Perspectives de l'inactivation des pathogènes: quand la fiction dépasse la réalité [Back to the future: a fiction dealing with pathogen inactivation of blood products].

AIM OF THE PAPER: Arouse the reflection with a fiction having a scientific appearance, presenting a late and unexpected complication of the universal inactivation of pathogens. CONCLUSION: Such a fiction story opens the debate on a series of fundamental questions that could be addressed during the paradigm shift that is expected by introducing universal pathogen inactivation of blood products.

Rotaviruses as a cause of nosocomial, infantile diarrhoea in Northern Brazil: pilot study

Faecal samples were obtained from 190 children, aged 0 to 5 years, admitted to a public hospital in Belém, Pará, Brazil. These patients were placed in a pediatric ward with 40 beds distributed in six rooms. Case were classified into three groups: (a) nosocomial: children who developed gastroenteritis 72 hr or later after admission; (b) community-acquired: patients admitted either with diarrhoea or who had diarrhoea within 72 hr following admission; (c) non-diarrhoeic: those children who had no diarrhoea three days before and three days after collection of formed faecal sample. Specimens were routinely processed for the presence of rotaviruses, bacteria and parasites. Rotaviruses were detected through enzyme-linked immunosorbent assay (ELISA) and subsequently serotyped/electrophoretyped. Rotaviruses were the most prevalent enteropathogens among nosocomial cases, accounting for 39 % (9/23) of diarrhoeal episodes; on the other hand, rotaviruses ocurred in 8.3 % (11/133) and 9 % (3/34) of community-acquired and non-diarrhoeic categories, respectively. Mixed infections involving rotavirus and Giardia intestinalis and rotavirus plus G. intestinalis and Entamoeba histolytica were detected in frequencies of 8.6 and 4.3 %, respectively, in the nosocomial group. The absence of bacterial pathogens in this category, and the unusual low prevalence of these agents in the other two groups may reflect the early and routine administration of antibiotics following admission to this hospital. Rotavirus serotype 2 prevailed over the other types, accounting for 77.8 % of isolates from nosocomial diarrhoeal episodes. In addition, at least five different genomic profiles could be observed, of which one displayed an unusual five-segment first RNA cluster. Dehydration was recorded in all cases of hospital-acquired, rotavirus-associated diarrhoea, whereas in only 57 % of nosocomial cases of other aetiology. It was also noted that nosocomial, rotavirus-associated diarrhoeal episodes occur earlier (7 days), following admission, if compared with those hospital-acquired cases of other aetiology (14 days).

Dried blood spots collected on filter paper: an international resource for the diagnosis and genetic characterization of human immunodeficiency virus Type-1

The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large-scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia.

Mucosal immunology and models of mucosal HIV infection

The mucosa associated lymphoid tissue regulates and coordinates immune responses against mucosal pathogens. Mucosal tissues are the major targets exposed to HIV during transmission. In this paper we describe in vitro models of HIV mucosal infection using human explants to investigate target cells within this tissue.

Conjugation by Mosquito Pathogenic Strains of Bacillus sphaericus

A mosquito pathogenic strain of Bacillus sphaericus carried out the conjugal transfer of plasmid pAMß1 to other strains of its own and two other serotypes. However, it was unable to conjugate with mosquito pathogens from three other serotypes, with B. sphaericus of other DNA homology groups or with three other species of Bacillus. Conjugation frequency was highest with a strain having an altered surface layer (S layer). Conjugal transfer of pAMß1 was not detected in mosquito larval cadavers. B. sphaericus 2362 was unable to mobilize pUB110 for transfer to strains that had served as recipients of pAMß1. These observations suggest that it is unlikely that genetically engineered B. sphaericus carrying a recombinant plasmid could pass that plasmid to other bacteria

Intracellular bacteria and adverse pregnancy outcomes.

