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Tracing Shigatoxigenic Escherichia coli O103, O145 and O174 Infections from 1
Farm Residents to Cattle 2
3
4
5
Sirpa Heinikainen,1* Tarja Pohjanvirta,
1 Marjut Eklund,
2 Anja Siitonen,
2 Sinikka Pelkonen
1 6
Kuopio Research Unit, Department of Animal Diseases and Food Safety Research, Evira, Finnish 7
Food Safety Authority, P.O. Box 92, 70701 Kuopio, Finland1, 8
Enteric Bacteria Laboratory, National Public Health Institute, Mannerheimintie 166, 00300 9
Helsinki, Finland2 10
11
12
13
14
15
Running title: Tracing non-O157 STEC infections to cattle 16
17
18
19
20
* Corresponding author. Mailing address: Finnish Food Safety Authority Evira, Kuopio Research 21
Unit, P.O. Box 92, FI-70701 Kuopio, Finland. 22
Phone: +358-20-7724963 Fax: +358-20-7724970. E-mail: [email protected]
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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00198-07 JCM Accepts, published online ahead of print on 5 September 2007
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Abstract 24
Severe diarrheal infections caused by shigatoxigenic Escherichia coli O103:H2:stx1:eae-ε:ehx, 25
O145:H28:stx1:eae-γ:ehx (two cases in a family), and O174:H21:stx2c in farm residents were traced 26
to cattle. Molecular methods were applied in the isolation and characterization of the strains. The 27
causative strains were also isolated from cattle samples one or four months later. 28
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Text 29
The most common and best-known shigatoxigenic Escherichia coli (STEC) causing human 30
infections is serotype O157:H7. However, increasing number of other STEC serotypes, especially 31
those of serogroups O26, O103, O111 and O145, are reported to cause severe diseases (9, 25, 26). 32
In cattle, non-O157 STEC are carried by about one third of the animals (20). In the outbreaks, 33
infection vehicle is usually contaminated or undercooked food or water, and person-to-person 34
transmission is often seen within families. In sporadic cases contact to cattle is one of the major risk 35
factors (21). Tracing non-O157 to animals is reported only in a few cases (2, 5). The high 36
prevalence and divergence of non-O157 STECs in cattle and the lack of selective culture media 37
require molecular methods for identifying the causative strain. In the outbreak or trace back 38
situations, the strain characteristics dictates the methodology. In this study, we describe tracing 39
human O103:H2, O145:H28 and O174:H21 STEC infections to cattle farms. 40
41
In Finland, all patients with STEC infection are asked for their connection to cattle farms, and the 42
contact farms are sampled for STEC. During 2003-2006, four human non-O157 STEC cases had 43
contact to cattle farms. A 7-year-old boy living on a farm raising beef cattle (Farm A) was 44
hospitalized with bloody diarrhea. STEC O103:H2:stx1:eae-ε:Ehly was isolated from his stool 45
sample. In a family of five persons living on a dairy farm (Farm B1) both parents had abdominal 46
symptoms and two children were hospitalized with bloody diarrhea. STEC O145:H28:stx1:eae-47
γ:Ehly was isolated from the 5-year-old son and the mother, other fecal samples tested negative for 48
Shiga toxins (Stx). The father also worked on another dairy farm (Farm B2). A man living on a 49
dairy farm (Farm C) had fallen ill with severe watery diarrhea and abdominal pains after cleaning 50
the cowshed with a pressure cleaner. STEC O174:H21:stx2c was isolated from his stool sample. 51
52
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The stool samples of the patients were tested for Stx in the local hospitals and the Stx positive fecal 53
cultures were sent to the National Public Health Institute (KTL) for further studies (14, 8). The 54
isolated strains were analyzed for the O:H serotype (14), enterohemolysin (Ehly) production (14), 55
the subtype of the stx (12) and eae genes (11), and pulsed-field gel electrophoresis (PFGE) profile 56
(11, 28) The data on characteristics including electronic PFGE profiles of the human STEC strains 57
were sent to the Finnish Food Safety Authority (Evira) for comparison with those of the farm 58
isolates. 59
60
Altogether, 303 samples were taken from the four contact farms (Table 1). The first fecal samples 61
were taken within 2-4 weeks after the first symptoms in the families. If the STEC strain causing 62
human infection was recovered from the first samples, samples were taken also from the farm 63
environment, and a risk management plan to reduce spreading of the infection was made. Follow-up 64
fecal and environmental samples were taken after 3-6 months. 65
66
The samples were analyzed for the presence of O103:H2:stx1:eae-ε:ehx (Farm A), 67
