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Occurrence of non-sorbitol fermenting, verocytotoxin-lacking O157 on cattle farms
Katrijn Cobbaut, Kurt Houf, Glenn Buvens, Ihab Habib, Lieven de Zutter
To cite this version:
Katrijn Cobbaut, Kurt Houf, Glenn Buvens, Ihab Habib, Lieven de Zutter. Occurrence of non-sorbitol
fermenting, verocytotoxin-lacking O157 on cattle farms. Veterinary Microbiology, Elsevier, 2009, 138
(1-2), pp.174. �10.1016/j.vetmic.2009.02.008�. �hal-00490544�
Accepted Manuscript
Title: Occurrence of non-sorbitol fermenting,
verocytotoxin-lacking Escherichia coli O157 on cattle farms Authors: Katrijn Cobbaut, Kurt Houf, Glenn Buvens, Ihab Habib, Lieven De Zutter
PII: S0378-1135(09)00085-6
DOI: doi:10.1016/j.vetmic.2009.02.008
Reference: VETMIC 4364
To appear in: VETMIC Received date: 10-10-2008 Revised date: 5-2-2009 Accepted date: 6-2-2009
Please cite this article as: Cobbaut, K., Houf, K., Buvens, G., Habib, I., De Zutter, L., Occurrence of non-sorbitol fermenting, verocytotoxin-lacking Escherichia coli O157 on cattle farms, Veterinary Microbiology (2008), doi:10.1016/j.vetmic.2009.02.008 This is a PDF file of an unedited manuscript that has been accepted for publication.
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Accepted Manuscript
Occurrence of non-sorbitol fermenting, verocytotoxin-lacking Escherichia coli
1
O157 on cattle farms
2 3
Katrijn Cobbaut
1, Kurt Houf
1, Glenn Buvens
2, Ihab Habib
1,3and Lieven De Zutter
14
5
1
Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Ghent 6
University, Salisburylaan 133, 9820 Merelbeke, Belgium 7
2
Belgian Reference Laboratory for E. coli, Department of Microbiology, University of Brussels, 8
Laarbeeklaan 101, 1090 Brussels, Belgium 9
3
Division of Food Hygiene and Control, High Institute of Public Health, Alexandria University, 165 El- 10
Horrya Avenue, Alexandria, Egypt 11
12 13
Corresponding author.
14
Lieven De Zutter 15
Department of Veterinary Public Health and Food Safety 16
Faculty of Veterinary Medicine 17
Salisburylaan 133 18
9820 Merelbeke 19
Belgium 20
Phone: +32-92647450 21
Fax: +32-92647491 22
E-mail: Lieven.dezutter@UGent.be 23
24
Manuscript
Accepted Manuscript
Abstract 25
Escherichia coli O157 is often associated with hemorrhagic colitis and the hemolytic uremic 26
syndrome (HUS). The verocytotoxins are considered to be the major virulence determinants.
27
However, vt-negative E. coli O157 were recently isolated from patients with HUS. Several 28
transmission routes to humans are described, but cattle feces are the primary source from which 29
both the food supply and the environment become contaminated with E. coli O157.
30
In a prevalence study performed on dairy, beef, mixed dairy/beef and veal farms in the summer of 31
2007, vt-negative isolates were detected on 11.7% (8/68) of the positive farms. From these eight 32
farms, a total of 43 sorbitol-negative E. coli O157:H7 were collected. On five farms, only strains 33
negative for the vt genes were present whereas both vt-negative and vt-positive strains could be 34
detected on three other farms. Further characterization revealed that all isolates carried the eaeA and 35
hlyA genes. Pulsed-field gel electrophoresis (PFGE) of all isolates resulted in nine different PFGE 36
types and within the vt-negative strains, four different genotypes were identified, indicating that 37
certain genetic clones are widespread over the cattle population.
