Polyphasic Study of Chryseobacterium Strains Isolated from Diseased Aquatic Animals Jean Francois Bernardet, M
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Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals Jean Francois Bernardet, M. Vancanneyt, O. Matte-Tailliez, L. Grisez, L. Grisez, Patrick Tailliez, Chantal Bizet, M. Nowakowski, Brigitte Kerouault, J. Swings To cite this version: Jean Francois Bernardet, M. Vancanneyt, O. Matte-Tailliez, L. Grisez, L. Grisez, et al.. Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals. Systematic and Applied Microbiology, Elsevier, 2005, 28 (7), pp.640-660. 10.1016/j.syapm.2005.03.016. hal-02681942 HAL Id: hal-02681942 https://hal.inrae.fr/hal-02681942 Submitted on 1 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ARTICLE IN PRESS Systematic and Applied Microbiology 28 (2005) 640–660 www.elsevier.de/syapm Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals J.-F. Bernardeta,Ã, M. Vancanneytb, O. Matte-Taillieza, L. Grisezc,1, P. Tailliezd, C. Bizete, M. Nowakowskie, B. Kerouaulta, J. Swingsb aInstitut National de la Recherche Agronomique, Unite´ de Virologie et Immunologie Mole´culaires, Jouy-en-Josas, France bGhent University, BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Ghent, Belgium cZoological Institute, Laboratory for Ecology and Aquaculture, Leuven, Belgium dInstitut National de la Recherche Agronomique, Universite´ Montpellier II, Unite´ d’ Ecologie Microbienne des Insectes et Interactions Hoˆte-pathoge`ne, Montpellier, France eInstitut Pasteur, De´partement Structure et Dynamique des Ge´nomes, Collection de l’Institut Pasteur, Paris, France Received 11 March 2004 Summary Members of most Chryseobacterium species occur in aquatic environments or food products, while strains of some other species are pathogenic to humans and animals. A collection of 52 Chryseobacterium sp. strains isolated from diseased fish, one frog isolate and 22 reference strains were included in a polyphasic taxonomy study. Fourteen clusters of strains were delineated following the comparison of whole-cell protein profiles. Most of these clusters were confirmed when the phenotypic and RAPD profiles and the 16S rRNA gene sequences were compared. Fatty acid composition helped differentiate the Chryseobacterium strains from members of related genera. None of the fish isolates could be allocated to the two species previously reported from fish but two isolates belonged to C. joostei, while the frog isolate was identified as Elizabethkingia meningoseptica, a human pathogen previously included in the genus Chryseobacterium. Three clusters grouping from 3 to 13 isolates will probably constitute the core of new Chryseobacterium species but all other isolates occupied separate or uncertain positions in the genus. This study further demonstrated the overall high similarity displayed by most Chryseobacterium strains whatever the technique used and the resulting difficulty in delineating new species in the genus. Members of this bacterial group should be considered potential emergent pathogens in various fish and frog species, farming conditions and geographical areas. r 2005 Elsevier GmbH. All rights reserved. Keywords: Chryseobacterium; Elizabethkingia; Fish disease; Frog disease; Polyphasic taxonomy; Phenotypic characteristics; Randomly amplified polymorphic DNA; Whole-cell protein profiles; SDS-PAGE; Fatty acid analysis; 16S rRNA gene sequence Abbreviation: RAPD, randomly amplified polymorphic DNA Introduction ÃCorresponding author. Tel.: +33 1 34 65 25 87; fax:+33 1 34 65 25 91. E-mail address: [email protected] The genus Chryseobacterium was proposed to accom- (J.-F. Bernardet). odate six bacterial species previously included in the 1Current address: Intervet Norbio Singapore Pte Ltd., Singapore. genus Flavobacterium [41]. Following an extensive 0723-2020/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2005.03.016 ARTICLE IN PRESS J.