Éditeur(s) : HAL CCSD Oxford University Press (OUP)
Résumé : International audience
Are bisphenol A (BPA) and BPA analogs (BPA-A) safe for male human reproductive function? The endocrine function of human testes explants [assessed by measuring testosterone and insulin-like factor 3 (INSL3)] was impacted by exposure of the human adult testis explants to BPA/BPA-A. The few epidemiologic studies performed suggest that bisphenols have potential endocrine disruptive properties, but they did not identify clear and direct patterns of endocrine disruption. Adult human testis explants in culture were exposed to BPA and the analogs bisphenol F (BPF), bisphenol S (BPS), bisphenol E (BPE), bisphenol B (BPB) and bisphenol A diglycidyl ether (BADGE) at 10(-9)-10(-5) M for 24 or 48 h. Human adult testes were obtained from prostate cancer patients who had no hormone therapy, or from multiorgan donors. After ex vivo exposure to the investigated bisphenols, the measured outcomes were related to histopathology (gross morphology and germ cell viability determined by anti-caspase three immunohistochemistry), and the levels of testosterone, INSL3 and inhibin B were measured using immunoassays. The levels of mRNA encoding key enzymes of bisphenol biotransformation were investigated by quantitative PCR: UGT2B15 UDP (glucuronosyltransferase two family, polypeptide B15), GUSB (glucuronidase beta), SULT1A1 and 3 (sulfotransferase family 1 A member 1 and 3) and STS (steroid sulfatase). A significant dose-dependent inhibition was found between testosterone levels measured in the culture medium and concentrations of BPA (P = 0.00778 at 24 h and P = 0.0291 at 48 h), BPE (P = 0.039) and BPF (P = 0.00663). The observed BPA and BPA-A-induced inhibition of testosterone production varied according to duration of exposure and BPA/BPA-A concentrations. BPA (10(-9) M; P < 0.05), BPB (10(-9) M; P < 0.05), BPS (10(-9) and 10(-8) M; P < 0.05) and BADGE (10(-5) M; P < 0.05) increased Leydig cell INSL3 production. By contrast, BPE dose dependently inhibited INSL3 (P = 0.0372). Conversely, Sertoli cell function (inhibin B) and germ cell viability were not significantly affected by either bisphenols. N/A. Environmental compounds cannot be deliberately administered to men, justifying the use of an ex vivo approach. A relatively low number of testes samples were available for analysis (n = 3, except for testosterone secretion with n = 5). The active concentrations of BPA and BPA-A used in the study were higher than those found in human biological fluids. Under our experimental conditions, direct exposure to BPA or BPA-A can result in endocrine disturbance in the adult human testis.
ISSN: 0268-1161

hal-01578582
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578582
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578582/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01578582/file/Parallel%20assessment%20of%20the%20effects%20of%20bisphenol%20A_accepted.pdf
DOI : 10.1093/humrep/dex093

Éditeur(s) : HAL CCSD Elsevier Masson
Résumé : International audience
The diagnosis of diabetic foot infections is difficult due to limitations of conventional culture-based techniques. The objective of this study was to evaluate the contribution of denaturing gradient gel electrophoresis (DGGE) in the microbiological diagnosis of diabetic foot ulcers in comparison to conventional techniques, and also to evaluate the need to perform a biopsy sample for this diagnosis. Twenty diabetic patients (types 1 and 2) with foot ulcers (grades 1-4) were included. After debridement of their wounds, samples were taken in duplicate by surface swabbing and deep-tissue biopsy. The samples were analyzed by conventional culture and by a new molecular biology tool, DGGE technology. Polymerase chain reaction (PCR)-DGGE led to the identification of more bacteria than did conventional cultures (mean: 2.35 vs 0.80, respectively). In 11 cases, the technology detected pathogenic species not isolated by classical cultures. PCR-DGGE also identified significantly more pathogenic species at deep levels compared with species detected at superficial levels (87% vs 58%, respectively; P = 0.03). In 9/20 cases, pathogenic bacteria were detected only in deep samples, revealing the need to perform tissue biopsy sampling. DGGE, achievable in 48h, could be a useful technique for the bacteriological diagnosis of diabetic foot infections. It may help to identify pathogenic bacteria in deeply infected ulcers, thereby contributing to a more appropriate use of antibiotics.
ISSN: 1262-3636

hal-01122240
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01122240
DOI : 10.1016/j.diabet.2014.03.002
PUBMED : 24751989

