bluuebunny/crossref_metadata_2025 huggingface dataset:
Sponge-Associated Bacteria Are Strictly Maintained in Two Closely Related but Geographically Distant Sponge Hosts
Authors: Montalvo Naomi F. , Hill Russell T.
Published: August 2011

The giant barrel spongesXestospongiamutaandXestospongiatestudinariaare ubiquitous in tropical reefs of the Atlantic and Pacific Oceans, respectively. They are key species in their respective environments and are hosts to diverse assemblages of bacteria. These two closely related sponges from different oceans provide a unique opportunity to examine the evolution of sponge-associated bacterial communities. Mitochondrial cytochrome oxidase subunit I gene sequences fromX.mutaandX.testudinariashowed little divergence between the two species. A detailed analysis of the bacterial communities associated with these sponges, comprising over 900 full-length 16S rRNA gene sequences, revealed remarkable similarity in the bacterial communities of the two species. Both sponge-associated communities include sequences found only in the twoXestospongiaspecies, as well as sequences found also in other sponge species and are dominated by three bacterial groups,Chloroflexi,Acidobacteria, andActinobacteria. While these groups consistently dominate the bacterial communities revealed by 16S rRNA gene-based analysis of sponge-associated bacteria, the depth of sequencing undertaken in this study revealed clades of bacteria specifically associated with each of the twoXestospongiaspecies, and also with the genusXestospongia, that have not been found associated with other sponge species or other ecosystems. This study, comparing the bacterial communities associated with closely related but geographically distant sponge hosts, gives new insight into the intimate relationships between marine sponges and some of their bacterial symbionts.
Role of Mrx Fimbriae of Xenorhabdus nematophila in Competitive Colonization of the Nematode Host
Authors: Snyder Holly , He Hongjun , Owen Heather , Hanna Chris , Forst Steven
Published: August 2011

Xenorhabdus nematophilaengages in mutualistic associations with the infective juvenile (IJ) stage of specific entomopathogenic nematodes. Mannose-resistant (Mrx) chaperone-usher-type fimbriae are produced when the bacteria are grown on nutrient broth agar (NB agar). The role of Mrx fimbriae in the colonization of the nematode host has remained unresolved. We show thatX. nematophilagrown on LB agar produced flagella rather than fimbriae. IJs propagated onX. nematophilagrown on LB agar were colonized to the same extent as those propagated on NB agar. Further, progeny IJs were normally colonized bymrxmutant strains that lacked fimbriae both when bacteria were grown on NB agar and when coinjected into the insect host with aposymbiotic nematodes. Themrxstrains were not competitively defective for colonization when grown in the presence of wild-type cells on NB agar. In addition, a phenotypic variant strain that lacked fimbriae colonized as well as the wild-type strain. In contrast, themrxstrains displayed a competitive colonization defectin vivo. IJ progeny obtained from insects injected with comixtures of nematodes carrying either the wild-type or themrxstrain were colonized almost exclusively with the wild-type strain. Likewise, when insects were coinjected with aposymbiotic IJs together with a comixture of the wild-type andmrxstrains, the resulting IJ progeny were predominantly colonized with the wild-type strain. These results revealed that Mrx fimbriae confer a competitive advantage during colonizationin vivoand provide new insights into the role of chaperone-usher fimbriae in the life cycle ofX. nematophila.
Ability of Bifidobacterium breve To Grow on Different Types of Milk: Exploring the Metabolism of Milk through Genome Analysis
Authors: Turroni Francesca , Foroni Elena , Serafini Fausta , Viappiani Alice , Montanini Barbara , Bottacini Francesca , Ferrarini Alberto , Bacchini Pier Luigi , Rota Claudio , Delledonne Massimo , Ottonello Simone , van Sinderen Douwe , Ventura Marco
Published: August 2011

We have investigated the occurrence of bifidobacteria in human milk samples, and we provide evidence regarding the predominance of members of theBifidobacterium brevespecies in this environment. Moreover, evaluation of the growth capabilities and transcriptomic analyses of one representative isolate of this species, i.e.,B. breve4L, on different milk types were performed.
Application of the Modular Approach to an In-House Validation Study of Real-Time PCR Methods for the Detection and Serogroup Determination of Verocytotoxigenic Escherichia coli
Authors: Kagkli Dafni-Maria , Weber Thomas P. , Van den Bulcke Marc , Folloni Silvia , Tozzoli Rosangela , Morabito Stefano , Ermolli Monica , Gribaldo Laura , Van den Eede Guy
Published: August 2011

