Natural Transmission of Zoonotic Babesia spp. by Ixodes ricinus Ticks

To determine characteristics of natural transmission of Babesia sp. EU1 and B. divergens by adult Ixodes ricinus ticks, we examined tick salivary gland contents. We found that I. ricinus is a competent vector for EU1 and that their sporozoites directly invade erythrocytes. We conclude that EU1 is naturally transmitted by I. ricinus

Detection of Wolbachia in the Tick Ixodes ricinus is Due to the Presence of the Hymenoptera Endoparasitoid Ixodiphagus hookeri

The identification of micro-organisms carried by ticks is an important issue for human and animal health. In addition to their role as pathogen vectors, ticks are also the hosts for symbiotic bacteria whose impact on tick biology is poorly known. Among these, the bacterium Wolbachia pipientis has already been reported associated with Ixodes ricinus and other tick species. However, the origins of Wolbachia in ticks and their consequences on tick biology (known to be very diverse in invertebrates, ranging from nutritional symbionts in nematodes to reproductive manipulators in insects) are unknown. Here we report that the endoparasitoid wasp Ixodiphagus hookeri (Hymenoptera, Chalcidoidea, Encyrtidae) – strictly associated with ticks for their development - is infested at almost 100% prevalence by a W. pipientis strain belonging to a Wolbachia supergroup that has already been reported as associated with other hymenopteran parasitoids. In a natural population of I. ricinus that suffers high parasitism rates due to I. hookeri, we used specific PCR primers for both hymenopteran and W. pipientis gene fragments to show that all unfed tick nymphs parasitized by I. hookeri also harbored Wolbachia, while unparasitized ticks were Wolbachia-free. We demonstrated experimentally that unfed nymphs obtained from larvae exposed to I. hookeri while gorging on their vertebrate host also harbor Wolbachia. We hypothesize that previous studies that have reported W. pipientis in ticks are due to the cryptic presence of the endoparasitoid wasp I. hookeri. This association has remained hidden until now because parasitoids within ticks cannot be detected until engorgement of the nymphs brings the wasp eggs out of diapause. Finally, we discuss the consequences of this finding for our understanding of the tick microbiome, and their possible role in horizontal gene transfer among pathogenic and symbiotic bacteria

Influence of lactate and acetate removal on the microbiota of French fresh pork sausages

International audienceThe microbiota of fresh French pork sausages were characterised in five batches of comminuted pork meat that were equally divided into two formulations either containing the acid-based preservatives lactate and acetate, or no preservatives. Conventional microbiological analysis and high-throughput 16S rDNA amplicon sequencing methods were performed on meat batches packed under modified atmosphere (70% oxygen and 30% carbon dioxide) during chilled storage. In addition, meat pH and colour, and gas composition of the packages were monitored until the end of the shelf-life. During storage, the population of mesophilic and lactic acid bacteria increased from 4 log CFU/g to 8 log CFU/g after 15 days of chilled storage, both with and without preservatives. Despite similar changes of the physical and chemical parameters, such as pH and package gas composition, spoilage was delayed in the meat containing the preservatives, suggesting that lactate and acetate are effective against spoilage. Metagenetic analysis showed that at the end of the shelf-life, the species distribution differed between both the formulations and the batches. Lactic acid bacteria were shown to dominate both with and without preservatives; however, samples containing no preservatives were characterised by the presence of an increased population of Brochothrix spp. and Pseudomonas spp. whereas, Leuconostoc mesenteroides/pseudomesenteroides and Lactobacillus curvatus/graminis were more abundant in the meat with preservatives

Apport de la génétique des populations pour déterminer le rôle respectif des mammifères et des oiseaux dans la dispersion passive de la tique Ixodes ricinus

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Antimicrobial resistance in the bacterial communities of the rainbow trout filet?

