Escherichia coli O157:H7 Shiga Toxin-Encoding Bacteriophages: Integrations, Excisions, Truncations, and Evolutionary Implications

Abstract

As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H ؊ presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx 1 (stx 1 -negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx 2 bacteriophage usually occupies wrbA in stx 1 ؉ /stx 2 ؉ E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx 1 -negative/stx 2 ؉ E. coli O157:H7 strains, the presumed progenitors of stx 1 ؉ /stx 2 ؉ E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx 2 bacteriophage insertion sites), suggest that stx 1 acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.Shiga toxins 1 and 2 (Stx1 and Stx2) are cardinal virulence factors of Escherichia coli O157:H7. Stx1 is nearly identical to Stx, the principal extracellular cytotoxin of Shigella dysenteriae serotype 1 (8). Stx2 has 56% identity to Stx1 (36). The stx 1 and stx 2 A and B subunit genes exist as tandem open reading frames (ORFs) in the central portion of lambdoid bacteriophages in E. coli O157:H7 (19). In Sakai and EDL933, the two E. coli O157:H7 strains that have been completely sequenced, the bacteriophage that encodes Stx1 is integrated into yehV (57), which encodes a protein that positively regulates curli expression (7), and is flanked by duplications of CCCTGT CACGTTACGCGCGTG. The bacteriophage that encodes Stx2 is integrated into wrbA (32, 40), which encodes a novel multimeric flavodoxin-like protein (13), and is flanked by duplications of GACATATTGAAAC. Almost all E. coli O157:H7 strains possess stx 2 , and approximately three-quarters contain, in addition, stx 1 (strains lacking stx 1 are referred to hereafter as stx 1 -negative strains) (23,38,45). Most human non-O157:H7 Stx-producing E. coli (STEC) strains possess stx 1 but lack stx 2 (5, 6, 28, 51). Except for an Stx2...

Optical maps distinguish individual strains of Escherichia coli O157 : H7

“…Although the frequency of inversions cannot be estimated from optical maps of temporally and geographically dispersed strains, the five sets of inversions in EDL933 identified in the laboratory studies arose during 20, 30 and 50 subcultures (Iguchi et al, 2006). Regarding the frequency of inversions and prophage changes in chromosomes, dissimilarities in the stx1 and stx2 gene profiles of particular O157 : H7 strains have been demonstrated to be not only the result of the heterogeneity in phages containing the toxin genes but also due to the unexpected variability in their integration sites (Shaikh & Tarr, 2003). Moreover, polymorphic PCR amplification patterns were an early indicator that insertions and deletions dominated the differences between O157 : H7 strains (Kudva et al, 2002a, b); many of these polymorphisms were found within prophages.…”

Genetic diversity of Shiga toxin-producing Escherichia coli O157 : H7 recovered from human and food sources

“…SBI genotypes of strains were determined as published (Besser et al, 2008;Shaikh & Tarr, 2003) with minor modification. Amplification of yehV or wrbA was performed in two multiplex PCRs for detection of bacteriophage integration and amplification of stx 1 and stx 2 genes was performed in two uniplex reactions as described (Botteldoorn et al, 2003).…”

“…Some investigators previously suggested that tir (255T) harbouring strains are more virulent in humans than tir (255A) harbouring strains Franz et al, 2012;Mellor et al, 2013). Shiga toxin bacteriophage insertion (SBI) site analysis relies on amplification of the stx toxin genes (stx 1 and stx 2 ) and the insertion site junctions of their encoding bacteriophages that can be used as a valuable genotyping technique to distinguish E. coli O157 : H7 strains, based on their distribution, gene expression and virulence potential (Besser et al, 2008;Shaikh & Tarr, 2003). Moreover, Shiga toxin 2 variants (stx 2a and stx 2c ) and stx 2 -Q antiterminator gene variants are clinically relevant genetic markers among E. coli O157 : H7 (Ahmad & Zurek, 2006;Persson et al, 2007).…”

Shiga toxins and stx phages: highly diverse entities

“…Considering the stx phages present in E. coli O157 : H7 strains, two insertion sites were first described as preferred: wrbA and yehV (Shaikh & Tarr, 2003;Besser et al, 2007). However, there are other integration sites described for stx phages in O157, such as sbcB, argW and yecE (De Greve et al, 2002;Mellor et al, 2012;Shringi et al, 2012).…”