Jpn. J. Infect. Dis., 54, 2001

Laboratory and Epidemiology Communications

Phylogenetic Analysis of Genotypic Variations ofEscherichia coli 0 1 57:H7
Isolates from Sporadic Infections by Using Pulsed‑Field Gel

Electrophoresis from March 1999 to February 2001
in Hyogo Prefecture
Tomohiro Oshibe, Hidetaka Tsuji and Kokichi Hamada*Division of Microbiology, Hyogo Prefectural Institute of Public Health,Arata‑cho 2‑1‑29, Hyogo‑ku, Kobe 652‑0032
Communicated by Takashi Kawamura(Accepted September 19, 2001)

Entero‑heヮorragic Escherichia coli (EHEC) is a highlyvariationsin 1997‑ 1998 isolates (4). We report here the genetic

virulent enterlC Pathogen and, since 1982, it has been founddiverslty and phylogenetic analysts Of the isolates in 1 999‑

worldwide including the United States, Europe, and Japan

early 2001. Eightyisolates aom human feces of sporadic and(I). E. coli 0157:H7 has been the most prevalent serotype

Pmall outbreak cases (Table) and one isolate from well water

Implicated in a familial transmission were analyzed･

among them (1). A large‑scale outbreak occu汀ed in 1996 inSakai City in Osaka Prefecture (2). The EHEC epidemic has

The isolates were examined for genes encoding verotoxin

continued ever.since (3)･ Even in the early epidemics, its high(VT) by polymerase chain reaction (PCR) using EVS‑ (forVTl) and EVT‑ (for VT2) primers purchased from Takara
Shuzo Co., Ltd., (Kyoto) (5). Six isolates were positive forgenetic diverslty Was revealed by pulsed‑field gel electro‑phoresis (PFGE) (2). We previously reported such genotypic

Table･ Infectional cases in small groups by Escherichia colt O157:H7 from 1999 to early 2001 in Hyogo Prefecture and some characters of the isolatesI
case

12No.

SamplingMarchJune

11date999

999

Relationships

famiIyfamilyamong

patients

members

members

Patient

No･Strain2Al (male, 4 y)
A2 (female, 67 y)
A3 (female, 5 y)

except A6, a舟iend ofA3 andA4 (male, 1 y)

A7, well wat￠㍉

A5 (male, 35 y)
A6 (female, 6 y)

A7A8 (male, 8 y)
3July 1999

A12,A13 andA14 were &omthe

A9 (male, 3 y)
AIO (male, 2 y)same family.

All (male,2y)

day nursery children except A14.

A12 (male, 2 y)
A13 (female, 4 y)
A14 (female, 7 y)
4August 1 999

fami1y members

5Scptember 1999

cmployees in a high school canteen

60ctober 1 999

cmployecs in a kindergarten canteen

A15 (female, 57 y)
A16 (male, 4 y)
A17 (female, 60 y)
A18 (female, 65 y)
A19 (female, 32 y)

A20 (female, 52 y)7

Apri1 2000

famiIy members

Bl (female, 62 y)
B2 (male, 31 y)

8July 2000

family members

B3 (male, 6 y)
B4 (male, 4 y)

9August 2000

fami1y members

B5 (female, 44 y)B6(male, 1 y)
1 0 August 2000

famiIy members

B7 (male, 2 y)

B8 (female, 5 y)
1 1 October 2000

famiIy members

B9 (female, 9 y)
BIO (female, 4 y)*Co汀eSpOnding author: Fax: +8ト78‑53 1 ‑7080

162

VT3type'98‑E.086'98‑E.087'99‑E.004

producedSM, TC
SM, TCV V V V V VT T T T T T 2 2 2 1 1 1

N N N N N N0 0 0 0 0 0n n n n n ne lC ら ら e e'99‑E.006'99‑E.007'99lE.008'99‑E.009'99‑E.01 1'99‑E.012'99‑B.013'99‑E.014'99‑E.015'99‑E.016'99‑E.025'99‑E.030'99‑E.03 1'99‑E.033'99‑E.034'00‑E.001'00‑E.002'00‑E.049'00‑E.050
一oo‑E.055

loo‑E.056
一oo‑E.057'00▲E.058'00‑E.068†oo‑E.069

resistance4VTl,2VTl,2'99‑E.005'99‑E.024

Drug

V V V V V V V VT T T T T T T T ‑1 2 2 2 2 2 2 22 2

Jpn. J. Infect. Dis., 54, 2001
Table‑Continued
Case

No･I 2Samplingdate

0ctober 2000

Relationships

amongpatients

PatientNo･

family membersBll

Strain2(male,9y)VT3'00‑E.071

B12(female,37y) '00lE.072

B13 (male, 7 y)
1 30ctober 2000

family members

B14 (female, 50 y)

B15 (female, 19 y)
14Novcmber2000

rcsidcnts in ahome fbrthe aged (BIG,B16 (female, 88 y)

B19, and B20) and family members

B17 (male, 63 y)

orB16 (817andB18)

