices. Bacteria are capable of escaping antibiotic treatment through encapsulation into biofilms. RNA III-inhibiting peptide (RIP) is a heptapeptide that inhibits staphylococcal biofilm formation by obstructing quorum-sensing mechanisms. K
4
-S4(1-13)
a
is a 13-residue dermaseptin derivative (DD
13
) believed to kill bacteria via membrane disruption. We tested each of these peptides as well as a hybrid construct, DD
13
-RIP, for their ability to inhibit bacterial proliferation and suppress quorum sensing in vitro and for their efficacy in preventing staphylococcal infection in a rat graft infection model with methicillin-resistant
Staphylococcus aureus
(MRSA) or
S. epidermidis
(MRSE). In vitro, proliferation assays demonstrated that RIP had no inhibitory effect, while DD
13
-RIP and DD
13
were equally effective, and that the chimeric peptide but not DD
13
was slightly more effective than RIP in inhibiting RNA III synthesis, a regulatory RNA molecule important for staphylococcal pathogenesis. In vivo, the three peptides reduced graft-associated bacterial load in a dose-dependent manner, but the hybrid peptide was most potent in totally preventing staphylococcal infections at the lowest dose. In addition, each of the peptides acted synergistically with antibiotics. The data indicate that RIP and DD
13
act in synergy by attacking bacteria simultaneously by two different mechanisms. Such a chimeric peptide may be useful for coating medical devices to prevent drug-resistant staphylococcal infections.
Single-Dose Pharmacokinetics and Safety of a Novel Broad-Spectrum Cephalosporin (BAL5788) in Healthy Volunteers
Authors: Schmitt-Hoffmann Anne , Roos Brigitte , Schleimer Michael , Sauer Jill , Man Anthony , Nashed Norman , Brown Thomas , Perez Antonio , Weidekamm Erhard , Kovács Péter 
Published: June 2004

ABSTRACTBAL5788 is the water-soluble prodrug of BAL9141, a novel broad-spectrum cephalosporin with potent bactericidal activities against methicillin-resistantStaphylococcus aureus(MRSA) and penicillin-resistantStreptococcus pneumoniae. We investigated the safety and pharmacokinetics of BAL5788 in a double-blind, single-ascending-dose study with 40 healthy male subjects. The subjects were randomized to receive placebo (n= 2 subjects per dose) or BAL5788 (n= 6 subjects per dose) as a 200-ml intravenous infusion over 30 min. The BAL5788 doses used were 125, 250, 500, 750, and 1,000 mg (BAL9141 equivalents). All doses were well tolerated, with no severe or serious adverse events (AEs). The most frequent AE was taste disturbance. No electrocardiographic abnormalities and no trends or clinically significant changes in laboratory parameters or vital signs were observed. The maximum concentration of drug in serum and the area under the concentration-time curve for BAL9141 were dose proportional over the dosing range. The elimination half-life of BAL9141 was about 3 h. The volume of distribution at steady state was equal to the volume of the adult extracellular water compartment, and the rate of renal clearance of free drug corresponded to the normal glomerular filtration rate for adults. More than 70% of the administered dose was excreted as BAL9141 in the urine, and almost no prodrug was detected. After the infusion of 750 mg, the mean plasma BAL9141 concentrations exceeded the MIC at which 100% of MRSA isolates are inhibited (4 μg/ml) for approximately 7 h, or 58% of a 12-h dosing interval. These results indicate that infusions of 750 mg twice a day should be adequate for the treatment of infections caused by MRSA.
Real-Time PCR for Dihydrofolate Reductase Gene Single-Nucleotide Polymorphisms inPlasmodium vivaxIsolates
Authors: Brega Sara , de Monbrison Frédérique , Severini Carlo , Udomsangpetch Rachanee , Sutanto Inge , Ruckert Paul , Peyron François , Picot Stéphane 
Published: June 2004

