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Póster presentado Online en el XXVIII Congreso Nacional de Microbiología 28 de junio al 2 de julio, 2021 .- https://hal.inrae.fr/hal-03337244/document, La brucelosis es una de las zoonosis bacterianas más ampliamente distribuidas en el mundo, contribuyendo de forma importante a la pobreza de muchos países en desarrollo. Ninguna de las vacunas actuales proporciona una protección completa, son virulentas para humanos e interfieren en el diagnóstico serológico. Por ello, para mejorar las vacunas actuales, es imprescindible seguirestudiando los mecanismos de virulencia de Brucella, el agente etiológico de la brucelosis. Ésta pertenece a las α-2 Proteobacteria, grupo que incluye desde bacterias ambientales hasta bacterias que han evolucionado a patógenos animales difícilmente detectados por el sistema inmune, como es el caso de Brucella. Para estudiar la evolución que se ha dado hacia la patogenicidad en esta familia y poder determinar mejor así los factores de virulencia de Brucella, es interesante comparar bacterias filogenéticamente próximas que son biológicamente diferentes. Por primera vez, se han aislado bacterias del género Pseudochrobactrum en un huésped natural de Brucella.Pseudochrobactrum es un género filogenéticamente cercano a Brucella que, hasta la fecha, sólo incluye bacterias ambientales. En este trabajo se han caracterizado estos nuevos aislamientos y teniendo en cuenta sus propiedades fisiológicas, su perfil de lípidos polares y ácidos grasos, y el análisis filogenético, se ha propuesto una nueva especie con el nombre de Pseudochrobactrum bovis. Los estudios comparativos con Brucella han revelado diferencias en la composición de la envoltura celular, el contenido genético y virulencia. Por ello, P. bovis puede representar un nuevo modelo para estudiar la evolución de Brucella hacia la virulencia

DESCRIPTION OF PSEUDOCHROBACTRUM ALGERIENSIS SP. NOV. Pseudochrobactrum algeriensis (al.ge.ri.en��sis. N.L. masc. adj. algeriensis of or belonging to Algeria, referring to the isolation place). Aerobic, Gram-stain-negative, oxidase- and catalase-positive, non-motile and rod-shaped. Non-pigmented, beige colonies with regular edges with a diameter of about 2 mm. Good growth occurs at 18���38��C within 24���48h on TSA, nutrient agar, MacConkey and CPSE. In API 20NE tests, urease, cytochrome oxidase, citrate utilization and assimilation of D-glucose,D-mannose,L-malate, and N -acetylD -glucosamine are positive. Denitrification, indole production, D-glucose fermentation, arginine dihydrolase, aesculin hydrolysis (��-glucosidase), gelatin hydrolysis (protease), ��-galactosidase and assimilation of gluconate, capric acid, adipic acid, phenylacetic acid, L-arabinose, maltose and D-mannitol are negative. The major fatty acids are C 18:0 and C 18:1. The polar lipid profile consists of the major compounds phosphatidylethanolamine, ornithine-lipids, phosphatidylglycerol, cardiolipin and phosphatidylcholine, plus moderate amounts of PMME, PDME and other aminolipids. The type strain is C130915_07 T (CECT 30232 T =LMG 32378 T) isolated from lymph nodes of an Algerian cow., Published as part of Loperena-Barber, Maite, Khames, Mammar, Leclercq, S��bastien O., Zygmunt, Michel S., Babot, Esteban D., Z����iga-Ripa, Amaia, Guti��rrez, Ana, Oumouna, Mustapha, Moriy��n, Ignacio, Cloeckaert, Axel & Conde-��lvarez, Raquel, 2022, Pseudochrobactrum algeriensis sp. nov., isolated from lymph nodes of Algerian cattle, pp. 1-7 in International Journal of Systematic and Evolutionary Microbiology (005223) (005223) 72 (2) on page 6, DOI: 10.1099/ijsem.0.005223, http://zenodo.org/record/6224086

DESCRIPTION OF PSEUDOCHROBACTRUM ALGERIENSIS SP. NOV. Pseudochrobactrum algeriensis (al.ge.ri.en´sis. N.L. masc. adj. algeriensis of or belonging to Algeria, referring to the isolation place). Aerobic, Gram-stain-negative, oxidase- and catalase-positive, non-motile and rod-shaped. Non-pigmented, beige colonies with regular edges with a diameter of about 2 mm. Good growth occurs at 18–38°C within 24–48h on TSA, nutrient agar, MacConkey and CPSE. In API 20NE tests, urease, cytochrome oxidase, citrate utilization and assimilation of D-glucose,D-mannose,L-malate, and N -acetylD -glucosamine are positive. Denitrification, indole production, D-glucose fermentation, arginine dihydrolase, aesculin hydrolysis (β-glucosidase), gelatin hydrolysis (protease), β-galactosidase and assimilation of gluconate, capric acid, adipic acid, phenylacetic acid, L-arabinose, maltose and D-mannitol are negative. The major fatty acids are C 18:0 and C 18:1. The polar lipid profile consists of the major compounds phosphatidylethanolamine, ornithine-lipids, phosphatidylglycerol, cardiolipin and phosphatidylcholine, plus moderate amounts of PMME, PDME and other aminolipids. The type strain is C130915_07 T (CECT 30232 T =LMG 32378 T) isolated from lymph nodes of an Algerian cow.

