Abstract
We identified the cytolethal distending toxin V (CDT-V) gene cluster in 19 (4.9%) of 391 enterohemorrhagic Escherichia coli O157:H7. cdt-V+ strains belonged to five phage types (PTs) and were most frequent within PTs 14 and 34. CDT-V was expressed in all but two cdt-V+ strains and was lethal to cultured endothelial cells. Subtyping schemes should include cdt-V as a marker to differentiate E. coli O157:H7 even within the same phage type.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes severe human disease (18), including painful bloody diarrhea and the hemolytic-uremic syndrome (HUS) (18). EHEC O157:H7 appears to have evolved from an enteropathogenic E. coli O55:H7 ancestor. This ancestor, which already contained the locus for enterocyte effacement, acquired the bacteriophage-encoded Shiga toxin 1 (stx1) and/or 2 (stx2) gene, a large virulence plasmid, and transitioned somatic antigens O55 to O157 (6). Sorbitol-fermenting (SF) EHEC O157:NM (nonmotile) strains subsequently separated from this lineage. We have shown (10) that the vast majority of SF EHEC O157:NM strains (87%) express cytolethal distending toxin (CDT) (10) and that cdt in SF EHEC O157:NM differs from the four cdt alleles (cdt-I, cdt-II, cdt-III, and cdt-IV) in E. coli of other pathogroups (19). We designated this new cdt allele cdt-V (2, 10).
As in the case of other members of the CDT family (19), CDT-V is encoded by three adjacent genes (cdtA, cdtB, and cdtC) which encode three subunits (CdtA, CdtB, CdtC), all of which are needed for biological activity. However, in some cases, CdtB and CdtC are sufficient to cause intoxication (19). CDT-V damages eukaryotic DNA, leading to G2/M cell cycle arrest and cell death (3). Other members of the CDT family can also cause G1 cell cycle arrest (5). Preliminary analysis of a limited number of clinical E. coli O157:H7 strains suggests that cdt genes are also present in these pathogens (10). This finding prompted us to investigate the prevalence of cdt-V in a large collection of well-defined E. coli O157:H7 strains and to characterize the structure of the toxin, its flanking regions, and its biological activity. We also determined the nature of the population of E. coli O157:H7 strains harboring cdt-V and investigated if the presence of cdt-V in these strains is associated with disease.
Frequency of cdt genes among E. coli O157:H7 strains.
Three hundred ninety-one EHEC O157:H7 strains, isolated between January 1988 and September 2005, were investigated for cdt-I, cdt-II, cdt-III, cdt-IV, and cdt-V using primers described previously (2). Three hundred twenty-nine previously described isolates (4, 7, 8, 11, 13) are from sporadic infections. Sixty-two additional strains were isolated as described previously (7). These strains from patients with HUS (n = 160) or diarrhea (n = 158), or from asymptomatic carriers (n = 73), were subjected to PCRs using primers targeting cdt-I, cdt-II, cdt-III, cdt-IV, and cdt-V (2, 10). None of the strains possessed cdt-I, cdt-II, cdt-III, and cdt-IV genes. However, 19 isolates (4.9%) tested positive for each of the cdtV-A, cdtV-B, and cdtV-C genes (Table 1); all others were negative for cdt-V.
TABLE 1.
