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J Clin Microbiol. Nov 2007; 45(11): 3817–3820.
Published online Sep 5, 2007. doi:  10.1128/JCM.00198-07
PMCID: PMC2168472

Tracing Shigatoxigenic Escherichia coli O103, O145, and O174 Infections from Farm Residents to Cattle[down-pointing small open triangle]

Abstract

Severe diarrheal infections caused by Shigatoxigenic Escherichia coli O103:H2:stx1:eae-epsilon:ehx, O145:H28:stx1:eae-γ:ehx (two cases in a family), and O174:H21:stx2c in farm residents were traced to cattle. Molecular methods were applied to the isolation and characterization of the strains. The causative strains were also isolated from cattle samples 1 or 4 months later.

The most common and best-known Shigatoxigenic Escherichia coli (STEC) serotype causing human infections is serotype O157:H7. However, increasing numbers of other STEC serotypes, especially those of serogroups O26, O103, O111, and O145, are reported to cause severe diseases (9, 26, 27). Among cattle, non-O157 STEC strains are carried by about one-third of the animals (21). In outbreaks of STEC infection, the infection vehicle is usually contaminated or undercooked food or water, and person-to-person transmission within families is often seen. In sporadic cases, contact with cattle is one of the major risk factors (22). Tracing of non-O157 strains to animals has been reported in only a few cases (2, 5). The high prevalence and divergence of non-O157 STEC strains in cattle and the lack of selective culture media require molecular methods for identifying the causative strain. In outbreak or trace-back situations, the strain characteristics dictate the methodology. In this study, we describe tracing human O103:H2, O145:H28, and O174:H21 STEC infections to cattle farms.

In Finland, all patients with STEC infection are asked about their connection to cattle farms, and the contact farms are sampled for STEC. During the period from 2003 to 2006, four cases of human non-O157 STEC infection involved contact with cattle farms. A 7-year-old boy living on a farm raising beef cattle (farm A) was hospitalized with bloody diarrhea. STEC O103:H2:stx1:eae-epsilon:ehx was isolated from his stool sample. In a family of five persons living on a dairy farm (farm B1), both parents had abdominal symptoms and two children were hospitalized with bloody diarrhea. STEC O145:H28:stx1:eae-γ:ehx was isolated from the 5-year-old son and the mother; other fecal samples tested negative for Shiga toxins (Stx). The father also worked on another dairy farm (farm B2). A man living on a dairy farm (farm C) fell ill with severe watery diarrhea and abdominal pains after cleaning the cowshed with a pressure cleaner. STEC O174:H21:stx2c was isolated from his stool sample.

The stool samples of the patients were tested for Stx in the local hospitals, and the Stx-positive fecal cultures were sent to the National Public Health Institute, Helsinki, Finland, for further studies (14, 19). The isolated strains were analyzed for the O:H serotype (14), enterohemolysin (Ehly) production (14), the subtypes of the stx (12) and eae (11) genes, and the pulsed-field gel electrophoresis (PFGE) profile (11, 29). The data on characteristics, including electronic PFGE profiles, of the human STEC strains were sent to the Finnish Food Safety Authority (Evira) for comparison with those of the subsequent farm isolates.

Altogether, 303 samples were taken from the four contact farms (Table (Table1).1). The first fecal samples were taken within 2 to 4 weeks after the onset of the first symptoms in the families. If the STEC strain causing human infection was recovered from the first samples, samples were also taken from the farm environment and a risk management plan to reduce the spreading of the infection was made. Follow-up fecal and environmental samples were taken after 3 to 6 months.

TABLE 1.
Isolation of the causative STEC strains from the contact farms

The samples were analyzed for the presence of O103:H2:stx1:eae-epsilon:ehx (farm A), O145:H28:stx1:eae-γ:ehx (farms B1 and B2), and O174:H21:stx2c (farm C). Instead of being analyzed for Ehly, the samples were examined for the ehx gene encoding Ehly (23). Samples were enriched with modified tryptone soy broth with a novobiocin supplement and cultured on sorbitol-MacConkey and/or tryptone bile X-glucuronide agar plates. For serogroups O103 and O145, immunomagnetic separation (IMS) from the enrichment broth was carried out according to the instructions of the IMS system manufacturer (Dynal Biotech, Smestad, Norway). The stx1, stx2, eae, ehx, and saa genes were detected on all primary culture plates by multiplex PCR (23). The last samples from farm A and samples from farm B1 were also analyzed by PCR to detect serogroup-specific genes of O103 and O145, respectively (25). In the case of O174, the stx2-positive colonies were first recognized by PCR or colony hybridization analysis and, after isolation, were O serotyped. For all strains, the O serogroup was confirmed by agglutination with antisera. The flagellar H antigens and stx2 subtypes were determined by PCR-restriction fragment length polymorphism analysis of the fliC and stx2 genes, respectively (10, 28). The eae gene was subtyped by PCR (7). XbaI PFGE was performed by following the PulseNet protocol (29). To overcome DNA degradation of the O174 strains, the electrophoresis was run in HEPES buffer (20).

All three causative STEC strains were isolated from cattle fecal samples from the first sampling (Table (Table1).1). The isolates were considered to be causative STEC strains if they were indistinguishable by their pheno- and genotypic characteristics from the corresponding human isolates (Fig. (Fig.1).1). The causative strains were isolated from environmental samples only once (farm A). None of the feed samples tested positive for the stx genes by PCR. All farms also yielded stx-positive fecal samples carrying strains not corresponding to the human strains, which indicates that multiple STEC strains were present in the cattle but not causing disease in the residents of these farms.