O145:H28:stx1:eae-γ:ehx (Farms B1 and B2) and O174:H21:stx2c (Farm C). Instead of analysis for 68
Ehly, the ehx gene encoding it, was searched for (22). Samples were enriched in modified tryptone 69
soy broth with novobiocin supplement (mTSBn) and cultured on sorbitol MacConkey (SMAC) 70
and/or tryptone bile X-glucuronide (TBX) agar plates. For serogroups O103 and O145, 71
immunomagnetic separation (IMS) was made from the enrichment broth according to the 72
manufacturer's (Dynal Biotech, Smestad, Norway) instructions. From all primary culture plates stx1, 73
stx2, eae, ehx and saa genes were detected by multiplex PCR (22). The last samples from Farm A 74
and samples from Farm B1 were also analyzed by PCR to detect serogroup specific genes for O103 75
and O145, respectively (24). In the O174 case, the stx2 positive colonies were first recognized by 76
PCR or colony hybridization and after the isolation were O serotyped. Of all strains, the O 77
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serogroup was confirmed by agglutination with antisera. The flagellar H antigens and stx2 subtypes 78
were determined by PCR-RFLP of the fliC or stx2 gene, respectively (10, 7). The eae gene was 79
subtyped by PCR (7). XbaI-PFGE was performed following the PulseNet protocol (28). To 80
overcome DNA degradation of the O174 strains, the electrophoresis was run in HEPES buffer (19). 81
82
All of the three causative STEC strains were isolated from cattle’s fecal samples in the first 83
sampling (Table 1). The isolates were considered as causative STEC strains if they were 84
indistinguishable by their pheno- and genotypic characteristics from the corresponding human 85
isolates (Figure 1). The causative strains were isolated from environmental samples only once 86
(Farm A). None of the feed samples tested positive for the stx genes in PCR. All farms had also stx 87
positive fecal samples not corresponding with the human strains, which indicates multiple STEC 88
strains present in the cattle, but not causing disease in the residents of these farms. 89
90
In Farm A, the causative strain O103:H2:stx1:eae-ε:ehx (Figure 1) was isolated with IMS from six 91
of the first fecal samples in May and one of the environmental (drinking cup) samples in June 92
(Table 1). In October, a closely related STEC O103:H2:stx1:eae-ε:ehx strain with only one band 93
difference in the PFGE profile compared to the causative strain (Figure 1) was isolated by colony 94
hybridization from one environmental sample from a calf feed alley. It is likely that the difference 95
in PFGE profile was due to the causative O103 STEC strain changing over the time. stx1 or stx2 96
genes were detected in 95% (19 of 20 samples) of the fecal samples in May and in 33% (5/15) of 97
the environmental samples in June. In October, one of the four pooled fecal (prevalence less than 98
25%) and one of the 18 environmental (6%) samples were stx positive. 99
100
In Farm B1, the causative STEC strain O145:H28:stx1:eae-γ:ehx (Figure 1) was isolated with IMS 101
from one of the three pooled fecal samples in the first sampling in February (Table 1). The strain 102
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was not recovered from subsequent samples in March and September. The strain was not recovered 103
from Farm B2. At Farm B1 in individual fecal samples stx genes were detected in 3% (1/33) in 104
March and in 33% (9/27) in September. In the environmental samples stx genes were detected in 105
0% (0/22) in March and 8% (2/26) in September. 106
107
In Farm B1, the father who was working with cattle was the first one to fell ill. Subsequently, 5-108
year-old son, mother and 4-year-old daughter developed symptoms and STEC O145 strain was 109
isolated from the son and the mother. STEC O145 identical with the human strains was isolated 110
from cattle at the family’s home farm. It is possible that the original transmission was from the 111
cattle to the father and the infection spread within the family through person-to-person contact. In 112
our previous study we found that one third of all STEC infections were secondary infections within 113
families (13). Although the STEC strain was isolated only from two family members it is possible 114
that all four persons with symptoms were infected with the same strain. In the follow-up samples 115
one month and six moths later the causative strain was no longer detectable in the farm, suggesting 116
transient colonization of STEC O145 in the cattle. The fact that STEC O145 still is one of the most 117
common serogroups infecting humans (29) but is only occasionally isolated from cattle (8, 17) 118
might be associated with its transient nature. Based on this, it is important to take the fecal samples 119
as soon as possible in trace back situations 120
