Abstract
The variability of the tir, espA, and espD genes of the locus of enterocyte effacement (LEE) in 185 attaching and effacing Escherichia coli (AEEC) strains isolated from healthy and diarrheic cattle, sheep, and goats was investigated by polymerase chain reaction. Nineteen of the strains were enterohemorrhagic E. coli (EHEC); the other 166 were enteropathogenic E. coli (EPEC). The combinations of the tir and esp genes were associated with the variants of the eae gene but not with a strain’s belonging to the EPEC or EHEC group, animal species, or health status (healthy or diarrheic) of the animal. In addition, most of the strains showed the same combinations of LEE genes and serogroups as have been found in AEEC strains isolated from humans, which indicates that ruminants seem to be an EPEC reservoir for humans.
Résumé
La variabilité des gènes tir, espA et espD du locus d’effacement des entérocytes (LEE) chez 185 souches d’Escherichia coli attachant et effaçant (AEEC) isolées de bovins, moutons et chèvres en santé et avec diarrhée, a été étudiée par réaction d’amplification en chaîne par la polymérase. Dix-neuf des souches étaient des E. coli entérohémorragiques (EHEC); les autres étaient des E. coli entéropathogènes (EPEC). Les combinaisons des gènes tir et esp étaient associées avec les variants du gène eae mais non avec une souche appartenant aux groupes EPEC ou EHEC, aux espèces animales, ou à l’état de santé (en santé ou diarrhéique) de l’animal. De plus, la plupart des souches présentaient les mêmes combinaisons de gènes LEE et de sérogroupes qui ont été trouvées chez les souches humaines d’AEEC, ce qui indique que les ruminants seraient un réservoir d’EPEC pour les humains.
(Traduit par Docteur Serge Messier)
Attaching and effacing Escherichia coli (AEEC) are a cause of diarrhea in humans and animals. These bacteria cause a characteristic attaching and effacing (AE) lesion in the gut mucosa because of the intimate bacterial adhesion to the enterocyte and effacement of the brush-border microvilli. Formation of the AE lesion is governed by the locus of enterocyte effacement (LEE) (1). Enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) cause AE lesions in the human intestinal mucosa. In contrast to EPEC, EHEC strains produce verotoxins (VTs) (1). The EPEC strains have been classified as typical (possessing the bfpA gene) or atypical (lacking the bfpA gene). Typical EPEC strains, a leading cause of infantile diarrhea in developing countries, are rare in industrialized countries, where the atypical EPEC strains seem to be a more important cause of diarrhea (1,2). By analogy with strains isolated from humans, AEEC strains isolated from animals are usually referred to as EPEC or EHEC preceded by the animal species from which the strain was isolated, for example, bovine EPEC. Ruminants are recognized as the main natural reservoir of EHEC strains that infect humans. However, the role of ruminants as a reservoir of EPEC for humans is not known (1).
The LEE genes are separated into 3 functional domains: a region encoding intimate adherence (Tir and intimin), a region encoding the EPEC-secreted proteins (EspA, EspB, and EspD) and the region encoding a type III secretion system (3). Tir, EspA, EspB, and EspD are essential for the subversion of host-cell signal transduction pathways, the delivery of proteins into the host cell, and the formation of AE lesion (3).
On the basis of antigenic variation, polymerase chain reaction (PCR) analysis, and sequencing, at least 16 variants of the eae gene, which encodes intimin, have been identified (4). Variants of the tir, espA, espB, and espD genes have also been described (4–11). Differentiation of eae, tir, and esp alleles is an important tool for EHEC and EPEC typing in routine diagnostics as well as in epidemiological and clonal studies (4). In contrast with the eae and espB genes, little is known about diversity in the tir, espA, and espD genes in AEEC from ruminants. The variants of these genes have been studied in a limited number of AEEC strains from cattle (4,5,7,8,10) and in only 1 AEEC strain from sheep (4). Moreover, to our knowledge, typing of the tir, espA, and espD genes of AEEC from goats has not been performed.
