bluuebunny/crossref_metadata_2025 huggingface dataset:
Hongiella mannitolivorans gen. nov., sp. nov., Hongiella halophila sp. nov. and Hongiella ornithinivorans sp. nov., isolated from tidal flat sediment
Authors: Yi Hana , Chun Jongsik
Published: January 2004

Three marine strains of the Cytophaga–Flavobacterium–Bacteroides group, designated JC2050T, JC2051T and JC2052T, were obtained from a single sediment sample of getbol, the Korean tidal flat. Comparative 16S rDNA sequence studies revealed that the test strains were not closely related to any validly published genera and that these strains were only distantly related to the genus Cyclobacterium (88·7–91·2 %). Phylogenetic analyses demonstrated that the three getbol isolates formed a distinct monophyletic clade within the family Cytophagaceae. Physiological, biochemical and chemotaxonomic data also indicated that these three getbol isolates differed significantly from members of other genera and were sufficiently different from each other to be recognized as separate species. On the basis of polyphasic evidence, a new genus, Hongiella gen. nov., is proposed, with three novel species, Hongiella mannitolivorans sp. nov. (type strain JC2050T=IMSNU 14012T=DSM 15301T), Hongiella halophila sp. nov. (type strain JC2051T=IMSNU 14013T=DSM 15292T) and Hongiella ornithinivorans sp. nov. (type strain JC2052T=IMSNU 14014T=DSM 15282T). Hongiella mannitolivorans is the type species of the genus.
Renaming of Agrobacterium larrymoorei Bouzar and Jones 2001 as Rhizobium larrymoorei (Bouzar and Jones 2001) comb. nov.
Authors: Young J. M.
Published: January 2004

In conformity with the nomenclature of the genus Rhizobium published by Young et al. (Int J
Syst Evol Microbiol
51, 89–103, 2001), it is proposed that Agrobacterium larrymoorei be named as Rhizobium larrymoorei comb. nov.
A novel psychrotolerant member of the hymenomycetous yeasts from Antarctica: Cryptococcus watticus sp. nov.
Authors: Guffogg Sharon P. , Thomas-Hall Skye , Holloway Paul , Watson Kenneth
Published: January 2004

Soil, snow and organic material, collected in November 1997 from the Vestvold Hills, Davis Base, Antarctica, were screened for yeasts. Two isolates, which were shown to be indistinguishable by rDNA sequencing and protein analysis by SDS-PAGE, are described in this communication as a novel species, Cryptococcus watticus sp. nov. (type culture, CBS 9496T=NRRL Y-27556T). Sequence analyses of the 26S rDNA D1/D2 region placed C. watticus in the hymenomycetous yeasts in a cluster with Holtermannia corniformis and Cryptococcus nyarrowii. This species has been allocated to the genus Cryptococcus on the basis of physiological and morphological characteristics.
Erysipelothrix inopinata sp. nov., isolated in the course of sterile filtration of vegetable peptone broth, and description of Erysipelotrichaceae fam. nov.
Authors: Verbarg Susanne , Rheims Holger , Emus Sabine , Frühling Anja , Kroppenstedt Reiner M. , Stackebrandt Erko , Schumann Peter
Published: January 2004

Based on 16S rRNA gene sequence comparison, an isolate that was detected in sterile-filtered vegetable broth was classified as a novel member of theErysipelothrixline of descent of theFirmicutes. Strain MF-EP02Tresembles members of the two species ofErysipelothrixwith validly published names,Erysipelothrix rhusiopathiaeandErysipelothrix tonsillarum, in morphology, fatty acid composition, lack of menaquinones in aerobically and anaerobically grown cultures, DNA G+C content and peptidoglycan amino acid composition. Distinct differences in physiological characteristics, however, support the allocation of this isolate to a novel species of the genusErysipelothrix, for which the nameErysipelothrix inopinatasp. nov. (type strain, MF-EP02T=DSM 15511T=CIP 107935T) is proposed. Members of theErysipelothrixline of descent are included in the familyErysipelotrichaceaefam. nov.
Reconsideration of Arthrobacter ilicis (Mandel et al. 1961) Collins et al. 1982 as a plant-pathogenic species. Proposal to emend the authority and description of the species. Request for an Opinion
Authors: Young J. M. , Watson D. R. W. , Dye D. W.
Published: January 2004

Strains now considered to represent the type strain of Arthrobacter ilicis, described as a pathogen of American holly, are not identical. The designated type strain does not represent this pathogen. However, one of the other strains sourced to the type strain of the pathogen does appear to be authentic, but is not a member of A. ilicis. It is proposed that A. ilicis is an unrelated species, not a pathogen of American holly. The nomenclature of A. ilicis can be rectified by emending the authority and by emending the species description to recognize this species as a novel species that is not a plant pathogen. The pathogen of American holly then becomes a novel pathovar, Curtobacterium flaccumfaciens pv. ilicis. The opinion of the Judicial Commission is sought.
Reclassification of Amycolatopsis orientalis subsp. lurida Lechevalier et al. 1986 as Amycolatopsis lurida sp. nov., comb. nov.
Authors: Stackebrandt Erko , Kroppenstedt Reiner M. , Wink Joachim , Schumann Peter
Published: January 2004

Amycolatopsis orientalissubsp.luridaDSM 43134Tdiffers significantly from the type strain ofA. orientalis,A. orientalissubsp.orientalisDSM 40040T, and from other species of the genus in genomic and metabolic properties. Its elevation to species status asAmycolatopsis luridasp. nov., comb. nov. is justified.
Status of strains that contravene Rules 27(3) and 30 of the Bacteriological Code. Request for an Opinion
Authors: Euzéby J. P. , Tindall B. J.
Published: January 2004

