bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling Household Transmission of American Trypanosomiasis
Authors: Cohen Joel E. , Gürtler Ricardo E.
Published: March 2006

American trypanosomiasis, or Chagas disease, caused by the protozoan parasiteTrypanosoma cruziand transmitted by blood-feeding triatomine bugs, is a chronic, frequently fatal infection that is common in Latin America. Neither adequate drugs nor a vaccine is available. A mathematical model calibrated to detailed household data from three villages in northwest Argentina shows that householders could greatly reduce the risk of human infection by excluding domestic animals, especially infected dogs, from bedrooms; removing potential refuges for bugs from walls and ceilings; and using domestically applied insecticides. Low-cost, locally practicable environmental management combined with intermittent use of insecticides can sustainably control transmission ofT. cruzito humans in rural Argentina and probably elsewhere.
The Composite Genome of the Legume Symbiont
Sinorhizobium meliloti
Authors: Galibert Francis , Finan Turlough M. , Long Sharon R. , Pühler Alfred , Abola Pia , Ampe Frédéric , Barloy-Hubler Frédérique , Barnett Melanie J. , Becker Anke , Boistard Pierre , Bothe Gordana , Boutry Marc , Bowser Leah , Buhrmester Jens , Cadieu Edouard , Capela Delphine , Chain Patrick , Cowie Alison , Davis Ronald W. , Dréano Stéphane , Federspiel Nancy A. , Fisher Robert F. , Gloux Stéphanie , Godrie Thérèse , Goffeau André , Golding Brian , Gouzy Jérôme , Gurjal Mani , Hernandez-Lucas Ismael , Hong Andrea , Huizar Lucas , Hyman Richard W. , Jones Ted , Kahn Daniel , Kahn Michael L. , Kalman Sue , Keating David H. , Kiss Ernö , Komp Caridad , Lelaure Valérie , Masuy David , Palm Curtis , Peck Melicent C. , Pohl Thomas M , Portetelle Daniel , Purnelle Bénédicte , Ramsperger Uwe , Surzycki Raymond , Thébault Patricia , Vandenbol Micheline , Vorhölter Frank-J. , Weidner Stefan , Wells Derek H. , Wong Kim , Yeh Kuo-Chen , Batut Jacques
Published: March 2006

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N
2
) to ammonium (NH
4
+
). We present here the annotated DNA sequence of the α-proteobacterium
Sinorhizobium meliloti
, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.
Calibration of the Lutetium-Hafnium Clock
Authors: Scherer Erik , Münker Carsten , Mezger Klaus
Published: March 2006

Well-defined constants of radioactive decay are the cornerstone of geochronology and the use of radiogenic isotopes to constrain the time scales and mechanisms of planetary differentiation. Four new determinations of the lutetium-176 decay constant (λ
176
Lu) made by calibration against the uranium-lead decay schemes yield a mean value of 1.865 ± 0.015 × 10
−11
year
−1
, in agreement with the two most recent decay-counting experiments. Lutetium-hafnium ages that are based on the previously used λ
176
Lu of 1.93 × 10
−11
to 1.94 × 10
−11
year
−1
are thus ∼4% too young, and the initial hafnium isotope compositions of some of Earth's oldest minerals and rocks become less radiogenic relative to bulk undifferentiated Earth when calculated using the new decay constant. The existence of strongly unradiogenic hafnium in Early Archean and Hadean zircons implies that enriched crustal reservoirs existed on Earth by 4.3 billion years ago and persisted for 200 million years or more. Hence, current models of early terrestrial differentiation need revision.
Topochemical Polymerization of C
70
Controlled by Monomer Crystal Packing
Authors: Soldatov Alexander V. , Roth Georg , Dzyabchenko Alexander , Johnels Dan , Lebedkin Sergei , Meingast Christoph , Sundqvist Bertil , Haluska Miro , Kuzmany Hans
Published: March 2006

Polymeric forms of C
60
are now well known, but numerous attempts to obtain C
70
in a polymeric state have yielded only dimers. Polymeric C
70
has now been synthesized by treatment of hexagonally packed C
70
single crystals under moderate hydrostatic pressure (2 gigapascals) at elevated temperature (300°C), which confirms predictions from our modeling of polymeric structures of C
70
. Single-crystal x-ray diffraction shows that the molecules are bridged into polymeric zigzag chains that extend along the
c
axis of the parent structure. Solid-state nuclear magnetic resonance and Raman data provide evidence for covalent chemical bonding between the C
70
cages.
Lysophosphatidylcholine as a Ligand for the Immunoregulatory Receptor G2A
Authors: Kabarowski Janusz H. S. , Zhu Kui , Le Lu Q. , Witte Owen N. , Xu Yan
Published: March 2006

Although the biological actions of the cell membrane and serum lipid lysophosphatidylcholine (LPC) in atherosclerosis and systemic autoimmune disease are well recognized, LPC has not been linked to a specific cell-surface receptor. We show that LPC is a high-affinity ligand for G2A, a lymphocyte-expressed G protein–coupled receptor whose genetic ablation results in the development of autoimmunity. Activation of G2A by LPC increased intracellular calcium concentration, induced receptor internalization, activated ERK mitogen-activated protein kinase, and modified migratory responses of Jurkat T lymphocytes. This finding implicates a role for LPC-G2A interaction in the etiology of inflammatory autoimmune disease and atherosclerosis.
Very-Long-Period Seismic Signals and Caldera Formation at Miyake Island, Japan
Authors: Kumagai Hiroyuki , Ohminato Takao , Nakano Masaru , Ooi Masahiro , Kubo Atsuki , Inoue Hiroshi , Oikawa Jun
Published: March 2006