Distribution of the cdt-V cluster within the phage types of E. coli O157:H7
| Phage type | No. (%) of strains
|
|
|---|---|---|
| Total | cdt-V positivea | |
| 1 | 6 (1.5) | 0 (0) |
| 2 | 108 (27.6) | 6 (5.6) |
| 3 | 1 (0.3) | 0 (0) |
| 4 | 21 (5.4) | 1 (4.8) |
| 8 | 118 (30.2) | 3 (2.5) |
| 14 | 32 (8.2) | 5 (15.6) |
| 21 | 1 (0.3) | 0 (0) |
| 28 | 11 (2.8) | 0 (0) |
| 31 | 8 (2.0) | 0 (0) |
| 32 | 28 (7.2) | 0 (0) |
| 33 | 4 (1.0) | 0 (0) |
| 34 | 13 (3.3) | 3 (23.1) |
| 39 | 1 (0.3) | 0 (0) |
| 45 | 4 (1.0) | 0 (0) |
| 46 | 1 (0.3) | 0 (0) |
| 51 | 1 (0.3) | 0 (0) |
| 54 | 16 (4.0) | 0 (0) |
| 70 | 1 (0.3) | 0 (0) |
| 78 | 2 (0.5) | 0 (0) |
| NTb | 14 (3.5) | 1 (7.1) |
| Total | 391 (100) | 19 |
Percentages represent the proportion of cdt-V-positive strains within the individual phage types.
NT, nontypeable with the spectrum of bacteriophages used (13).
Subtyping of cdt-V-harboring E. coli O157:H7 strains.
PCRs and fliC restriction fragment length polymorphism (7, 8, 17) demonstrated that all 391 EHEC O157:H7 strains shared eae, EHEC-hlyA, rfbO157, and fliCH7, which encode intimin, EHEC hemolysin, the O antigen 157, and the structural subunit of the H7 antigen, respectively. None contained sfpA, which encodes fimbriae and is a defining characteristic of SF EHEC O157:NM (8). The 391 EHEC O157:H7 strains investigated belonged to 19 phage types (PTs) (Table 1), which represent the spectrum of PTs within E. coli O157:H7 in Germany and are in concordance with other studies from Germany (1, 13) and other European countries (14, 20). The cdt-V-positive strains belonged to five different PTs (Table 1). To assess, if cdt-V is associated with a specific stx allele, stx genotyping (7) was performed and demonstrated that the 19 cdt-positive strains belonged to three different stx genotypes (Table 2). In addition, the genetic diversity of the cdt-V-positive strains was supported by pulsed field gel electrophoresis analysis (data not shown).
TABLE 2.
Characterization of CDT-positive E. coli O157:H7 strains
| Strain no. | Diagnosisa | Phage typeb | stx allele | CDT titerc
|
|
|---|---|---|---|---|---|
| CHO | EAhy.926 | ||||
| 4371/2/95 | D | 2 | stx2 | 1:16 | 1:8 |
| 4597/96 | D | 2 | stx2+ stx2c | 1:4 | 1:8 |
| 00-09850 | BD | 2 | stx2 + stx2c | 1:8 | 1:8 |
| 01-09698 | BD | 2 | stx2 | 1:8 | 1:8 |
| 3159/01 | HUS | 2 | stx2 | 1:2 | 1:4 |
| E-02-629 | D | 2 | stx2 | 1:4 | 1:8 |
| 01-00088 | D | 4 | stx2c | 1:2 | 1:2 |
| 00-10916 | D | 8 | stx2c | 1:4 | 1:8 |
| 02-11509 | D | 8 | stx2c | 1:8 | 1:8 |
| 05-03863 | D | 8 | stx2c | 1:2 | 1:2 |
| 5791/99 | HUS | 14 | stx2 | 1:8 | 1:8 |
| 00-04725 | D | 14 | stx2 | 1:8 | 1:16 |
| 00-05008 | D | 14 | stx2c | 1:2 | 1:4 |
| 03-00765 | D | 14 | stx2c | <1:2 | <1:2 |
| 05-06010 | D | 14 | stx2c | <1:2 | <1:2 |
| 3010/96 | D | 34 | stx2c | 1:8 | 1:2 |
| 5407/96 | D | 34 | stx2+ stx2c | 1:8 | 1:8 |
| 05-00404 | A | 34 | stx2c | 1:2 | 1:4 |
| 2403/92 | HUS | NT | stx2+ stx2c | 1:16 | 1:16 |
BD, bloody diarrhea; D, diarrhea without visible blood; A, asymptomatic.
NT, nontypeable with the spectrum of bacteriophages used (13).
