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Lab. Inmunoquímica y Biotecnología, Depto. Sanidad Animal y Medicina Preventiva, Fac. Cs. Veterinarias, Universidad Nacional del Centro Pcia. Buenos Aires. Pinto 399 (7000), Tandil, Argentina
Correspondence
Paula M. A. Lucchesi
paulaluc{at}vet.unicen.edu.ar
Received 23 March 2007
Accepted 20 July 2007
Abbreviations: VT, verotoxin; VTEC, verotoxin-producing Escherichia coli.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Several factors contribute to the pathogenicity of VTEC, but the verotoxins (VTs) are considered to be the main virulence factors. These toxins are classified into two principal types, verotoxin 1 (VT1) and verotoxin 2 (VT2) (Nataro & Kaper, 1998). The VT1 group consists of VT1, VT1c and VT1d variants (Paton et al., 1995; Zhang et al., 2002; Bürk et al., 2003; Friedrich et al., 2003). The VT2 group is more heterogeneous and includes the prototype VT2 (VT2-EDL933) (this VT2 subtype is referred to as VT2-EDL933 in this paper, as it is the VT2 produced by the EDL933 reference strain), a considerable number of variants grouped into VT2c, VT2d, VT2e and VT2f, and the recently identified VT2g variant (Schmitt et al., 1991; Piérard et al., 1998; Schmidt et al., 2000; Leung et al., 2003). Leung et al. (2003) described this novel VT2 variant in a VTEC strain isolated from the faeces of healthy cattle. They found the vt2g gene in 0.7 % of bovine VTEC isolates. Strains carrying vt2g have also been identified in cattle wastewater in a study of aquatic environments (García-Aljaro et al., 2005). When vt2g is aligned with published DNA sequences of vt2-EDL933 and vt2 variants, it exhibits the highest similarity with vt2 genes associated with human disease, and moreover VT2g cytotoxicity for HeLa and Vero cells is comparable to that of VT2-EDL933 (ATCC 43889) (Leung et al., 2003). This potential pathogenicity and the possibility that vt2g may be a newly emerged variant that has not yet extensively spread among cattle (Leung et al., 2003) indicate the necessity of studying the prevalence of vt2g in isolates from bovine, environmental and human samples.
It is known that the nomenclature of vt2 gene variants is confusing due to the use of different subtyping methods and designations. The continuous change of protocols to identify new variants adds more complexity. Therefore, the purpose of this study was to analyse the ability of commonly used vt2-subtyping methods to detect and discriminate vt2g, in order to facilitate its identification.
Two approaches were used for the analysis: the first was PCR amplification and PCR-RFLP subtyping, and the second was a virtual sequence analysis.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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VTEC strains or their DNA used as controls for vt2g (E. coli 7v), vt2-EDL933 (E. coli EDL933), vt2 vha (E. coli E32511), vt2 vhb (E. coli 1398-152; clinical isolate) and vt2d-Ount (E. coli EH250) were kindly supplied by Dr A. W. Friedrich (Institut für Hygiene, Universitätsklinikum Münster, Germany), Dr P. H. M. Leung (Queen Mary Hospital, The University of Hong Kong, People's Republic of China), Dr J. Blanco (Laboratorio de Referencia de E. coli, Spain) and Dr E. López (Hospital de Niños ‘Ricardo Gutiérrez’, Argentina).
Bacteria were grown overnight at 37 °C in Luria–Bertani (LB) broth with shaking. An aliquot of the culture was diluted 1/10 in water to determine OD600. At an OD600 of 0.5, a 500 µl aliquot of the stationary-phase culture was centrifuged (2 min at 12 000 g) and the pellet suspended in 500 µl double-distilled water. The suspension was then boiled for 10 min and centrifuged (2 min at 12 000 g). Five microlitres of a 1/10 dilution of the supernatant were used as the template for PCR amplification.
PCR products were obtained with the Lin primer set for the Bastian method, VT2-c/VT2-d for the Tyler protocol, and VT2-e/VT2-f and VT2-cm/VT2-f for the Piérard protocol. PCR protocols and cycling conditions corresponded to those published before (Tyler et al., 1991; Bastian et al., 1998; Piérard et al., 1998).
