International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3927–3929 DOI 10.1099/ijs.0.051524-0

Proposed reclassification of Pasteurella lymphangitidis Sneath & Stevens 1990 as pseudotuberculosis

Olivier Gaillot,1 Olivier Lemenand,2 Michae¨l Marceau3 and Michel Simonet1,3

Correspondence 1Lille University Medical Centre, F-59037 Lille, France Olivier Gaillot 2Centre Hospitalier de Saint-Nazaire, Saint-Nazaire F-44100, France [email protected] 3INSERM U1019, F-59000 Lille, France

The 16S rRNA gene sequences of Pasteurella lymphangitidis, Yersinia pseudotuberculosis and were found to be identical and multilocus sequence analysis could not discriminate between the three species. The susceptibility to a Y. pseudotuberculosis phage and the presence of the Y. pseudotuberculosis-specific invasin gene in P. lymphangitidis indicate that the latter should be reclassified as Y. pseudotuberculosis.

Pasteurella lymphangitidis was initially isolated from pseudotuberculosis genomes published at the time of our suppurative cervical lymphadenitis in zebus (Bos indicus) study. by Jayaraman & Sethumadavan (1974), who designated it However, P. lymphangitidis has yet to be formally as the ‘B. L. (bovine lymphangitis) organism’. In a reassigned to another genus. Therefore, as a Reference numerical taxonomic study, Sneath & Stevens (1985) Centre for and other ,we reported that although the B. L. organism shared some reassessed the bacterium’s taxonomic position. To that phenotypic features with members of the genus Yersinia,it end, we sequenced 1461 bp of the 16S rRNA (rrs) gene of was allied to the –Pasteurella complex. P. P. lymphangitidis obtained by PCR amplification, as lymphangitidis was then named as such by Sneath & described by Weisburg et al. (1991). The rrs sequence Stevens (1990). However, on the basis of DNA–DNA was found to be identical to those of Yersinia pestis CO92 hybridization, the type strain of P. lymphangitidis was and Y. pseudotuberculosis IP32953, in agreement with a found to exhibit less than 30 % DNA relatedness to recent study; Yarza et al. (2013) found that the rrs sequence members of the genus Pasteurella sensu stricto (Sneath of the type strain P. lymphangitidis CCUG 27188T & Stevens, 1990). Consequently, the meeting of the (GenBank accession number HF558372) was 99.93 % International Committee on Systematic Bacteriology’s identical to that of Y. pestis. We next performed multilocus subcommittee on Pasteurellaceae and related organisms in sequence analysis, as described by Kotetishvili et al. (2005); Osaka, Japan (held on 17 September 1990), stated that P. PCR-amplified segments of the glnA, gyrB, recA and groEL lymphangitidis should be included in the family (hsp60) housekeeping genes from P. lymphangitidis were , rather than the family Pasteurellaceae sequenced and compared with those of species in the genus (Fredericksen, 1991). In agreement with the latter finding, Yersinia, including three and five strains of Y. pestis and Y. we studied DNA extracted from the type strain P. lymphan- pseudotuberculosis, respectively. A phylogenetic tree built T T T gitidis CCUG 27188 (5Jayaraman strain I 5A82 5 from concatenated sequences showed that P. lymphan- T T T T 71 5ATCC 49635 5CIP 103823 5NCTC 10547 ) from gitidis, Y. pseudotuberculosis and Y. pestis were in the same the Culture Collection, University of Go¨teborg (CCUG), branch, which was distinct from the branch with the closely Go¨teborg, Sweden, by using degenerate primers to PCR- related Y. similis (Fig. 1). The fact that the concatenated amplify superoxide-dismutase-encoding genes in members nucleotide sequences of P. lymphangitidis, Y. pseudotu- of the family Pasteurellaceae (Gautier et al., 2005) and berculosis and Y. pestis were at least 98.4 % identical obtained a 428 bp amplicon that was 100 % identical to a prevented formal assignment of P. lymphangitidis to either sequence within the sodB gene in the four Yersinia Y. pseudotuberculosis or Y. pestis. We therefore amplified and sequenced the invasin-encoding gene inv that is disrupted by a 708 bp IS200-like insertion sequence The GenBank/EMBL/DDBJ accession number for the sodB, glnA, gyrB, recA, groEL (hsp60) and inv gene sequences of P. lymphangitidis (IS1541)inY. pestis but not in the enteroinvasive species CCUG 27188T are KC617876, KF322028, KF322029, KF322030, Y. pseudotuberculosis (Simonet et al., 1996). No insertion T KF322031 and KC679406, respectively . sequence was found in the P. lymphangitidis CCUG 27188