38 39
Keywords: E. coli O157, cattle, verocytotoxin-negative 40
41
1. Introduction 42
Escherichia coli O157:H7/- belongs to the group referred to as enterohemorrhagic E. coli (EHEC) 43
and was identified in 1982 as the cause of human illness associated with the consumption of 44
contaminated hamburgers (Riley et al., 1983). Ruminants, particularly cattle, have been identified 45
as the major reservoirs of E. coli O157:H7, and infections have been traced mostly to consumption 46
of raw or undercooked beef ((Ørskov et al., 1987; Chapman et al., 1993). However, humans can 47
also be infected by consumption of fecal contaminated vegetables (Cieslak et al., 1993), raw milk 48
(Lahti et al., 2002), dairy products (Morgan et al., 1993) and drinking water (Akashi et al., 1994).
49
Accepted Manuscript
Other transmission routes include animal-to-person (Crump et al., 2002) and person-to-person 50
contact (Reida et al., 1994). Clinical symptoms in humans range from mild diarrhoea to hemolytic 51
uremic syndrome (HUS), and can even lead to death (Griffin and Tauxe, 1991; Nataro and Kaper, 52
1998).
53
The pathogenicity of E. coli O157 is linked to the presence of several virulence factors such as the 54
phage-encoded verocytotoxins, intimin encoded by the chromosomal gene eaeA and 55
enterohemolysin encoded by the plasmid gene hlyA. The verocytotoxins VT1 and VT2 are 56
recognized as the most important virulence factors (Boerlin et al., 1999; Sandvig, 2001). The toxins 57
bind to the eukaryotic cell surface by the globotriosylceramide receptor (Lingwood, 1993). After 58
internalization and activation, the biological active part of the toxin inhibits the protein synthesis 59
leading to death of the host cell (Sandvig, 2001). Although verocytotoxins are considered as the 60
essential virulence factors of EHEC, strains lacking those genes have occasionally been isolated 61
from patients with HUS (Mellman et al., 2005;, Bielaszewska et al., 2007, Friedrich et al., 2007). It 62
is unclear whether these strains are either inherently vt-negative or whether they have lost their vt 63
gene during the course of infection (Friedrich et al., 2007).
64
Recently, non-sorbitol fermenting, vt-negative E. coli O157:H7 strains were also isolated from 65
cattle at the farm level (Nielsen and Scheutz, 2002; Wetzel and LeJeune, 2007). The role of these 66
vt-negative EHEC can not be neglected because the possibility exists that vt-negative isolates 67
acquire the vt2 gene again along the farm to fork production chain and that they emerge as a new 68
verocytotoxin-producing E. coli strain (Wetzel and LeJeune, 2007).
69
This study reports the isolation of sorbitol-negative, verocytotoxin lacking E. coli O157 in the cattle 70
population in the Northern part of Belgium.
71 72
2. Materials and methods 73
2.1. Bacterial isolates
74
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During the summer of 2007, an E. coli O157 prevalence study on 180 randomly selected Belgian 75
cattle farms was carried out. The farms were dairy (n=49), beef (n=75), mixed dairy/beef (n=45) or 76
veal farms (n=11). Bedding material of different cattle pens was sampled by the “overshoe method”
77
(Cobbaut et al., 2008) with a maximum of 10 samples per farm. When possible, three different age 78
categories were sampled: <8 months, 8-30 months, and >30 months.
79
To isolate E. coli O157 from the overshoes, 250 ml modified tryptone soya broth (Oxoid, 80
Basingstoke, United Kingdom) (mTSB) supplemented with 20 mg/L novobiocin (Sigma, Aldrich, 81
St-Louis, MO, USA) (mTSBn) was added to each pair of overshoes. After incubation in a warm 82
water bath at 42°C for 6 h, immunomagnetic separation (Dynal, Oslo, Norway) was performed 83
according to the manufacturer’s recommendations. One hundred microliter was spread onto 84
sorbitol-MacConkey agar (Oxoid) supplemented with cefixime (0.05 mg/l) (Dynal) and potassium 85
tellurite (2.5 mg/l) (Dynal) and incubated for 24 h at 42°C.
