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Pheno-genotypic characterization of O157:H7 isolates from domestic and wild ruminants
S. Sánchez, R. Martínez, J. Rey, A. García, J. Blanco, M. Blanco, J.E.
Blanco, A. Mora, S. Herrera-León, A. Echeita, et al.
To cite this version:
S. Sánchez, R. Martínez, J. Rey, A. García, J. Blanco, et al.. Pheno-genotypic characterization of O157:H7 isolates from domestic and wild ruminants. Veterinary Microbiology, Elsevier, 2010, 142 (3-4), pp.445. �10.1016/j.vetmic.2009.10.009�. �hal-00587282�
Accepted Manuscript
Title: Pheno-genotypic characterization ofEscherichia coli O157:H7 isolates from domestic and wild ruminants Authors: S. S´anchez, R. Mart´ınez, J. Rey, A. Garc´ıa, J.
Blanco, M. Blanco, J.E. Blanco, A. Mora, S. Herrera-Le´on, A.
Echeita, J.M. Alonso
PII: S0378-1135(09)00529-X
DOI: doi:10.1016/j.vetmic.2009.10.009
Reference: VETMIC 4637
To appear in: VETMIC
Received date: 29-4-2009 Revised date: 23-9-2009 Accepted date: 15-10-2009
Please cite this article as: S´anchez, S., Mart´ınez, R., Rey, J., Garc´ıa, A., Blanco, J., Blanco, M., Blanco, J.E., Mora, A., Herrera-Le´on, S., Echeita, A., Alonso, J.M., Pheno- genotypic characterization ofEscherichia coliO157:H7 isolates from domestic and wild ruminants,Veterinary Microbiology(2008), doi:10.1016/j.vetmic.2009.10.009
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Accepted Manuscript
SHORT COMMUNICATION 1
2
Pheno-genotypic characterization of Escherichia coli O157:H7 isolates from 3
domestic and wild ruminants 4
5 6
S. Sánchez a,*, R. Martínez a, J. Rey a, A. García b, J. Blanco c, M. Blanco c, J.E.
7
Blanco c, A. Mora c, S. Herrera-León d, A. Echeita d, J.M. Alonso a 8
9
a Patología Infecciosa y Epidemiología, Departamento de Sanidad Animal, 10
Facultad de Veterinaria, Universidad de Extremadura, 10071 Cáceres, Spain 11
b Producción Animal, Centro de Investigación Finca La Orden-Valdesequera, 12
Junta de Extremadura, 06187 Badajoz, Spain 13
c Laboratorio de Referencia de E. coli, Departamento de Microbiología y 14
Parasitología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 15
27002 Lugo, Spain 16
d Laboratorio de Enterobacterias, Servicio de Bacteriología, Centro Nacional de 17
Microbiología, Instituto de Salud Carlos III, 28220 Madrid, Spain 18
19 20
* Corresponding author. Tel.: +34 927257114; fax: +34 927257110.
21
E-mail address: sergiosp@unex.es (S. Sánchez).
22 23
*Manuscript
Accepted Manuscript
Abstract 24
Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major 25
public health concern worldwide, with ruminants recognized as their main natural 26
reservoir. The aim of this work was to determine the phenotypic features and 27
genetic relationships of 46 E. coli O157:H7 isolates obtained from sheep, cattle, 28
and deer faeces and from unpasteurized goat milk in Spain over a period of 11 29
years. Characterization was performed by PCR, phage typing, and pulsed-field gel 30
electrophoresis (PFGE). An atypical E. coli O157:H7 strain (sorbitol-fermenting 31
and β-glucuronidase positive) originating from deer faeces was detected. Genes 32
encoding Shiga toxins were detected in 69.6% of isolates, all of them carrying 33
only the stx2 gene. The isolates were from nine different phage types, although 34
67.4% were restricted to only three: PT14, PT34, and PT54. PT54 was the most 35
prevalent phage type and contained isolates from cattle, sheep, and deer. The 36
majority of isolates were from phage types previously found in strains associated 37
with human infection. XbaI-PFGE identified 33 different types and 11 groups of 38
closely related types (more than 85% similarity), one of which included 21 39
(45.7%) isolates originating from different animal species, including deer. These 40
results indicate common origin or inter-species spread of genetically similar E.
