Laboratory and Epidemiology Communications

Phylogenetic Analysis Of Salmonella Enteritidis Isolates fromFood PoisonlngS Using Pulsed‑Field Gel Electrophoresis over thePeriod of June 1997 to December 2000 in Hyogo Prefecture
Kokichi Hamada* and Tbmohiro Oshibe

Division of Microbiology, Hyogo Prefectural Institute of Public Health,Arata‑cho 2‑1129, Hyogo‑ku, Kobe 652‑0032
Communicated by Shinsaku lmashuku(Accepted April 5, 2001)

Since 1 989, Salmonella serovar Enteritidis has become theFourteen strains belonged to genotype A and three to subtype

most prevalent among the serotypes of Salmonella foodA'(3). Genotype A was further classified into two subtypes,poiPonings in Japan (1 ･2)I We previously reported the genetic
that occurred from 1 9971 I 999 in Hyogo Prefecture (3) together

A‑a and A‑b, according to the size of the largest band >630
kb; the band in A‑a moved more slowly than that in A‑b.
There were eight A‑a subtypes and six A‑b subtypes (Figs. 1with detailed analyses of three outbreak cases (4,5). In thatand 2). Others were classi丘ed as genotypes B, C, D, and E (3).

varlations of the 2 1 isolates obtained at the fわod poISOnlngS

study, pulsed‑field gel electrophoresis (PFGE) was used

We carried out a similar genotypIC analysis Of eight new

to identify six different genotypes, A, A', B, C, D, and E.

isolatesfrom seven food poISOnlng Cases in Hyogo Prefecture

Fig. L PFGE patterns ofBlnI‑digests of chromosomalDNA of Salmonella Enteritidis isolates from 28 cases of food poisonlng Outbreaks･(A) Salmonella Enteritidis from food poisoning outbreaks (cases 22‑28) in Hyogo Prefecture in 2000.

Lanes 1,7, and13: A DNA ladder. Lane 3: Hyogo‑SE160 (genotype A‑a) from a patient in case 22. Lane 4: Hyogo‑SE172 (A‑a) from thecausative food, omelet, inthe same case. Lane 5: Hyogo‑SE1 77 (nondescript) in case 23. Lane 6: Hyogo‑SE178 (nondescript) in case 24. Lane
8 : Hyogo‑SE179 (A‑a) in case 25, Lane 9: HyogoISE187 (A‑b) in case 26. Lane 10: Hyogo‑SE190 (A‑a) in case 27. Lane 1 1 : Hyogo‑SE193(A‑a) in case 28. Lanes 2 and 12: Hyogo‑SE186 (A‑b) and Hyogo‑SE189 (A‑b): two isolates from sporadic cases 53 and 54 in 2000 as ref. 7.
Chr: chromosome.
(B) Representative PFGE patterns of Salmonella Enteritidis isolates in food poisoning outbreaks in Hyogo Prefecture in I 99712000･

Lanes 1, 8, and 15: A DNA ladder. Lane 2: HyogoISEO17 (genotypeA‑a) in case 6. Lane 3: Nishinomiya‑01 (A‑b) in case 2. Lane 4: Hyogo‑
SE103 (A') in case 14. Lane 5: Hyogo‑SEOO8 (A') in case 5, Lane 6: Hyogo‑SE127 (〟) in case 17. Lan° 7: Hyogo‑SEO65 (良) in case 10.

Lane 9: Hyogo‑SE151 (C) in case 20. Lane 10: Kakogawa‑AO3 (D) in case 1. Lane ll: Sumot0‑21 (E) in case 3. Lane 12: Hyogo‑SE177(nondescript) in case 23. Lane 1 3: Hyogo‑SE178 (nondescript) in case 24. Lane 14 : Hyogo‑SE193 (A‑a) in case 28. Chr.: chromosome.
*Co汀eSpOnding author: Fax: +8 1‑78‑53ト7080

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

in 2000 (cases 22128 in Table)･ Three phage types (PTs) weredigests (Fig･ 1) revealed the presence of丘ve genotype A (3)identified: PT 1, PT 14b, and PT 47. The stralnS Were teSted(A‑a;.cases 22, 25, 27, 28, andA‑b; case 26), and two non‑for the sensitivityto 1 2 drugs, ampicillin (ABPC), cefotaxinIe

descnpts (3) (cases 23 and 24) (Fig. lA). No co汀elation wasobserved between PFGE pattems and phagetypes (Table) as(CTX), kanamycin (KM), gentamicin (GM), streptomycln(SM), tetracycline (TC), trimethoprim (TMP), ciplofloxacin(CPFX), fosfomycin (FOM), chloramphenicol (CP),in our previous obseⅣation (3). Figure lB shows representative

