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

Laboratory and Epidemiology Communications●Phylogenetic Analysis Of Genotypic Variations of Salmonella Enteritidis

Isolate's from Sporadic Infections Using Pulsed‑Field Gel Electrophoresis
from December 1 996 to November 2000 in Hyogo Prefecture
Tbmohiro Oshibe and Kokichi Hamada*Division of Microbiology, Hyogo Prefectural Institute of Public Health,Arata‑cho 2‑1‑29, Hyogo‑ku, Kobe 652‑0032
Communicated by Shinsaku lmashuku(Accepted April 5, 2001)

Since 1 989, Salmonella serovar Enteritidis has become theisolates were tested for sensitivity to 12 drugs (ampicillin

most prevalent among the Salmonella serotypes in Japan ( I ,2).

lABPC], cefotaxime lCTX], kanamycin lM】, gentamiein

In the previous (3‑5) and preceding papers (6) in which were

【GM】, streptomycin 【SM], tetracycline 【TC], trimethoprlm

reported 28 food poISOnlng Outbreaks caused by SalmonellalTMP], ciplofloxacin lCPFX], fosfon!ycin lFOM] , chloram‑

Enteritidis from June 1997 to December 2000 in Hyogo

phenicol lCP], sulfamethoxazole‑trlmethoprim lST], andPrefecture, we demonstrated, uslng pulsed‑fleld gel electro‑

nalidixic acid 【NA】) by Sensi Disk (Nippon Becton Dickinson

phoresis (PFGE), a clear phylogenetic relationship among theCo., Ltd., Tbkyo) as reported in a previous paper (7). The

causative strains for these food poisonings (6). We report here
the genotypic variations and phylogenetic analysis of the total

majority (3 1 strains) were sensitive to the drugs, while theremalnlng 24 strains were resistant to SM, ABPC, TC, and/or GM (Table).

55 isolates, including 50from human feces in sporadic infec‑tion cases, two from samples from chickens, and three fromAll of the 55 isolates were examined fb∫ PFGE pattems

using a gene path typing system (Program No. 2; Nippon

chicken egg samples (Table).

Twenty‑four of the 55 isolates were tested for phagetypes

Bio‑R礼d, Tbkyo) in a manner reported previously (3), except(PTs), the types being PTl, PT4, PT6, PT6a, PT9b, RDNC

that lysozomal lysates were further treated with 1 % sodium(甚eaction Qoes型ot Conform), and UT (iin圭ypable). The

laurylsulfate for 1 h at 500C for complete lysis. The typicalremaining 3 1 isolates were not tested (Table). All of the 55

PFGE pattems are shown in Figures 1 A and 1 B and summa‑
rized in Table. The PFGE pattems obtained with BlnI‑digested*Corresponding author: Fax +8 1‑78‑53 I‑7080

chromosomal DNAsshowed I 5 different pattems. A dominant

40Jpn. J. Infect. Dis., 54, 2001
Table･ List of Salmonella Enteritidis strains of sporadic occurrences over the past 4 years from December I 996 to November 2000 1)

cNa.S.e strain (sSeoxTracgee, (feS;e%tTeflion.fgds, Phagetype resl?tTngce2, :aF‑GeEa:attem:Sept. 1997

Not tested

SM

Case

12'96‑S. 070

Human (F, 0;ll)

Dec. 1996

Not tested

SM

Case

23'96‑S.

HumanJam. 1997

Nottested

None

Case

34'96‑S. 090

Human (F, 2)

Mar. 1997

Not tested

None

Case

35'97‑S.

HumanMar. 1997

Not tested

SM

Case

56'97‑S. 033

Human (F, 9)Åug. 1997

Not tested

SM

Case

17'97‑S.

039

Human(M,9)Åug. 1997

Not tested

SMCase78'97‑S.

040

Human(M,9)Åug. 1997

Not tested

SMCase79'971S.

041Åug. 1997

Not tested

SM

Case

91

Tatsun0‑03076

001

Human(F,20)(M,6)(M,Human(F,1)6)

10'97‑S. 047

Human (F, 3)

Sept. 1997

Not tested

None

11'97‑S.

