Variants include the subtype-specific prototypic toxins or related toxins within a subtype (that differ by one or more amino acids from the prototype). The variants are designated by toxin subtype, O group if the host strain is E. coli and generic name of the host bacterium if the host strain is not E. coli, followed by the strain name or number from which that toxin was described. These determinants are separated by hyphens, as in Stx1a-O157-EDL933 or Stx2c-O157-E32511. Nucleotide variants within a given Stx subtype are italicized (e.g., stx2a-O83-N1135 is a nucleotide variant that encodes Stx2a-O113-TS17-08). For identical sequences, the date of publication is given preference for choice of variant designation.
For reasons of simplicity and in order to minimize problems with database entries, only hyphens should be used for naming both amino acid and nucleotide variants, i.e., in species names and strain designations (e.g., Acinetobacter haemolyticus strain DS9B encodes Stx2a-Acinetobacter-haemolyticus-DS9B, strain T4/97 encodes Stx2f-O128-T4-97, strain H.I.8. encodes Stx2f-O89-HI8, etc.). In summary, the variant name includes the O serotype and strain name of the organism in which the toxin was detected. Toxin type 1 includes Stx and Stx1, but the prototypic Stx and Stx1 toxins were grouped within one new subtype, Stx/Stx1a. The other subtypes were those already described as Stx1c and Stx1d. Stx2 toxins were further defined with the addition of two new subtypes, Stx2a (the prototypic Stx2 sequence) and Stx2b (including the previously named VT2d variant), and the five existing subtypes, i.e., Stx2c, Stx2d (activation potential implied by sequence, see below), Stx2e, Stx2f, and Stx2g.
The sequence-based phylogenetic analyses included the intergenic regions and the identification of common motifs within each subtype and further supported the naming of subtypes. In particular, we hypothesized that two motifs in combination that are only present in variants of subtype Stx2d are related to the activatable property of this subtype. Subsequent testing for activation with intestinal mouse mucus confirmed this hypothesis in all nine strains within the Stx2d cluster, which contained these two motifs and were activatable by a factor of 6- to 28-fold. Only one strain, 06-5121, encoding Stx2c and Stx2d, did not meet the threshold for activatability. It is likely that the production of additional toxin Stx2c, which is more active on Vero cells than Stx2d, masked the activation phenotype in that strain.
The alignment of all known sequences also allowed us to evaluate some of the existing subtyping methods and identify theoretical pitfalls and possible misinterpretations of PCR-RFLP results. These methods have never really been validated against a representative number of strains. Bastian et al. (3
) used only nine strains to validate 14 PCR systems and create a subtyping scheme. Piérard et al. (51
) supplemented this with a method adding only one strain (EH250) to this panel. In a much more comprehensive study, Ziebell et al. (68
) used two PCR protocols, nine subtyping protocols, and three RFLP protocols on 12 reference strains and 496 field strains. They observed that the PCR-RFLP protocols gave contradictory results for approximately 20% of the strains tested and developed additional primers in order to allow for subtyping of all the studied subtypes and variants. None of these studies have used the same nomenclature for the toxins, nor have they addressed the problem of how to name the many variants using a systematic approach.
One of the cornerstones of RFLP typing has been the absence of the PstI site (position 908 to 913), which has been used as an indicator of the presence of the mucus-activatable stx2d
). However, the PstI site is also absent in 5 variants of stx2a
in E. coli
-O8-VTB178, and stx2a
-ONT-pEHEC400) and in stx2a
-E-cloacae-95MV2, in two variants of stx2c
-O171-EBC287 and stx2c
-ONT-EBC289), in stx2f
, and in all four variants of subtype stx2g
. Using the protocol developed in this study, HUSEC028 strain serotype O128:H2, previously described as stx2d
by classical typing (16
), was subtyped as stx2b
(). This can be explained by two point mutations within the PstI site (at position 909, T → A, and position 912, A → T). Similarly, strain MT71, encoding Stx2d, has acquired the PstI site, leading to misinterpretation as Stx2c. Thus, several variants within a given subtype may have single restriction enzyme (RE) site changes that would lead to misinterpretation by RFLP analysis. Furthermore, the primers often used for this RFLP typing method have been SLT-II-vc and CKS2, of which the latter is situated outside the locus of the stx2
gene itself. The primers developed and tested by us during this study have all been designed to lie within the structural gene for the A and B subunits in order to ensure that all the analyzed sequences contained the matching sequences.
