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Journal of Clinical Microbiology, May 2000, p. 1998-2000, Vol. 38, No. 5
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
A Three-Year Study of Campylobacter
jejuni Genotypes in Humans with Domestically Acquired Infections
and in Chicken Samples from the Helsinki Area
Marja-Liisa
Hänninen,1,*
Päivikki
Perko-Mäkelä,1
Anna
Pitkälä,2 and
Hilpi
Rautelin3
Department of Food and Environmental Hygiene,
Faculty of Veterinary Medicine,1 and
Department of Bacteriology and Immunology, The Haartman
Institute, University of Helsinki and Helsinki University Central
Hospital Diagnostics,3 00014 University of
Helsinki, and Laboratory of Food and Environment, City of
Helsinki, 000530 Helsinki,2 Finland
Received 29 October 1999/Returned for modification 30 December
1999/Accepted 26 February 2000
 |
ABSTRACT |
Campylobacter jejuni isolates from stool samples of
patients with domestically acquired sporadic infections and from
chicken from retail shops were studied during seasonal peaks from June to September over a 3-year period from 1996 to 1998. A large number of
pulsed-field gel electrophoresis (PFGE) genotypes (a combined SmaI-SacII pattern) were identified each year.
Certain genotypes persisted for the whole study period, and predominant
genotypes represented 28 to 52% of the strains during a restricted
period of time. The peak level of positive chicken samples was between July and August of each study year, when 10 to 33% of the samples were
positive for campylobacter. The same PFGE genotypes found in humans
were also detected in the chicken samples. This suggests that common
genotypes were circulating in the area.
| |
TEXT |
Campylobacter jejuni is
the most common bacterial enteropathogen in developed countries. The
sources and transmission routes of human campylobacteriosis are not
fully understood, but handling and eating poultry have been shown to be
important risk factors (10, 12, 14, 15). Several molecular
typing methods have been used to support studies of the epidemiology of
campylobacter infections during the 1990s, and pulsed-field gel
electrophoresis (PFGE) pattern analysis has been shown to be a highly
discriminatory method (3, 5). We have also recently used
PFGE typing for studies of the epidemiology of campylobacter infections
in humans in Finland (6). Little is known about the
persistence or fluctuation of the genotypes of C. jejuni
through the years. Identical genotypes might indicate common sources of
infection and provide data on the stability of the genotypes. This
paper describes PFGE genotypes of C. jejuni isolates
gathered over a three year period from fecal samples of enteritis
patients from the Helsinki, Finland, area with infections acquired in
Finland during the summer months. In addition, PFGE patterns of
C. jejuni strains isolated from chicken sold at the retail
level or from chicken fecal samples obtained at slaughterhouses during
the same study period were included for comparison.
Bacterial isolates.
C. jejuni isolates from fecal
samples of enteritis patients were collected from June through
September during each year of a 3-year period from 1996 to 1998. The
isolates were regarded as domestic in origin if the patients had not
travelled abroad prior to their illness. The fecal samples were
inoculated on campylobacter blood-free selective medium
(charcoal-cefoperazone-desoxycholate agar; Oxoid, Ltd., Basingstoke,
Hampshire, England) and incubated at 42°C in a microaerobic
atmosphere. Chicken samples were cultured after enrichment in Lab M
enrichment broth (Bury, United Kingdom) on
charcoal-cefoperazone-desoxycholate agar (6, 7) or according to the method outlined by the International Standardization
Organization (8). Fresh chicken pieces (legs and breasts)
sold at retail that were studied represented three major chicken
producers in Finland and were collected from shops in the Helsinki
area. For the year 1996, strains isolated from chicken cecal samples
taken during the slaughtering process were also included for PFGE
genotype analysis. Only C. jejuni isolates were included in
the study (6). After the original isolation, the isolates
were stored at 
70°C.
Typing C. jejuni isolates by PFGE.