Over a period of roughly 40 days, starting on 8 July 2000, a caldera structure 1.7 kilometers in diameter developed by means of gradual depression and expansion of the summit crater at Miyake Island, Japan. At the same time, very-long-period (VLP) seismic signals were observed once or twice a day. Source mechanism analyses of the VLP signals show that the moment tensor solutions are smooth step functions over a time scale of 50 seconds, with dominant volumetric change components. We developed a model to explain the caldera and the VLP signals, in which a vertical piston of solid materials in the conduit is intermittently sucked into the magma chamber by lateral magma outflow. This model offers potential for making quantitative estimations of the characteristic physical properties of magma systems.
β-Helical Polymers from Isocyanopeptides
Authors: Cornelissen Jeroen J. L. M. , Donners Jack J. J. M. , de Gelder René , Graswinckel W. Sander , Metselaar Gerald A. , Rowan Alan E. , Sommerdijk Nico A. J. M. , Nolte Roeland J. M.
Published: March 2006

Polymerization of isocyanopeptides results in the formation of high molecular mass polymers that fold in a proteinlike fashion to give helical strands in which the peptide chains are arranged in β-sheets. The β-helical polymers retain their structure in water and unfold in a cooperative process at elevated temperatures. The peptide architecture in these polymers is a different form of the β-helix motif found in proteins. Unlike their natural counterparts, which contain arrays of large β-sheets stacked in a helical fashion, the isocyanopeptide polymers have a central helical core that acts as a director for the β-sheet–like arrangement of the peptide side arms. The helical structure of these isocyanopeptide polymers has the potential to be controlled through tailoring of the side branches and the hydrogen-bonding network present in the β-sheets.
Noisy Clockwork: Time Series Analysis of Population Fluctuations in Animals
Authors: Bjørnstad Ottar N. , Grenfell Bryan T.
Published: March 2006

Both biotic interactions and abiotic random forcing are crucial influences on population dynamics. This frequently leads to roughly equal importance of deterministic and stochastic forces. The resulting tension between noise and determinism makes ecological dynamics unique, with conceptual and methodological challenges distinctive from those in other dynamical systems. The theory for stochastic, nonlinear ecological dynamics has been developed alongside methods to test models. A range of dynamical components has been considered—density dependence, environmental and demographic stochasticity, and climatic forcing—as well as their often complex interactions. We discuss recent advances in understanding ecological dynamics and testing theory using long-term data and review how dynamical forces interact to generate some central field and laboratory time series.
Egalitarianism in Female African Lions
Authors: Packer Craig , Pusey Anne E. , Eberly Lynn E.
Published: March 2006

Because most cooperative societies are despotic, it has been difficult to test models of egalitarianism. Female African lions demonstrate a unique form of plural breeding in which companions consistently produce similar numbers of surviving offspring. Consistent with theoretical predictions from models of reproductive skew, female lions are unable to control each other's reproduction because of high costs of fighting and low access to each other's newborn cubs. A female also lacks incentives to reduce her companions' reproduction, because her own survival and reproduction depend on group territoriality and synchronous breeding. Consequently, female relationships are highly symmetrical, and female lions are “free agents” who only contribute to communal care when they have cubs of their own.
Tauopathy in
Drosophila
: Neurodegeneration Without Neurofibrillary Tangles
Authors: Wittmann Curtis W. , Wszolek Matthew F. , Shulman Joshua M. , Salvaterra Paul M. , Lewis Jada , Hutton Mike , Feany Mel B.
Published: March 2006

The microtubule-binding protein tau has been implicated in the pathogenesis of Alzheimer's disease and related disorders. However, the mechanisms underlying tau-mediated neurotoxicity remain unclear. We created a genetic model of tau-related neurodegenerative disease by expressing wild-type and mutant forms of human tau in the fruit fly
Drosophila melanogaster
. Transgenic flies showed key features of the human disorders: adult onset, progressive neurodegeneration, early death, enhanced toxicity of mutant tau, accumulation of abnormal tau, and relative anatomic selectivity. However, neurodegeneration occurred without the neurofibrillary tangle formation that is seen in human disease and some rodent tauopathy models. This fly model may allow a genetic analysis of the cellular mechanisms underlying tau neurotoxicity.
Long-Term Studies of Vegetation Dynamics
Authors: Rees Mark , Condit Rick , Crawley Mick , Pacala Steve , Tilman Dave
Published: March 2006

By integrating a wide range of experimental, comparative, and theoretical approaches, ecologists are starting to gain a detailed understanding of the long-term dynamics of vegetation. We explore how patterns of variation in demographic traits among species have provided insight into the processes that structure plant communities. We find a common set of mechanisms, derived from ecological and evolutionary principles, that underlie the main forces shaping systems as diverse as annual plant communities and tropical forests. Trait variation between species maintains diversity and has important implications for ecosystem processes. Hence, greater understanding of how Earth's vegetation functions will likely require integration of ecosystem science with ideas from plant evolutionary, population, and community ecology.
An Autoinhibitory Mechanism for Nonsyntaxin SNARE Proteins Revealed by the Structure of Ykt6p
Authors: Tochio Hidehito , Tsui Marco M. K. , Banfield David K. , Zhang Mingjie
Published: March 2006

