View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library Antonie van Leeuwenhoek (2014) 105:731–741 DOI 10.1007/s10482-014-0129-0 ORIGINAL PAPER Frederiksenia canicola gen. nov., sp. nov. isolated from dogs and human dog-bite wounds Bozena_ M. Korczak • Magne Bisgaard • Henrik Christensen • Peter Kuhnert Received: 19 November 2013 / Accepted: 28 January 2014 / Published online: 8 February 2014 Ó Springer International Publishing Switzerland 2014 Abstract Polyphasic analysis was done on 24 strains branch with intraspecies sequence similarity of at least of Bisgaard taxon 16 from five European countries and 99.1, 90.8, 96.8 and 97.2 %, respectively. Taxon 16 mainly isolated from dogs and human dog-bite showed closest genetic relationship with Bibersteinia wounds. The isolates represented a phenotypically trehalosi as to the 16S rRNA gene (95.9 %), the rpoB and genetically homogenous group within the family (89.8 %) and the recN (74.4 %), and with Actinoba- Pasteurellaceae. Their phenotypic profile was similar cillus lignieresii for infB (84.9 %). Predicted genome to members of the genus Pasteurella. Matrix-assisted similarity values based on the recN gene sequences laser desorption/ionization time-of-flight mass spec- between taxon 16 isolates and the type strains of trometry clearly identified taxon 16 and separated it known genera of Pasteurellaceae were below the from all other genera of Pasteurellaceae showing a genus level. Major whole cell fatty acids for the strain T characteristic peak combination. Taxon 16 can be HPA 21 are C14:0,C16:0,C18:0 and C16:1 x7c/C15:0 iso further separated and identified by a RecN protein 2OH. Major respiratory quinones are menaquinone-8, signature sequence detectable by a specific PCR. In all ubiquinone-8 and demethylmenaquinone-8. We pro- phylogenetic analyses based on 16S rRNA, rpoB, infB pose to classify these organisms as a novel genus and and recN genes, taxon 16 formed a monophyletic species within the family of Pasteurellaceae named Frederiksenia canicola gen. nov., sp. nov. The type strain is HPA 21T (= CCUG 62410T = DSM 25797T). Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0129-0) contains supple- Keywords Bisgaard taxon 16 Á mentary material, which is available to authorized users. Pasteurellaceae Á Taxonomy Á Phylogeny Á Zoonosis B. M. Korczak Á P. Kuhnert (&) Vetsuisse Faculty, Institute of Veterinary Bacteriology, University of Bern, Laenggass-Strasse 122, 3001 Bern, Switzerland Introduction e-mail: [email protected] M. Bisgaard Presently, the family Pasteurellaceae is one of the Horsevaenget 40, 4130 Viby Sjælland, Denmark largest bacterial families, with many taxa still awaiting proper classification (Christensen and Bisgaard 2008; H. Christensen Gregersen et al. 2009; Kuhnert et al. 2010; Foster et al. Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, 2011; Christensen et al. 2011; Hansen et al. 2012). 4 Stigbøjlen, 1870 Frederiksberg C, Denmark Some members of the Pasteurellaceae are frequently 123 732 Antonie van Leeuwenhoek (2014) 105:731–741 found in the oral cavity and upper respiratory tract of 25797T also deposited under the number CCUG companion animals such as dogs and cats (Christensen 36444T = Him932-7T). and Bisgaard 2008). They are mainly considered commensals, however, under certain circumstances they may also act as opportunistic pathogens. Species Materials and methods obtained from dogs and cats include Pasteurella multocida, Pasteurella dagmatis, Pasteurella stomatis Bacterial strains and phenotypic characterization and Pasteurella oralis. Two additional species are more restricted in their host-specificity, Pasteurella Most of the 24 strains investigated were obtained from canis with dogs and [Haemophilus] felis with cats dog, mainly from the upper respiratory tract or vagina (Mutters et al. 1985; Inzana et al. 1992; Christensen (Table 1). Clinical cases included rhinitis, tracheitis, et al. 2012). In humans the aforementioned species chronic tracheobronchitis, facial swelling and genital may cause wound infections inflicted by dog- or cat tract infection. Four strains were isolated from humans, bites/scratches (Abrahamian and Goldstein 2011). three of them from dog-bite wounds while single Correct classification of these taxa has major impact isolates were obtained from cat, lion, hedgehog and on an unambiguous identification and is essential for banded mongoose (domesticated or zoo animals). proper medical treatment of patients, the development Primary phenotypic characterization identified them as of preventive measures and the performance of epide- taxon 16 or atypical P. canis, P. stomatis, P. dagmatis or miological studies. Identification of these