Impact des résidus de biocides sur la colonisation bactérienne de surfaces alimentaires

Le principal enjeu de ce stage était la détermination de l’impact des résidus de Triameen et de CDDAsur la colonisation d’ E. coli et de L. monocytogenes sur des supports en inox. Les résultats de cetteétude, ont montré une diminution de la colonisation des formes VC pour certaines concentrations deces produits. Cette diminution pourrait être due (a) au conditionnement des supports par des concentrationssupérieures aux concentrations sub-létales ; ou bien (b) au changement phénotypique desbactéries stressées qui passent à l’état VNC. Cette seconde hypothèse a été en partie vérifiée puisquedes formes VNC ont été détectées pour une concentration en Triameen résultant en une diminutiondes formes VC chez E. coli ATCC 25 922. Pour aucune autre souche et/ou conditions, des formes VNCont été mises en évidence. Cependant, ces résultats sont à prendre avec les limites de techniques utilisées.En effet, la méthode PCRq-PMA mise en place au laboratoire manque de sensibilité : le nombrede bactéries VT en particulier pour L. monocytogenes n’est pas assez important pour atteindre la limitede positivité de la PCRq. L’autre facteur important pouvant jouer sur les formes VT est la concentrationen PMA, le traitement d’une suspension d’environ 500 μL avec 50 μM de PMA pourrait être insuffisant.La quantification des formes VT par la PCRq-PMA doit être optimisée.La comparaison inter-souches de la capacité de multiplication, dans les mêmes conditions expérimentalesque les essais de colonisation, a montré que les souches isolées d’industries alimentaires possèdentune meilleure capacité de multiplication. Ces dernières pourraient s’adapter rapidement auxproduits désinfectants auxquels elles sont exposées. Par ailleurs, une étude comparative sur l’impactde la présence de BAC sur des surfaces en polystyrène pour des souches de P. aeruginosa adaptés ounon à des concentrations croissantes de BAC, a montré que la colonisation bactérienne était plus élevéepour les souches adaptées (Machado et al., 2011). Ces résultats nous laissent penser que si lessouches testées étaient préalablement adaptées au Triameen et au CDDA, des résultats différentspourraient être obtenus. Par ailleurs, ces expériences permettraient de se rapprocher davantage desconditions de terrain

Detection of Clostridium botulinum group III in environmental samples from farms by real-time PCR using four commercial DNA extraction kits

Abstract Objectives Few studies have tested DNA extraction methods to optimize the detection of Clostridium botulinum in environmental samples that can be collected during animal botulism outbreaks. In this study, we evaluated four commercial DNA extraction kits for the detection of C. botulinum group III in 82 various environmental samples (9 manure, 53 swabs, 3 insects, 8 water, 1 silage and 8 soil samples) collected in a context of animal botulism outbreaks. Results The PowerSoil® kit was the most efficient for almost all matrices (83.6% of the 73 tested samples), except manure for which the NucleoSpin® Soil kit was the most efficient. The NucleoSpin® Soil kit enabled detection in 75.3%, the QIAamp® DNA Mini Kit in 68.5%, and the QIAamp® Fast DNA Stool Mini Kit in 45.2%. However, the NucleoSpin® Soil kit detected C. botulinum in 9 of the 9 manure samples tested, while the PowerSoil® kit found C. botulinum in only two samples, and the other two kits in none of the samples. This study showed that PowerSoil® can be recommended for DNA extraction from environmental samples except for manure, for which the NucleoSpin® Soil kit appeared to be far more appropriate

Detection of undescribed ostreid herpesvirus 1 (OsHV-1) specimens from Pacific oyster, Crassostrea gigas

International audienceThe ostreid herpesvirus 1 (OsHV-1) and variants were implicated in mass mortality affecting the young Pacific cupped oysters, Crassostrea gigas, in European countries and those around the world. From 2008 onwards, oyster mortality had greatly increased on the French coast and was associated with the detection of a new OsHV-1 variant, entitled OsHV-1 μVar. The OsHV-1 μVar is predominant in oysters; however, other OsHV-1 variants have been detected in samples collected during mortality periods or collected out of mortality periods in France, Ireland, Spain, Portugal, Italy, Mexico, United States, South Korea, Australia, and New Zealand. A retrospective study conducted on 1047 OsHV-1 specimens sampled mainly in France between 2009 and 2012, revealed 17 undescribed OsHV-1 variants found in 65 oyster samples. These specimens presented point mutations situated downstream and upstream from the microsatellite area in the C region (ORF 4/5) which were different from the OsHV-1 reference and the OsHV-1 μVar. In the present work, investigation was performed to further characterize these OsHV-1 specimens by sequencing two habitually targeted regions to study genetic polymorphism of the virus: ORF 41/42 and ORF 35-38. An OsHV-1 variant detected in six oyster samples, contained a nucleotide substitution in the C region which impacted the amino acid sequence and might modify the function of the unknown protein encoding by ORF 4. For the ORF 41/42 region, only two specimens presented a synonymous mutation in comparison with the OsHV-1 μVar. All specimens contained the same deletion with the OsHV-1 μVar in ORF 35-38. Then, a phylogenetic analysis based on the C region was performed to investigate the distribution of undescribed specimens among 21 OsHV-1 DNA sequences notified in GenBank and collected from different countries (France, Japan, New Zealand, China, Ireland, and United States) between 1995 and 2012. All analyzed samples and the OsHV-1 μVar were placed in the same group, excepted for a Japan specimen. Our results contribute to improve the description of the genetic diversity of the OsHV-1 and the C region (ORF 4/5) appears to be a better target than ORF 42/42 and 35-38 to distinguish variants between themselves

Intra-Species and Inter-Species Differences in Cytokine Production by Porcine Antigen-Presenting Cells Stimulated by Mycoplasma hyopneumoniae, M. hyorhinis, and M. flocculare

Mycoplasma hyorhinis and M. flocculare are commonly co-isolated with M. hyopneumoniae (primary agent of swine enzootic pneumonia) in gross pneumonia-like lesions, but their involvement in the disease process remains unknown. T cells play an immuno-pathological role during mycoplasmal infections. Dendritic cells (DCs) are major antigen-presenting cells involved in T cell activation and differentiation. In this study, we investigated cytokine (IL-6, IL-8, IL-10, IL-12, and TNF-α) production by porcine bone-marrow-derived DCs (BM-DCs) stimulated by M. hyopneumoniae, M. hyorhinis, and/or M. flocculare. Results showed that cytokine production levels were relatively homogenous for all evaluated M. hyopneumoniae strains in contrast to M. hyorhinis and M. flocculare strains. The most noteworthy inter-species differences were the overall (i) lower IL-12 production capacity of M. hyopneumoniae, and (ii) higher TNF-α production capacity of M. flocculare. Co-stimulation of BM-DCs showed that M. hyorhinis dominated the IL-12 production independently of its association with M. hyopneumoniae or M. flocculare. In addition, a decreased BM-DC production of TNF-α was generally observed in the presence of mycoplasma associations. Lastly, M. flocculare association with M. hyopneumoniae increased BM-DC ability to secrete IL-10. A higher cytotoxicity level in BM-DCs stimulated by M. hyorhinis was also observed. Overall, this study demonstrated that the combination of M. hyorhinis or M. flocculare with M. hyopneumoniae may participate to the modulation of the immune response that might affect the final disease outcome