86
Following incubation, up to three non-sorbitol fermenting colonies with a typical morphology per 87
plate were transferred to plate count agar (Oxoid), incubated for 18-24 h at 37°C, and identified 88
serologically with the O157 antigen latex agglutination assay (Oxoid). From the isolates positive for 89
agglutination, a maximum of nine isolates per farm were selected (three of each sampled age 90
category) and examined by a multiplex PCR. The somatic rfb
O157and the flagellar fliC
H7gene 91
sequences were amplified using previously developed primer pairs by Maurer et al. (1999) and 92
Wang et al. (2000), respectively. The PCR assays were carried out in a 25 µl volume containing 1 93
µl of the lysate, 1 x Taq buffer (20 mM Tris-HCl, pH 8.0 and 50 mM KCl), 0.75 U Taq DNA 94
polymerase (Invitrogen, Carlsbad, California, USA), 500 µM dNTPs, 3 mM of MgCl
2, and 1.7 µM 95
of each primer.
96
Samples were subjected to an initial denaturation of 1 min by 95°C, 30 PCR cycles each consisting 97
of 15 s denaturation at 95°C, 15 s of annealing at 50°C and 30 s of elongation at 72°C. The final 98
cycle was followed by an elongation for 8 min at 72°C and hold at 4°C. The PCR amplification
99
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products were determined by analyzing 8 µl of product on 1.5% agarose gels, staining with 100
ethidium bromide and comparison with a molecular weight marker (TrackIt
TM100bp DNA ladder, 101
Invitrogen).
102 103
2.2. Detection of virulence genes 104
For each isolate identified as E. coli O157, a multiplex virulence PCR was performed using the 105
primers for vt1, eaeA and hlyA as described by Fagan et al. (1999) and for vt2 as described by Paton 106
and Paton (1998). PCR assays were carried out in a 25 µl volume containing 1 µl of the lysates, 1 X 107
Taq buffer (20 mM Tris-HCl, pH 8.0 and 50 mM KCl), 0.75 U Taq DNA polymerase, 500 µM 108
dNTPs, 3 mM of MgCl
2, 1.5 µM of the vt1, eaeA, hlyA and 0,6 µM of the vt2 primers.
109
Temperature conditions consisted of an initial 95°C denaturation step for 3 min followed by 30 110
cycles at 95°C for 20 s, 58°C for 40 s, and 72°C for 90 s. The final cycle was followed by a 8 min 111
hold at 72°C and a final hold at 4°C. The PCR amplification products were analyzed as described 112
above.
113
For confirmation, strains testing negative for the vt genes were retested for vt1 and vt2 using the 114
primer set described by Karch and Meyer (1989). Strains were considered as vt-negative if both 115
PCRs generated no amplicons for the vt genes.
116 117
2.3. Genotyping by pulsed-field gel electrophoresis 118
Pulsed-field gel electrophoresis (PFGE) was performed with 50 U of XbaI (Invitrogen) according to 119
the PulseNet protocol (2007). The Salmonella Braenderup standard strain H9812 was used as a 120
molecular size marker to produce the dendrogram. After electrophoresis, the gel was stained with 121
ethidium bromide and digitally captured under UV light. Band position differences were used to 122
allocate PFGE profiles using GelCompar version 3.0 (Applied Maths, Sint-Martens-Latem, 123
Belgium). The similarities between the fingerprints were calculated using the band-based Dice
124
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coefficient, with an optimization and position tolerance of 1%. The fingerprints were grouped 125
according to their similarities by use of the unweighted-pair group method using arithmetic 126
averages algorithm. Isolates showing identical XbaI-PFGE patterns were compared further using 30 127
U of NotI. For NotI, a linearly ramped switching time from 10 to 30 s for 18 h was used.
128
PFGE genotypes were assigned on the basis of major polymorphisms, defined as a difference in the 129
presence of at least one band in the XbaI fingerprint. The genotypes were indicated by a capital.
130
Some strains showed 100 % similarity after cluster analysis, but a small polymorphism, defined as a 131
small shift of one band, was visually seen. This was indicated by one or more apostrophes after the 132
capital.
133 134
2.4. Statistical analysis 135
Dependant variable of interest was the presence/absence of vt-negative E. coli O157 in a sample, 136
and that was correlated with the categorical explanatory variables (age and farm type). Statistical 137
analysis was carried out in Stata SE/8.0 using a logistic regression model with a logit function and a 138
binomial error distribution.
139 140
3. Results 141
E. coli O157 was present on 68 of the 180 participating farms. All isolates harboured the eaeA and 142
hlyA genes, but on eight of the 68 positive farms (11.8%) vt-negative E. coli O157 were detected.
