Downloaded from www.microbiologyresearch.org by IP: 54.237.57.119 On: Sun, 17 Apr 2016 10:32:32 Identification of a group of shigella-like isolates as Shigella boydii 20 Francine Grimont, 1 Monique Lejay-Collin, 1 Kaisar A. Talukder, 2 Isabelle Carle, 1 Sylvie Issenhuth, 1 Karine Le Roux 1 and Patrick A. D. Grimont 1 Correspondence Francine Grimont pfgrimont@free.fr 1 French National Reference Centre for Escherichia coli and Shigella, Unite ´ de Biodiversite ´ des Bacte ´ ries Pathoge ` nes Emergentes, Institut Pasteur, Paris, France 2 International Centre for Diarrhoeal Diseases Research, Dhaka-100, Bangladesh Received 4 July 2006 Accepted 15 January 2007 Infections by Shigella species are an important cause of diarrhoeal disease worldwide. Of 4198 Shigella isolates received by the French National Reference Centre for Escherichia coli and Shigella, 180 from patients with diarrhoea and dysentery in 2000–2004 did not react with any available polyclonal rabbit antisera used to identify the established Shigella serogroups. This study describes the molecular and phenotypic characteristics of these isolates in seroagglutination tests, molecular serotyping (rfb-RFLP and fliC-RFLP), ribotyping, detection of invasivity and enterotoxins genes, and antibiotic sensitivity. All isolates gave biochemical reactions typical of Shigella boydii, were mannitol-positive and indole-negative. They all carried invasion-associated genes, enterotoxin 2 [ShET-2] and an IS630 sequence. They had a unique ribotype that was distinct from all other Shigella and E. coli patterns. Further characterization by rfb-RFLP clearly distinguished this serogroup from all other Shigella or E. coli O-groups. The fliC-RFLP pattern corresponded to P4, an F-pattern which is associated with 10 different serogroups of S. boydii. A new antiserum prepared against strain 00-977 agglutinated all 180 isolates and cross-agglutination and absorption studies with anti-00-977 serum and anti-CDC 99-4528 (reference for the newly described S. boydii serogroup 20) serum showed identical antigenic structure. Furthermore, strains 00-977 and CDC 99-4528 had the same molecular serotype, ribotype and virulence genes. INTRODUCTION Shigellae cause diseases ranging from diarrhoea to bacillary dysentery, an invasive infection of the human colon affecting humans in developing countries, and are associated with poor hygiene conditions. In industrialized or developed countries, common-source outbreaks occur sporadically and several outbreaks have been reported (Butler, 2000; Legros, 2004). The diarrhoeal disease is highly contagious due to its low infectious dose. The genus Shigella comprises four subgroups that histor- ically have been treated as species (Enterobacteriaceae Subcommittee of the Nomenclature Committee of the International Association of Microbiological Societies, 1958): subgroup A is referred to as Shigella dysenteriae, subgroup B as Shigella flexneri, subgroup C as Shigella boydii and subgroup D as Shigella sonnei. In fact, these four so-called species constitute with Escherichia coli a single genomic species (Brenner et al., 1972, 1973), with the exce- ption of S. boydii 13, which is synonymous with Escherichia alberti (Brenner et al., 1982; Huys et al., 2003). Phylogen- etically, the shigellae (except S. boydii 13) are distributed in seven clusters within the E. coli branch (Lan & Reeves, 2002). Shigella species cannot be easily identified by biochemical properties alone, and definitive identification requires sero- typing (Ewing et al., 1958; Ewing & Lindberg, 1984). Based on somatic antigen (O-antigen) properties, S. dysenteriae is subdivided into 15 serogroups (Bopp et al., 2003) and 2 provisional serogroups (Ansaruzzaman et al., 1995; Coim- bra et al., 2001b; Melito et al., 2005). S. flexneri is subdivided into six serogroups and S. sonnei has a single serogroup which is divided into biotypes a, d, e, f and g (Janda & Abbott, 1988). Previously S. boydii consisted of 19 serogroups. In 1986, Ewing (1986) acknowledged 17 serogroups, including two provisional serogroups repre- sented by reference strain 2710-54 as S. boydii 16 and 3615- 53 as S. boydii 17. Gross et al. (1980) reported a new provisional serogroup with strain E10163, isolated in Bangladesh. Brenner and the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Enterobacteriaceae (Brenner, 1984) recommended that the strain be added to the scheme as S. boydii 18. More biochemical and serological studies were presented by Journal of Medical Microbiology (2007), 56, 749–754 DOI 10.1099/jmm.0.46818-0 46818 G 2007 SGM Printed in Great Britain 749