The aim of this study was to investigate the variability of the tir, espA, and espD genes in a large collection of AEEC strains isolated from diarrheic and healthy cattle, sheep, and goats.
A total of 185 AEEC strains isolated from diarrheic calves, lambs, and goat kids (25, 17, and 7, respectively) and from healthy cattle, sheep, and goats (38, 64, and 34, respectively) were used in this study. Nineteen of the strains were EHEC (eae+, VT+), and 166 were atypical EPEC (eae+, VT−, bfpA−). Of the 91 strains that had previously been tested in the rabbit ileal loop assay, 84 (92%) were able to induce AE lesions (12). These strains have been described previously (6,9,13) (Tables I to III).
Table I.
Serogroup, variants of genes of the locus of enterocyte effacement (LEE), and type of verotoxin (VT) of strains of attaching and effacing Escherichia coli (AEEC) isolated from cattle
| Number of strains | Gene and variant
|
Type of VTb | |||||
|---|---|---|---|---|---|---|---|
| Source and serogroup | eaeb | tir | espA | espBb | espD | ||
| Healthy cattle | |||||||
| O5 | 1 | β1 | β1 | β1 | β | β1 | 1 |
| O5a | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O10a | 3 | γ2/θ | γ2/θ | NT | α | NT | |
| O20 | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O26a | 6 | β1 | β1 | β1 | β | β1 | |
| O49 | 1 | ι | α1 | β2 | α | NT | |
| O51a | 1 | β1 | β1 | β1 | β | β1 | |
| O71 | 1 | ι | β1 | β1 | β | β1 | |
| O71 | 1 | ξ | β1 | β1 | β | β1 | |
| O84 | 1 | ζ | α1 | NT | α | NT | 1 |
| O85 | 1 | β1 | β1 | β1 | β | β1 | |
| O98 | 1 | ζ | α1 | NT | α | NT | 1 |
| O98 | 1 | ι | γ1 | NT | γ | γ1 | |
| O103 | 1 | β1 | β1 | β1 | β | β1 | 1 |
| O111 | 2 | γ2/θ | γ2/θ | NT | α | NT | 1, 2 |
| O145a | 2 | γ1 | γ1 | NT | γ | γ1 | |
| O146 | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O156 | 4 | γ2/θ | γ2/θ | NT | α | NT | |
| O172 | 1 | β1 | β1 | β1 | β | β1 | 2 |
| O177a | 2 | β1 | β1 | β1 | β | β1 | |
| NT | 1 | γ1 | γ1 | NT | γ | γ1 | |
| NT | 2 | ζ | α1 | NT | α | NT | |
| NT | 1 | ι | α1 | β2 | α | NT | |
| NT | 1 | ι | β1 | β1 | β | β1 | |
| Diarrheic calves | |||||||
| O4 | 3 | ι | β1 | β1 | β | β1 | |
| O4 | 2 | ξ | β1 | β1 | β | β1 | |
| O5 | 1 | β1 | β1 | β1 | β | β1 | 1 |
| O14 | 2 | β1 | β1 | β1 | β | β1 | |
| O17 | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O26 | 5 | β1 | β1 | β1 | β | β1 | 1 |
| O26a | 3 | β1 | β1 | β1 | β | β1 | |
| O111 | 1 | β1 | β1 | β1 | β | β1 | |
| O123 | 1 | ι | β1 | β1 | β | β1 | |
| O125 | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O128 | 1 | β1 | β1 | β1 | β | β1 | 1 |
| O145 | 1 | γ1 | γ1 | NT | γ | γ1 | 1 |
| O153a | 1 | β1 | β1 | β1 | β | β1 | |
| O180 | 1 | ρ | NT | NT | β | NT | |
| NT | 1 | ζ | α1 | NT | α | NT | |
Table III.