Éditeur(s) : Inter-Research
Résumé : Experimental infections of Penaeus (Litopenaeus) stylirostris were performed with a Vibrio penaeicida strain (AM101) isolated in New Caledonia from Syndrome 93 diseased shrimp. Cumulative mortalities resulting from intramuscular injection or immersion of shrimp in bacterial suspensions demonstrated high virulence for this bacterial strain and suggested that V. penaeicida could be the etiological agent of Syndrome 93. The median lethal dose (LD50) for AM101 was 1.3 x 104 CFU (colony forming units) ml-1 by immersion and less than 5 CFU shrimp-1 by intramuscular challenge, with mortality outbreaks at 48 and 22 h after challenge, respectively. A polymerase chain reaction (PCR) detection assay using a primer set designed from the 16S ribosomal RNA gene of V. penaeicida was developed. It gave an expected amplicon of approximately 310 bp in ethidium bromide-stained agarose gels. The specificity of these primers was assessed with different Vibrio species. Furthermore, DNA extracted by the ChelexTM method could be used to detect fewer than 20 cultured Vibrio cells in seawater or shrimp hemolymph by this assay. It appears to be a reliable screening method for detecting V. penaeicida in shrimp and from the aquatic environment.
Diseases of aquatic organisms (0177-5103) (Inter-Research), 2000-03 , Vol. 49 , N. 2 , P. 109-115

Droits : Inter-Research 2000
http://archimer.ifremer.fr/doc/2000/publication-4436.pdf
DOI:10.3354/dao040109
http://archimer.ifremer.fr/doc/00000/4436/

Éditeur(s) : Elsevier
Résumé : In a previous study, we demonstrated the existence of an emerging cluster of Vibrio nigripulchritudo that proved to be associated with shrimp mortality events in New Caledonia. Using sequence polymorphisms evidenced in this previous MultiLocus Sequence Typing study, we developed two new quantitative PCR assays permitting the detection and quantification of V. nigripulchritudo at the genospecies level using SYBR Green I chemistry and at the emerging cluster level using Fluorescence Resonance Energy Transfer technology with hybridization probes. The use of this molecular diagnostic tool evidenced the colonization of the shrimp pond ecosystem by the pathogenic cluster at least at the onset of the disease. This new tool will allow better investigation of the dynamics of this bacterial pathogen in the shrimp farm ecosystem. (C) 2007 Elsevier B.V. All rights reserved.
Journal of Microbiological Methods (0167-7012) (Elsevier), 2007-07 , Vol. 70 , N. 1 , P. 30-38

Droits : 2007 Elsevier B.V. All rights reserved
http://archimer.ifremer.fr/doc/2007/publication-2729.pdf
DOI:10.1016/j.mimet.2007.03.007
http://archimer.ifremer.fr/doc/00000/2729/

Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
BACKGROUND: Reliable molecular typing tools are required for a better understanding of the molecular epidemiology of Plasmodium vivax. The genes msp3a and msp1_block5 are highly polymorphic and have been used as markers in many P. vivax population studies. These markers were used to assess the genetic diversity of P. vivax strains from French Guiana (South America) and to develop a molecular typing protocol. METHODS: A total of 120 blood samples from 109 patients (including 10 patients suffered from more than one malaria episode, samples were collected during each episode) with P. vivax infection were genotyped. All samples were analysed by msp3a PCR-RFLP and msp1_b5 gene sequencing was performed on 57 samples. Genotyping protocol applied to distinguish between new infection or relapse from heterologus hypnozoites and treatment failure or relapse from homologus hypnozoites was based on analysing first msp3a by PCR-RFLP and secondly, only if the genotypes of the two samples are identical, on sequencing the msp1_b5 gene. RESULTS: msp3a alleles of three sizes were amplified by PCR: types A, B and C. Eleven different genotypes were identified among the 109 samples analysed by msp3a PCR-RFLP. In 13.8% of cases, a mixed genotype infection was observed. The sequence of msp1_b5 gene revealed 22 unique genotypes and 12.3% of cases with mixed infection. In the 57 samples analysed by both methods, 45 genotypes were found and 21% were mixed. Among ten patients with two or three malaria episodes, the protocol allowed to identify five new infections or relapses from heterologous hypnozoites and six treatment failures of relapses from homologous hypnozoites. CONCLUSION: The study showed a high diversity of msp3a and msp1_b5 genetic markers among P. vivax strains in French Guiana with a low polyclonal infection rate. These results indicated that the P. vivax genotyping protocol presented has a good discrimination power and can be used in clinical drug trials or epidemiological studies.
ISSN: 1475-2875

pasteur-00590981
https://hal-riip.archives-ouvertes.fr/pasteur-00590981
DOI : 10.1186/1475-2875-8-40
PUBMED : 19284592