In the period from January 2001, at least 207 new names proposed in articles in the International Journal of Systematic and Evolutionary Microbiology or cited in Validation Lists are not in accordance with Rules 27(3) and 30 of the Bacteriological Code. The purpose of the present Request for an Opinion is to clarify the status of the names listed and to provide a solution whereby they may be considered to be validly published.
Functional roles of cytoplasmic loops and pore lining transmembrane helices in the voltage‐dependent inactivation of HVA calcium channels
Authors: Stotz Stephanie C. , Jarvis Scott E. , Zamponi Gerald W.
Published: January 2004

Voltage‐dependent inactivation of calcium channels is a key mechanism for regulating intracellular calcium levels and neuronal excitability. In sodium and potassium channels, the molecular determinants that govern fast inactivation involve pore block by a cytoplasmic gating particle. As we discuss here, there is an increasing body of evidence that is consistent with a qualitatively similar inactivation mechanism in high‐voltage‐activated calcium channels. Work from a number of laboratories has implicated both cytoplasmic regions and the pore‐lining S6 transmembrane helices in the inactivation process. Together with our recent findings, this leads us to propose a model in which the intracellular domain I–II linker region acts as a ‘hinged lid’ that physically occludes the pore by docking to the cytoplasmic ends of the S6 segments. We further propose that the ancillary calcium channel β subunits differentially modulate inactivation kinetics by binding to and thereby regulating the mobility of the putative inactivation gate. Indeed, additional evidence suggests that the carboxy terminus, amino terminus and domain III–IV linker regions of the channel modulate inactivation rates through interactions with the I–II linker per se, or indirectly via the ancillary β subunits. Taken together, the fast voltage‐dependent inactivation of calcium channels appears reminiscent of that of sodium channels, but appears to show a more complex regulation through intramolecular interactions between the putative inactivation gate and other cytoplasmic regions.
Gene targeting approach to clarification of ion channel function: studies of Kir6.x null mice
Authors: Seino Susumu , Miki Takashi
Published: January 2004

ATP‐sensitive potassium (KATP) channels are present in many tissues, including pancreatic β‐cells, heart, skeletal muscle, vascular smooth muscle and brain, in which they couple the cell metabolic state to membrane potential. KATP channels are hetero‐octameric proteins composed of the pore‐forming subunits Kir6.x (Kir6.1 or Kir6.2) of the inwardly rectifying K+ channel family and the regulatory subunits SURx (SUR1, SUR2A or SUR2B), the receptor of the sulphonylureas widely used in treatment of type 2 diabetes mellitus. Different combinations of Kir6.x and SURx comprise KATP channels with distinct electrophysiological and pharmacological properties, but their physiological functions in the various tissues are unclear. Our studies of Kir6.2 null (knockout) and Kir6.1 null mice have shown that KATP channels are critical metabolic sensors in protection against acute metabolic stress such as hyperglycaemia, hypoglycaemia, ischaemia and hypoxia.
Physiological modulation of inactivation in L‐type Ca2+ channels: one switch
Authors: Findlay Ian
Published: January 2004

The relative contributions of voltage‐ and Ca2+‐dependent mechanisms of inactivation to the decay of L‐type Ca2+ channel currents (ICaL) is an old story to which recent results have given an unexpected twist. In cardiac myocytes voltage‐dependent inactivation (VDI) was thought to be slow and Ca2+‐dependent inactivation (CDI) resulting from Ca2+ influx and Ca2+‐induced Ca2+‐release (CICR) from the sarcoplasmic reticulum provided an automatic negative feedback mechanism to limit Ca2+ entry and the contribution of ICaL to the cardiac action potential. Physiological modulation of ICaL by β‐adrenergic and muscarinic agonists then involved essentially more or less of the same by enhancing or reducing Ca2+ channel activity, Ca2+ influx, sarcoplasmic reticulum load and thus CDI. Recent results on the other hand place VDI at the centre of the regulation of ICaL. Under basal conditions it has been found that depolarization increases the probability that an ion channel will show rapid VDI. This is prevented by β‐adrenergic stimulation. Evidence also suggests that a channel which shows rapid VDI inactivates before CDI can become effective. Therefore the contributions of VDI and CDI to the decay of ICaL are determined by the turning on, by depolarization, and the turning off, by phosphorylation, of the mechanism of rapid VDI. The physiological implications of these ideas are that under basal conditions the contribution of ICaL to the action potential will be determined largely by voltage and by Ca2+ following β‐adrenergic stimulation.
Cell signal control of the G protein‐gated potassium channel and its subcellular localization
Authors: Kurachi Yoshihisa , Ishii Masaru
Published: January 2004

G protein‐gated inward rectifier K+ (KG) channels are directly activated by the βγ subunits released from pertussis toxin‐sensitive G proteins, and contribute to neurotransmitter‐induced deceleration of heart beat, formation of slow inhibitory postsynaptic potentials in neurones and inhibition of hormone release in endocrine cells. The physiological roles of KG channels are critically determined by mechanisms which regulate their activity and their subcellular localization. KG channels are tetramers of inward rectifier K+ (Kir) channel subunits, Kir3.x. The combination of Kir3.x subunits in each KG channel varies among tissues and cell types. Each subunit of the channel possesses one Gβγ binding site. The binding of Gβγ increases the number of functional KG channels via a mechanism that can be described by the Monod–Wyman–Changeux allosteric model. During voltage pulses KG channel current alters time dependently. The KG current exhibits inward rectification due to blockade of outward‐going current by intracellular Mg2+ and polyamines. Upon repolarization, this blockade is relieved practically instantaneously and then the current slowly increases further. This slow current alteration is called ‘relaxation’. Relaxation is caused by the voltage‐dependent behaviour of regulators of G protein signalling (RGS proteins), which accelerate intrinsic GTP hydrolysis mediated by the Gα subunit. Thus, the relaxation behaviour of KG channels reflects the time course with which the G protein cycle is altered by RGS protein activity at each membrane potential. Subcellular localization of KG channels is controlled by several distinct mechanisms, some of which have been recently clarified. The neuronal KG channel, which contains Kir3.2c, is localized in the postsynaptic density (PSD) of various neurones including dopaminergic neurones in substantia nigra. Its localization at PSD may be controlled by PDZ domain‐containing anchoring proteins. The KG channel in thyrotrophs is localized exclusively on secretary vesicles, which upon stimulation are rapidly inserted into the plasma membrane and causes hyperpolarization of the cell. This mechanism indicates a novel negative feedback regulation of exocytosis. In conclusion, KG channels are under the control of a variety of signalling molecules which regulate channel activity, subcellular localization and thus their physiological roles in myocytes, neurones and endocrine cells.
P2X3 receptors and peripheral pain mechanisms
Authors: North R. Alan
Published: January 2004