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Yersinia intermedia CIP 80.28T Table 1. Key differential biochemical characteristics of P. Yersinia pestis CO92 lymphangitidis and Y. pseudotuberculosis, Y. pestis and Y. Yersinia pestis Antiqua similis Yersinia pestis KIM Taxa: 1, P. lymphangitidis (data from this study); 2, Y. pseudo- Yersinia pseudotuberculosis YPIII tuberculosis;3,Y. pestis;4,Y. similis. Data for taxa 2–4 from Sprague Yersinia pseudotuberculosis IP 32953 1000 et al. (2008). Characteristics of P. lymphangitidis strains CCUG T Pasteurella lymphangitidis CCUG 27188 27188T, NCTC 10548, NCTC 10549, NCTC 10550 and NCTC 10554 Yersinia pseudotuberculosis IP 31758 0.01 were determined by using API 20E and 50CH strips (bioMe´rieux) Yersinia similis DSM 18211T incubated at 28 uC for 48 h and 7 days, respectively. +, ¢90 % strains positive; 2, ¡10 % strains positive; V, 11–89 % strains Fig. 1. Phylogenetic tree based on concatenated glnA, gyrB, recA positive. and groEL (hsp60) sequences from P. lymphangitidis CCUG 27188T and the three most closely related species of the genus Characteristic 1 2 3 4 Yersinia (built using the neighbour-joining method). The only Fermentation of: bootstrap value above 90 % (calculated for 1000 subsets) is Melibiose ++22 indicated here. The concatenated sequence of Yersinia intermedia ++2 + T Rhamnose CIP 80.28 was used as an outgroup, in order to root the tree. Bar, Urease ++2 + 1 % difference in nucleotide sequence. Nitrate reductase 2 +++ b-Galactosidase 2 + V + inv gene. When compared to the inv gene from the Y. pseudotuberculosis IP32953 genome, 20 mutations were (Fig. 1). Besides uncommon Y. pseudotuberculosis bacteri- observed, representing a 0.7 % genetic variability, similar to ological features, B. L. organism isolates were all associated that recently reported among isolates of this species with suppurative cervical adenitis, a pseudotuberculosis (Palonen et al., 2013). However, two single-nucleotide presentation never reported in bovines, as abortion, mastitis deletions in the first third of the sequence were responsible and enterocolitis have been the only clinical expressions of Y. for a frame shift resulting in the non-expression of invasin pseudotuberculosis infection described in these animals in P. lymphangitidis. This was demonstrated in a Western (Bleul et al., 2002; Brown & Davis, 1989; Callinan et al., blot analysis using an anti-invasin monoclonal antibody as 1988; Hannam, 1993; Langford, 1969; Mair & Harbourne, previously described (Simonet & Falkow, 1992) (not 1963; Otter, 1996; Slee et al., 1988). Therefore, atypical shown). Nevertheless, the results of a lysis assay with a Y. phenotypic features and a clinical presentation that was not pseudotuberculosis phage (performed by the French suggestive of pseudotuberculosis may have led, 40 years ago, Reference Centre for Yersinia, Institut Pasteur, Paris, to the misidentification of the B. L. organisms as a new France) provided additional evidence that strain CCUG zoonotic species, rather than as Y. pseudotuberculosis. 27188T belongs to the species Y. pseudotuberculosis.We suggest that the International Committee on Systematic References Bacteriology’s subcommittee on Enterobacteriaceae should Bleul, U., Bu¨ hler, K., Stephan, R., Pospischil, A. & Braun, U. (2002). review a proposal to reclassify Pasteurella lymphangitidis Mastitis caused by Yersinia pseudotuberculosis in a cow. Vet Rec 151, Sneath & Stevens 1990 as Yersinia pseudotuberculosis. 767–769. In addition to the lack of invasin, which is essential for Brown, C. C. & Davis, F. N. (1989). Yersinia pseudotuberculosis enteroinvasiveness (Simonet & Falkow, 1992), strain enteritis in four calves. J Comp Pathol 101, 463–466. CCUG 27188T and other B.L. organisms display other Callinan, R. B., Cook, R. W., Boulton, J. G., Fraser, G. C. & Unger, phenotypic features that are unusual in Y. pseudotu- D. B. (1988). Enterocolitis in cattle associated with Yersinia berculosis (Jayaraman & Sethumadavan, 1974). In particu- pseudotuberculosis infection. Aust Vet J 65, 8–11. lar, we confirmed the absence of nitrate reductase and Fredericksen, W. (1991). International Committee on Systematic b-galactosidase activities in CCUG 27188T and the other B. Bacteriology, Subcommittee on Pasteurellaceae and Related Organ- isms. Minutes of the meeting, 17 September 1990, Osaka, Japan. Int J L. isolates NCTC 10548, NCTC 10549, NCTC 10550 and Syst Bacteriol 41, 592. NCTC 10554 (5Jayaraman strains II, III, IV and VIII, Gautier, A.-L., Dubois, D., Escande, F., Avril, J.-L., Trieu-Cuot, P. & respectively). Yersinia similis, a species closely related to Y. Gaillot, O. (2005). Rapid and accurate identification of human pseudotuberculosis, also lacks b-galactosidase activity; how- isolates of Pasteurella and related species by sequencing the sodA gene. ever, it is unable to ferment melibiose, unlike B. L. J Clin Microbiol 43, 2307–2314. organisms (Table 1). In addition, Y. similis displays a Hannam, D. A. (1993). Bovine abortion associated with Yersinia characteristic nucleotide sequence in its rrs genes (Sprague pseudotuberculosis. Vet Rec 133, 372. T et al., 2008) absent in CCUG 27188 . Lastly, our analysis of Jayaraman, M. S. & Sethumadavan, V. (1974). The B. L. organism - Y. similis glnA, gyrB, recA and groEL sequences confirmed the causal agent of bovine lymphangitis in Tamil Nadu. Indian Vet J that P. lymphangitidis and Y. similis are distinct species 51, 347–355.

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