143
On those eight farms, a total of 43 sorbitol-negative E. coli O157:H7 isolates were further 144
examined. Twenty-nine isolates had no vt genes and the remaining 14 isolates only possessed the 145
vt2 gene.
146
On five farms (6, 44, 101, 102 and 177), only strains negative for the vt genes were present whereas 147
both vt-negative and vt-positive strains could be detected on three other farms (93, 104 and 159) 148
(Fig. 1). The relationship between age category, cattle farm type and the presence of vt-negative
149
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isolates is shown in Table 1. The prevalence of vt-negative E. coli O157 in animals older than 30 150
months was higher (75% (15/20)) compared to the 8-to-30-month old animals (45% (5/11)) and 151
animals younger than 8 months (75% (9/12)). The statistical difference between the age groups was 152
not convincingly significant (P-value=0.084 and 0.086, respectively) due to the limited number of 153
recovered isolates, however this should not underestimate the biological significance of such 154
findings. In addition, E. coli O157 lacking the vt genes were detected on five dairy farms, two beef 155
cattle farms and one mixed cattle farm.
156
The restriction patterns of the 43 isolates could be categorized into nine XbaI-PFGE genotypes (Fig.
157
1). Restriction with NotI did not result in additional information and confirmed the results obtained 158
with XbaI (data not shown). Most genotypes were farm specific, however one PFGE type was 159
associated with five farms (44, 101, 102, 104, and 177). A small band shift was seen between the 160
genotypes of farms 44 and 177 on the one hand and farms 101, 102, and 104 on the other hand, 161
respectively assigned as subtype E’ and E’’. Two farms (104 and 159) harboured three different 162
genotypes. The vt-positive and vt-negative strains did not cluster together within the same genotype, 163
though they originate from the same farm (e.g. strain 786 and 787). A difference of at least four 164
bands and a similarity of maximum 86% was found between the profiles of vt-negative and vt- 165
positive strains originating from the same farm.
166 167
4. Discussion 168
The present study demonstrates that vt-negative E. coli O157:H7 are common on cattle farms. The 169
influence of age category and cattle farm type on the presence of vt-negative isolates was borderline 170
significant and only a large-scale study including more farms and isolates could clarify a possible 171
relationship. Tutenel et al. (2000) conducted a prevalence study in the period 1998-1999 on Belgian 172
cattle at the slaughterhouse level, using the same isolation method as the one that was applied in the 173
present study. Only three E. coli O157 lacking the vt genes were detected in a total of 82 isolates
174
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originating from 62 positive farms. Therefore, the present data may indicate that the prevalence of 175
vt-negative E. coli O157:H7 has increased over the years. Previously vt-negative strains have been 176
reported in cattle in other countries. During a 60-farm study in Denmark, vt-negative/eaeA-positive 177
strains were isolated from five farms out of 17 E. coli O157 positive farms (Nielsen and Scheutz, 178
2002). In a longitudinal study conducted on dairy farms in the USA, vt-negative strains were 179
isolated from four farms (Wetzel and Lejeune, 2007). In Italy, Bonardi et al. (1999) described the 180
isolation of 15 vt-negative strains from feedlot cattle at the slaughterhouse.
181
Two hypotheses can be postulated for the absence of the vt genes in strains carrying other virulence 182
genes such as eaeA and hlyA. Firstly, the vt-negative strains could represent strains that have lost 183
the vt genes during culturing. Loss of vt genes during in vitro culture has been demonstrated in 184
other verocytotoxin-producing E. coli serotypes (Karch et al., 1992). Secondly, the vt-negative 185
strains may already have lost the vt genes in the natural reservoir. In support of this hypothesis, 186
Mellmann et al. (2005) suggested that loss of the vt genes may provide an evolutionary advantage.
187
Survival might be favoured by loss of the phage, because such vt-negative progeny of vt-positive 188
progenitors are less prone to lysis. Although the loss of vt genes during isolation or subculturing in 189
this study can not be excluded, clustering of the strains lacking the vt genes (originating from up to 190
six samples from the same farm) supports the hypothesis that such strains are present in the natural 191
reservoir.
