Copyright © 1998, American Society for Microbiology
Epidemiological Study of a Food-Borne Outbreak of Enterotoxigenic
Escherichia coli O25:NM by Pulsed-Field Gel Electrophoresis
and Randomly Amplified Polymorphic DNA Analysis
TOSHIHIRO MITSUDA,
1,2* TETSUNORI MUTO,
3MIKIKO YAMADA,
3NOBUYOSHI KOBAYASHI,
3MASANORI TOBA,
3YUKOH AIHARA,
2AKIRA ITO,
1ANDSHUMPEI YOKOTA
2Division of Clinical Laboratory Medicine
1and Department of Pediatrics,
2School of Medicine, Yokohama City
University, 3-9 Fukuura, Kanazawa-Ku, Yokohama City, 236-0004, and Yokohama City Institute of Health,
1-2-17 Takigashira, Isogo-Ku, Yokohama City, 235-0012,
3Japan
Received 10 July 1997/Returned for modification 1 November 1997/Accepted 15 December 1997
This study investigated the applicability of molecular epidemiological techniques to the identification of the
causal agent of an outbreak of diarrhea caused by ingestion of food contaminated with enterotoxigenic
Esch-erichia coli (ETEC). The outbreak occurred at four elementary schools in July 1996 and affected more than 800
people. Illness was most strongly associated with eating tuna paste (relative risk, 1.79; 95% confidence
inter-val
5
1.16 to 2.79; P
5
0.0001). To evaluate the epidemiological characteristics of the pathogen, the DNAs from
numerous isolated ETEC strains were subjected to randomly amplified polymorphic DNA analysis,
pulsed-field gel electrophoresis of nuclease S1-treated plasmid DNA, and analysis of genomic DNA restriction
frag-ment length polymorphisms. All ETEC isolates were of the O25:NM (nonmotile) serotype, which carries a
heat-stable enterotoxin Ib gene. Genotypic analysis demonstrated that the strains isolated from the patients at
all four schools were identical. The isolates of ETEC O25:NM obtained from the tuna paste that had been
served for lunch at these schools were genetically indistinguishable from those isolated from the patients.
Results suggest that this outbreak was food borne. The molecular biology-based epidemiological techniques
used in this study were useful in characterizing the causal agent in this food-borne epidemic.
Enterotoxigenic Escherichia coli (ETEC) strains have been
etiologically associated with diarrheal illnesses that affect
indi-viduals of all age groups and at diverse locations around the
world. In underdeveloped countries, the organisms frequently
cause diarrhea in infants and in visitors from industrialized
countries. The etiology of this cholera-like illness has been
recognized for about 20 years. In Japan, ETEC infections were
previously thought to affect primarily travelers to foreign
coun-tries. More recently, however, these infections have
increas-ingly been identified among patients with diarrhea who had not
traveled abroad (9). For the past decade, approximately 800
ETEC strains have been isolated annually from Japanese
pa-tients, and about half of these patients had traveled to foreign
countries prior to their illness (9, 24). In 1996, a large outbreak
of ETEC serotype O25:NM (nonmotile) infections occurred at
four elementary schools in Yokohama, Japan. We here report
for the first time that ETEC O25:NM isolated from food (i.e.,
tuna paste) caused this outbreak. This report also describes a
practical and effective approach to investigating food-borne
ETEC outbreaks by combining several molecular
biology-based epidemiological methods, including randomly amplified
polymorphic DNA (RAPD) analysis (5), the generation of
large-plasmid profiles by nuclease S1 treatment and
pulsed-field gel electrophoresis (PFGE) (3), and restriction fragment
length polymorphism (RFLP) analysis with several restriction
enzymes (1, 2, 6, 8, 11, 14, 18, 20).
MATERIALS AND METHODS
Description of the diarrhea outbreak. The outbreak of food-borne ETEC O25:NM infections described here involved four elementary schools (designated schools A, B, C, and D) with a total of 2,019 students and 118 staff members. Among these, 737 students and 64 school staff exhibited symptoms of ETEC infection. The symptoms included abdominal cramps (84% of the patients), diarrhea (78%), fever (59%), and vomiting (10%). Most patients sought medical attention between 16 and 18 July 1996; the peak occurrence of symptoms among the patients was on 17 July 1996. Three patients were hospitalized due to dehydration, but all patients recovered without complications. Stool specimens from 641 symptomatic patients and 791 asymptomatic patients, as well as 296 environmental specimens including 34 school lunch meals stored in refrigerators, were examined. Routine cultures of the stool specimens were negative for Sal-monella, Shigella, Campylobacter, and Yersinia. Cultures of stool specimens ob-tained from symptomatic and asymptomatic patients were positive for ETEC strains (serotype O25:NM) that produced heat-stable toxin (ST).