FIG. 1.
PFGE profiles of the human and cattle STEC isolates. Lanes: M, molecular weight control Salmonella enterica serovar Braenderup H9812; 1, human O103 isolate; 2, farm A O103 isolate from May; 3, farm A O103 isolate from June; 4, farm A O103 isolate from ...

For farm A, the causative strain O103:H2:stx1:eae-epsilon:ehx (Fig. (Fig.1)1) was isolated with IMS from six of the first fecal samples in May and one of the environmental (drinking-cup) samples in June (Table (Table1).1). In October, a closely related STEC O103:H2:stx1:eae-epsilon:ehx strain with only one band of difference in the PFGE profile compared to that of the causative strain (Fig. (Fig.1)1) was isolated by colony hybridization from one environmental sample from a calf feed alley. It is likely that the difference in PFGE profiles was due to the changing of the causative O103 STEC strain over time. stx1 or stx2 genes were detected in 95% of the fecal samples (19 of 20) in May and in 33% of the environmental samples (5 of 15) in June. In October, 1 of the 4 pooled fecal samples (prevalence, less than 25%) and 1 of the 18 environmental samples (6%) were stx positive.

For farm B1, the causative STEC strain O145:H28:stx1:eae-γ:ehx (Fig. (Fig.1)1) was isolated with IMS from one of the three pooled fecal samples from the first sampling in February (Table (Table1).1). The strain was not recovered from subsequent samples obtained in March and September. The strain was not recovered from farm B2. stx genes were detected in 3% of individual fecal samples (1 of 33) obtained in March and in 33% of individual fecal samples (9 of 27) obtained in September from farm B1. Of the environmental samples, stx genes were detected in 0% (0 of 22) from March and 8% (2 of 26) from September.

On farm B1, the father who was working with cattle was the first one to fall ill. Subsequently, his 5-year-old son, the mother, and his 4-year-old daughter developed symptoms, and the STEC O145 strain was isolated from the son and the mother. A STEC O145 strain identical to the human strains was isolated from cattle at the family's home farm. It is possible that the original transmission was from the cattle to the father and that the infection spread within the family through person-to-person contact. In our previous study, we found that one-third of all STEC infections were secondary infections within families (13). Although the STEC strain was isolated from only two family members, it is possible that all four persons with symptoms were infected with the same strain. In the follow-up samples collected from the farm 1 and 6 months later, the causative strain was no longer detectable, suggesting transient colonization of the cattle by STEC O145. The fact that STEC O145 is still one of the most common serogroups infecting humans (30) but is isolated from cattle only occasionally (8, 17) may be associated with its transient nature. Based on this possibility, it is important to take the fecal samples as soon as possible in trace-back situations.

For farm C, the causative strain O174:H21:stx2c (Fig. (Fig.1)1) was isolated from one animal in the first sampling in June. Also, a strain of STEC O174:H21:stx2c with a four-band difference in the PFGE profile was isolated from the same animal (Fig. (Fig.1).1). The causative strain was not isolated from any of the samples taken in July. In October, the causative strain was again isolated from another animal but not from the environmental samples. The frequency of fecal samples positive for stx genes varied from 61% (11 of 18) in the first sampling in June to 4% (1 of 26) in July and 26% (9 of 35) in October (Table (Table1).1). Of the environmental samples, stx genes were detected in 4% (1 of 23) in July and 6% (1 of 18) in October. In a previous longitudinal study on a dairy farm, we detected a STEC O174:H21 strain with a virulence gene and a PFGE profile identical to those of the farm C human strain in this study. The strain persisted on the farm for 1 year with an unchanged PFGE profile (18). This particular STEC O174:H21 clonal line may be persistent in the cattle and difficult to eradicate from the farm. The causative strain was not observed in the samples taken from farm C 1 month after the first samples but was again isolated 3 months later. The O174 strain may have persisted on the farm even for years before the cleaning of the cowshed with a pressure cleaner exposed the farmer to the causative strain at concentrations high enough to lead to symptomatic infection.

Specific PCR methods (1, 4, 6, 7, 10, 15, 23, 25) and IMS (24, 31) are powerful tools when certain characteristics, including virulence-associated genes and O antigens, etc., must be screened. However, IMS is currently available only for the most common STEC serogroups, O26, O103, O111, O145, and O157. For O174, no specific PCR or IMS was available. Thus, the only virulence gene that could be detected from the mixed cultures was stx2, as the causative strain was negative for eae and ehx. The recently published O174-specific PCR method (6) helps in screening for STEC strains of this serogroup. The final trace back of the causative strains to cattle was made by PFGE, which is considered the “gold standard” for genetic comparison of outbreak-related STEC strains (3, 16).

The detection of non-O157 STEC bacteria still remains a challenge for diagnosing human infections and monitoring the prevalence of STEC in cattle. This study shows that there are already good methods available to trace back particular STEC infections to farms. In our experience, the most efficient method for strain isolation is IMS together with specific PCRs to detect O serogroup and virulence factors. For samples with low numbers of STEC bacteria, or if IMS is not available, colony hybridization increases the strain isolation efficiency.

Footnotes

[down-pointing small open triangle]Published ahead of print on 5 September 2007.

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