Jpn. J. Infect. Dis., 55, 19‑22, 2002

Short Communication

High Genomic Diverslty Of Enterohemorrhagic Escherichia colilsolates in Japan and Its Applicability fわr the Detectionof Di軌se Outbreak
Jun TeraJlma, Hidemasa Izumlya, Sunao Iyoda, Kazumichi Tamuraand Haruo Watanabe*
Department of Bacteriology, National Institute oflnfectious Diseases,Toyama 1‑23‑1, Shinjuku‑ku, Too,0 16218640, Japan(Received December 4, 2001. Accepted Febmary 19, 2002)

SUMMARY: Genotyping of 1,102 enterohemorrhagic Escherichia colt isolates by the use of pulsed‑field gelelectrophoresis (PFGE) carried out from January to November 2000 has revealed the high genomic diversity ofthese isolates in Japan. By combining the results of genotyplng Of the isolates with the information from otherepidemiologlCal investlgations of the cases, we identified a diffuse outbreak in Japan in the year 2000 that

seemed to be sporadic but was actually linked. Isolateswith only the Shiga toxin 2 gene derived from patient
specimens and the contaminated food involved in this difRISe Outbreak showed an indistinguishable PFGE profileand the same phage type･ Based on the diverslty Of genotypes among the isolates ofenterohemorrhagic E. colt
O157:H7/‑ in Japan, We suggest the presence of a few other possible di仇ISe Outbreaks due to the organisms,

showing indistinguishable genotypes.

The outbreak of enterohemorrhagic Escherichia colithat only appeared sporadic but were in fact linked to one(EHEC) 0157:H7 infection in 1996 that affected more thananother･ In 1998, analyses uslng PFGE and phage typlng

1 5,000 persons and caused the death of at least five children

combined with other epidemiologlCal data even山ally revealed

has led to the establishment or several measures fわr thea di恥se outbreak involving 49 symptomatic patients and 1 3control and prevention ofEHEC infection in Japan (I). EHEC

asymptomatic carriers dispersed in seven prefectures andinfection was listed as a specially designated communicablemunicipalities in Japan (7). It is urgent and important to havedisease and notification of both symptomatic and asymptomatica functionlng Surveillance network for the detection ofcases has become compulsory since August of the same year.
The disease was classified as a category III infectiousfoodborne diseases based on reliable and established methods(8‑ 1 0), because contaminations of mass‑produced or widelydisease under the Law ConcemIng the Prevention of lnfec‑

distributed fわod products are apparently emerglng aS a Serious

tious Diseases and Medical Care of Patients with Infections

threat to public health (I 1).

We report here that there exists genomic diverslty among

enacted inApril of 1999 in Japan (2).
Laboratory‑based surveillance, especially the molecular

the isolates ofEHEC in Japan and that the results orsubtyping

subtyping of EHEC isolates, is an established procedure thathas received increased support a洗er multiple large‑scaleof EHEC isolates, combinedwith other information fromepidemiologlCal investlgations, have enabled us to link widely

ouぬreaks occu汀ed in 1996 in Japan. Among several moleculardispersed outbreaks and sporadic cases to one another.

subtyplng techniques, we applied pulsed‑field gel electro‑Since 1996, we have been coHectlng Clinical isolates ofphoresis (PFGE) to obtain further discrimination of isolates

EHEC from each munlCIPal public health institute in Japan.
PFGE analysis has revealed 1,483 isolates ofEHEC 0157 inrequired fわr the investlgation of the outbreak; Other methods

1998, 1,605 in 1999, and 1,102 in 2000 as of November 17.
Fifty‑seven 0157:H‑ isolates were included among I,102
0 1 57:H7/‑. Biochemical identification of specimens that weresent to our instihte was reconfimed by standard tests such as

included evaluation of amplified‑fragment length polymor‑phism (AFLP) analysis (3) and random amplirlCation ofpolymorphic DNA methods.

PFGE analysis has proven to be useful in discriminatlng
isolates of the same serotype such as 0157:H7. This method

growth on MacConkey sorbitol agar with cefixime‑potassium

has led to an understanding of仇e prevalence throughout Japantellurite medium (CT‑SMAC) including serotyping fわrof the divergent clones ofO1 57:H7 (4). Furthemore, because

somatic and nagellar antlgen identification. Tn terms of Shiga

bacteriophage typlng lS easy tO perfbm and has already been

toxin production, 57% of the isolates were positive for bothstxl and stx2, 41% were positive only for stx2, and 2% wereintemationally standardized, We included phage typlng aS a

subtyping method, in addition to PFGEand strtyping to obtainmaximum strain discrimination; this approach has enhanced

positive only for stxl A All of the specimens were subjected toPFGE analysts according to a previously described method

the reliability of the analysis (5,6).