Ykt6p is a nonsyntaxin SNARE implicated in multiple intracellular membrane trafficking steps. Here we present the structure of the NH
2
-terminal domain of Ykt6p (Ykt6pN, residues 1 to 140). The structure of Ykt6pN differed entirely from that of syntaxin and resembled the overall fold of the actin regulatory protein, profilin. Like some syntaxins, Ykt6p adopted a folded back conformation in which Ykt6pN bound to its COOH-terminal core domain. The NH
2
-terminal domain plays an important biological role in the function of Ykt6p, which in vitro studies revealed to include influencing the kinetics and proper assembly of SNARE complexes.
Complex Species Interactions and the Dynamics of Ecological Systems: Long-Term Experiments
Authors: Brown James H. , Whitham Thomas G. , Morgan Ernest S. K. , Gehring Catherine A.
Published: March 2006

Studies that combine experimental manipulations with long-term data collection reveal elaborate interactions among species that affect the structure and dynamics of ecosystems. Research programs in U.S. desert shrubland and pinyon-juniper woodland have shown that (i) complex dynamics of species populations reflect interactions with other organisms and fluctuating climate; (ii) genotype x environment interactions affect responses of species to environmental change; (iii) herbivore-resistance traits of dominant plant species and impacts of “keystone” animal species cascade through the system to affect many organisms and ecosystem processes; and (iv) some environmental perturbations can cause wholesale reorganization of ecosystems because they exceed the ecological tolerances of dominant or keystone species, whereas other changes may be buffered because of the compensatory dynamics of complementary species.
Ecological Forecasts: An Emerging Imperative
Authors: Clark James S. , Carpenter Steven R. , Barber Mary , Collins Scott , Dobson Andy , Foley Jonathan A. , Lodge David M. , Pascual Mercedes , Pielke Roger , Pizer William , Pringle Cathy , Reid Walter V. , Rose Kenneth A. , Sala Osvaldo , Schlesinger William H. , Wall Diana H. , Wear David
Published: March 2006

Planning and decision-making can be improved by access to reliable forecasts of ecosystem state, ecosystem services, and natural capital. Availability of new data sets, together with progress in computation and statistics, will increase our ability to forecast ecosystem change. An agenda that would lead toward a capacity to produce, evaluate, and communicate forecasts of critical ecosystem services requires a process that engages scientists and decision-makers. Interdisciplinary linkages are necessary because of the climate and societal controls on ecosystems, the feedbacks involving social change, and the decision-making relevance of forecasts.
Structure-Activity Relationships of Aminocoumarin-Type Gyrase and Topoisomerase IV Inhibitors Obtained by Combinatorial Biosynthesis
Authors: Flatman Ruth H. , Eustaquio Alessandra , Li Shu-Ming , Heide Lutz , Maxwell Anthony
Published: March 2006

Novobiocin and clorobiocin are gyrase inhibitors produced by
Streptomyces
strains. Structurally, the two compounds differ only by substitution at two positions: CH
3
versus Cl at position 8′ of the aminocoumarin ring and carbamoyl versus 5-methyl-pyrrol-2-carbonyl (MePC) at the 3"-OH of noviose. Using genetic engineering, we generated a series of analogs carrying H, CH
3
, or Cl at 8′ and H, carbamoyl, or MePC at 3"-OH. Comparison of the gyrase inhibitory activities of all nine structural permutations confirmed that acylation of 3"-OH is essential for activity, with MePC being more effective than carbamoyl. Substitution at 8′ further enhanced activity, but the effect of CH
3
or Cl depended on the nature of the acyl group at 3": in the presence of carbamoyl at 3", CH
3
resulted in higher activity; in the presence of MePC at 3", Cl resulted in higher activity. This suggests that the structures of both natural compounds are highly evolved for optimal interaction with gyrase. In a second series of experiments, clorobiocin derivatives with and without the methyl group at 4"-OH of noviose, and with different positions of the MePC group of noviose, were tested. Again clorobiocin was superior to all of its analogs. The activities of all compounds were also tested against topoisomerase IV (topo IV). Clorobiocin stood out as a remarkably effective topo IV inhibitor. The relative activities of the different compounds toward topo IV showed a pattern similar to that of the relative gyrase-inhibitory activities. This is the first report of a systematic evaluation of a series of aminocoumarins against both gyrase and topo IV. The results give further insight into the structure-activity relationships of aminocoumarin antibiotics.
Organization of Tn
2610
Containing Two Transposition Modules
Authors: Takaya Akiko , Watanabe Masato , Yamamoto Tomoko
Published: March 2006

Transposon Tn
2610
, found in a conjugative plasmid from an
Escherichia coli
isolate recovered at a hospital in Chiba, Japan, in 1975, was completely sequenced. Tn
2610
is 23,883 bp long and is bracketed by two transposition modules, a Tn
1721
-like module and a Tn
21
-derived module, which correspond, respectively, to the long inverted repeats IRa and IRb previously described for this transposon. Although both
tnpA
genes are intact, only that in the Tn
21
-derived module (IRb) functions in the transposition, while that in the Tn
1721
-derived module (IRa) cannot recognize the 38-bp imperfect repeat at the end of the IRb element. Both
tnpR
and
res
are present in IRa, while the
tnpR
gene of IRb is interrupted by the insertion of an IS
26
insertion element. The intervening region, between the
res
site of the Tn
1721
module and IS
26
, carries multiple integron-associated resistance genes within a Tn
21
backbone, including a region identical to that found in the genome of
Salmonella enterica
serovar Typhimurium DT104. These findings suggest that Tn
2610
originated from Tn
1721
and Tn
21
, with extensive recombination events with other elements which have resulted in a complex mosaic structure.
Overexpression ofCandida albicans CDR1,CDR2, orMDR1Does Not Produce Significant Changes in Echinocandin Susceptibility
Authors: Niimi K. , Maki K. , Ikeda F. , Holmes A. R. , Lamping E. , Niimi M. , Monk B. C. , Cannon R. D.
Published: March 2006