European Commission regulation 2073/2005 on the microbiological criteria for food requires that
Escherichia coli
is monitored as an indicator of hygienic conditions. Since verocytotoxigenic
E. coli
(VTEC) strains often cause food-borne infections by the consumption of raw food, the Biological Hazards (BIOHAZ) panel of the European Food Safety Authority (EFSA) recommended their monitoring in food as well. In particular, VTEC strains belonging to serogroups such as O26, O103, O111, O145, and O157 are known causative agents of several human outbreaks. Eight real-time PCR methods for the detection of
E. coli
toxin genes and their variants (
stx
1
,
stx
2
), the intimin gene (
eae
), and five serogroup-specific genes have been proposed by the European Reference Laboratory for VTEC (EURL-VTEC) as a technical specification to the European Normalization Committee (CEN TC275/WG6). Here we applied a “modular approach” to the in-house validation of these PCR methods. The modular approach subdivides an analytical process into separate parts called “modules,” which are independently validated based on method performance criteria for a limited set of critical parameters. For the VTEC real-time PCR module, the following parameters are being assessed: specificity, dynamic range, PCR efficiency, and limit of detection (LOD). This study describes the modular approach for the validation of PCR methods to be used in food microbiology, using single-target plasmids as positive controls and showing their applicability with food matrices.
Pyrroloquinoline Quinone Biosynthesis GenepqqC, a Novel Molecular Marker for Studying the Phylogeny and Diversity of Phosphate-Solubilizing Pseudomonads
Authors: Meyer Joana Beatrice , Frapolli Michele , Keel Christoph , Maurhofer Monika
Published: August 2011

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify thepqqCgene of thePseudomonasgenus, were designed.pqqCfragments were amplified and sequenced from aPseudomonasstrain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based onpqqCsequences were compared to trees obtained with the two concatenated housekeeping genesrpoDandgyrB. For bothpqqCandrpoD-gyrB, similar main phylogenetic clusters were found. However, in thepqqCbut not in therpoD-gyrBtree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside thePseudomonas fluorescensgroup.pqqCsequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups thanpqqCsequences derived from the cultivation-independent approach. ComparingpqqCphylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the genepqqCis a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.
Comparing the Chlorine Disinfection of Detached Biofilm Clusters with Those of Sessile Biofilms and Planktonic Cells in Single- and Dual-Species Cultures
Authors: Behnke Sabrina , Parker Albert E. , Woodall Dawn , Camper Anne K.
Published: August 2011

Although the detachment of cells from biofilms is of fundamental importance to the dissemination of organisms in both public health and clinical settings, the disinfection efficacies of commonly used biocides on detached biofilm particles have not been investigated. Therefore, the question arises whether cells in detached aggregates can be killed with disinfectant concentrations sufficient to inactivate planktonic cells.
Burkholderia cepacia
and
Pseudomonas aeruginosa
were grown in standardized laboratory reactors as single species and in coculture. Cluster size distributions in chemostats and biofilm reactor effluent were measured. Chlorine susceptibility was assessed for planktonic cultures, attached biofilm, and particles and cells detached from the biofilm. Disinfection tolerance generally increased with a higher percentage of larger cell clusters in the chemostat and detached biofilm. Samples with a lower percentage of large clusters were more easily disinfected. Thus, disinfection tolerance depended on the cluster size distribution rather than sample type for chemostat and detached biofilm. Intact biofilms were more tolerant to chlorine independent of species. Homogenization of samples led to significantly increased susceptibility in all biofilm samples as well as detached clusters for single-species
B. cepacia
,
B. cepacia
in coculture, and
P. aeruginosa
in coculture. The disinfection efficacy was also dependent on species composition; coculture was advantageous to the survival of both species when grown as a biofilm or as clusters detached from biofilm but, surprisingly, resulted in a lower disinfection tolerance when they were grown as a mixed planktonic culture.
Methodologies for Salmonella enterica subsp. enterica Subtyping: Gold Standards and Alternatives
Authors: Wattiau Pierre , Boland Cécile , Bertrand Sophie
Published: August 2011

For more than 80 years, subtyping of
Salmonella enterica
has been routinely performed by serotyping, a method in which surface antigens are identified based on agglutination reactions with specific antibodies. The serotyping scheme, which is continuously updated as new serovars are discovered, has generated over time a data set of the utmost significance, allowing long-term epidemiological surveillance of
Salmonella
in the food chain and in public health control. Conceptually, serotyping provides no information regarding the phyletic relationships inside the different
Salmonella enterica
subspecies. In epidemiological investigations, identification and tracking of salmonellosis outbreaks require the use of methods that can fingerprint the causative strains at a taxonomic level far more specific than the one achieved by serotyping. During the last 2 decades, alternative methods that could successfully identify the serovar of a given strain by probing its DNA have emerged, and molecular biology-based methods have been made available to address phylogeny and fingerprinting issues. At the same time, accredited diagnostics have become increasingly generalized, imposing stringent methodological requirements in terms of traceability and measurability. In these new contexts, the hand-crafted character of classical serotyping is being challenged, although it is widely accepted that classification into serovars should be maintained. This review summarizes and discusses modern typing methods, with a particular focus on those having potential as alternatives for classical serotyping or for subtyping
Salmonella
strains at a deeper level.
Four-Year Evaluation of the Effect of Vaccination against Coxiella burnetii on Reduction of Animal Infection and Environmental Contamination in a Naturally Infected Dairy Sheep Flock
Authors: Astobiza Ianire , Barandika Jesús F. , Ruiz-Fons Francisco , Hurtado Ana , Povedano Inés , Juste Ramón A. , García-Pérez Ana L.
Published: August 2011