Éditeur(s) : HAL CCSD Saint-Maurice (Val de Marne) : Institut de veille sanitaire
Résumé : National audience
Introduction – Le nombre d’accès palustres diminue globalement en Guyane, alors que les orpailleurs travaillantsur les sites illégaux semblent particulièrement touchés par cette pathologie. Les objectifs de cette étudeétaient de déterminer la prévalence du paludisme dans cette population, les comportements associés ainsi quele niveau de résistance des parasites vis-à-vis des dérivés de l’artémisinine.Matériel et méthodes – Les inclusions ont eu lieu sur les sites de repli fréquentés par les orpailleurs le long dufleuve Maroni. Ont été effectués : un test de diagnostic rapide du paludisme, un questionnaire, un prélèvementsanguin pour PCR et un génotypage du gène pfK13 pour les PCR positives à Plasmodium falciparum.Résultats – De janvier à juin 2015, 421 orpailleurs ont été inclus, majoritairement des hommes (70,6%) brésiliens(93,8%). La prévalence du portage de plasmodies déterminée par PCR était en moyenne de 22,3%(IC95%: [18,3-26,3]), à 84% asymptomatiques. Les espèces identifiées étaient principalement P. falciparum(47,9%) puis P. vivax (37,2%), plus 10,6% de co-infections. Lors du dernier accès palustre, 52,4% des orpailleursavaient eu recours à l’automédication, majoritairement avec des dérivés de l’artémisinine (93,8%) avecune mauvaise observance (37,8%). L’analyse du gène pfK13 n’a pas mis en évidence de mutations associéesà la résistance à P. falciparumDiscussion – La prévalence élevée de porteurs asymptomatiques de plasmodies constitue un réservoir importantde transmission dans la région. L’utilisation massive de dérivés de l’artémisinine associée à une mauvaise observancedes traitements sont des facteurs de risque d’émergence de résistance imposant des mesures rapides
ISSN: 0245-7466

inserm-01491193
http://www.hal.inserm.fr/inserm-01491193
http://www.hal.inserm.fr/inserm-01491193/document
http://www.hal.inserm.fr/inserm-01491193/file/2017%2C%20Douine%20-%20Paludisme%20et%20orpaillage%20ill%C3%A9gal%20en%20Guyane.pdf

Éditeur(s) : HAL CCSD Elsevier Masson
Résumé : International audience
PURPOSE: Two hundred and fifty-three patients were included in a study on the etiology of encephalitis, carried out in France in 2007. Tuberculosis was the second most frequently identified cause, after HSV and with the same number of cases as VZV. The authors report the specific features of patients presenting with tuberculosis encephalitis (TE). METHODS: TE patients were defined as patients presenting with encephalitis, with positive culture or PCR for Mycobacterium tuberculosis, or the association of clinical, biological, imaging, and epidemiological evidence (possible cases). Clinical, microbiological, and brain imaging data was analyzed and compared to that of other included patients. RESULTS: Twenty cases of TE were identified. The M/F sex-ratio was 1.5, the mean age 53 years. Four (20%) patients had a history of tuberculosis before the encephalitis. The median delay between the onset of general and neurological symptoms was significantly longer for tuberculosis cases than for others (10 days vs. 2; P<10(-10)). The median CSF protein level was significantly higher for tuberculosis cases (2.1 g/L vs. 0.8 g/L, P=0.002). CT scan and MRI were normal on admission for eight patients out of 17. Fourteen isolated strains of M. tuberculosis were susceptible to first-line anti-tuberculosis drugs and one was rifampicin-resistant. Six (33%) patients died during hospitalization and two were lost to follow-up. Ten out of 12 (78.6%) had persisting neurological symptoms on discharge. DISCUSSION: Despite non-multiresistant MT strains, the case fatality rate among TE patients was high in our series. Early brain imaging is poor contributive for the diagnosis of TE.
ISSN: 0399-077X

hal-00874835
https://hal.archives-ouvertes.fr/hal-00874835
DOI : 10.1016/j.medmal.2013.05.003
PUBMED : 23816146

