bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-Acting Neuraminidase Inhibitor Laninamivir Octanoate (CS-8958) versus Oseltamivir as Treatment for Children with Influenza Virus Infection
Authors: Sugaya Norio , Ohashi Yasuo
Published: April 2010

We conducted a double-blind, randomized controlled trial to compare a long-acting neuraminidase inhibitor, laninamivir octanoate, with oseltamivir. Eligible patients were children 9 years of age and under who had febrile influenza symptoms of no more than 36-h duration. Patients were randomized to 1 of 3 treatment groups: a group given 40 mg laninamivir (40-mg group), a group given 20 mg laninamivir (20-mg group), and an oseltamivir group. Laninamivir octanoate was administered as a single inhalation. Oseltamivir (2 mg/kg of body weight) was administered orally twice daily for 5 days. The primary end point was the time to alleviation of influenza illness. The primary analysis included 184 patients (61, 61, and 62 in the 40-mg group, 20-mg group, and oseltamivir group, respectively). Laninamivir octanoate markedly reduced the median time to illness alleviation in comparison with oseltamivir in patients infected with oseltamivir-resistant influenza A (H1N1) virus, and the reductions were 60.9 h for the 40-mg group and 66.2 h for the 20-mg group. On the other hand, there were no significant differences in the times to alleviation of illness between the laninamivir groups and oseltamivir group for patients with influenza A (H3N2) or B virus infection. Laninamivir octanoate was well tolerated. The most common adverse events were gastrointestinal events. Laninamivir octanoate was an effective and well-tolerated treatment for children with oseltamivir-resistant influenza A (H1N1) virus infection. Further study will be needed to confirm clinical efficacy against influenza A (H3N2) or B virus infection. Its ease of administration is noteworthy, because a single inhalation is required during the course of illness.
In Vitro
Double and Triple Bactericidal Activities of Doripenem, Polymyxin B, and Rifampin against Multidrug-Resistant
Acinetobacter baumannii
,
Pseudomonas aeruginosa
,
Klebsiella pneumoniae
, and
Escherichia coli
Authors: Urban Carl , Mariano Noriel , Rahal James J.
Published: April 2010

In vitro
double and triple bactericidal activities of doripenem, polymyxin B, and rifampin were assessed against 20 carbapenem-resistant clinical isolates with different mechanisms of carbapenem resistance. Bactericidal activity was achieved in 90% of all bacteria assayed using combinations of polymyxin B, doripenem, and rifampin against five each of the carbapenem-resistant
Klebsiella pneumoniae
,
Acinetobacter baumannii
,
Pseudomonas aeruginosa
, and
Escherichia coli
isolates studied. Combinations with these antibacterials may provide a strategy for treatment of patients infected with such organisms.
Antimicrobial Susceptibilities of a Worldwide Collection of
Stenotrophomonas maltophilia
Isolates Tested against Tigecycline and Agents Commonly Used for
S. maltophilia
Infections
Authors: Farrell David J. , Sader Helio S. , Jones Ronald N.
Published: April 2010

Antimicrobial susceptibilities were determined for 1,586 isolates of
Stenotrophomonas maltophilia
from globally diverse medical centers using the Clinical Laboratory Standards Institute broth microdilution method. The combination trimethoprim-sulfamethoxazole (96.0% of isolates susceptible at ≤2 μg/ml trimethoprim and 38 μg/ml sulfamethoxazole) and tigecycline (95.5% of isolates sussceptible at ≤2 μg/ml) were the only antimicrobials tested with >94% susceptibility in all regions. Susceptibility rates for other commonly used were lower than expected and varied geographically. This
in vitro
data supports tigecycline as a potential candidate for clinical investigations into
S. maltophilia
infections.
Altered Growth, Pigmentation, and Antimicrobial Susceptibility Properties of
Staphylococcus
aureus
Due to Loss of the Major Cold Shock Gene
cspB
Authors: Duval Brea D. , Mathew Anselmo , Satola Sarah W. , Shafer William M.
Published: April 2010

An insertional mutation made in the major cold shock gene
cspB
in
Staphylococcus
aureus
strain COL, a methicillin-resistant clinical isolate, yielded a mutant that displayed a reduced capacity to respond to cold shock and many phenotypic characteristics of
S
.
aureus
small-colony variants: a growth defect at 37°C, a reduction in pigmentation, and altered levels of susceptibility to many antimicrobials. In particular, a
cspB
null mutant displayed increased resistance to aminoglycosides, trimethoprim-sulfamethoxazole, and paraquat and increased susceptibility to daptomycin, teicoplanin, and methicillin. With the exception of the increased susceptibility to methicillin, which was due to a complete loss of the type I staphylococcal cassette chromosome
mec
element, these properties were restored to wild-type levels by complementation when
cspB
was expressed in
trans
. Taken together, our results link a stress response protein (CspB) of
S
.
aureus
to important phenotypic properties that include resistance to certain antimicrobials.
Characterization of V36C, a Novel Amino Acid Substitution Conferring Hepatitis C Virus (HCV) Resistance to Telaprevir, a Potent Peptidomimetic Inhibitor of HCV Protease
Authors: Barbotte Laetitia , Ahmed-Belkacem Abdelhakim , Chevaliez Stéphane , Soulier Alexandre , Hézode Christophe , Wajcman Henri , Bartels Doug J. , Zhou Yi , Ardzinski Andrzej , Mani Nagraj , Rao B. Govinda , George Shelley , Kwong Ann , Pawlotsky Jean-Michel
Published: April 2010