Epidemiological studies and statistical methods.A retrospective cohort study was carried out on 24 July 1996 to identify food items that may have been associated with an increased risk of illness in schools A, B, and C. Relative risks with 95% confidence interval (CI) values are reported as measures of associa-tion. Also, thex2test was used to evaluate the data. A P value of,0.05 was accepted as statistically significant.
Bacterial strains.The E. coli isolates used in the molecular biology-based epidemiological study (strains 1 to 14) were obtained from symptomatic and asymptomatic people, including students, teachers, and licensed cooks, and from meals that included tuna paste served at lunch in the schools. The control strains of E. coli O25:NM (strains 15 and 16), isolated from two patients with sporadic diarrhea in July 1996 whose infections were unrelated to the school outbreak, were obtained from the Division of Pathology and Bacteriology of the Kanagawa Prefectural Institute of Health. Strain 16 was isolated from a patient who had previously traveled to the Maldives, whereas strain 15 was isolated from a patient who did not have a history of travel before the onset of diarrhea. For the isolation of E. coli from stool specimens, samples were plated onto a desoxycholate hydrogen sulfide lactose agar plate and a Salmonella-Shigella agar plate (BBL prepared plate medium; Becton Dickinson Microbiology Systems, Sparks, Md.). A total of five screened colonies were picked from either plate. After the iden-tification of each strain as E. coli by standard procedures, all E. coli isolates were reidentified with a Microscan instrument (DADE International, Tokyo, Japan) with Neg Combo 3J panels. Each isolate was serologically screened for both E. coli O- and H-antigen type by the slide agglutination technique with an antiserum kit (Escherichia coli Antisera Seiken; Denka-Seiken, Tokyo, Japan).
* Corresponding author. Mailing address: Department of Pediatrics,
School of Medicine, Yokohama City University, 3-9 Fukuura,
Kana-zawa-Ku, Yokohama City, 236-0004, Japan. Phone: 81-45-787-2671.
Fax: 81-45-784-3615. E-mail: tmitsuda@med.yokohama-cu.ac.jp.
652
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Detection of heat-stable enterotoxin protein and DNA by ELISA and PCR.
Heat-stable enterotoxin from each E. coli isolate was detected with the Colist enzyme-linked immunosorbent assay (ELISA) kit (Denka-Seiken), which can detect both STIa and STIb heat-stable enterotoxins (22). For PCR analysis, genomic DNA from each E. coli strain was prepared by using the SepaGene kit (Sanko Pharmaceutical Co., Tokyo, Japan). PCR was performed with the EC Nucleotides Mix (Nippon Shoji, Ltd., Tokyo, Japan), which includes four sets of primers for the detection of the ST gene (171-bp PCR product) and the heat-labile toxin gene (132-bp PCR product) of enterotoxigenic E. coli, the verotoxin 1 and 2 gene (228 bp) of enterohemorrhagic E. coli, and the invE gene (382 bp) of enteroinvasive E. coli (10, 12). The following were the primer sequences for these genes: for ST, 59-TTTATTTCTGTATTGTCTTT-39(forward) and 59-AT TACAACACAGTTCACAG-39(reverse); for the heat-labile toxin, 59-AGCAG GTTTCCCACCGGATCACCA-39(forward) and 59-GTGCTCAGATTCTGG GTCTC-39 (reverse); for verotoxin, 59-TTTACGATAGACTTCTCGAC-39
(forward) and 59-CACATATAAATTATTTCGCTC-39(reverse); and for the invE gene, 59-ATATCTCTATTTCCAATCGCGT-39(forward) and 59-GATGG CGAGAAATTATATCCCG-39(reverse). PCRs with sample DNA (10 ng) and the appropriate primers were performed with the Ready-To-GO PCR kit (Phar-macia Biotech AB, Uppsala, Sweden). Amplification was performed with a thermal cycler (TwinBlock System Easy Cycler; Ericomp, San Diego, Calif.) programmed for 1 cycle of 2 min at 95°C; 35 cycles of 30 s at 94°C, 1 min at 45°C, and 2 min at 72°C; and 1 cycle of 7 min at 72°C. Amplified products were resolved by electrophoresis in a 4.0% agarose gel and were detected by ethidium bromide staining. For size determination, a 100-bp DNA ladder (GIBCO BRL, Rockville, Md.) was used as a molecular size marker. The ST subtype was determined by HinfI digestion of the 171-bp PCR product. The STIa PCR product contains one HinfI restriction site resulting in two fragments of 120 and 51 bp, whereas the STIb PCR product contains two restriction sites, resulting in three fragments of 93, 51, and 27 bp (17).