B18 (female, 58 y)
B19 (female, 95 y)
B20 (female, 86 y)

1 5

Fcbruary 200 1

fami1y members

Cl (female, 2 y)
C2 (male, 59 y)

typeproducedVT1,

resistance4

VT 1,2'00‑E.073

VT 1, 2'00‑E.075

VT 2'00‑E.078

VT 2一oo‑E.081

Drug2

N N N N N N N0 0 0 0 0 0 0∩ ∩ ∩ ∩ n n ∩e e e C e e C

VT I, 2'00‑E.082

VT 1, 2'00‑E.083

VT 1, 2'00‑E.084

VT 1, 2'00‑E.085

VT 1, 2'00‑E.087

VT 1, 2None'00‑E.088

VT 1, 2ABPCl All the isolated bacteria were from human feces unless otherwise indicated. Bacteria were identified only ln Cases in which morethan two strains wereobtained.

2The isolates in each case exceptA4, A6, A16, A20, B6, and B19 showed the same pattem on PFGE (see Fig･ 2)･ The PFGE analysis of two strains in case No･
1 was unsuccessful due to DNA degradation.
･l verotoxin.

4ABPC: ampicillin, SM: streptomycin, TC: tctracycline.
5 well water used for purposes other than drinking water,strain), SM+TC (9 strains), or ABPC+SM+TC (2 strains)

VTl, 41 for VT2, and 34 for the both (Table)･ Sensitivities to
12 antibiotics, i.e., ABPC (ampicillin), CTX (cefbtaxlme),KM (kanamycin), GM (gentamicin), SM (streptomycin), TC(Table).

PFGE pattems of the 8 1 isolates were analyzed by uslng a(tetracycline), TMP (trimethoprim), CPFX (ciplofloxacin),FOM (fosfomycin), CP (chloramphenicol), ST (sulfamethoxa‑Bio‑Rad, Tokyo) as reported prev10usly (7). The typical PFGE

zole‑trimethoprim), and NA (nalidixic acid) were examinedpattems are shown in Figs. 1. The PFGE pattems ofmaL

by using Sensi Disk (Nippon Becton Dickinson Cot, Ltd･,

digested chromosomal DNA of the 81 isolates and cluster

gene path typing system (Progra.m No･ 5 or No･ 23; NipponTokyo) (6)･ Sixty‑two isolates.W.ere sensitive to au the anti‑analysis (Finger Printing PLUS, Bio‑比ad, Hercules, Calif･,biotics examined, but the remalnlng 1 9 strains were resistantUSA) of the 72 strains (Fig. 2) revealed a large strain‑to‑to either ABPC (6 strains), SM (1 strain), ABPC+SM (1strain or case‑to‑case variation. The dendrogram indicatedFig. I. PFGE patterns ofXbaI‑digests of chromosomal DNA ofEscherichia coli 01 57 isolates. PFGEs were performed using Pr0‑gram No. 5. Chr: chromosome.
a: Lanes 1, 8, and 15: A DNA ladder. Lane 2: '99‑E.0)4 (frompatient A12 in Table). Lane 3: '99lE.015 (A13). Lane 4: '99‑E.020.
Lane 5: '99‑E.021. Lane 6: '99‑E.024 (A15). Lane 7: '99‑E.025(A16). Lane 9: '99‑E.022. Lane 10: '99‑S.034. Lane H: '99‑E.026.
Lane 12: '99‑E.028. Lane 13: '99‑E.029. Lane 14: '99‑E.030(A17).
b: Lanes 1, 8, and 15:入DNA ladder. Lan° 2: '99‑E.044. Lan° 3:'00‑E.001(Bl). Lane 4: '00lE.002 (B2). Lane 5: '00lE.030. Lane

6: '00‑E.033. Lane 7: '00‑E.034. Lane 9: '00‑E.035. Lane 10: '00‑

E.048. Lane ll: '00‑E.049 (B3). Lane 12: '00‑E.050 (B4). Lane
13: '00‑E.051. Lane 14: 一oo‑E.053.
C: Lanes I, 8, and 15:入DNAladdcr. Lane 2: '00‑E.068 (89). Lan°

3: '00‑E,071 (all). Lane4: '00‑E.076. Lane 5: 一oo‑E.078 (B15).

Lane 6: '00‑E.079. Lane 7: '00‑E.080. Lane 9: '00‑E.081 (B16).

Lanc 10: '00‑E.082 (B17). Lane ll: '00‑E.083 (818). Lane 12:'00‑E.084 (819). Lane 13: '00‑E.085 (B20). La116 14: '00‑E.086.