For PFGE
analysis, the isolates were grown on brucella blood agar (Oxoid) for 2 days at 37°C in a microaerobic atmosphere. The bacterial cells were
harvested and treated with formaldehyde to inactivate endogeneous
nucleases (4). Otherwise, DNA plugs were prepared, and
electrophoresis conditions were as described earlier (6, 7,
11). A combined SmaI-SacII pattern was designated a genotype. Certain strains were also analyzed by digestion with KpnI. If the strains had one- to three-band differences
in their SmaI, SacII, and KpnI
patterns, they were designated subtypes and marked with a lowercase
letter (for example, VIa, VIb, and VIc).
There was a remarkable fluctuation in the number of domestically
acquired campylobacter cases in humans during the study. The summer
months of June through September were chosen for the follow-up period,
as there is an isolation peak from July to August in Finland, and
outside this season, domestic campylobacter isolates are uncommon
(6). In 1996, 89 domestic campylobacter cases were
identified, whereas the respective number in 1997 was only 36 and in
1998, 69. The reason for this fluctuation is not known, but the summer
in 1996 was hot and dry, whereas the summers of 1996 and 1998 were
especially rainy. During the same time period, campylobacter was also
isolated from chicken pieces sold at the retail level (Table
1). The isolation rates were low in the
beginning of the summer (0 to 10% in May; unpublished results),
increased to 10 to 30% from July to August, and dropped again in
September (8 to 14%). In comparison with other countries, the
isolation rate of campylobacter in chicken pieces was low. In a recent
study from the United Kingdom, approximately 80% of chicken pieces
sampled from retail shops were positive for campylobacter
(F. J. Bolton, J. K. Williamson, G. Allen, D. R. Wareing, and J. A. Frost, Abstr. 10th Intl. Workshop on
Campylobacter, Helicobacter, and Rel. Org., abstr. CF2, 1999). In a Danish study, 30 to 40% of chicken pieces sampled in one summer were positive (H. Rosenquist and N. L. Nielsen, Abstr. 10th Intl. Workshop on Campylobacter,
Helicobacter, and Rel. Org., abstr. CF20, 1999). Similar
seasonal variation as noted in infections in humans has also been
verified in the carriage rates of C. jejuni in live chickens
(2). This finding was confirmed in our study as well.
However, the annual variation in the incidence of campylobacter cases
in humans could not be explained only by the variable contamination
level of retail chicken pieces, because approximately the same
percentage of chicken pieces were found to be positive for
campylobacter each year. Most chickens in Finland are sold in fresh
cut-up pieces, which means that the time between slaughtering and
consumption is short. Although the peaks in infections in humans and
contamination rates of chicken meat are both seen from July to
August, it does not necessarily indicate that chicken would be the most
important direct source of infections in humans in Finland. Recent
case-control studies from other countries actually showed that eating
or handling chicken meat could explain less than 10% of the
human campylobacter cases (13, 14).
Along with the two restriction enzymes,
SmaI and
SacII, 30 PFGE genotypes were found among the human isolates
in 1996, 19
genotypes were found in 1997, and 28 were found in 1998. The overall
diversity of genotypes was similar each year. Seven
genotypes
(including variants) (I/E, I/J, I/K, II/A, IV, VIa, VIb, VIc,
and -/P) were identical for all three seasons (1996 to 1998),
and
genotypes I/B, I/L, and H were identified at two isolation
periods
(Tables
2 and
3). A total of 41 genotypes were unique
and typical for a certain time period only. PFGE patterns of five
persistent genotypes (I/B, I/K, IV, VI, and T101) and variants
of
genotypes VI (a, b, and c) and T101 (a and b) are shown in
Fig.
1. A large variation in genotypes was
also demonstrated for
the chicken isolates, as 15 genotypes were
identified in 1996,
19 genotypes were found in 1997, and 27 were found
in 1998 (Table
1). The same genotypes seen in human strains (for
example, I/B,
I/K, IV, VIa, VIb, VIc, T101a, T101b, and -/P) were
identified
among chicken strains as well (Table
1; Fig.