(4)･ PFGE profiles were recorded as digitalized data forwith infわ‑ation about other epidemiologlCal investlgations

further analysis by a computer software, GelCompar II fTrom
Applied Maths (Kortrijk, Belgium). As for the isolates ofof the cases, it became possible to detect diffuse outbreaks

EHEC 0157 from the indicated period in 2000, we have*Co汀eSpOnding author: Fax: +8ト3‑5285‑1 1 7 1 , E‑mail: hanlWata

were derived from 756 incidents in Japan during that period.

By combining the results of the subtyping of these isolates

analyzed a total of 1,102 ofEHEC 0157:H7/‑ isolates that
The PFGE pattems showed high diverslty Ofthese isolates,@nih.goJp19lad N凸dFig･ 1･ The dendrogram ofPFGE profiles ofEHEC 0157 isolates･ A total of 1,102 isolates ofO157 Were analyzed by PFGE and their profiles wereanalyzed to obtain the dendrogram using the computer software GelCompar II version 2･0 (Applied Maths)･ The isolates belonging to each cluster?reindicated as either DO, a diffuse outbreak, or as PD, one to four possible dimlSe Outbreaks. Dice coefrlCicnt was chosen to calculate the similarlty
matrix and the unweighted palr group metllOd using arithmetic averages was selected to specifythe clusterlng method.

20as shown in Figure 1.

By analyzlng 1,102 isolates or EHEC 0157:H7/‑, We
identified several isolates with indistinguishable PFGE

▲DO:Sか2,PTatyplCal

pattem, which may indicate epidemiologlCal linkage･ In fact,
one event that included three sporadic cases was dispersed

OpD2=slxl'stx2,PT21

into two widely separated prefectures; a patient was reported

LpD3:Sか2,PT2

in Wakayama Prefecture in August and two other patients

EjpD4:slx1+Sか2,PT14●pD1.=slx1.slx2,PT1/atypicalwere reported in Kanagawa Prefecture in September (Fig. 2,DO). EHEC 0157:H7 infected all three patients. Epidemi0‑
10glCal investlgation revealed that they consumed grilled beef

slices (yaki‑niku) at different steak houses b,aki‑niku restau‑rant). Strains ofO157:H7 were also isolated from a piece ofmeat unserved and le氏 at a restaurant in Kanagawa; this meat

was from the same lot of an unopened frozen stock of beef

imported from the United States･ The same brand of imported

frozen beef was used at the restaurant in Wakayama whereanother patient was reported to consume yaki‑niku. All of theisolates were of the same phage type, although it was atypicaland had only the stx2 gene. The PFGE profiles of the isolates
showed a similarlty Of lOO% in a dendrogram analysts Of

the profiles conducted by GelCompar II. We confirmed
indistinguishable PFGE profiles of the isolates by uslng threedifferent restriction endonucleases (XbaI, BlnI, and Spel).
Based on the genomic diverslty Of 0157:H7/‑ isolates
revealed by PFGE analysts, We Suggest that fわur more
possible di軌se outbreaks among 756 incidents occu汀ed,

whereby the isolates showed the same phage type, the same

six subtype, and identical PFGE profiles. Depending on thedate of isolation, strains ofpossible difRISe Outbreaks might beFig. 2. A map of Japanese prefectures showlng the distribution ofa

separately analyzed on different PFGE gels, and therefore,
the percentage of similarity on the dendrogram among Isolates

difRISe Outbreak and several other diffuse outbreaksin2000. Sporadic
cases and outbreaks compnslng a diffuse outbreak are represented

within the same cluster was not always lOO% when isolates

by the same symbol. Isolates &om the same incident showed in‑were analyzed by the computer software. However, when we

distinguishable PFGE proflles. stxl and stx2 represent Shiga toxinsl and 2, respectively. Atypical indicates an unclassified lysis pattern.

reanalyzed profiles belonglng tO the same cluster on the samePT, phagc type.

gel, they were actually indistinguishable斤om one another.