Éditeur(s) : Elsevier
Résumé : Shrimp farming is a small but growing industry in New Caledonia. Since 1993, "Syndrome 93" has been affecting New Caledonian shrimp farming industry every cold season, causing severe epizootic mortalities in grow-out ponds and significant losses. Highly pathogenic strains of Vibrio penaeicida are considered the etiological agent of the disease in Litopenaeus stylirostris. On one hand, studies demonstrated that healthy shrimp may carry V penaeicida for weeks with a high overall prevalence, regardless of any seasonal pattern or temperature conditions. On the other hand, larvae are free of V penaeicida and are also resistant to experimental infection. V penaeicida is frequently detected in incoming water pumped from the bays, which was shown, by a molecular typing study, to be the infectious source. This particular epidemiological pattern highlights the major role of the factors that trigger and aggravate the disease in grow-out ponds, where shrimp populations carry the pathogen all year round. In order to gain a better understanding of "Syndrome 93" epidemiology, quantification of V penaeicida both in shrimp and the shrimp farm ecosystem is necessary. This article describes the steps in the successful development of a real-time PCR quantification assay of V penaeicida in shrimp haemolymph, seawater (from ponds or bays) and sediment pore water, including the choice of an accurate extraction technique. The entire detection method; including sample processing, DNA extraction and real-time PCR amplification, can be completed within 4 h. (c) 2006 Elsevier B.V All rights reserved.
Journal of Microbiological Methods (0167-7012) (Elsevier), 2006-10 , Vol. 67 , N. 1 , P. 27-35

Droits : 2006 Elsevier
http://archimer.ifremer.fr/doc/2006/publication-1903.pdf
DOI:10.1016/j.mimet.2006.02.013
http://archimer.ifremer.fr/doc/00000/1903/

Éditeur(s) : Wiley-blackwell
Résumé : Aims To describe the diversity of the culturable mesophilic and potentially pathogenic vibrios isolated at 22 and 37 degrees C on TCBS medium, in September 2009 from seawater and surface sediments. Methods and Results q-PCR assays previously selected for the identification of bacterial strains isolated at 37 degrees C were used in combination with the partial sequencing of two housekeeping genes, pyrH and toxR, to identify 315 strains isolated at 22 degrees C. The great majority of the 37 degrees C strains was identified by q-PCR assays, (five of the six species) with the predominance of Vibrio alginolyticus (85 center dot 9%) and V.harveyi (10 center dot 7%). The human pathogens V.parahaemolyticus and V.cholerae were rarely detected (two strains each). The 22 degrees C strains were successfully identified by the phylogeny analysis of pyrH and toxR genes, revealing 20 Vibrio species, with the predominance of the clam pathogen V.celticus (36 center dot 8%). The Splendidus and the Harveyi groups represented the main Vibrio group at 22 degrees C (80%) and 37 degrees C (99 center dot 5%), respectively. Conclusions The combination of q-PCR assays and the sequencing of pyrH and toxR genes highlighted two different Vibrio communities at 22 and 37 degrees C both dominated by pathogenic species for marine organisms. Significance and Impact of the Study The sequencing of the pyrH gene revealed to be a valuable tool to identify environmental Vibrio spp. strains isolated at 22 degrees C, as 92 center dot 3% of them were identified in this study.
Journal Of Applied Microbiology (1364-5072) (Wiley-blackwell), 2013-06 , Vol. 114 , N. 6 , P. 1713-1724

Droits : 2013 The Society for Applied Microbiology
http://archimer.ifremer.fr/doc/00144/25502/24832.pdf
DOI:10.1111/jam.12181
http://archimer.ifremer.fr/doc/00144/25502/

Éditeur(s) : HAL CCSD
Résumé : Enamel formation requires the involvement of secretory calcium-binding phosphoproteins. Three of them, amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM), have been extensively studied in mammals. More recently, a fourth protein belonging to the same family has been identified: amelotin (AMTN). AMTN has only been studied in rodents and its role during amelogenesis remains unclear.The aim of this thesis is to extend the knowledge on AMTN by studying its evolution in amniotes and its expression in the lizard Anolis carolinensis.AMTN sequences from many amniote species have been extracted from databases or obtained by PCR, and analyzed. Those analyses allowed us to reveal differences in the gene structure and to highlight residues that were conserved during mammalian or amniote evolution. These conserved amino acids are essential to the structure and/or function of the protein.The expression of AMTN and of AMEL, AMBN and ENAM has been studied by in situ hybridization on lizard jaw sections. Comparison of the expression pattern of these genes during amelogenesis in the lizard with that described in rodents points to similarities (AMEL, AMBN and ENAM) but also to important differences, especially in the spatio-temporal expression of AMTN. In order to better understand AMTN evolution in tetrapods, I studied its expression in an amphibian (Pleurodeles waltl) and a marsupial (Monodelphis domestica).Taken together, these results suggest a link between the evolution of AMTN gene structure (loss of exons and of functional domains in mammals) and its expression (early in non-mammals versus late in mammals) with the emergence of prismatic structure of enamel in early mammals.
La formation de l'émail dentaire met en jeu des phosphoprotéines sécrétées liant le calcium. Parmi celles-ci figurent amélogénine (AMEL), améloblastine (AMBN) et énaméline (ENAM), qui ont fait l'objet de nombreuses études chez les mammifères. Plus récemment, une quatrième protéine de la même famille a été découverte, l'amélotine (AMTN). Elle n'a été étudiée que chez les rongeurs et son rôle au cours de l'amélogenèse reste mal défini. L'objectif de cette thèse est d'élargir les connaissances sur AMTN en étudiant son évolution chez les amniotes et son expression au cours de l'amélogenèse chez le lézard Anolis carolinensis. Les séquences d'AMTN d'un grand nombre d'amniotes ont été extraites des bases de données ou obtenues par PCR, et analysées. Ces analyses ont mis en évidence différentes structures géniques et des acides aminés très conservés au cours de l'évolution. Cette conservation indique qu'ils sont essentiels à la structure ou à la fonction de la protéine. L'expression d'AMTN ainsi que celle d'AMEL, AMBN et ENAM ont été étudiées par hybridation in situ sur des coupes de mâchoires de lézard. La comparaison des patrons d'expression chez le lézard avec ceux décrits chez la souris a révélé des différences dans l'expression spatio-temporelle d'AMTN. De ce fait, afin de mieux comprendre l'évolution de ce gène, son expression a été étudiée chez un amphibien (Pleurodeles waltl) et un marsupial (Monodelphis domestica).Ces travaux suggère un lien entre l'évolution de la structure génique d'AMTN (perte d'exons et de domaines fonctionnels) et de son expression (précoce versus tardive) avec l'émergence de la structure prismatique de l'émail chez les mammifères.
https://tel.archives-ouvertes.fr/tel-01154518

