MLST-v, multilocus sequence typing based on virulence genes, for molecular typing of Salmonella enterica subsp. enterica serovars B. Tankouo-Sandjong a , A. Sessitsch a , E. Liebana b , C. Kornschober c , F. Allerberger c , H. Hächler d , L. Bodrossy a, ⁎ a ARC Seibersdorf Research GmbH, Department of Bioresources, Seibersdorf, Austria b Department of Food and Environmental Safety, Veterinary Laboratories Agency, Weybridge, UK c Österreichische Agentur für Gesundheit und Ernährungssicherheit GmbH, Humanmedizin, Graz, Austria d NENT, Institute for Medical Microbiology, Kantonsspital, Luzern, Switzerland Received 19 July 2006; received in revised form 8 November 2006; accepted 20 November 2006 Available online 8 January 2007 Abstract Salmonella enterica subsp. enterica is one of the main causative agents of food-borne disease in man, and can also be the cause of serious systemic illness. Organisms belonging to this genus have traditionally been classified on the basis of the antigenic properties of the cell-surface lipopolysaccharide and of the phase 1 and phase 2 flagellar proteins. Primary isolation, biochemical identification, and serotyping are laborious and time consuming. Molecular identification based on suitable marker genes could be an attractive alternative to conventional bacteriological and serological methods. We have assessed the applicability of two housekeeping genes, gyrB, atpD, in combination with the flagellin genes fliC and fljB in multilocus sequence typing of Salmonella. Sequencing and comparative analysis of sequence data was performed on multiple strains from Austria, the United Kingdom, and Switzerland, representing all subspecies and 22 of the more prevalent non-typhoid S. enterica subsp. enterica serovars. A combination of these four marker genes allowed for a clear differentiation of all the strains analysed, indicating their applicability in molecular typing. The term MLST-v, for multilocus sequence typing based on virulence genes, is proposed to distinguish this approach from MLST based solely on housekeeping genes. An assortative recombination of the fliC gene was found in seven of the analysed serovars indicating multiple phylogenetic origin of these serovars. © 2006 Elsevier B.V. All rights reserved. Keywords: atpD; fliC; fljB; gyrB; Multilocus sequence typing; Salmonella 1. Introduction Salmonella is a large genus, and serotyping is widely used to classify isolates into serogroups according to their surface antigen variability. Serotyping is based on the immunological classification of the lipopolysaccharide moieties (O antigen), the flagellar protein (H antigen), and the capsular polysaccha- ride (Vi antigen). The Kauffmann–White scheme, generally used for the classification of Salmonella serotypes, recognises 46 O serogroups and 114 H antigens resulting in 2523 char- acterized serotypes (Popoff et al., 2003). The genus Salmonella is comprised of 2 species: S. enterica (with six subspecies — I, II, IIIa, IIIb, IV and VI), and S. bongori, now considered as a separate species (Brenner et al., 2000). Different genetic studies have indicated horizontal transfer events of chromosomal genes leading to the emergence of novel serovars (Beltran et al., 1988; Li et al., 1994; Porwollik et al., 2004). Serotyping is a reliable, epidemiologically congruent, well- established methodology for Salmonella typing, but is rather time consuming and requires a high number of specific antisera. Alternative molecular Salmonella typing methods are being developed in several laboratories (Echeit et al., 2002; Torpdahl et al., 2005; Kotetishvili et al., 2002; Sukhnanand et al., 2005; Herrera-Leon et al., 2004), aiming for higher sensitivity and specificity, better reproducibility. Multilocus sequence typing is one of the preferred methods, the existing MLST schemes being based on housekeeping genes. While these schemes reflect the Journal of Microbiological Methods 69 (2007) 23 – 36 www.elsevier.com/locate/jmicmeth ⁎ Corresponding author. Mailing address: ARC Seibersdorf Research GmbH, Department of Bioresources, A-2444 Seibersdorf, Austria. Tel.: +43 50550 3548; fax: +43 50550 3444. E-mail address: levente.bodrossy@arcs.ac.at (L. Bodrossy). 0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2006.11.013