The micafungin and caspofungin susceptibilities ofCandida albicanslaboratory and clinical isolates and ofSaccharomyces cerevisiaestrains stably hyperexpressing fungal ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters involved in azole resistance were determined using three separate methods. Yeast strains hyperexpressing individual alleles of ABC transporters or an MFS transporter fromC. albicansgave the expected resistance profiles for the azoles fluconazole, itraconazole, and voriconazole. The strains hyperexpressingCDR2showed slightly decreased susceptibility to caspofungin in agar plate drug resistance assays, as previously reported, but increased susceptibility to micafungin compared with either the strains hyperexpressingCDR1or the null parent deleted of seven ABC transporters. The strains hyperexpressingCDR1showed slightly decreased susceptibility to micafungin in these assays. AC. albicansclinical isolate overexpressing both Cdr1p and Cdr2p relative to its azole-sensitive isogenic progenitor acquired resistance to azole drugs and showed reduced susceptibility to caspofungin and slightly increased susceptibility to micafungin in agar plate drug resistance assays. None of the strains showed significant resistance to micafungin or caspofungin in liquid microdilution susceptibility assays. The antifungal activities of micafungin and caspofungin were similar in agarose diffusion assays, although the shape and size of the caspofungin inhibitory zones were affected by medium composition. The assessment of micafungin and caspofungin potency is therefore assay dependent; the differences seen with agar plate drug resistance assays occur over narrow ranges of echinocandin concentrations and are not of clinical significance.
Distribution of Florfenicol Resistance Genes
fexA
and
cfr
among Chloramphenicol-Resistant
Staphylococcus
Isolates
Authors: Kehrenberg Corinna , Schwarz Stefan
Published: March 2006

A total of 302 chloramphenicol-resistant
Staphylococcus
isolates were screened for the presence of the florfenicol/chloramphenicol resistance genes
fexA
and
cfr
and their localization on mobile genetic elements. Of the 114 isolates from humans, only a single
Staphylococcus aureus
isolate showed an elevated MIC to florfenicol, but did not carry either of the known resistance genes,
cfr
or
fexA
. In contrast, 11 of the 188 staphylococci from animal sources were considered florfenicol resistant and carried either
cfr
(one isolate),
fexA
(five isolates), or both resistance genes (five isolates). In nine cases we confirmed that these genes were carried on a plasmid. Five different types of plasmids could be differentiated on the basis of their sizes, restriction patterns, and resistance genes. The gene
fexA
, which has previously been shown to be part of the nonconjugative transposon Tn
558
, was identified in 10 of the 11 resistant isolates from animals. PCR assays were developed to detect different parts of this transposon as well as their physical linkage. Complete copies of Tn
558
were found in five different isolates and shown by inverse PCR to be functionally active. Truncated copies of Tn
558
, in which the
tnpA-tnpB
area was in part deleted by the integration of a 4,674-bp segment including the gene
cfr
and a novel 2,446-bp IS
21
-like insertion sequence, were seen in a plasmid present in three staphylococcal isolates.
Comparing Gatifloxacin and Clarithromycin in Pneumonia Symptom Resolution and Process of Care
Authors: Dean Nathan C. , Sperry Paul , Wikler Matthew , Suchyta Mary S. , Hadlock Carol
Published: March 2006

In looking for outcome differences beyond rates of cure, we prospectively compared the symptom resolution, side effects, and processes of care between the use of clarithromycin and gatifloxacin for the treatment of radiographically confirmed community-acquired pneumonia. We conducted a multicenter, randomized, open-label study comparing gatifloxacin monotherapy to clarithromycin alone or combined with ceftriaxone for patients with multiple risk factors. We measured the return to usual activities and symptoms over seven interviews ending 42 days after randomization. Admission and hospital discharge decision support were provided to treating physicians. We enrolled 266 patients over the age of 18 years between September 2000 and June 2003. The groups were similar in age and gender, with a mean age of 53.5 ± 19.4 years, and were 54% female. Patient severity as determined by the number of risk factors and the Pneumonia Severity Index was similar between groups; 95% of the patients were low risk. A total of 91% of patients completed at least five of seven symptom interviews. In the clarithromycin study arm, 64% received concomitant therapy with ceftriaxone. We found no significant difference in return to usual activities, pneumonia-specific symptom scores, and 12-item short-form health survey scores. Individual symptom scores were similar except for bad taste and injection site soreness, which were higher in clarithromycin patients. The rates of hospital admission and length of stay were similar. The cost of antibiotic was higher in the clarithromycin group: $257 versus $110 for gatifloxacin. We found that gatifloxacin monotherapy is similar to clarithromycin given with or without ceftriaxone for the treatment of community-acquired pneumonia, except that antibiotic cost, bad taste, and injection site soreness favor the use of gatifloxacin.
Determinants of Rifampin, Isoniazid, Pyrazinamide, and Ethambutol Pharmacokinetics in a Cohort of Tuberculosis Patients
Authors: McIlleron Helen , Wash Peter , Burger André , Norman Jennifer , Folb Peter I. , Smith Pete
Published: March 2006