41
coli O157:H7 isolates and contribute to earlier investigations identifying deer as a 42
natural source of E. coli O157:H7. The study also highlights the emergence of 43
phenotypic variants of E. coli O157:H7 which may not be identified by routine 44
culture methods or by biochemical tests used to characterize serotype O157:H7.
45 46
Keywords: E. coli O157:H7; STEC; Phenotypic variant; Virulence genes; Phage 47
typing; PFGE; Domestic and wild ruminants 48
Accepted Manuscript
1. Introduction 49
Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major 50
public health concern worldwide. Human diseases ranging from mild diarrhoea to 51
haemorrhagic colitis and the life-threatening haemolytic uraemic syndrome can be 52
caused by STEC O157:H7, typically affecting children, the elderly, and 53
immunocompromised patients (Centers for Disease Control and Prevention, 54
2001). The pathogenic capacity of STEC resides in a number of virulence factors, 55
including Shiga toxins (Stx1 and Stx2), intimin, and the enterohaemolysin (Gyles, 56
2007). Unlike other E. coli strains, O157:H7 strains neither ferment sorbitol nor 57
exhibit β-glucuronidase (GUD) activity after overnight incubation, and these 58
differences facilitate the detection of this organism (Kehl, 2002).
59
Healthy domestic ruminants can harbour STEC O157:H7 in their faeces and 60
are thus natural reservoirs of this pathogen (Rey et al., 2003; Blanco et al., 2004;
61
Orden et al., 2008), although STEC O157:H7 strains have also been isolated from 62
wild ruminants, especially from deer (Renter et al., 2001; García-Sánchez et al., 63
2007). Sources of human infection include undercooked meat, unpasteurized milk 64
and dairy products, vegetables or water, and contact with animal carriers or the 65
environment (Gyles, 2007).
66
The aim of the current study was to characterize a collection of E. coli 67
O157:H7 isolates obtained from domestic and free-ranging wild ruminants in 68
Spain over a period of 11 years, with the objective of determining their 69
phenotypic features and genetic relationships and therefore contributing to the 70
knowledge of the epidemiology of this pathogen.
71 72
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2. Materials and methods 73
2.1. E. coli O157:H7 isolates 74
A total of 46 E. coli O157:H7 isolates obtained from faeces of different 75
healthy ruminants [sheep, extensive and beef cattle, and red deer (Cervus 76
elaphus)] and from unpasteurized goat milk over a period of 11 years (1997 to 77
2008) were included in the present study. All of them originated from the 78
Extremadura region in the South-West of Spain and comprised (i) 24 isolates 79
epidemiologically related in different groups (15 isolates from sheep, 7 isolates 80
from extensive cattle, and 2 isolates from deer) and (ii) 22 isolates not known to 81
be epidemiologically related (14 isolates from sheep, 4 isolates from beef cattle, 3 82
isolates from deer, and 1 isolate from unpasteurized goat milk). Epidemiologically 83
related isolates were E. coli O157:H7 isolates obtained from different animals at a 84
single sheep flock, cattle herd, or game estate during the same periods. Some of 85
the isolates included in the present study were obtained from previously published 86
studies (Rey et al., 2003; Rey et al., 2006; García-Sánchez et al., 2007; Sánchez et 87
al., 2009), and the procedures for their isolation are described in detail in the 88
reports of those studies.
89 90
2.2. Biotyping, serotyping, and phage typing 91
All E. coli O157:H7 isolates were confirmed biochemically as E. coli by the 92
API 20E system (bioMérieux, Marcy L’Etoile, France). Fermentation of sorbitol 93
and GUD activity were investigated on sorbitol MacConkey agar (Oxoid, 94
Basingstoke, England) and Chromocult Coliform agar (Merck, Darmstadt, 95
Germany), respectively, after overnight incubation at 37 ºC.
96
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The identification of O and H antigens was carried out as described by Guinée 97
et al. (1981) using O157 antiserum and the full range of H antisera from H1 to 98
H56. All antisera were absorbed with corresponding cross-reacting antigens to 99
remove non-specific agglutinins. O157 antiserum was produced in the Laboratorio 100
de Referencia de E. coli (Lugo, Spain) and H antisera were obtained from the 101
Statens Serum Institut (Copenhagen, Denmark).
102
The phage typing was performed as described by Khakhria et al. (1990) in the 103
Centro Nacional de Microbiología (Instituto de Salud Carlos III, Madrid, Spain) 104
using phages provided by the National Microbiology Laboratory (Public Health 105
Agency of Canada, Winnipeg, Canada). The 16 different phages used were 106
capable of identifying 90 phage types.