We characterized a novel substitution conferring moderate resistance to telaprevir, a peptidomimetic inhibitor of hepatitis C virus protease. V36C conferred a 4.0-fold increase in the telaprevir 50% inhibitory concentration in an enzyme assay and a 9.5-fold increase in the replicon model. The replication capacity of a replicon harboring V36C was close to that of the wild-type protease. This case emphasizes the complexity of hepatitis C virus resistance to protease inhibitors.
Effects of Plant Genotype and Growth Stage on the Betaproteobacterial Communities Associated with Different Potato Cultivars in Two Fields
Authors: İnceoğlu Özgül , Salles Joana Falcão , van Overbeek Leo , van Elsas Jan Dirk
Published: April 2010

Bacterial communities in the rhizosphere are dynamic and susceptible to changes in plant conditions. Among the bacteria, the betaproteobacteria play key roles in nutrient cycling and plant growth promotion, and hence the dynamics of their community structures in the rhizosphere should be investigated. Here, the effects of plant cultivar, growth stage, and soil type on the communities associated with potato cultivars Aveka, Aventra, Karnico, Modena, Premiere, and Désirée were assessed for two different fields containing sandy soil with either a high or low organic compound content. Thus, bacterial and betaproteobacterial PCR-denaturing gradient gel electrophoresis analyses were performed to analyze the effects of plant cultivar and growth on the rhizosphere community structure. The analyses showed that in both fields all cultivars had a rhizosphere effect on the total bacterial and betaproteobacterial communities. In addition, the plant growth stage strongly affected the betaproteobacterial communities in both fields. Moreover, the community structures were affected by cultivar, and cultivars differed in physiology, as reflected in their growth rates, root development, and estimated tuber starch contents. Analyses of betaproteobacterial clone libraries constructed for two selected cultivars (one cultivar that produced low-starch-content tubers and one cultivar that produced high-starch-content tubers), as well as bulk soil, revealed that the rhizospheres of the two cultivars selected for specific bacteria, including plant-growth-promoting bacteria, such as
Variovorax
and
Achromobacter
spp. In addition, quantitative PCR-based quantification of the
Variovorax paradoxus
-specific functional gene
asfA
(involved in desulfonation) indicated that there were clear potato rhizosphere effects on the abundance of this gene. Interestingly, both cultivar type and plant growth stage affected the community under some circumstances.
Double-Blind Characterization of Non-Genome-Sequenced Bacteria by Mass Spectrometry-Based Proteomics
Authors: Jabbour Rabih E. , Deshpande Samir V. , Wade Mary Margaret , Stanford Michael F. , Wick Charles H. , Zulich Alan W. , Skowronski Evan W. , Snyder A. Peter
Published: April 2010

Due to the possibility of a biothreat attack on civilian or military installations, a need exists for technologies that can detect and accurately identify pathogens in a near-real-time approach. One technology potentially capable of meeting these needs is a high-throughput mass spectrometry (MS)-based proteomic approach. This approach utilizes the knowledge of amino acid sequences of peptides derived from the proteolysis of proteins as a basis for reliable bacterial identification. To evaluate this approach, the tryptic digest peptides generated from double-blind biological samples containing either a single bacterium or a mixture of bacteria were analyzed using liquid chromatography-tandem mass spectrometry. Bioinformatic tools that provide bacterial classification were used to evaluate the proteomic approach. Results showed that bacteria in all of the double-blind samples were accurately identified with no false-positive assignment. The MS proteomic approach showed strain-level discrimination for the various bacteria employed. The approach also characterized double-blind bacterial samples to the respective genus, species, and strain levels when the experimental organism was not in the database due to its genome not having been sequenced. One experimental sample did not have its genome sequenced, and the peptide experimental record was added to the virtual bacterial proteome database. A replicate analysis identified the sample to the peptide experimental record stored in the database. The MS proteomic approach proved capable of identifying and classifying organisms within a microbial mixture.
Involvement of the Mannose Phosphotransferase System of
Lactobacillus plantarum
WCFS1 in Peroxide Stress Tolerance
Authors: Stevens Marc J. A. , Molenaar Douwe , de Jong Anne , de Vos Willem M. , Kleerebezem Michiel
Published: April 2010

A
Lactobacillus plantarum
strain with a deletion in the gene
rpoN
, encoding the alternative sigma factor 54 (σ
54
), displayed a 100-fold-higher sensitivity to peroxide than its parental strain. This feature could be due to σ
54
-dependent regulation of genes involved in the peroxide stress response. However, transcriptome analyses of the wild type and the mutant strain during peroxide exposure did not support such a role for σ
54
. Subsequent experiments revealed that the impaired expression of the mannose phosphotransferase system (PTS) operon in the
rpoN
mutant caused the observed increased peroxide sensitivity.
Fate of aPseudomonas savastanoipv. savastanoi Type III Secretion System Mutant in Olive Plants (Olea europaeaL.)
Authors: Pérez-Martínez Isabel , Rodríguez-Moreno Luis , Lambertsen Lotte , Matas Isabel M. , Murillo Jesús , Tegli Stefania , Jiménez Antonio J. , Ramos Cayo
Published: April 2010