Vaccination is considered one of the best options for controllingCoxiella burnetiiinfection in livestock. The efficacy of a phase I vaccine was investigated over 4 years in a sheep flock with confirmedC. burnetiiinfection. Shedding was not detected in ewes and yearlings in the last 2 years, butC. burnetiistill persisted in the environment.
Microbial Inactivation for Safe and Rapid Diagnostics of Infectious Samples
Authors: Sagripanti Jose-Luis , Hülseweh Birgit , Grote Gudrun , Voß Luzie , Böhling Katrin , Marschall Hans-Jürgen
Published: August 2011

The high risk associated with biological threat agents dictates that any suspicious sample be handled under strict surety and safety controls and processed under high-level containment in specialized laboratories. This study attempted to find a rapid, reliable, and simple method for the complete inactivation of a wide range of pathogens, including spores, vegetative bacteria, and viruses, while preserving microbial nucleic acid fragments suitable for PCRs and proteinaceous epitopes for detection by immunoassays. Formaldehyde, hydrogen peroxide, and guanidium thiocyanate did not completely inactivate high titers of bacterial spores or viruses after 30 min at 21°C. Glutaraldehyde and sodium hypochlorite showed high microbicidal activity but obliterated the PCR or enzyme-linked immunosorbent assay (ELISA) detection of bacterial spores or viruses. High-level inactivation (more than 6 log
10
) of bacterial spores (
Bacillus atrophaeus
), vegetative bacteria (
Pseudomonas aeruginosa
), an RNA virus (the alphavirus Pixuna virus), or a DNA virus (the orthopoxvirus vaccinia virus) was attained within 30 min at 21°C by treatment with either peracetic acid or cupric ascorbate with minimal hindrance of subsequent PCR tests and immunoassays. The data described here should provide the basis for quickly rendering field samples noninfectious for further analysis under lower-level containment and considerably lower cost.
Identification of a Perchlorate Reduction Genomic Island with Novel Regulatory and Metabolic Genes
Authors: Melnyk Ryan A. , Engelbrektson Anna , Clark Iain C. , Carlson Hans K. , Byrne-Bailey Kathy , Coates John D.
Published: August 2011

A comparative analysis of the genomes of four dissimilatory (per)chlorate-reducing bacteria has revealed a genomic island associated with perchlorate reduction. In addition to the characterized metabolic genes for perchlorate reductase and chlorite dismutase, the island contains multiple conserved uncharacterized genes possibly involved in electron transport and regulation.
Norovirus Infectivity in Humans and Persistence in Water
Authors: Seitz Scot R. , Leon Juan S. , Schwab Kellogg J. , Lyon G. Marshall , Dowd Melissa , McDaniels Marisa , Abdulhafid Gwen , Fernandez Marina L. , Lindesmith Lisa C. , Baric Ralph S. , Moe Christine L.
Published: August 2011

To examine the long-term infectivity of human norovirus in water, 13 study subjects were challenged at different time points with groundwater spiked with the prototype human norovirus, Norwalk virus. Norwalk virus spiked in groundwater remained infectious after storage at room temperature in the dark for 61 days (the last time point tested). The Norwalk virus-seeded groundwater was stored for 1,266 days and analyzed, after RNase treatment, by reverse transcription-quantitative PCR (RT-qPCR) to detect Norwalk virus RNA contained within intact capsids. Norwalk virus RNA within intact capsids was detected in groundwater for 1,266 days, with no significant log
10
reduction throughout 427 days and a significant 1.10-log
10
reduction by day 1266. Purified Norwalk virus RNA (extracted from Norwalk virus virions) persisted for 14 days in groundwater, tap water, and reagent-grade water. This study demonstrates that Norwalk virus in groundwater can remain detectable for over 3 years and can remain infectious for at least 61 days. (ClinicalTrials.gov identifier NCT00313404.)
Effect of Sodium Bisulfite Injection on the Microbial Community Composition in a Brackish-Water-Transporting Pipeline
Authors: Park Hyung Soo , Chatterjee Indranil , Dong Xiaoli , Wang Sheng-Hung , Sensen Christoph W. , Caffrey Sean M. , Jack Thomas R. , Boivin Joe , Voordouw Gerrit
Published: August 2011

Pipelines transporting brackish subsurface water, used in the production of bitumen by steam-assisted gravity drainage, are subject to frequent corrosion failures despite the addition of the oxygen scavenger sodium bisulfite (SBS). Pyrosequencing of 16S rRNA genes was used to determine the microbial community composition for planktonic samples of transported water and for sessile samples of pipe-associated solids (PAS) scraped from pipeline cutouts representing corrosion failures. These were obtained from upstream (PAS-616P) and downstream (PAS-821TP and PAS-821LP, collected under rapid-flow and stagnant conditions, respectively) of the SBS injection point. Most transported water samples had a large fraction (1.8% to 97% of pyrosequencing reads) of
Pseudomonas
not found in sessile pipe samples. The sessile population of PAS-616P had methanogens (
Methanobacteriaceae
) as the main (56%) community component, whereas
Deltaproteobacteria
of the genera
Desulfomicrobium
and
Desulfocapsa
were not detected. In contrast, PAS-821TP and PAS-821LP had lower fractions (41% and 0.6%) of
Methanobacteriaceae
archaea but increased fractions of sulfate-reducing
Desulfomicrobium
(18% and 48%) and of bisulfite-disproportionating
Desulfocapsa
(35% and 22%) bacteria. Hence, SBS injection strongly changed the sessile microbial community populations. X-ray diffraction analysis of pipeline scale indicated that iron carbonate was present both upstream and downstream, whereas iron sulfide and sulfur were found only downstream of the SBS injection point, suggesting a contribution of the bisulfite-disproportionating and sulfate-reducing bacteria in the scale to iron corrosion. Incubation of iron coupons with pipeline waters indicated iron corrosion coupled to the formation of methane. Hence, both methanogenic and sulfidogenic microbial communities contributed to corrosion of pipelines transporting these brackish waters.
Effect of Glucose on the Fatty Acid Composition of Cupriavidus necator JMP134 during 2,4-Dichlorophenoxyacetic Acid Degradation: Implications for Lipid-Based Stable Isotope Probing Methods
Authors: Lerch Thomas Z. , Dignac Marie-France , Barriuso Enrique , Mariotti André
Published: August 2011