NNT : 2015PA066066
tel-01154518
https://tel.archives-ouvertes.fr/tel-01154518
https://tel.archives-ouvertes.fr/tel-01154518/document
https://tel.archives-ouvertes.fr/tel-01154518/file/2015PA066066.pdf

Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
Well studied in mammals, amelogenesis is less known at the molecular level in reptiles and amphibians. In the course of extensive studies of enamel matrix protein (EMP) evolution in tetrapods, we look for correlation between changes in protein sequences and temporospatial protein gene expression during amelogenesis, using an evo-devo approach. Our target is the major EMP, amelogenin (AMEL) that plays a crucial role in enamel structure. We focused here our attention to an amphibian, the salamander Pleurodeles waltl. RNAs were extracted from the lower jaws of a juvenile P. waltl and the complete AMEL sequence was obtained using PCR and RACE PCR. The alignment of P. waltl AMEL with other tetrapodan (frogs, reptiles and mammals) sequences revealed residue conservation in the N- and C-terminal regions, and a highly variable central region. Using sense and anti-sense probes synthetized from the P. waltl AMEL sequence, we performed in situ hybridization on sections during amelogenesis in larvae, juveniles, and adults. We demonstrated that (i) AMEL expression was always found to be restricted to ameloblasts, (ii) the expression pattern was conserved through ontogeny, even in larvae where enameloid is present in addition to enamel, and (iii) the processes are similar to those described in lizards and mammals. These findings indicate that high variations in the central region of AMEL have not modified its temporospatial expression during amelogenesis for 360 million years of tetrapod evolution. J. Exp. Zool. (Mol. Dev. Evol.) 320B:200209, 2013.
ISSN: 1552-5007

hal-01545483
https://hal.archives-ouvertes.fr/hal-01545483
DOI : 10.1002/jez.b.22494

Éditeur(s) : Elsevier
Résumé : A collection of 57 isolates of Vibrio nigripulchritudo from either diseased or healthy shrimp and from shrimp farms environment was studied in order to gain a better understanding of the epidemiology of this pathogen, notably isolated from two distinct shrimp disease complexes. Molecular typing using two different techniques, arbitrarily primed PCR (AP-PCR) and multi-locus sequence typing (MLST), studied together with experimental pathology data allowed a relevant epidemiological insight into this possibly emerging pathogen. Additionally, results obtained with the two molecular typing techniques were congruent and allowed discriminating the strains associated with the "Summer Syndrome" from strains isolated from other contexts, especially the other shrimp vibriosis "Syndrome 93". These results highlight that the "Summer Syndrome" is most probably caused by an emergent clonal pathogen that therefore deserves surveillance and that AP-PCR can satisfactorily be used for that purpose
Systematic and Applied Microbiology (0723-2020) (Elsevier), 2006-11 , Vol. 29 , N. 7 , P. 570-580

Droits : 2005 Elsevier
http://archimer.ifremer.fr/doc/2006/publication-1913.pdf
DOI:10.1016/j.syapm.2005.12.005
http://archimer.ifremer.fr/doc/00000/1913/