192
All vt-negative E. coli O157:H7 isolates of this study possessed two other virulence traits: the hlyA 193
and eaeA genes. Such strains may be considered as atypical enteropathogenic E. coli (aEPEC), 194
since they do not harbour the bpf gene encoding for the enteropathogenic E. coli adherence factor 195
(EAF). aEPEC have emerged in recent years as cause of diarrhea in both children and adults 196
(Bielaszeswska, 2008). Taking into account the importance of verocytotoxins in the pathogenesis of 197
EHEC-associated HUS (Boerlin et al., 1999; Sandvig, 2001), it seems unlikely that these isolates 198
could still play a role in the development of this syndrome. Since they have unoccupied vt-
199
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bacteriophage integration sites, vt-negative O157:H7 isolates could be transduced with such phages 200
and converted to EHEC. vt-negative attaching and effacing aEPEC presenting the same phenotype 201
of virulence factors and belonging to the most frequent EHEC serotypes have been isolated at a low 202
frequency from patients with diarrhea (Bielaszewska et al., 2008) and HUS (Bielaszewska et al., 203
2007). Within serotype O157:H7, most of these aEPEC fermented sorbitol, but two did not. The 204
authors of these papers suggest that these EHEC lost the vt gene during infection and are no simple 205
aEPEC but a potentially highly virulent group they call EHEC-LST.
206
The genotypes of vt-negative and vt-positive strains differed more than described by Feng et al.
207
(2001) who reported that when PGFE was performed, loss of vt genes resulted in a difference of 208
two bands and the strains without the vt gene had still a similarity of 90% compared to the vt- 209
positive strain. Possibly, the vt-negative strains in the present study do not originate from the vt- 210
positive strains present on the same farm. The vt-negative strains from five farms displayed the 211
same PFGE type (Fig. 1, PFGE type E). Three farms (101, 102, and 104) were situated within a 212
distance of maximum 10 km suggesting a transmission between farms (Wetzel and LeJeune, 2006).
213
However, transmission between farms by bovine animals seems unlikely because all farms were of 214
the closed farm type. Human contact may be the cause of transmission between farm 101 and 102 215
as they belonged to the same owner. Farm 44 and 177 were located at a distance of 150 km and 30 216
km from the other three farms, indicating the circulation of one vt-negative clone.
217
The high number of vt-negative E. coli O157:H7 isolates in this study stresses the importance of 218
testing E. coli O157:H7 isolates for the presence of other virulence factors, because pathogenic 219
strains could be overlooked by protocols that rely exclusively on vt genes or verocytotoxin 220
detection. Further studies are necessary to determine if these strains really can acquire vt genes by 221
transduction with vt-phages present in other E. coli strains in the intestinal flora in animal or human 222
intestine or in food and to examine the possible relationship between vt-negative isolates from 223
humans and cattle.
224
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Acknowledgements 225
We thank all the farmers involved in this study for their cooperation.
226 227
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Wetzel, A.N. and LeJeune J.T., 2006. Clonal dissemination of Escherichia coli O157:H7 subtypes 311
among dairy farms in Northeast Ohio. Appl. Environ. Microbiol. 72, 2621-2626.
312
Wetzel, A.N. and LeJeune, J.T., 2007. Isolation of Escherichia coli O157:H7 strains that do not 313
produce Shiga toxin from bovine, avian and environmental sources. Lett. Appl. Microbiol. 45, 314
504–507.
315
316
317
Accepted Manuscript
Table 1. Distribution of the vt-negative isolates according to the animals’ age (number of isolates 318
lacking the vt genes/total number of E. coli O157:H7 isolates).
319
Farm Farm Type Age category
<8 months 8-30 months >30 months
6 Dairy -
a2/2 (H)
b-
44 Dairy 3/3 (E’) - 3/3 (E’)
93 Mixed 3/3 (D) 0/3 (C) 0/3 (C)
101 Dairy 3/3 (E’’) 3/3 (E’’) 3/3 (E’’)
102 Dairy - - 3/3 (E’’)
104 Beef - 0/3 (G) 1/2 (E’’/I)
c159 Dairy 0/3 (A) - 2/3 (F/B)
177 Beef - - 3/3(E’)
Total 9/12 5/11 15/20
a
: not sampled 320
b
: PFGE-type 321
c