Serogroup, variants of LEE genes, and VT type of AEEC strains isolated from goats
| Number of strains | Gene and variant
|
Type of VTb | |||||
|---|---|---|---|---|---|---|---|
| Source and serogroup | eaeb | tir | espA | espBb | espD | ||
| Healthy goats | |||||||
| O2a | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O3 | 4 | β1 | β1 | β1 | β | β1 | |
| O4a | 3 | β1 | β1 | β1 | β | β1 | |
| O15a | 1 | β1 | β1 | β1 | β | β1 | |
| O35 | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O55a | 2 | γ1 | γ1 | γ1 | γ | γ1 | |
| O56a | 1 | β2 | α1 | β2 | α | NT | |
| O76 | 1 | ɛ | β1 | NT | β | β1 | |
| O91 | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O121 | 5 | ɛ | β1 | NT | β | β1 | |
| O127 | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O128 | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O145a | 2 | γ1 | γ1 | NT | γ | γ1 | |
| O149 | 2 | ρ | NT | NT | β | NT | |
| O153a | 3 | β1 | β1 | β1 | β | β1 | |
| O153a | 2 | γ2/θ | γ2/θ | NT | α | NT | |
| O153 | 1 | ɛ | β1 | NT | β | β1 | |
| O156 | 1 | ζ | α1 | NT | α | NT | 1 |
| NT | 1 | β1 | β1 | β1 | β | β1 | |
| Diarrheic goat kids | |||||||
| O3 | 2 | β1 | β1 | β1 | β | β1 | |
| O153a | 2 | β1 | β1 | β1 | β | β1 | |
| O163 | 2 | β1 | β1 | β1 | β | β1 | |
| NT | 1 | β1 | β1 | β1 | β | β1 | |
The AEEC strains were tested for the different variants of the tir, espA, and espD genes with a PCR scheme that has previously been described (4). The E. coli strains E2348/69 (O127:H6, tir α1, espAα1, espDα1), RDEC-1 (O15:H-, tirβ1, espAβ1, espDβ1), EPEC-359 (O119:H6, tirα1, espAβ2), EDL933 (O157:H7, tir γ1, espAγ1, espDγ1), and 95NR1 (O111:H-, tirγ2/θ) were used as positive controls.
Four variants of tir (α1, β1, γ1, and γ2/θ), 3 of espA (β1, β2, and γ1), and 2 of espD (β1 and γ1) were identified in the 185 ruminant AEEC strains analyzed. Tables I to III show the PCR results according to source of isolation, serogroup, and type of VT. Listed in Table IV are the 13 combinations found for the eae, tir, espA, espB, and espD genes.
Table IV.
Combinations (pathotypes) of LEE genes of the AEEC strains isolated from ruminants
| Number of strains | Gene and variant
|
|||||
|---|---|---|---|---|---|---|
| Pathotype | eae | tir | espA | espB | espD | |
| 1 | 83 | β1 | β1 | β1 | β | β1 |
| 2 | 1 | β1 | γ2/θ | NT | β | NT |
| 3 | 1 | β2 | α1 | β2 | α | NT |
| 4 | 2 | γ1 | γ1 | γ1 | γ | γ1 |
| 5 | 6 | γ1 | γ1 | NT | γ | γ1 |
| 6 | 47 | γ2/θ | γ2/θ | NT | α | NT |
| 7 | 21 | ɛ | β1 | NT | β | β1 |
| 8 | 7 | ζ | α1 | NT | α | NT |
| 9 | 8 | ι | β1 | β1 | β | β1 |
| 10 | 2 | ι | α1 | β2 | α | NT |
| 11 | 1 | ι | γ1 | NT | γ | γ1 |
| 12 | 3 | ξ | β1 | β1 | β | β1 |
| 13 | 3 | ρ | NT | NT | β | NT |
NT — Not typable.