Combining lipid biomarker profiling with stable isotope probing (SIP) is a powerful technique for studying specific microbial populations responsible for the degradation of organic pollutants in various natural environments. However, the presence of other easily degradable substrates may induce significant physiological changes by altering both the rate of incorporation of the target compound into the biomass and the microbial lipid profiles. In order to test this hypothesis,
Cupriavidus necator
JMP134, a 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium, was incubated with [
13
C]2,4-D, [
13
C]glucose, or mixtures of both substrates alternatively labeled with
13
C.
C. necator
JMP134 exhibited a preferential use of 2,4-D over glucose. The isotopic analysis showed that glucose had only a small effect on the incorporation of the acetic chain of 2,4-D into the biomass (at days 2 and 3) and no effect on that of the benzenic ring. The addition of glucose did change the fatty acid methyl ester (FAME) composition. However, the overall FAME isotopic signature reflected that of the entire biomass. Compound-specific individual isotopic analyses of FAME composition showed that the
13
C-enriched FAME profiles were slightly or not affected when tracing the 2,4-D acetic chain or 2,4-D benzenic ring, respectively. This batch study is a necessary step for validating the use of lipid-based SIP methods in complex environments.
Myxococcus xanthus Swarms Are Driven by Growth and Regulated by a Pacemaker
Authors: Kaiser Dale , Warrick Hans
Published: August 2011

The principal social activity of
Myxococcus xanthus
is to organize a dynamic multicellular structure, known as a swarm. Although its cell density is high, the swarm can grow and expand rapidly. Within the swarm, the individual rod-shaped cells are constantly moving, transiently interacting with one another, and independently reversing their gliding direction. Periodic reversal is, in fact, essential for creating a swarm, and the reversal frequency controls the rate of swarm expansion. Chemotaxis toward nutrient has been thought to drive swarming, but here the nature of swarm growth and the impact of genetic deletions of members of the Frz family of proteins suggest otherwise. We find that three cytoplasmic Frz proteins, FrzCD, FrzF, and FrzE, constitute a cyclic pathway that sets the reversal frequency. Within each cell these three proteins appear to be connected in a negative-feedback loop that produces oscillations whose frequencies are finely tuned by methylation and by phosphorylation. This oscillator, in turn, drives MglAB, a small G-protein switch, to oscillate between its GTP- and GDP-bound states that ultimately determine when the cell moves forward or backward. The periodic reversal of interacting rod-shaped cells promotes their alignment. Swarm organization ensures that each cell can move without blocking the movement of others.
Brucella melitensis Cyclic di-GMP Phosphodiesterase BpdA Controls Expression of Flagellar Genes
Authors: Petersen Erik , Chaudhuri Pallab , Gourley Chris , Harms Jerome , Splitter Gary
Published: August 2011

Brucella melitensis
encounters a variety of conditions and stimuli during its life cycle—including environmental growth, intracellular infection, and extracellular dissemination—which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in
B. melitensis
, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the
bpdA
and
bpdB
genes resulted in attenuation of virulence of the bacterium, while deletion of the
cgsB
gene produced a hypervirulent strain. In a
Vibrio
reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of
bpdA
resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the
bpdA
and
bpdB
mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in
Brucella melitensis
via cyclic-di-GMP.
The Yersinia enterocolitica Phage Shock Proteins B and C Can Form Homodimers and Heterodimers
In Vivo
with the Possibility of Close Association between Multiple Domains
Authors: Gueguen Erwan , Flores-Kim Josué , Darwin Andrew J.
Published: August 2011