Pseudomonas savastanoipv. savastanoi strain NCPPB 3335 is a model bacterial pathogen for studying the molecular basis of disease production in woody hosts. We report the sequencing of thehrpS-to-hrpZregion of NCPPB 3335, which has allowed us to determine the phylogenetic position of this pathogen with respect to previously sequencedPseudomonas syringae hrpclusters. In addition, we constructed a mutant of NCPPB 3335, termed T3, which carries a deletion from the 3′ end of thehrpSgene to the 5′ end of thehrpZoperon. Despite its inability to multiply in olive tissues and to induce tumor formation in woody olive plants,P.savastanoipv. savastanoi T3 can induce knot formation on young micropropagated olive plants. However, the necrosis and formation of internal open cavities previously reported in knots induced by the wild-type strain were not observed in those induced byP.savastanoipv. savastanoi T3. Tagging ofP.savastanoipv. savastanoi T3 with green fluorescent protein (GFP) allowed real-time monitoring of its behavior on olive plants. In olive plant tissues, the wild-type strain formed aggregates that colonized the intercellular spaces and internal cavities of the hypertrophic knots, while the mutant T3 strain showed a disorganized distribution within the parenchyma of the knot. Ultrastructural analysis of knot sections revealed the release of extensive outer membrane vesicles from the bacterial cell surface of theP.savastanoipv. savastanoi T3 mutant, while the wild-type strain exhibited very few vesicles. This phenomenon has not been described before for any other bacterial phytopathogen during host infection.
Carbohydrate Metabolism in Mutants of the Cyanobacterium
Synechococcus elongatus
PCC 7942 Defective in Glycogen Synthesis
Authors: Suzuki Eiji , Ohkawa Hajime , Moriya Katsuya , Matsubara Tatsuya , Nagaike Yukari , Iwasaki Ikuko , Fujiwara Shoko , Tsuzuki Mikio , Nakamura Yasunori
Published: April 2010

ADP-glucose pyrophosphorylase (AGPase) and glycogen synthase (GS) catalyze the first two reactions of glycogen synthesis in cyanobacteria. Mutants defective in each of these enzymes in
Synechococcus elongatus
PCC 7942 were constructed and characterized. Activities of the corresponding enzymes in the selected mutants were virtually undetectable, and their ability to synthesize glycogen was entirely abolished. The maximal activities of photosynthetic O
2
evolution and the rates of respiration in the dark were significantly decreased in the mutants compared to those in wild-type cells. Addition of 0.2 M NaCl or 3 mM H
2
O
2
to liquid cultures markedly inhibited the growth of the AGPase and GS mutants, while the same treatment had only marginal effects on the wild type. These results suggest a significant role for storage polysaccharides in tolerance to salt or oxidative stress.
Bacterial Capture by Peptide-Mimetic Oligoacyllysine Surfaces
Authors: Rotem Shahar , Raz Nili , Kashi Yechezkel , Mor Amram
Published: April 2010

Most procedures for detecting pathogens in liquid media require an initial concentration step. However, poor recovery efficiencies of conventional methods, such as filtration, often lead to low sensitivity. Here, we describe a strategy for concentrating bacteria using their binding affinity for an oligoacyllysine (OAK), a novel peptide-mimetic antimicrobial compound. We show that the resin-linked OAK (ROAK) efficiently captures a variety of pathogens in different media, upon brief incubation with ROAK beads or after continuous flow through a ROAK-packed column. Using
Escherichia coli
expressing green fluorescent protein, we show that binding occurs rapidly during incubation and persists after filtration as visualized by confocal microscopy. The high binding affinity of bacteria was confirmed by surface plasmon resonance technology using an OAK-linked chip. ROAK-bound bacteria remained viable and were readily identifiable by real-time PCR after ethanol elution. A single ROAK bead is estimated to capture about 3,000 bacterial cells in culture medium, in contaminated saline or tap water. ROAK beads can be regenerated for multiple uses after brief ethanol treatment. Collectively, the data support the notion that OAK-based coating of polymeric surfaces might represent a useful means for medium filtration as well as for concentration of bacteria.
Toxicogenomic Response of
Rhodospirillum rubrum
S1H to the Micropollutant Triclosan
Authors: Pycke Benny F. G. , Vanermen Guido , Monsieurs Pieter , De Wever Heleen , Mergeay Max , Verstraete Willy , Leys Natalie
Published: April 2010

In the framework of the Micro-Ecological Life Support System Alternative (MELiSSA) project, a pilot study was performed to identify the effects of triclosan on the MELiSSA carbon-mineralizing microorganism
Rhodospirillum rubrum
S1H. Triclosan is a biocide that is commonly found in human excrement and is considered an emerging pollutant in wastewater and the environment. Chronic exposure to MELiSSA-relevant concentrations (≥25 μg liter
−1
) of triclosan resulted in a significant extension of the lag phase of this organism but hardly affected the growth rate. Analytical determinations gave no indication of triclosan biodegradation during the growth experiment, and flow cytometric viability analyses revealed that triclosan is bacteriostatic and only slightly toxic to
R. rubrum
S1H. Using microarray analyses, the genetic mechanisms supporting the reversibility of triclosan-induced inhibition were scrutinized. An extremely triclosan-responsive cluster of four small adjacent genes was identified, for which there was up to 34-fold induction with 25 μg liter
−1
triclosan. These four genes, for which the designation
muf
(micropollutant-upregulated factor) is proposed, appear to be unique to
R. rubrum
and are shown here for the first time to be involved in the response to stress. Moreover, numerous other systems that are associated with the proton motive force were shown to be responsive to triclosan, but they were never as highly upregulated as the
muf
genes. In response to triclosan,
R. rubrum
S1H induced transcription of the phage shock protein operon (
pspABC
), numerous efflux systems, cell envelope consolidation mechanisms, the oxidative stress response, beta-oxidation, and carbonic anhydrase, while there was downregulation of bacterial conjugation and carboxysome synthesis genes. The
muf
genes and three efflux-related genes showed the most potential to be low-dose biomarkers.
Application of Fourier Transform Infrared Spectroscopy and Chemometrics for Differentiation of
Salmonella enterica
Serovar Enteritidis Phage Types
Authors: Preisner Ornella , Guiomar Raquel , Machado Jorge , Menezes José Cardoso , Lopes João Almeida
Published: April 2010