Éditeur(s) : HAL CCSD European Centre for Disease Prevention and Control
Résumé : International audience
Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally.
ISSN: 1560-7917

hal-01460743
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01460743
DOI : 10.2807/1560-7917.ES.2016.21.49.30418
PUBMED : 27983510

Éditeur(s) : HAL CCSD Oxford University Press
Résumé : International audience
Chronic rhinosinusitis (CRS) rank second at chronic inflammatory diseases in industrialized countries and are an important public health concern. Diagnosis relies on a set of arguments including clinical signs, imaging, histopathologic and mycological analyses of sinus specimens, collected during nasal endoscopy. The sensitivity of fungal cultures is reported to be poor, even when direct examination is positive, thus the epidemiology of fungal chronic sinusitis is ill-known. This study evaluated the sensitivity of molecular diagnosis in 70 consecutive samples (61 patients with CRS) analysed at the University Hospital of Rennes during a 3-year period. DNA detection was performed using a conventional PCR method targeting the ITS1/ITS2 sequence and the resulting amplification products were sequenced. Fungal CRS was proven in 42 patients (69%), of which only 20 (48%) had a positive culture. 37/42 (88%) patients were diagnosed with a fungus ball, 3 with allergic fungal CRS and 2 with undetermined fungal CRS. PCR was positive in all 42 cases and direct sequencing allowed to identify fungi in all cases but one, and detected multiple infection in 3. Aspergillus fumigatus was present in 69% of patients; Cladosporium cladosporoides in 9.5%, Scedosporium sp., A. nidulans and A. flavus in 7% each. In 2/19 patients with negative direct examination, sequencing analysis revealed the presence of Capnobotryella sp. and C. cladosporoides, in clinical settings compatible with fungal sinusitis. In conclusion, ITS1/ITS2 PCR had a twice better sensitivity than culture, and combined sequencing provides accurate epidemiological data on fungal CRS.
ISSN: 1369-3786

hal-01383632
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01383632
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01383632/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01383632/file/Contribution%20of%20molecular%20tools%20for%20the%20diagnosis.pdf
DOI : 10.1093/mmy/myw041

Éditeur(s) : HAL CCSD Wiley/Blackwell
Résumé : International audience
Microsatellites are widely used in population genetics to uncover recent evolutionary events. They are typically genotyped using capillary sequencer, which capacity is usually limited to 9, at most 12 loci for each run, and which analysis is a tedious task that is performed by hand. With the rise of next-generation sequencing (NGS), a much larger number of loci and individuals are available from sequencing: for example, on a single run of a GS Junior, 28 loci from 96 individuals are sequenced with a 30X cover. We have developed an algorithm to automatically and efficiently genotype microsatellites from a collection of reads sorted by individual (e.g. specific PCR amplifications of a locus or a collection of reads that encompass a locus of interest). As the sequencing and the PCR amplification introduce artefactual insertions or deletions, the set of reads from a single microsatellite allele shows several length variants. The algorithm infers, without alignment, the true unknown allele(s) of each individual from the observed distributions of microsatellites length of all individuals. MicNeSs, a python implementation of the algorithm, can be used to genotype any microsatellite locus from any organism and has been tested on 454 pyrosequencing data of several loci from fruit flies (a model species) and red deers (a nonmodel species). Without any parallelization, it automatically genotypes 22 loci from 441 individuals in 11 hours on a standard computer. The comparison of MicNeSs inferences to the standard method shows an excellent agreement, with some differences illustrating the pros and cons of both methods.
ISSN: 1755-098X

hal-01544754
https://hal.archives-ouvertes.fr/hal-01544754
DOI : 10.1111/1755-0998.12467

Éditeur(s) : HAL CCSD Taylor & Francis
Résumé : International audience
Abstract Indoor fungal contamination has been associated with a wide range of adverse health effects, including infectious diseases, toxic effects and allergies. The diversity of fungi contributes to the complex role that they play in indoor environments and human diseases. Molds have a major impact on public health, and can cause different consequences in hospitals, homes and workplaces. This review presents the methods used to assess fungal contamination in these various environments, and discusses advantages and disadvantages for each method in consideration with different health risks. Air, dust and surface sampling strategies are compared, as well as the limits of various methods are used to detect and quantify fungal particles and fungal compounds. In addition to conventional microscopic and culture approaches, more recent chemical, immunoassay and polymerase chain reaction (PCR)-based methods are described. This article also identifies common needs for future multidisciplinary research and development projects in this field, with specific interests on viable fungi and fungal fragment detections. The determination of fungal load and the detection of species in environmental samples greatly depend on the strategy of sampling and analysis. Quantitative PCR was found useful to identify associations between specific fungi and common diseases. The next-generation sequencing methods may afford new perspectives in this area.
ISSN: 1040-841X

hal-00874670
https://hal.archives-ouvertes.fr/hal-00874670
DOI : 10.3109/1040841X.2013.777687
PUBMED : 23586944