The
Yersinia enterocolitica
phage shock protein (Psp) stress response is essential for virulence and for survival during the mislocalization of outer membrane secretin proteins. The cytoplasmic membrane proteins PspB and PspC are critical components involved in regulating
psp
gene expression and in facilitating tolerance to secretin-induced stress. Interactions between PspB and PspC monomers might be important for their functions and for PspC stability. However, little is known about these interactions and there are conflicting reports about the ability of PspC to dimerize. To address this, we have used a combination of independent approaches to systematically analyze the ability of PspB and PspC to form dimers
in vivo
. Formaldehyde cross-linking of the endogenous chromosomally encoded proteins in
Y. enterocolitica
revealed discrete complexes corresponding in size to PspB-PspB, PspC-PspC, and PspB-PspC. Bacterial two-hybrid analysis corroborated these protein associations, but an important limitation of the two-hybrid approach was uncovered for PspB. A series of PspB and PspC proteins with unique cysteine substitutions at various positions was constructed.
In vivo
disulfide cross-linking experiments with these proteins further supported close association between PspB and PspC monomers. Detailed cysteine substitution analysis of predicted leucine zipper-like amphipathic helices in both PspB and PspC suggested that their hydrophobic faces could form homodimerization interfaces.
A Novel Factor Controlling Bistability in Bacillus subtilis: the YmdB Protein Affects Flagellin Expression and Biofilm Formation
Authors: Diethmaier Christine , Pietack Nico , Gunka Katrin , Wrede Christoph , Lehnik-Habrink Martin , Herzberg Christina , Hübner Sebastian , Stülke Jörg
Published: August 2011

Cells ofBacillus subtiliscan either be motile or sessile, depending on the expression of mutually exclusive sets of genes that are required for flagellum or biofilm formation, respectively. Both activities are coordinated by the master regulator SinR. We have analyzed the role of the previously uncharacterizedymdBgene for bistable gene expression inB. subtilis. We observed a strong overexpression of thehaggene encoding flagellin and of other genes of the σD-dependent motility regulon in theymdBmutant, whereas the two major operons for biofilm formation,tapA-sipW-tasAandepsA-O, were not expressed. As a result, theymdBmutant is unable to form biofilms. An analysis of the individual cells of a population revealed that theymdBmutant no longer exhibited bistable behavior; instead, all cells are short and motile. The inability of theymdBmutant to form biofilms is suppressed by the deletion of thesinRgene encoding the master regulator of biofilm formation, indicating that SinR-dependent repression of biofilm genes cannot be relieved in aymdBmutant. Our studies demonstrate that lack of expression of SlrR, an antagonist of SinR, is responsible for the observed phenotypes. Overexpression of SlrR suppresses the effects of aymdBmutation.
Structure and Host Recognition of Serotype 13 Glycopeptidolipid from Mycobacterium intracellulare
Authors: Naka Takashi , Nakata Noboru , Maeda Shinji , Yamamoto Reina , Doe Matsumi , Mizuno Seiko , Niki Mamiko , Kobayashi Kazuo , Ogura Hisashi , Makino Masahiko , Fujiwara Nagatoshi
Published: August 2011

TheMycobacterium avium-M. intracellularecomplex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfAandorfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2′-hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1→3)-4-O-methyl-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→3)-α-l-rhamnose-(1→2)-α-l-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only inO-methylations. We found a relationship between the structure and biosynthesis gene cluster.M. intracellulareserotypes 12 and 13 have a 1.95-kborfA-orfBgene responsible for 3-O-methylation at the terminal hexose,orfB, and 4-O-methylation at the rhamnose next to the terminal hexose,orfA. The serotype 13orfBhad a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.
Hydrogen-Stimulated Carbon Acquisition and Conservation in Salmonella enterica Serovar Typhimurium
Authors: Lamichhane-Khadka Reena , Frye Jonathan G. , Porwollik Steffen , McClelland Michael , Maier Robert J.
Published: August 2011

Salmonella entericaserovar Typhimurium can utilize molecular hydrogen for growth and amino acid transport during anaerobic growth. Via microarray we identified H2gas-affected gene expression changes inSalmonella. The addition of H2caused altered expression of 597 genes, of which 176 genes were upregulated and 421 were downregulated. The significantly H2-upregulated genes include those that encode proteins involved in the transport of iron, manganese, amino acids, nucleosides, and sugars. Genes encoding isocitrate lyase (aceA) and malate synthase (aceB), both involved in the carbon conserving glyoxylate pathway, and genes encoding the enzymes of thed-glucarate andd-glycerate pathways (gudT,gudD,garR,garL,garK) are significantly upregulated by H2. Cells grown with H2showed markedly increased AceA enzyme activity compared to cells without H2. Mutant strains with deletion of eitheraceAoraceBhad reduced H2-dependent growth rates. Genes encoding the glutamine-specific transporters (glnH,glnP,glnQ) were upregulated by H2, and cells grown with H2showed increased [14C]glutamine uptake. Similarly, the mannose uptake system genes (manX,manY) were upregulated by H2,and cells grown with H2showed about 2.0-fold-increased [14C]d-mannose uptake compared to the cells grown without H2. Hydrogen stimulates the expression of genes involved in nutrient and carbon acquisition and carbon-conserving pathways, linking carbon and energy metabolism to sustain H2-dependent growth.
The Bacillus subtilis Extracytoplasmic Function σ Factor σ
V
Is Induced by Lysozyme and Provides Resistance to Lysozyme
Authors: Ho Theresa D. , Hastie Jessica L. , Intile Peter J. , Ellermeier Craig D.
Published: August 2011