Fourier transform infrared (FT-IR) spectroscopy and chemometric techniques were used to discriminate five closely related
Salmonella enterica
serotype Enteritidis phage types, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a. Intact cells and outer membrane protein (OMP) extracts from bacterial cell membranes were subjected to FT-IR analysis in transmittance mode. Spectra were collected over a wavenumber range from 4,000 to 600 cm
−1
. Partial least-squares discriminant analysis (PLS-DA) was used to develop calibration models based on preprocessed FT-IR spectra. The analysis based on OMP extracts provided greater separation between the
Salmonella
Enteritidis PT1-PT1b, PT4b, and PT6-PT6a groups than the intact cell analysis. When these three phage type groups were considered, the method based on OMP extract FT-IR spectra was 100% accurate. Moreover, complementary local models that considered only the PT1-PT1b and PT6-PT6a groups were developed, and the level of discrimination increased. PT1 and PT1b isolates were differentiated successfully with the local model using the entire OMP extract spectrum (98.3% correct predictions), whereas the accuracy of discrimination between PT6 and PT6a isolates was 86.0%. Isolates belonging to different phage types (PT19, PT20, and PT21) were used with the model to test its robustness. For the first time it was demonstrated that FT-IR analysis of OMP extracts can be used for construction of robust models that allow fast and accurate discrimination of different
Salmonella
Enteritidis phage types.
Novel Technique for Quantifying Adhesion of
Metarhizium anisopliae
Conidia to the Tick Cuticle
Authors: Ment Dana , Gindin Galina , Rot Asael , Soroker Victoria , Glazer Itamar , Barel Shimon , Samish Michael
Published: April 2010

The present study describes an accurate quantitative method for quantifying the adherence of conidia to the arthropod cuticle and the dynamics of conidial germination on the host. The method was developed using conidia of
Metarhizium anisopliae
var.
anisopliae
(Metschn.) Sorokin (Hypocreales: Clavicipitaceae) and engorged
Rhipicephalus annulatus
(Say) (Arachnida: Ixodidae) females and was also verified for
M. anisopliae
var.
acridum
Driver et Milner (Hypocreales: Clavicipitaceae) and
Alphitobius diaperinus
(Panzer) (Coleoptera: Tenebrionidae) larvae. This novel method is based on using an organic solvent (dichloromethane [DCM]) to remove the adhered conidia from the tick cuticle, suspending the conidia in a detergent solution, and then counting them using a hemocytometer. To confirm the efficacy of the method, scanning electron microscopy (SEM) was used to observe the conidial adherence to and removal from the tick cuticle. As the concentration of conidia in the suspension increased, there were correlating increases in both the number of conidia adhering to engorged female
R. annulatus
and tick mortality. However, no correlation was observed between a tick's susceptibility to fungal infection and the amount of adhered conidia. These findings support the commonly accepted understanding of the nature of the adhesion process. The mechanism enabling the removal of the adhered conidia from the host cuticle is discussed.
Single-Cell Techniques Using Chromosomally Tagged Fluorescent Bacteria To Study
Listeria monocytogenes
Infection Processes
Authors: Balestrino Damien , Hamon Mélanie Anne , Dortet Laurent , Nahori Marie-Anne , Pizarro-Cerda Javier , Alignani Diego , Dussurget Olivier , Cossart Pascale , Toledo-Arana Alejandro
Published: April 2010

Listeria monocytogenes
is a Gram-positive facultative intracellular pathogen which invades different cell types, including nonphagocytic cells, where it is able to replicate and survive. The different steps of the cellular infectious process have been well described and consist of bacterial entry, lysis of the endocytic vacuole, intracellular replication, and spreading to neighboring cells. To study the listerial infectious process, gentamicin survival assays, plaque formation, and direct microscopy observations are typically used; however, there are some caveats with each of these techniques. In this study we describe new single-cell techniques based on use of an array of integrative fluorescent plasmids (green, cyan, and yellow fluorescent proteins) to easily, rapidly, and quantitatively detect
L. monocytogenes in vitro
and
in vivo
. We describe construction of 13 integrative and multicopy plasmids which can be used for detecting intracellular bacteria, for measuring invasion, cell-to-cell spreading, and intracellular replication, for monitoring
in vivo
infections, and for generating transcriptional or translational reporters. Furthermore, we tested these plasmids in a variety of epifluorescence- and flow cytometry-based assays. We showed that we could (i) determine the expression of a particular promoter during the cell cycle, (ii) establish in one rapid experiment at which step in the cell cycle a particular mutant is defective, and (iii) easily measure the number of infected cells
in vitro
and in mouse organs. The plasmids that are described and the methods to detect them are new powerful tools to study host-
Listeria
interactions in a fast, robust, and high-throughput manner.
Comparison of Culture Methods for Isolation of Nontuberculous Mycobacteria from Surface Waters
Authors: Radomski Nicolas , Cambau Emmanuelle , Moulin Laurent , Haenn Sophie , Moilleron Régis , Lucas Françoise S.
Published: April 2010