ATP released from damaged or inflamed tissues can act at P2X receptors expressed on primary afferent neurones. The resulting depolarization can initiate action potentials that are interpreted centrally as pain. P2X3 subunits are found in a subset of small‐diameter, primary afferent neurones, some of which are also sensitive to capsaicin. They can form homo‐oligomeric channels, or they can assemble with P2X2 subunits into hetero‐oligomers. Studies with antagonists selective for P2X3‐containing receptors, experiments with antisense oligonucleotides to reduce P2X3 subunit levels, and behavioural testing of P2X3 knock‐out mice, all suggest a role for the P2X2/3 receptor in the signalling of chronic inflammatory pain and some features of neuropathic pain. The availability of such tools and experimental approaches promises to accelerate our understanding of the other physiological roles for P2X receptors on primary afferent neurones.
Pharmacodynamics of S-3578, a Novel Cephem, in Murine Lung and Systemic Infection Models
Authors: Miyazaki Shuichi , Okazaki Kenichi , Tsuji Masakatsu , Yamaguchi Keizo
Published: January 2004

S-3578 is a novel beta-lactam with enhanced activity against drug-resistant gram-positive cocci such as methicillin-resistant
Staphylococcus aureus
(MRSA). We used murine penicillin-resistant
Streptococcus pneumoniae
lung infection and neutropenic murine systemic MRSA infection models to determine the pharmacokinetic (PK)-pharmacodynamic (PD) parameter that best correlated with efficacy. Pharmacokinetic studies revealed that the maximum concentration in serum/dose values for S-3578 and cefepime in plasma in the lung infection model were 1.21 to 1.54 and 0.97 to 1.29, respectively; those for S-3578 in plasma in the systemic infection model were 0.78 to 1.02. The area under the concentration-time curve (AUC)/dose values for S-3578 and cefepime in plasma in the lung infection model were 0.98 to 1.13 and 0.77 to 1.04, respectively, and those for S-3578 in plasma in the systemic infection model were 1.03 to 1.11. The half-lives of S-3578 and cefepime in plasma in the lung infection model were 0.29 to 0.38 and 0.29 to 0.34, respectively, and those of S-3578 in plasma in the systemic infection model were 0.40 to 0.61. The time above the MIC was the PK-PD parameter that best correlated with efficacy in the murine lung infection model (

R
2

= 84 and 92% for S-3578 and cefepime in plasma, respectively). There was a twofold increase in the dose of S-3578 in the systemic infection model compared to that in the pneumonia model, yet the AUCs were the same. This may be due to the different MICs for the two pathogens.
PBP 2a Mutations Producing Very-High-Level Resistance to Beta-Lactams
Authors: Katayama Yuki , Zhang Hong-Zhong , Chambers Henry F.
Published: January 2004

Resistance to the beta-lactam class of antibiotics in methicillin-resistant
Staphylococcus aureus
(MRSA) is mediated by PBP 2a, a synthetic bacterial cell wall penicillin-binding protein with a low affinity of binding to beta-lactams that is encoded by
mecA
. Beta-lactams that bind to PBP 2a with a high affinity and that are highly active against MRSA are under development. The potential for the emergence of resistance to such compounds was investigated by passage of homogeneous MRSA strain COL in L-695,256, an investigational carbapenem. A highly resistant mutant, COL52, expressed PBP 2a in which a two-amino-acid deletion mutation and three single-amino-acid substitution mutations were present. To examine the effects of these mutations on the resistance phenotype and PBP 2a production, plasmids carrying (i) PBP 2a with two or three of the four mutations, (ii) wild-type PBP 2a, or (iii) COL52 PBP 2a were introduced into methicillin-susceptible COL variants COLnex and COL52ex, from which the staphylococcus cassette chromosome
mec
(SCC
mec
) has been excised, as indicated by the “ex” suffix. Two amino acids substitutions, E→K
237
within the non-penicillin-binding domain and V→E
470
near the SDN
464
conserved penicillin-binding motif in the penicillin-binding domain in COL52, were important for high-level resistance. The highest level of resistance was observed when all four mutations were present. The emergence of PBP 2a-mediated resistance to beta-lactams that bind to PBP 2a with a high affinity is likely to require multiple mutations in
mecA
; chromosomal mutations appear to have a minor role.
Reemergence of Macrolide Resistance in Pharyngeal Isolates of Group A Streptococci in Southwestern Pennsylvania
Authors: Green Michael , Martin Judith M. , Barbadora Karen A. , Beall Bernard , Wald Ellen R.
Published: January 2004

