Acessibilidade / Reportar erro

Occurrence of subtilase cytotoxin and relation with other virulence factors in verocytotoxigenic Escherichia coli isolated from food and cattle in Argentina

Abstract

We investigated the presence of the gene of subtilase cytotoxin (SubAB), described in certain highly virulent verocytotoxigenic E. coli strains, in isolates from Argentina and its relation with other virulence factors. The gene subA was present in eae-negative strains mostly associated with saa, vt2 and ehxA genes.

subtilase; VTEC; megaplasmid; verotoxin


FOOD MICROBIOLOGY

Occurrence of subtilase cytotoxin and relation with other virulence factors in verocytotoxigenic Escherichia coli isolated from food and cattle in Argentina

Claudia V. Granobles Velandia; A. Mariel Sanso* * Corresponding Author. Mailing address: Lab. Inmunoquímica y Biotecnología, Dto. Sanidad Animal y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Pcia. de Buenos Aires. Pinto 399 (7000), Tandil, Pcia. de Buenos Aires, Argentina.; E-mail: msanso@vet.unicen.edu.ar ; Alejandra Krüger; Lorena V. Suárez; Paula M. A. Lucchesi; Alberto E. Parma

Lab. Inmunoquímica y Biotecnología, Dto. Sanidad Animal y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Pcia. de Buenos Aires. Pinto 399 (7000), Tandil, Pcia. de Buenos Aires, Argentina

ABSTRACT

We investigated the presence of the gene of subtilase cytotoxin (SubAB), described in certain highly virulent verocytotoxigenic E. coli strains, in isolates from Argentina and its relation with other virulence factors. The gene subA was present in eae-negative strains mostly associated with saa, vt2 and ehxA genes.

Key words: subtilase; VTEC; megaplasmid; verotoxin

Verocytotoxigenic Escherichia coli (VTEC) are a diverse group of E. coli strains characterized by the production of verotoxins (VT1 and/or VT2) which are regarded as their main virulence factors (6). VTEC are an important cause of gastrointestinal disease in humans (7, 12, 17) and life-threatening complications such as haemolytic uraemic syndrome (HUS).

More recently, it has been reported that some VTEC strains also produce another toxin called subtilase cytotoxin (SubAB). It was identified by Paton et al. (19) from an E. coli O113:H21 strain, which was responsible for an outbreak of HUS in South Australia in 1998, and since then has been detected in several other VTEC serotypes (5, 8, 14, 19, 20). Furthermore, Tozzoli et al. (25) found the first evidence that SubAB can also be produced by vt-negative E. coli isolated from cases of childhood diarrhoea. SubAB is encoded in the megaplasmid, and is the prototype of a new family of AB5 toxins comprising a single 35 kD A subunit which is a subtilase-like serine protease and a pentamer of B subunits, which mediates binding to glycolipid receptors on the target cell surface (19, 24).

SubAB was shown to be cytotoxic to Vero cells and lethal for mice, causing extensive microvascular thrombosis as well as necrosis in the brain, kidney, and liver (11, 19). The extreme cytotoxicity of this toxin for eukaryotic cells is due to a specific single-site cleavage of the essential endoplasmic reticulum chaperone BiP/GRP78 which is a master regulator of endoplasmic reticulum function (21). Its cleavage by subtilase cytotoxin represents a previously unknown trigger for cell death.

The cytotoxin SubAB has been described in certain highly virulent VTEC strains which are negative for the locus of enterocyte effacement (LEE), but the global distribution of SubAB-encoding VTEC strains is unknown. Furthermore, non-O157 VTEC including LEE negative strains predominate in Argentina, where HUS prevalence is the highest in the world. Consequently, our aim was to investigate the presence of the subA gene in strains isolated from various sources in Argentina. The relation with other VTEC virulence factors and with the serotype was also evaluated.

A total of 95 strains were selected from a well characterized, previously described strain collection of our Laboratory. To assess potential associations among subA, other virulence factors and serotypes, we selected representative strains taking into account the presence of vt1, vt2, eae, saa, and ehxAgenes determined in previous studies (15, 16, 22). In the present work, a previously described multiplex PCR, which detects subA, vt1 and vt2, was used for subAscreening (20). Samples were obtained by boiling a dilution 1:25 of the bacterial culture for 10 min. Amplification products were visualized in 2% agarose gels, stained with ethidium bromide.

