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Applied and Environmental Microbiology, April 2009, p. 2246-2249, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01957-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Molecular Subtyping and Distribution of the Serine Protease from Shiga Toxin-Producing Escherichia coli among Atypical Enteropathogenic E. coli Strains

Adrian L. Cookson,^1* Jenny Bennett,² Carolyn Nicol,³ Fiona Thomson-Carter,² and Graeme T. Attwood¹

Food, Metabolism and Microbiology Section, AgResearch, Grasslands Research Centre, Palmerston North 4442,¹ Institute of Environmental Science and Research Limited, Kenepuru Science Centre, Porirua 5240, New Zealand,² Enteric Reference Laboratory, Institute of Environmental Science and Research Limited, National Centre for Biosecurity & Infectious Diseases-Wallaceville, 66 Ward Street, Wallaceville, Upper Hutt 5018, New Zealand³

Received 21 August 2008/ Accepted 23 December 2008

Top ABSTRACT INTRODUCTION REFERENCES

 
Atypical enteropathogenic Escherichia coli (aEPEC) and Shiga toxin-producing E. coli (STEC) were examined to determine the prevalence and sequence of espP, which encodes a serine protease. These analyses indicated shared espP sequence types between the two E. coli pathotypes and thus provide further insights into the evolution of aEPEC and STEC.

Top ABSTRACT INTRODUCTION REFERENCES

 
The serine protease autotransporters of the Enterobacteriaceae (18) family of proteins have been identified in several different diarrheagenic Escherichia coli pathotypes and include EspP (7), EspI (23), and EpeA (16) in Shiga toxin-producing E. coli and EspC in enteropathogenic E. coli (EPEC) (11, 25). Typical EPEC strain, which possess the bfp operon involved in bundle-forming pilus expression, express EspC (25), but preliminary analysis of atypical EPEC (aEPEC) strains lacking bundle-forming pili indicates the presence of the espP gene (1, 2). Together with Shiga toxins and intimin (encoded by eae) (14, 15, 17), EspP and its proteolytic activity against pepsin A and human coagulation factor V is a significant marker for STEC and an established virulence factor (5, 7, 10). Its prevalence in aEPEC strains, however, has not been thoroughly investigated.

aEPEC strains have been isolated from ruminants (4, 13) and associated with human diarrheal disease (1-3, 19, 24, 26, 28), but their virulence determinants remain equivocal. Thus, EspP may contribute to the emergence of aEPEC as a virulent E. coli pathotype, and genetic analysis of espP is likely to provide an effective tool to assess pathogenicity. Thus, the primary aim of this study was to readily establish the presence of espP in aEPEC and to use rapid and convenient molecular-based methods to compare subtypes isolated from ruminants with those from locally sourced STEC.

Bacterial strains from cattle, sheep, and human diarrheal disease, positive for stx and/or eae (n = 376) were chosen for further study to detect the presence of espP by PCR amplification (8) and colony blot hybridization. The 1,830-bp amplicon from espP-positive strains was digested with the restriction enzyme AluI, and restriction fragment length polymorphism (RFLP) analysis was performed by agarose gel electrophoresis. Phylogenetic analysis was performed on the 1,830-bp espP gene sequenced from 11 STEC strains and 9 aEPEC strains.

espP-positive E. coli strains were isolated from 7 of 123 (5.7%) sheep compared to 59 of 120 (49.2%) (P < 0.001) cattle. Similarly, of the sheep (n = 215) and cattle (n = 139) E. coli strains that were stx and/or eae positive, 7 (3.3%) and 81 (58.3%) were espP positive, respectively (P < 0.001). Twenty of 25 (80%) human STEC isolates were espP positive. Almost all (38 of 39, 97.4%) eae-positive STEC isolates from either humans or ruminants were espP positive. Six distinct espP subtypes could be differentiated after AluI digestion and agarose gel electrophoresis (Fig. 1). aEPEC strains were subtype B, D, or E. Only espP PCR-RFLP type A was specific to a single E. coli serotype (O157:H7); the remaining five espP PCR-RFLP subtypes were heterogeneous with respect to E. coli serotypes (Table 1).

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FIG. 1. PCR-RFLP analysis of espP amplicons cleaved with AluI. Lanes: 1, AGR061 (ONT:H25), type B; 2, AGR373 (O26:H11), type B; 3, 1848 (O113:H21), type C; 4, AGR053 (O131:H25), type D; 5, AGR120 (O108:H25), type D; 6, AGR047 (O90:H8), type E; 7, AGR158 (O101:H–), type E; 8, AGR201 (O180:H–), type E; 9, 4238 (O157:H7), type A; 10, AGR609 (O9:H51), type G; 11, AGR087 (ONT:H–), type G.