Clin Microbiol Infect 2011; 17: 1312-1322 ABSTRACT: This review considers the role of intracellular bacteria in adverse pregnancy outcomes, such as miscarriage, stillbirths, and preterm labour. The cause of miscarriage, stillbirth and preterm labour often remains unexplained. Intracellular bacteria that grow either poorly or not at all on media used routinely to detect human pathogens could be the aetiological agents of these obstetric conditions. For example, Listeria monocytogenes and Coxiella burnetti are intracellular bacteria that have a predilection for the fetomaternal unit and may induce fatal disease in the mother and/or fetus. Both are important foodborne or zoonotic pathogens in pregnancy. Preventive measures, diagnostic tools and treatment will be reviewed. Moreover, we will also address the importance in adverse pregnancy outcomes of other intracellular bacteria, including Brucella abortus and various members of the order Chlamydiales. Indeed, there is growing evidence that Chlamydia trachomatis, Chlamydia abortus and Chlamydia pneumoniae infections may also result in adverse pregnancy outcomes in humans and/or animals. Moreover, newly discovered Chlamydia-like organisms have recently emerged as new pathogens of both animals and humans. For example, Waddlia chondrophila, a Chlamydia-related bacterium isolated from aborted bovine fetuses, has also been implicated in human miscarriages. Future research should help us to better understand the pathophysiology of adverse pregnancy outcomes caused by intracellular bacteria and to determine the precise mode of transmission of newly identified bacteria, such as Waddlia and Parachlamydia. These emerging pathogens may represent the tip of the iceberg of a large number of as yet unknown intracellular pathogenic agents.

Survey of Infectious Etiologies of Bovine Abortion during Mid- to Late Gestation in Dairy Herds.

Bovine abortion of unknown infectious etiology still remains a major economic problem. Thus, we investigated whether Brucella spp., Listeria monocytogenes, Salmonella spp., Campylobacter spp. and Coxiella burnetii are associated with abortion and/or stillbirth in Tunisian dairy cattle. Using a pan-Chlamydiales PCR, we also investigated the role of Chlamydiaceae, Waddlia chondrophila, Parachlamydia acanthamoebae and other members of the Chlamydiales order in this setting. Veterinary samples taken from mid to late-term abortions from twenty dairy herds were tested. From a total of 150 abortion cases collected, infectious agents were detected by PCR in 73 (48.66%) cases, 13 (8.66%) of which represented co-infections with two infectious agents. Detected pathogens include Brucella spp (31.3%), Chlamydiaceae (4.66%), Waddlia chondrophila (8%), Parachlamydia acanthamoebae (5.33%), Listeria monocytogenes (4.66%) and Salmonella spp. (3.33%). In contrast, Campylobacter spp. and Coxiella burnetii DNA were not detected among the investigated veterinary samples. This demonstrates that different bacterial agents may cause bovine abortion in Tunisia. This is the first report suggesting the role of Parachlamydia acanthamoebae in bovine abortion in Africa. Further studies with a larger number of samples are necessary to confirm whether this emerging pathogen is directly linked to abortion in cattle.

Separation and Mapping of Chromosomes of Parasitic Protozoa

Many protozoan parasites represent an important group of human pathogens. Pulsed Field Gradient Gel Electrophoresis (PFGE) analysis has been an important tool for fundamental genetic studies of parasites like Trypanosoma, Leishmania, Giardia or the human malaria parasite Plasmodium falciparum. We present PFGE conditions allowing a high resolution separation of chromosomes ranging from 500 to 4000 kb within a two day electrophoresis run. In addition, we present conditions for separating large chromosomes (2000-6000 kb) within 36 hr. We demontrate that the application of two dimentional PFGE (2D-PFGE) technique to parasite karyotypes is a very useful method for the analysis of dispersed gene families and comparative studies of the intrachomosomal genome organization

Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set.

Plant membrane compartments and trafficking pathways are highly complex, and are often distinct from those of animals and fungi. Progress has been made in defining trafficking in plants using transient expression systems. However, many processes require a precise understanding of plant membrane trafficking in a developmental context, and in diverse, specialized cell types. These include defense responses to pathogens, regulation of transporter accumulation in plant nutrition or polar auxin transport in development. In all of these cases a central role is played by the endosomal membrane system, which, however, is the most divergent and ill-defined aspect of plant cell compartmentation. We have designed a new vector series, and have generated a large number of stably transformed plants expressing membrane protein fusions to spectrally distinct, fluorescent tags. We selected lines with distinct subcellular localization patterns, and stable, non-toxic expression. We demonstrate the power of this multicolor 'Wave' marker set for rapid, combinatorial analysis of plant cell membrane compartments, both in live-imaging and immunoelectron microscopy. Among other findings, our systematic co-localization analysis revealed that a class of plant Rab1-homologs has a much more extended localization than was previously assumed, and also localizes to trans-Golgi/endosomal compartments. Constructs that can be transformed into any genetic background or species, as well as seeds from transgenic Arabidopsis plants, will be freely available, and will promote rapid progress in diverse areas of plant cell biology.

