Identification and characterization of a novel Shigella flexneri serotype Yv in China

Abstract

Shigella flexneri is the major cause of bacterial shigellosis in developing countries. S. flexneri is divided into at least 19 serotypes, the majority of which are modifications of the same basic O-antigen by glucosylation and/or O-acetylation of its sugar residues by phage encoded serotype-converting genes. Recently, a plasmid encoded phosphoethanolamine (PEtN) modification of the O-antigen has been reported, which is responsible for the presence of the MASF IV-1 determinant and results in conversion of traditional serotypes X, 4a and Y to novel serotypes Xv, 4av and Yv, respectively. In this study, we characterized 19 serotype Yv strains isolated in China. A variant of the O-antigen phosphoethanolamine transferase gene opt (formerly called lpt-O) carried by a pSFxv_2-like plasmid was found in serotype Yv strains, which specifies the phosphorylation pattern on the O-antigen of this serotype. For the majority of the O-antigen units, the PEtN modification occurs on Rha(III), while for a minority, modifications occur on both Rha(II) and Rha(III). Serotype-specific gene detection and PFGE analysis suggested that these serotype Yv isolates were originated from serotypes Y, Xv and 2a by acquisition of an opt-carrying plasmid and/or inactivation of serotype-specific gene gtrII or gtrX. These data, combined with those of serotypes Xv and 4av reported earlier, demonstrate that the plasmid-encoded PEtN modification is an important serotype conversion mechanism in S. flexneri, in addition to glucosylation and O-acetylation.

Figure 1. PFGE profiles of the 19 serotype Yv isolates studied and comparison with closest non-Yv strains.

The dendrogram constructed using the NotI-digested PFGE patterns was generated by the UPGMA algorithm with the Dice similarity value of two patterns at 1.0% pattern optimization and 0.8% band position tolerance. Serotype-specific genes were detected using multiplex-PCR as described previously .

Figure 2. The presence of opt-carrying plasmid in serotype Yv isolates, and genomic comparison with pSFxv_2 of strain 2002017 (accession No. CP001385).

A, plasmid profiles of the 19 serotype Yv isolates. Plasmid DNA was separated by electrophoresis with a Chef Mapper system (Bio-Rad) on a 1% SeaKem Gold agarose gel and visualized by EB staining. Red arrow indicates the position of opt-carrying plasmid. Plasmids isolated from strain 2002017 were used as control. Lambda DNA cleaved with HindIII (TaKaRa, Japan) was used as molecular mass markers. Southern hybridization detection of the opt gene in serotype Yv isolates is shown beneath the gel picture. B, genomic comparison of pSFyv_2 of strain HN006 and pSFxv_2 of 2002017. ORFs were shown as thick arrows, and coding regions were marked according to the genome annotations. The base changes were indicated using blue lines.

Figure 3. Multiplex-PCR detection of serotype-specific genes in the 19 serotype Yv isolates studied.

PCR products were electrophoresed on a 1.5% (wt/vol) agarose gel, stained with ethidium bromide, and photographed under UV light. Gene wzx was used as control for S. flexneri. The sizes of the PCR products are indicated on the right. M1 and M2, 150- and 100-bp DNA ladder markers (TaKaRa, Japan).

Figure 4. Schematic diagram of the putative origins of the serotype Yv isolates.

The serotypes, serotype-converting phages and the plasmid gene involved are indicated in ellipses. The O-antigen structures of different serotypes are shown below the ellipses. Sugar residues and modifications are displayed in symbols as shown. The order of occurrence of the gene inactivation and acquisition of the opt-carrying plasmid is unknown and alternative orders are thus depicted. Putative intermediate serotypes between 2a and Yv are indicated in dashed ellipses.