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TABLE 1. espP PCR-RFLP subtypes and serotypes of STEC and aEPEC strainsb

Using PCR-RFLP, subtype E represented the majority of the espP-positive STEC/aEPEC strains (56 of 108, 51.9%) (Tables 1 and 2). Overall 97.4%, 46.4%, and 8.0% of eae-positive STEC, aEPEC, and eae-negative STEC, respectively, were espP positive (P < 0.001). Sequencing of the PCR-RFLP profile of espP subtype G revealed a single DNA base pair difference at position 954, where a T was substituted for A, creating an additional AluI site. Using phylogenetic analysis of DNA sequences, distinct clades could be distinguished, corresponding to espP PCR-RFLP subtypes A, B, C, D, and E (Fig. 2a). Group A consisted exclusively of O157:H7 strains (n = 5). Phylogenetic analysis was also performed on the 610-amino-acid polypeptide derived from the 1,830-bp espP nucleotide sequence (Fig. 2b).

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TABLE 2. Distribution of espP subtypes among STEC and aEPEC strainsa

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FIG. 2. Phylogenetic positions of the partial 1,830-bp E. coli espP nucleotide (a) and corresponding 610-amino-acid (b) sequences from this study and the following espP sequences available in GenBank, based on neighbor joining: AB011549, AF074613, and AM691838 (O157:H7), corresponding to espP PCR-RFLP subtype A; AM691839 (O26:H11), Y13614 (O26:HNT), AM691840 (O111:H–), and AM691841 (O145:H–), corresponding to espP PCR-RFLP subtype B; AM691842 (O163:H19), AM691843 (O7:H18), AM691844 (O174:H2), and AY258503 (O113:H21), corresponding to espP PCR-RFLP subtype C; AM691845 (O77:H18), AM691847 (O84:H4), and AM691848 (O156:H–), corresponding to espP PCR-RFLP subtype D; and AM691846 (O127:HNT), corresponding to espP PCR-RFLP subtype E. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branch points. The number after the GenBank accession number corresponds to the branch length and indicates the genetic distance between the two nucleotide/amino acid sequences that the branch connects. Bar, 0.1% dissimilarity.

By targeting an 1,830-bp fragment of the espP gene, 52 of 112 (46.4%) of aEPEC strains were espP positive (Table 2). PCR-RFLP of the espP fragment indicated that aEPEC could be separated into three distinct subgroups (B, D, and E) (Fig. 2a). These specific groups also contained non-O157 eae-positive STEC, indicating parallel evolution or a likely common source of plasmid-borne espP through horizontal gene transfer. Like other virulence regions found on the large plasmids of STEC, such as the ehx (enterohemolysin) operon and the etp gene cluster (type II secretion pathway), espP is also flanked by insertion sequence-like elements (7, 10). However, sequence analysis of the espP gene and flanking regions from O157:H7 and O26:H11 (pssA) indicate different insertion sites within their respective large plasmids (7, 10). Thus, the presence of prospective virulence factors, such as ehx (20, 21), espP (5, 7, 10), katP (6), and etp (22), their sequence heterogeneity, and alternate insertion site are a testament to the variation of large plasmids in STEC strains (8) and likely also in aEPEC strains.

Although the expression and functionality of EspP were not examined in this study, it is our belief that sufficient similarity exists between espP alleles from aEPEC strains having espP PCR-RFLP types B and E and those previously described, to indicate that espP from these aEPEC strains is likely to give rise to a functionally active serine protease autotransporter of Enterobacteriaceae.

The immunogenicity of secreted EspP and antibody response in patients suffering from STEC O157:H7 infection have been noted previously (7). In comparison, the disease etiology and cause of diarrhea associated with human infection by aEPEC strains remain to be elucidated. However, aEPEC strains from human disease are heterogeneous both phylogenetically and in virulence profile (1, 2). The role of EspP in colonization and excretion of aEPEC in the ruminant gut also remains to be established (4, 13). Previous studies have indicated a role for EspP from STEC O26:H^– and O157:H7 in the adherence and colonization of calves (12, 27); however, the mechanism by which EspP may mediate intestinal colonization is unknown. Thus, the presence of espP, a recognized virulence factor of STEC, in aEPEC is likely to influence pathogenesis. Furthermore, the presence of espP in aEPEC may limit its use as a diagnostic virulence factor for STEC, but from an evolutionary perspective, the presence of both espP and ehxA (9) in aEPEC also demonstrates a close evolutionary relationship between the heterogeneous aEPEC and STEC pathotypes.

 
This work was supported by AgResearch Repositioning funds.

