Thus it seems that this novel serotype has already appeared in natural infections. Although serotype 1 d represented less than 1% of the isolates, it would be important to monitor this new serotype epidemiologically, considering that novel S. flexneri serotypes such as 1c and Xv achieved its dominance among the S. flexneri serotypes in a very short time frame [5, 16, 17] SfI and SfX integrated in tandem into the same site of host chromosome CT99021 supplier It has been observed that the serotype-converting phages, except for Sf6, usually integrate into the tRNA-thrW

gene of the host chromosome, which is adjacent to proA upstream [15]. However, the gene downstream the integrated phage have not been consistently identified [6, 7]. Genomic analysis of S. flexneri serotype 2a strain 301 (NC_004337), 2457 T (NC_004741) and serotype Xv strain 2002017 (CP001383) showed that the serotype-converting

phages were all integrated upstream of host gene yaiC. Thus cross-bridging PCR analyses of S. flexneri 036, 036_X, PD0332991 chemical structure and 036_1d across the proA-yaiC region were conducted using a series of primers and found that both phages SfX and SfI were integrated into the tRNA-thrW site, which is immediately downstream of gene proA, and upstream of gene yaiC (Figure 2). The phage SfI was found to be integrated immediately upstream of SfX genome, with an att site at both ends (Figure 2). By comparing the joining sequences between the serotype-converting phage genomes,

we found that the phage SfI was integrated at the attL site of phage SfX CYTH4 (see Additional file 1). The integration site for the 24 serotype X isolates converted by SfI was also found to be the same site and thus it appears that the integration is very site specific. Figure 2 Genetic organization of prophage genomes of SfX and/or SfI in S. flexneri 036_X and 036_1d. The prophage genomes of SfX and/or SfI are highlighted in yellow and pink respectively. The conserved genes of the host strain were shown in different colors: proA, gray; yaiC, yellow; IS600 ORF1 and ORF2, brown; IS629 ORF1 and ORF2, orange; the putative integrase gene (int), white. The integration sites attB, attL and attR are indicated in thick line. After strain name in brackets is the serotype of the strain. Conclusions A novel serotype 1 d was constructed by sequentially infecting a serotype Y strain of S. flexneri with phage SfX and SfI, or by infecting clinical serotype X isolates with SfI. These results indicate that serotype conversion with phages SfI and SfX could occur in nature. However, the observation that the order of infection by the 2 phages affects convertibility of a strain indicates that serotype conversion is not only determined by the modification specific genes but also constrained by the properties of the serotype-converting phages. Our findings provide possible mechanisms how new serotypes of S. flexneri could emerge in nature.