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018.01545/full#supplementary-material; International audience; Previous studies have shown the usefulness of MLVA16 as a rapid molecular identification and classification method for Brucella species and biovars including recently described novel Brucella species from wildlife. Most studies were conducted on a limited number of strains from limited geographic/host origins. The objective of this study was to assess genetic diversity of Brucella spp. by MLVA16 on a larger scale. Thus, 1404 animal or human isolates collected from all parts of the world over a period of 32 years (1974-2006) were investigated. Selection of the 1404 strains was done among the approximately 4000 strains collection of the BCCN (Brucella Culture Collection Nouzilly), based on classical biotyping and on the animal/human/geographic origin over the time period considered. MLVA16 was performed on extracted DNAs using high throughput capillary electrophoresis. The 16 loci were amplified in four multiplex PCR reactions. This large scale study firstly confirmed the accuracy of MLVA16 typing for Brucella species and biovar identification and its congruence with the recently described Extended Multilocus Sequence Analysis. In addition, it allowed identifying novel MLVA11 (based upon 11 slowly evolving VNTRs) genotypes representing an increase of 15% relative to the previously known Brucella MLVA11 genotypes. Cluster analysis showed that among the MLVA16 genotypes some were genetically more distant from the major classical clades. For example new major clusters of B. abortus biovar 3 isolated from cattle in Sub-Saharan Africa were identified. For other classical species and biovars this study indicated also genotypic expansion within the population structure of classical Brucella species. MLVA proves to be a powerful tool to rapidly assess genetic diversity of bacterial populations on a large scale, as here on a large collection of strains of the genomically homogeneous genus Brucella. The highly discriminatory power of MLVA appears of particular interest as a first step for selection of Brucella strains for whole-genome sequencing. The MLVA data of this study were added to the public Brucella MLVA database at http://microbesgenotyping.i2bc.paris-saclay.fr. Current version Brucella\₄\₃ comprises typing data from more than 5000 strains including in silico data analysis of public whole genome sequence datasets.

International audience; Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).

Session DP : Biological Protection and Environmental Hazards; International audience

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International audience; Culture is considered as the reference standard assay for diagnosis of spp. in humans and animals but it is time-consuming and hazardous. In this study, we evaluated the performances of newly designed real-time PCR assays using Taqman® probes and targeting the 3 following specific genes: () the insertion sequence IS, () and () genes for the detection of at genus level. The real-time PCR assays were compared to previously described conventional PCR assays targeting the same genes. The genus-specificity was evaluated on 26 strains, including all species and biovars. The analytical specificity was evaluated on a collection of 68 clinically relevant, phylogenetically related or serologically cross-reacting micro-organisms. The analytical sensitivity was assessed using decreasing DNA quantities of ovis, bv. 1, bv. 1 and reference strains. Finally, intra-assay repeatability and inter-assay reproducibility were assessed. All species DNA were amplified in the three tests. However, the earliest signal was observed with the IS real-time PCR, where it varied according to the IS copy number. No cross-reactivity was observed in all three tests. Real-time PCR was always more sensitive than conventional PCR assays. The real-time PCR assay targeting IS presented an identical or a greater sensitivity than the two other tests. In all cases, the variability was very low. In conclusion, real-time PCR assays are easy-to-use, produce results faster than conventional PCR systems while reducing DNA contamination risks. The IS-based real-time PCR assay is specific and highly sensitive and appears as an efficient and reproducible method for the rapid and safe detection of the genus .

International audience; Since the 1990s, Brucella strains have been isolated from a wide variety of marine mammals and were recently recognized as two different species, i.e. Brucella pinnipedialis for pinniped isolates and Brucella ceti for cetacean isolates. The aim of this study was to identify specific DNA fragments of marine mammal Brucella strains using a previously described infrequent restriction site-PCR (IRS-PCR) method but with three new couples of restriction enzymes applied on a larger panel of marine mammal Brucella isolates (n = 74) and one human isolate from New Zealand likely from marine mammal origin. This study revealed five DNA fragments specific of Brucella strains isolated from marine mammals. Among them two new DNA fragments were specific of B. pinnipedialis but were not detected in hooded seal isolates. DNA fragment I identified in the previous IRS-PCR study and fragment VI of this study were located on a cloned and sequenced 6 kb Sacl fragment. Its nucleotide sequence revealed that it is likely part of a putative genomic island. Sequence analysis showed that it carries four ORFs coding for putative metabolic functions. Although hooded seal isolates are classified within B. pinnipedialis it was shown in this study that they do not carry this genomic island and this raises the question about their evolutionary history within B. pinnipedialis. (C) 2008 Elsevier B.V. All rights reserved.