Evaluation of sources of pharmacokinetic variation can facilitate optimization of tuberculosis treatment regimens by identification of avoidable sources of variation and of risk factors for low or high drug concentrations in patients. Our objective was to describe the pharmacokinetics of rifampin, isoniazid, pyrazinamide, and ethambutol in a cohort of tuberculosis patients established on first-line treatment regimens and to evaluate the determinants of pharmacokinetic variation. Plasma concentration-time profiles were determined for each of the drugs in 142 patients with drug-sensitive pulmonary tuberculosis after 2 months of daily treatment in hospital. Pharmacokinetic measures were described by noncompartmental analysis. Multiple linear regression was used to evaluate the patient and the treatment factors associated with variation of the area under the concentration-time curve from 0 to 8 h. Several factors independently associated with variations in antituberculosis drug concentrations were identified: human immunodeficiency virus infection was associated with 39% and 27% reductions for rifampin and ethambutol, respectively; formulation factors were determinants of rifampin and isoniazid bioavailability; female patients had increased rifampin and isoniazid concentrations but reduced ethambutol concentrations; older patients had higher levels of isoniazid and ethambutol; patients with a history of previous antituberculosis treatment had lower ethambutol concentrations; and the dose per kilogram of body weight was associated with the concentrations of all four agents. Further studies are required to assess the implications of variations in antituberculosis drug concentrations for efficacy and safety before decisions are made to change the dosing strategy in patients at risk.
qnrB
, Another Plasmid-Mediated Gene for Quinolone Resistance
Authors: Jacoby George A. , Walsh Kelley E. , Mills Debra M. , Walker Victoria J. , Oh Herin , Robicsek Ari , Hooper David C.
Published: March 2006

A novel plasmid-mediated quinolone resistance gene,
qnrB
, has been discovered in a plasmid encoding the CTX-M-15 β-lactamase from a
Klebsiella pneumoniae
strain isolated in South India. It has less than 40% amino acid identity with the original
qnr
(now
qnrA
) gene or with the recently described
qnrS
but, like them, codes for a protein belonging to the pentapeptide repeat family. Strains with
qnrB
demonstrated low-level resistance to all quinolones tested. The gene has been cloned in an expression vector attaching a polyhistidine tag, which facilitated purification to ≥95% homogeneity. As little as 5 pM of QnrB-His
6
protected purified DNA gyrase against inhibition by 2 μg/ml (6 μM) ciprofloxacin. With a PCR assay
qnrB
has been detected in
Citrobacter koseri
,
Enterobacter cloacae
, and
Escherichia coli
isolates from the United States, linked to SHV-12 β-lactamase and coding for a product differing in five amino acids from the Indian (now QnrB1) variety. The
qnrB
gene has been found near Orf1005 in some, but not all, plasmids and in association with open reading frames matching known chromosomal genes, suggesting that it too was acquired by plasmids from an as-yet-unknown bacterial source.
Staphylococcus aureus
CcpA Affects Virulence Determinant Production and Antibiotic Resistance
Authors: Seidl Kati , Stucki Martin , Ruegg Martin , Goerke Christiane , Wolz Christiane , Harris Llinos , Berger-Bächi Brigitte , Bischoff Markus
Published: March 2006

Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the
ccpA
homologue (
saCOL1786
) in
Staphylococcus aureus
was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the Δ
ccpA
mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of
S. aureus
by down-regulating
RNAIII
, the effector molecule of the
agr
locus, and altering the transcription patterns of
hla
, encoding α-hemolysin, and
spa
, encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant
S. aureus
strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant
S. aureus
strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased
cap
operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.
Real-Time Reverse Transcription-PCR Quantification of Cytokine mRNA Expression in Golden Syrian Hamster Infected withLeishmania infantumand Treated with a New Amphotericin B Formulation
Authors: Rama Iñiguez S. , Dea-Ayuela M. A. , Sanchez-Brunete J. A. , Torrado J. J. , Alunda J. M. , Bolas-Fernández F.
Published: March 2006

A real-time quantitative reverse transcription-PCR assay was developed for the quantification of cytokine mRNA expression in the golden Syrian hamsterMesocricetus auratusinfected withLeishmania infantumand treated with amphotericin B (AMB) formulated in microspheres made of human serum albumin (HSA). Treatment was administered intravenously on days 69, 71, and 73 postinfection (p.i.) with 107metacyclic promastigotes, at doses of 2 and 40 mg/kg of AMB. High infection levels were recorded for untreated animals by day 76 p.i., with parasite loads always about 2 log10per gram higher in the liver than in the spleen. Treatment was highly effective with both doses, but at 40 mg/kg, almost complete parasite elimination was achieved. mRNA expression of gamma interferon (IFN-γ) and, to a lesser extent, tumor necrosis factor alpha (TNF-α) and transforming growth factor beta (TGF-β) in spleen cells was up-regulated in most animals of the untreated group. The mRNA expression of interleukin-4 was strongly down-regulated in untreated as well as treated infected animals. Treatment with the lower dose of AMB-HSA down-regulated the mRNA expression of IFN-γ and TNF-α, with no effect on the deactivating cytokine TGF-β. In contrast, treatment with the higher dose (40 mg/kg) of the formulation caused moderate up-regulation of IFN-γ and TNF-α and strong suppression of TGF-β. Treatment of noninfected animals did not alter the cytokine expression pattern with regard to untreated controls. Our results suggest that treatment ofL. infantum-infected Syrian hamsters with highly effective nontoxic doses of AMB-HSA causes deactivation of the anti-inflammatory cytokine TGF-β, which in turn results in up-regulation of the Th1 cytokines IFN-γ and TNF-α.
Bac 32, a Novel Bacteriocin Widely Disseminated among Clinical Isolates of
Enterococcus faecium
Authors: Inoue Takako , Tomita Haruyoshi , Ike Yasuyoshi
Published: March 2006

