Exposure to bioaerosols in poultry houses at different stages of fattening; use of real-time PCR for airborne bacterial quantification

Previous studies have demonstrated that poultry house workers are exposed to very high levels of organic dust and consequently have an increased prevalence of adverse respiratory symptoms. However, the influence of the age of broilers on bioaerosol concentrations has not been investigated. To evaluate the evolution of bioaerosol concentration during the fattening period, bioaerosol parameters (inhalable dust, endotoxin and bacteria) were measured in 12 poultry confinement buildings in Switzerland, at three different stages of the birds' growth; samples of air taken from within the breathing zones of individual poultry house employees as they caught the chickens ready to be transported for slaughter were also analysed. Quantitative polymerase chain reaction (Q-PCR) was used to assess the quantity of total airborne bacteria and total airborne Staphylococcus species. Bioaerosol levels increased significantly during the fattening period of the chickens. During the task of catching mature birds, the mean inhalable dust concentration for a worker was 26 +/- 1.9 mg m(-3) and endotoxin concentration was 6198 +/- 2.3 EU m(-3) air, >6-fold higher than the Swiss occupational recommended value (1000 EU m(-3)). The mean exposure level of bird catchers to total bacteria and Staphylococcus species measured by Q-PCR is also very high, respectively, reaching values of 53 (+/-2.6) x 10(7) cells m(-3) air and 62 (+/-1.9) x 10(6) m(-3) air. It was concluded that in the absence of wearing protective breathing apparatus, chicken catchers in Switzerland risk exposure beyond recommended limits for all measured bioaerosol parameters. Moreover, the use of Q-PCR to estimate total and specific numbers of airborne bacteria is a promising tool for evaluating any modifications intended to improve the safety of current working practices

Protection from lethal Gram-negative bacterial sepsis by targeting Toll-like receptor 4

Rapport de synthèse : L'immunité innée regroupe les mécanismes moléculaires et cellulaires formant la première ligne de défense contre les infections microbiennes. La détection des micro-organismes pathogènes est assurée par des cellules sentinelles (cellules dendritiques et macrophages) qui jouent un rôle fondamental dans l'initiation des mécanismes de défense de l'hôte. Au contact de produits microbiens, ces cellules produisent un large échantillonnage de molécules, dont des cytokines, impliquées dans le développement de la réponse inflammatoire. La régulation de cette réponse relève d'un équilibre délicat, son insuffisance tant que son excès pouvant compromettre le devenir des patients infectés. La sepsis sévère et le choc septique représentent les formes les plus sévères d'infection, et leur mortalité demeure élevée (25 à 30% pour la sepsis sévère et 50 à 60% pour le choc septique). De plus, l'incidence de la sepsis tend à augmenter, atteignant en 2000 plus de 240 cas pour 100'000 personnes en Grande-Bretagne. La sepsis est caractérisée dans sa phase aiguë par une réponse inflammatoire exubérante. La plupart des thérapies visant à la bloquer ont toutefois montré des bénéfices incertains lors de leur application clinique. Il est donc impératif d'identifier de nouvelles cibles thérapeutiques. Les "Toll-like receptors" (TLRs) sont une famille de récepteurs qui jouent un rôle fondamental dans la détection des micro-organismes par les cellules du système immunitaire inné. Parmi eux, TLR4 est indispensable à la reconnaissance du lipopolysaccharide (LPS) des bactéries Gram-négatives. L'interaction entre TLR4 et le LPS représentant un élément précoce de la réponse de l'hôte à l'infection, nous avons émit l'hypothèse que TLR4 pourrait représenter une cible de choix en vue du développement de nouvelles thérapies contre la sepsis. Dans l'objectif de valider ce concept, nous avons, dans un premier temps, démontré que des souris génétiquement déficientes en TLR4 étaient totalement résistantes au choc septique induit par Escherichia coli (E. coli), une bactérie Gram-négative fréquemment responsable de sepsis. Forts de cette observation, nous avons développé une molécule recombinante composée du domaine extracellulaire de TLR4 fusionné à la partie IgGi-Fc. Cette molécule soluble, qui inhibait la réponse des macrophages au LPS in vitro, a été utilisée pour générer des anticorps anti-TLR4 chez le lapin. La spécificité et l'efficacité de ces anticorps ont été prouvées en démontrant que les anti-TLR4 bloquaient les signaux d'activation intracellulaire et la production de TNF et d'IL-6 en réponse au LPS et aux bactéries Gram-négatives in vitro et in vivo. Enfin, l'efficacité des ces anticorps a été testée dans des modèles de sepsis chez la souris. Ainsi, l'injection prophylactique (-lh) ou thérapeutique (+3h) d'anticorps anti-TLR4 réduisait la production de TNF et protégeait les animaux de la mort. De manière spectaculaire, ces anticorps réduisaient également la production de TNF et protégeaient de la sepsis à E. coli lorsqu'ils étaient administrés de manière prophylactique (-4h) et thérapeutique, jusqu'à 13 heures après l'initiation de l'infection. Ces résultats indiquent donc qu'il est possible de bloquer le développement de la réponse inflammatoire et de protéger du choc septique à bactéries Gram-négatives en utilisant des thérapies ciblant TLR4. Par ailleurs, ils suggèrent qu'une fenêtre d'opportunité de plusieurs heures pourrait être mise à profit pour initier un traitement chez les patients septiques. Ces résultats devraient encourager la poursuite des essais cliniques en cours qui visent à tester l'efficacité de thérapies dirigées contre TLR4 comme traitement complémentaire de la sepsis.

