Shigella is the primary pathogen causing bacillary dysentery in developing countries. There are an estimated 164.7 million people worldwide infected by Shigella annually; resulting in 1.1 million deaths, most being children under five years . A more recent study estimated approximately 125 million annual shigellosis cases and 14,000 related deaths in Asia , suggesting that the death rate has decreased significantly in recent years. Among the four Shigella species, S. dysenteriae, S. flexneri, S. boydii, and S. sonnei, S. flexneri is the predominant species .
S. flexneri serotyping are based on structure of the O-antigen lipopolysaccharide. There are 15 known serotypes: 1a, 1b, 1c, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6, X, Xv and Y [4, 5]. Except for serotype 6, all share a common tetrasaccharide backbone of repeating units of N-acetylglucosamine-rhamnose-rhamnose-rhamnose . By adding glucosyl and/or O-acetyl groups to one or more of the sugars on the tetrasaccharide unit, various serotypes are formed. Serotype Y possesses the primary basic O-antigen without any modification of the tetrasaccharide backbone .
It is well known that S. flexneri serotype conversion is mediated by temperate bacteriophages [6, 7]. Six different serotype-converting phages or prophages, SfI, SfII, Sf6, SfIV, SfV and SfX, have been identified and characterized [8–12], which can convert serotype Y to serotype 1a, 2a, 3b, 4a, 5a and X respectively [8–12]. Except for Sf6 which carries a single gene, oac, for acetylation of the O-antigen , the other phages carry three genes, gtrA, gtrB, and gtr
type for O-antigen modification. The first two gtr genes are highly conserved and interchangeable in function, while the third gtr gene encodes a type-specific glucosyltransferase responsible for the addition of glucosyl molecules to sugar residue(s) on the basic O-antigen repeating unit [9, 12, 14]. These phages integrate into the S. flexneri host chromosome either at tRNA-thrW downstream of proA  or at tRNA-argW adjacent to yfdC . Once integrated, the int and O-antigen modification genes are located at the opposition ends of the prophage genome, flanked by an attL sequence on the left and an attR sequence on the right .
Recently, untypeable or novel serotypes of S. flexneri from natural infections had been reported worldwide [5, 16, 17]. A novel serotype 1c was identified in Bangladesh in the late 1980s and was a predominant serotype in Vietnam and other Asian countries [16, 17]. Serotype 1c was a result of modification of serotype 1a with addition of a glucosyl group by a cryptic prophage carrying a gtr1C gene cluster . More recently, a new serotype named as Xv emerged in China, and replaced 2a to become the most prevalent S. flexneri serotype . Although the antigenic determinant for the v variant is not yet known, the phage SfX, which is responsible for the group 7;8 antigenic determinant, was inducible from the sequenced S. flexneri Xv strain 2002017 . Therefore emergence and spread of novel S. flexneri serotypes in nature poses a significant public health threat globally and in particular in developing countries where S. flexneri is the predominant cause of shigellosis.
In order to reveal possible roles played by the serotype-converting phages in the emergence of new serotypes, and potential of emergence of novel serotypes through this mechanism in nature, we performed infection assays using SfI and SfX, the 2 most common serotype-converting bacteriophages carried by S. flexneri based on serotype frequency data [5, 19]. We demonstrate that a novel serotype, named serotype 1 d was created in laboratory by infecting S. flexneri serotype X strains with a SfI phage or by sequential infection of serotype Y strain with SfX and SfI.