A total of 636 vancomycin-resistant
Enterococcus faecium
(VRE) isolates that had been obtained between 1994 and 1999 from the Medical School Hospital of the University of Michigan, Ann Arbor, were tested for bacteriocin production. Two hundred seventy-seven (44%) of the strains were bacteriocinogenic; and 193 of these exhibited activity against
Enterococcus faecium
,
Enterococcus hirae
, and
Enterococcus durans
. Strain VRE200 harbors the highly efficient conjugative gentamicin resistance plasmid pG200 (70 kb) and bacteriocin plasmid pTI1 (12.5 kb). The bacteriocin encoded on pTI1 was designated bacteriocin 32 (Bac 32). Bacteriocin 32 was active against
E. faecium
,
E. hirae
, and
E. durans
but showed no activity against
Listeria monocytogenes
. The Bac 32 genetic locus consists of a bacteriocin gene (
bacA
) and an immunity gene (
bacB
). Neither of these genes showed significant homology to any known bacteriocin determinants. The deduced
bacA
product is 89 amino acids in length, with a putative signal peptide of 19 amino acids at the N terminus. The
bacB
gene encodes a deduced 55-amino-acid protein without a signal sequence. One hundred eighty-nine strains (97.9%) of the 193 strains with activity against the 3 test enterococcal strains gave rise to the expected specific PCR product with a primer specific for
bacA
, indicating that there is a high incidence of Bac 32 production among VRE clinical isolates. Data from Southern analyses of plasmid DNA from 189 of the Bac 32-producing strains with a plasmid pTI1-specific probe suggested that 137 (72.5%) of the strains harbored a pTI1-type plasmid. Bac 32 or Bac 32-type bacteriocin activity and the determinant genes were also identified in 22 (39.3%) of a total of 56 vancomycin-sensitive
E. faecium
clinical isolates, which suggests that this bacteriocin is widely disseminated among
E. faecium
strains.
Gentamicin Causes Apoptosis at Low Concentrations in Renal LLC-PK
1
Cells Subjected to Electroporation
Authors: Servais Hélène , Jossin Yves , Van Bambeke Françoise , Tulkens Paul M. , Mingeot-Leclercq Marie-Paule
Published: March 2006

Gentamicin accumulates in the lysosomes of kidney proximal tubular cells and causes apoptosis at clinically relevant doses. Gentamicin-induced apoptosis can be reproduced with cultured renal cells, but only at high extracellular concentrations (1 to 3 mM; 0.4 to 1.2 g/liter) because of its low level of uptake. We recently showed that gentamicin-induced apoptosis in LLC-PK
1
cells involves a rapid (2-h) permeabilization of lysosomes and activation of the mitochondrial pathway of apoptosis (10 h). We now examine whether the delivery of gentamicin to the cytosol by electroporation would sensitize LLC-PK
1
cells to apoptosis. Cells were subjected to eight pulses (1 ms) at 800 V/cm (square waves) in the presence of gentamicin (3 μM to 3 mM; 1.2 mg/liter to 1.2 g/liter); returned to gentamicin-free medium; and examined at 8 h for their Bax (a marker of mitochondrial pathway activation) contents by Western blotting and competitive reverse transcriptase PCR and at 24 h for apoptosis by 4′,6′-diamidino-2′-phenylindole staining (confirmed by electron microscopy) and for necrosis (by determination of lactate dehydrogenase release). Nonelectroporated cells were incubated with gentamicin for 8 and 24 h. Significant increases in Bax levels (8 h) and apoptosis (24 h) were detected with 0.03 mM (13.2 mg/liter) gentamicin in electroporated cells compared with those achieved with 2 mM (928 mg/liter) in incubated cells. The increase in the Bax level was not associated with an increase in the level of its mRNA but was associated with the accumulation of ubiquitinated forms (probably as a result of impairment of its degradation by the proteasome). Assay of cell-associated gentamicin showed a marked, immediate, but transient accumulation in electroporated cells, whereas a slow, steady uptake was detected in incubated cells. The data indicate that cytosolic gentamicin triggers apoptosis. Sequestration of gentamicin in lysosomes would, to some extent, protect against apoptosis.
Comparative Pharmacokinetics and Pharmacodynamic Target Attainment of Ertapenem in Normal-Weight, Obese, and Extremely Obese Adults
Authors: Chen M. , Nafziger A. N. , Drusano G. L. , Ma L. , Bertino J. S.
Published: March 2006