107 108
2.3. PCR of stx1, stx2, ehxA, eae, O157 rfbE, and fliCh7 genes 109
All isolates were tested as previously described (Mora et al., 2004) with 110
primers specific for the genes encoding Stx1 and Stx2 (stx1 and stx2), 111
enterohaemolysin (ehxA), intimin (eae and eae-γ1 variant), O157 antigen (O157 112
rfbE), and H7 antigen (fliCh7). Reference E. coli strains used as controls were 113
EDL 933 (human, O157:H7, stx1, stx2, eae, ehxA) (ATCC No. 43895) and K12- 114
185 (negative for stx1, stx2, eae, and ehxA genes) (Blanco et al., 2004).
115 116
2.4. Pulsed-field gel electrophoresis (PFGE) 117
PFGE was performed in accordance with the PulseNet-Europe protocol 118
(http://www.pulsenet-europe.org/docs.htm). Genomic DNA was digested with 119
XbaI (Roche Diagnostics, Mannheim, Germany) and analyzed in 1% agarose gels 120
(Bio-Rad, Hemel Hempstead, United Kingdom) in 0.5 × TBE buffer at 14 ºC 121
Accepted Manuscript
using the CHEF MAPPER system (Bio-Rad). The runtime was 21.3 h at 6 V/cm, 122
with initial and final switch times of 2.16 and 54.17 s, respectively. The XbaI- 123
digested DNA from Salmonella enterica Braenderup H9812 was used as a 124
molecular size marker. Resultant images were analyzed with the InfoQuestFP 125
software (Bio-Rad). Isolates were allocated a different PFGE type when a genetic 126
difference could be detected. Cluster analysis was performed using the Dice 127
coefficient and the unweighted pair group method with arithmetic averages 128
(UPGMA).
129 130
3. Results 131
3.1. Phenotypic properties and phage types 132
All but one of the isolates evaluated in the current study were biochemically 133
typical of E. coli O157:H7 (non-sorbitol-fermenting and GUD negative) (Fig. 1).
134
A single isolate, originating from deer faeces, fermented sorbitol and exhibited 135
GUD activity after overnight incubation.
136
The 46 E. coli O157:H7 isolates were from a total of nine different phage 137
types (Table 1). However, among those nine, three phage types accounted for 138
67.4% of isolates analysed: PT14 (4 isolates), PT34 (3 isolates), and PT54 (24 139
isolates). PT54 was the most prevalent phage type among both sheep (62.1%) and 140
deer (80.0%) isolates and one of the most frequently found among cattle isolates 141
(18.2%). Seven isolates reacted with typing phages but did not conform to a 142
recognized pattern (RDNC/NT = reacts but does not conform/non-typeable).
143
Forty-three (93.5%) of 46 E. coli O157:H7 isolates expressed the H7 antigen 144
and three (6.5%) were nonmotile (NM). These NM isolates belonged to PT14 (1 145
isolate) and PT34 (2 isolates).
146
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147
3.2. Gene detection: stx1, stx2, ehxA, eae, O157 rfbE, and fliCh7 148
The PCR procedure indicated that all E. coli O157:H7 isolates carried the eae- 149
γ1, O157 rfbE, and fliCh7 genes. In addition, genes encoding Shiga toxins were 150
detected in 32 (69.6%) isolates, all of them carrying only the stx2 gene, and 44 151
(95.7%) isolates contained the ehxA gene (Fig. 1).
152 153
3.3. PFGE types and cluster analysis 154
PFGE of XbaI-digested genomic DNA of the 46 E. coli O157:H7 isolates 155
produced a dendrogram indicating 33 different PFGE types with 15-21 discernible 156
fragments, ranging from approximately 33 to 1100 kb in molecular size (Fig. 1).
157
Twenty-one types were identified among 29 sheep isolates, six types in 11 cattle 158
isolates, and five types in five deer isolates, and no PFGE type contained isolates 159
from more than one origin. However, these 46 isolates could be divided into 11 160
groups (I to XI, containing between 1 and 21 isolates per group) of closely related 161
PFGE types (more than 85% similarity), according to the Dice coefficient of 162
similarity (Fig. 1). Forty isolates (87.0%) were grouped in only five clusters (IV, 163
V, VI, VII, and VIII), and one of them (group IV) included 21 (45.7%) isolates 164
originating from sheep, cattle, and deer faeces, with PT54 being the predominant 165
phage type in this cluster.