In previous studies (5,7,8,10), 4 variants of tir (α, β, γ, and θ), 4 variants of espA (α, β, βv, and γ), 3 variants of espD (α, β, and γ), and between 4 and 7 LEE profiles were identified. Later, Garrido et al (4) studied 25 AEEC strains with the PCR scheme used in this work and found 4 variants of tir (α1, β1, γ1, and γ2/θ), 4 variants of espA (α1, β1, β2, and γ1), 3 variants of espB (α1, β1, and γ1), 3 variants of espD (α1, β1, and γ1), and 12 combinations of these LEE genes.
As in the previous studies (4,5,7,8,10), we found that most of the AEEC strains with a specific eae variant, except for types γ1 and ι, showed the same combination of tir and esp genes. Our results also show that there is no correlation between combinations of the tir and esp genes and belonging to the EPEC or EHEC group, animal species, or health status (healthy or diarrheic) of the animal and that there is great genetic diversity among the LEE genes of the ruminant AEEC strains. In contrast to EHEC, the role of ruminants as a reservoir of EPEC for humans is not known (1). Most of the EHEC and EPEC strains studied in this work showed the same combinations of LEE genes as were previously found in AEEC strains isolated from humans (4). In addition, 59% (98 of 166) of the atypical EPEC strains from the ruminants presented eae variants and serogroups previously found in atypical EPEC strains from humans with diarrhea or other gastrointestinal alterations (Tables I to III). Thus, our results show that ruminants seem to be a reservoir of atypical EPEC for humans. However, further studies are necessary to support this hypothesis.
The results herein are in accordance with the findings of Garrido et al (4) concerning the pathotypes of the AEEC strains with the variants β1, β2, ɛ, ζ, and ξ of the eae gene. However, our results with strains with other eae variants do not fully agree with those obtained by these authors. Thus, Garrido et al (4), who found that the eaeγ1 strains possessed the variant γ1 of espA and that the espB gene of the eaeγ2/θ strains was not typable. We found that only 2 of the 8 eaeγ1 strains (both of serogroup O55) had the variant γ1 of espA [like the AEEC O157 strains described by Garrido et al (4)], whereas the espA gene of the other 6 eaeγ1 strains (5 O145 strains; O was not typable in the remaining strain) could not be typed. It is possible that this difference may be associated with the serogroups of the eaeγ1 strains and that the espA variant of the AEEC O55 and O157 strains with the eaeγ1 gene is different from that of the AEEC strains with the same eae variant but of other serogroups (such as O145). The EHEC O157 strains are believed to have evolved from EPEC O55 (15). The different results for the espB variant of the eaeγ2/θ strains in the 2 studies may be due to the difference in primers used to amplify the espB gene. Our results with this variant are in accordance with those obtained by Bertin et al (10).
None of the 3 pathotypes that were found for the 11 eaeι strains was identical to the pathotype of the only AEEC strain with this eae variant described by Garrido et al (4). These results show the high variability of associations between the eaeι gene and the variants of the other LEE genes.
In contrast, only 1 of the 84 eaeβ1 strains (isolated from a diarrheic lamb and of serogroup O75) showed a pathotype different from that of most of the AEEC strains having that eae variant.
Variants of tir, espA, and espD, of the eaep strains, a variant of eae recently described by our group (13), were not typable (Table IV).
Interestingly, we observed a relationship between the combination of tir and esp genes and the eae clusters described by Ito et al (16). That association was closer with the variants of tir than with the variants of esp. Ito et al (16) categorized the eae variants into 5 clusters (a, b, c, d, and x) by analysis of 5′-terminal nucleotide sequences of different intimin types and heteroduplex mobility assay. Thus, eaeβ2 and eaeζ were included in cluster a, eaeβ1, eaeɛ, and eaeξ in cluster b, eaeγ1 in cluster c, eaeγ2/θ in cluster d, and eaeρ in cluster x. In contrast, we did not find a relationship between the combination of tir and esp genes and the 6 groups of closely related intimins described by Blanco et al (14,17), which were determined by analysis of the complete nucleotide and amino acid sequences.