We found that 21 VTEC strains of diverse origins were positive for subA gene. They belonged to serotypes O2:H5, O20:H19, O39:H49, O79:H19, O88:H21, O113:H21, O141:H7, O141:H8, O178:H19. In O non-typable strains, subAwas detected among those expresing H7, H8 and H19. To our knowledge this is the first time subA gene is found in VTEC serotypes O2:H5, O20:H19, O79:H19, O88:H21, O141:H7 and O141:H8. Within serotypes O20:H19, O113:H21, O141:H8, O178:H19 and ONT:H19 both subA-negative and subA-positiveisolates were found (Table 1), in agreement with the reports of authors, such as Cergole-Novella et al. (1), Newton et al. (13) and Irino et al. (3).

In addition to SubAB, VTEC can present other megaplasmid-encoded virulence factors such as an enterohaemolysin (Ehx), considered as an indicator of megaplasmid presence, and the STEC autoagglutinating adhesin (Saa), only present in LEE-negative VTEC strains (2, 4, 9, 18, 23, 26). In our study, most of the subA-positive strains were also ehxA-positive with the exception of a strain belonging to O2:H5 serotype, positive for subA and negative for ehxA. We found two combinations among subtilase-positive strains: subA+/ehxA+/saa+ (20 strains) and subA+/ehxA-/saa+ (only one strain). Although subtilase gene was always detected in saa-positive strains, strains positive for saa were not necessarily subA-positive. Paton & Paton (20) and Karama et al. (5) also found some subtilase negative strains among saa-positive VTEC.

We have previously reported several saa variants among VTEC strains in our collection (10). Subtilase gene was found in strains carrying saa variants 1, 2, 3, 4 or 5, and therefore there was no evident association between saa variants and the presence/absence of subA.

As we expected, subAwas found in eae-negative strains (which were screened for the presence of all known eaevariants). These results are in accordance with those of Paton & Paton (20), Osek (14), Khaitan et al. (8), Cergole-Novella et al. (1), Karama et al. (5) and Irino et al. (3). However, Newton et al. (13) reported one VTEC strain harboring both subA and eae genes.

All subA-positive VTEC isolates carried vt2 either alone or with vt1 gene. Of 21 VTEC strains that were tested positive for subA, 14 strains had the gene encoding the vt2 toxin only and 7 strains were positive for both vt1 and vt2. Paton & Paton (20) demonstrated a strong association between the presence of subA and VTEC carrying the vt2 gene only, although they found a few VTEC strains that were subA-positive vt2-negative. Osek (14), Karama et al. (5) and Irino et al. (3) also found one vt1-positive vt2-negative VTEC isolate which was subA-positive.

The association of subA with vt2 and ehxA, in addition to the described potential of SubAB to augment clinical manifestations of VTEC infection or to cause disease in its own right, highlights the risk of some LEE-negative VTEC.

In summary, our results reveal the presence of subtilase cytotoxin gene in Argentine VTEC isolates belonging to several non-O157:H7 serotypes, and the existence of different combinations of megaplasmid encoded factors, confirming once again the great genetic variability of these plasmids.

ACKNOWLEDGEMENTS

The authors thank M.R. Ortiz for her technical assistance. This work was supported by grants from the Comisión de Investigaciones Científicas- Pcia. Buenos Aires (CIC), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), FONCYT and SECAT-UNICEN. C.V.G. is holder of a fellowship from FONCYT. A.M.S., A.K. and P.M.A.L. are members of the Research Career of CONICET. A.E.P. is a member of the Research Career of CIC.

Submitted: February 12, 2010; Returned to authors for corrections: June 08, 2010; Approved: July 04, 2010.