RAPD analysis.Genomic DNA for PCR analysis was prepared with the SepaGene kit (Sanko Pharmaceutical Co., Tokyo, Japan) and was quantified by spectrophotometry. PCR was performed with a primer with the sequence 59-T CACGATGCA-39(6) and the Ready-To-GO RAPD analysis kit (Pharmacia Biotech) which includes AmpliTaq and the Stoffel fragment of Taq DNA poly-merase. Sample DNA (10 ng) was mixed with 25 pmol of the primer in a standard PCR mixture. Amplification was performed with a thermal cycler (TwinBlock System Easy Cycler; Ericomp) programmed for 1 cycle of 4 min at 95°C; 40 cycles of 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C; and 1 cycle of 7 min at 72°C. The amplified products were resolved by electrophoresis in a 2.5% agarose gel and were detected by ethidium bromide staining. For size determination, a 100-bp DNA ladder (GIBCO BRL) was used as a molecular size marker.
Determination of plasmid profiles by nuclease S1 treatment and PFGE.To determine the molecular sizes of the bacterial plasmids accurately, plasmid profiles were established by using nuclease S1 treatment followed by PFGE as described previously (3). One colony of an ETEC isolate was grown at 37°C for 8 to 16 h in brain heart infusion broth (Oxoid, Hampshire, United Kingdom) and was then resuspended to 53109cells/ml in phosphate-buffered saline (PBS). Fifty microliters of each sample solution was used for plug preparation. After the serial extraction procedures were completed, the plugs were serially treated with proteinase K, followed by treatment with phenylmethylsulfonyl fluoride (PMSF) and washing buffer (50 mM Tris-HCl [pH 7.5]). One-eighth of each plug was rinsed with 200ml of 13nuclease S1 buffer (50 mM NaCl, 30 mM sodium acetate [pH 4.5], 5 mM ZnSO4) at room temperature for 20 min before being incubated with 1 U of Aspergillus oryzae nuclease S1 (Sigma, St. Louis, Mo.) in 200ml of 13
nuclease S1 buffer at 37°C for 45 min. The reaction was stopped by adding 10ml of 0.5 M EDTA (pH 8.0). Electrophoresis was performed on a GenePath PFGE apparatus (Bio-Rad Laboratories, Hercules, Calif.) under the gel running con-ditions used for Staphylococcus aureus in 0.53TBE (Tris-borate-EDTA) buffer for 20 h at 11°C. For size determination, a 48.5-kb bacteriophage lambda DNA ladder (FMC BioProducts, Rockland, Maine) was used as a molecular size marker. After gel electrophoresis, the gels were stained with ethidium bromide and were photographed under UV light at 302 nm.
Chromosomal RFLP analysis by PFGE.Genomic DNA for PFGE analysis was prepared by using the GenePath plug Kit 2 (Bio-Rad) by following the
manufacturer’s protocol, with some modifications (16). Disposable 100-ml scale plug molds were used to prepare agarose plugs for each sample. Overnight cultures of the ETEC isolates grown in brain heart infusion broth were resus-pended at a concentration of 53108cells/ml in PBS. Fifty microliters of each sample was used for plug preparation. After serial treatment with proteinase K, PMSF, and washing buffer (50 mM Tris-HCl [pH 7.5]), one-eighth of each plug was digested with 10 U of ApaI, SfiI, SpeI, or XbaI. The gels were processed with the CHEF-DR II PFGE apparatus (Bio-Rad) with the following electrophoresis conditions: 7 h at 170 V with an initial time of 5 s and a final time of 20 s, followed by 14 h at 170 V with an initial time of 5 s and a final time of 80 s. Electrophoresis was performed with 0.53TBE buffer at 11°C. A 48.5-kb lambda DNA ladder (FMC BioProducts) was used as a molecular size marker. After electrophoresis, the gels were stained with ethidium bromide and were photographed under UV light at 302 nm.