PFGE patterns observed among 28 Salmonella from foodpoISOnlngS in Hyogo Prefecture in 1997‑2000･
A dendrogram obtained by molecular analysts software

su胞methoxazole‑trimethoprim (ST), and nalidixic acid (NA),by Sensi Disk (Nippon Becton Dickinson Co･, Ltd･, Tbkyo)(6)･ All, e!?ept two SM‑resistan.t isolates (cases 23 and 24),(Finger Printing Plus; Bio‑Rad, Hercules, Calif., USA) for

were sensltlVe tO the 1 2 antibiotlCS.

the isolates in 1997‑2000 is shown in Figure 2. Two large

These eight isolates were examined by PFGE uslng a geneclusters were obseⅣed, each consistlng Of several subclusters.

path typing system (Program No.2; Nippon Bio‑Rad, Tbkyo)

Various genotypICally different characteristics including(4), and the pattems were compared with those of the previous

phage type (1‑5,7) and PFGE pattem (2‑5,7) Were observed

21 isolates in 1997‑1999. PFGE ofβJ〃I chromosomal DNA

in studies of a large‑scale epidemic in Japan (I ,2) as well asDo貞‑,cI)OfT.=

8.等･:

oooMI300.専{‑‥

0.豆.idL)責‖卓d机=‑ i‑I)【古l l■(古06 ‑‥‥=7(0日‥=…T‥‥‥…竿‥‥.".1㍗呈上｣

Hyogo‑SE151 (C)Kakogawa‑AO3 (D)Sumot0‑2 1 (E)Hyogo‑SE 1 78
Hyogo‑SEO65 (B)Hyogo‑SE 1 77
t‑It

Hyogo‑SEO17 (A‑a)
Nisinomiya‑0 1 (A‑b)

Hyogo‑SE127 (A')

Hyog6‑sE193 (A‑a)
Hyogo‑SEOO8 (A')

Hyogo‑SE103 (A‑)
Fig. 2. Cluster analysis of PFGE patterns of Salmonella Enteritidis.
Bands below 48.5 kb were omitted from the analysis. Genotypes are indicated in parenthcscs.

Table. Food poisonlng Outbreaks of Salmonella Enteritidis in 2000 in Hyogo Prefecture and causative strains
Causative strain usedCaseNo.

DateOccasion

ExposedPatients

Strain*22Apr.

2000

Lunchservedforvolunteer

23Aug. 2000

oldmengroupbya48

lnatown

Chinessdishes atahoteHn

20

Hyogo‑SE

Hyogo‑SE43318

140

I

Phage type Dmg rcsistamce

I60

72

Hyogo‑SE47

Nonc47177

1SMOsaka City(Osaka Prefecture)* *24

Aug. 2000

Funeral meal atan assemblyhal1

Hyogo‑SE178

1SMin Osaka City(Osaka Prefecture)* *25

Aug.

2000

260ct. 2000

Sushiatasushi‑barinKobeWedding recept10n at a rCStaurant

CityUnknown
29

69102

Hyogo‑SE179

Hyogo‑SE

187

141bNone
Nonein Kyoto City(Kyoto Prefecture)* *27Nov.

2000

Eggfromasupermarket

athomc22

Hyogo‑SEI

9047

Nonc

33

Hyogo‑SE

19347

Nomc

in Kakogawa City28

Dec.

2000

Raw‑leverand‑beeratarestaurant in Akoh City〜 The strains used were all human orlg]n except Hyogo‑SE172, an isolatefromthe causative food.

** A part of the patients related to the poISOmngS were residents in Hyogo Prefecture.

39

fTrom December 1996 to November 2000 in Hyogo Prefecture
Tbmohiro Oshibe and Kokichi Hamada*Division ofMicrobiology, Hyogo Prefectural Institute of Public Health,Arata‑cho 2‑1129, Hyogo‑ku, Kobe 652‑0032
Communicated by Shinsaku lmashuku(Accepted April 5, 2001)

Since I 989, Salmonella serovar Enteritidis has become theisolates were tested fわr sensitivity to 12血ugs (ampicillin

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

lABPC]･ cefotaxime lCTX]･ kanamycin lKM]･ 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], ciplonoxacin lCPFX], fosfo甲yCin lFOM], chloram‑

Enteritidis from June 1997 to December 2000 in Hyogo

phenicol lCP], sulfamethoxazole‑trlmethoprim lST], andnalidixic acid PA]) by Sensi Disk (Nippon Becton Dickinson

Prefecture, we demonstrated, uslng Pulsed‑fleld gel electro‑

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

causative strains for these food poisonings (6). We report here

majority (3 I strains) were sensitive to the drugs, while thethe genotypic variations and phylogenetic analysis of the totalremainlng 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 fわr 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 phase types

Bio‑Rad, Tokyo) 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
laurylsulfate for I h at 500C for complete lysis. The typical(基eaetion ⊇oes型ot Conform), and UT (坦n!ypable)･ The

PFGE pattems are showninFigures I A and I Band summa‑
rized in Table. The PFGE pattems obtained with BlnI‑digested

remalning 3 1 isolates were not tested (Table). All of the 55*Corresponding author: Fax +8 1 ‑78‑53 1 ‑7080

chromosomal DNAsshowed 1 5 different pattems. A dominant40