The environment is the likely source of most nontuberculous mycobacteria (NTM) involved in human infections, especially pulmonary, skin, and soft tissue infections. In order to measure the prevalence of NTM in different aquatic ecosystems, we tried to standardize the culture methods used for surface water testing since many procedures have been described previously. Cultivation of mycobacteria requires long-term incubation in rich media and inactivation of rapidly growing microorganisms whose growth impedes observation of mycobacterial colonies. Consequently, the two criteria used for evaluation of the methods examined were (i) the rate of inhibition of nontarget microorganisms and (ii) the efficiency of recovery of mycobacteria. We compared the competitive growth of
Mycobacterium chelonae
and
M. avium
with nontarget microorganisms on rich Middlebrook 7H11-mycobactin medium after treatment by several chemical decontamination methods that included acids, bases, detergent, or cetylpyridinium chloride (CPC) with and without an antibiotic cocktail, either PANTA (40 U/ml polymyxin, 4 μg/ml amphotericin B, 16 μg/ml nalidixic acid, 4 μg/ml trimethoprim, and 4 μg/ml azlocillin) or PANTAV (PANTA plus 10 μg/ml vancomycin). Our results showed that treatment for 30 min with CPC (final concentration, 0.05%) of water concentrated by centrifugation, followed by culture on a rich medium supplemented with PANTA, significantly decreased the growth of nontarget microorganisms (the concentrations were 6.2 ± 0.4 log
10
CFU/liter on Middlebrook 7H11j medium and 4.2 ± 0.2 log
10
CFU/liter on Middlebrook 7H11j medium containing PANTA [
P
< 0.001]), while the effect of this procedure on NTM was not as great (the concentrations of
M. chelonae
on the two media were 7.0 ± 0.0 log
10
CFU/liter and 6.9 ± 0.0 log
10
CFU/liter, respectively, and the concentrations of
M. avium
were 9.1 ± 0.0 log
10
CFU/liter and 8.9 ± 0.0 log
10
CFU/liter, respectively). We propose that this standardized culture procedure could be used for detection of NTM in aquatic samples.
A Predominant Multidrug-Resistant
Salmonella enterica
Serovar Saintpaul Clonal Line in German Turkey and Related Food Products
Authors: Beutlich Janine , Rodríguez Irene , Schroeter Andreas , Käsbohrer Annemarie , Helmuth Reiner , Guerra Beatriz
Published: April 2010

Recently,
Salmonella enterica
subsp.
enterica
serovar Saintpaul has increasingly been observed in several countries, including Germany. However, the pathogenic potential and epidemiology of this serovar are not very well known. This study describes biological attributes of
S
. Saintpaul isolates obtained from turkeys in Germany based on characterization of their pheno- and genotypic properties. Fifty-five
S
. Saintpaul isolates from German turkeys and turkey-derived food products isolated from 2000 to 2007 were analyzed by using antimicrobial agent, organic solvent, and disinfectant susceptibility tests, isoelectric focusing, detection of resistance determinants, plasmid profiling, pulsed-field gel electrophoresis (PFGE), and hybridization experiments. These isolates were compared to an outgroup consisting of 24
S
. Saintpaul isolates obtained from humans and chickens in Germany and from poultry and poultry products (including turkeys) in Netherlands. A common core resistance pattern was detected for 27 German turkey and turkey product isolates. This pattern included resistance (full or intermediate) to ampicillin, amoxicillin-clavulanic acid, gentamicin, kanamycin, nalidixic acid, streptomycin, spectinomycin, and sulfamethoxazole and intermediate resistance or decreased susceptibility to ciprofloxacin (MIC, 2 or 1 μg/ml, respectively) and several third-generation cephalosporins (including ceftiofur and cefoxitin [MIC, 4 to 2 and 16 to 2 μg/ml, respectively]). These isolates had the same core resistance genotype, with
bla
TEM-1
,
aadB
,
aadA2
,
sul1
, a Ser83→Glu83 mutation in the
gyrA
gene, and a chromosomal class 1 integron carrying the
aadB-aadA2
gene cassette. Their XbaI, BlnI, and combined XbaI-BlnI PFGE patterns revealed levels of genetic similarity of 93, 75, and 90%, respectively. This study revealed that a multiresistant
S
. Saintpaul clonal line is widespread in turkeys and turkey products in Germany and was also detected among German human fecal and Dutch poultry isolates.
Exploiting the Natural Diversity of Microviridin Gene Clusters for Discovery of Novel Tricyclic Depsipeptides
Authors: Ziemert Nadine , Ishida Keishi , Weiz Annika , Hertweck Christian , Dittmann Elke
Published: April 2010

Microviridins are ribosomally synthesized tricyclic depsipeptides produced by different genera of cyanobacteria. The prevalence of the microviridin gene clusters and the natural diversity of microviridin precursor sequences are currently unknown. Screening of laboratory strains and field samples of the bloom-forming freshwater cyanobacterium
Microcystis
via PCR revealed global occurrence of the microviridin pathway and an unexpected natural variety. We could detect 15 new variants of the precursor gene
mdnA
encoding microviridin backbones that differ in up to 4 amino acid positions from known isoforms of the peptide. The survey not only provides insights into the versatility of the biosynthetic enzymes in a closely related group of cyanobacteria, but also facilitates the discovery and characterization of cryptic microviridin variants. This is demonstrated for microviridin L in
M
icrocystis aeruginosa
strain NIES843 and heterologously produced variants.
The Case of Botulinum Toxin in Milk: Experimental Data
Authors: Weingart Oliver G. , Schreiber Tanja , Mascher Conny , Pauly Diana , Dorner Martin B. , Berger Thomas F. H. , Egger Charlotte , Gessler Frank , Loessner Martin J. , Avondet Marc-Andre , Dorner Brigitte G.
Published: April 2010

