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Journal of Clinical Microbiology, May 1999, p. 1570-1572, Vol. 37, No. 5
Institute of Food Safety and Hygiene, Faculty
of Veterinary Medicine, University of Zurich, CH-8057 Zurich,
Switzerland
Received 17 September 1998/Returned for modification 10 December
1998/Accepted 27 January 1999
Fourteen verotoxin-producing Escherichia coli strains
isolated from stool samples of 14 different asymptomatic human carriers were further characterized. A variety of serotypes was found, but none
of the strains belonged to serogroup O157. Only one isolate carried
most of the virulence genes that are associated with increased pathogenicity.
The importance of the verotoxin
(VT)-producing Escherichia coli (VTEC) group has increased
since a food-borne infection caused by enterohemorrhagic E. coli was first described (17). Other serogroups, like
O26, O111, and O103, have also been found in affected patients, in
addition to the classic serovars O157:H7 and O157:H In an ongoing study of routine stool samples from staff members of
meat-processing companies, 1,730 specimens from different persons in
all parts of Switzerland were examined in October and November 1997 by
using PCR for detection of VT-encoding genes and by culture methods for
detection of other pathogens relevant to food hygiene. All samples were
collected in urban areas, and each person was tested only once. The
population consisted of adults without diarrhea aged between 20 and 60 years, a quarter being female.
For the VTEC assay, samples were directly plated on sheep blood agar,
and after 24 h of incubation at 37°C, the colonies were washed
off in normal saline. The plate eluate obtained was then evaluated by
PCR with primers based on sequences targeting a region conserved
between the genes for which are VT1 and VT2 complementary to
nucleotides 439 to 462 and 943 to 962 of the sequence with EMBL/GenBank
accession no. M19473 for the VT1-encoding gene (20) and to
nucleotides 515 to 538 and 1016 to 1035 of the sequence with
EMBL/GenBank accession no. X07865 for the VT2-encoding gene
(8). The sequences of primers used and the cycling
conditions have been described by Burnens et al. (4).
Bacterial DNA was prepared by incubating 2 µl of washed-off cultures
in 42 µl of double-distilled water for 10 min at 100°C.
Amplifications were performed in a total volume of 50 µl containing
200 µM deoxynucleoside triphosphates, 30 pmol of each primer, 5 µl
of 10-fold-concentrated polymerase synthesis buffer, and 2.5 U of
Taq DNA polymerase (Promega) in a Perkin-Elmer Cetus DNA
cycler. The amplified products were visualized by gel electrophoresis
in 0.9% agarose agar stained with ethidium bromide. E. coli
EDL933 was used in each run as a positive control, and E. coli U4-41 was used as a negative control. The eluate was plated
again on MacConkey agar, and at least 18 single colonies each were
tested by the same PCR protocol in 27 positive samples in order to
obtain VTEC isolates. Only one VT-producing strain per sample was then
subjected to further typing. The strains were biochemically confirmed
as E. coli (acid production from mannitolol,
o-nitrophenyl- The PCR product of VT-encoding genes was detected in 79 (4.6%) (61 from males, 18 from females) of the 1,730 stool samples analyzed in
this study. Comparatively, we found Salmonella spp. in 3 (0.17%), Campylobacter spp. in 7 (0.4%),
Yersinia spp. in 10 (0.69%), and Listeria spp.
in 13 (0.75%) samples. No geographic clustering of VT
gene-positive samples was found, but if the distribution frequency of PCR-positive stool samples is compared to the workplaces of the employees, the following account can be found (Fig.
1). Slaughtering stands out with 9%.
Working in a slaughterhouse, where individuals would presumably
encounter VTEC bacteria more frequently and in higher numbers, may put
them at higher risk for carriage or excretion of these organisms. It
must be considered, however, that in this area a substantially smaller
number of persons (n = 74) could be examined.
Therefore, this observation must be backed up with further
examinations.