Éditeur(s) : HAL CCSD Wiley
Résumé :  
The symbiotic interactions between Frankia strains and their associated plants from the Casuarinaceae under controlled conditions are well documented but little is known about these interactions under natural conditions. We explored the symbiotic interactions between eight genotypically characterized Frankia strains and five Casuarinaceae species in long-term field trials. Characterization of strains was performed using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) for the nifD-nifK intergenic transcribed spacer (ITS) and 16S-23S ITS. Assessments of the symbiotic interactions were based on nodulation patterns using nodule dry weight and viability, and on actual N-2 fixation using the delta N-15 method. The PCR-RFLP patterns showed that the analyzed strains belonged to the same genotypic group (CeD group), regardless of the host species and environment of origin. The nodule viability index is introduced as a new tool to measure the viability of perennial nodules and to predict their effectiveness. The host Casuarinaceae species was a key factor influencing both the actual N-2-fixing activity of the associated Frankia strain and the viability of nodules within a location. This is the first study providing information on the symbiotic interactions between genotypically characterized Frankia strains and actinorhizal plants under natural conditions. The results revealed a way to improve a long-term management of the Casuarinaceae symbiosis.
ISSN: 0031-9317

hal-01032134
https://hal.archives-ouvertes.fr/hal-01032134
DOI : 10.1111/j.1399-3054.2007.00856.x

Éditeur(s) : HAL CCSD Wiley
Résumé : International audience
The deeply branching Deinococcus-Thermus lineage is recognized as one of the most extremophilic phylum of bacteria. In previous studies, the presence of Deinococcus-related bacteria in the hot arid Tunisian desert of Tataouine was demonstrated through combined molecular and culture-based approaches. Similarly , Thermus-related bacteria have been detected in Tunisian geothermal springs. The present work was conducted to explore the molecular diversity within the Deinococcus-Thermus phylum in these extreme environments. A set of specific primers was designed in silico on the basis of 16S rRNA gene sequences, validated for the specific detection of reference strains, and used for the polymerase chain reaction (PCR) amplification of metagenomic DNA retrieved from the Tataouine desert sand and Tunisian hot spring water samples. These analyses have revealed the presence of previously undescribed Deino-coccus-Thermus bacterial sequences within these extreme environments. The primers designed in this study thus represent a powerful tool for the rapid detection of Deinococcus-Thermus in environmental samples and could also be applicable to clarify the biogeography of the Deinococcus-Thermus phylum.
ISSN: 2045-8827

Droits : http://creativecommons.org/licenses/by/
hal-01560945
https://hal-amu.archives-ouvertes.fr/hal-01560945
https://hal-amu.archives-ouvertes.fr/hal-01560945/document
https://hal-amu.archives-ouvertes.fr/hal-01560945/file/Theodorakopoulos-2013.pdf
DOI : 10.1002/mbo3.119

Éditeur(s) : Springer
Résumé : Two recurring syndromes threaten the viability of the shrimp industry in New Caledonia, which represents the second largest export business. The "Syndrome 93" is a cold season disease due to Vibrio penaeicida affecting all shrimp farms, while the "Summer Syndrome" is a geographically restricted vibriosis caused by a virulent lineage of Vibrio nigripulchritudo. Microbiological procedures for diagnosis of these diseases are time-consuming and do not have the ability to discriminate the range of virulence potentials of V. nigripulchritudo. In this study, we developed a multiplex PCR method to simultaneously detect these two bacterial species and allow for pathotype discrimination. The detection limits of this assay, that includes an internal amplification control to eliminate any false-negative results, were determined at 10 pg purified DNA and 200 cfu/ml. After confirming the effectiveness of our method using experimentally infected animals, its accuracy was compared to standard biochemical methods during a field survey using 94 samples collected over 3 years from shrimp farms encountering mortality events. The multiplex PCR showed very high specificity for the detection of V. penaeicida and V. nigripulchritudo (inclusivity and exclusivity 100%) and allowed us to detect the spreading of highly pathogenic isolates of V. nigripulchritudo to a farm adjoining the "Summer Syndrome area." This assay represents a simple, rapid, and cost-effective diagnostic tool for implementing timely risk management decisions but also understanding the seasonal and geographical distribution of these pathogens.
Microbial Ecology (0095-3628) (Springer), 2012-01 , Vol. 63 , N. 1 , P. 127-138

Droits : Springer Science+Business Media, LLC 2011
http://archimer.ifremer.fr/doc/00063/17398/15169.pdf
DOI:10.1007/s00248-011-9951-3
http://archimer.ifremer.fr/doc/00063/17398/