Human(M,8)

Sept. 1997

Not tested

SM

12'97‑S.

Human(M,8)

13048
051

797‑S. 052

Human (M, 4)

Case

3

Case

13

Sept. 1997

Not tested

TC

Case

Oct. 1997

Nottested

SM

Case

3SM

Case

1414'97‑S. 055

Human (F, 6)

Oct. 1997

Nottested

15'97‑S. 064

Human (F, 4)

Nov. 1997

Not tested

None

Case

116'971S.

069

Human(M,5)

Nov. 1997

Not tested

None

Case

317'97‑S.

071

Human(M,ll)

Nov. 1997

Not tested

None

Case

318'98‑S. 015

Human (F, 2)

May 1998

Not tested

None

Case

3
319'98‑S. 018

Human (M, 3)

May 1998

Not tested

None

Case

20'98‑S.

023

Human(M,1)

June 1998

Not tested

ABPC

Case

321'98‑S.

024

Human(M,7)

June 1998

Not tested

None

Case

322'98‑S.

029

23'98‑S.

031

24'98‑S, 035

2526

Human(M,3)

June 1998

Not tested

SMCase22

Human(M,3)

July 1998

Nottested

SM

Case

22

Human (F, 9)

June 1998

Not tested

ABPC'98‑S. 036

Human (ど, 5)Åug. 1998'98‑S. 045

Human (F, 5)

Sept. 1998

Not tested

ABPC

Not tested

SMCase

Case

253
≒

Case

27'98‑S.

Sept, 1998

Not tested

None

Case

28'98‑S. 058

Human (F, 2)

Oct. 1998

Nottested

None

Case

329'98‑S.

059

Human5)Oct. 1998

Nottested

None

Case

330'98‑S.

0632)

331'98‑S.

071

32048

Human

Human
Human(M,14)(M,Nov. 1998

Not tested

None

Case

Dec. 1998

Not tested

None

Case

3May

1999

4

None

Case

3

Hyogo‑SEIOO

Human(M,11)

33

Hyogo‑SEIOIHuman(F,3)

June

1999

134

Hyogo‑SE102

Human (M, 1)

July

1999

635

Hyogo‑SEl 13

Human (M, 3)Åug. 1999

SM

6

Case

33

None

Case

3

None

Case

3

SM

Case

3636

Hyogo‑SEl 14

Human (M, 3)

Sept.

1999

137

Hyogo‑SEl 15

Human (F, 2)

Sept.

1999

4

None

Case

338

Hyogo‑SE155

Human (M, 4)

Nov. 1999

UT

None

Case

339

Hyogo‑SE156

Human (M, 1)

Nov. 1999

6None

Case

340

Hyogo‑SE157

Human (F, 2)

Dec. 1999

RDNC

None

Case

341

Hyogo‑SE158

ABPC, TC, SM Case 41 ≒ Case 34)

Human (F, 6)

Jam. 2000

6a42

Hyogo‑SE159

Human (F, 7)

Feb. 2000

9b

None

Case

43

Hyogo‑SE173

Human (M, 1)

July

6None

Case

344

Hyogo‑SE175

Human (F, 2)Åug. 2000

4None

Case

345

Hyogo‑SE176

Human (M, 2)Åug. 2000

4None

Case

46

Hyogo‑SE1 83

Human (M, 1)

Sept.

2000

2000

SMUT

Case

4234247

Hyogo‑SE184

Human (F, 5)

Oct. 2000

6None

Case

348

Hyogo‑SE185

Human (F, 1 1)

Sept. 2000

6None

Case48June

149

Kakogawa‑AO2

Chicken50

Kakogawa‑A 1 2

Chicken

5 1

Hyogo‑SEO6452199733)

38)(M,(F,Case26

ABPC, TC, SM, GM Case 49 ≒ Case 35)

June 1997

UTTC, SM Case 50

Chicken egg

Oct. 1998

1TC, SM Case 51

Hyogo‑SE 1 74

Chicken eggÅug. 2000

None

Case

353

Hyogo‑SE1 86

Human (F, 54)

Nov. 2000

6

None

Case

354

Hyogo‑SE1 89

Human (M, 22)0ct. 2000

4

None

Case

355

Hyogo‑SE 1 92

Chicken egg

Nov. 2000

UT

None

Case341)In three cases, the other serotypes of Salmonella were also isolated; that is, S･ Typhimurium in case No･ I , S･ Paratyphi‑B in casell, and S. Thompson in case 35.