We previously reported on the emergence of macrolide-resistant pharyngeal isolates of group A streptococci (GAS) in our community. The purpose of the present study was to track longitudinal trends in macrolide resistance in these isolates in southwestern Pennsylvania. Testing for susceptibility to erythromycin and clindamycin was performed for all pharyngeal GAS isolates recovered at the Children's Hospital of Pittsburgh and a local pediatric practice between September 2001 and May 2002. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion and PCR, respectively. Strain relatedness was determined by field inversion gel electrophoresis and
emm
gene sequence typing. A total of 708 isolates of GAS were recovered during the study period; 68 (9.6%) were macrolide resistant, while all isolates were sensitive to clindamycin. The monthly prevalence of macrolide resistance ranged from 0 to 41%. Only 21 of 573 (3.7%) strains recovered from September 2001 through March 2002 were macrolide resistant. A sudden increase in the rate of macrolide resistance (47 of 135 isolates [35%]) was seen in April and May 2002. Sixty-two isolates demonstrated the M phenotype (resistance to macrolide antibiotics), and six isolates demonstrated the MLS
B
phenotype (resistance to most macrolide, lincosamide, and streptogramin B antibiotics); these isolates were confirmed to be
mef
(A) and
erm
(A), respectively. Three unique
mef
(A) clones and four unique
erm
(A) clones were identified among the resistant isolates. The MIC at which 50% of isolates are inhibited (MIC
50
) for the
mef
(A) strains was 16 μg/ml, while the MIC
50
for
erm
(A) strains was 8 μg/ml. The finding of high levels of macrolide resistance among pharyngeal isolates of GAS for a second successive year in our community raises the concern that this problem may be more common in the United States than was previously appreciated. Longitudinal surveillance of isolates from multiple centers is needed to define the prevalence of antimicrobial agent-resistant GAS in the United States.
Mechanism of Action of NB2001 and NB2030, Novel Antibacterial Agents Activated by β-Lactamases
Authors: Stone Geoffrey W. , Zhang Qin , Castillo Rosario , Doppalapudi V. Ramana , Bueno Analia R. , Lee Jean Y. , Li Qing , Sergeeva Maria , Khambatta Gody , Georgopapadakou Nafsika H.
Published: January 2004

Two potent antibacterial agents designed to undergo enzyme-catalyzed therapeutic activation were evaluated for their mechanisms of action. The compounds, NB2001 and NB2030, contain a cephalosporin with a thienyl (NB2001) or a tetrazole (NB2030) ring at the C-7 position and are linked to the antibacterial triclosan at the C-3 position. The compounds exploit β-lactamases to release triclosan through hydrolysis of the β-lactam ring. Like cephalothin, NB2001 and NB2030 were hydrolyzed by class A β-lactamases (
Escherichia coli
TEM-1 and, to a lesser degree,
Staphylococcus aureus
PC1) and class C β-lactamases (
Enterobacter cloacae
P99 and
E. coli
AmpC) with comparable catalytic efficiencies (
k
cat
/

K
m

). They also bound to the penicillin-binding proteins of
S. aureus
and
E. coli
, but with reduced affinities relative to that of cephalothin. Accordingly, they produced a cell morphology in
E. coli
consistent with the toxophore rather than the β-lactam being responsible for antibacterial activity. In biochemical assays, they inhibited the triclosan target enoyl reductase (FabI), with 50% inhibitory concentrations being markedly reduced relative to that of free triclosan. The transport of NB2001, NB2030, and triclosan was rapid, with significant accumulation of triclosan in both
S. aureus
and
E. coli
. Taken together, the results suggest that NB2001 and NB2030 act primarily as triclosan prodrugs in
S. aureus
and
E. coli
.
Enhanced Saquinavir Exposure in Human Immunodeficiency Virus Type 1-Infected Patients with Diarrhea and/or Wasting Syndrome
Authors: Trout Hervé , Mentré France , Panhard Xavière , Kodjo Alissi , Escaut Lélia , Pernet Pascal , Gobert Jean-Gérard , Vittecoq Daniel , Knellwolf Anne-Laure , Caulin Charles , Bergmann Jean-François
Published: January 2004

The protease inhibitor saquinavir was administered to 100 human immunodeficiency virus type 1 (HIV-1)-infected patients as a single 600-mg oral dose (hard gelatin capsules) with a standard breakfast, including 200 ml of grapefruit juice, during an open-label trial to assess whether diarrhea and/or wasting syndrome has consequences on its pharmacokinetics. Three groups of patients were enrolled: group 1, asymptomatic patients (
n
= 30); group 2, AIDS symptomatic patients without body weight loss or diarrhea (
n
= 37); and group 3, AIDS symptomatic patients with severe body weight loss and/or diarrhea (
n
= 33). Clinical and biological data (covariates) were collected. A population approach was performed with three blood samples per patient to estimate the mean population pharmacokinetic parameters (clearance [CL]/oral bioavailability [F],
V
/F,
k
a
, and lag time) and the derived ones (
k
el
,
C
max
,
T
max
, and area under the curve [AUC]). The relationships between groups, exposure (i.e., estimated individual post hoc AUCs), and covariates were explored by using multiple linear regressions. A significant increase in median AUCs (165, 349, and 705 ng · h · ml
−1
for groups 1, 2, and 3, respectively [
P
< 0.0001]) was observed. The enhancement in saquinavir exposure could be due to the destruction of the transporters in enterocytes and/or to the enlargement of their tight junctions, allowing a paracellular crossing of saquinavir as the illness spreads. Because of grapefruit juice intake by every patient, no implication of CYP3A4 could be assessed. These results strongly suggest that, despite its low intrinsic oral bioavailability, saquinavir can be considered as a relevant treatment for HIV-1-infected patients with diarrhea and/or wasting syndrome. This must be evaluated in a long-term period.
Subinhibitory Concentrations of Linezolid Reduce
Staphylococcus aureus
Virulence Factor Expression
Authors: Bernardo Katussevani , Pakulat Norbert , Fleer Silke , Schnaith Annabelle , Utermöhlen Olaf , Krut Oleg , Müller Stefan , Krönke Martin
Published: January 2004