 
* Corresponding author. Mailing address: Food, Metabolism and Microbiology Section, Food and Health Group, AgResearch, Grasslands Research Centre, Tennent Drive, Private Bag 11008, Palmerston North 4442, New Zealand. Phone: 64 6 351 8229. Fax: 64 6 351 8003. E-mail: adrian.cookson{at}agresearch.co.nz

Published ahead of print on 9 January 2009.

Top ABSTRACT INTRODUCTION REFERENCES

 

1
1. Afset, J. E., G. Bruant, R. Brousseau, J. Harel, E. Anderssen, L. Bevanger, and K. Bergh. 2006. Identification of virulence genes linked with diarrhea due to atypical enteropathogenic Escherichia coli by DNA microarray analysis and PCR. J. Clin. Microbiol. 44:3703-3711.[Abstract/Free Full Text]
2
2. Afset, J. E., E. Anderssen, G. Bruant, J. Harel, L. Wieler, and K. Bergh. 2008. Phylogenetic backgrounds and virulence profiles of atypical enteropathogenic Escherichia coli strains from a case-control study using multilocus sequence typing and DNA microarray. J. Clin. Microbiol. 46:2280-2290.[Abstract/Free Full Text]
3
3. Aidar-Ugrinovich, L., J. Blanco, M. Blanco, J. E. Blanco, L. Leomil, G. Dahbi, A. Mora, D. L. Onuma, W. D. Silveira, and A. F. Pestana de Castro. 2007. Serotypes, virulence genes, and intimin types of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) isolated from calves in São Paulo, Brazil. Int. J. Food Microbiol. 115:297-306.[CrossRef][Medline]
4
4. Aktan, I., B. Carter, H. Wilking, R. M. La Ragione, L. Wieler, M. J. Woodward, and M. F. Anjum. 2007. Influence of geographical origin, host animal and stx gene on the virulence characteristics of Escherichia coli O26 strains. J. Med. Microbiol. 56:1431-1439.[Abstract/Free Full Text]
5
5. Brockmeyer, J., M. Bielaszewska, A. Fruth, M. L. Bonn, A. Mellmann, H.-U. Humpf, and H. Karch. 2007. Subtypes of the plasmid-encoded serine protease EspP in Shiga toxin-producing Escherichia coli: distribution, secretion, and proteolytic activity. Appl. Environ. Microbiol. 73:6351-6359.[Abstract/Free Full Text]
6
6. Brunder, W., H. Schmidt, and H. Karch. 1996. KatP, a novel catalase peroxidase encoded by the large plasmid of enterohemorrhagic Escherichia coli O157:H7. Microbiology 142:3305-3315.[Abstract/Free Full Text]
7
7. Brunder, W., H. Schmidt, and H. Karch. 1997. EspP, a novel extracellular serine protease of enterohemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V. Mol. Microbiol. 24:767-778.[CrossRef][Medline]
8
8. Brunder, W., H. Schmidt, M. Frosch, and H. Karch. 1999. The large plasmids of Shiga-toxin-producing Escherichia coli (STEC) are highly variable genetic elements. Microbiology 145:1005-1014.[Abstract/Free Full Text]
9
9. Cookson, A. L., J. Bennett, F. Thomson-Carter, and G. T. Attwood. 2007. Molecular subtyping and genetic analysis of the enterohemolysin gene (ehxA) from Shiga toxin-producing Escherichia coli and atypical enteropathogenic E. coli. Appl. Environ. Microbiol. 73:6360-6369.[Abstract/Free Full Text]
10
10. Djafari, S., F. Ebel, C. Deibel, S. Krämer, M. Hudel, and T. Chakraborty. 1997. Characterization of an exported protease from Shiga toxin-producing Escherichia coli. Mol. Microbiol. 25:771-784.[CrossRef][Medline]
11
11. Drag-Serrano, M., S. G. Parra, and H. A. Manjarrez-Hernández. 2006. EspC, an autotransporter protein secreted by enteropathogenic Escherichia coli (EPEC), displays protease activity on human hemoglobin. FEMS Microbiol. Lett. 265:35-40.[CrossRef][Medline]
12
12. Dziva, F., A. Mahajan, P. Cameron, C. Currie, I. J. McKendrick, T. S. Wallis, D. G. E. Smith, and M. P. Stevens. 2007. EspP, a type V-secreted serine protease of enterohaemorrhagic Escherichia coli O157:H7, influences intestinal colonization of calves and adherence to bovine primary intestinal epithelial cells. FEMS Microbiol. Lett. 271:258-264.[CrossRef][Medline]
13
13. Ewers, C., C. Schüffner, R. Weiss, G. Baljer, and L. H. Wieler. 2004. Molecular characteristics of Escherichia coli serogroup O78 strains isolated from diarrheal cases in bovines urge further investigations on their zoonotic potential. Mol. Nutr. Food Res. 48:504-514.[CrossRef][Medline]
14