Our multicenter validation of the PCR typing protocol revealed several areas of potential variability in results. Because Stx2a, Stx2c, and Stx2d are very closely related, they posed a special challenge to the design of specific primers and determination of optimal stringency. We identified 30 stx2a, 24 stx2c, and 26 stx2d nucleotide variants. The primers that we designed were discriminating of these variants in this study; however, other variants may exist that cannot be subtyped with these primers. Furthermore, we noted that cross-reactions occurred and appeared as ghost bands on gel electrophoresis, especially between stx2c- and stx2d-positive strains, so additional stringency may be needed to differentiate those toxin subtypes.
We observed differences in subtype results among the participating laboratories that we attributed to the use of different reagents and thermocyclers. A prototype protocol was adopted in an External Quality Assurance (EQA) scheme that involved both the networks of medical and veterinary/food National Reference Laboratories of the European Union (EU) member states and other laboratories outside the EU. The EQA was conducted jointly by the WHO Collaborating Centre for Reference and Research on Escherichia
in Copenhagen and by the EU Reference Laboratory for E. coli
in Rome, Italy, and also aimed at the harmonization of the typing methods used in both the networks, to favor the comparison of data referring to human and nonhuman isolates of STEC. The study, funded by the European Centre for Disease Prevention and Control (ECDC) and by the European Commission, was conducted in 2011, included 70 participating laboratories (unpublished data), and indicated that the prototype PCR subtyping protocol was subject to variability based on the accuracy of annealing temperatures in thermocyclers, primer quality, and manufacturer of the polymerases. Independently, the participating laboratories indicated that they had unwanted cross-reactions when subtyping strains with stx2a
, and stx2d
. In our study, this problem was resolved by raising the annealing temperature to 64 to 66°C. Gradient testing by two of the participating centers in this study [Istituto Superiore di Sanità (ISS) and SSI] on different thermocyclers showed that the maximum annealing temperatures may vary a little in different laboratories. Nonetheless, with careful standardization and use of the set of control strains provided to each lab, we showed that correct results were achieved by the majority of reference laboratories. The revised and final protocol for subtyping the Stx genes adopted from this study specifying the necessity for individual calibration of annealing temperatures on different brands of thermocyclers is described in and is available online (57
Among the unresolved discrepancies were the following observations. Unexpected results for strain 7279 were obtained in two laboratories and negative in four laboratories. Strain 7279 was negative for toxin by VCA but positive by Ridascreen EIA (8
). Our phylogenetic analysis placed this stx2
variant in the stx2d
cluster, but subtyping was positive for stx2c
. These results may be explained by the possible insertion of an IS element in the toxin gene, as indicated by the presence of a fragment larger than expected by the stx2
detection primers in two laboratories. The insertion of insertion sequence elements in the Stx-coding genes has been seen in other STEC O157 strains (31
A number of phylogenetic analyses have been published over the past decade (2
), but none of them provide the extensive sequence comparison shown here. Our phylogenetic trees correspond with trees in earlier publications (2
) except for our inclusion of Stx2g and our naming of subtypes Stx1a, Stx2a, and Stx2b. To avoid confusion with the toxin subunits A and B (uppercase), we propose that subtype names always be in lowercase letters. Hence, the A subunit protein of Stx1a should be designated StxA1a, the B subunit protein of Stx2a should be designated StxB2a, etc. We propose that toxin operons or open reading frames be written as stx
for holotoxin, stxA
for the A subunit gene, and stxB
for the B subunit gene in italics, with the type and subtype written as alleles without italics and in subscript. Thus, the gene encoding the A subunit of Stx1a should be written stxA1a
and the B subunit gene of the same toxin as stxB1a
. In databases that do not allow the use of italics or subscripts, the first letter will define toxin (Stx) or gene (stx).
Universal typing schemes such as serotyping of Salmonella and E. coli, pulsed-field gel electrophoresis, and multiple-locus variable-number tandem repeat analysis have demonstrated their usefulness in epidemiology, risk assessment, and outbreak detection for several bacterial species. Similarly, a universal language for Stx taxonomy is essential for the comparison of STEC strains among research and public health laboratories and for the surveillance of STEC strains regionally, nationally, and internationally. Standard methods and nomenclature are also necessary to elucidate associations between toxin subtypes and specific clinical features and to assess the risks of STEC in populations and agricultural reservoirs. One such important observation within the past 6 to 7 years has been the association of Stx2a with eae-positive strains and Stx2d with eae-negative strains and their links with HUS. By defining a common nomenclature and an accompanying subtyping protocol, we hope to strengthen these studies to achieve a better understanding of these associations and trends and the risks to public health.