Jpn. J. Infect. Dis., 54, 140‑143, 2001

Short Communication

Phage Typlng and DNA‑Based Comparison of Strains of EnterohemorrhaglC
Escherichia coli 0 1 57 from Apparently Sporadic Infections

in Osaka City, Japan, 1996
Yoshikazu Nishikawa*, Atsushi Hase, Jun Ogasawara, Tom Cheastyl, Geraldine A. Willshawl,
Henry 良. Smithl, Ybhichi Tatsumi2 and Akira Yasukawa

Department ofEpidemiology, Osaka City Institute of Public Health and Environmental Sciences,Tennouji‑ku, Osaka 543‑0026,
JLaboratofy OfEnteric Pathogens, Central Public Health Laboratory,London NW9 5HT United Kingdom and2Disease Prevention Department, Public Health Division,Environmental and Public Health Bureau, Osaka Municipal Government,
Kita‑ku, Osaka 530‑0005, Japan(Received June 13, 2001. AcceptedAugust 8, 2001)

SUMMARY : A marked increase in sporadic cases of enteritis due to enterohemorrhagic Escherichia colt serogroup
O157 occu汀ed in Osaka City, Japan, during 1996. Tb elucidate why the number of cases had increased, theisolates were classified using phage typlng, random amplified polymorphic DNA analysis, and pulsed‑field gelelectrophoresis (PFGE). Fifty‑seven percent of the isolates (I 05/1 84) belonged to the same phage type (PT‑32)
and gave the same PFGE pattem; the clone had been isolated during a 3‑Week period, with a peak on July 15. 1t

was concluded that the majorityof the cases identified in July 1 996 formed an outbreak, although epidemiologicallinks to a possible common source were not established. The possibility that this outbreak was part of a huge

reglOnal outbreak including children at prlmary schools in Sakai City was discussed.

The first outbreak due to enterohemorrhagic Escherichiation was tested by reversed‑passive latex agglutination withcolt (EHEC) 0157:H7 in Japan occurred in September 1990

the Verotox F kit (Denka Seiken Co., Ltd., Tokyo) according(1). This was fわllowed by a second outbreak in a day‑careto the manufacturer's instructions. Polymerase chain reac‑center in Osaka City in 1991 ; 161 individuals were identi丘ed astion (PCR) amplification yas used to test for the presence ofcases or healthy camiers. However, between 1992and 1995 Only
four sporadic cases were reported in Osaka City. From May

six genes. Two sets ofprlmerS (Cat. No. SOO6 and SOO7,Takara C0., Otsu) wereused according to the manufacturer's

1996, Outbreaks due to EHEC 0 I 57 were reported throughout

instructions to distinguish stxl and stx2.

Japan･ Then a massive outbreak occurred in the prlmary

Isolates were phage‑typed in the Laboratory or Entericschools in Sakai City, Osaka Prefecture (2). Sakai Cityborders

Pathogens, Central Public Health Laboratory, London, England,

Osaka City, and the number of sporadic cases in Osaka Cityuslng the extended scheme ofKhakhria et al., compnslng 1 6

also increased suddenly in July 1996.

typing phages (6)･ The scheme recognizes 90 phage types

The aim of this study was to investlgate in detail the isolates(PTs) (personal communication R. Khakhria).

from the apparently sporadic cases of EHEC 01 57 infectionin Osaka City, uslng phenotyplC and DNA‑based methods,
and to dete‑ine whether any of the cases constituted part of

PFGE was perf♭med by the method oHzumiya et al. (7,8)

at the National Institute of Infectious Diseases, Tokyo (NIID).
Xbal was used as the restriction endonuclease･ DNA bandinga bigger plCture･ The advent ormolecular typlng StrategleS
has benefited epidemiologlCal investlgations, by way, forpattems ofPFGE were classified as described by Izumlya etal. (7), according to the criteria ofNIID. In brief, each DNA

example, of the use of restriction fragment length polymor‑

pattem was classified into three parts depending on the size

phisms (3), pulsed‑field gel electrophoresis (PFGE)(4,5), andrandom amplified polymorphic DNA analysis (RAPD) (2).

of the DNA bands, then the ladder included in each part wasWe chose phage typing as an established phenotypic method,of standard strains designated by NIID. Pattems that did notanalyzed in comparison with that in the corresponding partand PFGE and RAPD as molecular methods fわr the analysュscorrespond to the standards in three or more bands wereof isolates from Osaka City. The effectiveness of these threeclassified as novel patterns. RAPD was performed by themethods is also discussed.

method ofWatanabe et al. (2). Classification of the pattems
was performed according to the criteria of NIID (Manual forE. colt 0 I 57 strains isolated from 1 85 individuals including