The influence of the antibiotic linezolid on the secretion of exotoxins by
Staphylococcus aureus
was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry and Western blot analysis.
S. aureus
suspensions were treated with grading subinhibitory concentrations of linezolid (12.5, 25, 50, and 90% of MIC) at different stages of bacterial growth (i.e., an optical density at 540 nm [OD
540
] of 0.05 or 0.8). When added to
S. aureus
cultures at an OD
540
of 0.05, linezolid reduced in a dose-dependent manner the secretion of specific virulence factors, including staphylococcal enterotoxin A (SEA) and SEB, bifunctional autolysin, autolysin, protein A, and alpha- and beta-hemolysins. In contrast, other presumably nontoxic exoproteins remained unchanged or even accumulated in supernatants in the presence of linezolid at a 90% MIC. Similarily, when added at OD
540
of 0.8, that is, after quorum sensing, linezolid reduced the release of virulence factors, whereas the relative abundance of nontoxic exoproteins such as triacylglycerol lipase, glycerol ester hydrolase, DnaK, or translation elongation factor EF-Tu was found to be increased. Consistently, linezolid reduced in a dose-dependent manner the tumor necrosis factor-inducing activity secreted by
S. aureus
into the culture supernatants. The results of our study suggest that the expression of virulence factors in
S. aureus
is especially sensitive to the inhibition of protein synthesis by linezolid, which should be an advantage in the treatment of infections with toxin-producing
S. aureus
.
Functional Angucycline-Like Antibiotic Gene Cluster in the Terminal Inverted Repeats of the
Streptomyces ambofaciens
Linear Chromosome
Authors: Pang Xiuhua , Aigle Bertrand , Girardet Jean-Michel , Mangenot Sophie , Pernodet Jean-Luc , Decaris Bernard , Leblond Pierre
Published: January 2004

Streptomyces ambofaciens
has an 8-Mb linear chromosome ending in 200-kb terminal inverted repeats. Analysis of the F6 cosmid overlapping the terminal inverted repeats revealed a locus similar to type II polyketide synthase (PKS) gene clusters. Sequence analysis identified 26 open reading frames, including genes encoding the β-ketoacyl synthase (KS), chain length factor (CLF), and acyl carrier protein (ACP) that make up the minimal PKS. These KS, CLF, and ACP subunits are highly homologous to minimal PKS subunits involved in the biosynthesis of angucycline antibiotics. The genes encoding the KS and ACP subunits are transcribed constitutively but show a remarkable increase in expression after entering transition phase. Five genes, including those encoding the minimal PKS, were replaced by resistance markers to generate single and double mutants (replacement in one and both terminal inverted repeats). Double mutants were unable to produce either diffusible orange pigment or antibacterial activity against
Bacillus subtilis
. Single mutants showed an intermediate phenotype, suggesting that each copy of the cluster was functional. Transformation of double mutants with a conjugative and integrative form of F6 partially restored both phenotypes. The pigmented and antibacterial compounds were shown to be two distinct molecules produced from the same biosynthetic pathway. High-pressure liquid chromatography analysis of culture extracts from wild-type and double mutants revealed a peak with an associated bioactivity that was absent from the mutants. Two additional genes encoding KS and CLF were present in the cluster. However, disruption of the second KS gene had no effect on either pigment or antibiotic production.
Genetic Environment and Transcription of
ampC
in an
Acinetobacter baumannii
Clinical Isolate
Authors: Segal Heidi , Nelson E. C. , Elisha B. Gay
Published: January 2004

An
ampC
gene was cloned from a clinical isolate of
Acinetobacter baumannii
(strain RAN). DNA sequencing and primer extension studies showed that
ampC
is transcribed from a promoter contained within a putative insertion sequence element which has been found to abut several different genes in
Acinetobacter
spp.
Penetration of Linezolid into the Anterior Chamber (Aqueous Humor) of the Human Eye after Intravenous Administration
Authors: Vázquez E. García , Mensa J. , López Y. , Couchard P. Díaz , Soy D. , Fontenla J. R. , Sarasa M. , Carné X. , Montull E.
Published: January 2004

We determined the linezolid concentrations in serum samples and aqueous humors (AHs) from 21 patients undergoing cataract extraction. Cataract removal was performed at various times (from 60 to 270 min) after the end of a 30-min infusion of 600 mg of linezolid. Serum samples were obtained 1 h after the end of linezolid administration to determine the maximum concentration of linezolid (Cmax); AHs and a second serum sample were taken simultaneously during the operation, and the concentrations of linezolid in AH (CAH) and serum (CS) were determined. The mean CAH1 h after linezolid administration was 4.94 μg/ml, and the mean ratio of CAHto CS(R= CAH/CS) was 0.43. All patients had a CAHof >2 μg/ml, which was higher than the MIC at which 90% ofStaphylococcus epidermidisstrains are inhibited.
Antimicrobial Activity and Bacterial-Membrane Interaction of Ovine-Derived Cathelicidins
Authors: Anderson Rachel C. , Hancock Robert E. W. , Yu Pak-Lam
Published: January 2004

Three ovine-derived cathelicidins, SMAP29, OaBac5mini, and OaBac7.5mini, were compared with respect to their antibacterial activities and interactions with membranes. SMAP29 was confirmed to be α-helical, broad spectrum, and able to disrupt both the outer and the cytoplasmic membranes at relatively low concentrations. In contrast, the two proline- and arginine-rich OaBac peptides had more-modest antibacterial activities, reduced levels of lipopolysaccharide binding, and a lesser ability to depolarize the cytoplasmic membrane, consistent with a cytoplasmic target.
Telithromycin Susceptibility and Genomic Diversity of Macrolide-Resistant Serotype III Group B Streptococci Isolated in Perinatal Infections
Authors: Bingen Edouard , Doit Catherine , Bidet Philippe , Brahimi Naima , Deforche Dominique
Published: January 2004