Nitric oxide mediates the microbicidal activity of eosinophils

There are several experimental evidences that nitric oxide (NO) is involved in the microbicidal activity of macrophages against a number of intracellular pathogens including Leishmania major, Trypanozoma cruzi, Toxoplasma gondii. It is also well known that eosinophils (EO) have microbicidal activity against many parasites such as Schistosoma mansoni, Trichinella spiralis, T. cruzi and L. amazonensis. The purpose of this study was to investigate if NO is involved in the microbicidal activity of EO against L. major. Eosinophils harvested from peritoneal cavity of rats released spontaneously after 24 and 48 hr a small amount of nitrite. This release was enhanced by the treatment of cells with IFN-gamma (200 IU/ml). This release was blocked by addition of the NO synthase inhibitor, L-NIO (100 mu M) into the culture. To determinate the leishmanicidal activity of eosinophils the parasites were incubated with activated eosinophils with IFN-gamma and the ability of surviving parasites to incorporate [³H]thymidine was evaluated. IFN-gamma-activated eosinophils were able to kill L. major and to release high levels of nitrite. The ability to destroy L. major and the release of NO were completely blocked by L-NIO. These results indicate that activated eosinophils release NO which is involved in the microbicidal activity of these cells against L. major.

Genomics of "chlamydia"-related bacteria

AbstractThe Chlamydiales order is an important bacterial phylum that comprises some of the most successful human pathogens such as Chlamydia trachomatis, the leading infectious cause of blindness worldwide. Since some years, several new bacteria related to Chlamydia have been discovered in clinical or environmental samples and might represent emerging pathogens. The genome sequencing of classical Chlamydia has brought invaluable information on these obligate intracellular bacteria otherwise difficult to study due to the lack of tools to perform basic genetic manipulation. The recent emergence of high-throughput sequencing technologies yielding millions of reads in a short time lowered the costs of genome sequencing and thus represented a unique opportunity to study Chlamydia-re\ated bacteria. Based on the sequencing and the analysis of Chlamydiales genomes, this thesis provides significant insights into the genetic determinants of the intracellular lifestyle, the pathogenicity, the metabolism and the evolution of Chlamydia-related bacteria. A first approach showed the efficacy of rapid sequencing coupled to proteomics to identify immunogenic proteins. This method, particularly useful for an emerging pathogen such as Parachlamydia acanthamoebae, enabled us to discover good candidates for the development of diagnostic tools that would permit to evaluate at larger scale the role of this bacterium in disease. Second, the complete genome of Waddlia chondrophila, a potential agent of miscarriage, encodes numerous virulence factors to manipulate its host cell and resist to environmental stresses. The reconstruction of metabolic pathways showed that the bacterium possesses extensive capabilities compared to related organisms. However, it is still incapable of synthesizing some essential components and thus has to import them from its host. Third, the genome comparison of Protochlamydia naegleriophila to its closest known relative Protochlamydia amoebophila revealed a particular evolutionary dynamic with the occurrence of an unexpected genome rearrangement. Fourth, a phylogenetic analysis of P. acanthamoebae and Legionella drancourtii identified several genes probably exchanged by horizontal gene transfer with other intracellular bacteria that might occur within their amoebal host. These genes often encode mechanisms for resistance to metal or toxic compounds. As a whole, the analysis of the different genomes enabled us to highlight a large diversity in size, GC percentage, repeat content as well as plasmid organization. The abundant genomic data obtained during this thesis have a wide impact since they provide the necessary bases for detailed investigations on countless aspects of the biology and the evolution of Chlamydia-related bacteria, whether in wet lab or by bioinformatical analyses.RésuméL'ordre des Chlamydiales est un important phylum bactérien qui comprend de nombreuses espèces pathogènes pour l'homme et les animaux, dont Chlamydia