166
The major genetic relatedness was observed in four groups containing isolates 167
with indistinguishable PFGE types (100% similarity): IV (6 isolates 168
epidemiologically related from sheep and 2 isolates not known to be 169
epidemiologically related from cattle), VI (2 isolates not known to be 170
epidemiologically related from sheep), VII (5 isolates epidemiologically related 171
Accepted Manuscript
from cattle), and VIII (3 isolates epidemiologically related from sheep). The most 172
divergent PFGE type was that of the atypical E. coli O157:H7 isolate obtained 173
from deer faeces, which was distantly related to the other typical isolates (less 174
than 64% similarity) (Fig. 1).
175 176
4. Discussion 177
The recognition of STEC O157:H7 has been largely facilitated by the 178
availability of classical microbiological diagnostic procedures that are based on 179
the characteristic phenotypic features of this pathogen, in particular, its inability to 180
ferment sorbitol and lack of GUD activity after overnight incubation. However, 181
other phenotypic variants of STEC O157 have been isolated during the last decade 182
in Germany (Ammon et al., 1999), the Czech Republic (Bielaszewska et al., 183
1998), Finland (Saari et al., 2001), Italy (Bonardi et al., 1999), the United States 184
(Hayes et al., 1995), Australia (Bettelheim et al., 2002), and Japan (Nagano et al., 185
2002). To our knowledge, the atypical STEC O157:H7 strain (sorbitol-fermenting 186
and GUD positive) originating from deer faeces detected in the present study is 187
the first phenotypic variant isolated in Spain and the first one originating from 188
ruminants other than cattle in Europe, since such variants have been isolated 189
before from deer in Japan and the United States (Dunn et al., 2004; Nagano et al., 190
2004). This atypical STEC O157:H7 strain was distantly related to the other 191
typical strains (less than 64% similarity) (Fig. 1). Similarly, GUD positive STEC 192
O157:H7 strains isolated in Japan between 1996 and 2001, including human and 193
deer strains, belonged to a single cluster only distantly related to the other typical 194
STEC O157:H7 strains (less than 60% similarity by PFGE) (Nagano et al., 2002;
195
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Nagano et al., 2004). Based on these findings the authors suggested that such 196
phenotypic variants may represent a distinct clone within STEC serogroup O157.
197
At least 90 phage types have been currently reported for STEC O157:H7 198
(Ahmed et al., 2001), but only seven of these (PT2, PT4, PT8, PT14, PT21/28, 199
PT32, and PT54) account for the majority (more than 75%) of human strains 200
isolated in Europe and Canada. Three of these phage types (PT4, PT14, and PT54) 201
were identified in the present study and they accounted for 63.0% of the isolates.
202
PT54 was the most prevalent phage type and contained isolates from cattle, sheep, 203
and deer. Actually, PT54 was the most prevalent phage type among isolates from 204
deer. To our knowledge, this is the first report of the phage typing of STEC 205
O157:H7 strains isolated from deer in the literature.
206
Fourteen (30.4%) E. coli O157:H7 isolates were stx-negative, although all of 207
them carried genes encoding other virulence-associated factors (enterohaemolysin 208
and/or intimin). Whether this finding resulted from loss of stx gene(s) from 209
initially stx-positive strains remains unclear. However, whilst the human health 210
risks associated with stx-negative E. coli O157:H7 strains may be less significant, 211
the acquisition of stx-carrying phages from the environment cannot be excluded 212
(Muniesa et al., 1999).
213
Among the 46 E. coli O157:H7 isolates characterized in the current study no 214
PFGE type contained isolates from more than one origin, findings in contrast to 215
those of Avery et al. (2004) and Mora et al. (2004), with some of the PFGE types 216
containing E. coli O157:H7 isolates from more than one origin, even though none 217
of the samples were known to be epidemiologically related. Nevertheless, the 218
PFGE group IV in the present study included closely related isolates (more than 219
85% similarity) obtained from different animal species, including deer, over a 220
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period of 11 years (isolated in 1997, 2003, 2004, 2007, and 2008) with and 221
without known epidemiological links (Fig. 1). These results indicate common 222
origin or inter-species spread of genetically similar E. coli O157:H7 isolates. On 223
the other hand, indistinguishable or closely related PFGE types were found in 224
isolates recovered from samples from different animals at a single sheep flock, 225
cattle herd, or game estate during the same periods (Fig. 1). This high similarity 226
suggests the existence of horizontal transmission among animals, which has been 227
demonstrated to be important in maintaining E. coli O157:H7 infections on farms 228
(Faith et al., 1996).