Epigenetic regulation of the bacterial cell cycle.

N(6)-methyl-adenines can serve as epigenetic signals for interactions between regulatory DNA sequences and regulatory proteins that control cellular functions, such as the initiation of chromosome replication or the expression of specific genes. Several of these genes encode master regulators of the bacterial cell cycle. DNA adenine methylation is mediated by Dam in gamma-proteobacteria and by CcrM in alpha-proteobacteria. A major difference between them is that CcrM is cell cycle regulated, while Dam is active throughout the cell cycle. In alpha-proteobacteria, GANTC sites can remain hemi-methylated for a significant period of the cell cycle, depending on their location on the chromosome. In gamma-proteobacteria, most GATC sites are only transiently hemi-methylated, except regulatory GATC sites that are protected from Dam methylation by specific DNA-binding proteins.

Bacterial lipopolysaccharide induces apoptosis in the trout ovary

BACKGROUND: In mammals it is well known that infections can lead to alterations in reproductive function. As part of the innate immune response, a number of cytokines and other immune factors is produced during bacterial infection or after treatment with lipopolysaccharide (LPS) and acts on the reproductive system. In fish, LPS can also induce an innate immune response but little is known about the activation of the immune system by LPS on reproduction in fish. Therefore, we conducted studies to examine the in vivo and in vitro effects of lipopolysaccharide (LPS) on the reproductive function of sexually mature female trout. METHODS: In saline- and LPS -injected brook trout, we measured the concentration of plasma steroids as well as the in vitro steroidogenic response (testosterone and 17alpha-hydroxyprogesterone) of ovarian follicles to luteinizing hormone (LH), the ability of 17alpha,20beta-dihydroxy-4-pregnen-3-one to induce germinal vesicle breakdown (GVBD) in vitro, and that of epinephrine to stimulate follicular contraction in vitro. We also examined the direct effects of LPS in vitro on steroid production, GVBD and contraction in brook trout ovarian follicles. The incidence of apoptosis was evaluated by TUNEL analysis. Furthermore, we examined the gene expression pattern in the ovary of saline- and LPS-injected rainbow trout by microarray analysis. RESULTS: LPS treatment in vivo did not affect plasma testosterone concentration or the basal in vitro production of steroids, although a small but significant potentiation of the effects of LH on testosterone production in vitro was observed in ovarian follicles from LPS-treated fish. In addition, LPS increased the plasma concentration of cortisol. LPS treatment in vitro did not affect the basal or LH-stimulated steroid production in brook trout ovarian follicles. In addition, we did not observe any effects of LPS in vivo or in vitro on GVBD or follicular contraction. Therefore, LPS did not appear to impair ovarian steroid production, oocyte final maturation or follicular contraction under the present experimental conditions. Interestingly, LPS administration in vivo induced apoptosis in follicular cells, an observation that correlated with changes in the expression of genes involved in apoptosis, as evidenced by microarray analysis. CONCLUSION: These results indicate that female trout are particularly resistant to an acute administration of LPS in terms of ovarian hormone responsiveness. However, LPS caused a marked increase in apoptosis in follicular cells, suggesting that the trout ovary could be sensitive to the pro-apoptotic effects of LPS-induced inflammatory cytokines.