trachomatis, responsable du trachome, la cause majeure de cécité d'origine infectieuse à travers le monde. Durant ces dernières décennies, de nombreuses bactéries apparentées aux Chlamydia ont été découvertes dans des échantillons environnementaux ou cliniques mais leur éventuel rôle pathogène dans le développement de maladies reste peu connu. Ces bactéries sont des intracellulaires obligatoires car elles ont besoin d'une cellule hôte pour se multiplier, ce qui rend leur étude particulièrement difficile. Le développement de nouvelles technologies permettant de séquencer le génome d'un organisme rapidement et à moindre coût ainsi que l'essor des méthodes d'analyse s'y rapportant représentent une opportunité exceptionnelle d'étudier ces organismes. Dans ce contexte, cette thèse démontre l'utilité de la génomique pour développer de nouveaux outils diagnostiques ainsi que pour étudier le métabolisme de ces bactéries, leurs facteurs de virulence et leur évolution.Ainsi, une première approche a illustré l'utilité d'un séquençage rapide pour obtenir les informations nécessaires à l'identification de protéines qui sont reconnues par des anticorps humains ou animaux. Cette méthode, particulièrement utile pour un pathogène émergent tel que Parachlamydia acanthamoebae, a permis de découvrir de bons candidats pour le développement d'un outil diagnostique qui permettrait d'évaluer à plus large échelle le rôle de cette bactérie notamment dans la pneumonie. L'analyse du contenu génique de Waddlia chondrophila, un autre germe qui pourrait être impliqué dans les avortements et tes fausses-couches, a en outre mis en évidence la présence de nombreux facteurs connus qui lui permettent de manipuler son hôte. Cette bactérie possède de plus grandes capacités métaboliques que les autres Chlamydia, mais elle est incapable de synthétiser certains composants et doit donc les importer de son hôte pour subvenir à ses besoins. La comparaison du génome de Protochlamydia naegleriophila à son plus proche parent, Protochlamydia amoebophila, a dévoilé une évolution dynamique particulière avec l'occurrence d'un réarrangement majeur inattendu après la séparation de ces deux espèces. En outre, ces études ont montré l'occurrence de plusieurs transferts de gène avec d'autres organismes plus éloignés, notamment d'autres intracellulaires d'amibes, souvent pour l'acquisition de mécanismes de résistances à des composés toxiques. Les données génomiques acquises durant ce travail posent les fondements nécessaires a de nombreuses analyses qui permettront progressivement de mieux comprendre de nombreux aspects de ces bactéries fascinantes.

Short term evolution of a highly transmissible methicillin-resistant Staphylococcus aureus clone (ST228) in a tertiary care hospital.

Staphylococcus aureus is recognized as one of the major human pathogens and is by far one of the most common nosocomial organisms. The genetic basis for the emergence of highly epidemic strains remains mysterious. Studying the microevolution of the different clones of S. aureus is essential for identifying the forces driving pathogen emergence and spread. The aim of the present study was to determine the genetic changes characterizing a lineage belonging to the South German clone (ST228) that spread over ten years in a tertiary care hospital in Switzerland. For this reason, we compared the whole genome of eight isolates recovered between 2001 and 2008 at the Lausanne hospital. The genetic comparison of these isolates revealed that their genomes are extremely closely related. Yet, a few more important genetic changes, such as the replacement of a plasmid, the loss of large fragments of DNA, or the insertion of transposases, were observed. These transfers of mobile genetic elements shaped the evolution of the ST228 lineage that spread within the Lausanne hospital. Nevertheless, although the strains analyzed differed in their dynamics, we have not been able to link a particular genetic element with spreading success. Finally, the present study showed that new sequencing technologies improve considerably the quality and quantity of information obtained for a single strain; but this information is still difficult to interpret and important investments are required for the technology to become accessible for routine investigations.

Redundant Notch1 and Notch2 signaling is necessary for IFNγ secretion by T helper 1 cells during infection with Leishmania major.