The reciprocal of the highest dilution of a culture filtrate that distended 50% of the cells after 4 days of incubation.
cdt sequencing.
The cdt genes cluster in six strains, including E-02-629 (PT2), E-01-88 (PT4), E-05-3863 (PT8), 5791/99 (PT14), 5407/96 (PT34), and 3159/01 (PT nontypeable), were 100% identical to each other and to that of the cdt-V gene cluster in SF E. coli O157:NM strain 493/89 (GenBank accession no. AJ508930).
cdt-V-flanking regions.
cdt-V in SF E. coli O157:NM strain 493/89 is located within the late gene region of bacteriophage P2 (10). To investigate if cdt-V flanks are conserved in E. coli O157:H7 strains, we performed PCR with primer pairs P2-A2 (5′-CACTGACAACGGCTGAAC-3′) and cdtA-F (5′-AAATGGGGAGCAGGATAC-3′) and cdtC-F (5′-GAACCCCAAATACAGACC-3′) and P2-C3 (5′-TGGTTGATGACGGTGTTA-3′), which amplify the cdt-VA- and cdt-VC-flanking regions, respectively, in strain 493/89. PCR products of 848 bp and 712 bp, respectively, produced by strain 493/89 were also elicited from each of the 19 cdt-V-positive E. coli O157:H7 strains, indicating that the cdt-V cluster in EHEC O157:H7 is also flanked by P2 phage-related sequences.
CDT-V expression in EHEC O157:H7.
Since a progressive cell distension is a hallmark of the biological activity of CDT (12), we investigated sterile filtered supernatants of the cdt-V-positive strains on Chinese hamster ovary (CHO) cells (10). Of 19 E. coli O157:H7 strains harboring cdt-V, 17 produced active CDT-V. Since endothelial cells are the major targets affected during HUS (18), we also investigated the effect of CDT on the endothelial cell line EA.hy926, which is derived from human umbilical vein endothelial cells (3). Both CHO and EA.hy926 cells progressively distended for up to 4 days after exposure to filtrates of the cultured strains and then died. CDT titers ranged from 1:2 to 1:16 on each of the cell lines (Table 2).
Clinical association.
cdt-V in the EHEC O157:H7 strains investigated was significantly more frequent in isolates from patients with diarrhea than in isolates from patients with HUS (Yate's corrected χ2 = 7.27, P = 0.007, 95% confidence interval [CI95] = 1.50 to 30.05) or asymptomatic carriers (Yate's corrected χ2 = 3.39, P = 0.047, CI95 = 11.11 to 322.28).
We describe the first systematic study of the prevalence of cdt among EHEC O157:H7 strains. It is noteworthy that the cdt cluster was not present in the chromosome sequences of two unrelated EHEC O157:H7 strains (9, 15). The cdt-V cluster is distributed among strains of three different stx genotypes and five different PTs, indicating considerable clonal diversity in E. coli O157:H7 strains harboring cdt-V. One explanation for this clonal diversity might be the independent acquisitions of cdt-V by EHEC O157:H7 via horizontal transfer of a mobile genetic element. Such a scenario is supported by our finding of bacteriophage P2 sequences flanking the cdt-V cluster in each of the 19 EHEC O157:H7 strains investigated in this study, raising the possibility that cdt-V is acquired by phage transduction. The association of cdt-V with several particular PTs might reflect the ability of such strains to be transduced by the cdt-containing phage, which, in turn, may depend on the presence of phage-specific receptors on the surface and/or specific phage integration sites within the chromosomes of these strains. It is worth noting that the frequency of cdt-V within the most prevalent E. coli O157:H7 PTs (PTs 2 and 8) is low (Table 1). In contrast, in PTs 14 and 34, the cdt-V frequencies were 15.6% and 23.1%, respectively. PT 14 is one of the most frequent E. coli O157:H7 PTs isolated in Canada (16) and was the third-most-frequent E. coli O157:H7 PT in our study. Thus, the proportion of cdt-positive EHEC O157:H7 strains may depend on the phage types tested, and secular trends can influence this frequency. Another interesting finding in this study is the relationship between an infection with E. coli O157:H7 strains harboring cdt-V and clinical disease, which confirms our previous findings on the presence of cdt-V in non-O157 EHEC strains and their pathogenicity (2). We demonstrated that among eae-negative non-O157 EHEC strains, cdt-V is significantly more frequent in isolates from patients with HUS or with diarrhea than in isolates from asymptomatic carriers (2).