1).
View this table:
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TABLE 3.
Distribution of five predominant domestic human
C. jejuni genotypes from June to September of 1996, 1997, and 1998
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FIG. 1.
SmaI, SacII, and KpnI
patterns of certain common Finnish C. jejuni genotypes. M,
molecular size marker.
|
|
Information on the PFGE patterns over a longer time span is limited. In
the present study, two predominant genotypes (VIb
and I/B in 1996 and
IV and T101a in 1997) represented 25 and 28%
of the human strains
isolated in 1996 and 1997, and three genotypes
(I/K, T101b, and the VI
variants VIa, VIb, and VIc) represented
52% of the strains isolated in
1998 (Table
3). Predominant PFGE
genotypes varied by year and also from
one month to another during
the same year (Table
2 and
3). For
instance, PFGE genotypes
I/B and VIb were the most common genotypes
among the
C. jejuni strains isolated in 1996, but no I/B
isolates and only one VIb
isolate were found in 1997. However, other
variants of genotype
VI (VIa and VIc), were common among the isolates
each year (Table
3). Similarly, PFGE genotype T101a was only found
among the strains
isolated in 1997, when it represented over 16% of
all isolates,
whereas another highly related genotype (T101b)
represented over
17% of the isolates in 1998. Predominant genotypes
persisted through
the 3-year follow-up, although their relative
proportion in patients
markedly varied from one year to another. This
phenomenon suggests
genomic
stability.
Most of the predominant human PFGE genotypes were found among chicken
isolates (Table
3), although not always among strains
isolated during
the same month. However, this does not prove that
the direct source for
the human infections was chicken but could
indicate that during summer
months campylobacters are common in
many sources and that certain
predominant genotypes circulate
and colonize various host animals.
Humans are probably infected
from many sources, direct contact with
chicken being only one
of them. Some chicken PFGE genotypes were never
found among human
isolates, and vice versa, which also suggests that
there are other
important sources than chicken for human
campylobacteriosis. Chickens
are raised in large units of approximately
10,000 animals and
slaughtered at the age of 6 weeks. Typing of
C. jejuni strains
from several subsequent breeding units has
shown that each unit
is usually contaminated with a different genotype
of
C. jejuni (
1,
2,
9). This explains the high
number of unique genotypes
identified from chicken
samples.
In many countries, as in Finland, the number of campylobacter cases in
humans has increased significantly in recent years
(
12,
14).
In Finland in 1998, the annual number of reported
campylobacter cases
(2,938) exceeded that of salmonella (2,735)
for the first time,
according to the National Infectious Disease
Registry, National Public
Health Institute, Helsinki, Finland).
Except for large outbreaks,
however, the source of human campylobacteriosis
is rarely identified,
and therefore the exact transmission routes
are poorly understood.
Precise molecular typing methods are necessary
epidemiological tools,
and PFGE has been shown to be a discriminatory
method with the
possibility of digitizing the pattern analysis
(
http://www.svs.dk/campynet/). Our present survey showed that
there was
a great fluctuation not only in the number of domestically
acquired
campylobacter cases in humans but also in the PFGE genotypes
identified
during the 3-year follow-up period. Some PFGE genotypes
were found
throughout the study period, although the predominant
genotypes usually
differed each year. At the same time, the prevalence
of campylobacter
among chicken meat samples at the retail level
was quite low. Although
most of the predominant PFGE genotypes
among human isolates were the
same as those seen among chicken
isolates during the same time period,
other sources for
Campylobacter infections in humans should
also be
considered.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Food and Environmental Hygiene, Faculty of Veterinary Medicine, P.O. Box 57, 00014 University of Helsinki, Finland. Phone: 358-9-19149704. Fax: 358-9-19149704. E-mail:
marja-liisa.hanninen{at}helsinki.fi.
| |
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0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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