We studied the telithromycin, erythromycin, azithromycin, and clindamycin susceptibilities of serotype III macrolide-resistant group B streptococci, together with genetic mechanisms of resistance and genomic diversity.
ermB
,
ermA
, and
mefA
were found in, respectively, 57, 32, and 9% of isolates. The telithromycin MIC at which 90% of isolates were inhibited was 0.5 μg/ml. Macrolide resistance was associated with dissemination of resistance determinants among isolates of different genetic backgrounds.
The Ferritin-Like Dps Protein Is Required for
Salmonella enterica
Serovar Typhimurium Oxidative Stress Resistance and Virulence
Authors: Halsey Thomas A. , Vazquez-Torres Andrés , Gravdahl Daniel J. , Fang Ferric C. , Libby Stephen J.
Published: January 2004

Resistance to phagocyte-derived reactive oxygen species is essential for
Salmonella enterica
serovar Typhimurium pathogenesis.
Salmonella
can enhance its resistance to oxidants through the induction of specific genetic pathways controlled by SoxRS, OxyR, σ
S
, σ
E
, SlyA, and RecA. These regulons can be found in a wide variety of pathogenic and environmental bacteria, suggesting that evolutionarily conserved mechanisms defend against oxidative stress both endogenously generated by aerobic respiration and exogenously produced by host phagocytic cells. Dps, a ferritin-like protein found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress by sequestering iron and limiting Fenton-catalyzed oxyradical formation. In
Escherichia coli
and some other bacterial species, Dps has been shown to accumulate during stationary phase in a σ
S
-dependent fashion, bind nonspecifically to DNA, and form a crystalline structure that compacts and protects chromatin from oxidative damage. In the present study, we provide evidence that Dps protects
Salmonella
from iron-dependent killing by hydrogen peroxide, promotes
Salmonella
survival in murine macrophages, and enhances
Salmonella
virulence. Reduced numbers of
dps
mutant bacteria in the livers and spleens of infected mice are consistent with a role of Dps in protecting
Salmonella
from oxidative stress encountered during infection.
A K
+
Uptake Protein, TrkA, Is Required for Serum, Protamine, and Polymyxin B Resistance in
Vibrio vulnificus
Authors: Chen Yu-Chung , Chuang Yin-Ching , Chang Chun-Chin , Jeang Chii-Ling , Chang Ming-Chung
Published: January 2004

Vibrio vulnificus
, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the world. To identify the genes required for resistance to human serum, we constructed a library of transposon mutants of
V. vulnificus
and screened them for hypersensitivity to human serum. Here we report that one of the isolated serum-susceptible mutants had a mutation in an open reading frame identified as
trkA
, a gene encoding an amino acid sequence showing high identity to that of TrkA of
Vibrio alginolyticus
, a protein required for the uptake of potassium. A
trkA
isogenic mutant was constructed via insertional inactivation, and it was significantly more easily killed by human serum, protamine, or polymyxin B than was the wild type. At K
+
concentrations of 1 to 20 mM, this isogenic mutant showed attenuated growth compared to the wild-type strain. In addition, infection experiments demonstrated virulence attenuation when this mutant was administered intraperitoneally or subcutaneously to both normal and iron-treated mice, indicating that TrkA may modulate the transport of potassium and resistance to host innate defenses and that it is important for virulence in mice.
Sequence Analysis ofp44Homologs Expressed byAnaplasma phagocytophilumin Infected Ticks Feeding on Naive Hosts and in Mice Infected by Tick Attachment
Authors: Felek Suleyman , Telford Sam , Falco Richard C. , Rikihisa Yasuko
Published: January 2004

The 44-kDa immunodominant outer membrane proteins (P44 proteins) ofAnaplasma phagocytophilumare encoded by thep44polymorphic multigene family. The present study examinedp44expression and analyzed the cDNA sequences of variousp44transcripts from the spleens and blood of mice infected by the bites of ticks infected with theA. phagocytophilumNTN-1 strain or of naturally infected nymphal ticks and in the salivary glands and midgut tissues of these ticks. A total of 300p44cDNAs were subjected to sequence analysis. Of these, 40 distinctp44species were found, and all of these had orthologs in theA. phagocytophilumHZ strain genome that shared 95 to 100% base sequence identity. The number of uniquep44species expressed in mouse blood was greater than that for mouse spleens. Higher numbers of differentp44transcripts were also expressed in the salivary glands of ticks than in the midgut tissues. Variations in the sequences of the samep44cDNA species within a singleA. phagocytophilumstrain and among different strains were concentrated in the conserved regions flanking the central hypervariable region ofp44genes. No mosaic sequences derived from two or morep44species were found within thep44hypervariable region. The conservation of the hypervariable region of eachp44cDNA species ofA. phagocytophilumin naturally infected ticks and in different geographic isolates suggests that eachA. phagocytophilumgenome carries a set ofp44paralogs to be expressed. Thus, a large but restricted repertoire ofp44hypervariable sequences exists inA. phagocytophilumstrains in the Northeastern United States.
Expression of Amoebapores Is Required for Full Expression ofEntamoeba histolyticaVirulence in Amebic Liver Abscess but Is Not Necessary for the Induction of Inflammation or Tissue Damage in Amebic Colitis
Authors: Zhang Xiaochun , Zhang Zhi , Alexander Diane , Bracha Rivka , Mirelman David , Stanley Samuel L.
Published: January 2004