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The Antibiotic Resistome of Farmed Rainbow Trout Filets Using Smartchip Real-time PCR

National audienceIntroduction: The role of food in the routes of transmission of resistant bacteria and antimicrobial resistance genes (ARG) is yet to be explored. There can be a risk of ARG presence on farmed fish filets because of their environmental exposures (animal farms, human activities, aqueous environment). Consequently, it is essential to determine the farmed fish antibiotic resistome. To analyse fresh fish filet resistomes, where bacterial load is known to be low, a highly efficient method like high-throughput qPCR arrays, able to detect and quantify hundreds of selected ARGs in a single run, can be useful. Purpose: The objective was to evaluate the significance of Smartchip RT-PCR technology to analyse the antibiotic resistome profile of rainbow trout filets. Methods: The analyses were performed on both fresh and spoiled rainbow trout filets. The filets were inoculated with antimicrobial-resistant bacterial strains (n=6) at various concentrations (104 to 108 CFU/g) to assess the capacity to detect a specific gene among the microbial communities of the filet. The ARG were detected and quantified using a 245 primer pair set. The set was chosen after bibliography analyses and In Silico verification. The amplification was realised thanks to the Smartchip Real-Time PCR technology (Takara). Results: The ARG detection threshold was determined to be 3.52 log CFU/g. Some ARG were detected at Ct values around 25 on fresh filets (tetL, tetB, sul1, qacEΔ1). In spoiled filets, the quantification of some ARG was enhanced (Ct around 18 to 20). Other genes such as tetS, tetB, strB, mexE, mexF were only detectable in spoiled filets. Significance: The Smartchip real-time PCR technology allows detecting ARG at different presence levels in the bacterial communities of fish filets. It may be interesting to use this tool to investigate and describe the resistome patterns of rainbow trout filets

Etude du résistome du filet de truite arc en ciel – étape 1 : faisabilité méthodologique ?

National audienceLe rôle de l'alimentation dans les voies de la transmission de bactéries ou de gènes de résistance (ARG) est suspecté depuis quelques temps (Woolhouse et al., 2015). Dans ce contexte, la denrée "filet de poisson frais truite arc en ciel" présente un intérêt. En effet, c'est un produit primaire pouvant être exposé à une contamination dès l'élevage par des antibiotiques en cas de maladies nécessitant traitement chez le poisson, par son environnement aquatique de type rivière, réservoir potentiel de gènes de résistance du fait d'expositions aux activités agricoles et urbaines voisines (autres élevages, STEP, hôpitaux, ...) et des équipements dans l'environnement des ateliers de transformation. Ces différents environnements de la denrée "filet de poisson frais truite arc en ciel" sont potentiellement générateurs de bactéries résistantes et/ou de résidus d'antibiotiques au sein du microbiote du filet. Le premier verrou méthodologique est la quantité de bactéries et donc la quantité d'ADN bactérien récupérable à partir d'une matrice alimentaire fraiche et complexe comme le filet (peau plus chair) de poisson. En effet, nous souhaitons nous affranchir le plus possible de l'ADN d'origine poisson pour avoir suffisamment d'ADN bactérien. De plus les matrices alimentaires "grasses" complexifient l'extraction par la présence d'inhibiteurs et par le colmatage des filtres. Deux techniques d'extraction d'ADN à partir de 30g de filet ont été éprouvées suite au stomachage ou au rinçage des filets pour arriver à une estimation de la présence de 3.5 log CFU / g de bactéries. Le second verrou était l'analyse haut-débit des gènes de résistance. Nous avons fait le choix d'utiliser la technique Smartchip Real Time PCR (Muzasiari et al., 2017) avec 248 couples d'amorces pour amplifier un large spectre de gènes décrits comme participant à la résistance aux antibiotiques. Pour valider la combinaison des méthodes, de l'extraction du microbiote et de son ADN à la détection par Smartchip Real Time PCR, une contamination expérimentale de filets à partir de concentrations connues de bactéries qui chacune possède un gène de résistance caractérisé a été conduite, avec des concentrations allant de 10⁸ à 10⁴ cfu/ml comme par exemple avec Enterococcus faecalis portant le gène de résistance à la vancomycine vanA. Les résultats obtenus valident l'extraction d'ADN à partir des filets rincés et la technique Smartchip montre un intérêt majeur pour analyser le resistome de filets de poissons. Des analyses d'un plus grand effectif de filets permettront d'avoir une première description du resistome du filet de poisso

Etude phénotypique (identification et antibiorésistance) de la flore listeria dans la matrice filet frais de poisson