Little is known of the effects of obesity on ertapenem drug disposition and pharmacodynamics. Thirty healthy volunteers in three body mass index (BMI) groups (10 per group), normal weight (BMI, 18.5 to 24.9 kg/m
2
), class I-II obesity (BMI, 30 to 39.9 kg/m
2
), and class III obesity (BMI, ≥40 kg/m
2
), were administered a 1-g dose of ertapenem. Serum concentrations were obtained over 24 h. Population pharmacokinetic data were obtained using a nonparametric adaptive grid followed by Monte Carlo simulation to determine the probability of obtaining the free drug exposure targets of the time that the free drug concentration remains above the MIC (
f
T
>MIC
) of 20% and 40% for bacteriostatic and maximal bactericidal activity, respectively. Compared to the subjects in the obese groups, area under the concentration-time curve from 0 h to infinity was significantly higher in the normal-weight subjects, whereas the total central compartment volume was higher in the class III obese subjects (
P
≤ 0.05). Achieving a bacteriostatic target of
f
T
>MIC
of 20% with a 90% probability was attained at MICs of ≤0.5 μg/ml for normal-weight subjects. Class I-II and class III obese subjects were able to achieve this target only at a MIC of ≤0.25 μg/ml. For maximal bactericidal activity (
f
T
>MIC
, 40%), no group attained the target at the 90% probability level at any tested MIC. The results suggest that the standard 1-g ertapenem dose may not provide adequate drug exposure for any body mass index classification for MICs in excess of 0.25 to 0.5 μg/ml.
In Vitro Characterization of the Antibacterial Spectrum of Novel Bacterial Type II Topoisomerase Inhibitors of the Aminobenzimidazole Class
Authors: Mani Nagraj , Gross Christian H. , Parsons Jonathan D. , Hanzelka Brian , Müh Ute , Mullin Steve , Liao Yusheng , Grillot Anne-Laure , Stamos Dean , Charifson Paul S. , Grossman Trudy H.
Published: March 2006

Antibiotics with novel mechanisms of action are becoming increasingly important in the battle against bacterial resistance to all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV (topoIV) are the familiar targets of fluoroquinolone and coumarin antibiotics. Here we present the characterization of two members of a new class of synthetic bacterial topoII ATPase inhibitors: VRT-125853 and VRT-752586. These aminobenzimidazole compounds were potent inhibitors of both DNA gyrase and topoIV and had excellent antibacterial activities against a wide spectrum of problematic pathogens responsible for both nosocomial and community-acquired infections, including staphylococci, streptococci, enterococci, and mycobacteria. Consistent with the novelty of their structures and mechanisms of action, antibacterial potency was unaffected by commonly encountered resistance phenotypes, including fluoroquinolone resistance. In time-kill assays, VRT-125853 and VRT-752586 were bactericidal against
Staphylococcus aureus
,
Streptococcus pneumoniae
,
Enterococcus faecalis
, and
Haemophilus influenzae
, causing 3-log reductions in viable cells within 24 h. Finally, similar to the fluoroquinolones, relatively low frequencies of spontaneous resistance to VRT-125853 and VRT-752586 were found, a property consistent with their in vitro dual-targeting activities.
Nanodisk-Associated Amphotericin B Clears
Leishmania major
Cutaneous Infection in Susceptible BALB/c Mice
Authors: Nelson Keith G. , Bishop Jeanette V. , Ryan Robert O. , Titus Richard
Published: March 2006

Nanometer-scale, apolipoprotein-stabilized phospholipid bilayer disk complexes (nanodisks [ND]) harboring the toxic and poorly soluble antileishmanial agent amphotericin B (AMB) were examined for efficacy in treatment of
Leishmania major
-infected BALB/c mice (
Mus musculus
).
L. major
-infected mice were intraperitoneally (i.p.) treated with AMB-ND in 0-, 1-, and 5-mg/kg doses at 24 h, 48 h, and 4, 7, 14, and 21 days postinfection in two experiments.
L. major
-infected mice were i.p. treated with phosphate-buffered saline, 5 mg/kg AMB-ND, or 5 mg/kg lipid-associated amphotericin B (liposomal amphotericin B, AmBisome) at 24 h, 48 h, and 10, 20, 30, and 40 days postinfection in one experiment. Parasite numbers, footpad lesion size progression, and development of cytokine responses were assayed at days 7, 15, 30, 50, 140, and 250 or at days 14, 30, 50, 95, and 140 postinfection. Mice administered AMB-ND in 1- or 5-mg/kg doses were significantly protected from
L. major
, displaying decreases in lesion size and parasite burden, particularly at the 5-mg/kg dosage level. In contrast to the i.p. treated AmBisome group, BALB/c mice treated with i.p. AMB-ND completely cleared an
L. major
infection by 140 to 250 days postinfection, with no lesions remaining and no parasites isolated from infected animals. Restimulated mixed lymphocyte culture cytokine responses (interleukin-4 [IL-4], IL-12, IL-10, NO, and gamma interferon) were unchanged by AMB-ND administration compared to controls. The marked clearance of
Leishmania
parasites from a susceptible strain of mice without an appreciable change in the cytokine response suggests that AMB-ND represent a potentially useful formulation for treatment of intrahistiocytic organisms.
Rapid Microbiologic and Pharmacologic Evaluation of Experimental Compounds against
Mycobacterium tuberculosis
Authors: Gruppo Veronica , Johnson Christine M. , Marietta Karen S. , Scherman Hataichanok , Zink Erin E. , Crick Dean C. , Adams Linda B. , Orme Ian M. , Lenaerts Anne J.
Published: March 2006