229
In conclusion, the current study contributes to earlier investigations 230
identifying deer as a natural source of E. coli O157:H7 and reports the isolation of 231
a sorbitol-fermenting and GUD positive strain from deer. This data highlights the 232
emergence of phenotypic variants of E. coli O157:H7 which may not be identified 233
by routine culture methods or by biochemical tests used to characterize serotype 234
O157:H7. The study reports that the most common phage type among strains 235
isolated from deer is also common among human strains, supporting the idea that 236
ruminants are a principal reservoir. The current study also shows the natural 237
occurrence of many genetic variants among E. coli O157:H7 isolated from 238
domestic and wild ruminants in Spain but indicate common origin or inter-species 239
spread of genetically similar isolates.
240 241
Conflict of interest statement 242
None of the authors of this paper has a financial or personal relationship with 243
other people or organisations that could inappropriately influence or bias the 244
content of the paper.
245
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246
Acknowledgements 247
The authors thank R. Rubio and A. Aladueña for their skilful technical 248
assistance. S. Sánchez acknowledges the Ministerio de Educación y Ciencia for 249
his research fellowship (AP2002-3286). R. Martínez acknowledges the Junta de 250
Extremadura for his research fellowship (PRE06053). A. Mora acknowledges the 251
Ramón y Cajal programme from the Ministerio de Educación y Ciencia. This 252
study was supported by grants from the Junta de Extremadura and FEDER (grant 253
3PR05A009-III Plan Regional de Investigación), from the Fondo de Investigación 254
Sanitaria (Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, grants 255
G03-025-COLIRED-O157 and RD06/0008-1018- REIPI), from the Xunta de 256
Galicia (grants PGIDIT05BTF26101P, PGIDIT065TAL26101P, 257
07MRU036261PR), and from the Ministerio de Educación y Ciencia (AGL-2008- 258
02129).
259 260
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47-56.
346
Rey, J., Sánchez, S., Blanco, J.E., Hermoso de Mendoza, J., Hermoso de 347
Mendoza, M., García, A., Gil, C., Tejero, N., Rubio, R., Alonso, J.M., 2006.
348
Prevalence, serotypes and virulence genes of Shiga toxin-producing 349
Escherichia coli isolated from ovine and caprine milk and other dairy 350
products in Spain. Int. J. Food Microbiol. 107, 212-217.
351
Saari, M., Cheasty, T., Leino, K., Siitonen, A., 2001. Phage types and genotypes 352
of Shiga toxin-producing Escherichia coli O157 in Finland. J. Clin.
353
Microbiol. 39, 1140-1143.
354
Sánchez, S., Martínez, R., García, A., Blanco, J., Echeita, A., Hermoso de 355
Mendoza, J., Rey, J., Alonso, J.M., 2009. Shiga toxin-producing Escherichia 356
coli O157:H7 from extensive cattle of the fighting bulls breed. Res. Vet. Sci., 357
in press.
358 359 360 361
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Tables 362
363
Table 1 364
Phage types of E. coli O157:H7 isolates from different origins 365
Phage type
No. of isolates
Total Sheep faeces Cattle faeces Deer faeces Goat milk
(n = 46) (n = 29) (n = 11) (n = 5) (n = 1)
PT4 1 0 1 0 0
PT14 4 4 0 0 0
PT31 1 1 0 0 0
PT34 3 1 2 0 0
PT42 2 0 2 0 0
PT43 1 1 0 0 0
PT54 24 18 2 4 0
PT63 2 2 0 0 0
PT82 1 1 0 0 0
RDNC/NTa 7 1 4 1 1
366
a Reacts but does not conform/non-typeable.
367 368 369
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Figure legends 370
371
Fig. 1. XbaI-PFGE dendrogram based on the Dice coefficient indicating the 372
genetic relatedness of the 46 E. coli O157:H7 isolates from domestic and wild 373
ruminants. The scales at the top indicate the similarity indices (in percentages) 374
and molecular sizes (in kilobases).
375 376
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Figure