The丘rst possible di軌se outbreak (PD 1 ) Occurred between

January and August 2000, and consisted ornine incidents

type of these isolates was PT2 1. The isolates clustered withinfrom five dispersed prefectures; this outbreak included six

a similarlty Of 96.3% in the dendrogram.

sporadic cases and two outbreaks. One of these was at a house

The third outbreak (PD3) involved eight incidents in thefor the elderly, where four symptomatic and four asympt0‑

eastem and westernparts of Japan: Tokyo Metropohtan City,

matic patients were reported, and the other outbreak was at a

matic patients were involved･ Neither documentations nor

Kanagawa Prefecture, Ibaragi Prefecture, and Fukuoka
Prefecture (Fig. 2, PD3). PD3 included two outbreaks inKanagawa and one in Fukuoka. One of the outbreaks was

daycare center, where three symptomatic and two asympto一isolates orthe source of contamination regarding each case werereported in June in a house fわr the elderly who were seen atreported. These isolates fbmed a cluster in the dendrogramofPFGE profiles. The similarlty Orthis cluster was 92.2%

the hospital within the facility in Kanagawa. We received 1 Iand the cluster included an isolate from the outbreak at thepatients; a specific contaminated food was never identified

isolates from the patients as well as from asymptomatic

house for the elderly; one band difference in the PFGE profilein this outbreak. Another outbreak in Kanagawa involved twowas obseⅣed. The phage type of these isolates was PTl, Withpatients who had eaten yaki‑niku at yaki‑niku restaurant in

the exceptlOn Of the abovementioned isolate fTrom the outbreakAugust. All six patients involved in another outbreak inat the house for the elderly that was of an atypICal phage

Fukuoka had traveled to Korea on the same tour for four days

type. The similarityofthe profiles of these isolates was 94.7%

in the middle of September and had eaten an ethnic dish ofby the exclusion of this isolate (Fig. 1, PDl).
The second possible diffuse outbreak (PD2) involved nine

beef in common in Korea. Isolates derived from sporadic cases

incidents dispersed among seven prefectures in the westernclustered with a simiIarlty Of 90.8% in the dendrogram. The

part of Japan. PD2 included two outbreaks in Fukuoka

relative lack or similarlty Was probably due to the various

Prefecture; one in a daycare center, and the other at a family

PFGEruns needed to identify the larger number of isolates.

barbecue party (Fig. 2, PD2)･ The cases were reported fromThe phage type ofall of these isolates was PT2.

from the end of July to themiddle of September were

August 18 to September 27. Four cases were related to theThe fわurth outbreak (PD4) involved eight independent

consumptlOn Ofyaki‑niku though the orlglnal source of beef

was unknown. There were 15 isolates from the nine incidents

incidents. PD4 included a yaki‑niku associated outbreak in
Kanagawa as well as other sporadic cases; all of these casesthat showed indistinguishable PFGE patterns, except for one

were reported in the westem part of Japan, except fわr one inisolatewith a slngle band difference from the isolate derived

GifuPrefecture,from the end of June to the end of September

from the outbreak at the family barbecue party. The phage(Fig. 2, PD4). All ten isolates什om these cases showed a21

similarityof 95.0% in the dendrogram, and all were ofphage
type 14.

E., Tucker, B･ and Watanabe, H. (1999): Evaluation ofAFLP, a high‑resolution DNA fingerprlntlng method, as

There appears to be a relatively high number of dimISe Out‑a tool for molecular subtyplng Of enterohemorrhagic

breaks depicted in Figure 1 , apart丘omthe four abovementioned

Escherichia coli 0 1 57:H7 isolates. Microbiol. Immunol.,43, 803‑806.

outbreaks, because a few clusters with relatively highthat the isolates belonglng tO the clusters with relatively high4･ Izumlya, H., TeraJlma, J., Wada, A., Inagaki, Y., Itoh, K.
I., Tamura, K. and Watanabe, H. (1997): Molecularsfmilarityin the dendrogram may represent a d胤se outbreak,typlng Of enterohemorrhagic Escherichia coli 01 57:H7

similarity in the dendrogram were observed. It is possible

slnCe they differed by only one or two bands in the PFGE

isolates in Japan by uslng Pulsed‑field gel electrophoresis.