RESULTS
Epidemiological study.
The association between clinically
defined illness and the consumption of specific foods on 15 July
1996 in three schools (schools A, B, and C) is presented in
Table 1. Results of interviews with all people in the three
schools, which included students, school staff, and licensed
cooks, suggested that the most significant association with
ill-ness was the ingestion of tuna paste, which was served for
lunch in these schools on 15 July 1996 (relative risk, 1.79; 95%
CI
5
1.16 to 2.79; P
5
0.0001). The tuna paste contained flakes
of tuna, shredded carrots, onions, and cucumbers, as well as
mayonnaise and mustard, but only the carrots, onions, and
cucumbers were ingredients commonly delivered to these
three schools by the same wholesaler. This same wholesaler
delivered carrots, onions, and cucumbers to the four schools
affected by the outbreak. Since uncooked foodstuff was not
routinely stored for bacterial examination, we could not
exam-ine identical uncooked vegetables. We collected food samples
from six schools that were unaffected by the ETEC O25:NM
outbreak but that served the same lunch menu on the same
day. For these schools unaffected by the outbreak, the carrots,
onions, and cucumbers used to prepare the tuna paste were
delivered by wholesalers different from the one that delivered
food to schools affected by the outbreak.
[image:2.612.51.549.81.177.2]Isolation of ETEC O25:NM, phenotyping, and toxin
detec-tion.
Examination of stool, food, and environmental samples
resulted in the isolation of E. coli strains of serotype O25:NM
producing ST, i.e., ETEC O25:NM. The samples had been
obtained from students, teachers, licensed cooks, and tuna
paste. ETEC O25:NM strains were isolated from a total of 393
people, including 270 of 641 symptomatic patients tested
(42%) and 123 of 791 asymptomatic patients tested (16%).
ETEC O25:NM was detected from tuna paste from schools A,
B, and C. All other foods tested were negative for ETEC
O25:NM. A total of 14 strains from all four schools were
selected at random for further genotypic analysis. Strains 1 to
4 were obtained from school A, strains 5 to 8 were obtained
from school B, strains 9 to 11 were obtained from school C,
and strains 12 to 14 were obtained from school D. Strains 1, 5,
TABLE 1. Association of clinically defined illness and consumption of specific foods on 15 July 1996 in three schools
Food
No. of subjects Attack rate (%) for the
following subjects: Relative
risk 95% CI P value byx2test
Affected Unaffected
Exposed Unexposed Exposed Unexposed Exposed Unexposed
Bread
646
23
983
58
39.7
28.4
1.40
0.85–2.29
0.0427
Milk
642
27
989
52
39.4
34.2
1.15
0.72–1.85
0.3564
Tuna paste
642
27
948
93
40.4
22.5
1.79
1.16–2.79
0.0001
Soup
640
29
953
88
40.2
24.8
1.62
1.05–2.50
0.0010
Ice cream
252
5
636
31
28.4
13.9
2.04
0.79–5.31
0.0572
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9, 12, and 13 were isolated from symptomatic students; strains
2, 6, and 10 were obtained from asymptomatic students; strains
3, 7, and 14 were obtained from cooks; and strains 4, 8, and 11
were obtained from tuna paste. Mixed-primer PCR of strain 1
detected an ST gene of the STIb subtype which was identical to
the toxin gene of two control ETEC O25:NM strains that had
been isolated from unrelated patients with sporadic infections
(Fig. 1). The toxin genes of the other ETEC O25:NM isolates
analyzed were identical to that of strain 1 (data not shown).
RAPD analysis, plasmid profiles, and chromosomal DNA
RFLP analysis.
RAPD analysis of DNA from 14 isolates from
the outbreak and the 2 control strains produced six to seven
bands by agarose gel electrophoresis (Fig. 2). All 14
food-borne outbreak-associated strains showed identical banding
patterns. The banding pattern of strain 15 is indistinguishable
from that of food-borne outbreak-associated strains 1 to 14.