Botulinum neurotoxin (BoNT) is the most toxic substance known to man and the causative agent of botulism. Due to its high toxicity and the availability of the producing organism
Clostridium botulinum
, BoNT is regarded as a potential biological warfare agent. Because of the mild pasteurization process, as well as rapid product distribution and consumption, the milk supply chain has long been considered a potential target of a bioterrorist attack. Since, to our knowledge, no empirical data on the inactivation of BoNT in milk during pasteurization are available at this time, we investigated the activities of BoNT type A (BoNT/A) and BoNT/B, as well as their respective complexes, during a laboratory-scale pasteurization process. When we monitored milk alkaline phosphatase activity, which is an industry-accepted parameter of successfully completed pasteurization, our method proved comparable to the industrial process. After heating raw milk spiked with a set amount of BoNT/A or BoNT/B or one of their respective complexes, the structural integrity of the toxin was determined by enzyme-linked immunosorbent assay (ELISA) and its functional activity by mouse bioassay. We demonstrated that standard pasteurization at 72°C for 15 s inactivates at least 99.99% of BoNT/A and BoNT/B and at least 99.5% of their respective complexes. Our results suggest that if BoNTs or their complexes were deliberately released into the milk supply chain, standard pasteurization conditions would reduce their activity much more dramatically than originally anticipated and thus lower the threat level of the widely discussed “BoNT in milk” scenario.
Partial Chromosome Sequence of
Spiroplasma citri
Reveals Extensive Viral Invasion and Important Gene Decay
Authors: Carle Patricia , Saillard Colette , Carrère Nathalie , Carrère Sébastien , Duret Sybille , Eveillard Sandrine , Gaurivaud Patrice , Gourgues Géraldine , Gouzy Jérome , Salar Pascal , Verdin Eric , Breton Marc , Blanchard Alain , Laigret Frédéric , Bové Joseph-Marie , Renaudin Joel , Foissac Xavier
Published: April 2010

The assembly of 20,000 sequencing reads obtained from shotgun and chromosome-specific libraries of the
Spiroplasma citri
genome yielded 77 chromosomal contigs totaling 1,674 kbp (92%) of the 1,820-kbp chromosome. The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsed-field gel electrophoresis and Southern blot hybridizations. Thirty-eight contigs were annotated, resulting in 1,908 predicted coding sequences (CDS) representing an overall coding density of only 74%. Cellular processes, cell metabolism, and structural-element CDS account for 29% of the coding capacity, CDS of external origin such as viruses and mobile elements account for 24% of the coding capacity, and CDS of unknown function account for 47% of the coding capacity. Among these, 21% of the CDS group into 63 paralog families. The organization of these paralogs into conserved blocks suggests that they represent potential mobile units. Phage-related sequences were particularly abundant and include plectrovirus SpV1 and SVGII3 and lambda-like SpV2 sequences. Sixty-nine copies of transposases belonging to four insertion sequence (IS) families (IS
30
, IS
481
, IS
3
, and IS
NCY
) were detected. Similarity analyses showed that 21% of chromosomal CDS were truncated compared to their bacterial orthologs. Transmembrane domains, including signal peptides, were predicted for 599 CDS, of which 58 were putative lipoproteins.
S. citri
has a Sec-dependent protein export pathway. Eighty-four CDS were assigned to transport, such as phosphoenolpyruvate phosphotransferase systems (PTS), the ATP binding cassette (ABC), and other transporters. Besides glycolytic and ATP synthesis pathways, it is noteworthy that
S. citri
possesses a nearly complete pathway for the biosynthesis of a terpenoid.
Comparison of Genotypes of
Salmonella enterica
Serovar Enteritidis Phage Type 30 and 9c Strains Isolated during Three Outbreaks Associated with Raw Almonds
Authors: Parker Craig T. , Huynh Steven , Quiñones Beatriz , Harris Linda J. , Mandrell Robert E.
Published: April 2010

In 2000 to 2001, 2003 to 2004, and 2005 to 2006, three outbreaks of
Salmonella enterica
serovar Enteritidis were linked with the consumption of raw almonds. The
S
. Enteritidis strains from these outbreaks had rare phage types (PT), PT30 and PT9c. Clinical and environmental
S
. Enteritidis strains were subjected to pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem repeat analysis (MLVA), and DNA microarray-based comparative genomic indexing (CGI) to evaluate their genetic relatedness. All three methods differentiated these
S
. Enteritidis strains in a manner that correlated with PT. The CGI analysis confirmed that the majority of the differences between the
S
. Enteritidis PT9c and PT30 strains corresponded to bacteriophage-related genes present in the sequenced genomes of
S
. Enteritidis PT4 and
S. enterica
serovar Typhimurium LT2. However, PFGE, MLVA, and CGI failed to discriminate between
S
. Enteritidis PT30 strains related to outbreaks from unrelated clinical strains or between strains separated by up to 5 years. However, metabolic fingerprinting demonstrated that
S
. Enteritidis PT4, PT8, PT13a, and clinical PT30 strains metabolized
l
-aspartic acid,
l
-glutamic acid,
l
-proline,
l
-alanine, and
d
-alanine amino acids more efficiently than
S
. Enteritidis PT30 strains isolated from orchards. These data indicate that
S
. Enteritidis PT9c and 30 strains are highly related genetically and that PT30 orchard strains differ from clinical PT30 strains metabolically, possibly due to fitness adaptations.
Interplay between Iron Homeostasis and the Osmotic Stress Response in the Halophilic Bacterium
Chromohalobacter salexigens
Authors: Argandoña Montserrat , Nieto Joaquín J. , Iglesias-Guerra Fernando , Calderón Maria Isabel , García-Estepa Raúl , Vargas Carmen
Published: April 2010