The assessment of physiochemical and pharmacological properties at early stages of drug discovery can accelerate the conversion of hits and leads into candidates for further development. A strategy for streamlined evaluation of compounds against
Mycobacterium tuberculosis
in the early preclinical stage is presented in this report. As a primary assay to rapidly select experimental compounds with sufficient in vitro activity, the growth inhibition microtiter plate assay was devised as an alternative to current methods. This microdilution plate assay is a liquid culture method based on spectrophotometric readings of the bacillary growth. The performance of this method was compared to the performance of two established susceptibility methods using clinical available tuberculosis (TB) drugs. Data generated from all three assays were similar for all of the tested compounds. A second simple bioassay was devised to assess the oral bioavailability of compounds prior to extensive in vivo efficacy testing. The bioassay estimates drug concentrations in collected serum samples by a microdilution MIC plate method using
M. tuberculosis
. In the same assay, the MIC of the compound is also determined in the presence of 10% mouse serum as an indication of protein binding. The method was validated using different clinically available TB drugs, and results are discussed in this report. With these methodological advances, screening of compounds against tuberculosis in the preclinical phase will be rapid, can be adapted to semi-high-throughput screening, and will add relevant physicochemical and basic pharmacological criteria to the decision process of drug discovery.
Activity of Cecropin A-Melittin Hybrid Peptides against Colistin-Resistant Clinical Strains of
Acinetobacter baumannii
: Molecular Basis for the Differential Mechanisms of Action
Authors: Saugar José María , Rodríguez-Hernández María Jesús , de la Torre Beatriz G. , Pachón-Ibañez María Eugenia , Fernández-Reyes María , Andreu David , Pachón Jerónimo , Rivas Luis
Published: March 2006

Acinetobacter baumannii
has successfully developed resistance against all common antibiotics, including colistin (polymyxin E), the last universally active drug against this pathogen. The possible widespread distribution of colistin-resistant
A. baumannii
strains may create an alarming clinical situation. In a previous work, we reported differences in lethal mechanisms between polymyxin B (PXB) and the cecropin A-melittin (CA-M) hybrid peptide CA(1-8)M(1-18) (KWKLFKKIGIGAVLKVLTTGLPALIS-NH
2
) on colistin-susceptible strains (J. M. Saugar, T. Alarcón, S. López-Hernández, M. López-Brea, D. Andreu, and L. Rivas, Antimicrob. Agents Chemother.
46:
875-878, 2002). We now demonstrate that CA(1-8)M(1-18) and three short analogues, namely CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH
2
), its N
α
-octanoyl derivative (Oct-KWKLFKKIGAVLKVL-NH
2
), and CA(1-7)M(5-9) (KWKLLKKIGAVLKVL-NH
2
) are active against two colistin-resistant clinical strains. In vitro, resistance to colistin sulfate was targeted to the outer membrane, as spheroplasts were equally lysed by a given peptide, regardless of their respective level of colistin resistance. The CA-M hybrids were more efficient than colistin in displacing lipopolysaccharide-bound dansyl-polymyxin B from colistin-resistant but not from colistin-susceptible strains. Similar improved performance of the CA-M hybrids in permeation of the inner membrane was observed, regardless of the resistance pattern of the strain. These results argue in favor of a possible use of CA-M peptides, and by extension other antimicrobial peptides with similar features, as alternative chemotherapy in colistin-resistant
Acinetobacter
infections.
Clinical and Economic Impact of Bacteremia with Extended- Spectrum-β-Lactamase-Producing
Enterobacteriaceae
Authors: Schwaber Mitchell J. , Navon-Venezia Shiri , Kaye Keith S. , Ben-Ami Ronen , Schwartz David , Carmeli Yehuda
Published: March 2006

We studied outcomes of extended-spectrum β-lactamase (ESBL) production in
Enterobacteriaceae
bacteremia. Inpatients with bacteremia caused by ESBL-producing
Escherichia coli
,
Klebsiella
spp., or
Proteus
spp. (cases) were compared with patients with bacteremia caused by non-ESBL producers (controls). Outcomes included mortality, mortality due to infection, length of stay (LOS), delay in appropriate therapy (DAT), discharge to a chronic care facility, and hospital cost. Ninety-nine cases and 99 controls were enrolled. Thirty-five percent of cases died, versus 18% of controls (odds ratio [OR], 2.5; 95% confidence interval [CI], 1.3 to 4.7;
P
= 0.01). Thirty percent of cases died due to infection, versus 16% of controls (OR, 2.3; 95% CI, 1.1 to 4.5;
P
= 0.03). The median LOS after bacteremia for cases was 11 days (interquartile range, 5 to 21), versus 5 days for controls (interquartile range, 3 to 9) (
P
< 0.001). DAT occurred in 66% of cases, versus 7% of controls (OR, 25.1; 95% CI, 10.5 to 60.2;
P
< 0.001). Cases were more likely than controls to be discharged to chronic care (52% versus 21%; OR, 4.0; 95% CI, 1.9 to 8.3;
P
< 0.001). The average hospital cost for cases was 65,509 Israeli shekels, versus 23,538 shekels for controls (
P
< 0.001). After adjusting for differences between groups by using multivariable analysis, ESBL production remained a significant predictor of mortality (OR, 3.6; 95% CI, 1.4 to 9.5;
P
= 0.008), increased LOS (1.56-fold;
P
= 0.001), DAT (OR, 25.1; 95% CI, 10.5 to 60.2;
P
< 0.001), and increased cost (1.57-fold;
P
= 0.003). The mean increase in equivalent cost attributable to ESBL production was $9,620. ESBL production was associated with severe adverse outcomes, including higher overall and infection-related mortality, increased LOS, DAT, discharge to chronic care, and higher costs.