Slight differences in the banding pattern of strain 16 are
ap-parent from those of strains 1 to 15. Compared with the RAPD
patterns of strains 1 to 15, strain 16 lacked the 1,050-bp band
and presented an additional 650-bp band. The isolates were
then compared by establishing plasmid profiles by using
nucle-ase S1 digestion and PFGE (Fig. 3). All 14 isolates exhibited
identical profiles, with each strain having five large plasmids
(100, 82, 67, 43, and 20 kb). The plasmid profiles of the
spo-radic ETEC O25:NM strains, in contrast, differed somewhat
from this pattern. In particular, both sporadic strains lacked
the 100-kb plasmid. In addition, strain 16 carried an additional
170-kb plasmid. The 14 ETEC O25:NM isolates also were
compared by RFLP analysis after digestion of the
chromo-somal DNA with SpeI (Fig. 4A), XbaI (Fig. 4B), and ApaI and
SfiI (data not shown). All isolates exhibited identical RFLP
[image:3.612.95.245.69.240.2]patterns. A comparison of one of the isolates (strain 1) with the
FIG. 1. Agarose gel electrophoresis of mixed-primer PCR fragments [image:3.612.321.533.70.233.2]ob-tained from three ETEC O25:NM strains. The PCR products were analyzed without (lanes 1 to 3) and with (lane 4 to 6) subsequent digestion with HinfI. The PCR fragments were from the following strains: strain 1, isolated from a symp-tomatic student (lanes 1 and 4); strain 15, isolated from a patient with a sporadic case of ETEC infection (lanes 2 and 5); and strain 16, also from a patient with a sporadic case of ETEC infection (lanes 3 and 6). All three strains contained an ST gene, characterized by a 171-bp fragment (lanes 1 to 3), of subtype Ib (characterized by three fragments of 93, 51, and 27 bp in lanes 4 to 6). Lanes M, molecular size marker (100-bp DNA ladder).
FIG. 2. RAPD analysis of 14 isolates from the ETEC O25:NM outbreak and 2 control strains. The lane numbers are the same as the strain numbers. Strains 1 to 4 were isolated from school A, strains 5 to 8 were from school B, strains 9 to 11 were from school C, and strains 12 to 14 were from school D. Strains 1, 5, 9, 12, and 13 were obtained from symptomatic students; strains 2, 6, and 10 were obtained from asymptomatic students; strains 3, 7, and 14 were from licensed cooks; and strains 4, 8, and 11 were from tuna paste. Strains 15 and 16 were obtained from two patients with sporadic cases of ETEC O25:NM infection. Lane M, molecular size marker (100-bp DNA ladder).
FIG. 3. Plasmid profiles of ETEC O25:NM isolates, determined by nuclease S1 treatment followed by PFGE analysis. For a description of strains 1 to 16 (lanes 1 to 16, respectively), see the legend to Fig. 2. Lanes M, molecular size marker (48.5 kb lambda DNA ladder).
FIG. 4. Chromosomal analysis of 14 ETEC O25:NM isolates by PFGE. (A) SpeI digestion patterns for the 14 isolates; (B) XbaI digestion patterns for the 14 isolates. For a description of strains 1 to 14 (lanes 1 to 14, respectively), see the legend to Fig. 2. Lanes M, molecular size marker (48.5-kb lambda DNA ladder).
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[image:3.612.311.547.496.689.2]two sporadic ETEC O25:NM isolates (strains 15 and 16)
dem-onstrated that all four restriction enzymes tested consistently
produced clearly distinguishable RFLP patterns for each of the
three strains (Fig. 5).
DISCUSSION
During the past 20 years, between 700 and 9,000 cases of
food poisoning due to E. coli have annually been reported in
Japan (9). In addition, occasional large outbreaks of ETEC
infections have occurred since 1967. ETEC has also been
iden-tified as the causative agent of illness in about 20% of patients
who developed diarrhea after traveling to foreign countries
(24). The ETEC serotypes most commonly associated with
large outbreaks in Japan are O6:NM and O6:H16, which
pro-duce both ST and heat-labile toxin, as well as O27:H7, O27:
H20, and O159:H20, which produce only ST. ETEC serotypes
O8, O25, O148, and O167 have also been reported in the
literature (4, 9, 23, 24). To our knowledge, this is the first
report of a large food-borne outbreak caused by ETEC
O25:NM in Japan. In the United States, in contrast, 13
out-breaks of gastroenteritis caused by ETEC have been reported
to the Centers for Disease Control and Prevention since 1975,
9 of which were food borne. Reported sources of the infections
included water contaminated with human sewage (19),
in-fected food handlers (21), dairy products such as semisoft
cheeses (13), and salads containing raw vegetables (15).