In this study, the connection between iron homeostasis and the osmostress response in the halophile
Chromohalobacter salexigens
was investigated. A decrease in the requirement for both iron and histidine and a lower level of siderophore synthesis were observed at high salinity, and these findings were correlated with a lower protein content in salt-stressed cells. A six-gene operon (
cfuABC
-
fur
-
hisI
-
orf6
operon) located downstream of the
ectABC
ectoine synthesis genes was characterized. A
fur
strain (in which the ferric iron uptake regulator Fur was affected) had the Mn resistance phenotype typical of
fur
mutants, was deregulated for siderophore production, and displayed delayed growth under iron limitation conditions, indicating that
fur
encodes a functional iron regulator.
hisI
was essential for histidine synthesis, which in turn was necessary for siderophore production. Fur boxes were found in the promoters of the
cfuABC
-
fur
-
hisI
-
orf6
and
ectABC
operons, suggesting that Fur directly interacts with DNA in these regions. Fur mediated the osmoregulated inhibition of
cfuABC
-
fur
-
hisI
-
orf6
operon expression by iron and functioned as a positive regulator of the
ectABC
genes under high-salinity conditions, linking the salt stress response with iron homeostasis. Excess iron led to a higher cytoplasmic hydroxyectoine content, suggesting that hydroxyectoine protects against the oxidative stress caused by iron better than ectoine. This study provides the first evidence of involvement of the iron homeostasis regulator Fur as part of the complex circuit that controls the response to osmotic stress in halophilic bacteria.
Evaluation of Lysine Biosynthesis as an Antifungal Drug Target: Biochemical Characterization of Aspergillus fumigatus Homocitrate Synthase and Virulence Studies
Authors: Schöbel Felicitas , Jacobsen Ilse D. , Brock Matthias
Published: April 2010

Aspergillus fumigatus
is the main cause of severe invasive aspergillosis. To combat this life-threatening infection, only limited numbers of antifungals are available. The fungal α-aminoadipate pathway, which is essential for lysine biosynthesis, has been suggested as a potential antifungal drug target. Here we reanalyzed the role of this pathway for establishment of invasive aspergillosis in murine models. We selected the first pathway-specific enzyme, homocitrate synthase (HcsA), for biochemical characterization and for study of its role in virulence.
A. fumigatus
HcsA was specific for the substrates acetyl-coenzyme A (acetyl-CoA) and α-ketoglutarate, and its activity was independent of any metal ions. In contrast to the case for other homocitrate synthases, enzymatic activity was hardly affected by lysine and gene expression increased under conditions of lysine supplementation. An
hcsA
deletion mutant was lysine auxotrophic and unable to germinate on unhydrolyzed proteins given as a sole nutrient source. However, the addition of partially purified
A. fumigatus
proteases restored growth, confirming the importance of free lysine to complement auxotrophy. In contrast to lysine-auxotrophic mutants from other fungal species, the mutant grew on blood and serum, indicating the existence of high-affinity lysine uptake systems. In agreement, although the virulence of the mutant was strongly attenuated in murine models of bronchopulmonary aspergillosis, virulence was partially restored by lysine supplementation via the drinking water. Additionally, in contrast to the case for attenuated pulmonary infections, the mutant retained full virulence when injected intravenously. Therefore, we concluded that inhibition of fungal lysine biosynthesis, at least for disseminating invasive aspergillosis, does not appear to provide a suitable target for new antifungals.
Single-Step Affinity Purification for Fungal Proteomics
Authors: Liu Hui-Lin , Osmani Aysha H. , Ukil Leena , Son Sunghun , Markossian Sarine , Shen Kuo-Fang , Govindaraghavan Meera , Varadaraj Archana , Hashmi Shahr B. , De Souza Colin P. , Osmani Stephen A.
Published: April 2010

A single-step protein affinity purification protocol using
Aspergillus nidulans
is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.
Fine Mapping of
Leishmania major
Susceptibility Locus
lmr2
and Evidence of a Role for
Fli1
in Disease and Wound Healing
Authors: Sakthianandeswaren Anuratha , Curtis Joan M. , Elso Colleen , Kumar Beena , Baldwin Tracey M. , Lopaticki Sash , Kedzierski Lukasz , Smyth Gordon K. , Foote Simon J. , Handman Emanuela
Published: April 2010

Genetic linkage studies of the host response to
Leishmania major
, the causative agent of cutaneous leishmaniasis, have identified significant genetic complexity in humans and mice. In the mouse model, multiple loci have been implicated in susceptibility to infection, but to date, the genes underlying these loci have not been identified. We now describe the contribution of a novel candidate gene,
Fli1
, to both
L. major
resistance and enhanced wound healing. We have previously mapped the
L. major
response locus,
lmr2
, to proximal chromosome 9 in a genetic cross between the resistant C57BL/6 strain and the susceptible BALB/c strain. We now show that the presence of the resistant C57BL/6
lmr2
allele in susceptible BALB/c mice confers an enhanced
L. major
resistance and wound healing phenotype. Fine mapping of the
lmr2
locus permitted the localization of the
lmr2
quantitative trait locus to a 5-Mb interval comprising 21 genes, of which microarray analysis was able to identify differential expression in 1 gene—
Fli1
. Analysis of
Fli1
expression in wounded and
L. major
-infected skin and naïve and infected lymph nodes validated the importance of
Fli1
in lesion resolution and wound healing and identified 3 polymorphisms in the
Fli1
promoter, among which a GA repeat element may be the important contributor.
The C-Terminal Tail of
Yersinia pseudotuberculosis
YopM Is Critical for Interacting with RSK1 and for Virulence
Authors: McCoy Melissa W. , Marré Meghan L. , Lesser Cammie F. , Mecsas Joan
Published: April 2010

