Ahead of Print -Clinical Isolates of Shiga Toxin 1a–Producing Shigella flexneri with an Epidemiological Link to Recent Travel to Hispañiola - Volume 20, Number 10—October 2014 - Emerging Infectious Disease journal - CDC

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Ahead of Print -Clinical Isolates of Shiga Toxin 1a–Producing Shigella flexneri with an Epidemiological Link to Recent Travel to Hispañiola - Volume 20, Number 10—October 2014 - Emerging Infectious Disease journal - CDC

Volume 20, Number 10—October 2014

Research

Clinical Isolates of Shiga Toxin 1a–Producing Shigella flexneri with an Epidemiological Link to Recent Travel to Hispañiola

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• Methods
• Results
• Discussion
• Suggested Citation

Figures

• Figure 1
• Figure 2
• Figure 3
• Figure 4
• Figure 5

Tables

• Table 1
• Table 2
• Table 3

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Miranda D. Gray, Keith A. Lampel, Nancy A. Strockbine, Reinaldo E. Fernandez, Angela R. Melton-Celsa, and Anthony T. Maurelli 

Author affiliations: Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA (M.D. Gray, R.E. Fernandez, A.R. Melton-Celsa, A.T. Maurelli); US Food and Drug Administration, College Park, Maryland, USA (K.A. Lampel); Centers for Disease Control and Prevention, Atlanta, Georgia, USA (N.A. Strockbine)

Suggested citation for this article

Abstract

Shiga toxins (Stx) are cytotoxins involved in severe human intestinal disease. These toxins are commonly found in Shigella dysenteriae serotype 1 and Shiga-toxin–producing Escherichia coli; however, the toxin genes have been found in other Shigella species. We identified 26 Shigella flexneri serotype 2 strains isolated by public health laboratories in the United States during 2001–2013, which encode the Shiga toxin 1a gene (stx1a). These strains produced and released Stx1a as measured by cytotoxicity and neutralization assays using anti-Stx/Stx1a antiserum. The release of Stx1a into culture supernatants increased ≈100-fold after treatment with mitomycin C, suggesting that stx1a is carried by a bacteriophage. Infectious phage were found in culture supernatants and increased ≈1,000-fold with mitomycin C. Whole-genome sequencing of several isolates and PCR analyses of all strains confirmed that stx1a was carried by a lambdoid bacteriophage. Furthermore, all patients who reported foreign travel had recently been to Hispañiola, suggesting that emergence of these novel strains is associated with that region.

Shiga toxins (Stx) are potent AB5 cytotoxins that inhibit eukaryotic protein synthesis, eventually leading to host cell death (1). Infections with bacteria that produce Stx result in hemorrhagic colitis and can lead to serious complications like hemolytic uremic syndrome (HUS) (2). Although 4 species of Shigella cause bacillary dysentery, historically only Shigella dysenteriae type 1 has been recognized as carrying the genes for Shiga toxin (stx). Likewise, S. dysenteriae 1 is the only Shigella species that causes HUS as a complication of infection (3).

The genes encoding the toxin are found in an operon consisting of stxA and stxB. The stx locus in S. dysenteriae 1 is surrounded by DNA sequence homologous to lambdoid bacteriophage sequence; however, the toxin genes are not associated with a complete prophage genome (4,5). Insertion sequences flanking the stx region suggest that gene rearrangements occurred and resulted in a defective phage. As a consequence, viable phage are not recovered from S. dysenteriae 1 cultures even under conditions that induce phage production (6).

Stx has been extensively studied in Shiga toxin–producing Escherichia coli (STEC), notably E. coli O157:H7. STEC produce 2 variants of Stx: Stx1a (which differs from S. dysenteriae 1 Stx by 1 aa), and Stx2 (which shares 56% identity with Stx1a) (7,8). In contrast to the toxin genes in S. dysenteriae 1, those in STEC are generally carried by lambdoid prophages, which integrate into the host bacterial chromosome (9). The phage remains in a lysogenic state until environmental conditions induce expression of phage lytic cycle genes, leading to new phage production and lysis of the host bacterium. The stx1a and stx2 loci are found within the late gene regions of the phage; therefore, induction of the lytic cycle increases expression of the toxin genes and host cell lysis allows for toxin release (10).

Recently, acquisition of stx genes in clinical isolates of other Shigella species has been reported (11,12). Three cases of infection with S. dysenteriae 4 were described, and all were shown to express stx1. No further characterization of the stx1-encoding S. dysenteriae 4 strains was reported; however, all 3 infected patients had reported recent travel to Hispañiola (11). An isolate of Shiga toxin–producing S. sonnei from a patient returning from the Ukraine was also characterized; the toxin genes were determined to be carried by a lambdoid prophage homologous to stx-encoding phages found in STEC (13).

We identified 26 clinical isolates of S. flexneri 2 that encode stx1a. DNA sequence and PCR analyses determined that stx1a is encoded by a lambdoid prophage. Characterization of the phage indicated that it behaves similarly to stx-encoding phages that infect STEC. Like the patients from whom stx1-encoding S. dysenteriae 4 was isolated, patients from whom stx1a-encoding S. flexneri 2 was isolated and who reported foreign travel had also recently visited Hispañiola. The potential consequences of an epidemiological link to this region are discussed.

Dr Gray is a postdoctoral research fellow at Uniformed Services University of the Health Sciences. Her primary research interests focus on understanding bacterial pathogens that cause intestinal diseases.

Acknowledgments

We thank the state health laboratories that provided strains, Charlie Wang and James Pettengill who provided sequencing assistance, and Stephen Darnell who provided technical assistance.

This work was supported by grants R01AI24656 and AI020148 from the National Institute of Allergy and Infectious Diseases.

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Figures

• Figure 1. Shiga toxin 1a (Stx1a) is produced and released from stx1a–encoding Shigella flexneri isolates. Overnight supernatants from BS937, BS938, and BS974 were serially diluted 10-fold in medium alone, medium containing...
• Figure 2. Mitomycin C induces production of Shiga toxin 1a (Stx1a) in a recA-dependent manner. Exponentially growing cultures of the indicated parental strains or recA mutants were grown with or without...
• Figure 3. Infectious phage in the supernatants of Shiga toxin 1a gene (stx1a)–encoding Shigella flexneri are induced with mitomycin C (mito C) treatment. Supernatants were collected from exponential cultures of BS937,...
• Figure 4. Schematic of PCR designed to determine that Shiga toxin 1a gene (stx1a) is phage encoded and inserted into the S1742 locus of Shigella flexneri. The genetic map shows the...
• Figure 5. PCR results from representative clinical isolates illustrate that the Shiga toxin 1a gene (stx1a) is phage encoded and inserted into the S1742 locus of Shigella flexneri. PCRs based on...

Tables

• Table 1. Isolation information for clinical strains of Shigella flexneri , USA, 2001–2013
• Table 2. Primer pairs used for PCR analysis
• Table 3. Virulence properties of Shigella flexneri strains

Suggested citation for this article: Gray MD, Lampel KA, Strockbine NA, Fernandez RE, Melton-Celsa AR, Maurelli AT. Clinical isolates of Shiga toxin 1a–producing Shigella flexneri with an epidemiological link to recent travel to Hispañiola. Emerg Infect Dis [Internet]. 2014 Oct [date cited].http://dx.doi.org/10.3201/eid2010.140292

DOI: 10.3201/eid2010.140292