ABSTRACTMutations in thedhfrgene ofPlasmodium vivax(pvdhfr) are associated with resistance to the antifolate antimalarial drugs. Polymorphisms in thepvdhfrgene were assessed by hybridization probe technology on the LightCycler instrument with 134P. vivax-infected blood samples from Turkey (n= 24), Azerbaijan (n= 39), Thailand (n= 16), Indonesia (n= 53), and travelers (n= 19). Double mutations (S58R and S117N) or quadruple mutations (F57L/I, S58R, T61M, and S117N) in thepvdhfrgenes were found in all Thai samples (100%).pvdhfrmutant-type alleles were significantly more common in samples from travelers (42%) than in those from patients from Indonesia (5%). Surprisingly, thepvdhfrsingle-mutation allele (S117N) was identified at a high frequency in parasites from Turkey and Azerbaijan (71 and 36%, respectively), where sulfadoxine-pyrimethamine is not recommended for the treatment ofP. vivaxmalaria by the World Health Organization and the Malaria National Programs.
Enzymatic Detachment of
Staphylococcus epidermidis
Biofilms
Authors: Kaplan Jeffrey B. , Ragunath Chandran , Velliyagounder Kabilan , Fine Daniel H. , Ramasubbu Narayanan 
Published: June 2004

ABSTRACT

The gram-positive bacterium
Staphylococcus epidermidis
is the most common cause of infections associated with catheters and other indwelling medical devices.
S. epidermidis
produces an extracellular slime that enables it to form adherent biofilms on plastic surfaces. We found that a biofilm-releasing enzyme produced by the gram-negative periodontal pathogen
Actinobacillus actinomycetemcomitans
rapidly and efficiently removed
S. epidermidis
biofilms from plastic surfaces. The enzyme worked by releasing extracellular slime from
S. epidermidis
cells. Precoating surfaces with the enzyme prevented
S. epidermidis
biofilm formation. Our findings demonstrate that biofilm-releasing enzymes can exhibit broad-spectrum activity and that these enzymes may be useful as antibiofilm agents.
Detection and Susceptibility Testing of Hypermutable
Pseudomonas aeruginosa
Strains with the Etest and Disk Diffusion
Authors: Maciá Maria D. , Borrell Nuria , Pérez José L. , Oliver Antonio 
Published: June 2004

ABSTRACT

Resistance development in
Pseudomonas aeruginosa
from chronically colonized cystic fibrosis (CF) patients has been linked to the presence of a high proportion of mismatch repair-deficient hypermutable strains. The detection of hypermutable strains by microbiology laboratories may be useful for establishing adequate antimicrobial therapies. In this work, we find that the Etest and disk diffusion can be used as simple methods for the detection and susceptibility testing of hypermutable
P. aeruginosa
isolates. Strain PAO1 and its hypermutable derivative strain PAOΔ
mutS
were used to standardize the procedure, which was tested with 35
P. aeruginosa
isolates from 21 CF patients. Mutation frequencies were estimated by standard methods, and 29% of the isolates were found to be hypermutable. MICs and inhibition zone diameters were determined for ceftazidime, imipenem, meropenem, ciprofloxacin, and tobramycin by using Etest strips and conventional disks, respectively. The presence (or absence) of resistant mutant subpopulations, as well as their relative numbers and the highest MICs for them (or smallest inhibition zone diameters), was recorded. The presence of resistant mutant subpopulations within the inhibition zones of three or more antibiotics clearly identified the strains as hypermutable (they were present in 10 of 10 hypermutable strains and 0 of 25 nonhypermutable strains) with both methods. Additionally, these methods allowed us to differentiate between dual effects of hypermutation in antibiotic resistance, namely, that (i) hypermutable isolates were substantially more resistant than nonhypermutable isolates and that (ii) the resistance of hypermutable isolates was dramatically increased by the presence of resistant mutant subpopulations. This differentiation may be relevant for the design of adequate treatments, since the second effect, in contrast to the first, may be overcome by antibiotic combinations.
Constitutive Activation of the
PDR16
Promoter in a
Candida albicans
Azole-Resistant Clinical Isolate Overexpressing
CDR1
and
CDR2
Authors: De Deken Xavier , Raymond Martine 
Published: June 2004

ABSTRACT

Candida albicans
azole-resistant clinical isolates overexpressing the
CDR1
and
CDR2
genes (multidrug transporters) also overexpress the
PDR16
gene (phosphatidylinositol transfer protein). We show here that the
PDR16
promoter displays higher transcriptional activity following integration in an azole-resistant isolate than in the matched azole-susceptible one. Thus, the upregulation of
PDR16
in the resistant strain results from a mutation acting in
trans
.