Because CDT is a potential virulence factor, subtyping schemes of E. coli O157:H7 should include typing for cdt-V as an additional marker gene, since this might enable the differentiation of clonally highly related EHEC O157:H7 strains even within the same PT. If cell cultures are not available, PCR detection of cdtB-V can be used as a surrogate test implying the presence of cdt-V in clinical isolates.
Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft (DFG) program “Infections of the Endothelium” SPP 1130, grant KA 717/4-2, a grant from the Interdisciplinary Center of Clinical Research Münster (IZKF, project no. Ka2/061/04), and 973 program grant 2005CB522904 from the Ministry of Science and Technology, People's Republic of China.
We thank M. Hülsmann for excellent technical assistance and P. I. Tarr (Washington University School of Medicine, St. Louis, Mo.) for critical reading of the manuscript and stimulating discussions.
REFERENCES
- 1.Beutin, L., S. Kaulfuss, T. Cheasty, B. Brandenburg, S. Zimmermann, K. Gleier, G. A. Willshaw, and H. R. Smith. 2002. Characteristics and association with disease of two major subclones of Shiga toxin (Verocytotoxin)-producing strains of Escherichia coli (STEC) O157 that are present among isolates from patients in Germany. Diagn. Microbiol. Infect. Dis. 44:337-346. [DOI] [PubMed] [Google Scholar]
- 2.Bielaszewska, M., M. Fell, L. Greune, R. Prager, A. Fruth, H. Tschäpe, M. A. Schmidt, and H. Karch. 2004. Characterization of cytolethal distending toxin genes and expression in Shiga toxin-producing Escherichia coli strains of non-O157 serogroups. Infect. Immun. 72:1812-1816. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Bielaszewska, M., B. Sinha, T. Kuczius, and H. Karch. 2005. Cytolethal distending toxin from Shiga toxin-producing Escherichia coli O157 causes irreversible G2/M arrest, inhibition of proliferation, and death of human endothelial cells. Infect. Immun. 73:552-562. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Bielaszewska, M., P. I. Tarr, H. Karch, W. Zhang, and W. Mathys. 2005. Phenotypic and molecular analysis of tellurite resistance among enterohemorrhagic Escherichia coli O157:H7 and sorbitol-fermenting O157:NM clinical isolates. J. Clin. Microbiol. 43:452-454. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Cortes-Bratti, X., C. Karlsson, T. Lagergard, M. Thelestam, and T. Frisan. 2001. The Haemophilus ducreyi cytolethal distending toxin induces cell cycle arrest and apoptosis via the DNA damage checkpoint pathways. J. Biol. Chem. 276:5296-5302. [DOI] [PubMed] [Google Scholar]
- 6.Feng, P., K. A. Lampel, H. Karch, and T. S. Whittam. 1998. Genetic and phenotypic changes in the emergence of Escherichia coli O157:H7. J. Infect. Dis. 177:1750-1753. [DOI] [PubMed] [Google Scholar]
- 7.Friedrich, A. W., M. Bielaszewska, W.-L. Zhang, M. Pulz, T. Kuczius, A. Ammon, and H. Karch. 2002. Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J. Infect. Dis. 185:74-84. [DOI] [PubMed] [Google Scholar]
- 8.Friedrich, A. W., K. V. Nierhoff, M. Bielaszewska, A. Mellmann, and H. Karch. 2004. Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H−. J. Clin. Microbiol. 42:4697-4701. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Hayashi, T., K. Makino, M. Ohnishi, K. Kurokawa, K. Ishii, K. Yokoyama, C. G. Han, E. Ohtsubo, K. Nakayama, T. Murata, M. Tanaka, T. Tobe, T. Iida, H. Takami, T. Honda, C. Sasakawa, N. Ogasawara, T. Yasunaga, S. Kuhara, T. Shiba, M. Hattori, and H. Shinagawa. 2001. Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res. 8:11-22. [DOI] [PubMed] [Google Scholar]