In conclusion, combinations of the tir and esp genes of ruminant AEEC strains are associated with variants of the eae gene but not with the origin of the strains. In addition, our results show that ruminants seem to be a reservoir of atypical EPEC for humans. However, further studies are necessary to support this hypothesis.
Table II.
Serogroup, variants of LEE genes, and VT type in AEEC strains isolated from sheep
| Number of strains | Gene and variant
|
Type of VTc | |||||
|---|---|---|---|---|---|---|---|
| Source and serogroup | eaec | tir | espA | espBc | espD | ||
| Healthy sheep | |||||||
| O2a | 13 | γ2/θ | γ2/θ | NT | α | NT | |
| O4a | 2 | β1 | β1 | β1 | β | β1 | |
| O20 | 1 | ζ | α1 | NT | α | NT | |
| O26 | 2 | β1 | β1 | β1 | β | β1 | 1, 2 |
| O55 | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O55, 91, 103b | 4 | β1 | β1 | β1 | β | β1 | |
| O55, 91, 103 | 2 | γ2/θ | γ2/θ | NT | α | NT | |
| O71a | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O76 | 1 | ɛ | β1 | NT | β | β1 | |
| O103a | 4 | γ2/θ | γ2/θ | NT | α | NT | |
| O103a | 13 | ɛ | β1 | NT | β | β1 | |
| O109 | 2 | β1 | β1 | β1 | β | β1 | |
| O153a | 12 | β1 | β1 | β1 | β | β1 | |
| O153a | 2 | γ2/θ | γ2/θ | NT | α | NT | |
| O177a | 3 | β1 | β1 | β1 | β | β1 | |
| NT | 1 | β1 | β1 | β1 | β | β1 | |
| Diarrheic lambs | |||||||
| O2a | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O4a | 1 | β1 | β1 | β1 | β | β1 | |
| O26a | 5 | β1 | β1 | β1 | β | β1 | |
| O71 | 2 | ι | β1 | β1 | β | β1 | |
| O73 | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O75 | 1 | β1 | γ2/θ | NT | β | NT | 1, 2 |
| O80a | 1 | β1 | β1 | β1 | β | β1 | |
| O103a | 1 | γ2/θ | γ2/θ | NT | α | NT | |
| O153a | 3 | β1 | β1 | β1 | β | β1 | |
| NT | 1 | β1 | β1 | β1 | β | β1 | |
Acknowledgments
This study was supported by grants from the Dirección General de Investigación (grant AGL2001-1476) and from the Instituto de Salud Carlos III (grants FIS G03-025-COLIRED-O157 and PI051428).
References
- 1.Nataro JP, Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998;11:142–201. doi: 10.1128/cmr.11.1.142. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Trabulsi LR, Keller R, Tardelli Gomes TA. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8:508–513. doi: 10.3201/eid0805.010385. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Chen HD, Frankel G. Enteropathogenic Escherichia coli: Unravelling pathogenesis. FEMS Microbiol Rev. 2005;29:83–98. doi: 10.1016/j.femsre.2004.07.002. [DOI] [PubMed] [Google Scholar]
- 4.Garrido P, Blanco M, Moreno-Paz M, et al. STEC-EPEC oligonucleotide microarray: a new tool for typing genetic variants of the LEE pathogenicity island of human and animal Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains. Clin Chem. 2006;52:192–201. doi: 10.1373/clinchem.2005.059766. Epub 2005 Dec. 29. [DOI] [PubMed] [Google Scholar]
- 5.China B, Goffaux F, Pirson V, Mainil J. Comparison of eae, tir, espA and espB genes of bovine and human attaching and effacing Escherichia coli by multiplex polymerase chain reaction. FEMS Microbiol Lett. 1999;178:177–182. doi: 10.1111/j.1574-6968.1999.tb13775.x. [DOI] [PubMed] [Google Scholar]