  • 1. Cergole-Novella, M.C.; Nishimura, L.C.; dos Santos, L.F.; Irino, K.; Vaz, T.M.I.; Bergamini, A.M.M.; Guth, B.E.C. (2007). Distribution of virulence profiles related to new toxins and putative adhesins in Shiga toxin-producing Escherichia coli isolated from diverse sources in Brazil. FEMS Microbiol. Lett 274, 329-334.
  • 2. Friedrich, A.W.; Borell, J.; Bielaszewska, M.; Fruth, A.; Tschäpe, H.; Karch, H. (2003). Shiga toxin 1c-producing Escherichia coli strains: phenotypic and genetic characterization and association with human disease. J. Clin. Microbiol 41, 2448-2453.
  • 3. Irino, K.; Vieira, M.A.; Gomes, T.A.; Guth, B.E.; Naves, Z.V.; Oliveira, M.G.; dos Santos, L.F.; Guirro, M.; Timm, C.D.; Pigatto, C.P.; Farah, S.M.; Vaz, T.M. (2010). Subtilase cytotoxin-encoding subAB operon found exclusively among Shiga toxin-producing Escherichia coli strains. J. Clin. Microbiol. 48, 988-990.
  • 4. Jenkins, C.; Perry, N.Y.; Cheasty, T.; Shaw, D.J.; Frankel, G.; Dougan, G.; Gunn, G.J.; Smith, H.R.; Paton, A.W.; Paton, J.C. (2003). Distribution of the saa gene in strains of Shiga toxin-producing Escherichia coli of human and bovine origins. J. Clin. Microbiol 41, 1775-1778.
  • 5. Karama M.; Johnson, R.P.; Holtslander, R.; McEwen, S.A.; Gyles, C.L. (2008). Prevalence and characterization of verotoxin-producing Escherichia coli (VTEC) in cattle from an Ontario abattoir. Can. J. Vet. Res 72, 297-302.
  • 6. Karmali, M.A.; Petric, M.; Lim, C.; Fleming, P.C.; Arbus, G.S.; Lior, H. (1985). The association between idiopathic hemolytic syndrome and infection by verotoxin-producing Escherichia coli J. Infect. Dis. 151, 775-782.
  • 7. Karmali, M.A. (1989). Infection by verotoxin-producing Escherichia coli Clin. Microbiol. Rev 2, 15-38.
  • 8. Khaitan, A.; Jandhyala, D.M.; Thorpe, C.M.; Ritchie, J.M.; Paton, A.W. (2007). The operon encoding SubAB, a novel cytotoxin, is present in Shiga toxin-producing Escherichia coli isolates from the United States. J. Clin. Microbiol. 45, 1374-1375.
  • 9. Kumar, H.S.; Karunasagar, I.; Teizou, T.; Shima, K.; Yamasaki, S. (2004). Characterization of Shiga toxin-producing Escherichia coli (STEC) isolated from seafood and beef. FEMS Microbiol. Lett. 233, 173-178.
  • 10. Lucchesi, P.M.A.; Krüger, A.; Parma, A.E. (2006). Distribution of saa gene variants in verocytotoxigenic Escherichia coli isolated from cattle and food. Res. Microbiol 157, 263-266.
  • 11. Morinaga, N.; Yahiro, K.; Matsuura, G.; Watanabe, N.; Moss, J.; Noda, M. (2007). Two distinct cytotoxic activities of subtilase cytotoxin produced by Shiga-toxigenic Escherichia coli. Infect. Immun 75, 488-496.
  • 12. Nataro, J.P.; Kaper, J.B. (1998). Diarrheagenic Escherichia coli Clinical Microbiol. Rev 11, 142-201.
  • 13. Newton, H.J.; Sloan, J.; Bulach, D.M.; Seemann, T.; Allison, C.C.; Tauschek, M.; Robins-Browne, R.M.; Paton, J.C.; Whittam, T.S.; Paton, A.W.; Hartland, E. L. (2009). Shiga toxin-producing Escherichia coli strains negative for Locus of Enterocyte Effacement. Emerging Infect. Dis. 15, 372-380.
  • 14. Osek, J. (2006). Identification of the subtilase cytotoxin gene among Shigatoxigenic Escherichia coli isolated from different sources. Bull. Vet. Inst. Pulawy 50, 29-33.