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Virulence Factors and Phenotypical Traits of Verotoxin-Producing
Escherichia coli Strains Isolated from Asymptomatic
Human Carriers
ABSTRACT
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(3, 5, 13). Apart from the ability to produce VT, these pathogroups may possess accessory virulence factors associated with the
capacity to colonize the gut, such as intimin and a 90-kbp virulence-associated plasmid (2, 12). In patients, VTEC
strains are associated with watery or bloody diarrhea, hemorrhagic
colitis, and the hemolytic-uremic syndrome (7, 9). Because
of the lack of a national surveillance program, the incidence of these diseases and the isolation rate of VTEC in Switzerland are not known.
Cattle are considered to be the main reservoir of VTEC (9,
22). Burnens et al. (4) described a Swiss prevalence of 21%. Therefore, the source of this food-borne infection was often
found to be foods of bovine origin or other fecally cross-contaminated foods. Person-to-person transmission has been reported during outbreaks
and may account for a significant number of sporadic cases (7,
16). An important question to address, however, is the role of
asymptomatic human carriers in food-producing companies as a source of
contamination. One study of Canadian dairy farm families (a group with
a high level of environmental exposure) detected carriage of VTEC in
about 6% of the individuals (23). The aim of this study was
to isolate VTEC strains of stool samples from staff members of
food-producing companies and to compare their serotype distribution and
the presence of virulence attributes.
-D-galactopyranoside [ONPG]
test, H2S and indole production, and proof of urease and
lysine decarboxylase). Moreover, they were tested for sorbitol
fermentation and -D-glucuronidase activity on Fluorocult
agar (Merck 4036) and for the hemolytic phenotype by using
CaCl2-washed blood agar. Production of VT by each isolated
strain was confirmed by cytotoxicity tests on Vero cells
(11). The genotype of the VT B subunit and the presence of
the E-hlyA, eae, and astA genes and
the 60-MDa plasmid was determined by separate PCRs. The sequences of
the primers used and the cycling conditions have been previously
described (6, 18, 19, 24). Serotyping of somatic and
flagellar antigens was performed at the Statens Serum Institute,
Copenhagen, Denmark.
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FIG. 1.
Distribution frequency of PCR-positive stool samples
compared to the workplaces of the employees (n = number
of employees examined).
Fourteen VTEC strains isolated from stool samples of 14 different
persons from all five work areas were further characterized. Serotyping
yielded seven different O:H serotypes comprising seven O serogroups and
seven H serogroups. Three strains were O nontypeable, and two were O
rough; all strains were motile (Table 1).
Compared to isolates found in patients (15), three strains
(O76:H19, O113:H4, and O146:H28) isolated from asymptomatic human
carriers had common serotypes, but the serotypes of these VTEC isolates are not those which have been convincingly and frequently associated with human disease. Further results of strain characterization are
summarized in Table 1. Phenotypically, all 14 strains were both
sorbitol and -D-glucuronidase positive and also positive in the Vero cell cytotoxicity test. Subtyping of the VT genes of the
isolated strains by using VT1- and VT2-specific primers showed that
three strains possessed VT1 alone, nine strains possessed VT2 genes,
and two strains possessed both VT1 and VT2. Since most patients
developing the hemolytic-uremic syndrome are infected with strains that
harbor the VT2 type (10, 14, 21), these findings in
asymptomatic human carriers are astonishing. The eae gene,
which is strongly correlated with symptomatic disease in humans
(1), was present in only one strain of serotype ONT:H25. The
60-MDa plasmid was found in seven strains. Although E-hlyA was proved in seven isolates, enterohemolysin production was found in
only six strains. The gene encoding EAST1, representing an additional
determinant in the pathogenesis of E. coli diarrhea, was
found in one strain. Two strains of serotypes O113:H4 and ONT:H25
harbored three of the four additional virulence factors tested.
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The aim of further studies that are in progress is to isolate and characterize more strains. Moreover, asymptomatic human carriers should be tested over prolonged periods of time.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Food Safety and Hygiene, Faculty of Veterinary Medicine, University of Zurich, Winterthurerstr. 270, CH-8057 Zurich, Switzerland. Phone: 1 635 86 54. Fax: 1 635 89 08. E-mail: ils{at}fsafety.unizh.ch.
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Journal of Clinical Microbiology, May 1999, p. 1570-1572, Vol. 37, No. 5
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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