- 6.Cid D, Ruiz-Santa-Quiteria JA, Marín I, et al. Association between intimin (eae) and EspB gene subtypes in attaching and effacing Escherichia coli strains isolated from diarrhoeic lambs and goat kids. Microbiology. 2001;147:2341–2353. doi: 10.1099/00221287-147-8-2341. [DOI] [PubMed] [Google Scholar]
- 7.Goffaux F, China B, Mainil J. Organisation and in vitro expression of esp genes of the LEE (locus of enterocyte effacement) of bovine enteropathogenic and enterohemorrhagic Escherichia coli. Vet Microbiol. 2001;83:275–286. doi: 10.1016/s0378-1135(01)00418-7. [DOI] [PubMed] [Google Scholar]
- 8.Nielsen EM, Andersen MT. Detection and characterization of verocytotoxin-producing Escherichia coli by automated 5′ nuclease PCR assay. J Clin Microbiol. 2003;41:2884–2893. doi: 10.1128/JCM.41.7.2884-2893.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Orden JA, Yuste M, Cid D, et al. Typing of the eae and espB genes of attaching and effacing Escherichia coli isolates from ruminants. Vet Microbiol. 2003;96:203–215. doi: 10.1016/s0378-1135(03)00238-4. [DOI] [PubMed] [Google Scholar]
- 10.Bertin Y, Boukhors K, Livrelli V, Martin C. Localization of the insertion site and pathotype determination of the locus of entero-cyte effacement of Shiga toxin-producing Escherichia coli strains. Appl Environ Microbiol. 2004;70:61–68. doi: 10.1128/AEM.70.1.61-68.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ogura Y, Ooka T, Whale A, et al. TccP2 of O157:H7 and non-O157 enterohemorrhagic Escherichia coli (EHEC): Challenging the dogma of EHEC-induced actin polymerization. Infect Immun. 2007;75:604–612. doi: 10.1128/IAI.01491-06. Epub 2006 Nov. 13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.de la Fuente R, García S, Ruiz-Santa-Quiteria JA, Cid D, Orden JA. Investigation of attaching and effacing activity of ruminant eae-positive Escherichia coli using rabbit and lamb ligated ileal loop assays. Vet Rec. 2004;154:565–568. doi: 10.1136/vr.154.18.565. [DOI] [PubMed] [Google Scholar]
- 13.Yuste M, De la Fuente R, Ruiz-Santa-Quiteria JA, Cid D, Orden JA. Detection of the astA (EAST1) gene in attaching and effacing Escherichia coli from ruminants. J Vet Med B Infect Dis Vet Public Health. 2006;53:75–77. doi: 10.1111/j.1439-0450.2006.00919.x. [DOI] [PubMed] [Google Scholar]
- 14.Blanco M, Blanco JE, Dahbi G, et al. Identification of two new intimin types in atypical enteropathogenic Escherichia coli. Int Microbiol. 2006;9:103–110. [PubMed] [Google Scholar]
- 15.Oswald E, Schmidt H, Morabito S, Karch H, Marchès O, Caprioli A. Typing of intimin genes in human and animal enterohemorrhagic and enteropathogenic Escherichia coli: characterization of a new intimin variant. Infect Immun. 2000;68:64–71. doi: 10.1128/iai.68.1.64-71.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Ito K, Iida M, Yamazaki M, et al. Intimin types determined by heteroduplex mobility assay of intimin gene (eae)-positive Escherichia coli strains. J Clin Microbiol. 2007;45:1038–1041. doi: 10.1128/JCM.01103-06. Epub 2007 Jan. 17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Blanco M, Blanco JE, Dahbi G, et al. Typing of intimin (eae) genes from enteropathogenic Escherichia coli (EPEC) isolated from children with diarrhoea in Montevideo, Uruguay: Identification of two novel intimin variants (μB and ξR/β2B) J Med Microbiol. 2006;55:1165–1174. doi: 10.1099/jmm.0.46518-0. [DOI] [PubMed] [Google Scholar]