-F. Bernardet et al. / Systematic and Applied Microbiology 28 (2005) 640–660 641 polyphasic study, the whole group of organisms related Materials and methods to the genera Chryseobacterium and Flavobacterium was included in the family Flavobacteriaceae [4,5]. The genus Bacterial strains Chryseobacterium currently comprises nine valid species and a non-valid one representing a variety of ecological The bacterial strains used in this study are listed in niches [3,20–22,41,44,46]. Five species, namely C. Table 1. Except one frog isolate, all isolates were indoltheticum [10], C. defluvii [21],‘‘C. proteolyticum’’ retrieved from diseased fish between 1994 and 2001. [44], C. formosense [46] and C. daecheongense [22] were The host fish represented a variety of fish species, isolated from various soil and aquatic environments. farming conditions, and geographical origins. About Chryseobacterium spp. strains are also part of the one-third of the strains were isolated from superficial psychrotrophic and proteolytic bacterial population lesions in otherwise apparently healthy fish while the that causes a variety of defects in food products such other strains were isolated from internal organs of as processed meats, chilled fish and shellfish, and dairy fish or frog suffering from hemorrhagic septicemia. products [14,18]. The polyphasic study of a group of Bacterial strains were isolated from samples inocu- dairy isolates recently resulted in the description of C. lated on trypticase soy agar (bioMe´ rieux sa, Marcy-- joostei [19,20]. Other Chryseobacterium species are l0E´ toile, France) incubated at 22 or 26 1C for 3 days. pathogenic to humans and animals. Chryseobacterium After the basic common characteristics (i.e., Gram- gleum and C. indologenes may cause nosocomial negative, non-motile rods, producing bright yellow- infections in humans, usually in neonates or immuno- orange flexirubin type pigments, a strong smell, compromised patients; they can also be retrieved from catalase and oxidase, and yielding a Chryseobacter- hospital environments and equipments and are usually ium spp. profile in API 20E and 20NE galleries) of resistant to many disinfectants and antibiotics [3]. The these strains were identified, a retrospective screen- 16S rRNA gene sequences of the putative new clinical ing of the culture collection of the Unite´ de Virologie species ‘‘C. massiliae’’ and ‘‘Candidatus Chryseobacter- et Immunologie Mole´ culaires yielded three poorly ium timonae’’ have recently been deposited in GenBank characterized fish isolates (i.e., JIP 105/82, JIP 108/ [12]. Finally, C. balustinum [6,16] and C. scophthalmum 83, and SAVU 12/90), tentatively labelled ‘‘Flavo [28,29] were isolated from diseased fish. Chryseobacter- bacterium-like’’ or ‘‘Flavobacterium sp.’’ at the time ium spp. strains were also detected among the bacterial of preservation, that shared the same basic char- flora in the gut of insects by direct amplification and acteristics and were consequently included in the sequencing of 16S rRNA genes [13], but they have not study. The type strain of six valid Chryseobacterium been fully characterized. Until recently, two more species and of E. meningoseptica were also studied, as bacterial species were allocated to the genus Chryseo- well as a second strain of each species. In the bacterium, i.e. C. meningosepticum and C. miricola. particular case of C. scophthalmum, a very high However, owing to their separate position compared to DNA homology had been demonstrated previously other Chryseobacterium species in 16S rRNA gene between the seven strains available [29]. Hence, all sequence comparison studies, their transfer to the new seven strains were included in this study in order to genus Elizabethkingia was proposed [23]. Hence, the test the repeatability of the techniques used there new combinations Elizabethkingia meningoseptica and after as well as their ability to reveal intra-specific E. miricola will be used thereafter. The latter species variability. Since Bergeyella zoohelcum, Riemerella was isolated from condensation water in the space anatipestifer and R. columbina [40,41] form a distinct sation Mir [25] whereas the former is considered the separate branch together with the Chryseobacterium most serious human pathogen in the group [3]. Since and Elizabethkingia species in published 16S rRNA E. meningoseptica was also isolated from diseased gene sequence similarity dendrograms [e.g., birds [39], frogs, turtles and cats [11,15 and referen- 3,4,20,22,25], the type strains of these three species ces therein,26], it may be the agent of zoonotic were also included in the study. The other members infections. of the Chryseobacterium–Elizabethkingia–Riemerella– Over the last few years, fish pathology laboratories in Bergeyella branch were not studied, either because Europe (Belgium, Finland, France) and Asia (Taiwan, they were published after this study was completed Singapore) have isolated an increasing number of (C. defluvii [21], C. formosense [46], C. daecheongense bacterial strains that shared the basic phenotypic [22], E. miricola [25], Kaistella koreensis [24], characteristics of the genus Chryseobacterium