Bacteria encounter numerous environmental stresses which can delay or inhibit their growth. Many bacteria utilize alternative σ factors to regulate subsets of genes required to overcome different extracellular assaults. The largest group of these alternative σ factors are the extracytoplasmic function (ECF) σ factors. In this paper, we demonstrate that the expression of the ECF σ factor σ
V
in
Bacillus subtilis
is induced specifically by lysozyme but not other cell wall-damaging agents. A mutation in
sigV
results in increased sensitivity to lysozyme killing, suggesting that σ
V
is required for lysozyme resistance. Using reverse transcription (RT)-PCR, we show that the previously uncharacterized gene
yrhL
(here referred to as
oatA
for
O
-acetyltransferase) is in a four-gene operon which includes
sigV
and
rsiV
. In quantitative RT-PCR experiments, the expression of
oatA
is induced by lysozyme stress. Lysozyme induction of
oatA
is dependent upon σ
V
. Overexpression of
oatA
in a
sigV
mutant restores lysozyme resistance to wild-type levels. This suggests that OatA is required for σ
V
-dependent resistance to lysozyme. We also tested the ability of lysozyme to induce the other ECF σ factors and found that only the expression of
sigV
is lysozyme inducible. However, we found that the other ECF σ factors contributed to lysozyme resistance. We found that
sigX
and
sigM
mutations alone had very little effect on lysozyme resistance but when combined with a
sigV
mutation resulted in significantly greater lysozyme sensitivity than the
sigV
mutation alone. This suggests that
sigV
,
sigX
, and
sigM
may act synergistically to control lysozyme resistance. In addition, we show that two ECF σ factor-regulated genes,
dltA
and
pbpX
, are required for lysozyme resistance. Thus, we have identified three independent mechanisms which
B. subtilis
utilizes to avoid killing by lysozyme.
Neisserial Correia Repeat-Enclosed Elements Do Not Influence the Transcription of
pil
Genes in Neisseria gonorrhoeae and Neisseria meningitidis
Authors: Lin Ya-Hsun , Ryan Catherine S. , Davies John K.
Published: August 2011

Two human-specific neisserial pathogens,
Neisseria gonorrhoeae
and
Neisseria meningitidis
, require the expression of type IV pili (tfp) for initial attachment to the host during infection. However, the mechanisms controlling the assembly and functionality of tfp are poorly understood. It is known that the gonococcal
pilE
gene, encoding the major subunit, is positively regulated by IHF, a multifunctional DNA binding protein. A neisserial specific repetitive DNA sequence, termed the Correia repeat-enclosed element (CREE) is situated upstream of three
pil
loci:
pilHIJKX
(
pilH-X
),
pilGD
, and
pilF
. CREEs have been shown to contain strong promoters, and some CREE variants contain a functional IHF binding site. CREEs might therefore be involved in the regulation of tfp biogenesis in pathogenic
Neisseria
. Site-directed and deletion mutagenesis on promoter::
cat
reporter constructs demonstrated that transcription of
pilH-X
and
pilGD
is from a σ
70
promoter and is independent of the CREE. The insertion of a CREE in the
pilF
promoter region in
N. meningitidis
generated a functional σ
70
promoter. However, there is also a functional promoter at this position in
N. gonorrhoeae
, where there is no CREE. These results suggest CREE insertion in these three
pil
loci does not influence transcription and that IHF does not coordinately regulate tfp biogenesis.
Inhibitory Effects of
d
-Amino Acids on Staphylococcus aureus Biofilm Development
Authors: Hochbaum Allon I. , Kolodkin-Gal Ilana , Foulston Lucy , Kolter Roberto , Aizenberg Joanna , Losick Richard
Published: August 2011

Biofilms are communities of cells held together by a self-produced extracellular matrix typically consisting of protein, exopolysaccharide, and often DNA. A natural signal for biofilm disassembly in
Bacillus subtilis
is certain
d
-amino acids, which are incorporated into the peptidoglycan and trigger the release of the protein component of the matrix.
d
-Amino acids also prevent biofilm formation by the related Gram-positive bacterium
Staphylococcus aureus
. Here we employed fluorescence microscopy and confocal laser scanning microscopy to investigate how
d
-amino acids prevent biofilm formation by
S. aureus
. We report that biofilm formation takes place in two stages, initial attachment to surfaces, resulting in small foci, and the subsequent growth of the foci into large aggregates.
d
-Amino acids did not prevent the initial surface attachment of cells but blocked the subsequent growth of the foci into larger assemblies of cells. Using protein- and polysaccharide-specific stains, we have shown that
d
-amino acids inhibited the accumulation of the protein component of the matrix but had little effect on exopolysaccharide production and localization within the biofilm. We conclude that
d
-amino acids act in an analogous manner to prevent biofilm development in
B. subtilis
and
S. aureus
. Finally, to investigate the potential utility of
d
-amino acids in preventing device-related infections, we have shown that surfaces impregnated with
d
-amino acids were effective in preventing biofilm growth.
The Bacillus subtilis GntR Family Repressor YtrA Responds to Cell Wall Antibiotics
Authors: Salzberg Letal I. , Luo Yun , Hachmann Anna-Barbara , Mascher Thorsten , Helmann John D.
Published: August 2011