163

Jpn. J. lnfect･ Dis･, 54, 2001
lV90057080901八手OS●0)守(ち.Cot000St00.CdC0)'OnN

Kb

00

1! lt.i"･ =‑99‑EO 3 4‑ ‑ ‑1‑ ‑ ‑ ‑ ‑1‑ ‑‑ 1 ‑ 1 ‑ 1 ‑ ■一 ‑ ‑00‑EO59‑ ‑ ̲1‑‑ ‑ 1‑ ̲1‑ ‑ ‑ ‑ ‑ ‑ ‑l1 ‑ ‑ ‑ ‑ ‑00‑EO55
00‑EO5600lEO58
ーーII I‑‑‑III‑‑‑‑‑

11■■■‑100 ‑EO 57‑I99‑EO35‑ 1‑1 ‑rl Ll r･1=‑ =hL

99‑EO3 899‑EO3600‑EO67‑‑

II‑‑‑

99‑EO43IIII0 0 ‑EO6399‑EO4299‑EO3700‑EO 62
一‑

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It1‑

I1‑‑‑‑‑1‑一‑‑

11‑‑‑‑‑‑

1‑

99‑EO24‑00‑EO7 7‑‑‑‑‑

11‑‑‑‑‑

99‑EOO4‑‑‑

99‑EOO899‑EOO599‑EOO799‑EOIO
1日

1rl=J卜‥=̲

99‑EO3 3･1.

99‑EO3 1‑ 1一 ‑99‑EO3 099‑EOO299‑EO2 1‑‑l■ 11 ‑99‑EO 1 4‑ IlI‑

99‑EO1 599‑EOl 199‑EO 1299‑EO 1 399‑EO3 999̲EO2699‑BOO I99‑EO2900‑EO3599‑EO3 2l一■ ‑ ‑ ‑99‑EO2200‑EO4900̲EO5000‑EO80
1‑1100‑EO3 0

100‑EO48ー‑

1‑
｢‑‑1‑

rl J=ー00‑EO8400‑EO3 300‑EO8 I00‑EO 8200‑EO5 1‑ー11‑
1‑‑00‑BOO 1100‑EO 02
00‑EO68
1‑‑‑‑

00 ‑EO7499‑EO20ー L ‑ Li I

00‑EO 5400‑EO87

I I1 ‑ ‑i L‖ = J 1 l｢001EO88
99‑EO2 599‑SO3400‑EO7 I00‑EO8699‑EO4499‑EO2800‑EO3400‑EO5300‑Eoヮ 500‑EO7800‑EO7900‑EO76Fig･ 2･ Cluster analysis ofPFGE patterns ofEscherichia colt 0157 isolates･ From 72 strains to be analyzed on PFGE figures,

three having degraded patterns were omittcd･ For the analysis,the bands far below 48･5 kb were omitted･164

Jpn. J. Infect. Dis., 54, 2001
the presence of more than ten small clusters･ Thus, it wasK. (1999): Molecular typing of enterohemorrhagic

concluded that EHEC 0 1 57:H7 epidemics in Hyogo PrefTectureEsherichia colt 0 I 57 isolates from sporadic infections

occurred in Hyogo Prefecture during 1997 and 1998 byin the past 2 years were caused by various EHECs of different

pulsed‑field gel electrophoresis. Bull. Hyogo Prefect･lnst. Public Health, 34, 1 181121 (in Japanese)･
5. Hamada, K., Shimada, K., Tsuji, H. and Ono, K. (1997):

genotypes ･The question arises as to why E. colt 0157 is so geneticallychangeable and what advantage this characteristic lends tobacteria. Emergence and rapid spread of this organism canlsolation and characterization of enterohemorrhagic

be explained by a high incidence ofhighly mutable variants

Escherichia colt in Hyogo Prefecture during outbreaks

which are found among lSOlates of EHEC and Salmonella
enterica; they may cause enhanced genetic variability of aof the bacteria throughout Japan 1996. BulL Hyogo
Prefect. lnst. Public Health, 32, 107‑1 10 (in Japanese).

population and accelerate adaptive evolution (8)･ But, this

6. Jorgensen, J. H., Tumidge, J. D. and Washington, J･ A･may not explain the observation that E. colt 0157 showed a( 1 999): Antibacterial susceptibility tests: dilution and disklarger PFGE diversitythan did Salmonella Enteritidis (9, 10)A
pattems is influenced by the number and mutability of thediffusion methods. p.1526‑1543. In P. R. Murray (ed),Manual of Clinical Microbiology, 7th ed. American
Society for Microbiology, Washington, D･C･sites recognized by the restriction enzymes. Therefわre, the7. Hamada, K., Tsuji, H., Shimada, K. and Hosoda, Y.

However, it should be mentioned that variability of PFGE

above mentioned different variabilitycould be more apparent( 1 999) : Epidemiological analysis of Salmonella serovarEnteritidis isolates from two food poISOnlng Outbreaks inthan real.

the Hyogo Prefecture by pulsed‑field gel electrophoresis･Bull. Hyogo Prer Inst. Public Health, 34, 113‑117 (inREFERENCE SJapanese).

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uslng Pulsed‑field gel electrophoresis over the period ofMicrobio1., 35, 1675‑1680.

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phoresis from December 1996 to November 2000 inHyogo Prefecture. Jpn. J. Infect. Dis･, 54, 40‑43･

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165