Entamoeba histolyticatrophozoites produce amoebapores, a family of small amphipathic peptides capable of insertion into bacterial or eukaryotic membranes and causing cellular lysis. Recently,E. histolyticatrophozoites that are totally deficient in the production of amoebapore-A were created through a gene silencing mechanism (R. Bracha, Y. Nuchamowitz, and D. Mirelman, Eukaryot. Cell 2:295-305, 2003). Here we tested the virulence of amoebapore A(−) trophozoites in models of the two major forms of amebic disease: amebic liver abscess and amebic colitis. We demonstrate that amoebapore expression is required for full virulence in the SCID mouse model of amebic liver abscess, butE. histolyticatrophozoites that do not express amoebapore-A can still cause inflammation and tissue damage in infected human colonic xenografts. These data are consistent with the concept that tissue damage may proceed by different mechanisms in amebic liver abscess compared to amebic colitis.
Sensitized Splenocytes Result in Deleterious Cytokine Cascade and Hyperinflammatory Response in Rats withPneumocystisPneumonia despite the Presence of Corticosteroids
Authors: Thullen Timothy D. , Ashbaugh Alan D. , Daly Kieran R. , Linke Michael J. , Steele Paul E. , Walzer Peter D.
Published: January 2004

The immune response to the opportunistic pulmonary pathogenPneumocystiscan have beneficial and harmful effects on the host despite the presence of corticosteroids. We hypothesized that this deleterious hyperinflammatory response is associated with exaggerated cytokine production. The adoptive transfer of at least 107immune splenocytes reduced the cyst count in rats with corticosteroid-induced pneumocystosis. About 18% of these rats developed clinical illness, an increased lung weight/body weight (LW/BW) ratio, and elevated levels of interleukin 1α (IL-1α), IL-1β, IL-6, tumor necrosis factor alpha, IL-5, IL-10, and gamma interferon in the lungs. This hyperinflammatory reaction was not observed in rats that remained clinically well or in control rats. Thus, in this model, corticosteroids have little effect on the cytokine cascade or other adverse effects of the host immune response toPneumocystis.
Investigation of a Novel DNase of
Streptococcus suis
Serotype 2
Authors: Fontaine Michael C. , Perez-Casal Jose , Willson Philip J.
Published: January 2004

A secreted nuclease, SsnA, was identified in the virulent
Streptococcus suis
isolate SX332 and subsequently in each of the type strains of capsular serotypes 1 through 9. Screening of 258 porcine clinical isolates from surface (nasal mucosa or palatine tonsil) or internal (joint, brain or other internal organ) locations revealed a significant relationship (
P
< 0.001) between expression of nuclease and isolation from an internal site. A 3,126-bp gene,
ssnA
, was identified from a phenotypically nuclease-negative pGh9:IS
S1
insertion mutant, and analysis of the predicted SsnA sequence revealed a 35-amino-acid (aa) secretion signal sequence, a 22-aa DNA-binding domain, and a typical gram-positive cell wall sorting motif. A requirement of Ca
2+
and Mg
2+
for SsnA activity was determined, and the substrate specificity was found to be for single- and double-stranded linear DNA. Reverse transcription-PCR experiments revealed that
ssnA
is expressed throughout all stages of
S. suis
growth, and Western blots with porcine anti-
S. suis
immune sera against a recombinant, truncated SsnA derivative (rSsnA
Δ
) confirmed that SsnA is expressed in vivo. Furthermore, anti-rSsnA
Δ
antibodies were sufficient to neutralize SsnA activity. Analyses of subcellular fractions of SX332 and derived mutants, on DNA-containing polyacrylamide gels and by Western blotting, suggest that SsnA is cell wall located.
Salivary Statherin Peptide-Binding Epitopes of Commensal and Potentially Infectious
Actinomyces
spp. Delineated by a Hybrid Peptide Construct
Authors: Niemi Liza Danielsson , Johansson Ingegerd
Published: January 2004

Adhesion of microorganisms to host receptor molecules such as salivary statherin molecules is a common event in oral microbial colonization. Here we used a hybrid peptide construct (with both a hydroxyapatite-binding portion and a test peptide portion) to map the interaction of
Actinomyces
species (and
Candida albicans
) with statherin. Adhesion to hybrid peptides and truncated statherin variants revealed three binding types, types I to III. (i) Type I strains of rat, hamster, and human infection origins bound C-terminal-derived QQYTF and PYQPQY peptides. The QQYTF peptide inhibited statherin binding for some strains but not for others. (ii) Type II strains of human and monkey tooth origins bound middle-region-derived YQPVPE and QPLYPQ peptides. Neither strain was inhibited by soluble peptides. (iii) Type III strains of human infection origins (and
C. albicans
) did not bind to either statherin-derived peptides or truncated statherin. Moreover, the type I strains inhibited by QQYTF were also inhibited by TF and QAATF peptides and were detached from statherin by the same peptides. In conclusion, it is suggested that commensal and potentially infectious microorganisms bind middle or C-terminal statherin differently and that other microbes might require discontinuous epitopes.
Role of theSalmonellaPathogenicity Island 1 Effector Proteins SipA, SopB, SopE, and SopE2 inSalmonella entericaSubspecies 1 Serovar Typhimurium Colitis in Streptomycin-Pretreated Mice
Authors: Hapfelmeier Siegfried , Ehrbar Kristin , Stecher Bärbel , Barthel Manja , Kremer Marcus , Hardt Wolf-Dietrich
Published: January 2004