Impairment of the Bacterial Biofilm Stability by Triclosan

The accumulation of the widely-used antibacterial and antifungal compound triclosan (TCS) in freshwaters raises concerns about the impact of this harmful chemical on the biofilms that are the dominant life style of microorganisms in aquatic systems. However, investigations to-date rarely go beyond effects at the cellular, physiological or morphological level. The present paper focuses on bacterial biofilms addressing the possible chemical impairment of their functionality, while also examining their substratum stabilization potential as one example of an important ecosystem service. The development of a bacterial assemblage of natural composition – isolated from sediments of the Eden Estuary (Scotland, UK) – on non-cohesive glass beads (,63 mm) and exposed to a range of triclosan concentrations (control, 2 – 100 mg L21) was monitored over time by Magnetic Particle Induction (MagPI). In parallel, bacterial cell numbers, division rate, community composition (DGGE) and EPS (extracellular polymeric substances: carbohydrates and proteins) secretion were determined. While the triclosan exposure did not prevent bacterial settlement, biofilm development was increasingly inhibited by increasing TCS levels. The surface binding capacity (MagPI) of the assemblages was positively correlated to the microbial secreted EPS matrix. The EPS concentrations and composition (quantity and quality) were closely linked to bacterial growth, which was affected by enhanced TCS exposure. Furthermore, TCS induced significant changes in bacterial community composition as well as a significant decrease in bacterial diversity. The impairment of the stabilization potential of bacterial biofilm under even low, environmentally relevant TCS levels is of concern since the resistance of sediments to erosive forces has large implications for the dynamics of sediments and associated pollutant dispersal. In addition, the surface adhesive capacity of the biofilm acts as a sensitive measure of ecosystem effects

Polyethylene glycol and polyvinylpirrolidone effect on bacterial rRNA extraction and hybridization from cells exposed to tannins

In order to detect fluctuations in ruminal microbial populations due to forage tannins using 16S ribosomal RNA (rRNA) probes, recovery of intact rRNA is required. The objective of this work was to evaluate the effect of polyethylene glycol (PEG) and polyvinylpirrolidone (PVP) on extraction of bacterial rRNA, in the presence of tannins from tropical legume forages and other sources, that hybridize with oligonucleotide probes. Ruminococcus albus 8 cells were exposed to 8 g/L tannic acid or 1 g/L condensed tannins extracted from Acacia angustissima, banana (Musa sp.) skin, Desmodium ovalifolium, red grape (Vitis vinifera) skin and Inga edulis, or no tannins. Cells were rinsed with Tris buffer pH 7 containing either 8% PEG or 6% PVP prior to cell lysis. Total RNA samples rinsed with either PEG or PVP migrated through denaturing agarose gels. The 16S rRNA bands successfully hybridized with a R. albus species-specific oligonucleotide probe, regardless of tannin source. The effect of rinsing buffers on the density of 16S rRNA bands, as well as on the hybridization signals was compared. There were significant effects (P<0.01) when the controls were compared to either buffer treatments due to tannin type, buffer used and the interaction of tannin type and buffer. The significant interaction indicates the influence of tannin type on the parameters evaluated.

Cutting edge: cell autonomous rather than environmental factors control bacterial superantigen-induced T cell anergy in vivo.

Anergic T cells display a marked decrease in their ability to produce IL-2 and to proliferate in the presence of an appropriate antigenic signal. Two nonmutually exclusive classes of models have been proposed to explain the persistence of T cell anergy in vivo. While some reports indicate that anergic T cells have intrinsic defects in signaling pathways or transcriptional activities, other studies suggest that anergy is maintained by environmental "suppressor" factors such as cytokines or Abs. To distinguish between these conflicting hypotheses, we employed the well-characterized bacterial superantigen model system to evaluate in vivo the ability of a trace population of adoptively transferred naive or anergized T cells to proliferate in a naive vs anergic environment upon subsequent challenge. Our data clearly demonstrate that bacterial superantigen-induced T cell anergy is cell autonomous and independent of environmental factors.

A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes.

The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor--Burkholderia sp. strain RP037--produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed.