J. Clin. Microbio1., 35, 1675‑1680.
5. Izumlya, H., Masuda, T., Ahmed, R., Khakhria, R., Wada,A., TeraJlma, J., Itoh, K., Johnson, W. M., Konuma, H.,
Shinagawa, K., Tamura, K. and Watanabe, H. (1998):
Combined use of bacteriophagetyping and pulsed‑field

pro丘les and also because they were of the same phage type andsix subtype. However, we did not include these cases among
the di軌se outbreaks due to a lack of thorough documenta‑tion from the epidemiologlCal investigations.

We established in this report that the EHEC 0157 strains
had a high genomic diverslty. The present丘ndings suggest

gel electrophoresis in the epidemiologlCal analysts Of
Japanese isolates of enterohemorrhagic Escherichia colithat it is important to make note of close epidemiologlCal

the capacity to find di凪ISe Outbreaks. It is suggested that more

O157:H7. Microbiol. Immuno1., 42, 515‑519.
6･ TeraJlma, J･, Izumlya, H., Wada, A., Tamura, K. andWatanabe, H. (2000): Molecular epidemiological inves‑prompt recognltlOn Of such cases be accomplished by reducing

tlgation of enterohaemorrhagic Escherichia coli isolates

linkage among cases showlng high values of similarity indendrogram ofPFGE profiles. The system described here hasin Japan. ∫. Appl. Microbio1., 88, 99S‑105S.

the time required to obtain decisive results a洗er an initialcase report. An intemational collaborative effort should be

7. TeraJlma, ∫., Izumlya, H., Iyoda, S., Tamura, K. andmade to block the dissemination of pathogen‑ContaminatedWatanabe, H. (1 999): Detection of a multi‑prefectual E.

food products across national borders to prevent foodbome

coli 01 57:H7 Outbreak caused by contaminated Ikura‑sushi ingestion. Jpn. J. Infect. Dis., 52, 52‑53.

diseases.

8･ Smith, D., Willshaw, G., Stanley, ∫. andAmold, C. (2000):

Genotyping of verocytotoxin‑producing Escherich ia colt

ACKNOWLEDGMENTS

O157: comparison ofisolates ofa prevalent phage type

We thank all of the investigators from a total of more than

byfluorescent amplifi ed‑fragment length polymorphism

80 prefectural and municipal public health institutes and pub‑and pulsed‑field gel electrophoresis analyses. J. Clin.

Microbio1., 38, 4616‑4620.

lic health centers in Japan fわr providing us with samples andinfbrmation･ We also thank Mrs. SumlyO Asahara and Mr.

9･ Stephenson, ∫, (1997): New approaches fわr detecting andTakahiro Takei for their help in conductlng the PFGE analysis.

curtailing foodbome microbial infections. JAMA, 277,

This work was supported by a grant from the Ministry ofHealth, Labour and Welfare of Japan.

10･ Willshaw, G.A., Smith, H, R., Cheasty, T., Wall, P. G. and1339‑1340.

Rowe, B. ( 1997): Vero cytotoxin‑producing Escherichia

coli 0157 Outbreaks in England and Wales, 1995: pheno‑REFERENCES

typIC methods and genotypIC Subtyplng. Emerg. Infect.

1 ･ Watanabe, H., Wada, A., Inagaki, Y., Itoh, K. and Tamura,Dis., 3, 561‑565.

K. ( 1 996): Outbreaks ofenterohaemorrhagic Escherichia

11･ Pebody, R･ G･, Furtado, C., Rqjas, A., McCarthy, N.,coli 01 57:H7 infection by two different genotype strains

Nylen, G･, Ruutu, P., Leino, T･, Chalmers, R･, de Jong,in Japan, 1996. Lancet, 348, 831‑832.

B., Donnelly, M., Fisher, Ⅰ., Gilham, C., Graverson,し.,2. National Institute of Infectious Diseases and Infectious

Cheasty, T., Willshaw, G., Navarro, M., Salmon, R.,
Leinikki, P･, Wall, P. and Bartlett, C. (1999): An inter‑

Diseases Control Divission, Ministry of Health andWelfare (2000): The status of enterohemorrhagic Esche‑richia coli infection in Japan, 19981March 2000. Infect.

nationaI outbreak of Vero cytotoxin‑producing Esche‑richia colt 0 1 57 infection amongst tourists; a challenge

Agents Surveillance Rep,, 21, 92'193'.

for the European infectious disease surveillance network.

Epidemiol. Infect., 123, 217‑223.

3･ Iyoda, S., Wada, A., Weller, ∫., Flood, S. ∫., Schreiber,22