taxa, how- Pasteurella sp. All strains had been kept frozen at ever, can be problematic as additional Pasteurella-like -80 °C for further investigation. The bacteria were organisms have been reported from the same niche subcultivated from frozen stocks on tryptone soya agar (Saphir and Carter 1976; Bisgaard and Mutters 1986; plates with sheep blood (TSA; Oxoid, Pratteln, Swit- Ganiere et al. 1993; Muhairwa et al. 2001; Forsblom zerland) for 24 h at 37 °C in an aerobic atmosphere. A et al. 2002). A group of bacteria, tentatively named number of different biochemical tests recommended for Bisgaard taxon 16, shows a phenotype, mol% G?Cin characterization of the phenotype of members of the DNA, genome size and cellular fatty acid composition Pasteurellaceae family were applied to all isolates as similar to the genus Pasteurella and might be misi- proposed in the ‘‘minimal standards’’ for the family dentified as P. canis, P. stomatis or P. dagmatis (Christensen et al. 2007). (Bisgaard and Mutters 1986; Forsblom et al. 2002). On the other hand, analysis of data provided by the DNA– Chemotaxonomy, MALDI-TOF MS and amino DNA and DNA-rRNA hybridization (De Ley et al. acid signature specific PCR 1990; Bisgaard and Mutters 1986), 16S rRNA sequencing (Olsen et al. 2005) and matrix-assisted Analysis of fatty acids and respiratory quinones of the laser desorption/ionization time-of-flight mass spec- strain HPA 21T were carried out by the Identification trometry (MALDI-TOF MS) technology (Kuhnert Service of the DSMZ (Deutsche Sammlung von et al. 2012) showed that this taxon differed consider- Mikroorganismen und Zellkulturen), Braunschweig, ably from the other known genera within the family and Germany. The analysis of whole cell fatty acid content should be classified as a new genus. was done using the Sherlock Microbial Identification Using a polyphasic approach we investigated 24 System (MIS) (MIDI, Microbial ID, Newark, DE isolates of taxon 16 that were diverse in geographical 19711, USA) according to the previously published location and host/tissue source. Based on phenotypic, protocols (Miller 1982; Kuykendall et al. 1988; genetic, as well as phylogenetic characteristics, includ- Kampfer and Kroppenstedt 1996). Analysis of respi- ing biochemistry, chemotaxonomy, MALDI-TOF MS, ratory quinones for the strain HPA 21T were performed amino-acid signature specific PCR, multilocus according to published protocols (Tindall 1990a, b). sequence analysis (MLSA), and recN-derived genome MALDI-TOF MS was performed according to similarity data, we propose classification of Bisgaard Kuhnert et al. (2012). Frederiksenia canicola (taxon taxon 16 as Frederiksenia canicola gen. nov., sp. nov., 16) specific peaks were determined in Biotyper 3.0 a new genus within the family of Pasteurellaceae. The software (Bruker Daltonik GmbH, Bremen, Ger- type strain is HPA 21T (= CCUG 62410T = DSM many). For diagnostic identification the direct plating 123 Antonie van Leeuwenhoek (2014) 105:731–741 Table 1 Frederiksenia canicola (Bisgaard taxon 16) strains investigated Strain No. Country Initial Isolated from Clinical signs GenBank accession no. phenotypic identification 16S infB recN rpoB rRNA 1 HPA 21T = CCUG 62410T = DSM 25797T also Denmark Pasteurella sp. Dog, pharynx Healthy dog JQ356598 JQ356622 JQ356650 JQ356678 known as CCUG 36444T and Him932-7T 2 Smith392 = P919 = CCUG 17204 UK Pasteurella sp. Dog Facial swelling JQ356599 JQ356623 JQ356651 JQ356679 3 M2500/96/3 UK N.a. Dog, eye N.a. JQ356600 JQ356624 JQ356652 JQ356680 4 D1949_98 = JF2157 Switzerland P. canis Dog, trachea Trachitis JQ356601 JQ356625 JQ356653 JQ356681 5 D2018_98 Switzerland P. canis Dog, nose Rhinitis JQ356602 JQ356626 JQ356654 JQ356682 6 D2941_98_JF2198 Switzerland Pasteurella sp. Dog, nose Rhinitis JQ356603 JQ356627 JQ356655 JQ356683 7 D227_99 = JF2221 France P. canis Dog, tracheo Chronical JQ356604 JQ356628 JQ356656 JQ356684 bronchial tracheobronchitis ichor 8 D262_99 = JF2223 Switzerland P. canis Dog, nose Rhinitis JQ356605 JQ356629 JQ356657 JQ356685 9 D452-3_99 = JF2240 Switzerland P. canis Dog, nose N.a. JQ356606 JQ356630 JQ356658 JQ356686 10 D536-2_99 = JF2247 Switzerland P. canis Dog, tonsil Chronical cough JQ356607 JQ356631 JQ356659 JQ356687 and tonsillitis 11 D597-2_99 = JF2247 Switzerland P. canis Dog, vagina Brown discharge JQ356608 JQ356632 JQ356660 JQ356688 12 D1071_99 Switzerland P. canis Dog, vagina N.a. JQ356609 JQ356633 JQ356661 JQ356689 13 KM1266_04 = JF4823 Switzerland Pasteurella sp. Dog, vagina N.a. JQ356610 JQ356634 JQ356662 JQ356690 14 KM1549_04 Switzerland P. stomatis Dog N.a. JQ356611 JQ356635 JQ356663 JQ356691 15 KM1721_06 Switzerland Pasteurella sp. Dog, nose Rhinitis JQ356612 JQ356636