Yersinia
spp. undermine the immune responses of infected animals by translocating Yops directly into host cells with a type III secretion system. YopM, a leucine-rich repeat protein, is a critical virulence factor in infection. YopM localizes to both the nucleus and the cytoplasm in cultured cells, interacts with mammalian p90 ribosomal S6 kinase 1 (RSK1), and causes a decrease in NK cell populations in spleens. Little is known about the molecular interaction between YopM and RSK1 and its significance in pathogenesis. We performed a systematic deletion analysis of YopM in
Yersinia pseudotuberculosis
to determine which regions are required for RSK1 interactions, nuclear localization, virulence, and changes in immune cell populations during infection of mice. Full-length YopM associated with RSK1 in at least two protein complexes in infected cells, and deletion of its C-terminal tail abrogated all RSK1 interactions. The C-terminal tail was required for tissue colonization, as
yopM
mutants that failed to interact with RSK1 were as defective for tissue colonization as was a Δ
yopM
mutant; however, nuclear localization of YopM was not dependent on its RSK1 interaction. Mutants expressing YopM proteins which do not interact with RSK1 caused more pathology than did the Δ
yopM
mutant, suggesting that there are other RSK1-independent functions of YopM. Histopathological and flow cytometric analyses of spleens showed that infection with wild-type
Y. pseudotuberculosis
caused an influx of neutrophils, while mice infected with
yopM
mutants had increased numbers of macrophages. Decreases in NK cells after
Y. pseudotuberculosis
infection did not correlate with YopM expression. In conclusion, the C terminus of YopM is essential for RSK1 interactions and for virulence.
Coupled Amino Acid Deamidase-Transport Systems Essential for
Helicobacter pylori
Colonization
Authors: Leduc Damien , Gallaud Julien , Stingl Kerstin , de Reuse Hilde
Published: April 2010

In addition to their classical roles as carbon or nitrogen sources, amino acids can be used for bacterial virulence, colonization, or stress resistance. We found that original deamidase-transport systems impact colonization by
Helicobacter pylori
, a human pathogen associated with gastric pathologies, including adenocarcinoma. We demonstrated that
l
-asparaginase (Hp-AnsB) and γ-glutamyltranspeptidase (Hp-γGT) are highly active periplasmic deamidases in
H. pylori
, producing ammonia and aspartate or glutamate from asparagine and glutamine, respectively. Hp-GltS was identified as a sole and specialized transporter for glutamate, while aspartate was exclusively imported by Hp-DcuA. Uptake of Gln and Asn strictly relies on indirect pathways following prior periplasmic deamidation into Glu and Asp. Hence, in
H. pylori
, the coupled action of periplasmic deamidases with their respective transporters enables the acquisition of Glu and Asp from Gln and Asn, respectively. These systems were active at neutral rather than acidic pH, suggesting their function near the host epithelial cells. We showed that Hp-DcuA, the fourth component of these novel deamidase-transport systems, was as crucial as Hp-γGT, Hp-AnsB, and Hp-GltS for animal model colonization. In conclusion, the pH-regulated coupled amino acid deamidase-uptake system represents an original optimized system that is essential for
in vivo
colonization of the stomach environment by
H. pylori
. We propose a model in which these two nonredundant systems participate in
H. pylori
virulence by depleting gastric or immune cells from protective amino acids such as Gln and producing toxic ammonia close to the host cells.
Comparative Study of the Ability ofLeishmania mexicanaPromastigotes and Amastigotes To Alter Macrophage Signaling and Functions
Authors: Abu-Dayyeh Issa , Hassani Kasra , Westra Edze R. , Mottram Jeremy C. , Olivier Martin
Published: April 2010

Leishmaniaalternates between two morphologically different stages, promastigotes and amastigotes. While the majority of reports focused on how the promastigote form can alter macrophage (Mφ) signaling and function, fewer reports investigated signaling alterations mediated by amastigotes, and there is a lack of comparative studies. In this study, we performed a comparison between the ability of both forms of the parasite to alter Mφ signaling and functions. Here, we show that both promastigotes and amastigotes were able to rapidly activate host protein tyrosine phosphatases (PTPs), importantly the Src homology 2 domain-containing PTP (SHP-1). However, we found that PTP-1B is specifically activated by promastigote but not amastigote infection and thatlmcpb−/−promastigotes were no longer able to activate PTP-1B. We also show a similarity in the way promastigotes and amastigotes inactivate the transcription factors (TFs) STAT-1α and AP-1, but we show differences in the modulation of NF-κB, with promastigotes cleaving the p65 subunit, generating a smaller p35 subunit, and amastigotes fully degrading the p65 subunit with no p35 production. Importantly, we show that the cysteine proteinase LmCPb plays a key role in the alteration of NF-κB, STAT-1α, and AP-1 by promastigote and amastigote infections, ultimately leading to the inability of these TFs to translocate to the nucleus in response to gamma interferon (IFN-γ) stimulation and thus contributing to the ability of both parasite forms to effectively block IFN-γ-mediated nitric oxide (NO) production in Mφs.