The transglycosylation step of cell wall synthesis is a prime antibiotic target because it is essential and specific to bacteria. Two antibiotics, ramoplanin and moenomycin, target this step by binding to the substrate lipid II and the transglycosylase enzyme, respectively. Here, we compare the ramoplanin and moenomycin stimulons in the Gram-positive model organism
Bacillus subtilis
. Ramoplanin strongly induces the LiaRS two-component regulatory system, while moenomycin almost exclusively induces genes that are part of the regulon of the extracytoplasmic function (ECF) σ factor σ
M
. Ramoplanin additionally induces the
ytrABCDEF
and
ywoBCD
operons, which are not part of a previously characterized antibiotic-responsive regulon. Cluster analysis reveals that these two operons are selectively induced by a subset of cell wall antibiotics that inhibit lipid II function or recycling. Repression of both operons requires YtrA, which recognizes an inverted repeat in front of its own operon and in front of
ywoB
. These results suggest that YtrA is an additional regulator of cell envelope stress responses.
Characterization of Invasive and Colonizing Isolates of Streptococcus agalactiae in East African Adults
Authors: Huber Charlotte A. , McOdimba Francis , Pflueger Valentin , Daubenberger Claudia A. , Revathi Gunturu
Published: August 2011

Ninety-five colonizing isolates and 74 invasive isolates of
Streptococcus agalactiae
from Kenyan adults were characterized by using capsular serotyping and multilocus sequence typing. Twenty-two sequence types clustering into five clonal complexes were found. Data support the view that
S. agalactiae
isolates belonging to a limited number of clonal complexes are invasive in adults worldwide.
Host Regulatory Network Response to Infection with Highly Pathogenic H5N1 Avian Influenza Virus
Authors: Li Chengjun , Bankhead Armand , Eisfeld Amie J. , Hatta Yasuko , Jeng Sophia , Chang Jean H. , Aicher Lauri D. , Proll Sean , Ellis Amy L. , Law G. Lynn , Waters Katrina M. , Neumann Gabriele , Katze Michael G. , McWeeney Shannon , Kawaoka Yoshihiro
Published: August 2011

During the last decade, more than half of humans infected with highly pathogenic avian influenza (HPAI) H5N1 viruses have died, yet virus-induced host signaling has yet to be clearly elucidated. Airway epithelia are known to produce inflammatory mediators that contribute to HPAI H5N1-mediated pathogenicity, but a comprehensive analysis of the host response in this cell type is lacking. Here, we leveraged a system approach to identify and statistically validate signaling subnetworks that define the dynamic transcriptional response of human bronchial epithelial cells after infection with influenza A/Vietnam/1203/2004 (H5N1, VN1203). Importantly, we validated a subset of transcripts from one subnetwork in both Calu-3 cells and mice. A more detailed examination of two subnetworks involved in the immune response and keratinization processes revealed potential novel mediators of HPAI H5N1 pathogenesis and host response signaling. Finally, we show how these results compare to those for a less virulent strain of influenza virus. Using emergent network properties, we provide fresh insight into the host response to HPAI H5N1 virus infection and identify novel avenues for perturbation studies and potential therapeutic interventions for fatal HPAI H5N1 disease.
Hospital cancer deaths: late diagnosis and missed opportunity: Table 1
Authors: Blaney JM , Crawford G , Elder TR , Johnston G , Gavin AT
Published: August 2011

Objectives

To establish factors that influence and contribute to the death of patients with cancer in acute hospitals in Northern Ireland.

Design

Retrospective clinical note review.

Setting

16 acute hospitals, covering 5 Health and Social Care Trusts across Northern Ireland.

Participants

793 adult patients with cancer who died in an acute hospital between July and December 2007 identified through the Northern Ireland Cancer Registry. Information was available for 695 (88%).

Results

Thee main reasons for acute hospital deaths were uncovered. First, 26.3% of patients were diagnosed with cancer during their last hospital admission. These patients were significantly different from the rest of the sample in being older, not partnered, having more comorbidities and fewer hospital admissions in their last year of life (all p<0.001). Second, patients were very ill with 78.7% admitted as an emergency, requiring medical attention as a result of cancer-related (37.4%) and urgent physical symptoms (33.5%). Third, despite 38.3% of patients specifically requesting discharge to their usual residence, hospice or other hospital, this was not achieved. For 76.3%, this was owing to a deterioration in their medical condition. However for 12.4% there was a lack of a suitable bed, a care package was not in place for 4.9% and 3.0% lacked the required family support. In addition, preferred place of death was only recorded for 41% of patients.

Conclusions

Late diagnosis of cancer is a problem which requires further research. Training should be in place to ensure that a patient's preferred place of death is discussed, recorded and made part of routine end of life care. To achieve this, all medical staff should know when a patient is dying. Further research is required to establish what enables patients with cancer to die at home.
Communication with survivors of motor vehicle crashes
Authors: Marco Catherine A , Wetzel Lindsay R
Published: August 2011

Background

Communication with survivors of severe motor vehicle crashes is challenging for emergency physicians. The appropriate timing of death notification to survivors of severe motor vehicle crashes is unknown.

Objective

To determine communication preferences among survivors of motor vehicle crashes.

Methods

In this cross-sectional survey study, eligible participants included adult survivors of motor vehicle crashes in which there was a death, between 2005 and 2009. Participants were interviewed and responses to 30 questions about communication were recorded verbatim. Responses were coded and grouped for statistical analysis.