Éditeur(s) : HAL CCSD
Résumé : Transposable elements play a major role in the evolution of eukaryotic genomes. Knowing the distribution of transposable elements between different species within the same taxon is essential to study their dynamics and to better understand their role in the evolution of species. Given their abundance, species diversity and living environment, crustaceans are an excellent model for studying comparative genomics of retrotransposons. It is notably in the squat lobsters that the GalEa clade of Superfamily Copia was defined. We studied the distribution of two well-known LTR retrotransposons superfamilies: Gypsy and Copia, in crustaceans. By combining PCRs with degenerate primers and in silico analysis, we identified 35 families of Copia retrotransposons and 46 families of Gypsy retrotransposons in 15 and 18 species of crustaceans (mainly Malacostraca: crabs, shrimp, krill ...). These elements have different distribution and diversity in crustaceans. Gypsy elements appear relatively commonly and diverse in all species. Conversely, the Copia elements seem rare, and consequently more difficult to detect, and are largely dominated by the elements of the clade GalEa. These results suggest two different dynamic strategies for retrotransposons Gypsy (the Red Queen theory) and retrotransposons GalEa (‘domino days spreading’ branching process). In addition, GalEa elements present a great evolutionary success being widely distributed in many branches of metazoans. They are also present in certain red algae and we have also detected them in Fungi. Taking advantage of the large amount of available genomic data, we have studied the distribution of GalEa elements of Fungi, in order to compare it with the results obtained in crustaceans. In fact, they appear only in a large phylum of Ascomycetes, in Pezizomycotina, and they form a monophyletic group within the GalEa. Finally, in the Fungi, the GalEa elements are not majority among Copia retrotransposons. We have therefore initiated a new study in molluscs, to define if the results obtained in crustaceans are a feature of GalEa elements, Malacostraca or metazoans.
Les éléments transposables jouent un rôle majeur dans l’évolution des génomes eucaryotes. La connaissance de la distribution des éléments transposables entre différentes espèces au sein d’un même taxon est une condition essentielle pour étudier leur dynamique et mieux comprendre leur rôle dans l'évolution des espèces. Compte tenu de leur abondance, de leur diversité spécifique et de milieu de vie, les crustacés sont un excellent modèle pour étudier la génomique comparative des rétrotransposons. C’est notamment chez les Galathées qu’a été défini le clade GalEa des éléments de la superfamille des Copia. Nous avons étudié la distribution de deux superfamilles de rétrotransposons à LTR bien connus: les Gypsy et les Copia, au sein des crustacés. En combinant des PCRs avec amorces dégénérées et des analyses in silico, nous avons identifié 35 familles de rétrotransposons Copia et 46 familles de rétrotransposons Gypsy dans respectivement 15 et 18 espèces de crustacés (principalement des malacostracés : crabes, crevettes, krill...). Ces éléments présentent une distribution et une diversité différentes au sein des crustacés. Les éléments Gypsy apparaissent relativement fréquents et diversifiés dans toutes les espèces. A l’inverse, les éléments Copia semblent rares, donc difficilement détectables, et sont largement dominés par les éléments du clade GalEa. Ces résultats suggèrent deux stratégies différentes de dynamique pour les rétrotransposons Gypsy (théorie de la Reine Rouge) et les rétrotransposons GalEa (‘domino days spreading’ branching process). De plus, les éléments GalEa présentent un grand succès évolutif en étant largement distribués dans de nombreuses branches de métazoaires. Ils sont aussi présents chez quelques algues rouges et nous en avons également détecté chez des Fungi. Profitant des nombreuses données génomiques disponibles, nous avons donc étudié la distribution des éléments GalEa de Fungi, dans le but de comparer celle-ci aux résultats obtenus chez les crustacés. En fait, ils n’apparaissent qu’au sein d’un grand embranchement d’ascomycètes, les Pezizomycotina, et ils forment un groupe monophylétique au sein des GalEa. Enfin, chez les Fungi, les éléments GalEa ne sont pas majoritaire parmi les rétrotransposons Copia. Nous avons donc initié une nouvelle étude chez les mollusques, afin de définir si les résultats obtenus chez les crustacés sont une caractéristique des éléments GalEa, des malacostracés ou des métazoaires.
https://tel.archives-ouvertes.fr/tel-01132413

NNT : 2015PA066017
tel-01132413
https://tel.archives-ouvertes.fr/tel-01132413
https://tel.archives-ouvertes.fr/tel-01132413/document
https://tel.archives-ouvertes.fr/tel-01132413/file/these_archivage_2600252o.pdf