NOTE TO THE EDITOR Genetic and phylogenetic evidence for misidentification of Vibrio species within the Harveyi clade M. Hoffmann 1,2 , S.R. Monday 1 , M. Fischer 2 and E.W. Brown 1 1 US Food and Drug Administration, Paint Branch Parkway, College Park, MD, USA 2 Department of Chemistry, Institute of Food Chemistry, University of Hamburg, Hamburg, Germany Vibrio is bacterial genus comprised of halophilic species showing remarkable biodiversity and persisting in a variety of geographic locales and eukaryotic marine hosts, includ- ing corals, molluscs, zooplankton and sponges (Thompson et al. 2004). Eleven Vibrio species are currently documented as members of the Harveyi clade (V. alginolyticus, V. para- haemolyticus, V. campbellii, V. harveyi, V. rotiferianus, V. natriegens, V. azureus, V. mytili, V. owensii, V. communis and V. sagamiensis) (Cano-Gomez et al. 2010; Chimetto et al. 2010; Lin et al. 2010; Yoshizawa et al. 2010). The Harveyi clade contains known pathogens of fishes, corals, mollusks and humans (Lin et al. 2010), making them an important concern to both public health and industry for the last 10 years. Recently, we revealed, in an unrelated study, several inconsistencies regarding the taxonomy and identification of vibrios contained within the Harveyi clade. The scope of this report was to investigate these taxonomic inconsistencies to make the Vibrio research community aware so as to avoid downstream complications. Forty-eight Vibrio isolates from the Harveyi clade were obtained from the American Type Culture Collection (ATCC) and the Belgium Co-ordinated Collection of Micro-organism (BCCM) and co-mingled with 39 additional sponge-derived (SD) Vibrio strains for this study (Table 1). All Vibrio strains were cultured on Trypticase Soy Agar (TSA; Oxoid, Basingstoke, UK) supplemented with 1% NaCl (w ⁄ v) at 28°C for 24 h. Additionally, growth on Thiosulfate-Citrate-Bile Salts- Sucrose (TCBS; DIFCO Laboratories, Sparks, MD) agar, which is highly selective for the isolation of vibrios, was applied to ensure culture quality. Multi-locus sequence analysis (MLSA) of six house- keeping genes, encoding topoisomerase I (topA), a cell division protein (ftsZ), actin-like cytoskeleton protein (mreB), RNA polymerase r 70 factor (rpoD), replication origin-binding protein (rctB) and transmembrane regula- tory protein (toxR), was carried out, as previously described for topA, ftsZ and mreB (Thompson et al. 2007) and for rctB, rpoD and toxR (Pascual et al. 2010). To vali- date clade, subclade and species assignments, data matri- ces were constructed using sequence data obtained from our own reference strains, as well as from reference strain sequence data from two previously validated three-gene MLSA studies of the Harveyi clade (Lin et al. 2010). Keywords identification, Harveyi clade, MLSA, molecular genetic, vibrios. Correspondence Maria Hoffmann, Division of Microbiology, Office for Regulatory Science, US Food and Drug Administration, 5100 Paint Branch Parkway, College Park, MD 20740, USA. E-mail: maria.hoffman@fda.hhs.gov 2011 ⁄ 1681: received 1 October 2011, revised 18 November 2011 and accepted 18 Novem- ber 2011 doi:10.1111/j.1472-765X.2011.03183.x Abstract Aim: This report describes the use of a six-gene multi-locus sequence analysis (MLSA) to correctly identify Vibrio strains of the Harveyi clade. Methods and Results: Vibrio isolates were characterized using a six housekeep- ing gene MLSA. The study provided evidence supporting: (i) a substantial number of reference strains maintained within commercial culture collections are misidentified taxonomically at the species level; (ii) two V. alginolyticus subclades retain species-level divergence; and (iii) V. communis and V. owensii likely are the same species. Conclusion: A significant number (n = 10) of Harveyi clade Vibrio strains have been inaccurately identified, including evidence that V. communis and V. owen- sii strains, two recently discovered species assigned to the Harveyi clade, comprise a single species. Significance and Impact of the study: As Harveyi clade vibrios have an enor- mous impact on human and aquatic animal health, it is of paramount impor- tance to identify members of the Harveyi clade correctly. Letters in Applied Microbiology ISSN 0266-8254 160 Letters in Applied Microbiology 54, 160–165 ª 2011 The Society for Applied Microbiology ª No Claim to Government Works