Results

Among 26 eligible participants, 21 consented to participate (81% participation rate). Survivors' relationship to the deceased included spouse/significant other (33%), friend (24%), child (5%) and no relationship (38%). Survivors had been notified of the death in the prehospital setting (14%), in the emergency department (43%), or later in the inpatient setting (43%). Survivors were notified of the death by family members (43%), indirect communication (14%), police (10%), prehospital provider (10%), or friend (10%). Most participants (88%) had to ask directly to obtain information about the status of others in the crash. Participants demonstrated variable opinions about the ideal time of death notification: some recommended immediately (24%), in the emergency department (24%), in the inpatient setting (29%), or it depends on the circumstances (24%).

Conclusions

Survivors of motor vehicles crashes are notified of fatalities most commonly by family members, most commonly in the hospital setting. Recommendations from survivors about the appropriate timing and setting for death notification varied significantly.
Distributing personal resuscitation manikins in an untrained population: how well are basic life support skills acquired?
Authors: Nielsen Anne Møller , Isbye Dan Lou , Lippert Freddy , Rasmussen Lars Simon
Published: August 2011

Background

Self-instruction with a DVD and a simple personal manikin is an effective alternative to traditional basic life support (BLS) courses.

Objective

To evaluate the effect of distributing DVD training kits to untrained laypersons. BLS skills were compared according to 2005 guidelines for resuscitation after 3.5 months with those obtained in untrained laypersons who completed the same course with instructor facilitation.

Methods

BLS skills of 55 untrained laypersons were assessed using the Laerdal ResusciAnne and PC Skill Reporting System in a 3 min test and a total score (12–48 points) was calculated. The participants received a DVD training kit without instructions. The test was repeated after 3.5 months. Data were compared with data from a previous published study where participants completed the same course in groups with instructor facilitation.

Results

There was no statistically significant difference in the total score after 3.5 months. The ‘DVD—self-instructor’ group obtained 33 (29–37) points and the ‘DVD—with instructor’ group obtained 34 (32–37) points, p=0.16. The ‘DVD—with instructor’ group performed significantly better in checking responsiveness and had a significantly shorter ‘total hands-off time’ (s) (85 (76–94) vs 96 (82–120), p=0.002) and delay until first compression or ventilation group (29 s (17–40) vs 33 s (22–48), p=0.04).

Conclusions

Since no significant difference in total BLS score was found after 3.5 months between untrained laypersons who either completed a DVD-based BLS course in groups with instructor facilitation or received the same DVD training kit without instruction, the latter seems more efficient.
A simple three-step dispatch rule may reduce lights and sirens responses to motor vehicle crashes
Authors: Isenberg Derek , Cone David C , Stiell Ian G
Published: August 2011

Introduction

Most patients involved in motor vehicle crashes (MVCs) are not seriously injured. However, dispatch protocols require an ambulance be sent with lights and sirens (L&S) to the vast majority of MVCs. L&S have been shown to reduce response times minimally. The rate of injuries among prehospital workers is nearly 15 times higher among ambulances operating with L&S than those without.

Objective

To derive a dispatch rule to reduce the need for L&S response by using MVC characteristics that could easily be described by a 9-1-1 caller. The US Centers for Disease Control Field Triage Guidelines were used as the standard for requiring L&S response; it was assumed that if a patient did not require transport to a trauma centre, he/she did not need an L&S response.

Methods

Data were extracted from prehospital patient care reports (PCRs) of patients transported by ambulance to a level I trauma centre between July 2007 and June 2008 with injuries sustained in MVCs. Patients with completed prehospital PCRs and hospital charts were included in the study. Five MVC characteristics were extracted that could easily be identified by a 9-1-1 caller. Using various permutations of these MVC characteristics, a dispatch rule was developed to determine when an ambulance should respond to an MVC without L&S. The sensitivity and specificity of this dispatch rule were calculated for both patients who met trauma centre triage criteria, and those who used trauma centre resources.

Results

509 patients were included in the analysis. The following dispatch rule was developed for an ambulance response without L&S to a MVC: (1) the MVC does not occur on an interstate/highway, (2) and the MVC involves more than one car. AND (3) all patients are ambulatory. This dispatch rule was 95.9% sensitive and 33.5% specific for patients who met trauma centre criteria, and 97.7% sensitive and 32.5% specific for patients who required trauma centre resources. The study was limited by the large number of patients for whom prehospital PCRs were not available.

Conclusions

A simple three-step dispatch rule for MVCs can safely reduce L&S responses by one-third, as judged by need for transport to a trauma centre or use of trauma centre resources. Prospective validation is needed.
The Segond fracture: a clue to intra-articular knee pathology: Figure 1
Authors: Cosgrave Ciaran H , Burke Neil G , Hollingsworth John
Published: August 2011

This short report presents the case of a 32-year-old man with a knee injury sustained while playing football. A plain radiograph revealed a Segond fracture and a subsequent MRI confirmed a complete anterior cruciate ligament rupture. While the Segond fracture, and its associated intra-articular pathology, is well recognised among orthopaedic surgeons, it is less well recognised among staff in the emergency department. The report aims to emphasise the importance of plain radiographs in patients with a history of knee injury and to highlight that this seemingly innocuous avulsion fracture may reveal more severe underlying pathology.