- 10.Janka, A., M. Bielaszewska, U. Dobrindt, L. Greune, M. A. Schmidt, and H. Karch. 2003. The cytolethal distending toxin (cdt) gene cluster in enterohemorrhagic Escherichia coli O157:H− and O157:H7: characterization and evolutionary considerations. Infect. Immun. 71:3634-3638. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Janka, A., G. Becker, A.-K. Sonntag, M. Bielaszewska, U. Dobrindt, and H. Karch. 2005. Presence and characterization of a mosaic genomic island which distinguishes sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H− from E. coli O157:H7. Appl. Environ. Microbiol. 71:4875-4878. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Johnson, W. M., and H. Lior. 1988. A new heat-labile cytolethal distending toxin (CLDT) produced by Escherichia coli isolates from clinical material. Microb. Pathog. 4:103-113. [DOI] [PubMed] [Google Scholar]
- 13.Liesegang, A., U. Sachse, R. Prager, H. Claus, H. Steinrück, S. Aleksic, W. Rabsch, W. Voigt, A. Fruth, H. Karch, J. Bockemühl, and H. Tschäpe. 2000. Clonal diversity of Shiga toxin-producing Escherichia coli O157:H7/H− in Germany—a ten-year study. Int. J. Med. Microbiol. 290:269-278. [DOI] [PubMed] [Google Scholar]
- 14.Mora, A., M. Blanco, J. E. Blanco, M. P. Alonso, G. Dhabi, F. Thomson-Carter, M. A. Usera, R. Bartolomé, G. Prats, and J. Blanco. 2004. Phage types and genotypes of Shiga toxin-producing Escherichia coli O157:H7 isolates from humans and animals in Spain: identification and characterization of two predominating phage types (PT2 and PT8). J. Clin. Microbiol. 42:4007-4015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Perna, N. T., G. Plunket III, V. Burland, B. Mau, J. D. Glasner, D. J. Rose, G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J. Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis, A. Lim, E. T. Dimalanta, K. D. Potamousis, J. Apodaca, T. S. Anantharaman, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409:529-533. [DOI] [PubMed] [Google Scholar]
- 16.Preston, M. A., W. Johnson, R. Khakhria, and A. Borczyk. 2000. Epidemiologic subtyping of Escherichia coli serogroup O157 strains isolated in Ontario by phage typing and pulsed-field gel electrophoresis. J. Clin. Microbiol. 38:2366-2368. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Sonntag, A.-K., R. Prager, M. Bielaszewska, W. Zhang, A. Fruth, H. Tschäpe, and H. Karch. 2004. Phenotypic and genotypic analyses of enterohemorrhagic Escherichia coli O145 strains from patients in Germany. J. Clin. Microbiol. 42:954-962. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Tarr, P. I., C. A. Gordon, and W. L. Chandler. 2005. Shiga toxin-producing Escherichia coli and the haemolytic uraemic syndrome. Lancet 365:1073-1086. [DOI] [PubMed] [Google Scholar]
- 19.Thelestam, M., and T. Frisan. 2004. Cytolethal distending toxins. Rev. Physiol. Biochem. Pharmacol. 152:111-133. [DOI] [PubMed] [Google Scholar]
- 20.Willshaw, G. A., H. R. Smith, T. Cheasty, P. G. Wall, and B. Rowe. 1997. Vero cytotoxin-producing Escherichia coli O157 outbreaks in England and Wales, 1995: phenotypic methods and genotypic subtyping. Emerg. Infect. Dis. 3:561-565. [DOI] [PMC free article] [PubMed] [Google Scholar]