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F. Allerberger1, M. Wagner2, P. Schweiger1,
H-P. Rammer1, A. Resch1, M.P. Dierich1, A.W. Friedrich3,
H. Karch3
1 National Reference Laboratory for Enterohaemorrhagic
Escherichia coli at the Federal Public Health Laboratory, Innsbruck, Austria
2 Institute for Milk hygiene and Food technology, University
of Veterinary medicine, Vienna, Austria
3 Institute for Hygiene, University of Münster,
Germany
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We report on two children with Escherichia coli
O157 infection, one of whom developed haemolytic uraemic syndrome (HUS).
Both had drunk raw cows’ or goats’ milk in the week before their illness.
Molecular subtyping identified a sorbitol fermenting Escherichia coli
O157:H isolate from a dairy cow. This isolate differed from Shiga toxin
producing O157:H strains isolated from the 6 year old boy with HUS. This
result underlines the need to search for other causes of infection, despite
documented consumption of unpasteurised milk. In the second patient, human
sorbitol non-fermenting O157:H isolates and animal isolates from goats
were indistinguishable. The isolation of indistinguishable sorbitol non-fermenting
Escherichia coli O157:H from contact animals supports the association
between HUS and consumption of raw goats’ milk, and re-emphasises the
importance of pasteurising milk.
Introduction
Illness caused by infection with enterohaemorrhagic Escherichia
coli (EHEC) is usually characterised by bloody diarrhoea. The usual
incubation period for illness is three to four days (range 1-8). The infectious
dose is very low. Haemolytic uraemic syndrome (HUS) complicates 5-14%
of enterohaemorrhagic E. coli O157 infections (1). Children younger
than 5 years are at greatest risk of developing HUS. In Austria, the incidence
of HUS due to EHEC in 2000 was 0.41 cases per 100 000 children aged <15
years. In continental Europe, cases of E. coli O157 infection are
observed less often than in the United Kingdom (UK). During the first
half of 2001, seven human cases of E. coli O157 infection were
documented in Austria, which has a total population of 8 million. Many
outbreaks of E. coli O157 have now been described, and the types
of foods associated with these include minced meat, unpasteurised milk,
unpasteurised apple juice, turkey meat, and water (2). Direct contact
with cattle or goats, and spread from person to person also have been
implicated in outbreaks (2). In cases of sporadic illness the source of
infection and vehicle of contamination are often not identified (3). We
report on two children with E. coli O157 infection, one of whom
developed HUS. Both had consumed raw cows’ or goats’ milk the week before
their illness.
Cases
Case A
A six year old boy from a rural village in Styria was
admitted to the surgical department of a hospital on 18 January 2001 for
weakness and anuria, having had severe, bloody diarrhoea in the preceding
days (onset 10 January). One week later the child was transferred to the
paediatric ward, and haemolytic uraemic syndrome was diagnosed. A stool
specimen, taken on the boy’s seventh day in hospital, yielded sorbitol
fermenting, Shiga toxin 2 producing, enterohaemorrhagic E. coli O157:H-
(isolate no. EH3). The child had regularly been given raw cows’ milk by
his parents. One of 13 faecal specimens taken on 31 January from epidemiologically
linked milk cows yielded sorbitol fermenting E. coli O157:H- (isolate
no. EH41), possessing the virulence genes eae and hly but lacking genes
for Shiga toxin production. The boy was discharged after 15 days in hospital;
he was an asymptomatic carrier at this point (isolates no. EH21 and EH42
from 6 and 19 February, respectively).
Case B
A nine year old boy from a major city in Tyrol was admitted
to hospital for bloody diarrhoea on 3 June 2001, two days after returning
from a school visit to a rural farm. A stool specimen from 5 June yielded
sorbitol non-fermenting, Shiga toxin 2c producing, enterohaemorhagic E.
coli O157:H- (isolate no. EH99). During his five days at the farm,
the child had drunk untreated goats’ milk supplied by the farmer, as had
13 others of the 19 children and one teacher. High numbers of sorbitol
non-fermenting, Shiga toxin 2c producing, enterohaemorrhagic E. coli
O157: H- (isolates no. EH119 and EH120) were cultured from two pooled
faecal specimens taken from seven epidemiologically linked goats at the
farm on 10 June. Milk specimens collected on the same day were negative
when tested for E. coli. None of four faecal specimens from nine
calves at this farm visit centre yielded EHEC isolates. One of the samples
was positive for the virulence genes stx1, stx2, eae, and hly when tested
by polymerase chain reaction (PCR). The patient recovered fully and was
discharged from hospital after four days, at which point he was an asymptomatic
carrier. On 12 June the boy’s sister (aged 27 months), who had not visited
the farm, was admitted for treatment of bloody diarrhoea. A stool specimen
gained on 13 June yielded sorbitol non-fermenting, Shiga toxin 2c producing,
E. coli O157:H- (isolate no. EH123). She was discharged after three
days.