The protective immune response to intracellular parasites involves in most cases the differentiation of IFNγ-secreting CD4(+) T helper (Th) 1 cells. Notch receptors regulate cell differentiation during development but their implication in the polarization of peripheral CD4(+) T helper 1 cells is not well understood. Of the four Notch receptors, only Notch1 (N1) and Notch2 (N2) are expressed on activated CD4(+) T cells. To investigate the role of Notch in Th1 cell differentiation following parasite infection, mice with T cell-specific gene ablation of N1, N2 or both (N1N2(ΔCD4Cre)) were infected with the protozoan parasite Leishmania major. N1N2(ΔCD4Cre) mice, on the C57BL/6 L. major-resistant genetic background, developed unhealing lesions and uncontrolled parasitemia. Susceptibility correlated with impaired secretion of IFNγ by draining lymph node CD4(+) T cells and increased secretion of the IL-5 and IL-13 Th2 cytokines. Mice with single inactivation of N1 or N2 in their T cells were resistant to infection and developed a protective Th1 immune response, showing that CD4(+) T cell expression of N1 or N2 is redundant in driving Th1 differentiation. Furthermore, we show that Notch signaling is required for the secretion of IFNγ by Th1 cells. This effect is independent of CSL/RBP-Jκ, the major effector of Notch receptors, since L. major-infected mice with a RBP-Jκ deletion in their T cells were able to develop IFNγ-secreting Th1 cells, kill parasites and heal their lesions. Collectively, we demonstrate here a crucial role for RBP-Jκ-independent Notch signaling in the differentiation of a functional Th1 immune response following L. major infection.

Early expression of the type III secretion system of Parachlamydia acanthamoebae during a replicative cycle within its natural host cell Acanthamoeba castellanii.

The type three secretion system (T3SS) operons of Chlamydiales bacteria are distributed in different clusters along their chromosomes and are conserved at both the level of sequence and genetic organization. A complete characterization of the temporal expression of multiple T3SS components at the transcriptional and protein levels has been performed in Parachlamydia acanthamoebae, replicating in its natural host cell Acanthamoeba castellanii. The T3SS components were classified in four different temporal clusters depending on their pattern of expression during the early, mid- and late phases of the infectious cycle. The putative T3SS transcription units predicted in Parachlamydia are similar to those described in Chlamydia trachomatis, suggesting that T3SS units of transcriptional expression are highly conserved among Chlamydiales bacteria. The maximal expression and activation of the T3SS of Parachlamydia occurred during the early to mid-phase of the infectious cycle corresponding to a critical phase during which the intracellular bacterium has (1) to evade and/or block the lytic pathway of the amoeba, (2) to differentiate from elementary bodies (EBs) to reticulate bodies (RBs), and (3) to modulate the maturation of its vacuole to create a replicative niche able to sustain efficient bacterial growth.

Insect eggs suppress plant defence against chewing herbivores.

Plants activate direct and indirect defences in response to insect egg deposition. However, whether eggs can manipulate plant defence is unknown. In Arabidopsis thaliana, oviposition by the butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens. In the present study, we found that the plant defence signal salicylic acid (SA) accumulates at the site of oviposition. This is unexpected, as the SA pathway controls defence against fungal and bacterial pathogens and negatively interacts with the jasmonic acid (JA) pathway, which is crucial for the defence against herbivores. Application of P. brassicae or Spodoptera littoralis egg extract onto leaves reduced the induction of insect-responsive genes after challenge with caterpillars, suggesting that egg-derived elicitors suppress plant defence. Consequently, larval growth of the generalist herbivore S. littoralis, but not of the specialist P. brassicae, was significantly higher on plants treated with egg extract than on control plants. In contrast, suppression of gene induction and enhanced S. littoralis performance were not seen in the SA-deficient mutant sid2-1, indicating that it is SA that mediates this phenomenon. These data reveal an intriguing facet of the cross-talk between SA and JA signalling pathways, and suggest that insects have evolved a way to suppress the induction of defence genes by laying eggs that release elicitors. We show here that egg-induced SA accumulation negatively interferes with the JA pathway, and provides an advantage for generalist herbivores.