National audienceLa listériose à Listeria monocytogenes est un enjeu de santé publique avec régulièrement des interdictions de ventes de produits contaminés voir de personnes infectées. Les produits incriminés sont les produits consommés crus, les produits au lait frais, les graines germées et aussi les charcuteries et les poissons (fumés et crus). Le genre de Listeria est ubiquitaire et peut se retrouver dans tous les types d'environnement : eau, sol, atelier de transformation,... Nous avons voulu étudier le portage au genre Listeria du filet de poisson avant et après transformation et nous avons caractérisé le niveau de résistance des isolats vis-à-vis d'un panel large d'antibiotiques. La présence de bactérie du genre Listeria a été recherchée dans différents filets de poisson, provenant directement de l'élevage (n=28) ou directement de l'atelier de transformation (n=28). La recherche des bactéries a été réalisée selon la norme ISO 11290-2 et l'utilisation d'un milieu de culture d'enrichissement spécifique puis d'un milieu d'isolement spécifique, le chromogenic listeria agar. Des tests biochimiques (Gram, catalase) ont également été réalisés. Sept filets (sur 56) présentaient un portage à Listeria, dont 4 en sortie d'élevage et 3 en sortie d'atelier de transformation. Vingt colonies spécifiques ont pu être isolées selon le protocole microbiologique. La confirmation de l'identification bactérienne a été réalisée par MALDITOF. La résistance des différents isolats a été caractérisée par CMI (Concentration Minimale Inhibitrice) selon les protocoles du CLSI. Les CMI de 9 antibiotiques ont été recherchées : acide oxolinique, oxytétracycline, florfénicol, triméthoprime/sulfaméthoxazole, fluméquine, ampicilline, ceftazidime, azithromycine, colistine et gentamicine. Les CMIs sont homogènes pour les antibiotiques oxytétracycline (CMI = 0.5ug/ml), ampiciline (CMI = 1ug/ml), l'association trimethoprim-sulfomethoxazole (CMI = 0.125/4.75 ug/ml). Elles sont plus étalées pour les antibiotiques type colistine, ceftazidine et azithromycine. Ainsi un siolat exprime une résistance intermédiaire à la gentamycine et deux isolats expriment une résistance clinique à l'azythromycine selon les critères CLSI M100 Staphylococcus spp. appliquées à Listeria. Les filets de poissons étudiés sont peu chargés en Listeria spp. et présentent une distribution homogène selon l'origine des échantillons. Les isolats étudiés expriment peu de résistance aux antibiotiques mais une surveillance peut être pertinente

Antimicrobial Resistance in the Bacterial Communities of the Rainbow-Trout Filet

National audienceThe role of food in the routes of transmission of resistant bacteria and antimicrobial resistance genes (ARG) is yet to be explored. Farmed fish filets can carry antibiotic-resistant bacteria because of their environmental exposure (animal farms, human activities, aquatic environment…). The presence of such bacteria on foodstuff must be evaluated as it might present a risk for the consumers. To extend our knowledge about the farmed fish filet resistome, ARG were sought in the bacterial communities of fresh rainbow-trout filets. Antimicrobial residues were also dosed in the filets. The analyses were performed on fishes originating from a single batch sampled in two conditions: a) filets excised in laboratory conditions (N = 14) and b) filets excised in an industrial facility (N = 14). Each filet was divided, one sample was rinsed and the DNA was extracted to perform the ARG investigation and one sample was analysed to perform the antimicrobial residues dosing. Insights about events occurring during the lifetime of the fishes, such as diseases, treatments, vaccines and pond cleaning frequency were provided by the breeders. The ARG were detected and quantified using a 245 primers pairs set. The set were chosen after bibliography analyses [1] and in silico verification. It was designed to detect resistances to 10 antimicrobial classes, including beta lactams, macrolides, tetracyclines, aminoglycosides, phenicols, as well as multidrug efflux pumps and biocides resistance genes. The amplification was realised thanks to the Smartchip Real-Time PCR technology (Takara). The residues of 75 antimicrobials, covering 10 antimicrobial classes, were looked for and quantified by LC-MS/MS. Some ARG were detected at Ct around 25 (tetL, tetB, sul1), as well as biocide resistance genes (qacEΔ1). Oxytetracycline residues were detected in 13 filets (out of 28) in concentrations lower than the authorized maximum residue limit. The concentrations ranged from 5,84 to 40,2 μg/kg and the authorized maximum residue limit is 100 μg/kg in the fish muscle. Fishes were treated with oxytetracycline more than 6 months before the sampling. Overall, few antimicrobial resistance genes were detected and the detected genes displayed high Ct, suggesting that this genes had low prevalence in the bacterial communities. Presence of tetracycline resistance genes and oxytetracycline residues have been observed in fish samples from a batch where an oxytetracycline treatment has been applied. No correlation has been established yet. Further investigations are necessary in order to understand the links between the presence of residues and genes, and the phenotypic resistance