2) sM: streptomycin, TC: tetracycline, ABPC: ampicillin, GM: gentamicin･3'The largest band ('630kb) had only a slightly upper location a.s compared to case 3 (see lane 4 in Fig･ lB)･

4)Two bands smaller than 48.5kb different from case 3 were exsISted (lane 6 in Fig. lB).
5)An extra band exsisted near 48.5kb as compared to case 3 (lane ll in Fig. lB).

41

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

Fig･ 1 ･ PFGE patterns ofBlnトdigests of chromosomal DNA of Salmonella Enteritidis isolates from sporadic infections･(A) Lanes 1, 8, and 15: ^DNA ladder. L?ne 2: Hyogo‑SE186 (case 53). Lane 3: Tatsun0‑03 (case 1). Lane 4: '96lS･070 (case 2). Lane 5: '96‑S･076(case 3). Lane 6: '97‑S. 001 (case 5). Lane 7: '971S.039 (case 7). Lane 9: '97‑S.041 (case 9)･ Lane 10: '98‑S･048 (case 27). Lane 1 I: Hyogo‑SEIOl (case 33). Lane 12: Hyogo‑SEl 14 (case 36). Lane 13: '97‑S.055 (case 14). Lane 14: '97‑S･064 (case 15)I Chr.: chromosome･(B) Lanes 1, 8, and 15: ^DNAladder. Lane 2: '971S.069 (case 16). Lane 3: '98‑S.029 (case 22)･ Lane 4: '98‑S･036 (case 25)･ Lane 5: '98‑S1045(case 26). Lane 6: Hyogo‑SE158 (case 41). Lane 7: Hyogo‑SE159 (case 42). Lane 9: Hyogo‑SE183 (case 46)･ Lane 10: Hyogo‑SE185 (case48). Lane 1 I: Kakogawa‑AO2 (case 49). Lane 12: Kakogawa‑A12 (case 50). Lane 13: Hyogo‑SEO64 (case 51). Lane 14: Hyogo‑SE189 (case54). Chr: chromosome.

dQ皇a)0{E00

HHuH川ふrt)○<]Hn一T

Sp‑Case4 8

Hyogo‑SEIO3 (A.)
Sp‑Case4 11‑‑I‑ ̲ ‑ I‑I‑t I‑1.‑‑.■‑1‑̲1

Sp‑Case49

Hyogo‑SEOO8 (A')
Sp‑Case 1 6
Sp‑Case25
Sp‑Case54

Sp‑Case5 3
Sp‑CaseO3

SpICase27

Hyogo‑SEO 1 7 (A‑a)
Nisinomiya‑0 1 (A‑b)
Hyogo‑SE I 27 (A.)
Hyogo‑SE1 93 (A‑a)
Sp‑Case5 0
Sp‑Case42
Sp‑Case46
Sp‑Case26

Hyogo‑SE15 1 (C)Sp‑Case 1 4
Kakogawa‑AO3 (D)Sp‑CaseO7

Sp‑Case5 1
Sp‑Case3 3
Sp‑Case22
Sp‑Case3 6

Hyogo‑SE 1 78
Sp‑CaseO9
Sumot0‑21 (E)Sp‑CaseO2

Hyogo‑SEO65 (B)Hyogo‑SE 1 77
Sp‑CaseO5
Sp‑CaseO 1

Sp‑Case 1 5
Fig.2. Cluster analysis ofPFGE patterns of Salmonella Enteritidis shown in Figure 1 and the preceding one (6)I FortheanalysIS, the bands below 48･5 kb were omitted. Sporadic infections are indicated by SP‑Case number･ Cases from foodpoisonings were indicated by strain names showing genotypes in parentheses according to refs･ 5, 6･42Jpn. J. Infect. Dis., 54, 2001
16, 1'‑2'.

