Structural Analysis of Phage-Borne stx Genes and Their Flanking Sequences in Shiga Toxin-Producing Escherichia coli and Shigella dysenteriae Type 1 Strains

Abstract

The stx-flanking regions of 49 Shiga toxin-producing Escherichia coli strains and nine Shigella dysenteriae serotype 1 strains containing either stx, stx 1 , stx 2 , or stx 2 variant genes, were examined. We analyzed these regions by PCR using a set of primers with one primer specific for the respective stx gene and a second primer complementary to sequences of Stx phages H-19B and 933W. We further characterized the amplification products by restriction endonuclease digestion and nucleotide sequencing. PCR products of stx 1 -containing E. coli strains of serogroups O157, O26, and 0103 showed the same lengths and similar restriction patterns. However, we failed to amplify the 3 stx-flanking region in stx 1 -harboring E. coli O111:H ؊ strains. Stx2-producing E. coli strains revealed amplification products of different lengths and restriction patterns, suggesting greater heterogeneity than in stx 1 -positive strains. We also obtained specific PCR products for two Stx2c-producing and seven Stx2f-producing E. coli strains when they were subjected to PCR analysis. In nine S. dysenteriae type 1 strains, H-19B-and 933W-specific primers amplified only the 3 stx-flanking region. The results of our study demonstrate that the stx genes of all strains investigated are continuous with phage sequences. Whereas almost all strains except E. coli O111:H ؊ strains were associated with a S-like gene, association with Q could not be demonstrated in nine S. dysenteriae type 1 strains and three E. coli strains. Furthermore, we showed that the organization of the stx-flanking regions is similar in all strains investigated, whereas fine-structure analysis showed subtle differences among the sequences examined. Our results support the hypothesis that stx genes in E. coli and S. dysenteriae are generally phage-borne.Shiga toxin (Stx)-producing Escherichia coli (STEC) can cause a broad spectrum of human diseases ranging from watery diarrhea to hemorrhagic colitis and the hemolytic-uremic syndrome (5). Shiga toxins have been generally accepted to be the main agent in the development of these diseases (5, 16).Two Stx subgroups have been described in E. coli: Stx1 and Stx2. The structural genes for Stx1 and Stx2 are encoded in the genome of temperate, lambdoid bacteriophages in E. coli O157 and O26 strains (9,(28)(29)(30). Such Stx-encoding phages of E. coli O157 and O26 strains were reported to have similar morphologies with regular hexagonal heads and short tails (20). E. coli O157 phages are similar in genome size and have highly related restriction fragment length polymorphism (RFLP) patterns (17). stx RFLP pattern analysis of O157 and non-O157 STEC strains suggested a more heterogeneous structure of phage DNA flanking the stx genes in E. coli O157:H7 than in non-O157 STEC strains (23). Recently, the whole genome of the Stx2-encoding phage 933W and a 17.3-kb fragment of the Stx1-encoding phage H-19B were determined by nucleotide sequencing (15,19).Sequence analysis of a part of the Stx1-encoding phage H-19B has shown that the stxA 1 and s...

Diversity of stx 2 converting bacteriophages induced from Shiga-toxin-producing Escherichia coli strains isolated from cattle

“…Phage infection of the different host strains was determined by positive hybridization. Phage infection of S. sonnei is noteworthy, as several authors have suggested that Shigella might be the origin of the dissemination of stx genes (Strauch et al, 2001;Unkmeir & Schmidt, 2000). Strain 866 of S. sonnei was also a suitable candidate for phage transduction, since two of the six phage lysogens were preferentially obtained with S. sonnei and we obtained a high number of lysogens with these two phages (data not shown).…”

“…According to the well-accepted theory of a common bacterial ancestor carrying the stx 2 gene, all stx 2 phages might share a common origin (Strauch et al, 2001;Unkmeir & Schmidt, 2000). However, in the case of the stx 2 gene, the presence of multiple variants argues against a single origin.…”

“…Various patterns of stx-phages have been described and since they present a wide range of DNA structures, restriction patterns, host spectra and morphology, they are considered to form a heterogeneous family (Unkmeir & Schmidt, 2000;Schmidt et al, 2000;Johansen et al, 2001). …”

Evaluation of Major Types of Shiga Toxin 2e-Producing Escherichia coli Bacteria Present in Food, Pigs, and the Environment as Potential Pathogens for Humans

“…Only five (4.5%) of the 110 Stx2e strains showed high production of Stx2e with noninduced and induced cultures, indicating that constitutive high expression of stx 2e genes is not common in natural isolates. The upstream regions flanking the 5Ј ends of the stx genes were reported to be conserved and continuous with phage sequences in a number of Shiga toxin-encoding bacteriophages (32,39,44). Genes for rare tRNAs are located in the neighborhood of the stx 2 genes, and it was suggested that these could play a role in effective expression of Stx2 (23,38).…”

Shiga toxins and stx phages: highly diverse entities

“…(2014) described a functional esterase encoded downstream of the stx 2a operon in the bacteriophage 933W, and homologue genes are present in many stx 2 -encoding phages (Unkmeir & Schmidt, 2000).…”