In 1996, large outbreaks of verotoxin-producing E. coli
(en-terohemorrhagic E. coli; EHEC) O157:H7 infection occurred
among students in several Japanese schools and day-care
cen-ters. The outbreaks affected 9,451 patients and caused 12
deaths and were thought to be caused by contaminated
lunches. In March 1997 there was another small outbreak of
EHEC O157:H7 in which the bacteria were tracked to
white-radish sprouts consumed by patients. In contrast to these
EHEC outbreaks, the ETEC strains causing the outbreak
de-scribed in this report produced only ST but no verotoxin.
Consequently, no cases of severe renal failure, brain damage,
or death were reported. Lunch is usually served at elementary
schools in Japan, and each student is served the same food.
Therefore, once the food served at lunch is contaminated with
a pathogen, a food-borne outbreak of illness can easily occur.
Efforts are now under way to improve the school lunch service
system and thereby avoid further food-borne bacterial
out-breaks. These efforts must take into consideration the
recom-mendations of the hazard analysis critical control point method
(7).
This study demonstrates that practical molecular
biology-based epidemiological approaches can be used to identify the
source and route of infection of food-borne ETEC poisoning
in the community. PFGE analysis is considered the most
reli-able and practical tool for molecular biology-based
epidemio-logical analyses of bacterial infections (1–3, 6, 8, 11, 14, 18, 20).
As demonstrated here, additional RAPD analysis (5, 6) and
the determination of plasmid profiles by nuclease S1 treatment
and PFGE (3) can increase the accuracy of these analyses.
PCR-based RAPD analysis is increasingly being used for
mo-lecular biology-based epidemiological applications, such as
subtyping of E. coli isolates. Although RAPD analysis can
provide RFLP data very rapidly, the resolution and
reproduc-ibility of these data are somewhat limited. Nevertheless,
RAPD analysis can provide valuable preliminary molecular
biology-based epidemiological information during bacterial
outbreaks while more time-consuming PFGE analyses are
per-formed. The analysis of plasmid profiles by conventional
elec-trophoresis has resolution problems including the instability of
the plasmids. However, nuclease S1 treatment and PFGE have
improved the resolution in analyzing large plasmids compared
with the resolution of conventional procedures. The major
limitation of PFGE analysis includes the requirements for
technical skill and an expensive apparatus and the long
dura-tion until the compledura-tion of the analysis (4 to 7 days).
More-over, the analysis of PFGE-RFLP data may be difficult. For
example, after isolating the bacterial strain responsible for an
epidemic, this strain must be compared to strains involved in
other outbreaks. Because PFGE conditions vary, however, it
may be difficult to compare PFGE data from different studies.
It is therefore necessary to establish databases that contain
reliable RFLP information on each bacterial strain obtained by
molecular biology-based methods involving several restriction
enzymes. Such information will allow international
collabora-tions for identifying the causative agents involved in large
out-breaks until rapid, automated partial or full genomic
sequenc-ing techniques replace the current RFLP technologies. In the
ETEC O25:NM outbreak described here, we used several
mo-lecular biology-based epidemiological techniques. PFGE is
one of the most informative tools for characterizing strains, but
it is not a perfect technique. To avoid misunderstanding the
molecular biology-based epidemiological results, we
recom-mend analysis of epidemic strains not only by PFGE but also
by other supportive techniques in the case of a similar
out-break.
ACKNOWLEDGMENTS
[image:4.612.78.265.68.319.2]We thank all the health care providers who dedicated their medical
service to the patients involved in the ETEC O25:NM outbreak
de-scribed here. We also thank Shiro Yamai, Department of Bacteriology
and Pathology, Kanagawa Prefectural Public Health Laboratory,
FIG. 5. Comparison of the chromosomal restriction patterns of three ETECO25:NM strains by PFGE. Strain 1 (lanes 1, 4, 7, and 10) was isolated from a symptomatic student. Strain 15 (lanes 2, 5, 8, and 11) and strain 16 (lanes 3, 6, 9, and 12) were isolated from two patients with sporadic cases of ETEC O25:NM infection. The isolates were digested with ApaI (lanes 1 to 3), SfiI (lanes 4 to 6), SpeI (lanes 7 to 9), and XbaI (lanes 10 to 12). Lanes M, molecular size marker (48.5-kb lambda DNA ladder).
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Yokohama City, Japan, for generously providing the two strains of
ETEC O25:NM derived from patients with sporadic diarrhea.
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