47 healthy carriers in Osaka City from January to December
1996 were examined. Ofthe 138 patients, 98 (71%) reported

detection and analysts Of EHEC 01 57, provided at a technicalseminar given by NIID on 12 May 1997).
Isolates were assigned to 10 different PTs (PT‑1, 2, 4, 8,

that their symptoms started in July. Shiga toxin (Stx) produc‑

14, 21, 23, 31, 32, 34). PT‑32 was the predominant type (1 1 1/Osaka 55818585, Japan. Tel and Fax: +8 1‑6‑6605‑2883, E‑mail:185; 60%), followedby PT‑8 (21/185; 11%), PT121 (ll/185;6%), PT‑4 (8/185; 4%), PT‑14 (6/185; 3%), PTll (5/185; 3%),PTl34 (4/185; 2%), PTl31 (2/185), PT‑2 (l/185), and PT‑23

nisikawa@life.osaka‑cu.ac.jp(1/185). Fifteen strains王eaCted with the phages but did塁Ot*Corresponding author: Present address: Faculty of Human LifTe

Science, Osaka CityUniversity, Sugimoto 3‑3‑ I 38, Sumiyoshi‑ku,

140

SaSe0‑0LaqUunN

ad^}aBeエdFig. 1. Date of onset of confirmed cases and the phagetypc of each isolate. Each PT‑32 strain, isolated from patients who

became ill on 18 June, 14, or 28 July, was distinguished fromthc dominant type based on the PFGE pattems.

Table 1. Discrimination by phage typing OfEHEC 01 57 isolates

旦Onformto a recognized type (RDNC). Eight different pattems

that possessed the same pulsed‑field gel electrophorcsis (PFGE)

were recognized among the 15 RDNC strains. The date ofpattem

onset of patients with the prevalent type of PT‑32 ranged fTrom

PFGE Phage Type RAPD No. of isolates

8 to 29 July, and showed a peak on 15 July (Fig. 1).

PFGE gave greater strain discrimination than any otherlaCX13CX124

method employed, and the 185 strains showed 33 different

21PFGE pattems. However, Some isolates showlng the same
PFGE pattern were divided into several different PTs, andvice versa (Table 1 ). Thus, by combining PFGE pattems andlIaIIbI

11111

Hc

14lIe

68

phagetyping, isolates yere classified into 42 different groups･IIc

14One hundred and five isolates (57%), all of which produced日￠32

both Stxl and Stx2, belonged to Plt32 and gave the sameR̲DNC

PFGE pattem (ⅠIa IIb 1); this type was the same strain as that1Iallb

CX3

in the outbreak in Sakai. However, six additional PT‑32 strains
were distinguished from the dominant type by their PFGE

*IIb

CX4patterns. The first PT‑32 strain, which was isolated from apatient who became ill on 18 June (Fig. 1), gave a pattern(CXI IIb CXl) different from that of the 105 PT‑32 isolates.
Similarly, two strains, isolated from patients whose symptoms

lIIc

CXICX2llc

31Ile

11Hc

21411e121IIc34

IJjRDNC

started on 1 4 and 28 July, respectively, were also distinguished

105IIc32lュa111c14

123IIjI*RDNC:旦eactswitll the phages but does旦Ot ￡Onform to a recognizedkom the dominanttype by PFGE. The other three strains weretypc･from healthy carriers. Seventeen strains belonglng tO PTl8
were isolated舟om patients or healthy camiers in an outbreakthat occurred in a day‑care center in December (Fig. 1). TheseAlthough phage typlng lS not aS discriminatory as PFGE,

strains were distinguishable by PFGE from the other four
strains ofPT‑8 isolated during July and August.

it is a rapid and economic technique (5), and a useful first

The 185 isolates were assigned to five types (I, IIe, lIf, lIj,

line‑screenlng method. In this study, phage typlng led to iden‑lIm) by RAPD pattems･ Although RAPD used in this study

tifying a widespread outbreak caused by EHEC 01 57 PT‑32

did not contribute to further discrimination among strainsin Osaka City (Fig. 1), despite the fact that most of the cases

showlng the same PFGE pattem, strains were classi丘ed into

had orlglnally been reported as sporadic cases. We suggest

20 different groups by combining phage typlng and RAPD.
This study showed that the use ofphage typlng and PFGE

that fresh food rather than preserved foods may have been

the vehicle, since EHEC 0157 PT‑32 was isolated only for 3
weeks with a peak on 15 July. This was 4 days later than theprovided good discrimination f♭r the study of EHEC 0 1 57.

141

City institute of Public Health and Environmental Sciences

start of the massive outbreak of EHEC 0157 infections thatoccurred in the primary schools in Sakai City (2). lf frozen

for their technical assistance throughout the investlgation offoods or other items suitable for preservation were involved,

the outbreak.

cases would have appeared over a longer period in a scatteredmanner. However, possible food vehicles linking the outbreaks

REFERENCES

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