  • 15. Padola, N.L.; Sanz, M.E.; Blanco, J.E.; Blanco, M.; Blanco, J.; Etcheverría, A.I.; Arroyo, G.H.; Usera, M.A.; Parma, A.E. (2004). Serotypes and virulence genes of bovine Shigatoxigenic Escherichia coli (STEC) isolated from a feedlot in Argentina. Vet. Microbiol 100, 3-9.
  • 16. Parma, A.E.; Sanz, M.E.; Blanco, J.E.; Blanco, J.; Viñas, M.R.; Blanco, M.; Padola, N.L.; Etcheverría, A.I. (2000). Virulence genotypes and serotypes of verotoxigenic Escherichia coli isolated from cattle and foods in Argentina. Importance in public health. Eur. J. Epidemiol. 16, 757-762.
  • 17. Paton, J.C.; Paton, A.W. (1998). Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev 11, 450-479.
  • 18. Paton, A.W.; Srimanote, P.; Woodrow, M.C.; Paton, J.C. (2001). Characterization of Saa, a novel autoagglutinating adhesin produced by locus of enterocyte effacement-negative Shiga toxigenic Escherichia coli strains that are virulent for humans. Infect. Immun 69, 6999-7009.
  • 19. Paton, A.W.; Srimanote, P.; Talbot, U.M.; Wang, H.; Paton, J.C. (2004). A new family of potent AB5 cytotoxins produced by Shiga toxigenic Escherichia coli J. Exp. Med 200, 35-46.
  • 20. Paton, A.W.; Paton, J.C (2005). Multiplex PCR for direct detection of Shiga toxigenic Escherichia coli producing the novel subtilase cytotoxin. J. Clin. Microbiol 43, 2944-2947.
  • 21. Paton, A.W.; Beddoe, T.; Thorpe, C.M.; Whisstock, J.C.; Wilce, M.C.J.; Rossjohn, J.; Talbot, U.M.; Paton, J.C. (2006). AB5 subtilase cytotoxin inactivates the endoplasmic reticulum chaperone BiP. Nature 443, 548-552.
  • 22. Sanz, M.E.; Villalobo, C.; Elichiribehety, E.; Arroyo, G. (2007). Prevalencia de Escherichia coli verotoxigenico en productos cárnicos de la ciudad de Tandil. La Ind. Cárnica Lat. 146, 56-58.
  • 23. Srimanote, P.; Paton, A.W.; Paton, J.C. (2002). Characterization of a novel type IV pilus locus encoded on the large plasmid of locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli strains that are virulent for humans. Infect. Immun 70, 3094-3100.
  • 24. Talbot, U.M.; Paton, J.C.; Paton, A.W. (2005). Protective immunization of mice with an active-site mutant of subtilase cytotoxin of Shiga toxin-producing Escherichia coli. Infect. Immun. 73, 4432-4436.
  • 25. Tozzoli, R.; Caprioli, A; Cappannella, S.; Michelacci, V.; Marziano, M.L.; Morabito, S. (2010). Production of the Subtilase AB5 cytotoxin by Shiga toxin-negative Escherichia coli. J. Clin. Microbiol 48, 178-183.
  • 26. Zweifel, C.; Blanco, J.E.; Blanco, M.; Blanco, J.; Stephan, R. (2004). Serotypes and virulence genes of ovine non-O157 Shiga toxin-producing Escherichia coli in Switzerland. Int. J. Food Microbiol 95, 19-27.
  • *
    Corresponding Author. Mailing address: Lab. Inmunoquímica y Biotecnología, Dto. Sanidad Animal y Medicina Preventiva, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la Pcia. de Buenos Aires. Pinto 399 (7000), Tandil, Pcia. de Buenos Aires, Argentina.; E-mail:
  • Publication Dates

    • Publication in this collection
      06 June 2011
    • Date of issue
      June 2011

    History

    • Accepted
      04 July 2010
    • Reviewed
      08 June 2010
    • Received
      12 Feb 2010
    Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
    E-mail: bjm@sbmicrobiologia.org.br