Subtyping of isolates
The eight isolates connected to cases A and B and the
remaining E. coli O157 strains isolated in Austria during the first
six months of 2001 were subtyped by pulsed field gel electrophoresis (PFGE),
using the restriction endonuclease XbaI as previously described (4).
The sorbitol non-fermenting Shiga toxin producing strains
originating from epidemiologically unrelated human cases of E. coli
O157:H7/H infection (including one case of HUS) (isolates no. EH53, EH92,
EH114, EH128) gave PFGE patterns clearly different from each other and
from the strains of cases A and B.
The three human sorbitol fermenting E. coli O157:H7
strains connected to case A (Shiga toxin 2 producing human isolates no.
EH3, EH21, EH42) gave PFGE patterns very similar to each other, but clearly
different from that of the cow’s isolate (non-toxigenic isolate no. EH41)
and of all other strains tested. Strain EH42 from a follow up stool specimen,
gained 25 days after the initial specimen was taken, differed in a single
restriction site from strains EH3 and EH21 (isolates from the first two
stool specimens of the case A).
Also the human and animal sorbitol non-fermenting, Shiga
toxin 2c producing E. coli O157:H isolates connected to case B
differed only in a single restriction site from each other, which shows
their close genetic relatedness.The clinical isolates (EH99 and 123) were
indistinguishable from each other; the same was true for the animal strains
(EH119 and EH120). This and the results from the toxin typing by PCR showed
that the sorbitol non-fermenting E. coli O157:H isolates were transmitted
by contaminated goats’ milk. The EHEC isolates related to case B were
clearly different from all control strains and the sorbitol fermenting
strains. The figure shows the PFGE patterns for the nine human and three
animal strains subtyped.
Discussion
Austrian law regulates the hygienic and microbiological
requirements concerning the production, processing, and direct merchandising
of raw milk at farm level in detail; for the few exceptions where delivery
of untreated milk is allowed, labelling the containers "Rohmilch,
vor dem Verzehr abkochen" ("raw milk, boil before consumption")
is mandatory (5). The sporadic cases of E. coli, O157 infections
reported here underline that consumption of unpasteurised raw milk nevertheless
poses a health risk in Austria in 2001. They also show that unpasteurised
milk is not necessarily always the cause of infection associated with
consumption of untreated milk. This report further reiterates the considerable
potential of profound epidemiological investigations including application
of genomic DNA typing methods to elucidate the sources of infection, even
in sporadic cases.
Although the child with HUS was given unpasteurised cows’
milk regularly by his parents, his severe illness caused by sorbitol fermenting
Shiga toxin producing E. coli O157:H7 was not related to consumption
of raw milk. The epidemiology of infections caused by sorbitol fermenting
E. coli O157 differs in some aspects from the epidemiology
of infections caused by sorbitol non-fermenting E. coli O157. Cattle
and other animals have been well established as major reservoirs of sorbitol
non-fermenting E. coli O157—these organisms can be found in the
intestines of 1—6% of healthy cattle (6,7). However, the rare isolation
of sorbitol-fermenting E. coli O157 in animals led to the assumption that
sorbitol fermenting E. coli O157 may be adapted to the human intestine
and that humans may be the major reservoir (8). This case also underlines
the need to search for other causes of infection despite documented consumption
of unpasteurised milk.
Although direct contact with animal manure may have been
a risk factor in the case B, our results suggest that the most likely
source of primary infection was the goats’ milk. Secondary cases, as a
result of transmission from person to person, are not uncommon. Cases
of infection with E. coli O157 related to raw milk have so far
not been reported in Austria, but are well known in the medical literature.
Bielaszewska et al showed that raw goats’ milk may serve as a vehicle
of E. coli O157 transmission when they reported a cluster of four
cases of HUS occurring in children in northern Bohemia in 1995 (9). Deschenes
et al reported a cluster of cases of HUS in children in the central region
of France after eating cheese made from unpasteurised mixed cows’ and
goats’ milk (10). Trevena et al documented a case of E. coli O157
infection in Cornwall (UK), in a 2 year old boy who had repeatedly drunk
untreated milk during a farm holiday and had developed severe bloody diarrhoea
(11). Transmission of bacterial pathogens other than E. coli O157
by raw milk has been documented in Austria repeatedly, including outbreaks
in children that were related to school activities (12-15).
Conclusions
In contrast to non-typhoidal salmonella and campylobacter,
the two most frequent causes of bacterial diarrhoea in Austria (16), EHEC
is highly contagious, as the transmission from person to person of sorbitol
non-fermenting E. coli O157:H- between the two siblings described
in this report illustrates. The ease with which E. coli O157
infection spreads from person to person reinforces the need to take precautions.
Teachers and parents of children with diarrhoea should be advised that
washing hands with soap after bowel movements is still the most important
measure in preventing the spread of infection (17). Furthermore, it is
prudent to remind them that children should not be given unpasteurised
milk.
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