14. Garmendia, J., G. Frankel, and V. F. Crepin. 2005. Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation. Infect. Immun. 73:2573-2585.[Free Full Text]
15
15. Kaper, J. B., J. P. Nataro, and H. L. T. Mobley. 2004. Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2:123-140.[CrossRef][Medline]
16
16. Leyton, D. L., J. Sloan, R. E. Hill, S. Doughty, and E. L. Hartland. 2003. Transfer region of pO113 from enterohemorrhagic Escherichia coli: similarity with R64 and identification of a novel plasmid-encoded autotransporter, EpeA. Infect. Immun. 71:6307-6319.[Abstract/Free Full Text]
17
17. Nataro, J. P., and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142-201.[Abstract/Free Full Text]
18
18. Restieri, C., G. Gariss, M.-C. Locas, and C. M. Dozois. 2007. Autotransporter-encoding sequences are phylogenetically distributed among Escherichia coli clinical isolates and reference strains. Appl. Environ. Microbiol. 73:1553-1562.[Abstract/Free Full Text]
19
19. Rosa, A. C. P., A. T. Mariano, A. M. S. Pereira, A. Tibana, T. A. Gomes, and J. R. Andrade. 1998. Enteropathogenicity markers in Escherichia coli isolated from infants with acute diarrhoea and healthy controls in Rio de Janeiro. J. Med. Microbiol. 47:781-790.[Abstract/Free Full Text]
20
20. Schmidt, H., L. Beutin, and H. Karch. 1995. Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933. Infect. Immun. 63:1055-1061.[Abstract]
21
21. Schmidt, H., C. Kernbach, and H. Karch. 1996. Analysis of the EHEC hly operon and its location in the physical map of the large plasmid of enterohaemorrhagic Escherichia coli O157:H7. Microbiology 142:907-914.[Abstract/Free Full Text]
22
22. Schmidt, H., B. Henkel, and H. Karch. 1997. A gene cluster closely related to type II secretion pathway operons of gram-negative bacteria is located on the large plasmid of enterohemorrhagic Escherichia coli O157 strains. FEMS Microbiol. Lett. 148:265-272.[CrossRef][Medline]
23
23. Schmidt, H., W. L. Zhang, U. Hemmrich, S. Jelacic, W. Brunder, P. I. Tarr, U. Dobrindt, J. Hacker, and H. Karch. 2001. Identification and characterization of a novel genomic island integrated at selC in locus of enterocyte effacement-negative, Shiga toxin-producing Escherichia coli. Infect. Immun. 69:6863-6873.[Abstract/Free Full Text]
24
24. Scotland, S. M., H. R. Smith, B. Said, G. A. Willshaw, T. Cheasty, and B. Rowe. 1991. Identification of enteropathogenic Escherichia coli isolated in Britain as enteroaggregative or as members of a subclass of attaching-and-effacing E. coli not hybridising with the EPEC adherence-factor probe. J. Med. Microbiol. 35:278-283.[Abstract/Free Full Text]
25
25. Stein, M., B. Kenny, M. A. Stein, and B. B. Finlay. 1996. Characterization of EspC, a 110-kilodalton protein secreted by enteropathogenic Escherichia coli which is homologous to members of the immunoglobulin A protease-like family of secreted proteins. J. Bacteriol. 178:6546-6554.[Abstract/Free Full Text]
26
26. Trabulsi, L. R., R. Keller, and T. A. Tardelli Gomes. 2002. Typical and atypical enteropathogenic Escherichia coli. Emerg. Infect. Dis. 8:508-513.[Medline]
27
27. van Diemen, P. M., F. Dziva, M. P. Stevens, and T. S. Wallis. 2005. Identification of enterohemorrhagic Escherichia coli O26:H^– genes required for intestinal colonization in calves. Infect. Immun. 73:1735-1743.[Abstract/Free Full Text]
28
28. Vieira, M. A., J. R. C. Andrade, L. R. Trabulsi, A. C. Rosa, A. M. Dias, S. R. Ramos, G. Frankel, and T. A. Gomes. 2001. Phenotypic and genotypic characteristics of Escherichia coli strains of nonenteropathogenic E. coli (EPEC) serogroups that carry EAE and lack the EPEC adherence factor and Shiga toxin DNA probe sequences. J. Infect. Dis. 183:762-772.[CrossRef][Medline]

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Applied and Environmental Microbiology, April 2009, p. 2246-2249, Vol. 75, No. 7
0099-2240/09/$08.00+0     doi:10.1128/AEM.01957-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cookson, A. L. Articles by Attwood, G. T. Search for Related Content PubMed PubMed Citation Articles by Cookson, A. L. Articles by Attwood, G. T. Agricola Articles by Cookson, A. L. Articles by Attwood, G. T.