For each PCR-RFLP method, 10 µl of each PCR product was incubated for 4 h at 37 °C in a water bath with 10 units of the appropriate enzyme in a final volume of 25 µl (HaeIII, RsaI, NciI and HincII) or 30 units in a final volume of 30 µl in the case of PvuII. PCR products and restriction fragments were separated by gel electrophoresis in 2 % agarose gels with ethidium bromide in Tris/borate/EDTA buffer.
In addition, 134 VTEC isolates from cattle in Argentina were tested with the primer set specific for the vt2g gene variant, 209F/781R (Leung et al., 2003). The serotypes and vt genotypes of these bacterial strains are listed in Table 1
and most of them have been described previously (Parma et al., 2000; Padola et al., 2004).
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All the vt2g-positive strains were also tested with the generic primer set for vt2 genes (stx2F/stx2R) designed by Paton & Paton (1998). PCR cycling conditions corresponded to those published by these authors.
Virtual sequence analysis.
Virtual analysis of two published sequences of the vt2g gene (GenBank accession nos AY286000 and AJ966782) was done to check PCR-RFLP results and assign fragment sizes. Sequences representing other vt2 variants were also included in this analysis to make a comparison with vt2g (Table 2).
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Examples of other vt variants are also shown. The computer-predicted RFLP patterns for vt2g in comparison with other vt2 sequences are summarized in Table 2
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, lane 2). No amplification was obtained with the VT2-cm/VT2-f primer set in the Piérard protocol; this was expected, as this set is specific for vt2d.
Although PCR was performed with standardized bacterial suspensions, it is important to emphasize that amplification with primers VT2-c/VT2-d in the Tyler protocol resulted in a less abundant product with vt2g than with the other variants (Fig. 1a). This situation can be explained by a 2 bp difference in the 3' reverse-priming region.
PCR-RFLP results with the vt2g-positive strain showed similarities between vt2g and vt2-EDL933 sequences (Fig. 1d–h, Table 2). Nevertheless, differences exist at some restriction sites:
(1) The vt2g PCR products remained unrestricted after incubation with RsaI (Tyler method) and with PvuII (Piérard method) (Fig. 1e, h, lane 3). The absence of these restriction sites in this vt2g sequence was confirmed with the virtual analysis (Table 2).
(2) In addition, when performing the Bastian method, vt2g lacked one of the restriction sites for HincII, in agreement with the virtual analysis. Therefore, a fragment of 819 bp was obtained instead of 556 and 263 bp fragments (Table 2).
These differences allow the identification of the vt2g variant with any of the analysed subtyping methods. The vt2g-positive VTEC strains (4/134) isolated from cattle in our country displayed the same patterns of bands as the control strain 7v in all these protocols. This variant was detected among isolates from cattle in feedlots belonging to serotypes O2 : H25, O15 : H21 and O175 : H8 (two isolates).
In addition, for all vt2g-positive VTEC strains, the ability of the generic primers stx2F/stx2R (Paton & Paton, 1998) to detect the vt2g gene was evaluated and corroborated. This is noteworthy because Leung et al. (2003) could not detect this variant with some vt2-specific primers and probes, and it is important to use initially a generic primer set to capture all possible variants before using the subtyping protocols (Ziebell et al., 2002).
The present work confirms the importance of reporting the sizes of atypical restriction fragments in vt2 typing, in order to be able to correlate with variants identified in other studies. Also, it is interesting that a consistently lower intensity of a PCR product, under optimal conditions, can alert to the presence of a new vt2 variant.
A different region of the vt2 gene is amplified in each of the analysed subtyping methods and this situation can generate contradictory results in the identification of vt2 gene variants (Ziebell et al., 2002). Nevertheless, all three protocols were able to identify the vt2g gene and differentiate it from the other vt2 variants.
These results show that it is possible to screen VTEC strains for the presence of vt2g without implementing new protocols that add further complexity to the analysis of vt2 variants. This variant has not been reported before in Argentina, and we found it in 3 % of VTEC strains isolated from cattle. Studies that determine the prevalence of the vt2g gene in strains isolated from bovine, human and environmental samples are important to improve the knowledge of the epidemiology and pathogenicity of VTEC strains that harbour this gene.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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