Salmonella entericasubspecies 1 serovar Typhimurium (serovar Typhimurium) induces enterocolitis in humans and cattle. The mechanisms of enteric salmonellosis have been studied most extensively in calf infection models. The previous studies established that effector protein translocation into host cells via theSalmonellapathogenicity island 1 (SPI-1) type III secretion system (TTSS) is of central importance in serovar Typhimurium enterocolitis. We recently found that orally streptomycin-pretreated mice provide an alternative model for serovar Typhimurium colitis. In this model the SPI-1 TTSS also plays a key role in the elicitation of intestinal inflammation. However, whether intestinal inflammation in calves and intestinal inflammation in streptomycin-pretreated mice are induced by the same SPI-1 effector proteins is still unclear. Therefore, we analyzed the role of the SPI-1 effector proteins SopB/SigD, SopE, SopE2, and SipA/SspA in elicitation of intestinal inflammation in the murine model. We found thatsipA,sopE, and, to a lesser degree,sopE2contribute to murine colitis, but we could not assign an inflammation phenotype tosopB. These findings are in line with previous studies performed with orally infected calves. Extending these observations, we demonstrated that in addition to SipA, SopE and SopE2 can induce intestinal inflammation independent of each other and in the absence of SopB. In conclusion, our data corroborate the finding that streptomycin-pretreated mice provide a useful model for studying the molecular mechanisms of serovar Typhimurium colitis and are an important starting point for analysis of the molecular events triggered by SopE, SopE2, and SipA in vivo.
Characterization of Immunoglobulin G and Its Subclass Response to Indian Kala-Azar Infection before and after Chemotherapy
Authors: Ravindran Rajesh , Anam Khairul , Bairagi Bibhas C. , Saha Bibhuti , Pramanik Netai , Guha Subhasis K. , Goswami Rama P. , Banerjee Dwijadas , Ali Nahid
Published: January 2004

Serologic parameters of kala-azar were evaluated by Western blot analysis. Sera from kala-azar patients with confirmed diagnoses were screened for immunoglobulin G (IgG) and IgG subclass-specific reactivity againstLeishmania donovanimembrane antigen (LAg). Heterogenous LAg-specific IgG reactivity with numerous proteins with molecular masses ranging from 18 to 190 kDa was observed. Though the individual band patterns were varied, seven polypeptides of approximately 31, 34, 51, 63, 72, 91, and 120 kDa were immunoreactive with all the sera tested from kala-azar patients. The band patterns of the immunoblots of sera from patients after treatment and clinical cure with sodium antimony gluconate revealed a decrease in the frequency of the bands. Still, recognition of the 63- and 120-kDa bands was 100%, and the 55- and 91-kDa fractions were recognized in 93% of the sera from cured individuals. Among the IgG subclasses, IgG1 reacted with the greatest number of polypeptides. The 63-kDa protein was again detected by all of the IgG subclasses of all the sera tested. Other fractions recognized by the subclasses of more than 70% of the serum samples included those of 47, 51, 55, and 78 kDa. Following treatment, 63- and 51-kDa bands were the most reactive with the IgG subclasses. LAg-associated cross-reaction with other reference human antisera revealed a mild reactivity of the 63-kDa polypeptide with some of the serum samples from leprosy, malaria, typhoid, tuberculosis, and healthy controls. Western blot analysis of LAg entrapped in liposomes, strong vaccine candidates against experimental visceral leishmaniasis, revealed a more restricted band pattern. The 63-kDa fraction revealed by all pre- and posttreatment sera showed almost negligible levels of cross-reaction with sera from patients with other diseases or from healthy controls. These observations provide insight into induced immunity during kala-azar infection for future application.
CpG Oligodeoxynucleotide and Montanide ISA 51 Adjuvant Combination Enhanced the Protective Efficacy of a Subunit Malaria Vaccine
Authors: Kumar Sanjai , Jones Trevor R. , Oakley Miranda S. , Zheng Hong , Kuppusamy Shanmuga P. , Taye Alem , Krieg Arthur M. , Stowers Anthony W. , Kaslow David C. , Hoffman Stephen L.
Published: January 2004

Unmethylated CpG dinucleotide motifs present in bacterial genomes or synthetic oligodeoxynucleotides (ODNs) serve as strong immunostimulatory agents in mice, monkeys and humans. We determined the adjuvant effect of murine CpG ODN 1826 on the immunogenicity and protective efficacy of theSaccharomyces cerevisiae-expressed 19-kDa C-terminal region of merozoite surface protein 1 (yMSP119) of the murine malaria parasitePlasmodium yoelii. We found that in C57BL/6 mice, following sporozoite challenge, the degree of protective immunity against malaria induced by yMSP119in a formulation of Montanide ISA 51 (ISA) plus CpG ODN 1826 was similar or superior to that conferred by yMSP119emulsified in complete Freund's adjuvant (CFA/incomplete Freund's adjuvant). In total, among mice immunized with yMSP119, 22 of 32 (68.7%) with ISA plus CpG 1826, 0 of 4 (0%) with CFA/incomplete Freund’s adjuvant, 0 of 4 (0%) with CpG 1826 mixed with ISA (no yMSP119), and 0 of 11 (0%) with CpG 1826 alone were completely protected against development of erythrocytic stage infection after sporozoite challenge. The adjuvant effect of CpG ODN 1826 was manifested as both significantly improved complete protection from malaria (defined as the absence of detectable erythrocytic form parasites) (P= 0.007, chi square) and reduced parasite burden in infected mice. In vivo depletions of interleukin-12 and gamma interferon cytokines and CD4+and CD8+T cells in vaccinated mice had no significant effect on immunity. On the other hand, immunoglobulin G (IgG) isotype levels appeared to correlate with protection. Inclusion of CpG ODN 1826 in the yMSP119plus ISA vaccine contributed towards the induction of higher levels of IgG2a and IgG2b (Th1 type) antibodies, suggesting that CpG ODN 1826 caused a shift towards a Th1 type of immune response that could be responsible for the higher degree of protective immunity. Our results indicate that this potent adjuvant formulation should be further evaluated for use in clinical trials of recombinant malarial vaccine candidates.