Sequencing of the PCR products from these 9 serotype Xv strains showed that all were identical to that of 2002017

Sequencing of the PCR products from these 9 serotype Xv strains showed that all were identical to that of 2002017. Plasmid pSFXv_2 is usually a double-stranded circular plasmid of 6,850 bp in length. one of the rhamnose residues. A plasmid carried gene, (LPS phosphoethanolamine transferase for OCantigen), mediates the addition of PEtN for serotype Xv and additional MASF IV-1 positive strains. These findings reveal a novel serotype conversion mechanism in and display the necessity of further extension of the serotype classification plan realizing the MASF IV-1 positive strains as unique subtypes. Introduction is the major pathogen causing bacillary dysentery in developing countries. It is estimated that is responsible for approximately 164. 7 million shigellosis instances yearly worldwide, resulting in 1,100,000 deaths, with the majority including children under five years old [1]. is divided into numerous serotypes based Timp2 on the combination of antigenic Z-LEHD-FMK determinants present in the O-antigen of the cell envelope lipopolysaccharide (LPS). To day, at least 16 serotypes have been acknowledged [2], [3], [4], [5]. Except for serotype 6, all have the same fundamental repeating tetrasaccharide unit, comprised of 2)–L-Rha(type)] have been recognized [3], [8], [9], [10], [11]. They may be arranged in one operon known as the cluster [12]. and are highly conserved and interchangeable, while (type) is unique and encodes the glucosyltransferase responsible for the attaching of a glucosyl group to a specific sugars in the tetrasaccharide repeat unit of the O-antigen [3], [12]. The O-acetylation of RhaI depends on the presence of the gene for O-acetyl transferase [6], [7]. All the O-antigen changes genes known to day are encoded by seven temperate bacteriophages or prophages (SfI, SfIC, SfII, Sf6, SfIV, SfV and SfX), which are integrated into the conserved sites of the sponsor genome [3], [6], [8], [9], [10], [11], [13], [14]. In 2001, a novel serotype called serotype Xv appeared in Henan province, China. In the following years (2002C2006), it replaced serotype 2a and became probably the most common serotype in Henan province, accounting for 14%, 35%, 47%, 48%, and 27% of the isolations in the respective years [2]. Serotype Xv was also found to become the most common serotype in Shanxi province in 2006 (67%) and 2007 (33%) and in Z-LEHD-FMK Gansu, Anhui, and Shanghai in 2007 with 67%, 54%, and 35% of the isolations of serotypes 4a, Y and 6 strains [15], [17]. The genes responsible for the group 7,8 antigen have been identified on a SfX prophage integrated into the sponsor genome [2]. However, the element(s) responsible for the presence of MASF IV-1 or E1037 antigenic determinant in serotype Xv and additional MASF IV-1 Z-LEHD-FMK positive serotypes had not yet been recognized. In this study, we analyzed the O-antigen structure of serotype Xv strains and found Z-LEHD-FMK a phosphoethanolamine (PEtN) residue attached at position 3 of RhaII, which is definitely absent from the typical serotype X O-antigen. This changes was shown to confer the MASF IV-1 positive phenotype in serotype Xv strains. The gene named as strains were acquired with the written informed consent of the diarrhea individuals and with the authorization of the ethics committee of National Institute for Communicable Disease Control and Prevention, according to the medical study regulations of Ministry of Health (permit quantity 2007-17-3). Bacterial Strains, Plasmid and Culturing Condition Serotype Xv strains 2002017 [2] and 2003055 were utilized for LPS structure analysis. Serotype X strain 51580 (amps) and 4a strain NCTC 9725 (amps) were used as hosts for gene manifestation. pMD20-T Vector (TaKaRa) was utilized for DNA sequencing and manifestation vector. JM109 was utilized for plasmid propagation. strains utilized for plasmid profiling and gene detection analysis were isolated from diarrheal individuals in China or purchased from National Collection of Type Ethnicities (NCTC), UK. Strains were grown inside a 37C incubator or orbital shaker in Luria-Bertani broth (LB) supplemented with ampicillin (100 g ml?1) when appropriate. Isolation of Lipopolysaccharides and Polysaccharides LPS were isolated from dried bacterial cells from the phenol-water method [18]. The crude extract without separation of the layers was dialyzed against distilled water, nucleic acids and proteins were precipitated by adding aqueous 50% trichloroacetic acid at 4C to reduce pH to 2, the supernatant was dialyzed against distilled water and freeze-dried to give purified LPS in yields of 5.6C7.6% of dried cells mass. Delipidation of the LPS was performed with aqueous 2% acetic acid (6 ml) at 100C until precipitation of lipid A. The precipitate was eliminated by centrifugation (13,000strains were determined by slip agglutination test using two serotyping packages specific for those type- and group-factor antigens: (i) a commercially available monovalent antisera kit (Denka Seiken, Japan) and (ii) monoclonal antibody reagents (Reagensia Abdominal, Sweden), as explained in the manufacturers protocols. DNA Techniques Chromosomal DNA was isolated from strains using.