pattem was that seen in Pase 3 (at least 32 cases of the 55cases) supposedly belonglng tO genOtype A‑b (6). There wasno correlation between phage types and PFGE pattems (Table).
The absence of correlation between PFGE pattems and phage

2. Terajima, J., Nakamura, A. and Watanabe, H. (1998):

types was previously reported f♭r fわod poisoning cases (5,6).

gel electrophoresis. Epidemiol. Infect., 120, 223‑229.

In summary, out of55 isolates, 32 cases (about 60%) belongedto A‑b (6), and 2 isolates (cases 41 and 49) had a pattem
close to A‑b･ There were one isolate ofA‑a type (6) (case 25),
one lSOlate similar to D type (5)(case 14), and 19 belonging

3. Hamada, K., Tsuji, H., Shimada, K. and Hosoda, Y.

EpidemiologlCalanalysis of Salmonella enterica Enterit‑idis isolates in Japan by phase‑typlng and pulsed‑field( 1 999): Epidemiological analysis of Salm?nella serovarEnteritidis isolates from two food poISOnlng Outbreaks

in Hyogo Prefecture by pulsed‑field gel electrophoresis.

Bull. Hyogo Prefect. Inst. Public Health, 34, I 13‑I 17 (into other nondescript genotypes (5,6).

Thus, the Salmonella Enteritidis isolates from variousJapanese).

sporadic infection.s showed a great genetic diversityas in the

4. Tsuji, H., Shimada, K., Hamada, K. and Nakajima, H.

cases of fわod poISOning outbreaks (5,6). This was clearly(2000): Outbreak of Salmonella Enteritidis caused bydemonstrated by clusteranalysis (Finger Printing Plus; Bio‑Rad,Hercules, Calif･, USA) (Fig1 2)･ The dendrogra.ms indicatedcontaminated buns peddled by a producer uslng travel‑

the presence or two large clusters, each contalnlng Several

an epidemiological study uslng Pulsed‑field gel electro‑

ing Cars in Hyogo and neighboring Prefectures in 1999:

subclusters as reported for 28 food poisoning cases (6). Thephoresis. Jpn. J. Infect. Dis., 53, 23‑24.

5. Hamada, K., Tsuji, H., Izumlya, H. and Watanabe, H.

sporadic infections of Salmonella Enteritidis in Hyogo
Prefecture over the past 4 years appear to have been caused(2000): Comparison by pulsed‑field gel electrophoresis

by genotypically different bacteria as in food poisoning casesof Salmonella Enteritidis genotypes from various food(6). The data appears to renect the large‑scale epidemic due
to Salmonella Enteritidis since 1 989 in Japan (1 ,2) including

poISOnlng Outbreaks kom 1997 to 1999 in Hyogo Prefec‑ture. Jpn. J. Infect. Dis., 53, 25‑27.

6･ Hamada, K･ and Oshibe, T･ (2001): Phylogenetic.analysis

Hyogo Prefecture (3‑6).

of Salmonella Enteritidis isolates from food poISOnlngS

The authors are grateful to Drs. Hidemasa Izumlya anduslng Pulsed‑field gel electrophoresis over the period ofHaruo Watanabe, National Institute of Infectious Diseases,
Tokyo, for the phagetyping of our isolates and for a criticalJune 1997 to December 2000 in Hyogo Prefecture. Jpn.
J. Infect. Dis., 54, 38‑40.
7. Hamada, K., Oshibe, T., Tsuji, H., Yoshida, S. andAoki, Y.

reading of the manuscrlpt.

(2000)‥ Outbreaks of heat stable enterotoxfn‑producing

Esherichia coli 0169 in the Kinki distrlCt in Japan:

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43