Maximum likelihood Linear Programming Data Fusion for Speaker Recognition

Biometric system performance can be improved by means of data fusion. Several kinds of information can be fused in order to obtain a more accurate classification (identification or verification) of an input sample. In this paper we present a method for computing the weights in a weighted sum fusion for score combinations, by means of a likelihood model. The maximum likelihood estimation is set as a linear programming problem. The scores are derived from a GMM classifier working on a different feature extractor. Our experimental results assesed the robustness of the system in front a changes on time (different sessions) and robustness in front a change of microphone. The improvements obtained were significantly better (error bars of two standard deviations) than a uniform weighted sum or a uniform weighted product or the best single classifier. The proposed method scales computationaly with the number of scores to be fussioned as the simplex method for linear programming.

A genomic island present along the bacterial chromosome of the Parachlamydiaceae UWE25, an obligate amoebal endosymbiont, encodes a potentially functional F-like conjugative DNA transfer system.

BACKGROUND: The genome of Protochlamydia amoebophila UWE25, a Parachlamydia-related endosymbiont of free-living amoebae, was recently published, providing the opportunity to search for genomic islands (GIs). RESULTS: On the residual cumulative G+C content curve, a G+C-rich 19-kb region was observed. This sequence is part of a 100-kb chromosome region, containing 100 highly co-oriented ORFs, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem are present at the proximal end of this genetic element. Several mobility genes encoding transposases and bacteriophage-related proteins are located within this chromosome region. Thus, this region largely fulfills the criteria of GIs. The G+C content analysis shows that several modules compose this GI. Surprisingly, one of them encodes all genes essential for F-like conjugative DNA transfer (traF, traG, traH, traN, traU, traW, and trbC), involved in sex pilus retraction and mating pair stabilization, strongly suggesting that, similarly to the other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. A close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of gly-tRNA distribution in 140 GIs suggest a proteobacterial origin of the parachlamydial tra unit. CONCLUSIONS: A GI of the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydia bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceae are emerging agents of pneumonia, this GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for Chlamydiales.

Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA) and an eucalyptus arboretum (EAA). PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains.

Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.

Genomic islands: tools of bacterial horizontal gene transfer and evolution.

Abstract Bacterial genomes evolve through mutations, rearrangements or horizontal gene transfer. Besides the core genes encoding essential metabolic functions, bacterial genomes also harbour a number of accessory genes acquired by horizontal gene transfer that might be beneficial under certain environmental conditions. The horizontal gene transfer contributes to the diversification and adaptation of microorganisms, thus having an impact on the genome plasticity. A significant part of the horizontal gene transfer is or has been facilitated by genomic islands (GEIs). GEIs are discrete DNA segments, some of which are mobile and others which are not, or are no longer mobile, which differ among closely related strains. A number of GEIs are capable of integration into the chromosome of the host, excision, and transfer to a new host by transformation, conjugation or transduction. GEIs play a crucial role in the evolution of a broad spectrum of bacteria as they are involved in the dissemination of variable genes, including antibiotic resistance and virulence genes leading to generation of hospital 'superbugs', as well as catabolic genes leading to formation of new metabolic pathways. Depending on the composition of gene modules, the same type of GEIs can promote survival of pathogenic as well as environmental bacteria.

Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles

The objective of this work was to determine the shifts on the PCR-DGGE profiles of bacterial communities associated to the rhizosphere of potato cultivars, in order to generate baseline information for further studies of environmental risk assessment of genetically modified potato plants. A greenhouse experiment was carried out with five potato cultivars (Achat, Bintje, Agata, Monalisa and Asterix), cultivated in pots containing soil from an integrated system for agroecological production. The experiment was conducted in a split plot randomized block design with five cultivars, three sampling periods and five replicates. Rhizosphere samples were collected in three sampling dates during plant development. DNA of rhizosphere microorganisms was extracted, amplified by PCR using bacterial universal primers, and analyzed through DGGE. Shifts on the rhizosphere bacterial communities associated to rhizosphere of different cultivars were related to both cultivar and plant age. Differences among rhizosphere bacterial communities were clearest at the earliest plant age, tending to decrease in later stages. This variation was detected among bacterial communities of the five tested cultivars. The characterization of soil microbial communities can be part of plant breeding programs to be used on studies of environmental risk assessment of genetically modified potatoes.