121
In Farm C, the causative strain O174:H21:stx2c (Figure 1) was isolated from one animal in the first 122
sampling in June. Also a strain of STEC O174:H21:stx2c with a four-band difference in PFGE 123
profile was isolated from the same animal (Figure 1). The causative strain was not isolated from any 124
of the samples taken in July. In October, the causative strain was again isolated from an other 125
animal, but not from the environmental samples. Fecal samples positive for stx genes varied from 126
61% (11/18) in the first sampling in June, to 4% (1/26) in July and 26% (9/35) in October (Table 1). 127
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From the environmental samples stx genes were detected in 4% (1/23) in July and 6% (1/18) in 128
October. In previous longitudinal study on a dairy farm, we detected a STEC O174:H21 strain with 129
a virulence gene and PFGE profile identical with the human strain in this study. The strain persisted 130
on the farm for one year with unchanged PFGE profile (S. Heinikainen, V. Seppänen, T. 131
Pohjanvirta, and S. Pelkonen, Abstr. Verocytotoxigenic E. coli in Europe. Epidemiology of 132
Verocytotoxigenic E. coli., abstr. 134, 2001). This particular STEC O174:H21 clonal line may be 133
persistent in the cattle and difficult to eradicate from the farm. In Farm C, the causative strain was 134
not observed in the samples taken after one month from the first samples, but was again isolated 135
three months later. The O174 strain might have persisted on the farm even for years, before the 136
cleaning of the cowshed with a pressure cleaner exposed the farmer to the causative strain in 137
numbers enough to lead in symptomatic infection. 138
139
Specific PCR methods (1, 4, 6, 7, 10, 15, 22, 24) and IMS (23, 30) are a powerful help when certain 140
characteristics, including virulence-associated genes, O antigens etc. must be screened. However, 141
IMS is currently available only for the most common STEC serogroups O26, O103, O111, O145 142
and O157. For O174, no specific PCR or IMS were available. Thus, the only virulence gene that 143
could be detected from the mixed cultures was stx2 as the causative strain was negative for eae and 144
ehx. The recently published O174 specific PCR (6) helps in screening for STEC of this serogroup. 145
The final trace back of the causative strains to cattle was made by PFGE, which is considered the 146
golden standard for genetic comparison of the outbreak related STEC strains (3, 16). 147
148
Detection of non-O157 STEC bacteria still remains a challenge for diagnosing of human infections 149
and monitoring the prevalence of STEC in cattle. This study shows that there are already good 150
methods available to trace back particular STEC infections to farms. To our experience, the most 151
efficient method for the strain isolation is IMS together with specific PCRs to detect O serogroup 152
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and virulence factors. For samples with low numbers of STEC bacteria, or if IMS is not available, 153
colony hybridization increases the strain isolation efficiency. 154
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257
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Figure legends 258
259
Figure 1 260
PFGE profiles of the human and cattle STEC isolates. Lanes: M, molecular weight control S. 261
Braenderup H9812; 1, human O103 isolate; 2, farm A O103 isolate in May; 3, farm A O103 isolate 262
in June; 4, farm A O103 isolate in October; 5, human O145 isolate; 6, farm B1 O145 isolate in 263
February; 7, human O174 isolate; 8 and 9, farm C O174 isolates in June; 10, farm C O174 isolates 264
in October. 265
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Table 1. Isolation of the causative STEC strains from the contact farms.
No. of samples
causative strain isolated / total (stx positive primary culture) Causative human
STEC strain
Farm
Sampling
month Cattle fecal,
individual
(n = 159)
Cattle fecal,
pooleda
(n = 17)
Environment
(n = 122)
Feed
(n = 5)
May 6 / 20 (19)
June 1 / 15 (5) 0 / 1 (0)
O103:H2
stx1:eae-ε:ehx
Farm A
October 0 / 4 (1) 0b / 18 (1)
February 1 / 3 (1)
March 0 / 33 (1) 0 / 4 (0) 0 / 22 (0) 0 / 3 (0)
O145:H28
stx1:eae-γ:ehx
Farm B1
September 0 / 27 (9) 0 / 3 (1) 0 / 26 (2)
Farm B2 February 0 / 2 (2)
June 1c / 18 (11)
July 0 / 26 (1) 0 / 1 (0) 0 / 23 (1) 0 / 1 (0)
O174:H21
stx2c
Farm C
October 1 / 35 (9) 0 / 18 (1)
a From 3-13 animals
b A strain related (a one-band difference in PFGE) to the causative strain was recovered
c An additional O174 strain with a four-band difference in PFGE with the causative strain was recovered
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Figure 1
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